( PDF ) Avaliação bioquímica-fisiológica de clones de batata em relação ao alumínio

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UNIVERSIDADE FEDERAL DE SANTA MARIA

CENTRO DE CIÊNCIAS RURAIS

PROGRAMA DE PÓS-GRADUAÇÃO EM AGRONOMIA

AVALIAÇÃO BIOQUÍMICA-FISIOLÓGICA DE

CLONES DE BATATA EM RELAÇÃO AO ALUMÍNIO

TESE DE DOUTORADO

Luciane Almeri Tabaldi

Santa Maria, RS, Brasil

2008

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AVALIAÇÃO BIOQUÍMICA-FISIOLÓGICA DE CLONES DE

BATATA EM RELAÇÃO AO ALUMÍNIO

por

Luciane Almeri Tabaldi

Tese apresentada ao Curso de Doutorado do Programa de Pós-

Graduação em Agronomia, da Universidade Federal de Santa Maria

(UFSM, RS), como requisito parcial para obtenção do grau de

Doutor em Agronomia.

Orientador: Prof. Fernando Teixeira Nicoloso

Santa Maria, RS, Brasil

2008

ads:

Universidade Federal de Santa Maria

Centro de Ciências Rurais

Programa de Pós-Graduação em Agronomia

AVALIAÇÃO BIOQUÍMICA-FISIOLÓGICA DE CLONES DE BATATA

EM RELAÇÃO AO ALUMÍNIO

Elaborada por

Luciane Almeri Tabaldi

Comissão Examinadora:

__________________________________________

Fernando Teixeira Nicoloso, Dr. (UFSM)

(Presidente/Orientador)

___________________________________________

Maria Rosa Chitolina Schetinger, Drª. (UFSM) (Co-orientador)

____________________________________________

Dílson Antônio Bisognin, Dr. (UFSM) (Co-orientador)

____________________________________________

Arthur Germano Fett Neto, Dr. (UFRGS)

____________________________________________

Fernando Irajá Félix de Carvalho, Dr. (UFPEL)

Santa Maria, 11 de abril de 2008.

Viver e não ter a vergonha de ser feliz.

Cantar e cantar e cantar

a beleza de ser um eterno aprendiz!!!

Gonzaguinha

Aos meus queridos pais

Edenir e Joice,

Pela educação, apoio, dedicação,

compreensão e total incentivo

para realizar este trabalho

OFEREÇO

Ao meu grande amor Márcio,

pelo amor,

incentivo, paciência,

apoio e compreensão

DEDICO

AGRADECIMENTOS

Sou grata a

todos que, indistintamente, através de sua vivência e

compreensão, contribuíram para que eu chegasse até aqui. Agradeço em especial:

Ao professor Fernando Teixeira Nicoloso, pela orientação, amizade, confiança

e compreensão recebida e acima de tudo pelo exemplo de profissional, que

contribuiu incansavelmente para o meu crescimento pessoal e profissional. Obrigada

pela paciência e apoio em todos os momentos que marcaram o curso.

Aos professores Maria Rosa Schetinger e Dílson A. Bisognin, pela orientação,

acompanhamento e participação constante na realização deste trabalho.

Um agradecimento especial à Denise Cargnelutti, à Jamile F. Gonçalves, ao

Gabriel Y Castro, à Renata Rauber e à Liana Rossato, pessoas competentes que

além de grandes amigos, são meus colegas de laboratório e me auxiliaram no

desenvolvimento desse trabalho: vocês são “três vezes ótimos”!!!

Às amigas Joseila Maldaner, Etiane Skrebski e Luciane Pereira que da

mesma forma tiveram participação ativa e importante na realização deste trabalho: o

auxílio de vocês foi valioso.

Aos demais colegas do Laboratório de Biotecnologia Vegetal e do Laboratório

de Enzimologia Toxicológica, pela amizade e carinho.

A todo corpo docente do Programa de Pós-Graduação em Agronomia que

proporcionaram minha formação.

Aos colegas e funcionários do Programa de Pós-Graduação em Agronomia

pela convivência amiga e enriquecedora.

À toda minha família, em especial meus pais Edenir e Joice, irmãos Tarciane

e Ronaldo e sobrinhos Michele e Rodrigo pelo carinho, compreensão, ajuda,

paciência e essencialmente pelo incentivo para que eu vencesse mais essa etapa.

Ao meu amor Márcio, pelo amor e pela ajuda, por sempre me estimular,

acompanhar e entender os momentos de ausência.

À minha grande amiga Rosélia: “tudo o que sempre esperei de uma amiga

encontrei em ti”. Obrigada por tudo!!!

Igualmente sou grata a CAPES, pelo apoio financeiro à realização deste

trabalho.

A todas as pessoas que colaboraram, de forma direta ou indireta, para a

realização deste trabalho, os meus sinceros agradecimentos.

SUMÁRIO

INTRODUÇÃO GERAL..............................................................................................19

OBJETIVOS...............................................................................................................21

REVISÃO BIBLIOGRÁFICA......................................................................................22

1 Os solos ácidos no mundo...................................................................................22

2 Alumínio (Al)..........................................................................................................23

2.1 Química do Al.......................................................................................................23

2.2 Efeitos bioquímicos e fisiológicos do alumínio.....................................................25

2.3 Estresse oxidativo................................................................................................28

2.4 Sistema antioxidante de plantas …….………………………………………………29

2.4.1 Sistema antioxidante enzimático.......................................................................30

2.4.2 Sistema antioxidante não enzimático................................................................31

3 Fosfatases ácidas………………….……………………………………...……………32

4 Batata......................................................................................................................33

RESULTADOS E DISCUSSÃO.................................................................................35

CAPÍTULO I - Respostas fisiológicas e bioquímicas de quatro clones de batata

expostos ao Al..........................................................................................................36

Artigo I - Physiological and oxidative stress responses of four potato clones to

aluminum in nutrient solution………..……………………..……………...…...…......37

ABSTRACT ...............................................................................................................38

RESUMO ...................................................................................................................39

INTRODUÇÃO ..........................................................................................................41

MATERIAL E MÉTODOS ..........................................................................................43

RESULTADOS...........................................................................................................47

DISCUSSÃO .............................................................................................................54

REFERÊNCIAS..........................................................................................................59

Manuscrito I - Oxidative stress is an early symptom triggered by aluminum in

Al-sensitive potato plantlets...................................................................................65

ABSTRACT................................................................................................................66

INTRODUÇÃO...........................................................................................................67

MATERIAL E MÉTODOS...........................................................................................70

RESULTADOS...........................................................................................................74

DISCUSSÃO .............................................................................................................82

REFERÊNCIAS..........................................................................................................88

CAPÍTULO II - Efeito do alumínio na atividade in vitro de fasfatases ácidas em

quatro clones de batata...........................................................................................94

Manuscrito II - Aluminum on the in vitro activity of acid phosphatases of four

potato clones cultivated in three growth systems…………...……………………..95

ABSTRACT ...............................................................................................................96

RESUMO....................................................................................................................97

INTRODUÇÃO...........................................................................................................98

MATERIAIS E MÉTODOS..........................................................................................99

RESULTADOS.........................................................................................................102

DISCUSSÃO............................................................................................................104

REFERÊNCIAS........................................................................................................110

CAPÍTULO III - Efeito do alumínio no teor de micronutrientes em plântulas de

batata.......................................................................................................................115

Manuscrito III - Micronutrient concentration in potato clones with distinct

physiological sensitivity to Al stress…………………………..……………………116

ABSTRACT .............................................................................................................117

RESUMO..................................................................................................................118

INTRODUÇÃO.........................................................................................................119

MATERIAIS E MÉTODOS........................................................................................121

RESULTADOS E DISCUSSÃO................................................................................122

REFERÊNCIAS........................................................................................................128

CAPÍTULO IV - Respostas localizadas e sistêmicas de estresse oxidativo

induzidas por alumínio em batata (Solanum tuberosum L.) cultivadas em

sistema de raízes divididas...................................................................................133

Manuscrito IV - Local and systemic oxidative stress responses induced by

aluminum in two potato clones (Solanum tuberosum L.) that differ in Al-

avoidance………………………………………………………...……………………….134

ABSTRACT .............................................................................................................135

INTRODUÇÃO.........................................................................................................136

MATERIAIS E MÉTODOS........................................................................................139

RESULTADOS.........................................................................................................143

DISCUSSÃO............................................................................................................153

REFERÊNCIAS........................................................................................................162

CONSIDERAÇÕES FINAIS.....................................................................................167

REFERÊNCIAS........................................................................................................169

APÊNDICE A -.........................................................................................................178

APÊNDICE B -.........................................................................................................179

APÊNDICE C -.........................................................................................................181

LISTA DE FIGURAS

FIGURA 1 – Distribuição das atividades relativas de Al

e das espécies

mononucleares de Al-OH em função do pH...............................................................24

FIGURA 2 – Representação dos passos intermediários da redução do

oxigênio......................................................................................................................28

FIGURA 3 – Relação entre mecanismos celulares pró-oxidantes, antioxidantes e

estresse oxidativo.......................................................................................................29

FIGURA 4 – Representação dos mecanismos de defesa usando as enzimas

antioxidantes SOD, CAT e APX contra as EROs.......................................................30

Artigo I

FIGURA 1 - Aluminum concentration in roots (A) and shoot (B) of potato plants

(Macaca, S. microdontum, SMIC148-A and Dakota Rose clones) submitted to

increasing Al levels for 7 days…………......................................................................48

FIGURA 2 - Relative Root Growth (A), shoot length (B) and total number of nodal

segments (C) in potato plants (Macaca, S. microdontum, SMIC148-A and Dakota

Rose clones) submitted to increasing Al levels for 7

days……………………………………………….……………………………………….…49

FIGURA 3 - Concentration of H

in roots (A) and shoot (B) and catalase activity of

roots (C) and shoot (D) in potato plants (Macaca, S. microdontum, SMIC148-A and

Dakota Rose clones) submitted to increasing Al levels for 7

days…..………………………….……………………..…..……………………..…………51

FIGURA 4 - Chlorophyll (A) and carotenoid (B) concentrations in potato plants

(Macaca, S. microdontum, SMIC148-A and Dakota Rose clones) submitted to

increasing Al levels for 7 days…..…………………………………………………...……52

FIGURA 5 - Lipid peroxides of roots (A) and shoot (B) and protein carbonyl of roots

Rose clones) submitted to increasing Al levels for 7

days………………………………………………………..……………………………...…53

Manuscrito I

FIGURA 1 – Time-course of Al inhibition of root length (Macaca (A) and SMIC148-A

(B) clones) of potato plantlets exposed to different concentrations of Al in nutrient

solution………………………………………………..……………………………………..75

FIGURA 2 – Effect of Al on lipid peroxidation over time in Macaca (Root (A) and

Shoot (B)) and SMIC148-A (Root (C) and Shoot (D)) potato

clones……………………………………….……………………………………………….76

FIGURA 3 – Effect of Al on catalase activity over time in Macaca (Root (A) and Shoot

(B)) and SMIC148-A (Root (C) and Shoot (D)) potato

clones………………………………………………………………………………………..77

FIGURA 4 – Effect of Al on ascorbate peroxidase (APX) activity over time in Macaca

(Roots (A) and Shoot (B)) and SMIC148-A (Roots (C) and Shoot (D)) potato clones.

APX activity was expressed as µmol ascorbate oxided mim

-1

protein…………………………………………………………………………………..…...79

FIGURA 5 – Effect of Al on non-protein thiol groups (NPSH) contents over time in

Macaca (Roots (A) and Shoot (B)) and SMIC148-A (Roots (C) and Shoot (D)) potato

clones……………………………………………………...………………...………………80

FIGURA 6 – Effect of Al on Ascorbic acid content over time in Macaca (Roots (A) and

Shoot (B)) and SMIC148-A (Roots (C) and Shoot (D)) potato

clones………………………………………………………………………………………..81

Manuscrito II

FIGURA 1 - Effect of increasing Al concentration on the in vitro acid phosphatase

activity of roots (A) and shoot (B) of potato plantlets (Macaca, S. microdontum,

SMIC148-A and Dakota Rose clones) grown in

vitro…………………………………………………………………………………………102

FIGURA 2 - Effect of increasing Al concentration on the in vitro acid phosphatase

activity of roots (A) and shoot (B) of potato plantlets (Macaca, S. microdontum,

SMIC148-A and Dakota Rose clones) grown in

hydroponics………………………..………………………………………………………104

FIGURA 3 - Effect of increasing Al concentration on the in vitro acid phosphatase

activity of roots (A) and shoot (B) of potato plantlets (Macaca, S. microdontum,

SMIC148-A and Dakota Rose clones) grown in

greenhouse………………………………...………………………………………………105

Manuscrito III

FIGURA 1 - Effect of increasing Al concentration on the root (A) and shoot (B) zinc

(Zn) concentration and root (C) and shoot (D) iron (Fe) concentration of potato

clones, Macaca, S. microdontum, SMIC148-A and Dakota Rose, submitted to

increasing Al concentrations for 7 days………………………………………..……….124

FIGURA 2 - Effect of increasing Al concentration on the root (A) and shoot (B)

manganese (Mn) concentration and root (C) and shoot (D) copper (Cu) concentration

of potato clones, Macaca, S. microdontum, SMIC148-A and Dakota Rose, submitted

to increasing Al concentrations for 7 days…………...…………………………………126

Manuscrito IV

FIGURA 1 - Effect of varying Al concentrations on chlorophyll concentration of the (A)

Al-sensitive (Macaca) and (B) Al-tolerant (SMIC148-A) clones, and carotenoid

concentration of the (C) Al-sensitive (Macaca) and (D) Al-tolerant (SMIC148-A)

clones in a split-root system……..….………………………………………...…………144

FIGURA 2 - Effect of varying Al concentrations on shoot catalase (CAT) activity of the

(A) Al-sensitive (Macaca) and (B) Al-tolerant (SMIC148-A) clones, and protein

oxidation of the (C) Al-sensitive (Macaca) and (D) Al-tolerant (SMIC148-A) clones in

a split-root system…………………………………………………………………………146

FIGURA 3 - Hydrogen peroxide concentration in shoot of the (A) Al-sensitive

(Macaca) and (B) Al-tolerant (SMIC148-A) clones, and inof roots in the (C) Al-

sensitive (Macaca) and (D) Al-tolerant (SMIC148-A) clones under varying Al

concentrations in a split-root system………………………………………………...….147

FIGURA 4 - Lipid peroxidation in shoot of the (A) Al-sensitive (Macaca) and (B) Al-

tolerant (SMIC148-A) clones, and in roots of the (C) Al-sensitive (Macaca) and (D) Al-

tolerant (SMIC148-A) clones under varying Al concentrations in a split-root

system………………………………………………………………...……………………149

FIGURA 5 - Non-protein thiol groups (NPSH) concentration in shoot of the (A) Al-

sensitive (Macaca) and (B) Al-tolerant (SMIC148-A) clones, and in roots of the (C) Al-

sensitive (Macaca) and (D) Al-tolerant (SMIC148-A) clones under varying Al

concentrations in a split-root system…………………………………………..………..150

FIGURA 6 - Effect of varying Al concentrations on acid phosphatase (APases)

activity in shoot of the (A) Al-sensitive (Macaca) and (B) Al-tolerant (SMIC148-A)

clones, and in roots of the (C) Al-sensitive (Macaca) and (D) Al-tolerant (SMIC148-A)

clones in a split-root system……………………………………………………..………152

FIGURA 7 – Shoot and roots of potato plants, Macaca clone (Al-sensitive), exposed

to 0/0 mg Al L

-1

(A,B), 50/50 mg Al L

-1

(C,D), 0/100 mg Al L

-1

(E,F), 100/100 mg Al L

-1

(G,H) and 0/200 mg Al L

-1

(I,J), in split-root system…………………….…………….155

FIGURA 8 – Shoot and roots of potato plants, SMIC148-A clone (Al-tolerant),

exposed to 0/0 mg Al L

-1

(A,B), 50/50 mg Al L

-1

(C,D), 0/100 mg Al L

-1

(E,F), 100/100

mg Al L

-1

(G,H) and 0/200 mg Al L

-1

(I,J), in split-root system……………………….156

RESUMO

Tese de Doutorado

Programa de Pós-graduação em Agronomia

Universidade Federal de Santa Maria

AVALIAÇÃO BIOQUÍMICA-FISIOLÓGICA DE CLONES DE BATATA

EM RELAÇÃO AO ALUMÍNIO

AUTORA: LUCIANE ALMERI TABALDI

ORIENTADOR: FERNANDO TEIXEIRA NICOLOSO

Data e Local: Santa Maria, 11 de abril de 2008.

O alumínio (Al) é o metal mais abundante na crosta terrestre, afetando o

crescimento e desenvolvimento das plantas. O objetivo deste trabalho foi investigar

e comparar respostas bioquímicas e fisiológicas de clones de batata, Macaca,

SMIC148-A, Dakota Rose e Solanum microdontum, expostos a 0, 50, 100, 150 e 200

mg Al L

-1

em solução nutritiva (pH 4,0). Após sete dias, o conteúdo de Al foi em

média 3,9, 2,8, 3,6 e 3,7 vezes maior nas raízes que na parte aérea nos clones

Macaca, S. microdontum, SMIC148-A e Dakota Rose, respectivamente. Baseado no

crescimento relativo da raiz, S. microdontum e SMIC148-A foram considerados

tolerantes ao Al e Macaca e Dakota Rose sensíveis ao Al. Foi observado inibição no

crescimento da parte aérea somente no clone Macaca. Vários parâmetros

bioquímicos foram afetados, principalmente nos clones sensíveis ao Al, como o

aumento na concentração de H

, a atividade da catalase (CAT) e a peroxidação

lipídica, e a redução no conteúdo de clorofila e carotenóides. A concentração de

zinco, manganês, ferro e cobre foi maior nas raízes que na parte aérea em todos os

clones. Um aumento na concentração desses micronutrientes foi observado

somente no clone S. microdontum, enquanto uma redução foi observada nos clones

Macaca, SMIC148-A e Dakota Rose com o suprimento de Al. Com o objetivo de

analisar o efeito do Al na atividade in vitro de fosfatases ácidas (APases), os quatro

clones de batata cresceram in vitro, em hidroponia ou em casa de vegetação. Em

plântulas in vitro, APases de raízes foram inibidas por Al em S. microdontum e

Dakota Rose e ativadas em Macaca em todos os níveis de Al. Em plântulas de

hidroponia, APases de raízes aumentaram em Macaca em 50 mg L

-1

, enquanto

diminuíram em S. microdontum em todos os níveis de Al. Em plântulas de casa de

vegetação, APases de raízes foram inibidas em 200 mg L

-1

em S. microdontum e

SMIC148-A, e em 100, 150 e 200 mg L

-1

em Dakota Rose. APases de parte aérea

foram inibidas em Macaca e SMIC148-A e ativadas em 50 e 100 mg L

-1

Dakota

Rose. Os clones Macaca (sensível ao Al) e SMIC148-A (tolerante ao Al) foram

utilizados em um outro experimento com o objetivo de analisar se o estresse

oxidativo causado por Al é um sintoma primário que pode desencadear inibição do

crescimento da raiz. Em 24, 72, 120 e 168 horas após a adição de Al, foi observado

inibição do crescimento da raiz e peroxidação lipídica somente no clone sensível ao

Al. No clone tolerante, há sempre pelo menos um componente do sistema

antioxidante protegendo as plantas do estresse de Al, o mesmo não acontecendo

com o clone sensível. Com o objetivo de checar se o estresse oxidativo provocado

pelo Al difere entre os clones, Macaca (sensível ao Al) e SMIC148-A (tolerante ao

Al), os quais apresentam distinto grau de escape ao Al, esses clones foram

cultivados em sistema de raízes divididas por 10 dias, com cinco tratamentos de

variação de concentração e localização de Al. Em 200 mg Al L

-1

, uma redução na

concentração de clorofila e aumento na oxidação de proteínas foi observada

somente na Macaca. Na presença de 200 mg L

-1

em metade do sistema radicular, a

concentração de H

na parte aérea foi menor que com ambas as metades da raiz

tratadas com 100 mg L

-1

. A peroxidação lipídica na parte aérea aumentou com o

aumento do suprimento de Al na Macaca, enquanto foi menor em plantas tratadas

com 100 e 200 mg Al L

-1

em somente metade do sistema radicular em SMIC148-A.

Quando ambas as metades da raiz foram tratadas com 100 mg Al L

-1

, Macaca

apresentou resposta de tolerância ineficiente, baseado na atividade da CAT,

oxidação protéica, peroxidação lipídica, concentração de H

e atividade de

APases. Esses resultados mostram que o SMIC148-A, embora apresentou menor

reação de escape ao Al que a Macaca, mostrou uma resposta antioxidante local e

sistêmica mais eficiente frente ao suprimento de Al. Portanto, os clones Macaca e

SMIC148-A diferiram na expressão da quantidade e tipo de antioxidante, sugerindo

que o estresse oxidativo pode ser um importante mecanismo para toxicidade de Al,

principalmente nos clones sensíveis ao metal. Esta toxicidade depende não somente

da disponibilidade de Al, mas também do clone e do sistema de crescimento. Além

disso, os efeitos adversos do Al não desaparecem quando parte do sistema radicular

não está em contato com o Al, principalmente no clone sensível ao alumínio.

Palavras-chave: Solanum tuberosum; alumínio; crescimento; estresse oxidativo.

ABSTRACT

Doctoral Thesis

Post-Graduate Program in Agronomy

Federal University of Santa Maria

BIOCHEMICAL AND PHYSIOLOGICAL EVALUATION OF POTATO

CLONES IN RELATION TO ALUMINUM

AUTHOR: LUCIANE ALMERI TABALDI

ADVISOR: FERNANDO TEIXEIRA NICOLOSO

Place and date of defense: Santa Maria, April 11, 2008.

Aluminum (Al) is the most abundant metal in the earth’s crust, affecting the

growth and development of plants. The objective of this work was to investigate and

compare biochemical and physiological responses of potato clones, Macaca,

SMIC148-A, Dakota Rose and Solanum microdontum, exposed to 0, 50, 100, 150

and 200 mg Al L

-1

in nutrient solution (pH 4.0). After 7 days, Al content in roots was

on average 3.9, 2.8, 3.6, and 3.7 fold greater than in shoot, in Macaca, S.

microdontum, SMIC148-A and Dakota Rose clones, respectively. Based on the

relative root growth, the S. microdontum and SMIC148-A were considered Al-tolerant

while Macaca and Dakota Rose were considered Al-sensitive. Inhibition in shoot

growth was observed only in Macaca clone. After 7 d of Al exposure, several

biochemical parameters were affected, mainly in Al-sensitive clones, such as

increased H

concentration, catalase (CAT) activity and lipid peroxidation, and

decreased chlorophyll and carotenoid content. In addition, zinc (Zn), manganese

(Mn), iron (Fe) and copper (Cu) concentrations were higher in roots than in shoot of

all potato clones tested. An increase in the concentration of most of the

micronutrients analyzed was observed only in S. microdontum, while a decrease was

observed in Macaca, SMIC148-A and Dakota Rose. Macaca, SMIC148-A, Dakota

Rose and S. microdontum were grown in vitro, in hydroponics or in greenhouse to

evaluate the effect of Al on the in vitro activity of acid phosphatases (APases). In

plantlets grown in vitro, root APases were inhibited by Al in all clones, while shoot

APases were inhibited by Al in S. microdontum and Dakota Rose and increased in

Macaca at all Al levels. In plantlets grown in hydroponics, root APases increased in

Macaca at 50 mg L

-1

, but decreased at all Al levels in S. microdontum. In greenhouse

plantlets, root APases were reduced at 200 mg L

-1

in S. microdontum and SMIC148-

A, and at 100, 150 and 200 mg L

-1

in Dakota Rose. Shoot APases were reduced in

Macaca and SMIC148-A. Conversely, in Dakota Rose, APases increased at 50 and

100 mg L

-1

. Macaca (Al-sensitive) and SMIC148-A (Al-tolerant) clones were utilized

in another experiment with the objective of evaluating whether the oxidative stress

caused by Al is an early symptom than can trigger root growth inhibition. At 24, 72,

120 and 168 hours after Al addition, root growth inhibition and lipid peroxidation was

observed only for the Al-sensitive clone. In the Al-tolerant clone, there was always at

least one component of the antioxidant system protecting the plant against Al stress,

which did not occur in the Al-sensitive clone. With the objective of checking whether

Al oxidative stress differs in potato clones, Macaca (Al-sensitive) and SMIC148-A (Al-

tolerant), which present distinct degrees of Al- avoidance, were cultivated in a split-

root system for 10 days with five treatments of varying concentrations and locations

of Al. At 200 mg Al L

-1

, a significant decrease in chlorophyll concentration and

increase in protein oxidation was observed only for Macaca. At 200 mg L

-1

supplied

to half of the root system, shoot H

concentration was lower than that with both

root halves treated by 100 mg L

-1

. Shoot lipid peroxidation in Macaca increased with

increasing Al supply. In SMIC148-A, plants treated with 100 and 200 mg Al L

-1

in only

one root half showed lower shoot lipid peroxidation. The 200 half of 0/200 plants

presented significantly greater lipid peroxidation than the half untreated by Al, mainly

in Macaca. At 100 mg Al L

-1

supplied to both root halves, Macaca showed an

inefficient tolerance response, based on CAT activity, protein oxidation, lipid

peroxidation, H

concentration and APase activity. These results show that

SMIC148-A, even though presenting lower Al-avoidance than Macaca, showed a

stronger local and systemic antioxidant response to Al supply. Therefore, potato

clones differed in their expression of antioxidant responses in terms of amount and

type, suggesting that oxidative stress is an important mechanism for Al toxicity,

mainly in Al-sensitive clones. This toxicity depends not only on Al availability but also

on the clone and the growth system. In addition, it was observed that the adverse

effects of Al do not disappear when part of the root system is not in contact with Al,

mainly in the Al-sensitive clone.

Keywords: Potato; aluminum; growth; acid phosphatases; oxidative stress.

INTRODUÇÃO

A acidez do solo é um dos principais fatores limitantes à produção agrícola.

Os solos ácidos estão presentes em muitas partes do mundo, os quais provocam a

inibição do crescimento das plantas devido a uma combinação de vários fatores,

incluindo a toxidez do alumínio (Al) e a de manganês, bem como a deficiência de

elementos essenciais, particularmente cálcio, magnésio, fósforo e molibdênio.

Portanto, os problemas impostos às plantas pela acidez do solo não dependem de

um só fator ou agente, mas de uma série de fatores que afetam o crescimento das

mesmas por meio de diferentes mecanismos bioquímicos e fisiológicos. As relações

entre a acidez do solo e a solubilidade do Al, assim como os efeitos tóxicos desse

metal sobre as plantas, começaram a ser estudados nas primeiras décadas do

século passado. Mesmo assim, a compreensão dos mecanismos causais da toxidez

e da tolerância ao Al em plantas ainda é bastante limitada.

O Al é o terceiro elemento mais abundante na litosfera, após o oxigênio e o

silício, participando em aproximadamente 8% na composição da crosta terrestre.

Dessa forma, as raízes das plantas estão quase sempre expostas ao Al de alguma

forma. A especiação de Al em solução é complexa, e somente recentemente foi

demonstrado que Al

é a espécie de Al mais rizotóxica. Devido ao fato que o Al

outras formas monoméricas de Al são potencialmente reativas com ligantes

biológicos, pesquisadores têm especulado que a toxicidade do Al (inibição do

crescimento da raiz) pode resultar de interações do Al com vários sítios diferentes

dentro da parede celular, membrana plasmática e protoplasma. O excesso de Al,

além de inibir a formação normal da raiz, interfere nas reações enzimáticas e na

absorção, transporte e uso de nutrientes pelas plantas. Além disso, o Al causa

estresse oxidativo, levando a oxidação de biomoléculas como lipídios, proteínas,

pigmentos e ácidos nucléicos.

Embora o Al possa produzir alguns efeitos comuns sobre as plantas em

geral, como inibição do crescimento da raiz, na maioria dos casos há efeitos

específicos sobre diferentes genótipos de plantas. Assim, tanto dentro como entre as

espécies vegetais pode haver uma ampla variação genética na tolerância ao Al,

sugerindo que espécies ou cultivares tolerantes possuem vários mecanismos para

destoxificar o Al. Mesmo após anos de pesquisa sobre os efeitos do Al no

crescimento e no desenvolvimento de plantas, os mecanismos primários de sua

toxidez e de tolerância ainda precisam ser esclarecidos. Em vista dessa situação,

muitos pesquisadores, em diferentes lugares do mundo, postulam que a seleção de

variedades produtivas e tolerantes à toxidez do Al seja considerada um componente

de grande importância dentro das estratégias de manejo dos solos ácidos. Para isso,

uma forma adequada de avaliação de genótipos para a tolerância ao Al pode ser

realizada em sistemas hidropônicos sob condições controladas, oferecendo várias

vantagens, como o pronto acesso ao sistema radicular e a possibilidade de

monitoramento e controle do pH e das concentrações de Al e de outros íons

relevantes à expressão das reações de sensibilidade e tolerância.

Apesar de a batata ser um alimento empregado em todo o mundo como fonte

de energia, sendo a quarta cultura mais importante do mundo, depois do arroz, trigo

e milho, há uma carência de estudos com relação a alguns aspectos nutricionais e

bioquímicos nesta espécie. Neste trabalho, quatro clones de batata foram

analisados, três adaptados e tetraplóides, da espécie Solanum tuberosum (Macaca,

SMIC148-A e Dakota Rose) e um diplóide, da espécie selvagem Solanum

microdontum (PI595511-5).

Tendo em vista a característica ácida dos solos do Rio Grande do Sul e sendo

a batata cultivada em grande escala neste estado, o objetivo do presente trabalho foi

analisar o comportamento de diferentes clones de batata em relação ao Al, bem

como a interação Al-planta nestes clones, tanto em aspectos nutricionais

(disponibilidade de nutrientes e crescimento) quanto bioquímicos (atividade de

enzimas envolvidas no metabolismo de nutrientes e no sistema antioxidante das

plantas).

OBJETIVOS

Objetivo geral

Avaliar as respostas bioquímicas e fisiológicas de clones de batata em

relação ao alumínio.

Objetivos específicos

1) Identificar clones de batata sensíveis e tolerantes ao alumínio.

2) Investigar e comparar respostas fisiológicas e de estresse oxidativo de

clones de batata expostos ao alumínio em solução nutritiva.

3) Avaliar se o estresse oxidativo causado por toxicidade de alumínio é

um sintoma inicial que pode desencadear inibição do crescimento da raiz em clones

de batata.

4) Avaliar o efeito do alumínio na atividade in vitro de fosfatases ácidas de

quatro clones de batata cultivados em três sistemas de crescimento.

5) Analisar a influência do estresse de alumínio no conteúdo de

micronutrientes em clones de batata.

6) Examinar os efeitos locais e/ou sistêmicos do alumínio em parâmetros

bioquímicos de clones de batata crescendo em sistema de raízes divididas.

REVISÃO BIBLIOGRÁFICA

1 Os solos ácidos no mundo

Os solos ácidos, os quais apresentam pH menor ou igual a 5,5, promovem

inibição do crescimento de plantas, especialmente devido a deficiência de fósforo e

ao estresse causado pelo alumínio (Al). Considera-se a toxidez do Al um dos

principais fatores limitantes da produtividade agrícola em solos ácidos (FOY et al.,

1978). O Al afeta aproximadamente 40% das terras aráveis do mundo que são

potencialmente usadas para a produção de biomassa e alimentos (MA et al., 2001).

Ainda mais relevante é o fato de que muitas dessas áreas estão localizadas em

países em desenvolvimento na América do Sul, África Central e Sudoeste da Ásia. A

acidez de solos é uma ocorrência natural em áreas tropicais e subtropicais e pode

resultar de desbalanços nos ciclos de nitrogênio, enxofre e carbono (BOLAN;

HEDLEY, 2003; TANG; RENGEL, 2003); maior captação de cátions em comparação

com ânions (TANG; RENGEL, 2003); e menor fixação de nitrogênio por leguminosas

(BOLAN et al., 1991; TANG; RENGEL, 2003). Além disso, em várias partes do

mundo, os níveis de acidez dos solos estão aumentando, em decorrência de

atividades humanas. Entre os motivos da acidificação antropogênica dos solos estão

a liberação atmosférica de poluentes industriais, associada à lixiviação de solos com

chuvas ácidas, as atividades de mineração e, no setor agrícola, a nitrificação

subseqüente à aplicação de altas doses de fertilizantes amoniacais (RENGEL;

ZHANG, 2003).

Dentro da faixa intertropical, 37% dos solos do sudeste asiático, 40% dos

solos da África e 55% dos solos da América do Sul apresentam limitações ao seu

uso agrícola por excesso de acidez (SANCHEZ; SALINAS, 1981). A maioria dos

solos do Rio Grande do Sul e do Brasil são ácidos (VOLKWEISS, 1989). No Brasil,

cerca de 500 milhões de hectares são cobertos por solos ácidos, compreendendo

em torno de dois terços de seu território total – a maior área de solos ácidos dentro

de um único país (VITORELLO et al., 2005). Um estudo abrangendo 26 solos de

regiões brasileiras mostrou que 75% dos valores de pH da camada superficial

variaram entre 3,78 e 5,52 e que o Al

foi o cátion trocável predominante em mais

de um terço dos solos com pH inferior a 5,6 (ABREU Jr. et al., 2003).

A produção de alimentos, principalmente de grãos, é afetada negativamente

por solos ácidos. Por exemplo, 20% da produção de milho e 13% da produção de

arroz do mundo estão em solos ácidos (von UEXKÜLL; MUTERT, 1995). Assim,

solos ácidos limitam a produtividade de culturas em muitos países em

desenvolvimento onde a produção de alimentos é crítica (KOCHIAN et al., 2004).

2 Alumínio (Al)

O Al é um metal leve que compõe aproximadamente 8% da crosta terrestre,

ocorrendo como óxidos e aluminosilicatos inofensivos, precipitando em pH alcalino

ou neutro na forma de sais insolúveis, como por exemplo, Al

SiO

, Al(OH)

, AlPO

, e

formas organicamente complexas de Al. Entretanto, sob certas condições, o Al pode

tornar-se solúvel, por exemplo, quando o ambiente torna-se ácido ou quando os

níveis de matéria orgânica no solo são altos (JANSEN et al., 2002). Quando o pH do

solo torna-se ácido, como é o caso de 30% a 40% das terras aráveis do mundo (von

UEXKÜLL; MUTERT, 1995), formas solúveis de Al podem se acumular em

concentrações que inibem o crescimento e o funcionamento da raiz. Portanto, à

medida que os solos se acidificam, íons Al passam a ocupar as posições de troca

catiônica, em superfícies eletronegativas dos colóides, em substituição aos cátions

removidos pela lixiviação, onde concentrações de espécies de Al podem alcançar

níveis tóxicos para os organismos (RENGEL; ZHANG, 2003). Dessa forma, a

toxicidade do Al é fortemente dependente das espécies predominantes, onde a

concentração de Al total não pode ser usada como um índice real de toxicidade de

Al.

2.1 Química do Al

Evidências atuais e reinterpretação das primeiras literaturas sugerem que Al

monomérico tais como Al

, hidróxidos de Al (AlOH

; Al(OH)

) e espécies

de AlSO

são as formas mais tóxicas para as plantas, juntamente com os polímeros

(KINRAIDE, 1991; JANSEN et al., 2002). Como pode ser observado na Figura 1,

em pHs menores que 5,0, o Al

fitotóxico domina (ROSSIELLO; NETTO, 2006). O

Al monomérico também forma complexos de baixo peso molecular com vários

ligantes, tais como grupos carboxilato, sulfato e fosfato. Assim, o Al

forma

complexos com os ácidos orgânicos, o fosfato inorgânico e o sulfato, e também se

ligará a esses grupos em macromoléculas tais como as proteínas e os nucleotídeos.

Devido ao fato que o Al

e outras formas monoméricas de Al são potencialmente

reativas com ligantes biológicos, pesquisadores têm especulado que a toxicidade do

Al (inibição do crescimento da raiz) pode resultar de interações do Al com vários

sítios diferentes dentro da parede celular, membrana plasmática e protoplasma.

Figura 1. Distribuição das atividades relativas de Al

e das espécies mononucleares de Al-OH em

função do pH. (Fonte: KINRAIDE; PARKER, 1989).

Apesar de sua presença ubíqua, o Al não é essencial para os organismos

vivos. Algumas possíveis razões para isso foram resumidas por Williams (1999).

Com exceção daqueles cátions que podem sofrer mudanças na valência, tais como

o Fe e o Co, os sistemas biológicos são aparentemente incapazes de utilizar

efetivamente os cátions trivalentes livres. Os dois fatores que aparentemente

determinam isso são o pequeno tamanho desses cátions, os quais determinam

limitações à estequiometria de complexação, e as lentas taxas de troca de ligantes

desses metais (WILLIAMS, 1999).

Por ser um cátion trivalente, o Al é retido firmemente e, assim, sua

concentração, na solução do solo, é baixa, dentro da faixa de µmol L

-1

(HAYNES;

MOKOLOBATE, 2001). Entretanto, essas baixas concentrações de Al em solução

são tóxicas para a maioria das espécies vegetais, primariamente por lesar o

funcionamento normal das raízes, inibindo o seu crescimento e bloqueando os

mecanismos de aquisição e transporte de água e nutrientes.

2.2 Efeitos bioquímicos e fisiológicos do alumínio

Como as raízes são os primeiros órgãos a entrar em contato com o Al do

solo, desde as primeiras observações tem sido registrado que os sintomas de

toxidez ao Al expressavam-se de forma mais acentuada no sistema radicular

(ROSSIELLO; NETTO, 2006). A inibição do crescimento da raiz é um dos primeiros

e mais dramáticos sintomas de toxicidade do Al exibidos pelas plantas (RENGEL;

ZHANG, 2003). O excesso de Al além de inibir a formação normal da raiz, interfere

nas reações enzimáticas e na absorção, transporte e uso de nutrientes pelas plantas

(TAMÁS et al., 2006). Salvador et al. (2000), constataram que doses crescentes de

Al reduziram a absorção e o transporte de P, Ca, Mg, S, Fe e Mn para a parte aérea,

sugerindo que a redução de Ca e Mg deve-se a uma inibição interiônica desses

cátions pelo Al. A inibição do crescimento da raiz foi observada dentro de horas, ou

até minutos, de exposição a concentrações micromolares de Al em solução

(ZHANG; RENGEL, 1999; MA et al., 2002). Entretanto, com a exposição prolongada

ao Al, as plantas exibem vários outros sintomas de toxidez, tanto na raiz como na

parte aérea (FOY, 1988; RENGEL, 1996).

A inibição do crescimento da raiz induzida por Al frequentemente precede ou

coincide com um declínio nas divisões celulares (HORST, 1995). Entretanto, a

rápida inibição do crescimento da raiz induzida por Al é comumente causada pela

inibição do alongamento celular mais do que pela divisão celular (RENGEL; ZHANG,

2003).

O confinamento do crescimento radicular ao volume do horizonte superficial

tem conseqüências restritivas para o crescimento da parte aérea, assim como para o

pleno crescimento e desenvolvimento da planta, o que resultará em reduções na

produtividade das culturas. Essa limitação adquire ainda maior relevância durante

períodos de deficiência hídrica (FAGERIA; ZIMMERMANN, 1979), quando a

aquisição de água e nutrientes das camadas mais profundas pode ser crucial para a

sobrevivência das plantas. Nesse sentido, o estresse hídrico e a toxidez de Al

tendem a reforçar os seus efeitos negativos. Entre os relatos sobre os mecanismos

envolvidos na tolerância ao Al, Giannakoula et al. (2008) relataram que a tolerância

ao Al em milho está correlacionada com maiores níveis de nutrientes minerais.

Entretanto, Furlani & Furlani (1991), destacaram que o maior teor de nutrientes

verificado nas plantas tolerantes ao Al pode ser devido ao efeito indireto do maior

aprofundamento e crescimento das raízes, explorando maior volume de solo. Na

parte aérea das plantas, os sintomas resultantes da toxidez de Al não são

claramente identificáveis, e as injúrias provocadas pelo Al podem ser confundidas

com aquelas decorrentes de desbalanço ou deficiência nutricional, especialmente do

fósforo (ROSSIELLO; NETTO, 2006).

A extensão da inibição do crescimento da raiz é comumente usada como uma

medida de toxicidade do Al (FOY, 1988). Entretanto, o crescimento da raiz é um

processo complexo e dinâmico. Provavelmente, vários processos bioquímicos e

fisiológicos podem já ter sido alterados antes da inibição do crescimento da raiz

induzida por Al (RENGEL; ZHANG, 2003). A literatura é rica em relatos mostrando

que numerosos processos bioquímicos e fisiológicos são afetados em várias

espécies dentro de minutos ou horas após a exposição ao Al, entre eles o

exacerbado estresse oxidativo (CAKMAK; HORST, 1991; YAMAMOTO et al., 2002).

Uma característica comum a vários tipos de estresse, incluindo toxicidade do Al, é a

perturbação da homeostase redox celular, e como uma conseqüência, o aumento da

produção de espécies reativas de oxigênio (EROs) (CAKMAK; HORST, 1991),

incluindo radical superóxido (O

•-

), radical hidroxil (

•

OH) e peróxido de hidrogênio

) (CHAOUI; FERJAN, 2005). Essas espécies de oxigênio altamente citotóxicas

podem causar dano oxidativo a biomoléculas tais como lipídios, proteínas,

pigmentos e ácidos nucléicos, levando a peroxidação de lipídeos de membrana,

perda de íons, hidrólise de proteínas, e até mesmo dano ao DNA (GUO et al., 2007).

A membrana plasmática, o último obstáculo para o acesso livre de íons Al no

simplasto, pode ser o alvo primário do Al rizotóxico (BARCELÓ et al., 1996). Estudos

de toxicidade de Al em raízes sugerem que a produção de EROs pode contribuir

significativamente para a inibição induzida por Al do alongamento da raiz

(YAMAMOTO et al., 2003; TAMÁS et al., 2004).

Embora os termos “resistência”, “tolerância” e “mecanismos de escape” sejam

frequentemente usados na literatura como sinônimos, quando se referem a

estresses abióticos, o termo “resistência” se refere a mecanismos que impedem o Al

de entrar na planta, enquanto o termo “tolerância” se refere a mecanismos que

destoxificam ou seqüestram o Al internamente (DELHAIZE et al., 2007). Além disso,

alguns autores relataram também que algumas espécies de plantas desenvolvem

um mecanismo chamado de “escape ao Al”. Nesse mecanismo, quando as raízes

das plantas crescem em um ambiente heterogêneo com diferentes níveis de Al

tóxico, há o desenvolvimento preferencial das raízes nos locais com menor

concentração de Al, acompanhado por uma maior inibição do crescimento das

raízes em contato com altas concentrações de Al (HAIRIAH et al., 1993).

Há uma ampla variação genética, tanto dentro como entre espécies na

tolerância de plantas ao Al (BONA et al, 1993; MA; FURUKAWA, 2003). Algumas

plantas são capazes de tolerar até mesmo concentrações fitotóxicas de alumínio

(POLAK et al., 2001). A variedade de processos celulares nos quais o Al pode

interferir potencialmente sugere que espécies ou cultivares tolerantes ao alumínio

possuem vários mecanismos de detoxificação, entre os quais de tolerância internos

e externos (TAYLOR, 1988, 1991).

Os mecanismos externos (os quais minimizam a captação de alumínio)

incluem a formação de quelados não tóxicos de alumínio com ânions ácidos

orgânicos (malato, citrato e oxalato) secretados pelos ápices radiculares ou

alcalinização do apoplasto radicular e rizosfera, os quais substituem as espécies

tóxicas de alumínio por formas menos tóxicas (WENZL et al., 2001). Há fortes

correlações entre tolerância ao Al e liberação de ácidos orgânicos ativada por Al em

numerosas espécies de plantas (KOCHIAN et al., 2004).

Recentemente, alguns pesquisadores têm voltado sua atenção para espécies

de plantas que podem acumular altos níveis de Al na parte aérea. Essas espécies,

consequentemente, possuem mecanismos internos de destoxificação do Al. Dentre

os mecanismos para destoxificar o alumínio internamente está a formação de

complexos de ácidos orgânicos e alumínio, sendo que essa quelação reduz

efetivamente a atividade do alumínio no citosol, prevenindo a formação de

complexos entre o alumínio e os componentes celulares (MA et al., 2001).

Dada à natureza do estresse de Al, o meio hidropônico oferece óbvias

vantagens aos estudos da interação desse elemento com as plantas, como o pronto

acesso ao sistema radicular e a possibilidade de monitoramento e controle do pH e

das concentrações de Al e de outros íons relevantes à expressão das reações de

sensibilidade e tolerância (ROSSIELLO; NETTO, 2006).

2.3 Estresse oxidativo

O oxigênio molecular (O

) é necessário para a sobrevivência de todos os

organismos aeróbicos. Assim, a obtenção de energia por estes organismos é feita

na mitocôndria através da fosforilação oxidativa, onde o O

é reduzido por quatro

elétrons a H

O. Quando o oxigênio é parcialmente reduzido, tanto na fosforilação

oxidativa quanto em outras reações, há a formação de radicais livres, que

constituem moléculas com coexistência independente e que contém um ou mais

elétrons não pareados na camada de valência. Esta configuração faz dos radicais

livres espécies altamente instáveis, de meia vida relativamente curta e quimicamente

muito reativas (SALVADOR; HENRIQUES, 2004).

→ O

•

→ H

→ OH

•

→ H

Figura 2. Passos intermediários da redução do oxigênio. A redução por 4 elétrons do oxigênio até a

água ocorre em etapas sucessivas de redução por 1 elétron. Neste processo são formados os

intermediários: ânion radical superóxido, peróxido de hidrogênio e radical hidroxila, que

correspondem à redução por um, dois e três elétrons, respectivamente (SALVADOR; HENRIQUES,

2004).

O estresse oxidativo corresponde a um estado em que há uma elevada

produção de espécies reativas de oxigênio, onde os mecanismos celulares pró-

oxidantes superam os antioxidantes, como esquematizado na Figura 3.

A terminologia Espécies Reativas de Oxigênio (EROs ou ROS:“reactive

oxygen species”) inclui as espécies chamadas de radicais livres e outras que,

embora não possuam elétrons desemparelhados, são muito reativas em decorrência

de sua instabilidade (MARRONI, 2002). Um dos principais representantes de EROs

é o anion radical superóxido (O

•-

), o qual é produzido através de uma redução

monoeletrônica do oxigênio. Nas células o O

•-

é rapidamente convertido à peróxido

de hidrogênio (H

) através de sua dismutação espontânea ou enzimática

(superóxido dismutase). O H

é menos reativo que o O

•-

, porém na presença de

metais como o ferro (Fe

) ou o cobre (Cu

), ele pode gerar radicais hidroxila (OH

•

O OH

•

é provavelmente um dos radicais mais reativos dentre os EROs.

Condições Normais

Estresse Oxidativo

Figura 3. Relação entre mecanismos celulares pró-oxidantes, antioxidantes e estresse oxidativo.

2.4 Sistema antioxidante de plantas

Para atenuar o dano oxidativo iniciado pelas EROs, as plantas desenvolveram

um complexo sistema de defesa antioxidante, incluindo antioxidantes de baixo peso

molecular, como a glutationa, o ácido ascórbico e os carotenóides, assim como as

enzimas antioxidantes, tais como a superóxido dismutase (SOD), a ascorbato

peroxidase (APX) e a catalase (CAT). Essas enzimas reduzem eficientemente as

EROs sob circunstâncias normais, mas se a redução completa não ocorrer, como

sob condições de alta produção de EROs, o resultado pode ser um estado de

estresse oxidativo levando à oxidação de biomoléculas (BOSCOLO et al., 2003).

2.4.1 Sistema antioxidante enzimático

As principais enzimas envolvidas na defesa de plantas contra as EROs

incluem as superóxido dismutases (SOD), a ascorbato peroxidase (APX) e a

catalase (CAT) (SHAH et al., 2001). As SODs estão localizadas em vários

compartimentos celulares e catalisam a conversão de radicais superóxido (O

•-

) à

uma espécie de oxigênio menos destrutiva, e O

. A CAT e APX estão

envolvidas na conversão do H

à H

O (Figura 4).

Figura 4. Representação dos mecanismos de defesa usando as enzimas antioxidantes SOD, CAT e

APX contra as EROs (FOYER et al., 1994).

As catalases estão localizadas em peroxissomos/glioxissomos e mitocôndrias,

enquanto que a APX, a qual utiliza ascorbato como doador de elétrons, está

primariamente localizada em cloroplastos e no citosol (HEGEDÜS et al., 2001). As

diferentes afinidades da APX (variação µM) e CAT (variação mM) ao H

sugerem

que elas pertençam a duas diferentes classes de enzimas removedoras de H

APX pode ser responsável pela fina modulação de EROs, enquanto CAT pode ser

responsável para a remoção do excesso de EROs durante situações de estresse

(MITTER, 2002). O balanço entre as atividades da SOD, CAT e APX é crucial para

determinar o estado estável de radicais superóxido e peróxido de hidrogênio nas

células. A importância dessas enzimas é baseada no fato de que a atividade de uma

ou mais dessas enzimas em geral aumenta em plantas quando as mesmas são

expostas a condições de estresse, e esta atividade aumentada está relacionada com

um aumento na tolerância ao estresse ou à produção aumentada de EROs.

2.4.2 Sistema antioxidante não enzimático

Além do sistema de defesa antioxidante enzimático, as defesas antioxidantes

não-enzimáticas são de fundamental importância para as células. Os antioxidantes

não enzimáticos incluem, entre outros, o ácido ascórbico, a glutationa, o α-tocoferol

e os carotenóides. O ácido ascórbico e a glutationa são encontrados em altas

concentrações nos cloroplastos e outros compartimentos celulares (5-20 mM de

ácido ascórbico e de 1-5 mM de glutationa) e são cruciais para a defesa da planta

contra o estresse oxidativo (NOCTOR; FOYER, 1998). Além de seu papel como

substratos de enzimas, eles podem reagir quimicamente com quase todas as formas

de O

ativadas (HALLIWELL; GUTTERIDGE, 1999). O ácido ascórbico é sintetizado

nas mitocôndrias e é transportado para todos os compartimentos sub-celulares

incluindo o apoplasto, onde é o principal tampão redox modulando respostas

fisiológicas e de estresse. Está associado com a remoção do H

via ascorbato

peroxidase (SAIRAM et al., 1998), além de reagir com radicais superóxido e radicais

hidroxil (REDDY et al., 2004). Está também envolvido na regeneração de um outro

antioxidante não enzimático, o α-tocoferol (SAIRAM et al., 2005).

Os grupos tióis não protéicos, entre estes a glutationa (GSH), são conhecidos

por possuírem um papel central nos mecanismos de resposta aos metais em plantas

terrestres (ZENK, 1996; RAUSER, 1999). A GSH é um tripeptídeo contendo enxofre,

e tem sido considerado como um antioxidante muito importante envolvido na defesa

celular contra agentes tóxicos (SCOT et al., 1993). Em resposta a estresses, as

plantas aumentam a atividade de enzimas biossintéticas de GSH e,

consequentemente, as concentrações de GSH (NOCTOR et al., 2002). Além disso,

a GSH é precursora na síntese de fitoquelatinas (COBBETT; GOLDSBOROUGH,

2002) e mantém o estado redox celular. Um alto nível de grupos tióis pode

proporcionar aos metabólitos funcionarem na detoxificação de EROs e de radicais

livres.

Nos últimos anos, uma grande diversidade de resultados, obtidos em estudos

fisiológicos e de mapeamento molecular mostraram que a tolerância vegetal ao

estresse causado pelo Al é uma característica multigênica complexa, que pode

envolver vários mecanismos de tolerância. Além disso, estudos relativos aos

mecanismos de resposta vegetal a estresses ambientais comprovaram que os

agentes estressantes são percebidos de forma diferenciada pelos sistemas de

sinalização das plantas, de acordo com a intensidade da sua ação (PASTORI;

FOYER, 2002). No caso do estresse de Al, a situação deve ser similar uma vez que

o tempo de exposição e a atividade do Al interagem tanto na manifestação dos

sintomas de toxidez quanto na expressão dos mecanismos de tolerância ao estresse

(PARKER, 1995; BARCELÓ; POSCHENREIDER, 2002; KOCHIAN et al., 2004).

Embora os mecanismos causais da toxidez do Al possam parecer

complicados, não se deve esquecer que eles resultam, na sua essência, da ligação

do Al com substâncias situadas na parede celular, na membrana plasmática ou no

citoplasma, devido ao fato que o Al possui forte afinidade por compostos doadores

de oxigênio (ROSSIELLO; NETTO, 2006). Isso significa um amplo leque de

oportunidades de ligação a diversos sítios nos domínios apoplástico e simplástico.

3 Fosfatases ácidas

As enzimas fosfatases ácidas (APases) (E.C.3.1.3.2) catalisam a hidrólise de

uma ampla variedade de monoésteres de fosfato, liberando fosfato inorgânico (Pi)

de substratos fosforilados em pH abaixo de 7,0 (VINCENT et al., 1992). APases são

ubíquas e abundantes em plantas, animais, fungos e bactérias, e exibem baixa

especificidade de substratos (VINCENT et al., 1992; DUFF et al., 1994). Estão

presentes em vários órgãos e também em diferentes compartimentos celulares,

sugerindo que essas enzimas estão envolvidas em vários compartimentos celulares

(YONEYAMA et al., 2007).

O controle da expressão de APases é mediado por uma variedade de fatores

ambientais e de desenvolvimento (DUFF et al., 1994). As APases são induzidas sob

vários estresses, incluindo deficiência de água, salinidade e ataque de patógenos

(BOZZO et al., 2002), assim como na germinação de sementes, florescimento,

formação de tubérculos e amadurecimento de frutos (DUFF et al., 1994; GELLATLY

et al., 1994; TURNER; PLAXTON, 2001), dificultando a definição de sua função nas

células (PENHEITER et al., 1997; BOZZO et al., 2002). Entretanto, a ativação das

APases em resposta a deficiência de Pi é bem documentada (DUFF et al., 1994).

As APases existem como isoenzimas específicas de compartimentos

celulares ou tecidos, as quais se diferenciam quanto à massa molecular,

especificidades quanto a substratos, sensibilidade à inibidores e a presença e

número de carboidratos ligados à cadeia polipeptídica (VINCENT et al., 1992; DUFF

et al., 1994). Além disso, estão envolvidas na produção, transporte e reciclagem de

Pi, o qual é crucial para o metabolismo celular e para processos de transdução de

energia (BOZZO et al., 2002). As APases intracelulares normalmente controlam a

homeostase interna de Pi enquanto as APases secretadas controlam a aquisição

externa de Pi (DUFF et al., 1994). Além disso, estão envolvidas em situações de

estresse oxidativo, atuando no metabolismo de espécies reativas de oxigênio

(EROs) (del POZZO et al., 1999).

4 Batata

A batata (Solanum tuberosum L.) é uma planta dicotiledônea, da família

Solanaceae, do gênero Solanum. A batata cultivada, com exceção daquela da

região dos Andes da América do Sul, pertence à sub-espécie tuberosum. É um dos

alimentos mais consumidos no mundo como fonte de energia, devido à composição,

versatilidade gastronômica e tecnológica e baixo custo de comercialização dos

tubérculos (COELHO et al., 1999), sendo a hortaliça de maior importância

econômica no Brasil (BISOGNIN, 2006). Os principais estados produtores são Minas

Gerais, São Paulo, Paraná e Rio Grande do Sul, responsáveis por mais de 90% da

produção nacional (IBGE, 2004). A produção mundial de batata representa,

aproximadamente, a metade da produção mundial de todas as raízes e tubérculos.

Utilizada desde tempos ancestrais pelos povos americanos, o processamento

é tão antigo quanto o uso direto na alimentação humana (MELLO, 1997). A batata é

plantada em, pelo menos, 125 países e consumida por mais de um bilhão de

pessoas em todo o mundo; dentre estes, 500 milhões de consumidores são de

países em desenvolvimento e, na sua dieta básica, está incluída a batata (SALLES,

1997). Nenhuma outra cultura pode competir com a batata como alimento energético

e em termos de valor alimentar por unidade de área (SIECZA; THORTON, 1993).

Possui também uma alta quantidade de vitamina C, niacina e vitamina B6. No Brasil,

é a hortaliça mais importante (BISOGNIN, 1996), sendo que o hábito de utilizar

batata na alimentação foi trazido pelos imigrantes europeus. O estado do Rio

Grande do Sul figura entre os principais estados brasileiros em área cultivada com

batata.

A cultura da batata se desenvolve sob uma variedade de altitudes, latitudes, e

condições climáticas, desde o nível do mar até 4000 metros de elevação (DAVIES et

al., 2005). Tolera uma acidez moderada no solo, produzindo bem na faixa de pH 5,0

a 6,5 (PREZOTTI et al., 1986). Acima desta faixa, pode ocorrer aumento da

suscetibilidade dos tubérculos a certos patógenos presentes no solo, como é o caso

da sarna. Por outro lado, nos solos excessivamente ácidos (pH abaixo de 5,0)

ocorrem decréscimos de produção, uma vez que este pH prejudica o crescimento da

planta pela própria ação da acidez, além de diminuir a disponibilidade de nutrientes

e aumentar a concentração de alumínio trocável no solo (CASTRO, 1983).

As espécies cultivadas de batata são muito sensíveis a estresses abióticos,

enquanto várias espécies primitivas ou selvagens de diferentes níveis de ploidia são

bem adaptadas a crescer sob condições desfavoráveis tais como seca, frio,

salinidade e alta radiação (LI; FENNEL, 1985; MENDOZA; ESTRADA, 1979). A

descoberta que espécies de Solanum possuem diferenças genéticas na resistência

ao estresse abiótico não é somente interessante para programas de melhoramento

da batata, mas também fornece um bom material para se estudar outros aspectos

dos mecanismos de resistência ao estresse abiótico.

RESULTADOS E DISCUSSÃO

Os resultados e discussão deste trabalho serão apresentados em cinco

artigos científicos, distribuídos em quatro capítulos, como segue:

Capítulo I: Respostas fisiológicas e bioquímicas de quatro clones de batata expostos

ao alumínio.

Artigo I: Physiological and oxidative stress responses of four potato clone to

aluminum in nutrient solution.

Manuscrito I: Oxidative stress is an early symptom triggered by aluminum in

Al-sensitive potato plantlets.

Capítulo II: Efeito do alumínio na atividade in vitro de fosfatases ácidas em quatro

clones de batata.

Manuscrito II: In vitro activity of acid phosphatases of four potato clones

cultivated in three growth systems: effect of aluminum.

Capítulo III: Influência do estresse de alumínio no teor de micronutrientes em

plântulas de batata.

Manuscrito III: Micronutrient concentration in potato clones with distinct

physiological sensitivity to Al stress.

Capítulo IV: Respostas localizadas e sistêmicas de estresse oxidativo induzidas por

alumínio em batata (Solanum tuberosum L.) cultivadas em sistema de raízes

divididas.

Manuscrito IV: Local and systemic oxidative stress responses induced by

aluminum in two potato clones (Solanum tuberosum L.) that differ in Al-avoidance.

CAPÍTULO I

Respostas fisiológicas e bioquímicas de quarto clones de

batata expostos ao alumínio

Artigo I

(Publicado no Periódico “Brazilian Journal of Plant Physiology”)

Physiological and oxidative stress responses of four potato clones to

aluminum in nutrient solution

Luciane Almeri Tabaldi

1,4

, Gabriel Y Castro

, Denise Cargnelutti

2,5

, Jamile Fabbrin

Gonçalves

1,4

, Renata Rauber

, Etiane Caldeira Skrebsky

1,4

, Vera Maria Morsch

2,5

, Dilson

Antônio Bisognin

3,4

, Maria Rosa Chitolina Schetinger

2,5

, Fernando Teixeira Nicoloso

1,4*

Departamento de Biologia,

Química e

Fitotecnia,

Programa de Pós-Graduação em

Agronomia e

Bioquímica Toxicológica, Centro de Ciências Naturais e Exatas, Universidade

Federal de Santa Maria, 97105-900, Fax:0+555532208339, Santa Maria, RS, Brasil.

*Corresponding author: [email protected]

Aluminum toxicity is a serious problem in Brazilian soils and selecting potato clones is an

important strategy to produce this crop on these kinds of soils. Potato clones, Macaca,

SMIC148-A, Dakota Rose, and Solanum microdontum, were grown in a nutrient solution (pH

4.00±0.1) with 0, 50, 100, 150 and 200 mg Al L

-1

. After 7 days, Al concentration in both root

system and shoot of all clones increased linearly with increasing Al levels. Based on relative

root growth, S. microdontum and SMIC148-A were considered Al-tolerant clones, whereas

Macaca and Dakota Rose were considered Al-sensitive. Shoot growth in Macaca linearly

decreased with increasing Al levels. Root H

concentration in both Al-sensitive clones

increased with increasing Al supply, whereas in Al-tolerant clones it either decreased

(SMIC148-A) or demonstrated no alteration (S. microdontum). Shoot H

concentration

increased linearly in Macaca, whereas for Dakota Rose it showed a quadratic relationship with

Al levels. On the other hand, shoot H

concentration in the Al-tolerant clones either

demonstrated no alteration (S. microdontum) or presented lower levels (SMIC148-A). Root

catalase (CAT) activity in both Al-sensitive clones increased with increasing Al levels, whereas

in Al-tolerant clones it either demonstrated no alteration (SMIC148-A) or presented lower levels

(S. microdontum). Shoot CAT activity in the S. microdontum increased curvilinearly with

increasing Al levels. In all potato clones, chlorophyll concentration showed a curvilinear

response to Al supply, where in Al-sensitive clones it decreased upon addition of Al exceeding

100 mg L

-1

, but in SMIC148-A it increased at levels between approximately 100 and 150 mg L

, and decreased in S. microdontum regardless of the Al level. Carotenoid concentrations in the

Al-sensitive clones were linearly decreased with increasing Al levels. Al supply caused root

lipid peroxidation only in the Al-sensitive clones, whereas in the shoot it increased linearly in

the Al-sensitive clones and in S. microdontum it only increased at around 50 mg L

-1

. Most of

root protein oxidation was only observed in the Al-sensitive clones. However, shoot protein

oxidation was increased with increasing Al levels for all potato clones. These results indicate

that oxidative stress caused by Al in potato may harm several components of the cell, mainly in

Al-sensitive clones.

Keywords: aluminum toxicity, antioxidative enzymes, growth, oxidative stress, Solanum

tuberosum

Respostas fisiológicas e de estresse oxidativo de quatro clones de batata ao alumínio em

solução nutritiva: A toxicidade do alumínio é um problema sério em solos brasileiros e a

seleção de clones de batata é uma estratégia importante para produzir esta cultura em tais solos.

Clones de batata, Macaca, SMIC148-A, Dakota Rose e Solanum microdontum, foram cultivados

em solução nutritiva (pH 4,0±0,1) com 0, 50, 100, 150 e 200 mg Al L

-1

. Após 7 d, o teor de Al

em raízes e parte aérea em todos clones aumentou linearmente com o suprimento de Al.

Baseado no crescimento relativo da raiz, os clones S. microdontum e SMIC148-A foram

considerados tolerantes ao Al, enquanto os clones Macaca e Dakota Rose foram considerados

sensíveis. O crescimento da parte aérea do clone Macaca diminuiu linearmente com o Al. A

concentração de H

nas raízes de ambos os clones sensíveis ao Al aumentou com o

suprimento de Al, enquanto nos clones tolerantes houve declínio (SMIC148-A) ou falta de

resposta (S. microdontum). A concentração de H

na parte aérea aumentou linearmente em

Macaca, enquanto em Dakota Rose houve uma relação quadrática com os níveis de Al. Por

outro lado, nos clones tolerantes ao Al a concentração de H

não foi alterada (S.

microdontum) ou foi reduzida (SMIC148-A). A atividade da catalase (CAT) nas raízes de

ambos os clones sensíveis ao Al aumentou com o suprimento de Al, enquanto nos clones

tolerantes não houve alteração (SMIC148-A) ou, então, redução (S. microdontum). Na parte

aérea, a atividade da CAT em S. microdontum aumentou com o suprimento de Al. Em todos os

clones de batata, a concentração de clorofila variou curvelinearmente em relação ao suprimento

de Al; nos clones sensíveis, a concentração de clorofila diminuiu pela adição de Al em níveis

acima de 100 mg L

-1

, porém em SMIC148-A houve aumento na presença de Al (na faixa

próxima a 100 e 150 mg L

-1

) e diminuição em S. microdontum, independentemente do

tratamento de Al. A concentração de carotenóides nos clones sensíveis ao alumínio diminuiu

linearmente, em resposta ao Al. O Al aumentou a peroxidação lipídica em raízes dos clones

sensíveis, enquanto na parte aérea houve aumento linear nesses clones e também em S.

microdontum (próximo a 50 mg Al L

-1

). Em raízes, a oxidação protéica foi observada

principalmente nos clones sensíveis ao alumínio. Entretanto, na parte aérea, foi observada

oxidação protéica em todos os clones de batata em resposta ao Al. Esses resultados indicam que

o estresse oxidativo causado por Al em batata pode prejudicar vários componentes celulares,

principalmente nos clones sensíveis ao metal.

Palavras-chave: crescimento, enzimas antioxidativas, estresse oxidativo, Solanum tuberosum,

toxicidade de Al

INTRODUCTION

Most tropical soils present an acid characteristic that decreases nutrient availability and

increases aluminum (Al) toxicity, affecting plant growth and development (Marschner, 1991).

Aluminum is the most abundant metal and the third most common element in the earth’s crust.

Aluminum toxicity is considered a major abiotic stress factor in low pH soils. Aluminum stress

impairs root growth, decreasing the absorption, transport and use of several nutrients such as P,

Ca, Mg, S, Fe and Mn and diminishing biomass production (Brondani and Paiva, 1996).

The initial and most evident symptom of Al-toxicity is a rapid inhibition of root

elongation (Dipierro et al., 2005), which can occur within minutes after exposing roots to Al,

with less marked effects on shoot development. Severe Al-toxicity reduces and damages the

root system, causing plant drought susceptibility and mineral nutrient deficiency. The principal

sites of Al-toxicity are the actively dividing and expanding cells of the root apex (Ryan et al.,

1993). Aluminum can rapidly enter into the cytoplasm (Lazof et al., 1994), but it is still

unknown whether the primary site(s) of toxicity is external (interactions with the cell wall or

external face of plasma membrane) or internal (affecting cytoplasmic functions or activities in

internal membranes/compartments). After prolonged exposure (e.g. 12 h), Al can affect many

physiological processes either directly or indirectly (Kochian, 1995). Genetic variability to Al-

tolerance exists among and within plant species.

Many environmental stresses induce the formation of reactive oxygen species (ROS) in

plant cells (Schützendübel and Polle, 2002). Al-toxicity in plants is a well-known example of

such environmental stress (Kochian, 1995; Ma et al., 2001). Under normal conditions, the

production and destruction of these radicals is regulated by cell metabolism. To prevent cellular

compartments from the damaging effects of ROS, organisms have evolved multiple

detoxification mechanisms, including synthesis of antioxidant molecules (ascorbic acid,

glutathione and carotenoids) and enzyme systems such as superoxide dismutases (SOD,

E.C.1.15.1.1), ascorbate peroxidase (APX, E.C.1.11.1.11) and catalase (CAT, E.C.1.11.1.6).

These ROS can attack membranes, proteins and nucleic acids causing lipid peroxidation, protein

denaturation and DNA mutation (Schützendübel and Polle, 2002). Oxidative stress is probably

an important component of plant response to Al-toxicity.

It has been suggested that Al

, the most toxic of the soluble forms of Al (Parker et al.,

1988), induces oxidative stress, since this ion is involved in various process, including an

increase in SOD activity and lipid peroxidation in soybeans (Cakmak and Horst, 1991), peas

(Yamamoto et al., 2001) and tobacco plants (Ikegawa et al., 2000). Moreover, alterations in the

expression of various genes induced by Al in Arabidopsis (Richards et al., 1998), tobacco

(Ezaki et al., 2000) and wheat (Snowden and Gardner, 1993) have been reported.

Potatos are grown world-wide under a wider range of altitudes, latitudes, and climatic

conditions than any other major food crop – from sea level to over 4000 m elevation. No other

crop can match potato in its production of food energy and food value per unit area (Sieczka and

Thornton, 1993). The widely cultivated potato (Solanum tuberosum subsp. tuberosum) is very

sensitive to abiotic stresses, whereas several wild or primitive cultivated species of different

ploidy levels are well adapted to growth under unfavorable conditions such as drought, cold,

salinity and high irradiation (Li and Fennell, 1985). The fact that the Solanum species possess

genetic variation for abiotic stresses is not only interesting for potato breeding but also as a

model plant to study other aspects of physiological resistance. An appropriate approach to

evaluate the Al stress response is a genotype evaluation in nutrient solution under controlled

conditions (Schmohl et al., 2000; Jorge et al., 2001; Boscolo et al., 2003). In spite of the

importance of potato, there is no report in the literature on its antioxidant system under Al stress

conditions. The antioxidant system is responsible for scavenging excess free radicals caused by

environmental stresses. Studying the major components of the antioxidant system under Al

stress, it is possible to ascertain whether Al induces oxidative stress, and whether it is involved

in Al-tolerance mechanisms.

The objective of the present study was, therefore, to investigate and compare some

physiological and oxidative stress responses of four potato clones, Macaca, SMIC148-A,

Dakota Rose (all of S. tuberosum) and Solanum microdontum, , exposed to Al in nutrient

solution.

MATERIAL AND METHODS

Plant materials and growth conditions: Three adapted (2n=4x=48) clones (Macaca, SMIC148-

A and Dakota Rose) and one wild species (2n=2x=24) clone (PI595511-5/ S. microdontum)

were evaluated. The S. microdontum clone was identified as highly resistant to Phytophora

infestans (Bisognin et al., 2005) and has been used in our breeding program. This clone will be

referred to as S. microdontum. Tissue culture plantlets were obtained from the Potato Breeding

and Genetics Program, Federal University of Santa Maria, Brazil. Nodal segments (1.0 cm long)

were micropropagated in MS medium (Murashige and Skoog, 1962), supplemented with 30 g L

of sucrose, 0.1 g L

-1

of myo-inositol and 6 g L

-1

of agar. Twenty-day-old plantlets from in vitro

culture were transferred into plastic boxes (10 L) filled with aerated full nutrient solution of low

ionic strength. The nutrient solution had the following composition (in µM): 6090.5 of N; 974.3

of Mg; 5229.5 of Cl; 2679.2 of K; 2436.2 of Ca; 359.9 of S; 0.47 of Cu; 2.00 of Mn; 1.99 of Zn;

0.17 of Ni; 24.97 of B; 0.52 of Mo; 47.99 of Fe (FeSO4/Na-EDTA). Treatments consisted of the

addition of 0, 50, 100, 150 or 200 mg L

-1

of Al as AlCl

.6H

O. The solution pH was adjusted

daily to 4.0 ± 0.1 by titration with HCl or NaOH solutions of 0.1 M. Both in vitro and ex vitro

cultured plants were grown in a growth chamber at 25 ± 2ºC on a 16/8-h light/dark cycle with

35 µmol m

-2

-1

of irradiance. Aluminum-treated plantlets remained in each treatment for 7 d. At

harvest, the plants were divided into shoot and roots. Roots were rinsed twice with distilled

water. Subsequently, growth and biochemical parameters were determined. Three replicates

with nine seedlings were made for each treatment.

Aluminum determination: After Al treatment, samples (roots and shoot) were separated and

washed in deionized water twice and dried at 60ºC until reaching a constant weight. The dried

tissues were weighed and ground into a fine powder before nitric-perchloric digestion.

Aluminum concentrations were determined by atomic absorption spectrometry. A standard

calibration curve was prepared for the 0-200 mg L

-1

Al concentration range.

Growth parameters: To access different responses to Al sensitivity the relative root growth

(RRG) of four clones was determined. Before Al treatment, the length of the main root of each

plantlet was measured and recorded. Afterwards, the plantlets returned to the nutrient solution.

At the end of the experiment (7 d after Al application), the length of the main root was

measured again. The RRG was calculated by dividing the root growth of each seedling under a

given treatment by the mean root growth of all plantlets grown in the control solution (Jorge et

al., 2001). Shoot length and total number of nodal segments per plantlet were also determined.

Determination of hydrogen peroxide: The H

concentration was determined according to

Loreto and Velikova (2001). Approximately 0.1 g of both roots and shoots was homogenized at

4ºC in 2 mL of 0.1% trichloroacetic acid (TCA) (w/v). The homogenate was centrifuged at

12,000 x g for 15 min at 4ºC. Then, 0.5 mL of the supernatant was added to 0.5 mL of 10 mM

K-phosphate buffer (pH 7.0) and 1 mL of 1M KI. The H

concentration of the supernatant

was evaluated by comparing its absorbance at 390 nm with a standard calibration curve.

Hydrogen peroxide concentration was expressed as µmol g

-1

FW.

Catalase assay: Catalase activity was assayed following the modified Aebi (1984) method.

Fresh roots and shoot samples (1 g) were homogenized in 5 mL of 50 mM K-phosphate buffer

(pH 7.0), 10 g L

-1

PVP, 0.2 mM EDTA and 10 mL L

-1

Triton X-100. The homogenate was

centrifuged at 12,000 x g for 20 min at 4ºC and the supernatant was used for enzyme assay.

Activity of CAT was determined by measuring the decrease in absorbance at 240 nm of a

reaction mixture with a final volume of 2 mL containing 15 mM H

in K-phosphate buffer

(pH 7.0) and 30 µL extract. Activity was expressed as ∆E min

-1

protein.

Chlorophyll and carotenoid determination: Chlorophyll and carotenoids were extracted

following the method of Hiscox and Israelstam (1979) and estimated with the help of Arnon’s

formulae (Arnon, 1949). Fresh leaves (0.1 g) were incubated at 65ºC in dimethylsulfoxide

(DMSO) until pigments were completely bleached. Absorbance of the solution was then

measured at 663 and 645 nm for chlorophyll and 470 nm for carotenoids. Chlorophyll and

carotenoid concentrations were expressed as µg g

-1

FW and mg g

-1

FW, respectively.

Estimation of lipid peroxides: The degree of lipid peroxidation was estimated following the

method of El-Moshaty et al. (1993). Fresh roots and shoot samples of 0.1 g were homogenized

in 20 mL of 0.2 M citrate-phosphate buffer (pH 6.5) containing 0.5% Triton X-100, using

mortar and pestle. The homogenate was filtered with two paper layers and centrifuged for 15

min at 20,000 x g. One milliliter of the supernatant fraction was added to an equal volume of

20% (w/v) TCA containing 0.5% (w/v) of thiobarbituric acid (TBA). The mixture was heated at

95ºC for 40 min and then quickly cooled in an ice bath for 15 min, and centrifuged at 10,000 x g

for 15 min. The absorbance of the supernatant at 532 nm was read and corrected for unspecific

turbidity by subtracting the value of the absorbance at 600 nm. The lipid peroxides were

expressed as nmol MDA mg

-1

protein, by using an extinction coefficient of 155 L mmol

-1

Protein oxidation: Samples of roots and shoot (1 g) were homogenized with 25 mM K-

phosphate buffer (pH 7.0) containing 10 mL L

-1

Triton X-100, at a proportion of 1:2 (w/v)

(Levine et al., 1990). After the homogenate was centrifuged at 15,000 x g for 10 min at 4ºC, the

supernatant was used for immediate determination of protein oxidation, which was expressed as

nmol carbonyl mg

-1

protein.

Protein determination: In all the enzyme preparations, protein was determined following

Bradford (1976) using BSA for constructing the standard curves.

Statistical analysis: All data were analyzed by ANOVA procedures. The effects of Al on

growth and biochemical parameters in potato plantlets were quantified using regression analysis

with the SOC statistic package (Software Científico: NTIA/EMBRAPA). Coefficients were

included in a regression equation when their values were significant (P < 0.05).

RESULTS

Al concentration: Regression analysis showed that the concentration of Al in both the roots and

shoot of all clones studied increased linearly with increasing Al levels, and the increase in tissue

Al was much steeper for Macaca and SMIC148-A (Figure 1A,B).

Aluminum accumulated more in roots than in shoot (on average of 3.9-, 2.8-, 3.6-, and

3.7-fold greater in roots than in shoot, respectively in Macaca, S. microdontum, SMIC148-A and

Dakota Rose clones). The maximum concentration of Al in roots and shoot was 49,300 and

17,900 mg kg

-1

, as respectively found in the Dakota Rose clone at 200 mg L

-1

. In Macaca and

SMIC148-A clones, Al concentration was lower at levels above 50 mg L

-1

when compared with

the Dakota Rose and S. microdontum clones (Figure 1A, B).

Growth analysis: The response of root growth in the Al-sensitive clones (Macaca and Dakota

Rose) to Al levels was linear and negative (Figure 2A), whereas in the Al-tolerant clones there

was no alteration. At 200 mg L

-1

of Al, root growth of Macaca and Dakota Rose clones

decreased by about 95 and 70%, respectively, when compared to the control. Therefore, Macaca

and Dakota Rose were classified as Al-sensitive clones, the and S. microdontum and SMIC148-

A as Al-tolerant clones.

Aluminum negatively affected shoot length only in Macaca plantlets (Figure 2B). At 200

mg L

-1

of Al, shoot length was decreased by 74% when compared to the control. Also, Al

treatments linearly reduced the total number of nodal segments in Macaca, SMIC148-A and

Dakota Rose clones (Figure 2C).

10000

20000

30000

40000

50000

60000

0 50 100 150 200

Al (m g L¯¹)

A l c o n c en tra tio n (m g k g ¯¹)

(Macaca) y=8889.26+139.51x (R²=0.94)

(S. microdontum) y=60.13+256.6x (R²=0.90)

(SMIC148-A) y=6972.53+143.99x (R²=0.93)

(Dakota Rose) y=9813.6+221.12x (R²=0.93)

3000

6000

9000

12000

15000

18000

21000

0 50 100 150 200

Al (m g L¯¹)

A l c o n c e n tr a tio n (m g k g ¯¹)

(Macaca) y=1459.5+53.35x (R²=0.97)

(S. microdontum) y=667.86+71.22x (R²=0.99)

(SMIC148-A) y=841.2+57.28x (R²=0.99)

(Dakota Rose) y=1319.2+86.1x (R²=0.99)

Figure 1. Aluminum concentration in roots (A) and shoot (B) of potato plants (Macaca, S. microdontum,

SMIC148-A and Dakota Rose clones) submitted to increasing Al levels for 7 d. Each point is the mean of three

replicates.

(A)

(B)

-0.2

0.2

0.4

0.6

0.8

1.2

0 50 100 150 200

Al (mg L¯¹)

Relative root growth

(Macaca) y=0.75-0.0044x (R²=0.72)

(S. microdontum) Mean=1.0 (n.s.)

(SMIC148-A) Mean=1.0 (n.s.)

(Dakota Rose) y=0.68-0.0025x (R²=0.34)

0.2

0.4

0.6

0.8

1.2

1.4

1.6

1.8

0 50 100 150 200

Al (mg L¯¹)

Sh o o t le n g th ( cm )

(Macaca) y=1.72-0.0067x (R²=0.94) (S. microdontum) Mean=1.88 (n.s.)

(SMIC148-A) Mean=1.45 (n.s.) (Dakota Rose) Mean=1.48 (n.s.)

-0.5

0.5

1.5

2.5

3.5

4.5

5.5

0 50 100 150 200

Al (mg L¯¹)

Total num ber of nodal segm ents

(Macaca) y=4.42-0.014x (R²=0.86) (S. microdontum) Mean=2.67 (n.s.)

(SMIC148-A) y=5.06-0.0099x (R²=0.84) (Dakota Rose) y=4.11-0.014x (R²=0.83)

Figure 2. Relative root growth (A), shoot length (B) and total number of nodal segments (C) in potato plants

(Macaca, S. microdontum, SMIC148-A and Dakota Rose clones) submitted to increasing Al levels for 7 d. Each

point is the mean of three replicates. n.s. = not significant.

(B)

(C)

(A)

Catalase activity and hydrogen peroxide concentration: Root H

concentration in the two Al-

sensitive clones increased with increasing Al levels, whereas in the Al-tolerant clones it either

decreased (SMIC148-A) or did not demonstrate any alteration (S. microdontum) (Figure 3A).

Shoot H

concentration increased linearly in Dakota Rose, whereas for Macaca it

showed a quadratic relationship with Al levels. On the other hand, H

concentration in the Al-

tolerant clones either did not demonstrate any alteration (S. microdontum) or presented lower

levels (SMIC148-A) (Figure 3B).

Root CAT activity increased curvilinearly with increasing Al levels in Macaca, whereas

in Dakota Rose it decreased at ca. 50 mg L

-1

and increased at levels exceeding 100 mg L

-1

(Figure 3C). Root CAT activity in the Al-tolerant clones either did not demonstrate any

alteration (SMIC148-A) or presented lower levels (S. microdontum). Shoot CAT activity was

only altered in S. microdontum, where it increased curvilinearly with increasing Al levels

exceeding 100 mg L

-1

(Figure 3D).

Chlorophyll and carotenoids levels: In all potato clones, chlorophyll concentration showed a

curvilinear response to Al supply, where it increased at ca. 50 mg Al L

-1

, except for S.

microdontum, and decreased at levels exceeding 100 mg L

-1

in the Al-sensitive clones.

However, in SMIC148-A, it increased between approximately 100 and 150 mg L

-1

, and in S.

microdontum it decreased regardless of the Al level (Figure 4A). Carotenoid concentrations in

the Al-sensitive clones linearly decreased with increasing Al levels, whereas in the Al-tolerant

clones there was no alteration (Figure 4B).

0.4

0.8

1.2

1.6

0 50 100 150 200

Al (mg L¯¹)

Hydrogen peroxide

concentration (µmol g¯¹ FW)

(Macaca) y=0.23-0.00075x+0.0000072x² (R²=0.81)

(S. microdontum) Mean=0.76 (n.s.)

(SMIC148-A) y=0.443-0.003x+0.0000098x² (R²=0.66)

(Dakota Rose) y=1.063+0.002x (R²=0.71)

0.2

0.4

0.6

0.8

1.2

1.4

0 50 100 150 200

Al (mg L¯¹)

Hydrogen peroxide

concentration (µmol g¯¹ FW)

(Macaca) y=0.1737+0.0023x-0.000011x² (R²=0.62)

(S. microdontum) Mean=0.743 (n.s.)

(SMIC148-A) y=0.365-0.00051x (R²=0.53)

(Dakota Rose) y=1.099+0.00054x (R²=0.66)

0 50 100 150 200

Al (mg L¯¹)

CAT (E min¯¹ mg¯¹ protein)

(Macaca) y=0.152+0.0254x-0.00011x² (R²=0.78)

(S. microdontum) y=6.97-0.113x+0.00053x² (R²=0.97)

(SMIC148-A) Mean=0.52 (n.s.)

(Dakota Rose) y=3.5-0.138x+0.0022x²-0.0000076x³ (R²=0.98)

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 50 100 150 200

Al (mg L¯¹)

CAT (E min¯¹ mg¯¹ protein)

(Macaca) Mean=0.34 (n.s.)

(S. microduntum) y=0.3642-0.0044x+0.000033x² (R²=0.86)

(SMIC148-A) Mean=0.39 (n.s.)

(Dakota Rose) Mean=0.23 (n.s.)

Figure 3. Concentration of H

in roots (A) and shoot (B) and catalase activity of roots (C) and shoot (D) in

potato plants (Macaca, S. microdontum, SMIC148-A and Dakota Rose clones) submitted to increasing Al levels for

7 d. Each point is the mean of three replicates. n.s. = not significant.

(A)

(B)

1000

1100

1200

1300

1400

1500

1600

0 50 100 150 200

Al (mg L¯¹)

Chlorophyll (µg g¯¹ FW)

(Macaca) y=1318.4+1.48x-0.013x² (R²=0.36)

(S. microdontum) y=1435.095-2.59x+0.012x² (R²=0.47)

(SMIC148-A) y=1201.41-1.42x+0.044x²-0.00018x³ (R²=0.64)

(Dakota Rose) y=1425.34+8.42x-0.133x²+0.00043x³ (R²=0.61)

0.2

0.4

0.6

0.8

1.2

1.4

0 50 100 150 200

Al (mg L¯¹)

C a rote noids (m g g¯¹ FW )

(Macaca) y=1.143-0.0012x (R²=0.30)

(S. microdontum) Mean=0.5135 (n.s.)

(SMIC148-A) Mean=0.4325 (n.s.)

(Dakota Rose) y=1.311-0.00214x (R²=0.6)

Figure 4. Chlorophyll (A) and carotenoid (B) concentrations in potato plants (Macaca, S. microdontum, SMIC148-

A and Dakota Rose clones) submitted to increasing Al levels for 7 d. Each point is the mean of three replicates. n.s.

= not significant.

Lipid peroxidation and protein oxidation: Concentration of MDA in roots and shoot of both

Macaca and Dakota Rose increased linearly with increasing Al levels, indicating enhanced lipid

peroxidation for these Al-sensitive clones (Figure 5A, B). In Macaca and Dakota Rose, the

increase of lipid peroxidation in roots was of ca. 55% and 73%, respectively, and in the shoot it

(B)

(A)

was of about 72% and 149%, respectively. Interestingly, the basal level of lipid peroxidation

both in roots and shoot of S. microdontum (Al-tolerant clone) was significantly higher than that

of the others. In spite of this, only at lower Al levels did an increase in lipid peroxidation occur

(65%) in the shoot of this clone (Figure 5B).

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0 50 100 150 200

Al (mg L¯¹)

MDA (nmol mg¯¹ protein)

(Macaca) y=0.0299+0.000113x (R²=0.89)

(S. microdontum) Mean=0.118 (n.s.)

(SMIC148-A) Mean=0.06 (n.s.)

(Dakota Rose) y=0.0615+0.00029x (R²=0.72)

0.05

0.1

0.15

0.2

0.25

0.3

0 50 100 150 200

Al (mg L¯¹)

M D A (n m o l m g¯¹ p ro tein )

(Macaca) y=0.028+0.000123x (R²=0.86)

(S. microdontum) y=0.16+0.005x-0.00006x²+0.0000002x³ (R²=0.99)

(SMIC148-A) Mean=0.042 (n.s.)

(Dakota Rose) y=0.0342+0.000283x (R²=0.75)

0 50 100 150 200

Al (mg L¯¹)

C a r b o n y l ( n m o l m g ¯ ¹ p r o te in )

(Macaca) y=13.2+0.067x (R²=0.68)

(S. microdontum) y=14.88+0.0087x-0.00007x² (R²=0.56)

(SMIC148-A) y=12.52-0.046x+0.00036x² (R²=0.86)

(Dakota Rose) y=16.34+0.039x (R²=0.91)

0 50 100 150 200

Al (mg L¯¹)

C a r b o n y l (n m o l m g ¯¹ p r o t e in )

(Macaca) y=14.44-0.069x+0.00043x² (R²=0.90)

(S. microdontum) y=7.10+0.1085x-0.0004x² (R²=0.55)

(SMIC148-A) y=6.85+0.029x (R²=0.65)

(Dakota Rose) y=8.86+0.053x (R²=0.89)

Figure 5. Lipid peroxides in roots (A) and shoot (B) and protein carbonyl in roots (C) and shoot (D) in potato

plants (Macaca, S. microdontum, SMIC148-A and Dakota Rose clones) submitted to increasing Al levels for 7 d.

Each point is the mean of three replicates. n.s. = not significant

(A) (B)

The carbonyl concentrations in the roots of Al-sensitive clones increased linearly with

increasing Al levels, while in the Al-tolerant clones there was a curvilinear response in which

SMIC148-A presented increased carbonyl concentrations at levels exceeding 100 mg L

-1

and, in

contrast, S. microdontum presented a decline in the same Al range (Figure 5C). In the shoot, an

increase in protein oxidation occurred for all clones except in the Macaca clone, where it

decreased at levels below 150 mg L

-1

(Figure 5D). In SMIC148-A and Dakota Rose clones, Al

caused a linear increase in carbonyl concentrations, whilst in the S. microdontum clones the

carbonyl concentrations increased curvelinearly with increasing Al levels.

DISCUSSION

In the present study, the significant, variable reduction of root growth in potato clones

exposed to Al suggests a distinct physiological sensitivity to Al stress. The phytotoxic effects of

Al to the root system, in turn, can cause susceptibility to drought stress and mineral nutrient

deficiencies (Degenhardt et al., 1998). This, consequently, might negatively affect growth and

development of Al-sensitive plants. Since Al also induced root damage, and roots are the main

site of cytokinin synthesis, the reduction in shoot length may therefore be a consequence of

impaired cell division in the root meristem (Meriga et al., 2004). Since Al primarily affects the

root tips, effects on shoot development may be expressed only at later stages as a result of

altered water and nutrient uptake as well as phytohormone production (Collet and Horst, 2001).

Nutrient solutions used as a substrate contain divalent cations which can compete with

Al and influence their availability for plant uptake. Yet, the high Al concentrations of the

nutrient solution overcame these limiting factors, since Al concentrations in root tissues showed

a significant 3.5-fold increase between Al levels of 0 and 200 mg L

-1

. Almost all the

adsorbed/precipitated Al on the roots’ outer surface and in root cortical cells is not removed

after washing with water. Thus, as roots were only washed with deionized water before Al

analysis, the values obtained for Al concentrations are related to both absorbed and adsorbed

mechanisms. The sharp increase in root Al concentrations was closely related to the level of Al

in the nutrient solution, as has been reported elsewhere (Lidon et al., 1999). Moreover, the quite

high Al concentration measured in root tissues in the control was related to a direct uptake from

the water/tray substrate. Root Al concentrations for the Al treatments were mostly associated

with an increase in the level of Al in the nutrient solution. Accumulation of Al was lower in the

shoot, which indicates that the absorbed Al was mostly retained in root tissues.

A common feature of several stresses, including Al toxicity, is the perturbation of cell

redox homeostasis, enhancing ROS production, which is generally considered harmful to plant

cells (Richards et al., 1998). Studies on Al-toxicity in roots suggest that the production of ROS

may significantly contribute to Al-induced inhibition of root elongation (Tamás et al., 2004).

Furthermore, several reports have shown that Al stress can increase the production of ROS, and

activate several oxidative enzymes in plant and animal cells (Cakmak and Horst, 1991). Thus,

oxidative stress is possibly an important component of Al-toxicity plant responses.

In the present study, Al stress increased H

concentration in roots and shoot of both

Al-sensitive clones. Elevated H

production due to Al has also been observed in barley

(Simonovicová et al., 2004), wheat (Darkó et al., 2004) and pumpkin roots (Dipierro et al.,

2005). Tamás et al. (2004) also reported elevated H

production in intact germinating barley

seeds during Al stress. In relation to roots and shoot of Al-tolerant clones, in the SMIC148-A

clone reduced H

concentration was observed with increasing Al levels. This decline of H

concentration might be due to the scavenging action of the antioxidant system. It can be

suggested that Al treatment mainly induced an oxidative burst in both roots and shoot of the Al-

sensitive clones, where the antioxidant system was not able to protect these clones from Al

toxicity.

Among the enzymatic systems considered to play an important role in the cellular

defense strategy against oxidative stress, CAT plays a pivotal role as it decomposes H

water and O

. Interestingly, the greater CAT activity in roots than in shoot might indicate higher

oxidative stress in roots. In fact, the marked increase of CAT activity in the roots with

increasing Al levels in Macaca and Dakota Rose clones may indicate enhanced production of

ROS under an excess of Al. In contrast, at lower levels of Al exposure, CAT activity was

inhibited in roots of S. microdontum and Dakota Rose clones. This lower activity in Al-stressed

plants is suggestive of a possible delay in removal of H

and toxic peroxides mediated by

CAT and in turn an enhancement in the free radical mediated lipid peroxidation under Al-

toxicity (Shi et al., 2006). Aluminum increased the activity of CAT in the shoot only in the S.

microdontum clone at higher Al concentrations. This enhanced activity seems to be related to

increased oxidative stress tolerance (Allen, 1995).

As a visible symptom, the reduced chlorophyll concentration can be used to monitor Al

induced damage in green leaves. In the present study, the reduction in chlorophyll concentration

observed for Macaca, S. microdontum and Dakota Rose clones indicates oxidative damage

induced by Al exposure, possibly due to the inhibition of aminolevulinic acid dehydratase, an

important enzyme in chlorophyll biosynthesis (Pereira et al., 2006). Carotenoid concentration

decreased in Macaca and Dakota Rose (Al-sensitive clones). Although the principal recognized

role of carotenoids is to act as photoreceptive antenna pigment for photosynthesis, collecting

wavelengths of light that are not absorbed by chlorophylls, their protective function against

oxidative damage has also been recognized for several decades (Larson, 1988). Perhaps the

most important function of carotenoid is the dissipation of excess energy of excited chlorophyll

and the elimination of ROS (Lawlor, 2001).

Lipid peroxidation is a metabolic process that can occur under normal aerobic conditions

and is one of the most investigated ROS effects on membrane structure and function (Blokhina

et al., 2003). It is widely reported that ROS bring about peroxidation of membrane lipids leading

to membrane damage. Since cell membranes are the first targets of many plant stresses, the

maintenance of their integrity and stability under stress conditions is a major component of Al

tolerance in plants. In the present study, MDA concentration in both roots and shoot was

significantly increased with increasing Al levels in the two Al-sensitive clones, indicating

enhanced lipid peroxidation for these clones and, therefore, the presence of poisoning ROS. In

S. microdontum (Al-tolerant clone), an increased in shoot lipid peroxidation occurred only at Al

levels of approximately 50 mg L

-1

, indicating that the active stress was lower and growth

inhibition was smaller. These results also indicate that the antioxidative system in Al-tolerant

clones was more efficient to protect the membrane lipids from Al stress. Several studies have

shown increased lipid peroxidation in plants exposed to Al. In wheat, the increase of MDA

concentration in the Al-sensitive cultivar was greater than in Al-tolerant cultivar (Dong et al.,

2002). Yamamoto et al. (2001) showed the induction of lipid peroxidation in pea plants after 4 h

of exposure to Al

. Cakmak and Horst (1991) also observed an increase in lipid peroxidation of

a sensitive soybean cultivar after 24 h of treatment. Basu et al. (2001) found a correlation

between decreased lipid peroxidation and increased resistance to Al in Brassica napus.

The possible connection between Al stress and oxidative stress had also been previously

suggested by Cakmak and Horst (1991) based upon the fact that the Al-induced inhibition of

root elongation was correlated with enhanced lipid peroxidation. The interpretation of these

findings was that the primary effects of Al could be the induction of free radical generation and

related alterations in the membrane structure. Further evidence corroborating the relation

between Al stress and oxidative stress in plants has been obtained with transgenic arabidopsis

plants (Ezaki et al., 2000).

In the present study, protein damage due to increased stress suggests Al-induced

formation of ROS. Halliwell and Gutteridge (1999) suggested that the oxidation of proteins to

form carbonyls occurs via the hydroxyl radical, since neither H

nor superoxide is reactive

enough to provoke oxidation. The accumulation of carbonyls in the shoot of all potato clones

studied indicates that the quantity of radicals generated exceeded the capacity of the antioxidant

defensive system, whereas in roots of the Al-tolerant clones, ROS were eliminated by plant

defenses more efficiently. Interestingly, Boscolo et al. (2003) found that the onset of protein

oxidation in two inbred lines of maize took place after the reduction of RRG observed in the

sensitive line, indicating that oxidative stress is not the primary cause of root growth inhibition.

In addition, the presence of Al did not induce lipid peroxidation in either line, contrasting with

the observations made in other species. In order to characterize four potato clones for their Al

sensitivity in hydroponics, using root elongation, Al-induced callose formation and Al

concentrations of root tips as parameters, Schmohl et al. (2000) found that the higher genotypic

Al sensitivity was related to enhanced Al accumulation in root tips, and that the transgenic

potato mutant that overexpressed pectin methylesterase proved to be more Al-sensitive than the

wild type. These data clearly demonstrate the importance of apoplast properties for the

expression of Al-toxicity.

Although potato is a commercially important plant, there is not much knowledge about

its Al stress tolerance and the physiological consequences of this stress. Thus, the finding that

Solanum species possess genetic differences in abiotic stress resistance shows that it is good

plant material for studying other aspects of abiotic stress resistance mechanisms. Based on the

present work, it can be suggested that toxic concentrations of Al cause oxidative stress, as

evidenced by increased H

formation, lipid peroxidation and oxidation of proteins in roots

and shoot of plants, mainly in Al-sensitive clones. In this study, a significant reduction in

different parameters such as length of shoot and roots, chlorophyll and carotenoid

concentrations coupled with lipid peroxidation and protein oxidation indicated that high Al

levels in nutrient solution produced toxic effects. It was proposed that the reduced growth in Al-

sensitive clones of potato exposed to toxic levels of Al might be induced by an enhanced

production of toxic oxygen species and subsequent lipid peroxidation. Moreover, it was possible

to observe that Al-tolerant plants developed some defense mechanisms against oxidative stress.

Further studies are required to investigate whether the oxidative stress caused by Al toxic levels

is an early symptom that can trigger root growth inhibition.

Acknowledgements: The authors thank the Coordenação e Aperfeiçoamento de Pessoal de

Nível Superior, Conselho Nacional de Desenvolvimento Científico e Tecnológico, and

Fundação de Amparo à Pesquisa de Estado do Rio Grande do Sul for the research fellowships.

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Borher D, Rocha JBT, Schetinger MRC (2006) Effect of aluminum on aminolevulinic acid

dehydratase (ALA-D) and the development of cucumber (Cucumis sativus). Environ. Exp. Bot.

57:106-115.

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oxidative stress genes in Arabidopsis thaliana. Plant Physiol. 116:409-418.

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spatial sensitivity and the role of the root cap. J. Exp. Bot. 44:437-446.

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sensitivity in Zea mays and Solanum tuberosum. Physiol. Plant. 109:419-427.

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oxidative stress and protection by mycorrhization. J. Exp. Bot. 53:1351-1365.

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probably caused by cell death due to peroxidase-mediated hydrogen peroxide production.

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aestivum) roots. Plant Physiol. 103:855-861.

Tamás L, Simonovicová M, Huttová J, Mistrík I (2004) Aluminium stimulated hydrogen

peroxide production of germinating barley seeds. Environ. Exp. Bot. 51:281-288.

Yamamoto Y, Kobayashi Y, Matsumoto H (2001) Lipid peroxidation is an early symptom

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Physiol. 125:199-208.

Manuscrito I

Oxidative stress is an early symptom triggered by aluminum in Al-

sensitive potato plantlets

OXIDATIVE STRESS IS AN EARLY SYMPTOM TRIGGERED BY ALUMINUM IN Al-

SENSITIVE POTATO PLANTLETS

Luciane Almeri Tabaldi

1,4

, Denise Cargnelutti

2,5

, Jamile Fabbrin Gonçalves

1,4

, Luciane

Belmonte Pereira

2,5

, Gabriel Y Castro

, Joseila Maldaner

1,4

, Renata Rauber

, Liana

Verônica Rossato

, Dilson Antônio Bisognin

3,4

, Maria Rosa Chitolina Schetinger

2,5

Fernando Teixeira Nicoloso*

1,4

Departamento de Biologia

, Química

e Fitotecnia

, Programa de Pós-Graduação em

Agronomia

e Bioquímica Toxicológica

, Centro de Ciências Naturais e Exatas,

Universidade Federal de Santa Maria, 97105-900, Santa Maria, RS, Brasil.

*[email protected]

ABSTRACT

The objective of this study was to evaluate whether the oxidative stress caused

by aluminum (Al) toxicity is an early symptom that can trigger root growth inhibition in

Macaca and SMIC148-A potato clones. Potato plantlets were grown in a nutrient

solution (pH 4.00±0.1) with 0, 100 and 200 mg Al l

-1

. At 24, 72, 120 and 168 h after Al

addition, growth (root length) and biochemical parameters (lipid peroxidation, catalase

(CAT) and ascorbate peroxidase (APX) activity, and ascorbic acid (AsA) and non-

protein thiol group (NPSH) concentration) were determined. Regardless of exposure

time, root length of the Macaca clone was significantly lower at 200 mg Al l

-1

. For the

SMIC148-A clone, root length did not decrease with any Al treatments. Therefore,

potato clones were classified as Al-sensitive (Macaca) and Al-tolerant (SMIC148-A). Al

supply caused lipid peroxidation only in the Al-sensitive clone (Macaca), in both roots

(at 24, 72, 120 and 168 h) and shoot (at 120 and 168 h). In roots of the Al-sensitive

clone, CAT and APX activity decreased at 72 and 120 h, and at 24, 72 and 120 h,

respectively. At 168 h, both activities increased upon addition of Al. In the shoot, CAT

activity decreased at 72 and 168 h and increased at 120 h, whereas APX activity

increased at 120 and 168 h. In roots of the Al-tolerant clone, CAT activity increased at

72 and 168 h, whereas APX activity decreased at 72 h and increased at 24, 120 and

168 h. In the shoot, CAT activity increased only at 120 h, whereas APX activity

decreased at 24 h. However, APX activity increased at 72, 120 and 168 h after Al

supply. The Al-sensitive clone showed lower root NPSH concentration at 200 mg Al l

-1

in all evaluations, but the Al-tolerant clone either did not demonstrate any alterations at

24 and 72 h or presented higher levels at 120 h. This pattern was also observed in root

AsA concentration at 24 and 120 h. In the shoot, NPSH concentration in the Al-tolerant

clone did not increase with Al supply for up to 120 h of exposure, whereas in the Al-

sensitive clone it increased at 24, 120 and 168 h with the addition of 200 mg Al l

-1

. The

cellular redox status of these potato clones seems to be affected by Al. Therefore,

oxidative stress may be an important mechanism for Al toxicity, mainly in the Al-

sensitive Macaca clone.

Keywords: Solanum tuberosum; Aluminum; Growth; Antioxidants.

1. INTRODUCTION

Potatoes are grown world-wide under a wider range of altitudes, latitudes, and

climatic conditions than any other major food crop - from sea level to over 4000 m

elevation. No other crop can match the potato in its production of food energy and food

value per unit area (Sieczka & Thornton, 1993). The widely cultivated species of

potato, Solanum tuberosum subsp. tuberosum, is very sensitive to abiotic stress (Li &

Fennell, 1985). However, further studies indicated that the Solanum species possess

genetic diversity to abiotic stress (Martinez et al., 2001).

On a global scale, acid soils comprise a surface estimated at 37.8 million Km

(Eswaran et al., 1997). Moreover, soil acidification has been increased around the

world as a result of human activities, atmospheric liberation of industrial contaminants,

and continuous use of ammonia- and amide-containing fertilizers (Rengel & Zhang,

2003). Such soils are a major constraint to agricultural production in Latin America.

They tend to contain low levels of essential cations, nitrogen and plant-available

phosphate, in combination with high levels of phytotoxic aluminum (Al) (Rao et al.,

1993) (in this paper we will refer to Al

as Al). As soil pH decreases, active Al in the

soil shows a great increase, resulting in toxicity to plants. In Brazil, roughly 60% of soils

with potential for agricultural activity present Al toxicity (Abreu Jr. et al., 2003).

The Al

cation is toxic to many plants at micromolar concentrations, affecting

primarily the normal functioning of roots, with a rapid inhibition of growth (Ryan et al.,

1993), which can occur within minutes or hours of exposure of roots to Al. Severe Al

phytotoxicity reduces and damages the root system, which results in poor nutrient and

water acquisition and transport, consequently leading to nutrient deficiencies and crop

yield reduction (Kochian, 1995).

During evolution, plants have developed numerous mechanisms that allow for

survival in acid soils with higher availability of Al (Kochian, 1995). As a result of

selection pressure, inter- and intra-species differences in response to Al are widely

observed in the plant kingdom. Moreover, great differences in tolerance to Al have

been reported among genotypes of the same species. Over the last several years, a

great diversity of results obtained in physiologic and molecular mapping studies have

shown that plant tolerance to Al toxicity is a complex multigenic characteristic that can

involve several mechanisms of tolerance (Kochian, 1995; Barceló & Poschenraider,

2002; Kochian et al., 2004). These include external (or exclusion) and internal

detoxification mechanisms, such as the immobilization of Al by cell wall components

(Zhang et al., 1997), the exudation of organic acids for the detoxification of Al in the

apoplast (Ryan et al., 2001), Al compartmentation in the vacuole, etc. In this context,

selection of varieties that are productive and tolerant to Al toxicity must be considered a

very important component of strategies for dealing with acid soils.

A wide range of cellular responses occur when plants are exposed to a variety of

environmental stresses such as freezing, drought, salinity and metal toxicity. It has

been suggested that Al

, the most toxic of the soluble forms of Al (Parker et al., 1988),

induces oxidative stress, since this ion is related to an increased ascorbate peroxidase

activity and an increased level of ascorbic acid and H

concentration (Dipierro et al.,

2005). Richards et al. (1998) reported that oxidative stress genes, including peroxidase

and glutathione-S-transferase, were induced in Arabidopsis thaliana in the presence of

Al. Further evidence corroborating the relation between Al stress and oxidative stress

in plants has been obtained with transgenic Arabidopsis plants (Ezaki et al., 2000).

Induction of oxidative stress-related genes by Al stress confirmed the important role of

reactive oxygen species (ROS) in this stress (Tamás et al., 2003).

Oxidative stress is characterized by the production of ROS and reactive nitrogen

species (RNS), including the superoxide radical (O

•−), hydroxyl radical (•OH),

hydrogen peroxide (H

) and nitric oxide (NO), that are able to initiate a free radical

chain reaction. ROS and RNS can cause oxidative damage to the biomolecules such

as lipid, protein and nucleic acids, leading to cell membrane peroxidation, loss of ions,

protein hydrolysis, and even DNA strand breakage (Schützendübel & Polle, 2002).

Studies of Al toxicity in roots suggest that production of ROS may significantly

contribute to Al-induced inhibition of root elongation (Tamás et al., 2004). Both plants

and animals possess antioxidant systems that counteract the action of ROS. To

mitigate the oxidative damage initiated by ROS, plants have developed a complex

defense anti-oxidative system, including low-molecular mass antioxidants (ascorbic

acid, glutathione and carotenoids) as well as antioxidative enzymes, such as

superoxide dismutase (SOD), catalase (CAT) and peroxidases (Koca et al., 2006).

There are several indications that oxidative stress is involved in the plant

responses to Al stress. Therefore, the objective of this study was to evaluate whether

oxidative stress caused by Al toxicity is an early symptom that can trigger root growth

inhibition in two potato clones that differ in Al tolerance.

2. MATERIALS AND METHODS

2.1. Plant materials and growth conditions

Two clones (Macaca (Al-sensitive) and SMIC148-A (Al-tolerant)) of Solanum

tuberosum subsp. tuberosum were evaluated. These clones were chosen after

preliminary tests revealed that they differed in Al-tolerance. Tissue culture plantlets

were obtained from the Potato Breeding and Genetics Program, Federal University of

Santa Maria, RS, Brazil. Nodal segments (1.0 cm long) were micropropagated in MS

medium (Murashige & Skoog, 1962), supplemented with 30 g l

-1

of sucrose, 0.1 g l

-1

myo-inositol and 6 g l

-1

of agar.

Twenty-day-old plantlets from in vitro culture were transferred into plastic boxes

(10 L) filled with aerated full nutrient solution of low ionic strength. The nutrient solution

contained the following composition (in µM): 6090.5 of N; 974.3 of Mg; 5229.5 of Cl;

2679.2 of K; 2436.2 of Ca; 359.9 of S; 0.47 of Cu; 2.00 of Mn; 1.99 of Zn; 0.17 of Ni;

24.97 of B; 0.52 of Mo; 47.99 of Fe (FeSO

/Na-EDTA). Al stress was induced by

adding 0, 100 or 200 mg l

-1

of Al as AlCl

.6H

O. The pH of the solutions was adjusted

to 4.00±0.1 daily by titration with HCl or NaOH solutions 0.1 M. Both in vitro and ex

vitro cultured plants were grown in a growth chamber at 25±2°C on a 16/8 h light/dark

cycle with 35 µmol m

-2

-1

of irradiance. Al-treated plantlets remained in the respective

solutions for 24, 72, 120 and 168 h.

At harvest (24, 72, 120 and 168 h after Al application), all living plantlets from

each container were divided into two sub-samples. Plants were partitioned into shoot

and roots. During each sampling, a new set of plantlets was used. Roots were rinsed

twice with fresh aliquots of distilled water. Subsequently, growth and biochemical

parameters were determined. Three replicates were made for each treatment, with

twenty five plantlets per replicate.

2.2. Growth parameters

To assess different responses to Al sensitivity, the root length of two clones was

determined before and after the treatments, at different times (24, 72, 120 and 168 h

after Al application).

2.3. Estimation of lipid peroxides

The degree of lipid peroxidation was used to calculate membrane integrity and

was estimated following the method of El-Moshaty et al. (1993). Fresh root and shoot

samples were collected at regular intervals (24, 72, 120 and 168 h after Al application),

weighed (0.1 g fresh weight) and ground in 10 ml of 0.2 M citrate-phosphate buffer (pH

6.5) containing 0.5% Triton X-100, using mortar and pestle. The homogenate was

filtered with two paper layers and centrifuged for 15 min at 20,000 x g. One milliliter of

the supernatant fraction was added to an equal volume of 20% (w/v) trichloroacetic

acid (TCA) containing 0.5% (w/v) of thiobarbituric acid (TBA). The mixture was heated

at 95°C for 40 min and then quickly cooled in an ice bath for 15 min, and centrifuged at

10,000 x g for 15 min. The absorbance of the supernatant at 532 nm was read and

corrected for unspecific turbidity by subtracting the value at 600 nm. The lipid peroxides

were expressed as nmol MDA mg

-1

protein.

2.4. Catalase assay

Catalase (CAT) activity was assayed following the modified Aebi (1984) method.

Fresh root and shoot samples (1 g) were collected at regular intervals (24, 72, 120 and

168 h after Al application) and homogenized in 3 ml of 50 mM KH

/ K

HPO

(pH

7.0), 10 g l

-1

PVP, 0.2 mM EDTA and 10 ml l

-1

Triton X-100. The homogenate was

centrifuged at 12,000 x g for 20 min at 4°C and supernatant was then used for the

enzyme assay. CAT activity was determined by monitoring the disappearance of H

by measuring the decrease in absorbance at 240 nm of a reaction mixture with a final

volume of 2 ml containing 15 mM H

in KPO

buffer (pH 7.0) and 30 µl extract.

Activity was expressed as ∆E min

-1

protein.

2.5. Ascorbate Peroxidase assay

Ascorbate peroxidase (APX) was measured according to Zhu et al. (2004). The

reaction mixture, at a total volume of 2 ml, contained 25 mM (pH 7.0) sodium

phosphate buffer, 0.1 mM EDTA, 0.25 mM ascorbate, 1.0 mM H

and 100 µl enzyme

extract. H

-dependent oxidation of ascorbate was followed by a decrease in the

absorbance at 290 nm (E= 2.8 l mM

-1

) and activity was expressed as µmol

oxidized ascorbate min

-1

protein.

2.6. Ascorbic acid (AsA) and non-protein thiol group (NPSH) concentrations

Potato plantlets were homogenized in a solution containing 50 mM l

-1

Tris-HCl

and 10 ml l

-1

Triton X-100 (pH 7.5), centrifuged at 6,800 x g for 10 min. To the

supernatant was added 10% TCA at a proportion of 1:1 (v/v) followed by centrifugation

(6,800 x g for 10 min) to remove protein. Ascorbic acid determination was performed as

described by Jacques-Silva et al. (2001). An aliquot of the sample (300 µl) was

incubated at 37°C in a medium containing 100 µl TCA 13.3%, 100 µl deionized water

and 75 µl 2,4-Dinitrophenylhydrazine (DNPH). The DNPH solution contained 2%

DNPH, 0.23% thiourea, 0.27% CuSO

diluted in 49% H

. After 3 h, 500 µl of 65%

was added and samples were read at 520 nm. A standard curve was

constructed using L(+) ascorbic acid. Non-protein thiol concentration in plantlets was

spectrophotometrically measured with Ellman’s reagent (Ellman, 1959). An aliquot of

the sample (400 µl) was added to a medium containing 550 µl 1 M l

-1

Tris-HCl (pH 7.4).

The reaction was read at 412 nm after the addition of 10 mM l

-1

5-5-dithio-bis 2-

nitrobenzoic acid (DTNB) (0.05 ml). A standard curve using cysteine was used to

calculate the thiol groups concentration in the samples.

2.7. Protein extraction

In all the enzyme preparations, protein was determined following the method of

Bradford (1976) using bovine serum albumin as standard.

2.8. Statistical analysis

Data were submitted to variance analyses and treatment means were compared

by Tukey’s range test at 5% of error probability. Treatments were presented as mean ±

S.D. of three replicates.

3. RESULTS

3.1. Aluminum effects on growth

The effects of Al on the root growth of potato clones are shown in Fig. 1.

Significant differences were observed in root length between the potato clones under Al

stress. After 24 h of Al exposure, root length in the Macaca clone was significantly

lower (about 15%) at 200 mg Al l

-1

(Fig. 1A). This data clearly showed that in the

Macaca clone, root elongation was inhibited by Al at the first point of time after Al

exposure. The same behavior was observed at 72, 120 and 168 h of metal exposure,

with inhibitions of 15%, 20% and 17%, respectively, compared to the control. On the

other hand, in the SMIC148-A clone Al treatment did not affect root length (Fig. 1B).

This difference in root growth indicates a distinct sensitivity to Al between the two

clones.

24 72 120 168

Ti m e (h )

Root Le ngth (c m)

0 mg l

- 1

100 mg l

- 1

200 mg l

- 1

24 72 120 168

Ti m e (h )

Ro ot L eng th (c m)

0 mg l

- 1

100 mg l

- 1

200 mg l

- 1

Figure 1- Time-course of Al inhibition of root length (Macaca (A) and SMIC148-A (B) clones) of potato

plantlets exposed to different concentrations of Al in nutrient solution. The data are the mean ± S.D. of

three different replicates.

3.2. Al effect on lipid peroxidation level

A significant increase in MDA content in both roots and shoot were observed

only in the Al-sensitive clone (Macaca) (Fig. 2A and 2B). The highest increase in MDA

content was seen in roots under Al treatment. In roots, an increase of 80% in MDA

content was observed in the first 24 h at 200 mg Al l

-1

(Fig. 2A). Such an effect was

also observed at later times. At 168 h after Al-treatments, an increase of 45% and 78%

in MDA content occurred at 100 and 200 mg Al l

-1

, respectively, indicating that, after

(A)

(B)

prolonged incubation at these highly toxic Al levels, the toxicity remained severe. In the

shoot (Fig. 2B), lipid peroxidation increased only at 120 and 168 h after Al treatment.

In the SMIC148-A clone, Al treatment did not provoke lipid peroxidation in either

roots or shoot at any time of exposure (Fig. 2C and 2D, respectively).

24 72 120 168

Ti m e (h )

0. 00

0. 06

0. 12

0. 18

0. 24

0. 30

MDA (n mo l mg

- 1

p ro tein)

0 mg l

- 1

100 mg l

- 1

200 mg l

- 1

24 72 120 168

Ti m e (h)

0. 00

0. 04

0. 08

0. 12

0. 16

0. 20

MDA (nmo l mg

- 1

pro te in)

0 mg l

- 1

100 mg l

- 1

200 mg l

- 1

24 72 120 168

Ti m e (h)

0. 00

0. 06

0. 12

0. 18

0. 24

0. 30

MDA (n mo l mg

- 1

pro tei n)

0 mg l

- 1

100 mg l

- 1

200 mg l

- 1

24 72 120 168

Ti m e (h )

0. 00

0. 06

0. 12

0. 18

0. 24

0. 30

MDA (nmol mg

- 1

prote in )

0 mg l

- 1

100 mg l

- 1

200 mg l

- 1

Figure 2- Effect of Al on lipid peroxidation over time in Macaca (Root (A) and Shoot (B)) and SMIC148-A

(Root (C) and Shoot (D)) potato clones. The data are the mean ± S.D. of three different replicates.

3.3. Effects on antioxidative systems

3.3.1. Enzymes

(C)

(D)

(A) (B)

Among the enzymatic antioxidants in plants, catalase (CAT) can transform

peroxides into non-reactive species. A time-, organ- and clone-dependent response to

Al stress was observed (Fig. 3).

24 72 120 168

Ti m e (h )

0. 0 0

1. 8 0

3. 6 0

5. 4 0

7. 2 0

9. 0 0

CA T (³ E mi n

- 1

p ro te i n )

0 m g l

- 1

100 mg l

- 1

200 mg l

- 1

24 72 120 168

Ti m e (h )

0. 0 0

1. 2 0

2. 4 0

3. 6 0

4. 8 0

6. 0 0

CAT (³ E mi n

- 1

p ro te in )

0 m g l

- 1

100 m g l

- 1

200 m g l

- 1

a b

24 72 120 168

Ti m e (h )

0. 0 0

1. 2 0

2. 4 0

3. 6 0

4. 8 0

6. 0 0

CAT (³ E min

- 1

p ro te i n )

0 m g l

- 1

100 mg l

- 1

200 m g l

- 1

a b

24 72 120 168

Ti m e (h )

0. 0 0

1. 2 0

2. 4 0

3. 6 0

4. 8 0

6. 0 0

CA T (³ E mi n

- 1

p ro te i n )

0 m g l

- 1

100 mg l

- 1

200 mg l

- 1

Figure 3- Effect of Al on catalase activity over time in Macaca (Root (A) and Shoot (B)) and SMIC148-A

(Root (C) and Shoot (D)) potato clones. The data are the mean ± S.D. of three different replicates.

In general, CAT activity was greater for the Al-sensitive clone (Macaca) than for

the Al-tolerant clone (SMIC148-A). Moreover, in both potato clones, root CAT activity

was greater than that of the shoot. At 24 h after Al treatment, root and shoot CAT

activities remained unchanged, except for the Al-sensitive clone, which showed a

significant decrease in shoot tissue at 200 mg Al l

-1

, in comparison with 100 mg Al l

-1

At 72 and 120 h, the presence of Al caused a decrease in root CAT activity for the

(C)

(D)

(A) (B)

Macaca clone. A similar pattern in root CAT activity was observed at 120 h for the

SMIC148-A clone (Fig. 3C). Conversely, after prolonged exposure (168 h) to both

levels of 100 mg Al l

-1

and 200 mg Al l

-1

, root CAT activity was increased for both

clones.

In the Al-sensitive clone, shoot CAT activity decreased significantly upon

exposition of Al after 72 and 168 h. On the other hand, at 120 h shoot CAT activity

increased at 100 mg Al l

-1

, whereas no significant difference in shoot CAT activity was

found between 200 mg Al l

-1

and the control. In the Al-tolerant clone, shoot CAT activity

was altered after prolonged incubation. At 120 h, CAT activity increased at all levels of

Al, whereas at 168 h, CAT activity was slightly, but not significantly, increased by 100

mg Al l

-1

Ascorbate peroxidase (APX) activity (Fig. 4) was greater in roots than in shoots

for both potato clones. In roots of the Macaca clone, APX activity was inhibited at 24 h

(100 and 200 mg l

-1

), 72 h (100 and 200 mg l

-1

) and 120 h (200 mg l

-1

) of Al exposure.

On the other hand, at 168 h, APX activity increased by about 80% at 200 mg l

-1

, when

compared to the control (Fig. 4A). In the shoot (Fig. 4B), APX activity was increased

upon addition of Al at the later points of time (120 and 168 h). The effect of Al on APX

activity in roots and shoot of the SMIC148-A clone is shown in Fig. 4C and 4D,

respectively. The presence of 200 mg Al l

-1

induced the activity of root APX at 120 h

(184%) and 168 h (50%). Conversely, at 72 h of Al-exposure, root APX activity was

inhibited by about 60% (Fig. 4C). In the shoot, after 24 h Al exposure, at 200 mg l

-1

APX activity was inhibited by 46%, when compared to the control. On the other hand,

at 72, 120 and 168 h shoot APX activity was induced upon addition of both 100 and

200 mg Al l

-1

24 72 120 168

Ti m e (h )

0. 00

1. 60

3. 20

4. 80

6. 40

8. 00

A P X (µmol ascorbat e oxi ded mi n

- 1

prot ei n)

0 mg l

- 1

100 mg l

- 1

200 mg l

- 1

a b

24 72 120 168

Ti m e (h )

0. 00

0. 60

1. 20

1. 80

2. 40

3. 00

AP X (µmol ascorbat e oxi ded mi n

- 1

prot ei n)

0 mg l

- 1

100 mg l

- 1

200 mg l

- 1

24 72 120 168

Ti m e (h )

A P X (µmol ascorbat e oxi ded mi n

- 1

prot ei n)

0 mg l

- 1

100 mg l

- 1

200 mg l

- 1

24 72 120 168

Ti m e (h )

0. 00

0. 80

1. 60

2. 40

3. 20

4. 00

A P X (µmol ascorbate oxi ded mi n

- 1

prot ei n)

0 mg l

- 1

100 mg l

- 1

200 mg l

- 1

a b

Figure 4- Effect of Al

on ascorbate peroxidase (APX) activity over time in Macaca (Roots (A) and Shoot

(B)) and SMIC148-A (Roots (C) and Shoot (D)) potato clones. APX activity was expressed as µmol

oxidized ascorbate mim

-1

protein. The data are the mean ± S.D. of three different replicates.

3.3.2. Non-enzymatic antioxidants

Non-protein thiol group (NPSH) concentration was higher at 72, 120 and 168 h,

when compared to 24 h of Al exposure, in both roots and shoot. The content of NPSH

was significantly reduced in roots of the Al-sensitive clone at both 24 and 72 h after Al

treatment (Fig. 5A). At 120 h of Al-exposure, NPSH in roots of the Al-tolerant clone

(Fig. 5C) increased significantly with increasing Al concentration, whereas for the Al-

sensitive clone it was slightly, but not significantly, reduced by 200 mg Al l

-1

, when

compared to control.

(C)

(D)

(A)

(B)

24 72 120 168

Ti m e (h )

0. 00

0. 50

1. 00

1. 50

2. 00

2. 50

NPSH (µmol -SH g

- 1

FW)

0 mg l

- 1

100 mg l

- 1

200 mg l

- 1

a b

24 72 120 168

Ti m e (h)

0. 00

0. 50

1. 00

1. 50

2. 00

2. 50

NPSH (µmol -SH g

- 1

FW)

0 mg l

- 1

100 mg l

- 1

200 mg l

- 1

a b

24 72 120 168

Ti m e (h )

0. 00

0. 50

1. 00

1. 50

2. 00

2. 50

NPSH (µmol -SH g

- 1

FW)

0 mg l

- 1

100 mg l

- 1

200 mg l

- 1

24 72 120 168

Ti m e (h )

0. 00

0. 50

1. 00

1. 50

2. 00

2. 50

NPSH (µmol -SH g

- 1

FW)

0 mg l

- 1

100 mg l

- 1

200 mg l

- 1

Figure 5- Effect of Al on non-protein thiol group (NPSH) concentration over time in Macaca (Roots (A)

and Shoot (B)) and SMIC148-A (Roots (C) and Shoot (D)) potato clones. The data are the mean ± S.D.

of three different replicates.

For both potato clones, at 168 h after Al exposure, root NPSH concentration

decreased at 200 mg Al l

-1

when compared to control. Shoot NPSH concentration in

the Al-tolerant clone (Fig. 5D) was not affected by Al-treatment for up to 120 h,

whereas at 168 h it was significantly increased for all Al levels, when compared to

control. By contrast, shoot NPSH concentration in the Al-sensitive clone increased at

200 mg Al l

-1

at 24 h (65%), 120 h (18%) and 168 h (13%). At 72 h, while the Al-tolerant

clone did not demonstrate NPSH concentration alteration, in the Al-sensitive clone, it

was significantly decreased at all Al levels.

(C)

(D)

(A)

(B)

Regardless of the Al level, the shoot presented more ascorbic acid (AsA) than

roots (Fig. 6). The concentration of AsA was significantly reduced (about 20%) in roots

of the Al-sensitive clone at 24, 72 and 120 h at 200 mg Al l

-1

, when compared to control

(Fig. 6A). On the other hand, AsA concentration at 24 and 168 h at 100 mg Al l

-1

was

about 41% and 23% higher than that of the control, respectively. In roots of the Al-

tolerant clone (Fig. 6C), AsA concentration increased at 24 h upon addition of Al in the

substrate. However, at 72 and 168 h, a significant decrease in AsA concentration was

observed in the presence of Al. Shoot AsA concentration in the Al-sensitive clone (Fig.

6B) increased significantly at 24, 72 and 168 h upon addition of Al.

24 72 120 168

Ti m e (h )

120

150

As A (µg g

- 1

FW)

0 mg l

- 1

100 mg l

- 1

200 mg l

- 1

a b

24 72 120 168

Ti m e (h )

102

136

170

As A (µg g

- 1

FW)

0 mg l

- 1

100 mg l

- 1

200 mg l

- 1

24 72 120 168

Ti m e (h )

120

150

As A (µg g

- 1

FW)

0 mg l

- 1

100 mg l

- 1

200 mg l

- 1

a b

24 72 120 168

Ti m e (h )

102

136

170

As A (µg g

- 1

FW)

0 mg l

- 1

100 mg l

- 1

200 mg l

- 1

Figure 6- Effect of Al on Ascorbic acid concentration over time in Macaca (Roots (A) and Shoot (B)) and

SMIC148-A (Roots (C) and Shoot (D)) potato clones. The data are the mean ± S.D. of three different

replicates.

(C)

(D)

(A)

(B)

In contrast, shoot AsA concentration in the Al-tolerant clone (Fig. 6D) only

increased at 72 and 168 h at 200 mg Al l

-1

. Moreover, at 24 and 120 h, AsA

concentration decreased at 200 mg Al l

-1

DISCUSSION

Our study revealed the importance of the effects of oxidative stress and the

antioxidant defense mechanisms after Al exposure in two potato clones. It is clear that

Al toxicity depends not only on the concentration and exposure time but also on the

clone used.

Inhibition of root elongation is the primary Al toxicity symptom (Dipierro et al.,

2005) and has been used as a suitable parameter for assessing genotypic differences

in Al tolerance (Collet et al., 2002; Jemo et al., 2007). The presence of 200 mg l

-1

of Al

in solution inhibited significantly root growth only in the Macaca clone (Al-sensitive)

(Fig. 1), indicating a distinct genetic sensitivity to Al supply. This effect was observed at

the first point of time after Al exposure (24 h) and was observed throughout the

experiment. Thus, these two potato clones were classified as Al-sensitive (Macaca)

and Al-tolerant (SMIC148-A), confirming our previous results (Tabaldi et al., 2007).

Root growth reductions may, therefore, be the result of elevated Al concentrations in

the rooting medium, leading to disruption of cell functioning as proposed by Kochian

(1995). This is believed to be caused by a number of different mechanisms, including

apoplastic lesions, interactions within the cell wall, the plasma membrane, or the root

symplasm (for review, see Marschner, 1995), resulting in mineral nutrient and water

acquisition deficiency, and, consequently, leading to shoot nutrient deficiencies and

poor crop yields.

The imposition of several abiotic stresses, including Al presence, can give rise to

excess concentrations of reactive oxygen species (ROS) in plant cells (Noctor & Foyer,

1998; Lin & Kao, 2000), which are potentially harmful since they initiate the

peroxidation and destruction of lipids, nucleic acids and proteins (Yamaguchi-Shinozaki

& Shinozaki, 2006). The most prominent indicator of plasma membrane damage is Al-

stimulated lipid peroxidation (Silva et al., 2002). In the present study, data for root

growth (Fig. 1) and lipid peroxidation (Fig. 2) suggest that lipid peroxidation in the

Macaca clone (Al-sensitive) might be a direct effect of Al toxicity on root growth,

indicating Al induced oxidative stress and, as a result, irreversible damage to tissue

development and function. A higher increase in MDA content was seen in roots when

compared to the shoots at all times of exposure. In addition, in the shoot, Al provoked

lipid peroxidation only after prolonged exposure (120 and 168 h), hence these data

show that the active stress in roots was higher. Thus, similar to results obtained with

peas (Yamamoto et al., 2001), soybeans (Cakmak & Horst, 1991) and maize (Boscolo

et al., 2003), in the present study, lipid peroxidation in the Al-sensitive potato clone

seems to be an early symptom induced by Al toxicity, indicating that lipids are the

primary cellular target of oxidative stress. In the SMIC148-A clone, Al treatment did not

provoke lipid peroxidation in either the roots or shoot at any time of exposure (Fig. 2C

and 2D, respectively). This indicates that the antioxidant system in this clone might be

more efficient to protect membrane lipids of reactive oxygen species (ROS).

Plants respond to metal stress by physiological and biochemical strategies. Anti-

oxidation mechanisms of the cell include the enzymatic ROS-scavenging system and

non-enzymatic antioxidants (ascorbic acid (AsA) and non-protein thiol groups (NPSH)),

which function to interrupt the cascades of uncontrolled oxidation in each organelle.

Among these enzymatic systems, catalase (CAT) and ascorbate peroxidase (APX) can

transform peroxides into non-reactive species, but APX has a very high affinity for H

as compared with CAT (Graham & Patterson, 1982). Ascorbate is the primary

antioxidant reacting directly with ROS and also acts as secondary antioxidant by

reducing the oxidized form of α-tocopherol and preventing membrane damage

(Demirevska-Kepova et al., 2004). Glutathione is the predominant NPSH, redox-buffer,

phytochelatin precursor and substrate for keeping ascorbate in its reduced form in the

ascorbate-glutathione pathway (Noctor & Foyer, 1998).

At the first point of time after Al exposure (24 h), both APX activity and non-

enzymatic antioxidant levels (NPSH and AsA) were reduced in roots of the Al-sensitive

clone, except at 100 mg Al l

-1

, at which AsA concentration was increased. These

alterations occurred concomitantly with the inhibition of root growth and the increase of

lipid peroxidation (Fig. 1 and 2). This might indicate that, at that moment, the reductions

in concentration and activity of antioxidants were contributing to enhance damage

provoked by Al treatment. The inhibition in root APX activities observed almost

exclusively for the Al-sensitive potato clone at times ranging from 24 to 120 h can be

attributed to the blockage of essential functional groups, like -SH in the enzymes, or the

displacement of essential metal ions from the enzyme, as suggested by Schutzendubel

& Polle (2002). On the contrary, in the shoot, an increase in AsA concentration was

observed and NPSH concentration increased significantly at 200 mg Al l

-1

at 24, 72 and

168 h. These antioxidants are present in plant tissue in milimolar concentrations and in

stress conditions their levels increase (Noctor & Foyer, 1998) as an attempt to defend

the plant from this stress.

Plants can also tolerate Al toxicity by inducing antioxidant defense systems. In

roots of the Al-tolerant clone, Al presence for 24 h provoked an increase only in AsA

concentration. This increase might have contributed to free radicals and ROS

detoxification and suggests their active participation in Al detoxification, since root

growth inhibition and lipid peroxidation were not observed in this clone. As suggested

by Wu et al. (2004), AsA can reverse metal toxicity through two possible mechanisms:

1) AsA may bind metals, thereby affecting their movement across biological

membranes; or 2) AsA may act as a reducing agent, protecting the oxidation of the

mercapto (-SH) group by contributing electron or reducing power for photo-system II.

On the other hand, shoot APX activity and AsA concentration decreased at 24 h after

addition of Al. Therefore, other antioxidant systems might also be acting, as shoot

growth was not affected (data not shown).

At prolonged times of Al exposure (72 h and 120 h), the activity of both

enzymatic antioxidant and non-enzymatic antioxidant concentration in roots of the Al-

sensitive clone were reduced, except NPSH concentration at 120 h, which remained

unaltered. This decrease in CAT and APX activity could be due to the blocking of

essential functional groups like –SH in the enzymes or the displacement of essential

metal ions from enzymes, as suggested for other metals (Shah & Dubey, 1995;

Schutzendubel & Polle, 2002). In the shoot, the same behavior was observed for CAT

activity and NPSH concentration at 72 h. On the other hand, at 24 and 72h, AsA

concentration was increased. This increase could be explained by the induction of

protective mechanisms for detoxifying excess Al, which seems to be efficient in the

protection of membrane lipids. At 120 h, CAT and APX activity and NPSH

concentration increased. Even so, this activation of the antioxidant system was not

enough to avoid cellular damage, since membrane lipids were injured.

In roots of the Al-tolerant clone, the inadequate response of APX activity to Al

was compensated by the increased activity of CAT at 72 h. However, at 120 h, a

contrary effect was observed. Alscher et al. (2002) reported that lower expression of a

member of one gene family related to antioxidant defense leads to an increase in

expression of another member of the family. To that effect, the enzymatic and non-

enzymatic antioxidants seem to compensate for each other at 120 h. At 72 h, while the

NPSH level remained unaltered, AsA concentration decreased. On the other hand, at

120 h, while NPSH level increased, AsA concentration remained unaltered. Even with

these variations, the antioxidant system was efficient, protecting membrane lipids from

oxidation.

In the shoot of the Al-tolerant clone at 72 h, while CAT activity remained

unaltered, APX activity was activated by the presence of Al. At the same time, AsA

concentration increased. On the other hand, enzymatic antioxidants were active, while

AsA concentration decreased at 120 h after Al exposure. Therefore, in the Al-tolerant

clone, there was always at least one component of the antioxidant system protecting

the plant against Al stress. On the other hand, in roots of the Al-sensitive clone, the

presence of Al negatively affected the antioxidant system, diminishing activity and

concentration of these antioxidants. A decline in both CAT and APX activities and non-

enzymatic antioxidant concentrations suggests a possible delay in the removal of ROS,

hence an increase of lipid peroxidation and, consequently, root growth inhibition.

At the final point of time (168 h), the presence of antioxidants did not prevent

root growth inhibition and lipid peroxidation by Al in the Al-sensitive clone. In this clone,

enzymatic activity and the concentration of non-enzymatic antioxidants increased over

time, and were not enough to avoid both root and shoot damage. On the other hand, in

the Al-tolerant clone, only an increase in enzymatic antioxidants was sufficient to

protect the roots from Al stress. In the shoot, both enzymatic (APX) and non-enzymatic

(NPSH and AsA) antioxidants increased with Al exposure. Therefore, the Macaca and

SMIC148-A clones differed in the expression of the amount and type of antioxidants,

suggesting a varying capacity of these clones to deal with oxidative stress, which

resulted in varying sensitivity and tolerance to Al.

These results show that the cellular redox status of potato clones seems to be

affected by Al, and oxidative stress may be an important mechanism involved in Al

toxicity, mainly in the Al-sensitive Macaca clone. Biochemical (lipid peroxidation) and

morphological (root growth) alterations were observed in the first hours of Al exposure.

This could indicate that the root elongation impairment observed in the Al-sensitive

Macaca clone might be caused by oxidative stress. Unlike redox-active metals (Cu,

Fe), Al is not able to induce the production of ROS through a Fenton-like reaction. Al

causes oxidative stress probably through indirect mechanisms such as interaction with

the antioxidant defense, disruption of the electron transport chain and induction of lipid

peroxidation. Further research on the indirect mechanisms of Al-induced oxidative

stress is required to reveal the underlying molecular and biochemical events involved.

Acknowledgments

This work was supported by the Conselho Nacional de Desenvolvimento

Científico e Tecnológico (CNPq-Brazil) and Coordenação de Aperfeiçoamento de

Pessoal de Nível Superior (CAPES-Brazil).

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Capítulo II

Efeito do alumínio na atividade in vitro de fosfatases ácidas em

quatro clones de batata

Manuscrito II

In vitro activity of acid phosphatases of four potato clones cultivated

in three growth systems: aluminum effects

Aluminum on the in vitro activity of acid phosphatases of four potato clones cultivated in

three growth systems

Luciane Almeri Tabaldi

(1)

, Denise Cargnelutti

(2)

, Gabriel Y Castro

(3)

, Jamile Fabbrin

Gonçalves

(4)

, Renata Rauber

(5)

, Dilson Antônio Bisognin

(6)

Maria Rosa Chitolina Schetinger

(7)

Fernando Teixeira Nicoloso

(8*)

(1,3,4,6)

Programa de Pós-Graduação em Agronomia,

(2,7)

Programa de Pós-Graduação em

Bioquímica Toxicológica,

(5,8)

Departamento de Biologia. * Corresponding author:

[email protected]

Abstract - The aim of this study was to evaluate the effect of aluminum (Al) on the in vitro

activity of acid phosphatases (APases) of four potato clones cultivated in three growth systems.

Potato clones Macaca, SMIC148-A, Dakota Rose and Solanum microdontum were grown in

vitro, in hydroponics or in a greenhouse. The enzyme was assayed in the presence of 0, 50, 100,

150 and 200 mg Al L

-1

. In plantlets grown in vitro, root APases were inhibited by Al in all

clones, while shoot APases were inhibited by Al in S. microdontum and Dakota Rose and

increased in Macaca at all Al levels. In plantlets grown in hydroponics, root APases increased in

Macaca at 50 mg L

-1

, whereas decreased at all Al levels in S. microdontum. In greenhouse

plantlets, root APases were reduced at 200 mg L

-1

in S. microdontum and SMIC148-A, and at

100, 150 and 200 mg L

-1

in Dakota Rose. Shoot APases were reduced in Macaca and SMIC148-

A. Conversely, in Dakota Rose, APases increased at 50 and 100 mg L

-1

. These results show that

the effect of Al toxicity on in vitro APase activity depends not only on Al availability but also

on the clone and the growth system evaluated.

Index terms: acid phosphatases, aluminum, phosphorus, Solanum tuberosum, S. microdontum.

Atividade in vitro de fosfatases ácidas de quatro clones de batata cultivados em três

sistemas de cultivo: efeitos do alumínio

Resumo: O objetivo deste trabalho foi avaliar os efeitos do alumínio (Al) na atividade in vitro

de fosfatases ácidas (APases) de quatro clones de batata cultivados em três sistemas de cultivo.

Os clones Macaca, SMIC148-A, Dakota Rose e Solanum microdontum foram cultivados in

vitro, em sistema hidropônico e em vasos em casa de vegetação. A enzima foi analisada na

presença de 0, 50, 100, 150 e 200 mg Al L

-1

. Em todos os clones, as APases de raízes de plantas

derivadas do cultivo in vitro foram inibidas por Al. Na parte aérea, o Al inibiu a atividade de

APases somente nos clones S. microdontum e Dakota Rose, enquanto no clone Macaca, um

aumento foi observado em todos os níveis de Al. Em raízes de plantas cultivadas em sistema

hidropônico, a atividade de APases aumentou em 50 mg Al L

-1

no clone Macaca, enquanto em

S. microdontum a atividade foi reduzida em todos os níveis de Al. Em raízes de plantas

crescendo em casa de vegetação, a atividade de APases foi reduzida nos clones S. microdontum

e SMIC148-A em 200 mg Al L

-1

, enquanto no clone Dakota Rose, a atividade foi reduzida em

100, 150 e 200 mg L

-1

. A atividade de APases de parte aérea foi reduzida nos clones Macaca e

SMIC148-A, enquanto aumentou em 50 e 100 mg Al L

1 no clone Dakota Rose. Esses

resultados mostram que a toxicidade do Al na atividade in vitro de APases depende não somente

da concentração de Al, mas também do clone e do sistema de crescimento utilizado.

Termos para indexação: fosfatases ácidas, alumínio, fósforo, Solanum tuberosum, S.

microdontum.

Introduction

Phosphorus (P) plays important roles as a structural and regulatory element in plant

growth and development. P deficiency limits plant growth more frequently than any other

nutrient except nitrogen (Raghothama & Karthikeyan, 2005). In natural ecosystems, P

availability is seldom optimal for plant growth because of limited P content in minerals,

chemical and biological reactions. The inorganic phosphorus (Pi) level in soil solutions is

regulated mainly by its interaction with organic or inorganic surfaces in the soil. Aluminum and

iron ions in acid soils and calcium ions in alkaline soils interact strongly with Pi and render it

unavailable to plants (Sousa et al., 2007).

Organic phosphorus compounds in the soil, representing approximately 50% of total soil

phosphate, are mainly present as phosphoric esters such as inositol phosphates (Halstead &

McKercher, 1975), which must be hydrolyzed to Pi by hydrolases of phosphoric esters

(phosphatases, E.C.3.1.3) before P can be taken up by plants. The hydrolysis of phosphoric

organic esters is catalyzed by both acidic and alkaline phosphatases (Marzadori et al., 1998).

Synthesis of phosphatase enzymes is dependent on environmental conditions and the

physiological state of the plant, among other factors (Grierson & Comerford, 2000).

Acid phosphatases (orthophosphoric-monoester phosphohydrolases, E.C.3.1.3.2) are a

group of enzymes that catalyze the hydrolysis of a variety of phosphate esters releasing Pi from

phosphorylated substrates (Yoneyama et al., 2007) in acidic environments and are widely

distributed in plants. They appear to be important in the production, transport and recycling of

Pi (Tejera García et al., 2004). Phosphorus deficiency in higher plants has been shown to

increase the activity of acid phosphatases (Duff et al., 1994). Moreover, metals such as Hg and

Zn can affect acid phosphatase activity (Tabaldi et al., 2007a).

Toxic concentrations of aluminum (Al), generally found in acid soils (pH < 5.0), inhibit

root growth (Dong et al., 2002), restricting water and nutrient uptake and leading to poor growth

and yield. Aluminum can bind to the pectic residues or proteins in the cell wall, displacing other

ions from critical sites on the cell wall or membranes (Emmanuel & Peter, 1995). Aluminum

can also be transported across the root plasma membrane and interact with components in the

cell sap such as nuclear DNA, enzymes, calmodulin, tubulin and ATP (Emmanuel & Peter,

1995). Aluminum causes oxidative stress (Tabaldi et al., 2007b) and mineral deficiency in

higher plants (Guo et al., 2007), probably because it affects enzyme-mediated reactions,

especially those involving carbon, nitrogen and phosphorus metabolisms (Dong et al., 2002). In

addition, Al can interfere with PO

binding (Rai et al., 1998). Therefore, it is important to

study key enzymes involved in these processes, such as acid phosphatases (APases).

Plant protein and enzyme responses to a variety of environmental factors may be useful

in predicting the survival capacity of a plant species or variety to stress conditions. There are

many reports in the literature showing the enzymatic activity level of several processes

involving plant metabolism (Dong et al., 2002; Tejera Garcia et al., 2004). Hydrolysis of

phosphate esters is a crucial process in the energy metabolism and metabolic regulation of plant

cells and the expression of their respective enzymes (phosphatases). This process is regulated by

a variety of developmental and environmental factors (Murata & Los, 1997).

The aim of this work was to evaluate the effect of Al on the in vitro activity of acid

phosphatases of four potato clones cultivated in three growth systems.

Material and Methods

Three adapted (2n=4x=48) (Macaca, SMIC148-A and Dakota Rose) and one diploid

(2n=2x=24) clone (PI595511-5 of Solanum microdontum) potato clones were evaluated. The S.

microdontum clone was identified as highly resistant to Phytophora infestans (Bisognin et al.,

2005) and has been used in our breeding program. This clone will be referred to as S.

microdontum. These clones were obtained from the Potato Breeding and Genetics Program,

Federal University of Santa Maria, RS, Brazil. The effect of the aluminum on APases was

determined by assays in the presence of 0, 50, 100, 150 and 200 mg L

-1

of Al as AlCl

.6H

These concentrations were chosen after preliminary tests in our laboratory (data not shown).

To in vitro culture system, nodal segments (1.0 cm long) were micropropagated in MS

medium (Murashige & Skoog, 1962), supplemented with 30 g L

-1

of sucrose, 0.1 g L

-1

of myo-

inositol and 6 g L

-1

of agar. Plantlets were grown in a growth chamber at 25±2ºC on a 16/8-h

light/dark cycle with 35 µmol m

-2

-1

of irradiance during 30 days. At harvest, plantlets were

colleted, divided into roots and shoot and utilized for enzymatic assay.

To hydroponic system, twenty-day-old plantlets from in vitro culture were transferred

into plastic boxes (10 L) filled with aerated full nutrient solution of low ionic strength. The

nutrient solution had the following composition (in µM): 6090.5 of N; 974.3 of Mg; 4986.76 of

Cl; 2679.2 of K; 2436.2 of Ca; 359.9 of S; 243.592 of P; 0.47 of Cu; 2.00 of Mn; 1.99 of Zn;

0.17 of Ni; 24.97 of B; 0.52 of Mo; 47.99 of Fe (FeSO

/Na-EDTA). These ex vitro cultured

plantlets were grown in a growth chamber at 25±2ºC on a 16/8-h light/dark cycle with 35 µmol

-2

-1

of irradiance. After 10 days of culture in hydroponic system, plantlets were colleted,

divided into roots and shoot and utilized for enzymatic assay.

To greenhouse culture system, ten tubers of each clone, with approximately 1.0 cm

were sowed separately in plastic pots of 300 mL, employing sand as substrate. The irrigation

was made with the same nutritive solution utilized in the hydroponic culture. After 30 days of

cultivation, all plantlets were colleted and divided into shoot and roots for enzymatic analysis.

Roots were rinsed with aliquots of distilled water to remove the substrate.

Enzyme extraction and assay were carried out at 4ºC. For extraction, fresh potato

samples of roots and shoot were centrifuged at 43200 x g for 30 min and the resulting

supernatant was used for enzyme assay. Acid phosphatases activity was determined according

to Tabaldi et al. (2007a) in a reaction medium consisting of 3.5 mM sodium azide, 2.5 mM

calcium chloride, 100 mM citrate buffer, pH 5.5, and 20 µL of metals, except in controls, at a

final volume of 200 µL. A 20 µL aliquot of the enzyme preparation (10-20 µg protein) was

added to the reaction mixture, except in controls, and preincubated for 10 min at 35°C. The

reaction was started by the addition of substrate and stopped by the addition of 200 µL of 10%

trichloroacetic acid (TCA) to a final concentration of 5%. Inorganic phosphate (Pi) was

measured at 630 nm using malachite green as the colorimetric reagent and KH

as standard

for the calibration curve. Controls were carried out to correct for nonenzymatic hydrolysis by

adding enzyme preparation after TCA addition. Enzyme specific activities are reported as nmol

Pi released min

-1

protein. All assays were performed in triplicate using PPi as substrate at a

final concentration of 3.0 mM. In all the enzyme preparations, protein was determined by the

method of Bradford (1976) using bovine serum albumin.

Data were submitted to variance analyses and treatment means compared by Tukey’s

range test at 5% of error probability. Treatments were presented as mean ± S.D. of at least three

independent replicates.

Results

The growth systems were shown to have significant effects on acid phosphatase

(APases) activity. Under Al stress, APase activity of plantlets grown in vitro was affected in

both roots and shoot (Fig. 1). APase activity was higher in the shoot (Fig. 1B) than in roots of

all clones studied (Fig. 1A). Root APases were inhibited by Al in all clones, but the degree of

inhibition differed (Fig. 1A).

0 50 100 150 200

Al (mg L

-1

)

140

280

420

560

700

nmol Pi released min

-1

protein

Macaca S. microdontum SMIC148-A Dakota Rose

0 50 100 150 200

Al (mg L

-1

)

180

360

540

720

900

nmol Pi released min

-1

protein

Macaca S. microdontum SMIC148-A Dakota Rose

Figure 1. Effect of increasing Al concentration on the in vitro acid phosphatase activity of roots (A) and shoot (B)

of potato plantlets (Macaca, S. microdontum, SMIC148-A and Dakota Rose clones) grown in vitro. Data represent

the mean ± S.D. of three different replicates. *Different from control at p<0.05.

(B)

(A)

The Macaca clone was the most Al-sensitive, where APase activity was reduced at all Al

levels. At 200 mg Al L

-1

, there was a reduction of about 17%. In the S. microdontum clone, root

and shoot APase activity was only reduced (30%) at 200 mg Al L

-1

. In the SMIC148-A clone,

root APases were reduced by 16% at 150 and 200 mg Al L

-1

, and in the Dakota Rose clone there

was a reduction of 16% at 100, 150 and 200 mg Al L

-1

. In S. microdontum and Dakota Rose

clones, shoot APase activity was only reduced (7%) at the 200 mg Al L

-1

(Fig. 1B). This

inhibition was lower than that observed in roots. On the other hand, APase activity in the shoot

of Macaca increased (10%) at all Al levels. In the SMIC148-A clone, APase activity was not

altered with increasing Al levels.

APase activity of plantlets grown in hydroponics was higher in shoot than in roots (Fig.

2A and 2B). In Macaca, root APases increased by about 35% at 50 mg Al L

-1

(Fig. 2A), whereas

in S. microdontum, there was a reduction at all Al levels, and inhibition was about 43% at 200

mg Al L

-1

. APases of SMIC148-A and Dakota Rose were not affected by Al treatment. In the

shoot (Fig. 2B), Al did not affect the APase activity in any of the clones evaluated.

APase activity was higher in roots than in shoot of greenhouse grown plantlets (Fig. 3A

and 3B). In roots (Fig. 3A), Al reduced APase activity in S. microdontum (24%) and SMIC148-

A (13%) only at 200 mg Al L

-1

. In Dakota Rose, APase activity was reduced at 100, 150 and

200 mg Al L

-1

, and inhibition was of about 15% at 200 mg L

-1

. In roots of Macaca, Al did not

affect APase activity. On the other hand, shoot APase activity (Fig. 3B) was reduced in Macaca

(200 mg Al L

-1

) and SMIC148-A (50, 100 and 200 mg Al L

-1

). At 200 mg Al L

-1

, the inhibition

was about 22% and 11% for Macaca and SMIC148-A clones, respectively. Conversely, in

Dakota Rose, APase activity increased at 50 and 100 mg Al L

-1

, while in S. microdontum, no

alteration was observed.

0 50 100 150 200

Al (mg L

-1

)

100

150

200

250

nmol Pi released min

-1

protein

Macaca S. microdontum SMIC148-A Dakota Rose

0 50 100 150 200

Al (mg L

-1

)

100

200

300

400

500

nmol Pi released min

-1

protein

Macaca S. microdontum SMIC148-A Dakota Rose

Figure 2. Effect of increasing Al concentration on the in vitro acid phosphatase activity of roots (A) and shoot (B)

of potato plantlets (Macaca, S. microdontum, SMIC148-A and Dakota Rose clones) grown in hydroponics. Data

represent the mean ± S.D. of three different replicates. *Different from control at p<0.05.

Discussion

Phosphorus (P) is qualitatively and quantitatively one of the most important nutrients of

many organisms, making up about 0.2% of plant dry weight. It forms part of key biomolecules

and, in the form of Pi, PPi, ATP, ADP or AMP, plays a crucial role in energy transfer and

metabolic regulation (Raghothama & Karthikeyan, 2005). In this context, higher plants possess

(A)

(B)

the innate ability to mineralize organic P compounds through acid phosphatases, which

encompass a broad group of hydrolytic enzymes that catalyze the

0 50 100 150 200

Al (mg L

-1

)

180

360

540

720

900

nmol Pi released min

-1

protein

Macaca S. microdontum SMIC148-A Dakota Rose

0 50 100 150 200

Al (mg L

-1

)

100

200

300

400

500

nmol Pi released min

-1

protein

Macaca S. microdontum SMIC148-A Dakota Rose

Figure 3. Effect of increasing Al concentration on the in vitro acid phosphatase activity of roots (A) and shoot (B)

of potato plantlets (Macaca, S. microdontum, SMIC148-A and Dakota Rose clones) grown in greenhouse. Data

represent the mean ± S.D. of three different replicates. *Different from control to p<0.05.

breakdown of P-monoesters with acid pH optima (Duff et al., 1994). Therefore, phosphatase

activity plays a significant role in P bioavailability to plants from native organic P compounds.

(B)

(A)

Acid phosphatases (APases) are expressed under a variety of conditions and in response

to many stimuli (Zhang et al., 2001). It is believed that APases play an important role during

cell starvation for P scavenging and remobilization, and in some other conditions that promote

phosphate mobilization and/or oxidative stress (del Pozo et al., 1999).

The mode of action of metals varies with enzymes and little is known about the exact

mechanisms by which metals interact with the multitude of enzymes that exist. In this study, Al

stress provoked significant effects on APase activity in the four potato clones, but these effects

depended on the growth system utilized. In the Macaca clone, Al-mediated APase inhibition

occurred either in roots of in vitro grown plantlets or in the shoot of plantlets grown in the

greenhouse. On the other hand, in the S. microdontum clone, Al inhibited APase activity in roots

of plantlets grown in all three systems used, as well as in the shoot of in vitro grown plantlets. In

the SMIC148-A clone, APase activity was inhibited only in roots of in vitro grown plantlets and

in roots and shoot of plantlets grown in the greenhouse. In the Dakota Rose clone, Al inhibited

APase activity in roots and shoot of in vitro grown plantlets and in shoot of plantlets grown in

the greenhouse.

Enzyme reactions can be inhibited by metals, which can form a complex with the

substrate, combine with protein-active groups of the enzymes, or react with the enzyme-

substrate complex. Purified APase from leaves and nodules of common bean was strongly

inhibited by Al (Tejera Garcia et al., 2004). Phosphatases are generally metalloenzymes

dependent on Ca

or Mg

. One possible mechanism explaining Al-toxicity may be the

replacement of Mg

by Al in the active site of the enzyme. Another possibility, suggested by

Rai et al. (1998), is that Al may interfere with the PO

binding sites. Other metals, such as Hg

and Zn, also inhibited in vitro APase activity in cucumber seedlings, possibly replacing Mg

the active site of enzyme, or interfering with the PO

binding sites (Tabaldi et al., 2007a). In

addition, once within the cell, Al may affect a range of mechanisms, such as the complexation

of ligands required by Ca

-dependent enzymes (Rengel, 1992). Therefore, it is possible to

suggest that Al-mediated inhibition of APase activity in potato may impair phosphate

mobilization, since this enzyme is involved in the metabolism of P, an essential element for

plant growth and development (Duff et al., 1994).

In the Macaca clone, Al induced an increase in APase activity in shoot of in vitro

plantlets, as well as in roots of hydroponic plantlets. Conversely, in shoot of the Dakota Rose

clone, APase activity increased only in greenhouse plantlets. Intra- and/or extracellular APases

of plants are induced under various environmental and developmental conditions (Duff et al.,

1994), including exposure to cations, salt stress and in response to phosphate starvation

(Gabbrielli et al., 1989; Yoneyama et al., 2007). In the present study, it was shown that in vitro

APase activity was affected by Al supply, but it depended on other factors such as: the growth

system, genetic background, and plant organ analyzed. Therefore, all of these factors must be

considered in the development of protocols for the characterization of Al tolerant potato clones.

In Arabidopsis, it was suggested that purple acid phosphatase had a bifunctional role, acting in

phosphate mobilization and in the metabolism of reactive oxygen species (del Pozo et al., 1999).

SAP

and SAP

, two secreted purple acid phosphatase isozymes from Lycopersicum esculentum

may also be multifunctional proteins that operate as: (a) scavengers of Pi from extracellular

phosphate-esters during Pi deprivation, or (b) alkaline peroxidases that participate in the

production of extracellular reactive oxygen species during the oxidative burst associated with

the defense response of the plant to pathogen infection (Bozzo et al., 2002). Moreover, Dong et

al. (2002) reported an increase in APase activity in plants exposed to Al. These authors

suggested that the increased activity of acid phosphatase was an indication of membrane

damage, which leads to liberation of enzymes from lysosomes, mitochondria and other

phosphate-containing cell structures.

In all clones studied, the APase activity of in vitro and hydroponic plantlets was higher

in shoot than in roots. On the contrary, in greenhouse plants, APase activity was higher in roots

than in shoot. This result might be related to different physiological characteristics of plants

grown in these three environments. Zimmermann et al. (2004) reported a different expression of

three purple acid phosphatases (PAP) from potato plants grown aeroponically either with or

without Pi, where StPAP

was expressed more abundantly in root and stem than in young

leaves, stolons and flowers. This gene was not responsive to P deprivation. StPAP

, in contrast,

responded strongly but locally to P deficiency stress and also showed a higher expression in

roots. Similar to StPAP

, StPAP

was induced by P starvation, but showed a higher expression

in the stem than in roots and leaves.

Major differences exist between the environment of plants grown in tissue culture and

those grown in a greenhouse. These include differences in lighting, both quantity and quality;

relative humidity; nutrients and other growth promoters; the gaseous composition; and the

medium substrate (Hazarika, 2003). Differences between these two environments and their

effect on plants have been recognized in numerous studies (Pospísilová et al., 1999; Hazarika,

2003). In the present study, greenhouse plants, grown in uncontrolled temperatures, had a higher

transpiration rate than in vitro and hydroponic plants, where the temperature was maintained at

25ºC. During acclimatization to ex vitro conditions, the transpiration rate usually decreases

gradually because stomatal regulation of water loss becomes more effective and cuticle and

epicuticular waxes develop (Pospísilová et al., 1999). In addition, chlorophyll a and b contents

increase after transplantation (Pospísilová et al., 1998). The net photosynthetic rate in Solanum

tuberosum decreased in the first week after transplantation and increased thereafter (Baroja et

al., 1995). An increase in the transpiration rate may enhance the uptake and translocation of

mineral elements in the xylem (Marschner, 1995). As APases are enzymes involved in the

production, transport and recycling of Pi (Bozzo et al., 2002), it might be suggested that Pi taken

up by roots was not efficiently transported to the shoot in plants grown in vitro and in

hydroponics due to the lower transpiration rate. Therefore, it can be suggested that APase

activity in the shoot was higher than in the roots because Pi was more available in the latter. In

addition, these data also suggest that the pool of acid phosphatases in roots and shoot is either

different or that the mechanism of regulation of these enzymes is tissue specific. Navarro-De la

Sancha et al. (2007) found the presence of at least three Mg-dependent inorganic

pyrophosphatase activity groups in roots and leaves of 3-week-old Arabidopsis plants. These

authors also presented interesting data about the expression of the AtPPa genes

(https://www.genevestigator.ethz.ch/), as follow: AtPPa

showed a high expression throughout

plant development, with a slight reduction during senescence, whereas AtPPa

and AtPPa

presented a lower level of expression, though present at all ages, and their expression was lower

in young plants and reached a maximum during flowering.

Conclusions

1. Aluminum interferes with APase activity in potato clones.

2. Such effects are related to the three factors tested: the growth system utilized, the

genetic background, and the plant organ analyzed.

Acknowledgements

To Coordenação e Aperfeiçoamento de Pessoal de Nível Superior, Conselho Nacional de

Desenvolvimento Científico e Tecnológico and Fundação de Amparo à Pesquisa de Estado do

Rio Grande do Sul for the financial support.

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Capítulo III

Influência do estresse de alumínio no teor de micronutrientes em

plântulas de batata

Manuscrito III

Micronutrient concentration in potato clones with distinct

physiological sensitivity to Al stress

Micronutrient concentration in potato clones with distinct

physiological sensitivity to Al stress

Luciane Almeri Tabaldi

, Gabriel Y Castro

, Denise Cargnelutti

, Etiane Skrebski

, Jamile

Fabbrin Gonçalves

, Renata Rauber

, Liana Rossato

, Dílson Antônio Bisognin

III

, Maria Rosa

Chitolina Schetinger

, Fernando Teixeira Nicoloso

Departamento de Biologia, Universidade Federal de Santa Maria, Santa Maria, RS, Brasil.

E-mail: [email protected] *Corresponding author.

Departamento de Química, Universidade Federal de Santa Maria, Santa Maria, RS, Brasil.

III

Departamento de Fitotecnia, Universidade Federal de Santa Maria, Santa Maria, RS,

Brasil.

This study aimed to evaluate the effects of aluminum (Al) on zinc (Zn), manganese (Mn), iron

(Fe) and copper (Cu) concentrations in potato clones, Macaca, SMIC148-A, Dakota Rose and

Solanum microdontum, grown in a nutrient solution (pH 4.00) with 0, 50, 100, 150 and 200 mg

Al L

-1

. Root Zn and Fe concentrations decreased linearly with increasing Al levels in Macaca,

SMIC148-A and Dakota Rose and increased linearly in S. microdontum. Shoot Zn concentration

showed a quadratic relationship with Al in S. microdontum and SMIC148-A, but a curvilinear

response in Dakota Rose. Shoot Fe concentration showed a quadratic relationship with Al in S.

microdontum, SMIC148-A and Dakota Rose. Root Mn concentration decreased linearly in

Macaca and SMIC148-A, and increased linearly in S. microdontum. Mn concentration showed a

quadratic relationship with Al in roots of Dakota Rose and in shoot of SMIC148-A, and

increased curvilinearly with Al levels in shoot of Dakota Rose. In shoot, there was no alteration

in Zn, Fe and Mn in Macaca or in Mn concentration in S. microdontum. Root and shoot Cu

concentration increased linearly in Dakota Rose, and showed a quadratic relationship in

Macaca. Root Cu concentration showed a quadratic relationship with Al levels in S.

microdontum and SMIC148-A. Shoot Cu concentration increased linearly in S. microdontum

and decreased linearly in SMIC148-A. Therefore, Al accumulation in potato tissues affects the

rate of uptake and distribution of certain micronutrients in roots and shoot of potato clones,

which may affect the growth and plant yield.

Key words: Aluminum; copper; iron; manganese; potato; zinc.

Concentração de micronutrientes em clones de batata com distinta sensibilidade

fisiológica ao estresse de alumínio: O objetivo deste estudo foi caracterizar o efeito do

alumínio (Al) na concentração de zinco (Zn), manganês (Mn), ferro (Fe) e cobre (Cu) em quatro

clones de batata (Macaca, SMIC148-A, Dakota Rose e Solanum microdontum) crescendo em

solução nutritiva (pH 4,00) com 0, 50, 100, 150 e 200 mg Al L

-1

. A concentração de Zn e Fe em

raízes diminuiu linearmente com o aumento nos níveis de Al nos clones Macaca, SMIC148-A e

Dakota Rose e aumentou linearmente em S. microdontum. Na parte aérea, a concentração de Zn

mostrou resposta quadrática ao Al em S. microdontum e SMIC148-A, enquanto no clone Dakota

Rose, houve uma resposta cúbica. Nos clones S. microdontum, SMIC148-A e Dakota Rose, a

concentração de Fe mostrou resposta quadrática ao Al. A concentração de Mn em raízes

diminuiu linearmente em relação ao Al nos clones Macaca e SMIC148-A, e aumentou

linearmente em S. microdontum. Para Dakota Rose e SMIC148-A, a concentração de Mn

mostrou uma resposta quadrática em relação ao suprimento de Al em raízes e parte aérea. A

concentração de Mn na parte aérea aumentou de forma cúbica com os níveis de Al no clone

Dakota Rose. Na parte aérea, não houve alteração na concentração de Zn e Fe na Macaca e de

Mn nos clones Macaca e S. microdontum. Em raízes e parte aérea, a concentração de Cu

aumentou linearmente no clone Dakota Rose, e mostrou resposta quadrática no clone Macaca. A

concentração de Cu mostrou resposta quadrática com os níveis de Al em raízes dos clones S.

microdontum e SMIC148-A. Na parte aérea, a concentração de Cu aumentou linearmente no

clone S. microdontum e diminuiu linearmente no clone SMIC148-A com o aumento nos níveis

de Al. Portanto, o Al afeta a taxa de absorção e distribuição de alguns micronutrientes em raízes

e parte aérea de clones de batata, podendo consequentemente afetar o crescimento e a

produtividade das plantas.

Palavras-chave: Alumínio; batata; cobre; ferro; manganês; zinco.

INTRODUCTION

The mineral nutrient status of plants is directly related to their growth and productivity.

Micronutrients are basic requirements for plant growth and development and their status is

controlled by their genetically fixed nutrient uptake potential, availability of the nutrient in the

soil, and other environmental factors (Mengel and Kirkby, 2001). Environmental variation of

nutrient availability is expected to result in changes for plant physiology and morphology, and

consequently in changes in the yield.

On the other hand, availability of most micronutrients depends, among other factors, on the pH

of the soil solution, as well as the nature of binding sites on organic and inorganic particle

surfaces (Tuna et al., 2008). Trace elements are adsorbed by inorganic constituents such as iron

(Fe) and aluminum (Al) oxides and form complexes with organic matter (Omil et al., 2007).

Acid soils are found throughout the world. It is estimated that about 40% of the world’s

arable soils and 12% of the land in crop production have a pH below 5.5 (von Uexküll and

Mutert, 1995). Moreover, soil acidification is increasing globally. These soils are often

characterized by reduced availability of several nutrients (Kamprath, 1984; Foy, 1992). In fact,

soil acidification may bring about many other changes in the physical and chemical properties

of the soil, which in turn affect plant growth and development.

In acid soil, some nutrients, such as P, Ca, and Mg, may be deficient, whereas others,

such as Mn and B, could be toxic to plants. In addition, the continuing acidification of soils with

low buffering capacity leads to an increase of Al mobilization in the environment and may be

potentially hazardous to all terrestrial and aquatic systems (Matús et al., 2006). These effects are

further complicated by interactions of Al with other ions in different plant genotypes and under

stress conditions (Foy, 1992). Although Al is the most abundant metal in the Earth’s crust, it is

nonessential for plants. Although Al occurs in various chemical species, the Al cation Al

regarded as the most toxic soluble form of Al (Parker et al., 1988). Al

is toxic to many plants

at micromolar concentrations, primarily affecting the normal functioning of roots. The rapid

inhibition of Al-mediated root growth (Ryan et al., 1993) results in poor nutrient and water

acquisition and transport, consequently leading to nutrient deficiencies and decreasing crop

yields (Kochian, 1995). Aluminum toxicity may be manifested as a deficiency of essential

nutrients such as Ca, Mg, Fe, Zn or Mo; decreased availability of P or as toxicity of Mn and H

(Guo et al., 2007; Schöll et al., 2005). Aluminum at high levels competes with cationic (mono

or bivalents) ions for absorption sites in channels or transporters (Kochian, 1995).

Al’s interference in uptake, transport and utilization efficiency of most of the

macronutrients has been well documented (McColl et al., 1991; Guo et al., 2007). On the other

hand, there is a lack of studies connecting the effects of Al on the uptake and transport of

micronutrients in plants. Thus, the objective of this study was to analyze the influence of

exposure to Al in a nutrient solution on micronutrient concentrations in roots and shoot of four

potato clones.

MATERIAL AND METHODS

Plant materials and growth conditions: Three adapted (2n=4x=48) clones (Macaca, SMIC148-

A and Dakota Rose) and one wild species (2n=2x=24) clone (PI595511-5 of Solanum

microdontum) were evaluated. S. microdontum was identified as highly resistant to Phytophora

infestans (Bisognin et al., 2005) and has been used in our breeding program. This clone will be

referred to as S. microdontum. Tissue culture plantlets were obtained from the Potato Breeding

and Genetics Program, Federal University of Santa Maria, Brazil. Nodal segments (1.0 cm long)

were micropropagated in MS medium (Murashige and Skoog, 1962), supplemented with 30 g L

of sucrose, 0.1 g L

-1

of myo-inositol and 6 g L

-1

of agar.

Twenty-day-old plantlets from in vitro culture were transferred into plastic boxes (10 L)

filled with aerated full nutrient solution of low ionic strength. The nutrient solution had the

following composition (in µM): 6090.5 of N; 974.3 of Mg; 5229.5 of Cl; 2679.2 of K; 2436.2 of

Ca; 359.9 of S; 0.47 of Cu; 2.00 of Mn; 1.99 of Zn; 0.17 of Ni; 24.97 of B; 0.52 of Mo; 47.99 of

Fe (FeSO

/Na-EDTA). Treatments consisted of the addition of 0, 50, 100, 150 or 200 mg Al L

-1

as AlCl

.6H

O. The solution pH was adjusted daily to 4.0±0.1 by titration with HCl or NaOH

solutions of 0.1 M. Both in vitro and ex vitro cultured plants were grown in a growth chamber at

25±2ºC on a 16/8-h light/dark cycle with 35 µmol m

-2

-1

of irradiance. Aluminum-treated

plantlets remained in each treatment for 7 days. At harvest, the plantlets were divided into shoot

and roots. Roots were rinsed twice with distilled water. Subsequently, micronutrient

concentrations were determined, according to Tedesco et al., 1995.

Determination of micronutrient concentration: After Al treatment, samples (roots and shoot)

were dried at 60ºC until reaching a constant weight. The dried tissues were weighed and ground

into a fine powder before nitric-percloric digestion. Micronutrient concentrations were

determined by atomic absorption spectrometry.

Statistical analysis: All data were analyzed by ANOVA procedures. The effects of Al on

micronutrient concentrations in roots and shoot of potato plantlets were quantified using

regression analysis with the SOC statistic package (Software Científico: NTIA/EMBRAPA).

Coefficients were included in a regression equation when their values were significant (P <

0.05).

RESULTS AND DISCUSSION

In our previous study (Tabaldi et al., 2007), based on relative root growth, S.

microdontum and SMIC148-A were shown to be Al-tolerant clones, whereas Macaca and

Dakota Rose were shown to be Al-sensitive clones. In addition, regression analysis showed that

the concentration of Al in both roots and shoot of these clones increased linearly with increasing

Al levels, and the increase in tissue Al was much steeper for Macaca and SMIC148-A.

However, the maximum concentrations of Al, 49,300 mg kg

-1

in roots and 17,900 mg kg

-1

shoot, were found in Dakota Rose at 200 mg Al L

-1

Plants require an adequate supply of micronutrients for their normal physiological and

biochemical functions. Deficiencies of essential micronutrients induce abnormal pigmentation,

size, and shape of plant tissues, reduce leaf photosynthetic rates, and lead to various detrimental

conditions (Masoni et al., 1996). In the present study, the concentration of some micronutrients

in the tissue of roots and shoot of four potato clones was examined after 7 days of Al exposure.

A micronutrient- and organ-dependent response to Al toxicity was observed in all potato clones.

Micronutrient concentrations were higher in roots than in shoot of all potato clones tested,

suggesting that more micronutrients were retained in the roots and smaller amounts were

transported to the shoot.

Regression analysis showed that the concentration of zinc (Zn) decreased linearly with

increasing Al levels in roots of Macaca, SMIC148-A and Dakota Rose clones (Fig. 1A). This

result is similar to that reported by Kolawole et al. (2000) and Jemo et al. (2007), who observed

a reduction in nutrient acquisition in cowpea genotypes exposed to Al. High concentrations of

Al in the substrate decreased the uptake of Ca, K, P, Fe, and Zn in birch seedlings (Betula

pendula Roth.), limiting the growth of roots and shoot (Bojaczuk et al., 2002).

As was observed for Zn concentration, root iron (Fe) concentration decreased linearly

with increasing Al levels in Macaca, SMIC148-A and Dakota Rose clones (Fig. 1C). At 200 mg

Al L

-1

, root Fe concentration decreased by about 20%, 47% and 30%, in Macaca, SMIC148-A

and Dakota Rose clones, respectively, when compared to the control. Metal-metal interactions

may occur when cations compete for negatively charged binding sites at the cell surface

(Kinraide and Parker, 1987; Kinraide et al., 1992). Since the cell wall is the major site of metal

accumulation (Kochian, 1995; Taylor, 1988) and provides the bulk of charged surfaces in the

apoplasm, the metal-metal interactions should affect total metal accumulation. These data might

indicate a direct competition between Al and essential nutrients for the same uptake site. At high

levels, Al competes with cationic (mono or bivalent) ions for absorption sites in channels or

transporters (Kochian, 1995). This competition may reduce ion absorption and utilization. In

addition, Al ions may bind to the phospholipid heads of the plasma membrane, alter the lipid-

protein interaction, and modify transporter activity (Suhayda and Haug, 1986). Another

possibility is that Al binds directly to the transport proteins, thereby impairing their function

(Schroeder, 1988).

0 50 100 150 200

250

500

750

1000

1250

1500

1750

(Macaca) y=1222.2-3.573x (R

=0.81)

(S. microdontum) y=696.87+5.305x (R

=0.6)

(SMIC148-A) y=603.87-1.36x (R

=0.83)

(Dakota Rose) y=621.53-1.32x (R

=0.75)

Zn concentration (mg kg

-1

)

Al (mg L

-1

)

0 50 100 150 200

100

200

300

400

500

(Macaca) Mean=324.0 (n.s.)

(S. microdontum) y=340.99+2.80x-0.015x

=0.85)

(SMIC148-A) y=351.89+1.012x-0.0073x

=0.72)

(Dakota Rose) y=334.9-3.93x+0.056x

-0.00019x

=0.87)

Zn concentration (mg kg

-1

)

Al (mg L

-1

)

0 50 100 150 200

750

1500

2250

3000

(Macaca) y=2581.73-2.103x (R

=0.79)

(S. microdontum) y=1858.93+6.63x (R

=0.75)

(SMIC148-A) y=1448-2.95x (R

=0.78)

(Dakota Rose) y=1176.6-1.51x (R

=0.79)

Fe concentration (mg kg

-1

)

Al (mg L

-1

)

0 50 100 150 200

300

600

900

1200

1500

1800

(Macaca) Mean=480.8 (n.s.)

(S . microdontum) y=596.06+2.54x-0.013x

=0.72)

(SMIC148-A) y=552.99-2.94x+0.013x

=0.87)

(Dakota Rose) y=608.8-412x+0.028x

=0.99)

Fe concentration (mg kg

-1

)

Al (mg L

-1

)

Figure 1- Effect of increasing Al concentration on the root (A) and shoot (B) zinc (Zn) concentration and root (C) and

shoot (D) iron (Fe) concentration of potato clones, Macaca, S. microdontum, SMIC148-A and Dakota Rose, submitted

to increasing Al concentrations for 7 days. n.s.: not significant.

Root Zn and Fe concentrations increased linearly with increasing Al levels in S.

microdontum (Fig. 1A and Fig. 1C, respectively). In this study, S. microdontum presented

greater concentrations of most micronutrients analyzed. Therefore, as this potato clone is Al-

(A)

(C)

(B)

(D)

tolerant (Tabaldi et al., 2007), it seems that the Al levels tested were not high enough to cause

severe alteration in the metabolism. Thus, higher levels of mineral nutrients may be connected

with Al tolerance, as suggested by Giannakoula et al. (2008). Shoot Zn concentration showed a

quadratic relationship in the S. microdontum and SMIC148-A clones, increasing at intermediary

Al levels (Fig. 1B). In Dakota Rose, shoot Zn concentration showed a curvilinear response,

decreasing at approximately 50 mg Al L

-1

and increasing at approximately 150 mg Al L

-1

(Fig.

1B).

In SMIC148-A clone, shoot Fe concentration decreased between Al levels of

approximately 50 and 150 mg Al L

-1

and showed a quadratic increase in Dakota Rose. On the

other hand, shoot Fe concentration slightly increased at Al levels between approximately 50 and

100 mg Al L

-1

in S. microdontum.

Root manganese (Mn) concentration decreased linearly in both Macaca and SMIC148-A

clones, and increased linearly with increasing Al levels in S. microdontum (Fig. 2A). However,

in Dakota Rose, Mn concentration showed a quadratic relationship to Al supply, decreasing at

levels between approximately 50 and 100 mg Al L

-1

and increasing at 200 mg Al L

-1

. Shoot Mn

concentration showed a quadratic relationship with Al levels in SMIC148-A (Fig. 2B),

decreasing at levels between approximately 50 and 100 mg Al L

-1

and increasing at 200 mg Al

-1

. For Dakota Rose, shoot Mn concentration showed a curvilinear response to Al supply,

increasing at levels between approximately 100 and 150 mg Al L

-1

, while in S. microdontum

there was no alteration in Mn concentration.

In our previous study (Tabaldi et al., 2007), shoot growth in Macaca decreased linearly

with increasing Al levels. However, there was no alteration in shoot Zn (Fig. 1B), Fe (Fig. 1D)

or Mn (Fig. 2B) concentrations in this clone. Therefore, the interference of Al in root growth

and absorption and transport of water and other nutrients may have brought about lower shoot

growth.

0 50 100 150 200

150

300

450

600

750

(Macaca) y=645.67-2.078x (R

=0,85)

(S. microdontum) y=437.0+1.804x (R

=0.89)

(SMIC148-A) y=414.13-0.871x (R

=0.89)

(Dakota Rose) y=412.45-5.12x+0.0299x

=0.95)

Mn concentration (mg kg

-1

)

Al (mg L

-1

)

0 50 100 150 200

100

200

300

400

500

(Macaca) Mean=331.7 (n.s.)

(S. microdontum) Mean=334.2 (n.s.)

(SMIC148-A) y=300.09-1.67x+0.011x

=0.78)

(Dakota Rose) y=219.33-2.53x+0.056x

-0.00022x

=0.98)

Mn concentration (mg kg

-1

)

Al (mg L

-1

)

0 50 100 150 200

(Macaca) y=45.93+0,403x-0,002x

=0.98)

(S. microdontum) y=43.81-0.211x+0.0013x

=0.73)

(SMIC148-A) y=25-0.15x+0.00067x

=0.91)

(Dakota Rose) y=17.8+0.253x (R

=0.83)

Cu concentration (mg kg

-1

)

Al (mg L

-1

)

0 50 100 150 200

(Macaca) y=20.18+0,06x-0,0004x

=0.81)

(S. microdontum) y=16.8+0.012x (R

=0.78)

(SMIC148-A) y=12.87-0.017x (R

=0.85)

(Dakota Rose) y=11+0.135x (R

=0.71)

Cu concentration (mg kg

-1

)

Al (mg L

-1

)

Figure 2- Effect of increasing Al concentration on the root (A) and shoot (B) manganese (Mn) concentration and root

Rose, submitted to increasing Al concentrations for 7 days. n.s.: not significant.

Copper (Cu) is an essential plant micronutrient that plays an important role in both

photosynthetic and respiratory electron transport, being a cofactor for many enzymes (Owen,

(B) (A)

1982). However, when present at elevated concentrations it affects different parameters of plant

metabolism, such as dry mass accumulation (Ali et al., 2002; Zheng et al., 2004), chlorophyll

(Lou et al., 2004), and water content (Burzynski and Klobus, 2004) and the balance of macro-

and micronutrient levels (Ali et al., 2002; Bernal et al., 2007). Being a redox active metal, Cu

generates reactive oxygen species (ROS) by Fenton reaction, which may result in oxidative

stress leading to peroxidation of membrane lipids (Stohs and Bagchi, 1995). The response of

root Cu concentration to Al in Dakota Rose was linear and positive (Fig. 2C), whereas in

Macaca it increased only at levels between approximately 50 and 150 mg Al L

-1

. By contrast, in

both Al-tolerant clones, root Cu concentration generally showed an inverse relationship with

increasing Al levels, with exception of S. microdontum, which showed an increased Cu

concentration at 200 mg Al L

-1

when compared to the control (Fig. 2C). Shoot Cu concentration

in Dakota Rose increased linearly with increasing Al levels (Fig. 2D), while in Macaca it

slightly increased at Al levels between approximately 50 and 100 mg Al L

-1

. The response of

shoot Cu concentration to Al in S. microdontum was linear and positive (Fig. 2D), while in

SMIC148-A it was linear and negative (Fig. 2D). However, shoot Cu concentration was less

altered in both of these Al-tolerant clones than was in the Al-sensitive clones. Therefore, the

increase in tissue Cu concentration observed in the potato clones exposed to Al might have

caused a disturbance in the metabolism, and hence in plant growth and development, mainly in

the Al-sensitive clones.

Plants vary in their sensitivity to toxic compounds in the soil. Many studies have shown

that sensitivity depends on many factors, such as physicochemical properties of the soil,

concentration of organic matter and nutrients, but primarily on soil pH (Göransson and

Eldhuset, 2001; Rengel, 1996). Aluminum is widespread in the Earth’s crust and its availability

to plants increases with decreasing pH of the soil (Boudot et al., 1994). The toxic Al ions

present in the substrate can damage root cells, which become inefficient in absorption and

translocation of both nutrients and water (Mossor-Pietraszewska et al., 1997), blocking their

participation in important metabolic processes, such as photosynthesis and respiration (Rengel,

1996).

Therefore, in the present work, the excessive Al accumulation observed could have

affected the rate of uptake and distribution of certain micronutrients in roots and shoot of potato

clones, and consequently would be responsible for mineral deficiencies/imbalance and

depression of the plant growth. Selection of plants tolerant to toxic ions contained in the soil

may enable more effective management of degraded habitats.

Acknowledgements: The authors thank the Coordenação e Aperfeiçoamento de Pessoal de

Nível Superior, Conselho Nacional de Desenvolvimento Científico e Tecnológico, and

Fundação de Amparo à Pesquisa de Estado do Rio Grande do Sul for the research fellowships.

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Capítulo IV

Respostas localizadas e sistêmicas de estresse oxidativo

induzidas por alumínio em clones de batata (Solanum

tuberosum L.) cultivados em sistema de raízes divididas

Manuscrito IV

Local and systemic oxidative stress responses induced by aluminum

in two potato clones (Solanum tuberosum L.) that differ in Al-

avoidance

Local and systemic oxidative stress responses induced by aluminum in two

potato clones (Solanum tuberosum L.) that differ in Al-avoidance

Luciane Almeri Tabaldi

1,4

, Denise Cargnelutti

2,5

, Jamile Fabbrin Gonçalves

1,4

, Luciane

Belmonte Pereira

2,5

, Gabriel Y Castro

, Joseila Maldaner

1,4

, Renata Rauber

, Liana

Verônica Rossato

, Dilson Antônio Bisognin

3,4

, Maria Rosa Chitolina Schetinger

2,5

Fernando Teixeira Nicoloso*

1,4

Departamento de Biologia

, Química

e Fitotecnia

, Programa de Pós-Graduação em

Agronomia

e Bioquímica Toxicológica

, Centro de Ciências Naturais e Exatas,

Universidade Federal de Santa Maria, Santa Maria, RS, Brasil. *Corresponding author:

[email protected]

Abstract

The objective of this study was to check whether Al oxidative stress differs in the

potato clones Macaca (Al-sensitive) and SMIC148-A (Al-tolerant), which present

distinct degrees of Al-avoidance. Plants were cultivated in a split-root system for 10

days with five treatments of varying concentrations and locations of Al (in mg L

-1

): T1 -

pot 1: 0.0, pot 2: 0.0; T2- pot 1: 50, pot 2: 50; T3- pot 1: 0.0, pot 2: 100; T4- pot 1: 100,

pot 2: 100; T5- pot 1: 0.0, pot 2: 200. At 200 mg Al L

-1

, a significant decrease in

chlorophyll concentration and increase in protein oxidation was observed only for

Macaca. At 200 mg L

-1

supplied to half of the root system, shoot H

concentration

was lower than that with both root halves treated by 100 mg L

-1

for both clones, but this

effect was much less pronounced in Macaca. Shoot lipid peroxidation in Macaca

increased with increasing Al supply. In SMIC148-A, plants treated with 100 and 200 mg

Al L

-1

in only one root half showed lower shoot lipid peroxidation. The 200 side of 0/200

plants demonstrated significantly greater lipid peroxidation than that untreated with Al,

mainly in Macaca. The increase observed in the concentration of NPSH in shoot of

SMIC148-A seemed to present a higher correlation with lipid peroxidation than that for

Macaca. At 100 mg Al L

-1

at both root halves, Macaca showed an inefficient tolerance

response in terms of CAT activity, protein oxidation, lipid peroxidation, H

concentration and APase activity. These results show that SMIC148-A, despite lower

Al-avoidance when compared to Macaca, presented a stronger local and systemic

antioxidant response to Al supply.

Keywords: aluminum; oxidative stress; Solanum tuberosum; split-root.

Introduction

Potatoes (Solanum tuberosum L.) rate fourth in world production among various

agricultural products, following wheat, rice and corn (FAO, 2004), with an overall

annual production of nearly 327 million tons and about 19 million ha planted. The most

widely cultivated species of potato are very sensitive to abiotic stress, whereas several

wild or primitive cultivated species from different ploidy levels adapt well to grow under

unfavorable conditions (Li and Fennel, 1985). Potato is the main horticultural crop in

Brazil in terms of area and food preference, with about 98% of the producers located in

the southern states of Minas Gerais, São Paulo, Paraná and Rio Grande do Sul. Potato

crops tolerate moderate acidity in the soil, growing well at a pH of 5.0 to 6.5. On the

other hand, in very acid soils (pH below 5.0) a decrease in yield occurs (Castro, 1983).

Acid soils, which comprise 30–40% of the world’s arable lands (Vitorello et al.,

2005) are a limiting factor to crop growth and are usually associated to low inherent

levels of plant-available phosphorus (P) (Jemo et al., 2007) and high levels of

aluminum (Al) (von Uexküll and Mutert, 1995), which is solubilized in acidic pH into the

toxic cation Al

. Aluminum is known to inhibit plant growth (Ciamporova, 2002), mainly

that of the root (Balestrasse et al., 2006; Tabaldi et al., 2007b). Symptoms of Al toxicity

are also manifested in the shoot and are regarded as a consequence of injuries to the

root system (Vitorello et al., 2005). In addition, Al also alters water relations (Barceló

and Poschenrieder, 2002), reduces stomatal opening, decreases photosynthetic

activity and causes chlorosis and necrosis of leaves, decreasing carbon sequestration

and biomass formation (Vitorello et al., 2005). Potential alternatives to the direct

amelioration of subsoil acidity include the use of Al-tolerant germplasm (Foy, 1988).

Although the physiological mechanism of Al toxicity is still unclear, several

reports suggest a role of Al in the induction of oxidative stress (Yamamoto et al., 2002;

Tabaldi et al., 2007b) and, consequently, formation of reactive oxygen species (ROS)

in plants, including superoxide radical (O

•−

), hydroxyl radical (

•

OH) and hydroxygen

peroxide (H

). These ROS can cause oxidative damage to the biomolecules such as

lipids, proteins (Tabaldi et al., 2007b), photosynthetic pigments and nucleic acids,

which leads to cell membrane peroxidation, loss of ions, protein hydrolysis, and even

DNA strand breakage (Guo et al., 2007). To mitigate the oxidative damage initiated by

ROS, plants have developed a complex defense antioxidant system, including low-

molecular mass antioxidants as well as antioxidant enzymes. Many plant species vary

in their ability to withstand Al toxicity. The antioxidant defense system might possibly

protect cells from Al toxicity.

Plant roots are characterized by very high adaptability. Their growth and

development involve complex interactions with both the soil environment and the shoot

(Marschner, 1996). Under natural soil conditions, roots are able to respond to the

heterogeneous soil environment by improving root growth in more favorable pockets

(Kerley et al., 2000), which is described as a plastic response of the root system

(Feldman, 1984). Hairiah et al. (1993) showed that velvet bean (Mucuna pruriens) was

Al-resistant when the whole root system was exposed to homogeneous Al supply.

However, when Al was supplied to only one part of the root system, roots avoided Al by

preferential development of roots not in contact with Al, accompanied by marked

inhibition of roots exposed to Al. This relative Al avoidance, rather than absolute Al

tolerance or toxicity, explains root response to acid subsoil conditions in the field. Al-

avoidance reactions in this sense may help to explain why selection of Al-tolerant

genotypes based on experiments with homogeneous media may fail to be successful

for field trials.

In our previous study (Tabaldi et al., 2007b), utilizing a homogeneous supply of

Al to the roots of potato clones grown in a hydroponic growth system, it was

demonstrated that the SMIC148-A clone was Al-tolerant, whereas the Macaca clone

was Al-sensitive. Moreover, it was observed that Al supply induced oxidative stress,

mainly in the Al-sensitive clone. Therefore, we formulated the hypothesis that potato

clones with distinct physiological sensitivity to Al stress and growing in a

heterogeneous root environment (split-root experiment) would show contrasting Al-

avoidance responses. A consequence of this hypothesis is that both Al avoidance and

Al oxidative stress should be less pronounced for the Al-tolerant clone, since the

response to local supply of Al is reduced under this condition. The aim of the present

paper is to test this hypothesis.

Material and Methods

Plant materials and growth conditions: Microtubers of potato clones (Solanum

tuberosum L.) Macaca (Al-sensitive) and SMIC148-A (Al-tolerant) were obtained from

the Potato Breeding and Genetics Program, Federal University of Santa Maria, Santa

Maria, RS, and were sowed in plastic pots of 300 mL, employing sand as substrate.

The plants were irrigated with a complete nutrient solution. The nutrient solution had

the following composition (in µM): 6090.5 of N; 974.3 of Mg; 4986.76 of Cl; 2679.2 of K;

2436.2 of Ca; 359.9 of S; 243.592 of P; 0.47 of Cu; 2.00 of Mn; 1.99 of Zn; 0.17 of Ni;

24.97 of B; 0.52 of Mo; 47.99 of Fe (FeSO

/Na-EDTA). After about 3 weeks, uniform

plants were chosen and transferred to a split-root system, in which the two halves of

the root system, each in a pot of 1 L, were exposed to an aerated complete nutrient

solution for 1 week. After that acclimatization period, these plants with split-roots were

cultivated for 10 days in a new nutrient solution (without P and pH 4.0±0.1) with five

treatments (six replicates for each treatment) of varying concentrations and locations of

Al, as follows: Treatment 1 (control) - pot 1: 0.0 mg Al L

-1

, pot 2: 0.0 mg Al L

-1

;

Treatment 2- pot 1: 50 mg Al L

-1

, pot 2: 50 mg Al L

-1

; Treatment 3- pot 1: 0.0 mg Al L

-1

pot 2: 100 mg Al L

-1

; Treatment 4- pot 1: 100 mg Al L

-1

, pot 2: 100 mg Al L

-1

; Treatment

5- pot 1: 0.0 mg Al L

-1

, pot 2: 200 mg Al L

-1

. With exception of Al, the concentrations of

the other mineral elements in the nutrient solution were the same for all treatments.

Nutrient solutions were replaced every 48 hours and pH was evaluated daily.

Aluminum-treated plantlets remained in each treatment for 10 d. At harvest, the plants

of both clones were divided into shoot, left root and right root to evaluate biochemical

parameters.

Chlorophyll and carotenoid determination: Chlorophyll (a+b) and carotenoids were

extracted following the method of Hiscox and Israeslstam (1979) and estimated with

the help of Lichtenthaler’s formulae (Lichtenthaler, 1987). Fresh leaves (0.1 g) were

incubated at 65ºC in dimethylsulfoxide (DMSO) until tissues were completely bleached.

Absorbance of the solution was then measured at 663 and 645 nm for chlorophyll and

470 nm for carotenoids on a spectrophotometer (Celm E-205D). Chlorophyll and

carotenoid concentrations were expressed as mg g

-1

fresh weight.

Catalase assay: Catalase (CAT) activity was assayed following the modified Aebi

(1984) method. Fresh roots and shoot samples (1 g) were homogenized in 5 mL of 50

mM KH

/ K

HPO

(pH 7.0), 10 g L

-1

of polyvinylpyrrolidone (PVP), 0.2 mM EDTA

and 10 mL L

-1

Triton X-100. The homogenate was centrifuged at 12000 x g for 20 min

at 4ºC and the supernatant was used for enzyme assay. Activity of CAT was

determined by monitoring the disappearance of H

by measuring the decrease in

absorbance at 240 nm of a reaction mixture with a final volume of 2 mL containing 15

mM H

in KPO

buffer (pH 7.0) and 30 µL extract. Activity was expressed as ∆E min

-1

protein.

Protein oxidation: Samples of roots and shoot (1 g) were homogenized with 25 mM

HPO

(pH 7.0) containing 10 mL L

-1

Triton X-100, at a proportion of 1:2 (w/v) (Levine

et al., 1990). After the homogenate was centrifuged at 15000 x g for 10 min at 4ºC, the

supernatant was used for immediate determination of protein oxidation, which was

expressed as nmol carbonyl mg

-1

protein.

Determination of hydrogen peroxide: The H

concentration was determined

according to Loreto and Velikova (2001). Approximately 0.1 g of both roots and shoot

was homogenized at 4ºC in 2 mL of 0.1% trichloroaceti acid (TCA) (w/v). The

homogenate was centrifuged at 12000 x g for 15 min at 4ºC. Then, 0.5 mL of the

supernatant was added to 0.5 mL of 10 mM potassium phosphate buffer (pH 7.0) and 1

mL of 1M KI. The H

concentration of the supernatant was evaluated by comparing

its absorbance at 390 nm with a standard calibration curve. Hydrogen peroxide

concentration was expressed as µmol g

-1

fresh weight.

Estimation of lipid peroxides: The degree of lipid peroxidation was estimated

following the method El-Moshaty et al. (1993). Fresh roots and shoot samples of 0.1 g

were homogenized in 20 mL of 0.2 M citrate-phosphate buffer (pH 6.5) containing 0.5%

Triton X-100, using mortar and pestle. The homogenate was filtered with two paper

layers and centrifuged for 15 min at 20000 x g. One milliliter of the supernatant fraction

was added to an equal volume of 20% (w/v) TCA containing 0.5% (w/v) of thiobarbituric

acid (TBA). The mixture was heated at 95ºC for 40 min and then quickly cooled in an

ice bath for 15 min, and centrifuged at 10000 x g for 15 min. The absorbance of the

supernatant at 532 nm was read and corrected for unspecific turbidity by subtracting

the value of the absorbance at 600 nm. The lipid peroxides were expressed as nmol

MDA mg

-1

protein, by using an extinction coefficient of 155 L mmol

-1

Non-protein thiol groups (NPSH) contents: Roots and shoot of potato plants were

homogenized in a solution containing 50 mmol L

-1

Tris-HCl and 10 mL L

-1

Triton X-100

(pH 7.5), centrifuged at 6800 x g for 10 min. To the resulting supernatant 10% TCA

was added at proportion 1:1 (v/v) followed by centrifugation (6800 x g for 10 min) to

remove protein. Non-protein thiols content was measured spectrophotometrically with

Ellman’s reagent (Ellman, 1959). An aliquot of the sample (400 µL) was added in a

medium containing 550 µL 1 mol L

-1

Tris-HCl (pH 7.4). The developed color was read

at 412 nm after the addition of 10 mmol/L 5-5-dithio-bis 2-nitrobenzoic acid (DTNB)

(0.05 mL). A standard curve using cysteine was used to calculate the content of thiol

groups in samples.

Acid phosphatases assay: Fresh root and shoot samples were centrifuged at 43200

x g for 30 min at 4ºC and the supernatant was used for enzyme assay. Acid

phosphatases activity was determined according to Tabaldi et al. (2007a) in a reaction

medium consisting of 3.5 mM sodium azide, 2.5 mM calcium chloride, 100 mM citrate

buffer, pH 5.5, at a final volume of 200 µL. A 20 µL aliquot of the enzyme preparation

(10-20 µg protein) was added to the reaction mixture, and preincubated for 10 min at

35°C. The reaction was started by the addition of PPi as substrate and stopped by the

addition of 200 µL of 10% trichloroacetic acid (TCA) to a final concentration of 5%.

Inorganic phosphate (Pi) was measured at 630 nm using malachite green as the

colorimetric reagent and KH

as standard for the calibration curve. Controls were

carried out to correct for nonenzymatic hydrolysis by adding enzyme preparation after

TCA addition. Enzyme specific activities were reported as nmol Pi released min

-1

of protein. All assays were performed in triplicate using PPi as substrate at a final

concentration of 3.0 mM.

Protein determination: In all the enzyme preparations, protein was determined

following the method of Bradford (1976) using bovine serum albumin.

Statistical analysis: Data were submitted to variance analyses and treatment means

compared by Tukey’s range test at 5% of error probability. Treatments were presented

as mean ± S.D. of three replicates.

Results

Chlorophyll and carotenoid concentrations: After 10 d in a split-root system, a

significant decrease in the chlorophyll concentration was observed in the Al-sensitive

clone (Macaca) both when plants were treated at 100 mg Al L

-1

in both halves of the

root system (100/100; decrease of 40%) and with Al supplied to only half of the root

system at 200 mg L

-1

(0/200; decrease of 34%) (Fig. 1A). On the other hand, no

significant difference was observed in chlorophyll concentration in the Al-tolerant clone

(SMIC148-A) exposed to varying Al concentrations (Fig. 1B).

In the Al-sensitive clone, there was no alteration in carotenoid concentration at

any Al concentration (Fig. 1C). In the Al-tolerant clone, it increased significantly, by

about 60%, both when only one root half of the plant was supplied with Al at 100 mg L

-1

(0/100) and when both sides of the root system were supplied (100/100 mg Al L

-1

) (Fig.

1D). Moreover, plants in which only half of the root system was treated with 200 mg Al

-1

(0/200) showed a slight, but not significant, increase in carotenoid concentration.

0/0 50/50 0/100 100/100 0/200

Al (mg L

-1

)

0.00

0.36

0.72

1.08

1.44

1.80

Chlorophyll (mg g

-1

FW)

0/0 50/50 0/100 100/100 0/200

Al (mg L

-1

)

0.00

0.36

0.72

1.08

1.44

1.80

Chlorophyll (mg g

-1

FW)

0/0 50/50 0/100 100/100 0/200

Al (mg L

-1

)

0.00

0.10

0.20

0.30

0.40

0.50

Carotenoids (mg g

-1

FW)

0/0 50/50 0/100 100/100 0/200

Al (mg L

-1

)

0.00

0.10

0.20

0.30

0.40

0.50

Carotenoids (mg g

-1

FW)

Figure 1. Effect of varying Al concentrations on chlorophyll concentration of the (A) Al-sensitive

(Macaca) and (B) Al-tolerant (SMIC148-A) clones, and carotenoid concentration of the (C) Al-sensitive

(Macaca) and (D) Al-tolerant (SMIC148-A) clones in a split-root system. Data are means ± SD at p<0.05,

Tukey test.

Catalase activity and protein oxidation: In general, shoot catalase (CAT) activity

was greater for the Al-sensitive clone than for the Al-tolerant clone. In the Al-sensitive

clone, shoot CAT activity was significantly decreased in the treatments with both sides

of the root system exposed to Al (50/50 and 100/100 mg L

-1

) (Fig. 2A), whereas for

plants where Al was supplied to only half of the root system (0/100 and 0/200 mg Al L

), it was slightly, but not significantly, reduced when compared to the control plants

(C)

(D)

(A)

(B)

(0/0 mg Al L

-1

). In the Al-tolerant clone, CAT activity decreased when plants were

supplied either with 100 mg Al L

-1

or 200 mg Al L

-1

to only half of the root system (0/100

and 0/200), as well as when both halves of the root system were treated with 100 mg

Al L

-1

(100/100) (Fig. 2B). In addition, plants in which both halves of the root system

were treated with 50 mg Al L

-1

(50/50) showed a slight, but not significant, reduction in

CAT activity when compared to the control plants.

A significant increase in shoot carbonyl concentration (i.e. protein oxidation) was

only observed in the Al-sensitive clone at all Al concentrations (Fig. 2C). The highest

increase in protein oxidation (about 114%) was seen in the treatments with both halves

of the root system exposed to 100 mg Al L

-1

(100/100) and when only half of the root

system (0/200) was supplied with 200 mg Al L

-1

. In the Al-tolerant clone, no significant

difference was observed in shoot protein oxidation when the roots were either

completely or partly exposed to Al, as compared to the control (0/0) (Fig. 2D).

Interestingly, the basal level of protein oxidation was significantly lower for this clone

than for the Al-sensitive clone.

Hydrogen peroxide concentration: In general, shoot H

concentration was higher

in the Al-sensitive clone (Fig. 3A) than in the Al-tolerant clone (Fig. 3B). In both clones,

an increase in shoot H

concentration was found both upon supply of Al to only one

half of the root system and to the entire root system. When both sides of the root

system were treated with 100 mg Al L

-1

(100/100), an increase of about 150% and

101% in shoot H

concentration was observed in the Al-sensitive and Al-tolerant

clones, respectively, while in the treatment where plants were supplied with 200 mg Al

-1

to only half of the root system (0/200), an increase of about 122% and 28% was

found in the Al-sensitive and Al-tolerant clones, respectively.

0/0 50/50 0/100 100/100 0/200

Al (mg L

-1

)

0.00

0.14

0.28

0.42

0.56

0.70

CAT activity (E min

-1

protein)

0/0 50/50 0/100 100/100 0/200

Al (mg L

-1

)

0.00

0.14

0.28

0.42

0.56

0.70

CAT activity (E min

-1

protein)

0/0 50/50 0/100 100/100 0/200

Al (mg L

-1

)

0.00

0.90

1.80

2.70

3.60

4.50

Carbonyl (nmol mg

-1

protein)

0/0 50/50 0/100 100/100 0/200

Al (mg L

-1

)

0.00

0.90

1.80

2.70

3.60

4.50

Carbonyl (nmol mg

-1

protein)

Figure 2. Effect of varying Al concentrations on shoot catalase (CAT) activity of the (A) Al-sensitive

(Macaca) and (B) Al-tolerant (SMIC148-A) clones, and protein oxidation of the (C) Al-sensitive (Macaca)

and (D) Al-tolerant (SMIC148-A) clones in a split-root system. Data are means ± SD at p<0.05, Tukey

test.

(C)

(D)

(A)

(B)

0/0 50/50 0/100 100/100 0/200

Al (mg L

-1

)

0.00

0.70

1.40

2.10

2.80

3.50

concentration (µmol g

-1

FW)

0/0 50/50 0/100 100/100 0/200

Al (mg L

-1

)

0.00

0.70

1.40

2.10

2.80

3.50

concentration (µmol g

-1

FW)

0/0 50/50 0/100 100/100 0/200

Al (mg L

-1

)

0.00

0.10

0.20

0.30

0.40

0.50

concentration (µmol g

-1

FW)

Left root half Right root half

0/0 50/50 0/100 100/100 0/200

Al (mg L

-1

)

0.00

0.30

0.60

0.90

1.20

1.50

concentration (µmol g

-1

FW)

Left root half Right root half

cde

bcd

cde

bcd

bcde

Figure 3. Hydrogen peroxide concentration in shoot of the (A) Al-sensitive (Macaca) and (B) Al-tolerant

(SMIC148-A) clones, and roots of the (C) Al-sensitive (Macaca) and (D) Al-tolerant (SMIC148-A) clones

under varying Al concentrations in a split-root system. Data are means ± SD at p<0.05, Tukey test.

In contrast to that observed in shoot, root H

concentration was greater in the

Al-tolerant clone than in the Al-sensitive clone. Root H

concentration in the Al-

sensitive clone increased in one root half of plants supplied with Al at 100 mg L

-1

both root halves (100/100) (Fig. 3C). In addition, in plants supplied with Al at 200 mg L

to only one half of the root system (0/200), root H

concentration was slightly, but

not significantly, increased in that root half treated by Al, when compared to the control

plants (0/0). In roots of the Al-tolerant clone, a significant increase in H

(C)

(D)

(A)

(

)

concentration was only observed in one root half of plants supplied with Al at 50 mg L

-1

to both root halves (50/50) (Fig. 3D).

Lipid peroxidation: The basal level of shoot MDA concentration (i.e. lipid

peroxidation) in the Al-tolerant clone was higher than in the Al-sensitive clone. On the

other hand, a greater increase in shoot lipid peroxidation in response to Al treatment

was observed in the Al-sensitive clone. A significant increase in shoot lipid peroxidation

in the Al-sensitive clone was observed either by applying Al to only one half of the root

system (0/100 and 0/200) or when the entire root system (50/50 and 100/100) was

exposed to Al (Fig. 4A). The highest increase in lipid peroxidation (of about 162%) was

seen in the Al-sensitive clone when Al was supplied to both sides of the root system at

100 mg L

-1

(100/100). On the other hand, in the Al-tolerant clone, a significant increase

in shoot lipid peroxidation (about 40%) was observed only in treatments with both sides

of the root system exposed to Al (50/50 and 100/100), when compared to the control

plants (Fig. 4B).

In roots of the Al-sensitive clone, lipid peroxidation increased significantly in both

halves of the root system supplied with 100 mg Al L

-1

(100/100) and in one half of the

root treated with 200 mg Al L

-1

(0/200) (Fig. 4C). In the Al-tolerant clone, a significant

increase in root lipid peroxidation was only observed in one half of the root in all Al

treatments (50/50, 0/100, 100/100 and 0/200) (Fig. 4D).

0/0 50/50 0/100 100/100 0/200

Al (mg L

-1

)

0.00

0.20

0.40

0.60

0.80

1.00

(E-1)

MDA (nmol mg

-1

protein)

0/0 50/50 0/100 100/100 0/200

Al (mg L

-1

)

0.00

0.20

0.40

0.60

0.80

1.00

(E-1)

MDA (nmol mg

-1

protein)

0/0 50/50 0/100 100/100 0/200

Al (mg L

-1

)

0.00

0.12

0.24

0.36

0.48

0.60

MDA (nmol mg

-1

protein)

Left root half Right root half

cde

0/0 50/50 0/100 100/100 0/200

Al (mg L

-1

)

0.00

0.06

0.12

0.18

0.24

0.30

MDA (nmol mg

-1

protein)

Left root half Right root half

bcd

Figure 4. Lipid peroxidation in shoot of the (A) Al-sensitive (Macaca) and (B) Al-tolerant (SMIC148-A)

clones, and roots of the (C) Al-sensitive (Macaca) and (D) Al-tolerant (SMIC148-A) clones under varying

Al concentrations in a split-root system. Data are means ± SD at p<0.05, Tukey test.

Non-protein thiol groups (NPSH) concentration: In the Al-sensitive clone, a

significant increase in shoot NPSH concentration was observed upon supply of Al both

to one half of the root system and to the entire root system (Fig. 5A). The greatest

increases in NPSH concentration, of about 207% and 153%, were seen when Al was

supplied to both sides of the root system at 100 mg L

-1

(100/100) and to only one half

of the root system at 200 mg L

-1

(0/200), respectively. In the Al-tolerant clone (Fig. 5B),

(D)

(C)

(A)

(B)

shoot NPSH concentration increased significantly (about 100%) only in the treatments

with both root halves exposed to Al (50/50 and 100/100).

0/0 50/50 0/100 100/100 0/200

Al (mg L

-1

)

NPSH (µmol -SH g

-1

FW)

0/0 50/50 0/100 100/100 0/200

Al (mg L

-1

)

NPSH (µmol -SH g

-1

FW)

0/0 50/50 0/100 100/100 0/200

Al (mg L

-1

)

0.00

0.10

0.20

0.30

0.40

0.50

NPSH (µmol -SH g

-1

FW)

Left root half Right root half

0/0 50/50 0/100 100/100 0/200

Al (mg L

-1

)

0.00

0.10

0.20

0.30

0.40

0.50

NPSH (µmol -SH g

-1

FW)

Left root half Right root half

Figure 5. Non-protein thiol group (NPSH) concentration in shoot of the (A) Al-sensitive (Macaca) and (B)

Al-tolerant (SMIC148-A) clones, and roots of the (C) Al-sensitive (Macaca) and (D) Al-tolerant

(SMIC148-A) clones under varying Al concentrations in a split-root system. Data are means ± SD at

p<0.05, Tukey test.

In roots of the Al-sensitive clone, NPSH concentration decreased significantly in

the right root half of the 50/50 and 0/100 treatments. On the other hand, in the 0/200

treatment, a significant increase in NPSH concentration was observed in the root half

supplied at 200 mg Al L

-1

(Fig. 5C). In the Al-tolerant clone, NPSH concentration

(C)

(D)

(A)

(B)

decreased significantly only in one half of the root supplied with 50/50 and 100/100

(Fig. 5D).

Acid phosphatase (APases) activity: In roots and shoot of potato plants, the basal

activity of APases in the Al-tolerant clone was higher than in the Al-sensitive clone. A

significant decrease (about 21%) in shoot APase activity was observed in the Al-

sensitive clone only when plants were supplied with 200 mg Al L

-1

to half of the root

system (0/200) (Fig. 6A). On the other hand, in the Al-tolerant clone, shoot APase

activity decreased in all Al treatments, either by applying Al only to one half of the root

system or when the entire root system was exposed to Al (Fig. 6B).

In roots of both clones, in general, an inhibition of APase activity was observed

in all Al treatments, but this effect was less pronounced in the Al-tolerant clone (Fig. 6C

and 6D). Plants of the Al-sensitive clone which had only half of the root system

exposed to Al (treatments 0/100 and 0/200 mg Al L

-1

) showed lower APase activity in

that root half treated by Al. Moreover, plants with both root halves exposed to Al at 100

mg L

-1

presented reduced APase activity in both root halves, whereas plants treated

with Al at 50 mg L

-1

(50/50) showed a decrease in APase acitivity only in one root half,

when compared to the control.

In the Al-tolerant clone, root APase activity was either reduced at least in one

root half exposed to Al (treatments 50/50 and 100/100) or in both root halves

(treatments 0/100 and 0/200), when compared to the control. Moreover, the 200 half of

0/200 plants presented significantly higher root APase activity than that half untreated

with Al, whereas the 0 half of 0/100 plants presented higher root APase activity than

did the 100 side.

0/0 50/50 0/100 100/100 0/200

Al (mg L

-1

)

180

270

360

450

APase activity (nmol Pi min

-1

protein)

0/0 50/50 0/100 100/100 0/200

Al (mg L

-1

)

180

270

360

450

APase activity (nmol Pi min

-1

protein)

0/0 50/50 0/100 100/100 0/200

Al (mg L

-1

)

100

200

300

400

500

APase activity (nmol Pi min

-1

protein)

Left root half Right root half

0/0 50/50 0/100 100/100 0/200

Al (mg L

-1

)

300

600

900

1200

1500

APase activity (nmol Pi min

-1

protein)

Left root half Right root half

Figure 6. Effect of varying Al concentrations on acid phosphatase (APases) activity in shoot of the (A)

Al-sensitive (Macaca) and (B) Al-tolerant (SMIC148-A) clones, and roots of the (C) Al-sensitive (Macaca)

and (D) Al-tolerant (SMIC148-A) clones in a split-root system. Data are means ± SD at p<0.05, Tukey

test.

(A)

(B)

Discussion

Growth inhibition, which is a physiological response to acidic soils, especially

due to aluminum (Al) toxicity, is a serious problem for the production of most important

crops in the world. Some screening techniques can be used to readily quantify the

response of different species or different clones of the same species towards

environmental stresses. In a previous study, utilizing a homogeneous supply of Al to

the roots of potatoes grown in a hydroponic growth system and based on relative root

growth, it was demonstrated that there was a significant difference in Al tolerance

among the four potato clones. In addition, it was shown that Al treatment induced

oxidative stress, mainly in the Al-sensitive clones (Tabaldi et al., 2007b). Hairiah et al.

(1993) observed that the response of root growth for Mucuna pruriens to the presence

of Al in its environment was positive when no other choice was given, and negative in

the absence of Al around other parts of the same root system. This relative Al

avoidance was related to the response of local P sources of plants with an overall

insufficient P supply. This hypothesis is actually based on internal P shortage in Al-

exposed roots, due to precipitation of Al phosphates. In the present study, to avoid the

interaction between P and Al in the nutrient solution, the experimental setup

determined that plants be grown for about 4 weeks in the presence of 250 µM of P,

and, subsequently, during the Al exposure (for 10 days), P was omitted from the

nutrient solution. In a previous experiment, we observed that potato plants that were

very well nourished with P could withstand 10 days in the absence of P in the nutrient

solution without showing visible symptoms of P deficiency. In this study, calculations

with ‘Visual MINTEQ’ showed that 83-90% of the nominal Al concentration (based on

initial ion concentration) are in the monomeric form (Supplement A).

Based on the results, a stronger visible (based on biomass) Al avoidance was

seen in the 0/100 and 0/200 plants of the Al-sensitive clone (Macaca) than in the Al-

tolerant clone (SMIC148-A) (Fig. 7 and 8). The 0 side of 0/100 and 0/200 plants

showed a significantly higher fresh biomass than that half of the root system supplied

with Al. Moreover, shoot growth of the Al-sensitive clone was significantly affected by

the Al treatments, while it was slightly decreased in the Al-tolerant clone. The nutrient

solutions were renewed every 48 h and the pH of the nutrient solutions was evaluated

at 24 h intervals. From the onset of the Al treatments, the pH of the nutrient solutions

decreased similarly for both potato clones upon presence of Al. However, in general,

within 10 d of Al treatment, the difference in solution pH between root halves in 0/100

and 0/200 was less pronounced for the Al-tolerant clone (Supplement B). Interestingly,

in these treatments, at 10 d of Al exposure, that root half of the Al-tolerant clone

supplied with either 100 or 200 mg Al L

-1

showed higher capacity to buffer the pH of the

nutrient solution (Supplement B).

Changes in the degree of lipid, protein and pigment oxidation, in the

concentration of non-enzymatic antioxidants and in the activity of antioxidant enzymes

are symptomatic of the plant oxidative stress response in relation to several biotic and

abiotic factors (Smirnoff, 1993). In the present study, potato plants grown under varying

Al concentrations revealed signs of oxidative stress. After 10 days of Al exposure in a

split-root system, Al supply at 100 mg L

-1

in both halves of the root system (100/100)

and at 200 mg L

-1

in only one root half (0/200) resulted in a significant decrease in the

shoot chlorophyll concentration

Figure 7 – Shoot and roots of potato plants, Macaca clone (Al-sensitive), exposed to 0/0 mg Al L

-1

(A,B),

50/50 mg Al L

-1

(C,D), 0/100 mg Al L

-1

(E,F), 100/100 mg Al L

-1

(G,H) and 0/200 mg Al L

-1

(I,J), in split-

root system.

(A)

(B)

(C)

(D)

(E)

(F)

(G)

(H)

(I)

(J)

Figure 8 – Shoot and roots of potato plants, SMIC148-A clone (Al-tolerant), exposed to 0/0 mg Al L

-1

(A,B), 50/50 mg Al L

-1

(C,D), 0/100 mg Al L

-1

(E,F), 100/100 mg Al L

-1

(G,H) and 0/200 mg Al L

-1

(I,J), in

split-root system.

(A)

(B)

(C)

(D)

(E)

(F)

(G)

(H)

(I)

(J)

only for the Al-sensitive clone (Fig. 1A). In contrast, in the Al-tolerant clone, no

significant difference in chlorophyll concentration was observed (Fig. 1B). This

decrease in the chlorophyll concentration in the Al-sensitive clone demonstrates the

poisoning effect of high concentrations of Al. According to Smirnoff (1993) and Brito et

al. (2003), the decrease in chlorophyll concentration is a typical symptom of oxidative

stress and may either be the result of chlorophyll degradation or lower chlorophyll

synthesis associated with changes in the thylakoid membrane structure. Carotenoids,

besides acting as accessory light harvesting pigments, show antioxidant properties (de

Pascale et al., 2001). In the present study, the increase in carotenoid concentration

with increasing Al supply observed only for the Al-tolerant clone (Fig. 1D) suggests that

this clone has greater potential for detoxification of toxic oxidation radicals formed in

response to Al treatments.

is a toxic reactive oxygen species (ROS), which has deleterious effects on

plant tissue (Salin, 1988). For both potato clones tested, shoot H

concentration

increased in a similar pattern upon Al treatment. This effect was dependent on the Al

concentration and, to a lesser degree, on the distribution of roots exposed to Al. At the

highest Al concentration supplied (200 mg L

-1

) to only half of the root system (0/200),

shoot H

concentration was lower than that with both root halves treated by 100 mg

-1

(100/100). However, this effect was less pronounced in the Al-sensitive clone.

Therefore at levels of Al that are toxic to potato, based on shoot chlorophyll

concentration (Fig. 1A), the amount of root exposed to Al was well correlated with

several biochemical alterations linked to oxidative stress.

Shoot CAT activity in the Al-sensitive clone was significantly reduced in the

treatments with both sides of the root system exposed to Al (50/50 and 100/100) when

compared to treatments where Al was supplied to only one root half (0/100 and 0/200

mg L

-1

). This result was correlated with the shoot H

concentration. The decreased

activity of the H

scavenging enzyme CAT upon Al treatment might have contributed

to the observed increases in shoot H

concentration. Thus, a decrease in shoot CAT

activity would result in accumulation of H

, which can react with O

to produce

hydroxyl-free radicals via the Herbert–Weiss reaction (Bowler et al., 1992). The

hydroxyl-free radicals can directly damage the membrane by attacking unsaturated

fatty acids of lipids to induce lipid peroxidation (Okuda et al., 1991). During oxidative

stress, H

is a strong toxic oxidant causing cell damage or even cell death and can

also contribute to the carbonylation of proteins (Bienert et al., 2006). The decrease, at

least, in CAT activity could be due to the blocking of essential functional groups in the

enzyme such as –SH or the displacement of essential metal ions from enzymes, as

suggested for other metals (Schützendübel and Polle, 2002).

It is worthy to note that plants respond to Al stress by various antioxidant

mechanisms, including the enzymatic ROS-scavenging system and by non-enzymatic

antioxidants, which function to interrupt the cascades of uncontrolled oxidation in each

organelle. In a previous study (Tabaldi et al., unpublished), it was observed that in

roots of the SMIC148-A potato clone (Al-tolerant), the inadequate response of CAT

activity to Al was compensated by the increased activity of ascorbate peroxidase.

Moreover, the enzymatic and non-enzymatic (acid ascorbic and non-protein thiol group

concentrations) antioxidants seem to compensate for each other.

Interestingly, in the 100/100 and 0/200 mg Al L

-1

treatments, the Al-sensitive

potato clone showed a significant increase in H

concentration in only one part of the

root system (right root half in the 100/100 treatment) when compared to the control

plants. Such an effect could be related to an uneven split of roots used in the split-root

experiment, even though care was taken in the division of the root system. Moreover,

root H

concentration was slightly, but not significantly, increased in the root half

treated by 200 mg Al L

-1

(treatment 0/200). These data suggest that, despite the fact

that the potato roots accumulate more Al than does the shoot (Tabaldi et al., 2007b),

some antioxidant systems in the roots were more efficient than those of shoot in

scavenging Al stress side effects. In addition, in those Al treatments (100/100 and

0/200) where shoot chlorophyll concentration was decreased in the Al-sensitive clone,

the H

concentration in the roots seemed to respond locally to Al supply.

Protein carbonylation is one of the markers of oxidative stress that results from

excessive production of ROS in the cell which is not balanced by an increased

efficiency of the antioxidant system (Juszczuk et al., in press). Although the basal level

of root H

concentration was higher for the Al-tolerant clone than for the Al-sensitive

clone (Fig. 3), the former showed no alteration in shoot protein oxidation at any Al

concentration (Fig. 2). On the other hand, in the Al-sensitive clone, shoot protein

oxidation was strongly correlated to Al concentrations in the nutrient solution rather

than to the distribution of roots exposed to Al.

Independently of the Al concentration and distribution of roots exposed to Al

treatments, shoot MDA concentration (i.e. lipid peroxidation) significantly increased in

the Al-sensitive clone, even in those treatments (50/50 and 0/100) which did not alter

the shoot chlorophyll concentration. However, root lipid peroxidation presented a

different pattern, where it increased upon Al supply either with both root halves

exposed at 100 mg Al L

-1

(100/100) or only with one root half exposed at 200 mg Al L

-1

(0/200). This indicates that Al toxicity of one half of the roots resulted in a local

response. In the Al-tolerant clone, the effect of Al on shoot lipid peroxidation was

dependent on the concentration and location of Al. Plants treated with 100 and 200 mg

Al L

-1

in only one root half (0/100 and 0/200) showed lower shoot lipid peroxidation than

those which had both root halves treated by Al at concentrations of 50 (50/50) and 100

(100/100) mg L

-1

. Such data suggest, to a certain degree, that potato roots responded

locally to Al stress, whereas the shoot showed a systemic response.

The major pool of non-protein thiol groups (NPSH) in most plant species is

represented by reduced glutathione. NPSH are known to be affected by the presence

of several metals (Xiang and Oliver, 1998). In the present study, the effect of Al on

shoot NPSH concentration in both potato clones (Fig. 5A, 5B) showed a pattern similar

to that shown for H

concentration (Fig. 3A, 3B). However, the increase observed in

shoot NPSH concentration in the Al-tolerant clone (Fig. 5B) seemed to have a higher

correlation with lipid peroxidation (Fig. 4B) than that presented for the Al-sensitive

clone. The increase in NPSH concentration is associated with tolerance against several

stresses (Agrawal and Rathore, 2007), and they may function as reducers of oxidative

damage (Ali et al., 2005). Therefore, the antioxidant system of the Al-sensitive clone

was less efficient to remove the excess of ROS than that of Al-tolerant clone.

In the Al-tolerant clone, the Al treatment which caused the highest negative

effect on both shoot and root APase activity was that with only one root half exposed at

100 mg Al L

-1

(0/100). Although the root half not exposed to Al showed higher APase

activity than that treated with 100 mg Al L

-1

, the levels of APase activities were lower

than those of the control plants. Therefore, this result suggests that roots of potato

responded both locally and systemically to Al stress. This assumption is corroborated

by the effect of other Al treatments, suggesting that the Al transported to the shoot

might be retranslocated to roots not treated by Al. This hypothesis should be further

tested.

The expectation that the Al-tolerant clone (SMIC148-A) would be more tolerant

to Al than the Al-sensitive clone (Macaca) was confirmed by the results. At 100 mg Al

-1

supplied to both root halves, the Al-sensitive clone showed an inefficient tolerance

response, based on tissue CAT activity, protein oxidation, lipid peroxidation, H

concentration and APase activity. In contrast, the Al-tolerant clone showed a

comparatively small negative effect on both shoot and root biochemical parameters at

this concentration. However, an apparently significant Al-avoidance reaction was

presented, though less pronounced than for the Al-sensitive clone at the higher Al

concentrations. The higher Al tolerance of SMIC148-A might be related to a more

efficient oxidative scavenging capacity and the higher basal APase activity level than

that found in Macaca. Acid phosphatases are a group of enzymes involved in the

production, transport and recycling of inorganic phosphate (Yoneyama et al., 2007). In

natural ecosystems, P availability is seldom optimal for plant growth because of limited

P content in the soil solution. Aluminum and iron ions in acid soils interact strongly with

P and render it unavailable to plants (Sousa et al., 2007). Indeed, Hairiah et al. (1993)

observed that Al avoidance for Mucuna pruriens and Centrosema pubescens was

related to a response to local P sources in plants with an overall insufficient P supply.

Moreover, the present study shows that the Al-tolerant potato clone presented a

stronger local and systemic antioxidant response to Al supply. Therefore, the

SMIC148-A clone, which is more Al-tolerant than the Macaca clone, might show a

deeply rooted system in surroundings with toxic levels of Al.

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CONSIDERAÇÕES FINAIS

O uso do alumínio em solução nutritiva mostrou-se uma técnica adequada para

estudos bioquímicos e fisiológicos de toxicidade desse metal. Esse estudo contribuiu

para uma melhor compreensão dos mecanismos causais da toxicidade do alumínio

(Al) em clones de batata. Além disso, a determinação de parâmetros bioquímicos

mostrou ser uma ferramenta importante para correlacionar inibição do crescimento

com alterações bioquímicas provocadas pelo Al em plantas de batata.

O Al foi absorvido pelas raízes e transportado para a parte aérea de todos os

clones, mas os efeitos tóxicos desse metal foram diferenciados entre os clones. Os

maiores sintomas de toxidez se manifestaram na raiz, justamente onde foram

detectadas as maiores concentrações desse metal.

A inibição do crescimento da raiz é o primeiro sintoma visível de toxicidade do

Al. Utilizando-se o crescimento relativo da raiz como critério, foi possível separar os

clones de batata em sensíveis (Macaca e Dakota Rose) e tolerantes (Solanum

microdontum e SMIC148-A) ao Al. Os dados sugerem que o(s) mecanismo(s) de

tolerância ao Al existentes nos clones tolerantes (S. microdontum e SMIC148-A)

é(são) interno(s), uma vez que o alumínio foi absorvido pelas raízes e também

transportado para a parte aérea das plantas. O crescimento da parte aérea foi bem

menos afetado. Entretanto, vários parâmetros bioquímicos mostraram-se alterados

pela presença de Al, tanto em clones tolerantes como sensíveis ao Al.

As diferenças entre os clones foram observadas especialmente em nível de

dano oxidativo a biomoléculas e na expressão da quantidade e tipo de antioxiante.

Esse dano pode ser uma conseqüência do aumento na concentração de peróxido de

hidrogênio, ou de outras espécies reativas formadas a partir do peróxido de

hidrogênio, observado nesses clones.

Nos clones tolerantes ao Al, as biomoléculas como lipídios de membrana, as

proteínas e os pigmentos sofreram menor dano oxidativo, comparado com os clones

sensíveis ao Al. Nesses clones, pode-se observar que há sempre um componente do

sistema antioxidante protegendo as plantas do estresse de Al, o mesmo não

acontecendo com os clones sensíveis ao Al. Além disso, o clone S. microdontum

manteve uma concentração maior de micronutrientes em raízes e parte aérea,

sugerindo um mecanismo adicional de tolerância.

Da mesma forma, quando os clones Macaca (sensível ao Al) e SMIC148-A

(tolerante ao Al) foram cultivados em sistema de raízes divididas com variação na

concentração e distribuição de Al ao sistema radicular, as plantas apresentaram sinais

de estresse oxidativo, os quais foram observados principalmente no clone sensível ao

Al. Baseado em parâmetros bioquímicos de raízes e parte aérea, o clone tolerante ao

Al sofreu danos oxidativos menores, em comparação com o clone sensível,

apresentando respostas antioxidativas sistêmicas e locais mais evidentes ao

suprimento de Al, mesmo tendo uma reação de escape ao Al menor que o clone

sensível ao Al. Portanto, a maior tolerância ao Al do clone SMIC148-A pode ser

relacionada à sua eficiente capacidade antioxidante. Além disso, por apresentar uma

reação de escape ao Al menor, o clone tolerante pode apresentar um sistema de

enraizamento mais profundo em solos com níveis tóxicos de Al.

Baseado nessas observações pode-se afirmar que o estresse oxidativo pode

ser um importante mecanismo de toxicidade do Al, principalmente em clones sensíveis

ao metal. Essa toxicidade depende da disponibilidade de Al, do clone e do sistema de

crescimento analisado. Além disso, pode-se observar que os efeitos adversos do Al

não desapareceram quando parte do sistema radicular não está em contato com o Al.

Portanto, todos esses fatores devem ser considerados no desenvolvimento de

protocolos para a caracterização de clones de batata tolerantes ao Al.

Experimentos futuros devem ser realizados para investigar se esses clones

possuem outros mecanismos de tolerância, como por exemplo, a complexação interna

do Al com ácidos orgânicos. Além disso, é interessante observar o comportamento

desses clones em um solo caracteristicamente ácido e com alta saturação em Al.

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APÊNDICES

APÊNDICE A - Porcentagem de distribuição de espécies de alumínio (Al) dissolvidas e adsorvidas, segundo programa Visual

Minteq.

Espécies de alumínio

Concentrações de Al

AlOH

Al(OH)

(OH)

AlCl

AlSO

Al(SO

)

…………………………………………………….……..%...................................................................................

50 mg L

-1

83,413 4,124 0,136 0,058 0,392 0,075 11,76 0,041

100 mg L

-1

87,716 3,941 0,123 0,261 0,835 0,070 7,038 0,016

150 mg L

-1

89,255 3,705 0,110 0,599 1,257 0,065 5,0 0

200 mg L

-1

89,772 3,485 0,099 1,054 1,651 0,061 3,872 0

APÊNDICE B – Valores de pH da solução nutritiva durante o período de exposição ao Al (10 dias) de clones de batata

(Macaca e SMIC148-A) em sistema de raízes divididas. As soluções foram trocadas a cada 48 horas.

1º dia (24 h) 2º dia (48 h)

Tratamentos Macaca SMIC148-A Macaca SMIC148-A

(mg Al L

-1

) RE RD RE RD RE RD RE RD

RE 0, RD 0

RE 50, RD 50

RE 0, RD 100

RE 100, RD 100

RE 0, RD 200

4,11 a

3,78 c

4,0 ab

3,84 bc

3,96 abc

4,13 a

3,78 c

3,84 bc

3,83 bc

3,95 abc

4,26 a

3,90 c

4,3 a

3,88 c

4,1 b

4,26 a

3,87 c

3,85 c

3,93 c

4,41 ab

3,80 c

4,1 bc

3,84 c

4,07 bc

4,45 a

3,79 c

3,84 c

3,82 c

3,91 c

4,88 a

4,03 b

4,58 a

3,96 b

4,0 b

4,89 a

3,97 b

3,96 b

3,97 b

3º dia (24 h) 4º dia (48 h)

Tratamentos Macaca SMIC148-A Macaca SMIC148-A

(mg Al L

-1

) RE RD RE RD RE RD RE RD

RE 0, RD 0

RE 50, RD 50

RE 0, RD 100

RE 100, RD 100

RE 0, RD 200

4,16 ab

3,94 ab

3,91 ab

3,89 ab

3,93 ab

4,18 a

3,94 ab

3,95 ab

3,88 b

3,96 ab

4,12 n.s.

4,15 n.s.

4,13 n.s.

4,09 n.s.

4,12 n.s.

4,10 n.s.

4,12 n.s.

4,09 n.s.

4,14 n.s.

4,46 a

4,18 ab

4,14 ab

4,07 b

4,04 b

4,46 a

4,16 ab

4,06 b

4,14 ab

4,23 ab

4,01 cd

4,11 bc

3,96 cd

3,95 cd

4,32 a

3,96 cd

3,93 cd

3,92 d

5º dia (24 h) 6º dia (48 h)

Tratamentos Macaca SMIC148-A Macaca SMIC148-A

(mg Al L

-1

) RE RD RE RD RE RD RE RD

RE 0, RD 0

RE 50, RD 50

RE 0, RD 100

RE 100, RD 100

RE 0, RD 200

4,21 a

3,98 ab

4,07 ab

4,01 ab

3,94 b

4,22 a

4,0 ab

3,97 ab

4,01 ab

4,0 ab

4,23 ab

3,95 d

4,21 abc

4,0 bcd

4,04 bcd

4,27 a

3,95 d

3,98 cd

4,0 bcd

3,96 d

4,34 a

3,95 b

3,97 b

3,93 b

3,74 b

4,35 a

3,95 b

3,91 b

3,87 b

4,21 ab

3,91 bc

4,14 abc

3,9 bc

3,87 c

4,25 a

3,91 bc

3,9 bc

3,88 c

3,83 c

7º dia (24 h) 8º dia (48 h)

Tratamentos Macaca SMIC148-A Macaca SMIC148-A

(mg Al L

-1

) RE RD RE RD RE RD RE RD

RE 0, RD 0

RE 50, RD 50

RE 0, RD 100

RE 100, RD 100

RE 0, RD 200

4,2 a

4,0 b

3,97 b

4,0 b

3,8 c

4,21 a

4,03 b

3,96 b

4,0 b

3,94 b

4,21 a

4,04 b

4,09 ab

4,02 b

4,0 b

4,22 a

4,01 b

4,0 b

3,99 b

4,02 b

4,29 a

4,13 b

4,02 b

4,09 b

3,86 c

4,39 a

4,13 b

4,04 b

4,07 b

4,0 bc

4,3 a

3,95 b

4,0 b

3,89 b

3,87 b

4,44 a

3,91 b

3,88 b

3,87 b

3,85 b

9º dia (24 h) 10º dia (48 h)

Tratamentos Macaca SMIC148-A Macaca SMIC148-A

(mg Al L

-1

) RE RD RE RD RE RD RE RD

RE 0, RD 0

RE 50, RD 50

RE 0, RD 100

RE 100, RD 100

RE 0, RD 200

4,08 a

3,93 cd

3,75 e

3,96 bc

3,73 e

4,11 a

3,91 d

3,89 d

3,95 bc

3,98 b

4,09 a

3,87 bc

3,99 ab

3,9 bc

3,86 bc

4,09 a

3,84 c

3,87 bc

3,88 bc

3,91 bc

4,28 a

3,95 cd

4,16 ab

3,92 d

4,07 bc

4,29 a

3,95 cd

3,87 d

3,91 d

4,23 ab

4,02 abc

4,07 abc

4,04 abc

3,91 c

4,28 a

3,98 bc

4,02 abc

4,0 bc

4,01 bc

*Médias seguidas de mesma letra não diferem entre si pelo teste de Tukey em nível de 5% de probabilidade de erro.

n.s. – Não significativo.

RE: metade esquerda da raiz; RD: metade direita da raiz.

APÊNDICE C – Cultivo in vitro de clones de batata em câmara climatizada (A); Sistema hidropônico em câmara

climatizada (B); Clones de batata crescendo em areia em casa de vegetação (C); Clones de batata em sistema de raízes

divididas em casa de vegetação (D).

(A)

(B)

(C)

(D)

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