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DANIEL NOGUEIRA MARTINS
IN SITU
HYBRIDIZATION OF SUCROSE SYNTHASE
TRA
N
SCRIPTS AND POSTHARVEST STUDIES IN TUBEROUS
ROOTS
Tese apresentada à
Universidade Federal de
Viçosa, como parte das
exig
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Ficha catalográfica preparada pela Seção de Catalogaç
ão e
Classificação da Biblioteca Central da UFV
T
Martins, Daniel Nogueira, 1976
-
M386i
In situ
hybridization of sucrose synthase transcripts and
2007
postharvest studies in tuberous roots / Daniel Nogueira
Martins.
–
Viçosa, MG, 2007.
xii, 92f.
: il. col. ; 29cm.
Orientador: Fernando Luiz Finger.
Tese (doutorado)
-
Universidade Federal de Viçosa.
Inclui bibliografia.
1. Biologia molecular. 2. Hortaliças
-
Fi
siologia pós
-
colheita. 3.
Arracacia xanthorrhiza
. 4.
Beta vulgaris
.
5. Alimentos
-
Conservação. 6. Alimentos
-
Resfriamento.
7. Alimentos
-
Embalagens. I. Universidade Federal de
Viçosa. II.Título.
CDD 22.ed. 572.8
ads:
DANIEL N
OGUEIRA MARTINS
IN SITU
HYBRIDIZATION OF SUCROSE SYNTHASE TRANSCRIPTS AND
POSTHARVEST STUDIES IN TUBEROUS ROOTS
Tese apresentada à
Universidade Federal de Viçosa,
como parte das exigências do
Programa de Pós-Graduação em
Fisiologia Vegetal, para obtenção
do título de
Doctor Scientiae
.
APROVADA: 29 de março de 2007.
________________________________
_________________________________
Prof. Raimundo Santos Barros
(Co
-
Orientador)
Prof. Mário Puiatti
(Co
-
Orientador)
____________
____________________
_________________________________
Pesq. Karen L. Klotz
Prof. Wagner Campos Otoni
_______________________________
Prof. Fernando Luiz Finger
(Orientador)
ii
To my wife
, Fernanda
iii
ACKNOWLEDGEMENTS
Firstly,
I would like to thank God for the care, consolation and comfort in
every moment and for giving me the strength to finish this study.
Special thanks are also owed to:
Viçosa Federal University (UFV), in special the Vegetal Biology
Department
, for the oppo
rtunity of carrying out the course.
United States Department of Agriculture (USDA), through the Northern
Crop Science Laboratory (NCSL) and the North Dakota State University for
receiving, supporting and providing me with the necessary infrastructure for
performing
part of
the experiments of this thesis.
CNPq
and CAPES for the financial support.
Fernanda, my wife, who, besides giving me love, tenderness and
encouragement, was essential in carrying out the experiments of this thesis.
Prof. Fernando Luiz Finger, for his excellent advice, availability,
practicality, competence, efficiency, teachings, professional and competence
example and friendship.
Dr.
Karen Klotz, NCSL-USDA researcher, for her valuable teachings and
patience, for offering her lab for my PhD internship and for enriching the
defense of my thesis with her presence.
My
co
-
advisers
, Professors Mário Puiatti and Raimundo
Santos
Barros,
for their suggestions and constructive criticism.
Prof. Wagner
Campos
Otoni, for accepting to participate in this thesis’s
defense, contributing significantly with many suggestions.
John White, lab
technician
of the Sugarbeet Laboratory - NCSL, for his
experience and valuable help in carrying out the molecular biology experiments.
The
trainees
,
Janaína e Lucile
ne,
for the friendship and important
support in the experiments.
My parents, Genário and Vanda, and all my family for their
encouragement and unlimited and constant support.
iv
The Postharvest lab colleagues for their companionship, continuous
knowledge and learning exchange, and for the laughter that made the
workplace always pleasant.
The
Postharvest
lab
technicians,
Geraldo e Sebastião,
for the experience
and logistic support at the lab
oratory
.
And to all those who, direct or indirectly, contributed to realizing this
work
.
v
BIOGRAPHY
DANIEL NOGUEIRA MARTINS, son of Genário Martins and Vanda
Nogueira Martins, was born on December 12
th
, 1976, in Ribeirão das Neves,
MG
-
Brazil.
In 1995, he entered Viçosa Federal University, graduating in agron
omy
engineering in October 2000.
In 2003, he got his Master’s degree in
Fitotecnia
from Viçosa Federal
University.
In March 2003, he started his PhD course in Plant Physiology at Viçosa
Federal University. He worked for 10 months at the Northern Crop Scien
ce
Laboratory
- USDA, Fargo, ND, in a doctoral training program, defending his
thesis on March 29, 2007.
vi
CONTENTS
RESUMO …………………………….…………………………….……………
ix
ABSTRACT
………………………….………………………….………………
xi
1. GENERAL INTRODUCTION
…….…….……………….………………….
1
2. LITERATURE
…………………………………………………………………
3
CHAPTER 1
…………………………………………………………………
..
..
In situ
hybridization of riboprobes
for
two sucrose synthase
transcripts on
sugarbeet
root tissue
5
ABSTRACT
………………………………………………………………….…..
5
1. INTRODUCTION
…………………
…………………………………………. 7
1.1 Economical importance
……………………………………………………
7
1.2 Plant and root anatomy
……………………………………………………
7
1.3 Sucrose
accumulation
……………………………………………………..
8
1.4 Sucrose degradation
…………………………………………………….…
9
1.5 Sucrose synthase
………………………………
……………………….….
10
1.6 Non-
radioactive
in situ
hybridization considerations
……………………
11
1.7 Objective
…………………………………………………………………….
14
2. MATERIAL
S
AND METHODS
……………………………………………..
15
2.1 Precautions to work with RNA
…..………………………………………..
15
2.2 Probe prepar
ation
…………………………………………………………..
15
2.2.1 RNA extraction
……………………………………………………………
15
2.2.2 Probe production by RT
-
PCR
…………………………………………..
16
2.2.2.1 First str
and cDNA synthesi
s
………………………………………….
16
2.2.2.2 Primers design
……………………………………………………...
16
2.2.2.3
cDNA
amplification by PCR
…………………………………………..
17
2.2.3 Extraction and purification of PCR probe products
…………………..
18
2.2.4 Probe
cloning and cell transformation
………………………………..
18
2.2.5 Plasmid isolation …………………………………………………………
19
2.2.6 Verification of
plasmid
-
probe quality and sequencing
……………….
19
2.3 Labeled RNA probe synthesi
s
……………………………………………
20
2.3.1 Plasmid linearization
……………………………………………………..
20
2.3.2 Linearized plasmid purification
………………………………………….
21
vii
2.3.3
In vitro
transcription
………………………
………………………………
22
2.4 Labeled probe purification
…………………………………………………
22
2.5 Estimation of the labeled probe concentration
………………………….
22
2.6 RNA dot blot hybridization
…………………………………………………
23
2.6.1 Non
-
labeled RNA preparation
…………………………………………..
24
2.6.1.
1 Cloning SBSS1 fragment into pLITMUS 38i
………………………..
24
2.6.1.1.1 Double restriction reaction ………………………………………….
24
2.6.1.1.2 Fragment cloning and cell transformation
………………………...
25
2.6.1.2 Plasmids linearization
…………………………………………………
26
2.6.1.3
In vitr
o
transcription of non
-
labeled RNA
……………………………
26
2.6.1.4 Non
-
labeled RNA purification
…………………………………………
27
2.6.2 Blotting RNA in the membrane
…………………………………………
27
2.6.3 Pre
-
hybridization procedures
…………………………………………...
27
2.6.4 Hybridization of the label
ed RNA probe to
R
NA on the blot
…………
28
2.6.5 Chromogenic detection of the probe on the blot
……………………...
28
2.7
In situ hybridization procedures
…………………………………………..
29
2.7.1 Tissue preparation
……………………………………………………….
29
2.7.1.1 Fixation of the tissue
………
…………………………………………..
29
2.7.1.2 Embedding of tissue in paraffin
………………………………………
30
2.7.1.3 Sectioning of tissue
……………………………………………………
31
2.7.2 Pre treatment of slides before hybridization
…………………………..
31
2.7.2.1 De
-
paraffinization and rehydratation of the
slides
…………………
31
2.7.2.2 Protese digestion, post fixation and acetylation
……………………
31
2.7.2.3 Dehydratation
…………………………………………………………..
32
2.7.3 Hybridization of the labeled probe on tissue
………………………….
32
2.7.4 Treatment after hybridization
………………………………
……………
33
2.7.5 Immunohistochemical detection of DIG probes
………………………
33
2.7.6 Slide set up
……………………………………………………………….
34
3. RESULTS AND DISCUSSION
……………………………………………..
35
3.1 Probe production
…………………………………………………………...
35
3.2 RNA dot blot hybridization
…
………………………………………………
38
3.3 The empirical art of
in situ
hybridization
…………………………………
39
3.3.1 Tissue fixation
…………………………………………………………….
39
viii
3.3.2 Protease treatment ………………………………………………………
40
3.3.3 Conditions of hybridization
………………………………………………
41
3.4
In si
tu
hybridization of labeled probes in
sugarbeet
root tissue
……….
41
4. CONCLUSION
……………………………………………………………….
53
5. LITERATURE
………………………………………………………………...
54
CHAPTER 2
……………………………………………………………………..
Effect of the temperature and PVC
film cover on
fresh
m
ass loss
and sucrose synthase activity
of stored arracacha roots
60
ABSTRACT
……………………………………………………………………...
60
1. INTRODUCTION
…………………………………………………………….
62
1.1 General aspects
…………………………………………………………….
62
1.2 Culture limitations
………………………………………………………….
.
63
1.3 Refrigeration
………………………………………………………………...
64
1.4 Chilling injury
………………………………………………………………..
65
1.5 Sweetening
………………………………………………………………….
66
1.6 Objectives
…………………………………………………………………...
68
2. MATERIAL
S
AND METHODS
……………………………………………..
69
2.1 Ha
rvesting and root preparing
…………………………………………….
69
2.2 Experiment
………………………………………………………………….
69
2.3
Fresh m
ass loss
…………………………………………………………….
70
2.4 Vapor pressure deficit
……………………………………………………...
70
2.5 Determining of the sucrose synthase activity
………………
……………
70
2.5.1 Extraction
………………………………………………………………….
70
2.5.2 Sucrose synthase activity ……………………………………………….
71
3. RESULTS AND DISCUSSION
……………………………………………..
72
3.1
Fresh m
ass loss and
vapor
pressure deficit
………………………
.
……
72
3.2 Activity of sucrose synt
hase
………………………………………………
78
3.3 Diseases incidence
………………………………………………………...
84
4. CONCLUSION
……………………………………………………………….
86
5. LITERATURE
………………………………………………………………...
87
ix
RESUMO
MARTINS, Daniel Nogueira, D.
Sc
. Universidade Federal de Viçosa, março de
200
7,
Hibridização
in situ de transcritos da sintase da sacarose e
estudos de pós-colheita em raízes tuberosas.
Orientador
: Fernando Luiz
Finger. Co
-
orientadores:
Raimundo Santos Barros e Mário Puiatti.
O presente trabalho foi dividido em dois capítulos, se
ndo
cada um deles
destinado à descrição dos estudos desenvolvidos com um tipo de rai
z
tuberosa, a saber: beterraba açucareira e m
andioquinha
-
salsa.
Foram
investigados diferentes aspectos de cada uma destas raízes relacionados ao
metabolismo de carboidratos e mais especificamente à ação da sintase da
sacarose, enzima sacarolítica, a qual desempenha importante papel no
metabolismo de carboidratos das plantas em geral. Enquanto a beterraba
açucareira
armazena sacarose nas raízes, o amido é o principal carboidrato de
reserva armazenado nas raízes da mandioquinha
-
salsa. A beterraba açucareira
é uma espécie temperada, cujas raízes tuberosas são utilizadas para produção
de açúcar, sendo portanto, primordial evitar a degradação da sacarose,
tanto
no cultivo quanto d
urante
a
pós
-colheita. Por outro lado, a mandioquinha-
salsa
é cultivada em regiões tropicais e sub-tropicais e suas raízes são utilizadas na
alimentação humana, sendo de extrema importância a extensão da vida pós-
colheita e a manutenção da qualidade das raízes durante a armazenagem.
Na
beterraba açucareira, a sintase da sacarose é uma enzima de importância tanto
no deselvolvimento quanto na pós
-
colheita das raízes. O estudo baseado nesta
cultura teve por objetivo a produção de sondas marcadas com alta
espec
ificidade para os mRNAs de duas isoformas da sintase da sacarose
, para
fins de utilização na técnica de hibridização in situ
adaptada
para os tecidos de
raízes da beterraba açucareira, e deste modo, verificar a expressão de dois
tipos de transcritos da sintase da sacarose em diferentes tecidos da raiz. Para
este propósito, sondas de DNA foram produzidas por RT-
PCR
, e
subseqüentemente,
usadas para produzir sondas de RNA marcadas com
digoxigenina
visando as reações de transcrição in vitro. A especificidade da
s
sondas foi testada em ensaios de hibridização via
dot
-
blot
. A técnica de
hibridização
in situ foi adaptada para os tecidos da raíz da beterraba
açucareir
a, principalmente no que tange às condições de fixação do tecido,
às
x
etapas de pré-
tra
tamento do tecido a fim de torná-lo mais acessível à sonda, e
às condições de hibridização. Foram obtidas, com sucesso, s
ondas
marcadas
de RNA com alta especificidade para dois mRNAs expressos
nas
raízes da
beterraba açucareira. Verificou-se que, os dois tipos de transcritos da sintase
da sacarose estavam concentrados no tecido de reserva do parênquima
,
apontando para um possível papel destes tecidos como centro de
armazenagem e distribuição de açúcares nas raízes, e para o papel regulatório
da sintase da sacarose neste processo. Em mandioquinha-
salsa
, os estudos
tiveram por objetivo verificar o efeito da temperatura e do filme plástico de PVC
na armazenagem das raízes, considerando a perda de massa fresca, o dé
ficit
de pressão de vapor e a atividade da sintase da sacarose
.
Raízes
foram
submetidas à armazenagem em temperatura ambiente e
nas
temperaturas de
5º C, 10º C e 15º C, com e sem a cobertura do filme de PVC durante o período
de armazenagem. A perda de massa fresca foi diretamente afetada pelo filme
de PVC e pelo dé
fi
cit de pressão de vapor, o qual resultou da combinação
entre temperatura e umidade relativa. A atividade da sintase da sacarose foi
induzida pelo estresse hídrico e possivelmente pela injúria por frio, pondendo
estar envolvida, também, em um processo de adoçamento na mandioquinha-
salsa. Este trabalho demonstrou que, a sintase da sacarose desempenha um
importante papel na regulação do metabolismo de carboidratos em ambas as
raízes tuberosas estudadas.
xi
ABSTRACT
MARTINS,
Daniel Nogueira, D.Sc
.
Universidad
e Federal de Viçosa, March,
2007.
In
situ
hybridization of sucrose synthase transcripts and
postharvest studies in tuberous roots.
Adviser
:
Fernando Luiz Finger.
Co
-
advisers
: Raimundo Santos Barros
and
M
ário Puiatti.
The present work was divided into two chapters in which studies
developed with two tuberous roots were considered separately.
Sugarbeet
and
arracacha
were investigated focusing different aspects of each one considering
the carbohydrate metabolism, more specifically involving the sucrose synth
ase,
a sucrolytic enzyme that performs an important role on carbohydrate
metabolism of plants in general. While
sugarbeet
stores sucrose in roots, starch
is the mainly storage car
xii
aimed to verify the effect of temperature and PVC film cover on root storage
based in
fresh
mass loss, vapor pressure deficit and sucrose synthase activity.
Roots were submitted to storage at room te
mperatures,
5
o
C, 10
o
C and 15
o
C
with and without PVC cover during
the
storage period. Fresh mass loss was
directly affected by PVC film cover and vapor pressure deficit which resulted
from the combination between temperature and relative humidity. Sucros
e
synthase activity decreased in roots covered with PVC film independently of
storage temperature except at 5
o
C
in which the enzyme activity remained high.
Sucrose synthase activity was induced by water stress and possibly by chilling
injury and can be involved also in
the
sweetening process in arracacha.
This
work demonstrated that sucrose synthase
plays
an important role in
carbohydrate metabolism regulation in both tuberous roots studied.
1
1. GENERAL INTRODUCTION
Tuberous roots include many kinds of plants broadly spread
worldwide
.
In general these crops develop a relevant role in agricultural systems since they
provide
energetic foods, rich in carbohydrates.
As
happens with a variety of horticultural products, tuberous roots show
high levels of postharvest loss due to many causes such as metabolic
modifications associated with respiration and senescence processes, loss of
fresh matter, mechanic damages, diseases and physiological disturbs among
other factors related to inappropriate handling (Kays
, 1991).
Arracacha (Arracacia xanthorrhiza
Bancroft
) is a plant from tropical and
sub
-tropical origin with typical tuberous roots that demonstrates great rusticity,
demanding low input expenses. However its life cycle is long, 8 to 11 months
(Filgueira, 20
03), and postharvest life is very short, reaching 6 days of self life at
most (Scalon et al., 1998).
Studies have demonstrated the significant effect of plastic films on
extension of the postharvest life of arracacha, mainly when associated with
refrigera
tion (Casali et al., 1988; Câmara, 1984). However, arracacha
demonstrated to be sensitive to chilling injury (Ribeiro, 2005). Furthermore,
alterations on carbohydrate contents of the roots were observed under
refrigerated storage conditions (Ribeiro, 2003)
.
Arracach
a roots are rich in starch like other tuberous roots such as
cassava and sweet potato (Cereda, 2002) and the breakdown of starch as well
as sugar metabolism is a relevant aspect of root quality to be considered in
postharvest conditions mainly during long refrigerated storage conditions.
However, few works have been developed concerning this issue.
The
sugarbeet
(Beta vulgaris
L.
) is a biannual crop from temperate origin
wit
h a life cycle of 4 to 6 months in general, depending on varieties and cl
imate
conditions (Bloch and Hoffmann, 2005; Tsialtas and Karadimos, 2003). It
diverges from table cultivars (Filgueira, 2003) by root size and absence of
be
taine on roots characterized by creamy white, elongated, tapering, and
conical st0.09187 0 0 -0.09187 3874 140 0 -0.09187 2517 11132 Tm(l)
2
carbohydrate stored is sucrose, whose concentration can reach 20% of fresh
weight (Wyse, 1979).
After harvest
sugarbeet
roots are stored up to 200 days in large outdoor
piles prior to processing in the Northern United States, Canada, Northern
Europe and Russia. During this postharvest period substantial loss of sucrose
and increase of glucose and fructose occur (Klotz and Finger, 2004). Sucrose
loss results in decreasing of
the
sugar production and consequent reduction of
gains by
sugarbeet industries (Wiltshire and Cobb, 2000).
The four enzymes in
sugarbeet
roots known to hydroly
ze sucrose to form
reducing sugar are insoluble acid invertase, vacuolar acid invertase, alkaline
invertase and sucrose synthase (Wiltshire and Cobb, 2000).
To better understand the process that leads sucrose degradation by
sucrolytic
enzymes, studies have been developed to verify the role of the these
enzy
mes during the development of roots (Klotz and Finger, 2002;
Giaquinta,
1979), postharvest storage (Klotz and Finger, 2004; Wyse, 1974) and under
stress conditions (Rosenkranz et al., 2001). Furthermore, characterization of the
sucrolytic
enzymes isoforms were performed (Klotz et al., 2003; Klotz and
Finger, 2001) as well as gene expression patte
rn were investigated (Haagenson
et al., 2006). However, the role of each enzyme on the complex carbohydrate
metabolic pathway and the factors governing
sucrolytic
activity are not
completely understood, requiring further studies.
3
2. LITERATURE
BLOCH, D.; HOFFMANN, C. Seasonal development of genotypic differences in
sugar beet (
Beta vulgaris L.) and their interaction with water supply.
Journal of
Agronomy & Cro
p Science
, v. 191, p. 263
-
272, 2005.
CÂMARA, F. L. A. Manejo pós-colheita da mandioquinha-
salsa.
Informe
Agropecuário
, v.
10, n.
120, p. 70
-
72, 1984.
CASALI, V. W. D.; KIMURA, S.; AVELAR FILHO, J. A. de. Tempo de
frigorificação e conservação da mandioquinha-salsa após a retirada da câmara
fria.
Horticultura Brasileira
, v.
6, n.
1
, p. 49, 1988. (Resumo).
CEREDA, M. P. Importância das tuberosas tropicais. In: Cereda, M. P.
Agricultura: tuberosas amilaceas
latinoamericanas
. São Paulo: Fundação
Cargill, 2002. p
. 1
-
16. (Série Tuberosas Amilaceas Latino Americanas, v.
2).
FILGUEIRA, F. A. R
.
Novo manual de olericultura: agrotecnologia moderna
na produção e comercialização de hortaliças. 2ª edição revista e ampliada.
Viçosa:
Editora UFV, 2003. 412
p. il.
GIAQUINTA,
R.T. Sucrose translocation and storage in the sugar beet.
Plant
Physiology
, v. 63, p. 828
-
832, 1979.
HAAGENSON, D.M.; KLOTZ, K.L.; McGRATH, J.M. Sugarbeet sucrose
synthase genes differ in structure and organ-specific and developmental
expression.
Journal
of Plant Physiology
, v. 163, p. 102
-
106, 2006.
KAYS, S. J. Postharvest physiology of perishable plant products
.
New
York: Van Nostrand Reinhold, 1991. 453 p.
KLOTZ, K.L.; FINGER, F.L
.
Activity and stability of a soluble acid invertase from
sugarbeet roots. Journal of Sugar Beet Research, v. 38, n. 2, p. 121-
138,
2001.
KLOTZ, K. L.; FINGER, F. L. Contribution of invertase and sucrose synthase
isoforms to sucrose catabolism in developing sugarbeet roots. Journal of
Sugar Beet Research
,
v.
39, n.
1-
2, p. 1
-
24,
2002.
KLOTZ, K. L.; FINGER, F. L. Impact of temperature, length of storage and
postharvest disease on sucrose catabolism in sugarbeet. Postharvest Biology
and Technology
, v. 34, p. 1
-
9, 2004.
KLOTZ, K.L.; FINGER, F.L.; SHELVER, W.L. Characterization of two sucrose
synthase isoforms in sugarbeet root. Plant Physiology and Biochemistry, v.
41, p. 107
-
115, 2003.
RIBEIRO, R. A. Conservação pós-colheita e metabolismo de carboidratos
em raízes de dois clones de mandioquinha-salsa (Arracacia xanthorrhiza
Bancroft
).
Viçosa MG: UFV, 2003. 88 p.: il. Tese de Doutorado – Universidade
Federal de Viçosa, 2003.
4
RIBEIRO, R.A.; FINGER, F.L.; PUIATTI, M.; CASALI, V.W.D. Chilling injury
sensitivity in arracacha (Arracacia xanthorrhiza) root
s.
Tropical Science, v. 45,
p. 55
-
57, 2005.
ROSENKRANZ
, H
.;
VOGEL, R
.;
GREINER, S
.;
RAUSCH, T
.
In wounded sugar
beet (Beta vulgaris L.) tap-root, hexose accumulation correlates with the
induction of a vacuolar invertase isoform. Journal of Experimental Botany, v.
52, p. 2381
-
2385, 2001.
SC
ALON, S. P. Q.; HEREDIA, Z. N. A.; VIEIRA, M. C. Conservação pós-
colheita de mandioquinha-salsa em atmosfera modificada.
Horticultura
Brasileira
, v.
16, n.
1, 1998. Resumo 303.
TSIALTAS, J. T.; KARADIMOS, D. A. Leaf carbon isotope discrimination and
its relation with qualitative root traits and harvest index in sugar beet (
Beta
vulgaris
L.),
Journal of
Agronomy & Crop Science
, v. 189, p. 286-
290, 2003.
WILTSHIRE, J.J.J.; COBB, A.H. Bruising of sugar beet roots and consequential
sugar loss: current understanding and research needs. Annals of Applied
Biology
, v. 136, p. 159
-
166, 2000.
WYSE, R. Enzymes involved in the postharvest degradation of sucrose in
Beta
vulgaris
L
. root tissue.
Plant Physiology
, v.
53
, p.
507
-
508
, 1974.
WYSE, R. Sucrose uptake by sugar beet tap root tissue. Plant Physiology, v.
64, p. 837
-841, 1979.
5
CHAPTER 1
IN SITU HYBRIDIZATION OF RIBOPROBES
FOR
TWO SUCROSE
SYNTHASE TRANSCRIPTS ON SUGARBEET ROOT TISSUE
ABSTRACT
Sucrose synthase is an
important
enzyme
on
sucrose metabol
ism in
sugar
beet roots. Its activity rises during root development
and
it
is
correlated
with sucrose accumulation. It is also the main sucrolytic enzyme during the
postharvest storage period. Sucrose synthase has two active isoforms
in
sugarbeet
roots. S
uc
rose synthase
isoforms
are common in many plants and
they show, in general, organ specificity.
Multiple
genes (
isoforms
)
also
can
demonstrate
differential expression
in
tissues and at developmental stages of
the organs. The
goals
of this work were:
to
prod
uce labeled probes with
specificity
for both SBSS1 and SBSS2 sucrose synthase genes; to use
the
probes
for
in situ hybridization procedures; and to study spatial distribution of
sucrose synthase mRNA
in
young
sugarbeet
roots. RNA was isolated
from
roots
, a
nd
RT-PCR with specific primers for each sucrose synthase gene was
performed
to produce DNA probes. DNA
probes
were used in in vitro
transcriptase reactions to produce DIG (digoxigenin) labeled RNA probes in
sense and anti-sense direction for both genes. Specificity of each probe was
verified by hybridization with mRNA of SBSS1 and SBSS2 genes fixed
6
of 1 µg/mL for 25 minutes and 5 µg/mL for 30 minutes, both at 37 ºC,
respectively
to
tissues fixed for 12 and 24 hours with FAA.
Separate
slides with
tissue were used for the hybridization reaction with each labeled probe,
sense
and anti-
sense
.
Hybridizati
on was performed at 55 ºC for 18 hours. Increase of
stringency conditions during hybridization did not
generate
improvement in the
results. After hybridization,
tissue was submitted to high stringenc
y washing and
RNase treatment. Immunohistochemical assay was performed with anti-
DIG
antibody conjugated with alkaline phosphatase. Final color was
developed
by
incubation with NBT/BCIP substrate. High specific RNA labeled probe was
successfully produced and used on in situ hybridization protocol adapted to
suga
rbeet
roots tissue. Transcripts of SBSS1 and SBSS2 genes did not differ in
relation to localization on tissue. They were found mainly
in
storage
parenchyma. No sucrose synthase mRNA was detected in accessory cambium
or in vascular bundles. The results suggest that parenchyma develops a central
role in sucrose storage and
distribution
, and sucrose synthase can be involved
in the regulation of these processes. However, spatial distribution of invertases,
as well as
the
distribution
pattern of sucrose synthase in other developmental
stage
s of the roots should be
further
investigate
d to better understanding
of
the
real r
ole
of sugar metabolism on
sugarbeet
roots.
7
1. INTRODUCTION
1.1
Economical importance
Sugar
beet (Beta vulgaris L.) is an important industrial crop of the
temperate zone, the worldwide production of which exceeded
248
million
ton
nes
in 2004 (FAO, 2004
).
France is the biggest producer of s
ugar
beet in the
world with about
31
million
tones in 2005. However, the biggest planted area
is
found in
The
United State of America
with
more than 501
thousands
of hectare
cultivated
with this crop in 2005 (FAO, 2007).
Sucrose from sugarbeet is an important dietary supplement worldwide.
With production of about
35
,000,
000 tonnes in 2002, just less than
one
-
thir
d of
world sucrose supplies are derived from
the
sugarbeet
. From this, nearly a
half
is
originated from countries in the European Union.
Sugarbeet
production is
distributed across continental regions characterized by temperate climates,
complementing
sugar
cane production in more tropical clime (Weiland
and
Koch, 2004)
.
1.2
Plant
and root a
natomy
Sugar
beet is
an
herbaceous dicotyledon and a member of
Chenopodiaceae
, a family characterized morphologically by inconspicuous,
radially symmetric, petalless flo
wers and non
-
fleshy fruit (Klotz, 2005).
The
sugarbeet
taproot is a vegetative storage organ formed by the main
root and to a smaller extent of the hypocotyl, from which the leaf rosette is
formed. The taproot shows an atypical growth with repeated concentric rings of
cambium producing secondary phloem, xylem and parenchymous cells, leading
to a potentially unlimited growth (Milford, 2006). The alternating concentric rings
surround a central star-shaped core which contains the vascular and ground
tissue th
at developed mainly during root primary growth (Klotz, 2005).
The rings of growth are more or less equidistant except near the
periphery where they are very close together. Often, instead of a complete ring,
smaller or large segments of
the
ring appear here or there being connected by
their margin to the next inner ring.
The b
undles of mature rings are widest in the
8
region of the cambium and taper gradually toward the phloem and xylem
pole
giving to them appearance of a double
wedge (Artschwager, 1926).
Th
e concentric rings of vascular tissue are separated from one another
by broad bands of storage parenchyma. The cells are large and almost
spherical, the
walls
thin
and extensively pitted. The outer and inner peripheries
of this interzonal parenchyma contain vascular elements in addition; the outer
periphery scattered xylem cells, the inner periphery obliterated phloem
(Artschwager, 1926)
The six innermost
growth
rings are particularly active in producing cells
which will expand. This part of the root accounts for 75% of the mature storage
organ (Schneider et al., 1999).
1.3
Sucrose
accumulation
Parenchyma cells are the mains sinks for sucrose storage (Berghall et
al., 1997) which can reach as high as 20% of fresh weigh for mature
sugarbeet
roots (Wyse, 19
79).
Sucrose is synthesized in leaves by the photosynthesis process and the
sucrose accumulation depends of the amount of assimilates produced and
efficiency of transportation and storage of them. The triose phosphates
produc
ed
by
the
Calvin Cycle are used to
generate
either starch in the
chloro
plast or sucrose in the cytosol (Taiz and Zeiger, 2004
).
Then,
sucrose
synthesized
in leaf mesophyll cells is
dif
fus
ed
symplastically towards the
phloem (Williams et al., 2000). However,
sugarbeet
exhibit
s
apoplasti
c phloem
loading
due to the lack of
symplastical
connections between the mesophyll cells
and the conducting complex (Sovonick et al., 1974).
The active loading of sucrose into the phloem mediates the long-
distance
transport in the phloem cells of the
vascu
lar system by a positive hydrostatic
pressure difference between the source and sink tissues that drives mass flow
of solution
(
Taiz and Zeiger, 200
4).
In mature
sugarbeet
taproot, sucrose unloading is apoplastic. The
uptake
and accumulation in the vacuole occurs in majority without hydrolysis (Wyse,
1979)
.
Intact
sugarbeet
plants
can
export
70% of the translocate to the beet,
greater than 90% of which is retained as sucrose with little
subsequent
9
conversions
(Giaquinta, 1979). Wyse (1979) proposed that sucrose enters the
storage parenchyma cell via a nonsaturating site at the plasmalemma before
entering
in
the vacuole. The mechanism of sucrose transport through the
tonoplast
is an alkali cation influx/proton efflux reaction coupled to the active
uptake of s
ucrose (Saft
n
er
and
Wyse, 1980
; Saftner et al., 1983
).
An
alternative hypothesis proposes the
cleavage
of sucrose by cell wall
invertase
during
an
unloading
step either in the apoplast of taproot phloem
elements or in the cytoplasm of adjacent cells. Glucose and fructose resulted of
the sucrose hydrolysis would be then, easily transported across the plasma
membrane by active carrier systems
(Fieuw
s
and Willenbrink, 1990).
1.4
Sucrose degradation
Sucrose provides carbon skeletons for use in biosynthesis, and acts as
respiratory substrate. Plant roots
consume
about 50% of the carbon imported
from photosynthetic tissues to provide adenosine triphosphate (ATP) to drive
energy consuming processes like ion uptake, maintenance and turnover of
existing and biosyn
thesis of new tissues (Farrar, 1985)
Sucrose catabolism is a fundamental metabolic process that is essential
for the growth, development and maintenance of the
sugarbeet
crop. Four
enzyme activities, one sucrose synthase
(UDP
-glucose: D-fructose 2-
glucosy
ltransferase, EC 2.4.1.13) and three invertases (ß-
fructofuranosidase;
EC 3.2.1.2
6)
, soluble acid invertase
,
insoluble acid invertase and alkaline
invertase are responsible for nearly all sucrose catabolism in
sugarbeet
roots
(Klotz
and
Finger, 2001).
Sucr
ose accumulation in
sugarbeet
roots is inversely proportional to
soluble and insoluble acid invertase activities (Klotz
and
Finger, 2002). These
authors
observed
that sucrose accumulation was not evident in the roots of
seedlings when the activities of those enzymes were maximal.
Like
wise
, in
matured plants,
in
which acid invertase activit
ies
had precipitously declined, the
sucrose content of the roots increased.
10
1.5
Sucrose synthase
Sucrose
synthase which catalyses the reversible conversion of sucrose
an
d uridine diphosphate (UDP) into fructose and UDP
-
glucose, is a key enzyme
involved in carbohydrate metabolism (Rouhier
and
Usuda, 2001). This enzyme
has been studied in various plants and
seems
to play a major role in energy
metabolism,
controlling the mobilization of sucrose into various
pathways
important for the metabolic, structural, and storage functions of the plant cell
(Hesse and Willmitzer,
1996
).
Sucrose synthase is
found
in cytoplasmic and a
membrane
associated
form, both creating sucrose gradients by their activity and thus enhancing
sucrose transport (Wittich
and
Willemse, 1999). Cytoplasmic sucrose synthase
supplies UDP-
glucose
and it is
associated
with respiration (Xu et al., 1989),
cell
wall
biosynthesis in
conjunction
with the cellulose sy
nthase
complex (Ruan et
al., 2003) and protein synthesis (Doehlert, 1990). Fu
rther
more,
sucrose
synthase
cleavage activity is related with the sink strength of storage organs,
providing substrates for starch synthesis in rice seeds, carrot taproots,
maize
kernels and
tomato
fruits (Wang et al., 1999; Sturm et al., 1999;
Wittich
and
Vreugdenhil, 1998, Wang et al., 1994
).
The membrane-associated form of
sucrose synthase is involved in the synthesis of cellulose and probably callose
(Amor et al., 1995).
Recentl
y a tonoplast-associated form of sucrose synthase
has
been described in red beet (Etxeberria and
Gonzalez, 2003).
Activity of sucrose synthase during development of
sugarbeet
roots w
as
investigated
by Klotz
and
Finger (
2002
). The enzyme activity was evident at all
stages of
sugarbeet
root development and was
the
predominant
sucroly
tic
activity at all but the earliest stages of growth. A high correlation between
sucrose synthase activity and
accumulation
of
mass
,
size
and sucrose also was
observed.
Similar
results were seen by Giaquinta (1979) who proposed that
sucrose synthase
plays
an
important role in regulating the partitioning of
translocate storage and utilization.
In
sugarbeet
root, two sucrose synthase isoform, Susy I and Susy II,
were identified showing differences in physical and biochem
ical
properties and
in
developmental patterns of expression (Klotz
and
Finger, 2002; Klotz et al.,
2003). Multiple isoforms can provide to
sugarbeet
roots more flexibility in
11
regulating sucrose metabolism to changes
in
development,
and metabolic or
environmental conditions by differential regulation of expression of the two
isoforms and by modulation of their activities by changes in cellular pH
(K
lotz et
al., 2003).
Two
full
length
cDNAs of the sucrose synthase isoforms were
described
and
designated as SBSS1 and SBSS2
in
Genebank. SBSS1 has predominant
mRNA expression
in
sugarbeet
taproots. Expression also was induced by cold
and anaerobiosis conditions, whereas wounding of taproot slices leads to
a
repression (Hesse
and
Willmitzer, 1996). In the same manner, SBSS2 is
expressed mainly in root tissues. In comparison with SBSS1, SBSS2 seems to
be more strongly expressed in early stages of taproot development (Haagenson
et al., 2006)
.
These authors observed that the changes in transcript level
reflected on the protein level with significant delay,
indicating
post-
transcriptional regulatory mechanism
s.
Based on enzyme
activity
verified on above mentioned works
,
sucrose
synthase
appears to be the major enzyme of the sucrose catabolism in
sugarbeet
roots. The expression pattern of the two enzyme isoforms
demonstrates
differentiated
accumulation of transcripts during beetroot
development.
However, little is known about
the
spatial expression of these
genes in root tissues.
1.
6
Non
-
radioactive
i
n situ
hybridization considerations
In situ hybridization (ISH) is a technique that allows for precise
localization of a specific segment of nucleic acid within a histologic section
(Silva
-
Valenzuela et al., 2006)
. Successful utilizati
on of the technique requires a
basic knowledge of molecular biology in combination with an ability to
appreciate subtle hi
stomorphologic
changes.
Furthermore
, it represents a
perfect synergy between fundamental molecular biology and traditional
histologica
l interpretation (Brown, 1998).
ISH techniques in plants are used as a diagnostic tool to detect plant
pathogens,
to
analyze
genomic organization of plant genes and to
analyse
s
of
tissue expression patterns of mRNA. The great advance of ISH over other
con
ventional nucleic acid detection techniques like Northern and Southern blot
12
hybridization is the possibility to detect the precise spatial and temporal
expression of mRNA (Zachgo, 2002).
The ISH method consists in annealing specific labeled DNA or RNA
prob
es, complementary to target sequences of the fixed tissue, followed
visualization of the probe location (Urbanczyk
-
Wochniak
et al.
, 2002).
In general, the
ISH
technique
has
five
steps:
tissue preparation,
tissue
pretreatment
, hybridization, p
ost
-
hybridizat
ion and detection. Production of
labeled probes is not a direct step of the ISH
,
but it is essential for application of
this technique.
Choosing
appropriate probes
directly
affects specif
icity
and quality of
hybridization as well as
the
final result
.
Some
issues are very important to
consider before
desig
n of
the probe
,
because they will influence
the
efficiency of
hybridization, such as: probe sequence (
percent
of homology with the template
and percent of CG base pairs
),
probe
length
, kind of probe (RNA, DNA or
oligonucleotides
) and kind of labeled agent. Furthermore, the concentration of
probe into hybridization solution also affects the hybridization reaction (Kenny-
Moynihan
and
Unger, 2002).
Tissue preparation step
s
include
fixation of tissue, embe
d
ding
of tissue in
paraffin, section
ing
of paraffin-tissue blocks and fixation of tissues on slide.
Th
ese steps interfere directly on
the
quality of results which are directly
dependent
on
nucleotide fixation in situ and morphological conservation of the
tissue
(Nardone, 1999)
Pretreatment of tissue after fixation-paraffinization procedures is a
needful issue to get success
with
ISH. Pretreatment includes two main
procedures: protease and acetylation treatments. The first consist of treatment
of tissue with proteinase K, to remove excess of
protein
linked
to
nucleotides
after
the
fixation step, and to facilitate probe access to the target nucleotides
.
Acety
lation
aims
to transform the reactive amino group
to
a neutral amino group
by reaction with acetic anhydride. It reduces non-specific binding of negatively
charged probes due to electrostatic forces (Morel
and
Cavalier, 2001).
The hybridization step consists
of
incubation
of
the slide, during a
specific time, with
an
appropriate solution containing probes which w
ill
anneal
with
the
target template forming a duplex. Many parameters can be used to
control and optimize the hybridization, such as: temperature, formamide
13
concentration, monovalent cation concentrations
,
inclusion of dextran sulphate
and blocking reagent
s, and pH (Zachgo, 2002).
During th936 Tm(r)Tj0.09187 0 0 -0.09187 2840 182Tj( )Tj0.09187 0 0 -0.09187 310oDa
20 1612 Tm100.04 Tz(s, and pH (844 3211 1936-Tm(20 1612 Tm100.04 Tz(s, and pH (90-0.09187 3/F7 2048 Tfhybridization48 Tf0.09187 0 0 -0.09187 25n)Tj0.09187 0 0 -0.09132187 5126 3211 1936 Tm(h936 Tm(r)Tj0.091875187 809187 310oD)Te)Tj0.09187 0 0 -0.09187 2986 1936p7 5976 1287 Tm(s)Tj/F7 2048 Tf0.09138-0.09187 31,7 5976 1287 Tm(187 2537 1936 Tm(D)Tj0.09187 0 0 -0.0918-0.09187 310 r30.091wash0 0 7 6195 1287 Tm100 Tz(i)Tj0.09187 0 2650.09187 3/F70.091 Tm(n)Tj( )Tj0.09187 0 0370 -0.09187 8 1287 Tm(d)Tj0.09187 0 0 -0.06490 3211 1936 Tm(h936 Tm(r)Tj0.091876542 -0.09187 2840 182Tj( )Tj0.09187 0664-0.09187 310oD)T76 1287 Tm(187 25n)Tj( )Tj0.09187 0 0 -0 -0.09187 2840 182Tj( )Tj0.09187 06926 3211 1936yTm(s)Tj0.09187 0 0 -0.09019187 bTm(f)Tj( )Tj0.09187 0 0 23 2840 1936 Tm(i)Tj0.09187 0 0 -0.071-0.09187 315iTm(d)Tj0.09187 0 0 -0.09187 79187 315 Tm(e)Tj0.09187 0 0 -0.09187 79187 315iTm(d)Tj0.09187 0 0 -0.093750.09187 3(zTm(x)Tj0.09187 0 0 -0.09167 809187 310oD)Te)Tj0.09187 0 0 -0.07587 2777 1936d 1287 Tm(s)Tj0.09187 0 0 -0.09746187 7187 2882 19360oD)TecTm(u)Tj0.09187 0 0 -0.09137 3211 1936 Tm(h936 Tm(r)Tj0.091878097 2882 1936 Tm(n)Tj0.09187 0 0 -0.0813Tj/F7 2048 Tf0.09187 010oD 1281 0 -8287 2673 1936nTm(a)Tj0.09187 0 0 -0.09142187 7 Tm(s)Tj0.09187 0 0 -0.08918 2882 1936 Tm(n)Tj0.09187 0 0 -0.0854-0.09187 31s0.09187 1972 1612 Tm100.07 Tz(and blocking rea2261 1287 Tm(e)Tj0.09187 0 0 -0.09187 2261 1287aTm(n)Tj0.09187 0 0 -0.0918ea2261 1287 Tm(i)Tj0.09187 0 0 -0.09234a2261 1287 Tm(i)Tj0.09187 0 0 -0.09297a2261 1287 Tm(n)Tj0.09187 0 0 -0.09338a2261 1287 Tm(n)Tj0.09187 0 0 -0.09187 2261 1287d 1287 Tm(s)Tj0.09187 0 0 -0.02649 2261 1287 Tm(n)Tj0.09187 0 0 -0.09918 2261 1287uTm(i)Tj0.09187 0 0 -0.09188a2261 12871 1287 Tm(c)Tj0.09187 0 0 -0.03010a2261 12871 128c)Tj0.09187 0 0 -0.03062 2261 1287 Tm(7 Tm(m)Tj0.09187 0 0 -0.09168a2261 1287 Tm(i)Tj0.09187 0 0 -0.01871a2261 1287 Tm(n)Tj0.09187 0 0 -0.03187 2261 1287m0 0 -0.09187 2799 1287 Tent)1 2261 1287 Tm(n)Tj0.09187 0 0 -0.00.07a2261 1287 Tm(a)Tj0.09187 0 0 -0.09180a2261 1287 Tm(187 1972 1612 Tm100.07 Tz(and blockin3997 2261 12810 8ea.091the87 1972 1612 Tm100.07 Tz(and blockin1877a2261 128/F70.091pTm(t)Tj0.09187 0 0 -0.09163a2261 1287 Tm(i)Tj0.09187 0 0 -0.04525 2261 1287 Tm(n)Tj0.09187 0 0 -0.01630a2261 1287bTm(f)Tj( )Tj0.09187 0 4734a2261 12870oD)Tj0.09187 0 0 -0.09187 267m9187 2261 12871 128c)Tj0.09187 0 0 -0.04992 2261 1287h7 25n)Tj0.09187 0 0 -0.09107a2261 1287 Tm(t)Tj0.09187 0 0 -0.09202a2261 12871 1287 Tm(c)Tj0.09187 0 0 -0.00.4 2261 1287h7 25n)Tj0.09187 0 0 -0.09459a2261 1287 Tm(t)Tj0.09187 0 0 -0.09187 2261 12877 Tm(s)Tj0.09187 0 0 -0.05788a2261 1287bTm(n)Tj/F7 2048 Tf0.091862 2261 1287 Tm(s)Tj/F7 2048 Tf0.091868a2261 1287 Tm(s)Tj0.09187 0 0 -0.0908ea2261 1287 Tm(c)Tj0.09187 0 0 -0.09177 2261 1287d 1287 Tm(s)Tj0.09187 0 0 -0.06187 2261 12871 128c
7 Tm(m)Tj0.09187 0 0 -0.06640a2261 1287 Tm(i)Tj0.09187 0 0 -0.06703a2261 1287 Tm(n)Tj0.09187 0 0 -0.06807 2261 1287 Tm(u)Tj0.09187 0 0 -0.09918a2261 1287 Tm(t)Tj0.09187 0 0 -0.069.4 2261 1287 Tm(r)Tj0.09187 0 0 -0.09006a2261 1287 Tm(n)Tj0.09187 0 0 -0.07111 2261 1287d 1287 Tm(s)Tj0.09187 0 0 -0.070 -02261 1287x
14
1.7
O
bjective
The
work is part of a big
ger
project developed in the
Sugar
b
eet
Laboratory NCSL - USDA to understand the processes that regulate the sugar
metabolism
of
sugarbeet
roots. The aims of the present study were:
- To produce probes for both SBSS1 and SBSS2 isoforms of sucrose
synthas
e from
sugarbeet
roots and to verify the specificity of each probe
by
dot blot hybridization
assays
.
- To adapt procedures of in situ hybridization to
sugarbeet
root tissue and
to evaluate, using this technique, the spatial distribution of the transcripts
of
two sucrose synthase isoforms in
immature
roots of
sugar
beet
.
15
2.
MATERIAL
S
AND METHODS
2.1
Precautions
to work
with RNA
To avoid RNA degradation by RNase contaminat
ion
, all materials were
kept RNase free following procedures suggested by Sambrook
and
Russel
(200
1). Gloves were used during all procedures. Solutions were prepared
with
diethyl pyrocarbonate (DEPC) treated water.
For
DEPC treatment, water was
mix
ed
with 0.1% DEPC
,
incubated overnight
at 37
o
C
and
autoclaved.
Reagents and plasticwares were from fresh packages set aside for RNA
work.
Glas
sware,
spatulas and stir bars were
decontaminated
by baking
overnight at 180
o
C
.
Gel boxes, comb and old glassware were washed either in
0.1 M NaOH, 1mM EDTA pH 8.0 or in 3 % hydrogen peroxide for 10 minutes, at
room temperature,
and finally rinsed with 3 washes
of DEPC treated water.
2.2
Probe p
reparation
2.2.1
RNA e
xtraction
RNA was isol
ate
d from 9 week old VanDerHave hybrid H66156
sugarbeet
roots by washing and slicing the roots into 1
cm
cubes and flash
freezing
in liquid nitrogen. The frozen root pieces were lyophilized in a Virtis
XL35 freezemobile freeze dryer. After this, the RNA was isolated by single-
step
methodology using Trizol reagent (Invitrogen) in agreement with Simms et al.
(1993)
with modifications
.
Therefore, 0.5 g of
sugarbeet
root powder wa
s
placed
in
a 50
mL
centrifuge tube with 5
mL
Trizol and ground with a PCU11 Polytron
for two 15 seconds bursts. The tubes
were
kept on ice for 5 minutes, then spu
n
at 10,000 x g for 10 minutes. The supernatant was transferred t
o
a clean
centrifuge tube and 2
mL
of chloroform was added. The mixture was vortex
ed
and placed on ice for 10 minutes, then centrifuged at 10,000 x g for 10 minutes.
The upper
colorless
aqueous
phase
was removed and added to 2
mL
isopropanol
and 2
mL
0.8 M Na citrate, 1.2 M NaCl
solu
tion
, vortex
ed and
left at
room temperature for 10 minutes. Then it was centrifuged
once
more
at 10,000
x
g for 10 minutes. Subsequently the supernatant was decanted and the tube
inverted for 5 minutes. The remain
ing
pellet was washed with 75% ethanol,
16
mix
ed and centrifuged for 10 minutes. The supernatant was
discarded
and the
pellet was air dried. The RNA was suspended in DEPC treated water. To
check
the RNA concentration and purity, the absorbance of the solution
was
mensured
at 260 and 280
nm in
a
NanoDr
op
®
ND
-1000 Spectrophotometer.
2.2.2
Probe production by RT
-
PCR
The RT
-
PCR reaction was performed with
RNA extracted from
sugarbeet
tissue using the AccuScript
TM
High Fidelity RT-PCR System (Stratagene) as
directed by manufacturer.
2.2.2.1
First strand
cDNA s
ynthesis
The cDNA was synthesized from total RNA in a reaction primed with
oligo(dT)
using AccuScript RT. Each reaction was prepared
initially
by adding,
in order, to one microcentrifuge tube:
30
µL
RN
ase free water, 5 µL
10X
AccuScript RT buffer, 3 µL oligo(dT) primer (100 ng/
µL
),
5
µL
10 mM dNTP mix
and 1 µL RNA (673 ng/µL isolated from
sugarbeet
roots). This solution was
incubated at 65
o
C for 5 minutes and cooled at room temperature
.
Then
,
5
µl
of
100 mM DTT and 1
µL
AccuScript RT
was added
to ea
ch reaction
tube
, and the
tubes were placed in a temperature-controlled thermal block at 42
o
C for 30
minutes.
Completed reaction, with first-strand cDNA synthesized, was kept on
ice until PCR amplification reaction. The control reaction followed the same
procedures
substituting
the
sugarbeet
RNA
with
mRNA (50 ng/
µL
) from
the
kit.
2.2.2.2
Primer design
The primers used in the cDNA
template
amplification
aimed
to get
specific probes to SBSS1 and SBSS2 genes. Because they have very similar
sequences,
it was necessary to choose primers
which
would allow the
amplific
ation of sequences unable to
annealing with the other
non
-
target gene.
The complete cDNA sequences of SBSS1 and SBSS2 genes,
respectively access numbers
X81974
and
AY457173
, were
obtained
from
GeneBank from
the
National Center of
Biotechnology
Information – NCBI.
SBSS1 sequence
was
not
a
complete
sequence, then it was compared with
17
complete SBSS1 cDNA from two clones (3C and 3D)
isolated
at Sugar
beet
Laboratory (NCSL
–
USDA) which have exactly the same sequence.
The sequences of SBSS1 and SBSS2 cDNA were co
mpared
using
the
sequence alignment
function
(AlignX)
from
Vector NTI
10
(I
nvitrogen
). The low
conserved sequence region was
chosen
for
probe production. Then the PCR
primers
were selected at low conserved region between SBSS1 and SBSS2
cDNAs to get specific probes. The primers were
constructed
with
the
help of
a
large set of parameters (
Gorelenkov
et al., 2001) included in the PCR prime
design
function
of Vector NTI
10
program
.
For
each probe (SBSS1 and
SBSS2)
,
two different
primers were obtained:
Probe
1
–
F1: AATGACATTGGCAGGGGTGT
R1: CGGAATGTCGCAACTGCATC
Probe
2
–
F2: AGGGAGGATTGCAACGAATT
R2: TGCGTCCTCGATAGCCAAAG
2.2.2.3
cDNA amplification by PCR
The
probes were produced by
a
cDNA first strand amplification. Each
reaction had 40
µL
RNase free water, 5
µL
10X PCR Buffer (Stratagene kit), 1
µL
10 mM dNTP mix, 1
µL
of each primer (
10 µM
)
for
each probe reaction, 1
µL
first strand cDNA reaction solution and 1
µL
Pfu Ultra HF DNA polymerase (2.5
U/
µL
). A control reaction was run in the same conditions using control primer
and control first strand cDNA
from
the
kit
.
The amplification reactions were performed in 8 strip thin walled PCR
tubes, 0.2
mL
capacity, in a MJ Research PTC 200 thermocycler. The
cycle’s
steps were: initial denaturation step
at
95
o
C for 1 minute; 35 cycles composed
of
: one denaturation step at 95
o
C for 30 seconds, one template-
primer
annealing
step
at 60
o
C for 30 seconds and one extension step at 68
o
C for 6
minutes;
and one last extra extension step at 68
o
C for 10 minutes. After PCR
procedures
were finished,
the reaction tubes were kept at 4
o
C.
18
2.2.3
Extraction and purification of PCR probe products
The probes produced
by
PCR reaction were isolated through
electrophoretic
purification
in
1 % Seakem
LE
agarose gel in TBE buffer (100
mM
Tris base,
100 mM Boric acid and 20 mm EDTA, pH 8.3)
with 1
µL
ethidium
bromide (10 mg/
mL
).
Wells
were loaded with PCR product and Loading B
uffer
(5% glycerol, 10 mM EDTA, 0.1% sodium dodecyl sulfate and 0.01%
bromophenol blue). One molecular weight marker, Hyperladder I (Bioline) was
used to permit size identification of the gel bands. Electrophoresis was
performed
at 120 volts, constant voltage, for 1 hour. A FotoPrep Ultraviolet
transilluminator was used to permit visualization of probe bands in the agarose
gel.
Parts
19
Yeast Extract
,
10 mM NaCl
,
2.5 mM KCl
,
10 mM MgCl
2
,
10 mM MgSO
4
and
20
mM glucose
)
was
added
to the tubes
with cell
s s
olutions and agitated
for 1 hour
at 200 rpm. After this, 10 to 50
µL
of cells in SOC media were spread on LB
agar plate [
Luri
a Bertani pH 7.0 containing 100 µg/
mL
ampicillin and 26 µg/
mL
5-
bromo
-4-
chloro
-3-
indolyl
-ß-D-galactoside (X-
gal)]
and incubated overnight at
37
o
C.
The white colonies
grown
on LB agar plates
were
isolated,
inoculated
in
10
mL
LB broth media containing 100 µg/
mL
ampicillin and incubated at 37
o
C
overnight in agitation at 200 rpm in a Brinkman G24 shaker.
2.2.5
Plasmid isolation
Plasm
id isolation was performed with QIAprep Spin Miniprep kit
(QIAGEN) following manufacturer instructions.
Purified pCRII-
TOPO
plasmids
containing probes were kept in sterilized
water at 4
o
C.
Plasmid concentration and purity was measured by absorbance
of the
solution at 260 and 280 nm in
NanoDrop
®
ND
-
1000 Spectrophotometer
.
2.2.6
Verification of plasmid
-
probe quality and sequencing
The efficiency of the cloning and transformation steps were tested with
restriction enzyme
reactions.
Recombinant plasmids were submitted to restriction reaction assay, with
E
co
RI enzyme (supplied together with specific buffer by New England Biolabs)
.
This enzyme was used
,
because
the pCRII
-
TOPO plasmid
has two restrictions
sites flanking the multiple cloning site. The assay con
tained
N
E
Eco
R
I
Buffer
(50 mM NaCl, 100 mM Tris-HCl pH 7.5, 10 mM MgCl
2
, 0.025% Triton X – 100),
recombinant plasmid with SBSS1 or SBSS2 probes (1 µg), and
Eco
R
I
(20 U) in
30 µL of total volume
and digestion was
carried out
at 37
o
C overnight.
The products of enzyme restriction assay
were
electrophoresed in an
agarose gel as described in the item 2.2.
4.
Plasmids containing probes at
a
concentration of
0.1
µg/µL were sent to
Northwoods DNA for sequencing.
20
2.3
Labeled
RNA probe
synthesis
RNA probe was label during its production by in vitro
transcription
reaction using nucleotides label
ed
with d
igoxige
nnin (DIG). Recom
binant
plasmids were linearized to permit
RNA
synthesized
by RNA
polymerase
to
terminate at the end of the gene. I
t
was necessary
to
produce
labeled
probes in
anti
-sense and sense (control) direction.
This
was
possible because pCRII –
TOPO
vector contains two RNA polymerase promoter (T7 and SP6) sites,
one
at each end
of
the
multiple cloning site
.
Two different restriction enzymes,
Hind
III and
Xho
I,
were used
for
plasmid linearization. Then anti-
sense
and
sense
labeled
probes were obtained
respectively
with
T7 RNA polymerase and
SP6 RNA polymerase (Figure 1).
2.3.1
Plasmid l
inearization
Linearization of recombinant plasmid,
aimed
at
anti
-
sense
labeled
probe
production, was performed in
an
assay containing NE Buffer 2 (50 mM NaCl, 10
mM Tris-HCl pH 7.9, 10 mM MgCl
2
, 1 mM dithiothreitol), recombinant plasmid
with SBSS1 or SBSS2 probes (4
µg) and
Hind
III (40 U) in 50 µL of total volume.
Reaction was carried out at 37
o
C overnight.
The same reaction conditions were
used
for
sense
labeled
probe, changing restriction enzyme to
Xho
I (both of
enzymes were supplied together with NE Buffer 2 by New England Biolabs).
Bot
h of restrictions enzymes,
Hind
III and
Xho
I, were used to generate 5’ sticky
ends after cleavage of plasmid. This is important to avoid production of artifacts
during RNA synthesis when cleavage result in either 3’ stick ends or blunt ends
(Zachgo, 2002; Melton et al., 1984).
21
Anti
-sense direction
Sense direction
DNA Probe
Anti
-sense direction
Sense direction
DNA Probe
Figure
1 – Scheme of the circularized pCRII-TOPO vector highlighting adjacent
regions of multiple cloning site, where DNA probe fragment was inserted.
Yellow box indicates T7 promoter sequences. Yellow arrows indicate T7 RNA
polymerase direction until e
ig
22
2.3.3
In
vitro
t
ranscription
After linearization of the template DNA (plasmid) at
a
suitable site, the
RNA polymerases were used to produce “run off” transcripts. DIG-UTP was
used as a substrate and incorporated into the transcript. Every 20 – 25
th
nucleotide
of the newly synthesized RNA was a DIG-UTP. Since the nucleot
ide
concentration did not become limiting in the standard transcription assay, a
large amount of labeled RNA was generated.
The transcription assay was performed in vitro with DIG RNA Labeling
Kit (Roche Diagnostics GmbH). As described by the manufacturer
DNA
templat
e (1µg), NTP labeling mixture (1 mM ATP, 1 mM CTP, 1mM GTP, 0.65
mM UTP and 0.35 mM DIG-
11
-UTP), transcription buffer, RNase inhibitor (20
U)
and SP6 RNA polymerase (40 U) or T7 RNA polymerase (40 U) were mixed
in 20 µL of total volume and incubated for 2 hours at 37 ºC. After this time
,
DNase I (40 U) was added and incubated for 15 minutes at the same
temperature, to remove DNA template. Reaction was stopped with addition of 2
µL 0.2
M EDTA (pH 8.0).
2.4
Labeled probe purification
The purification of labeled RNA probe was executed
with
E.Z.N.A.
®
RNA
Probe Purification Kit (Omega Bio-tek, Inc.) following manufacturer
’s
protocol.
This kit contained
HiBind
®
spin cartridges and optimized buffers
that
allowe
d
facilitated
isolation of pure RNA from nucleotides, unincorporated label,
enzymes, and salts. Labeled RNA was eluted with 30 µL DEPC treated water
and stored
at
-
80 ºC.
2.5
Estimati
on of the labeled probe
concentration
The
estimation of the labeled RNA probe
concentration
was
done
with
electrophoretic
running in 2% Seakem LE agarose gel
dissol
ved in TBE buffer
(100 mM Tris base, 100 mM Boric acid and 20 mm EDTA, pH 8.3) with 1 µL of
ethidium bromide (10 mg/
mL
).
The g
el’s
well
s were loaded with labeled RNA
sample
and
RNA Gel Loading Buffer II (95% formamide, 0.025% xylene cyanol,
0.025% SDS, 0.025% bromophenol blue, 18 mM EDTA; Ambion) previously
heated for 2 minutes at 95 ºC and kept on ice. Labeled control
RNA
(from DIG
23
RNA labeling Kit) 100 ng/µL in DEPC treated water was used
as
a
concentration
standard
.
Electrophoresis was performed at 100 volts, constant
voltage, for 1 hour and 10 minutes. A FotoPrep Ultraviolet transilluminator was
used to permit visualization of probe bands in the agarose gel.
2.6
RNA dot blot hybridization
Dot blot h
ybridization
was performed to verify the specificity of the
labele
d RNA probes. The mRNA from SBSS1 and SBSS2 genes were
transferred and
fixed
on to nylon membranes as
a
dot blot.
Membranes
containing both SBSS1 and SBSS2 mRNAs into different dots were hybridized
separately
with each labeled RNA probe.
After
membrane
blocking
, alkaline
phosphatase
(AP)
conjugate
d
with
anti-dioxigenin antibody
was
joined
with
the
Hybridized
DIG-labeled RNA probes. Then, DIG-labeled RNA linked to
antibody
-AP was visualized with chromogenic AP substrates which produced a
colored
signal directly on the membrane (Figure 2).
Figure 2 - Diagram showing an overview of the RNA dot blot hybridization
reaction.
24
Pre
-
hybridization,
hybridization and
chrom
ogenic detection steps were
ad
apted from Eisel et al. (
2000
). These steps wer
e
performed with DIG Easy
Hyb solution (Roche Diagnostics GmbH), Washing buffer, Maleic acid buffer,
Blocking solution and Detection buffer from DIG Wash and Block Buffer Set
(Roche
Diagnostics GmbH
).
The Saline-Sodium Citrate (SSC) buffer was
prepared 20 fold concentrated (3 M NaCl, 0.3 M C
6
H
5
Na
3
O
7
) and used at
different dilutions during pre
-
hybridization and hybridization steps
2.6.1
Non
-
labeled RNA preparation
To generate non
labeled
mRNA for dot blot experiments, SBSS1 and
SBSS2 cDNA previously cloned in the Sugarbeet Laboratory (NCSL –
USDA)
respectively into pCRII
- TOPO and pBlueScript SK
vectors
were used
.
During dot blot hybridization only complementary sequences form
annealing
pairs and consequently produce
color
on
membrane
at
the
detection
step
.
T
hroughout
in vitro transcription reaction aimed at producing
labeled
probe
s, part of
the
plasmid is tra
25
The assay contained NE EcoR I Buffer (50 mM NaCl, 100 mM Tris-
HCl
pH 7.5, 10 mM MgCl
2
, 0.025% Triton X – 100), BSA (0.1
µg/µ
L
),
recombinant
pCRII
– TOPO containing SBSS1 cDNA (
7.5
µg)
or pLITMUS 38i (5 µg), EcoR I
(
60
U) and BamH I (60 U) in 50 µL of total volume. Reaction was carried out at
37
o
C overnight.
Double restriction reaction produced
a linearized pLITMUS 38i with
sticky
ends (two different overhangs). In the recombinant pCRII - TOPO the double
cleavage resulted in 4 bigger fragments: 3 fragments from SBSS1 cDNA
(274,
1181 and 1114
bp
) and 1 from plasmid (3955
bp
)
(
Figure 3
).
Figure 3
–
Scheme of linear
ized recombinant pCRII
–
TOPO
with SBSS1 cDNA
showing the fragments originated from double restriction reaction with both
Eco
RI and
Bam
HI
enzymes
. The 274 bp fragment of the 3’ end cDNA region
highlighting probe region is showed.
The
274 pb cDNA SBSS1 fragment and linearized pLITMUS 38i were
isolated by agarose gel
as
describe
d
in the item 2.3 and purified using the same
methodology of the item 3.2. The 274 bp fragment was chosen because it was
homologous to the labeled
RNA probe
.
2.
6.1.1.2
Fragment clo
ning and cell transformation
The
274 bp fragment was cloned in only one direction into pLITMUS 38i
because both of them were cleaved with
Eco
RI and
Bam
HI
generating
complementary
cohesive
ends.
Probe Region (174 pb)
1181 pb
274 pb
1014 pb
3955 pb
pCRII
-
TOPO
Probe Region (174 pb)
1181 pb
274 pb
1014 pb
3955 pb
pCRII
-
TOPO
Probe Region (174 pb)
1181 pb
274 pb
1014 pb
3955 pb
pCRII
-
TOPO
26
Ligation reaction assay was performed with T4 DNA Ligase Buffer (50
mM Tris-
HCl
pH 7.5, 10 mM MgCl
2
, 10 mM dithiothreitol, 1 mM ATP, 0.025
µg/µL of BSA), SBSS1 cDNA fragment (27.4 ng), pLITMUS 38i (70 ng) and T4
DNA ligase in 20 µL of total volume. Reaction was carried out at 25
o
C for 10
minutes.
The cell transformation was effected with One Shot TOP10 Competent
Cells (Invitrogen) like described in the item 2.4.
Plasmid isolation was
done
using
methodology
described on section 2.5.
2.
6.1.2 Plasmid
linearization
Linearization of recombinant pBluescript
(contai
ning SBSS2 cDNA) was
carried out
in reaction containing NE Buffer 2 (50 mM NaCl, 10 mM Tris-
HCl pH
7.9, 10 mM MgCl
2
, 1 mM dithiothreitol), recombinant plasmid (4 µg) and
Hind
III
(40 U). Linearization of recombinant pLITMUS 38i was done in reaction
containing
NE Buffer 3 (100 mM NaCl, 50 mM Tris-HCl pH 7.9, 10 mM MgCl
2
, 1
mM dithiothreitol), BSA (0.1 µg/µL), recombinan
t
plasmid (4 µg) and
Sal
I (40 U).
Both assays were performed at 37 ºC for 1 hour.
Linearized
plasmids were purified
as described
on
section
3.2.
2.
6.1.3
In vitro
transcription of non
-
labeled RNA
After
the
cleavage (linearization) of both pBluescript (
con
taining
SBSS2
cDNA) and pLITMUS 38i (containing SBSS1 cDNA end fragment) at
appropriate
site, the RNA polymerases were used to produce “run off”
transcripts.
With the recombinant pBluescript T7 RNA polymerase was used
and
with
the
recombinant pLITMUS 38i T
3 RNA
polymerase
was used
.
The transcription reaction was assembled at room temperature using
reagents of the
MAXIscript
®
T7/T3 Kit
(Ambion
). As described by the
manufacturer DNA template (1
µg),
transcription buffer, 0.5 mM ATP, 0.5 mM
CTP, 0.5 mM GTP, 0.5 mM UTP,
and
T3
RNA polymerase (60 U) or T7 RNA
polymerase (
30
U), in 20 µL total volume
were
then
incubated for 1 hour at 37
ºC. After this time, TURBO DNase (2 U) was added and incubated for 15
minutes at the same temperature,
aiming
to remove DNA template. Reaction
was stopped
by adding
of 2 µL 0.2 M EDTA (pH 8.0).
27
2.6.1.4
Non
-
labeled RNA purification
The n
on
-
labeled RNA from in vitro transcription reaction was purified with
MEGAclear
TM
Kit (Ambion) following the manufacture
r’s
instructions. The RNA
was eluted in 100
µL
DEPC
treated water.
RNA was concentrated by addition of 1:10 volumes of 5 M ammonium
a
cetate
(pH 4.8)
and 2.5 volumes of 100% ethanol. This solution was mixed and
incubate
d at -20 ºC for 30 minutes. Then, it was microcentrifuged at 14000 rpm
for 15 minutes at 4 ºC. The supernatant was discarded and the pellet washed
with 70% cold ethanol. The solution was microcentrifuged again an
d
the ethanol
was removed. The pellet was air dried and r
esuspend
ed in 20 µL DEPC treated
water.
Non
labeled RNA was quantified by spectrophotometry and its quality
was verified by agarose gel.
2.6.2
Blotting RNA in the membrane
For the nonlabeled RNA blotting, positively charged Nylon membranes
(Roche
Diagnostics GmbH) positively charged with high
binding
capacity for
RNA
were used
.
Membranes (5 x 7 cm) were pre-
treated
with
100% ethanol with shaking
,
DEPC
treated water and 10 x SSC B
uffer
respectively for 5, 10 and 10 minutes
at room temperature. After this,
the
wet
membrane
was placed on Whatman
paper 3 previously soaked in 2 x SSC
B
uffer
.
The transcribed SBSS1 and SBSS2 mRNAs, previously boiled for 5
minutes and kept on ice, were blotted
at
different locations on the same wet
membrane.
While the membrane was stil
l
d
amp
, t
he
mRNAs were fixed by UV
crossl
inking with one
Stratal
inker
®
2400 UV Crosslinker (Stratagene)
,
through
exposure
of
the membrane (RNA side f
acing up) to UV light.
2.6.3
Pre
-h
ybridization procedures
The labeled SBSS1 and SBSS2 RNA probes were separately boiled for
5
minutes
and kept on ice. After t
r .09187 0 0 -0.09187 3918 11673 Tm(i)Tj0.0 0 -0.0918l49187 3313 96170.09187 7116 11348 Tm(p)Tj0.09187 0 0 -0a5
28
concentration)
.
At the same time, the blots (membrane with fixed mRNAs) w
ere
placed in
different
hybridization tubes with pre-warmed DIG Easy Hyb s
olution
at 68 ºC for 30 minutes
with
shaking
.
2.6.4
Hybridization of the labeled RNA probe
to RNA on the blot
The DIG Easy Hyb solution in the blot was poured out of the
hybridization tubes and they
were
replaced with pre-warmed hybridization
solution. Each
membrane
in
the hybridization solution
was
incubated at 68 ºC
for 17 hours. After this, the blots were
incubated
twice
in
low s
tringency
b
uffer
(2 x SSC, 0.1% SDS), 5 minutes each time, at room temperature in agitation.
Then
the membranes were
incubated
twice
more
in
high stringency b
uffer (0.1 x
SSC, 0.1 % SDS), 15 minutes each time, at 68
ºC with shaking
.
2.6.5
Chromogenic detection of the probe on the blot
All the procedures for chromogenic detection steps were performed at
room temperature.
The membranes were transferred to plastic container containing wa
shing
buffer (
50
mL) and
washed
in agitation for 2 minutes. Then, blots were
incubated in blocking solution (30 mL) for 45 minutes under agitation. After this,
membrane was submitted to immunological detection, by incubation in 20 mL of
antibody solution (Anti-
DIG
-Alcaline phosphatase 1:5000 in blocking solution)
for 30 minutes with shaking.
Following,
membranes were washed twice with
washing buffer (50 mL) 15 minutes each time, and equilibrated 3 minutes with
detection b
uffer.
For the
color reaction
step,
NTB/BCIP stock solution (
500 µL), containing
18.75 mg/mL Nitro blue tetrazolium chloride and 9.4 mg/mL 5-b
romo
-4-
chloro
-
3-indolyl phosphate
,
was
mixed with detection buffer (25 mL) to produce the
color substrate solution. The blots w
ere
incubated with color s
ubstrate
solution
until
colo
r formation (1 hour) in the dark without shaking. After complete color
development, reaction was stopped by washing the membranes twice with
water
, 10 minute each time, with shaking. Finally, the membranes were air
dried.
29
2.7
In
situ
hybridization procedu
res
All the methodology
used
to perform the in situ hybridization, in
sugarbeet fixed tissue, was applied
with
adaptation of various protocols (
Roche
Applied Science, 2006; Balasubramanian et al. 2005; Zachgo, 2002; Shu et al.
,
1999
;
Schneitz et al., 199
8
; Jackson, 199
1).
2.
7.1 Tissue preparation
2.7.1.1
Fixation of the tissue
Roots
of
6 week old sugarbeet h
ybrid
, Beta 6225,
sugarbeet
were
harvested, washed and sliced
for
tissue extraction. Root was sectioned at
middle
just below hypocotyl region (
abo
ut 15 - 20 mm
below
apical extremity)
.
From the transversal sectioned areas of the roots were extracted many pieces
(3
-
5 x 6
- 8 x 3 mm) to be used in the fixation
process
.
Fixation was done with 4% paraformaldehyde in PBS (130 mM NaCl, 30
mM NaH
2
PO
4
, 70 mM Na
2
HPO
4
, pH 7.0) with Tween 20 (0.02%) for 12 hours,
FAA (50% ethanol, 5% glacial acetic acid, 3.7% formaldehyde) for 12
or
24
hours
,
or
Histochoice
Tm
,
30
The last dehydration steps were performed sequentially before
pre
-
embedding
steps: 100% Ethanol for 1 hour thrice at room temperature with
shaking
.
2.7.1.2
Embedding of tissue in paraffin
Embedding
treatment includes all paraffinization steps and previous
treatment used to
substitute
ethanol
for
Histoclear (National Diagnostics), a
harmless substitute of xylene.
The pre-embedding steps were performed
sequentially
at room
temperature with shaking: 25% Histoclear, 50% Histoclear and 75% Histoclear
in
100%
ethanol for 30 minutes each step. After, the vials were replaced with
100% Histoclear twice for 1 hour. In the last step 100% Histoclear was
subst
ituted for a mix of Histoclear and Paraplast Plus (Fisher Scientific), a
highly purified paraffin with plastic polymers and dimethyl sulfoxide
,
respectively
in
a
proportion of 2/4 and 1/4 (v/v) of each vial. This mix was incubated
overnight at room tempera
ture without agitation.
T
he
next
day,
embedding procedures were started. Vials containing
Paraplast Plus chips and Histoclear w
ere
placed in bath at 42 ºC
until
chip
s
were completely melted. Then, more 1/4 of vials volume were fill
ed
up with
Paraplast Plu
s
chips
and kept at 60 ºC for several hours. Then, Paraplast-
Histoclear solution was replaced by new freshly melted Paraplast and kept at 60
ºC overnight. After this, the melted Paraplast from each vial contai
ning
tissues
was changed for new melted Parapla
st twice a day during more three days.
After the embedding period, tissues were transferred into the
HistoPrep
Stainless
-Steel Base Molds (Fisher Scientific), previously treated with Mold
Release
(Tissue-
Tek)
. Using a
Reichert
-Jung T
issue
E
mbedding
C
ente
r
the
mold was fill
ed
up with melted
Paraplast
and paraffin blocks for each piece of
tissue w
ere
formed into the saran wraps (Tissue Path Disposable Embedding
Rings
-Fisher Scientific). After solidification of the paraffin blocks, they were
stored at 4 ºC.
31
2.7.1.3
Sectioning of tissue
The
sections on paraffin blocks were performed with a Reichert/Leica
Jung 2040 Autocut Multi-Purpose Microtome
.
The ribbons were cut with 8 µm
thick sections.
Sections were placed on water at 42 ºC for about 20 minutes to remove
compression and captured on Superfrost Plus
Microscope
slide
(Fisher
Scientific), a pre-cleaned and electrostatically ac
tiv
ate
d slide to bind the tissue
section on t
he
slide surface. Then the slides were placed on
a
42 ºC heated
plate set (Fisher Slide Warmer) for 24 hours to dry the sections. After this,
slides
were stored at 4 ºC.
2.7.2
Pretreatment of slides before hybridization
All the pretreatment stages were executed sequentially until the end step
without stops during the procedures. Except
for
protease reaction, all steps
were performed at room temperature.
2.7.2.1
De
-
paraffinization and
rehydratation of the slides
To remove the paraffin from tissues and to
rehydrate
them , slides were
placed on slide rack and submitted to
sequential
dipping into the following
solutions: 100% Histoclear for
20
minutes twice, 100% ethanol for 5 minutes
twice, 95% e
thanol
for 2 minutes, 90% e
thanol
for 2 minutes, 80% e
than
ol in
0.85% NaCl for 5 minutes, 60% ethanol in 0.85% NaCl for 2 minutes, 30%
ethanol in 0.85% NaCl for 2 minutes, 0.85% NaCl for 5 minutes and PBS for 5
minutes
.
2.7.2.2
Protease
digestion, post fixation and acetylation
Protease
treatment was the next step after dehydratation. It consisted
of
immersi
ng
the
rack
into
the
proteinase buffer (100 mM Tris-HCl pH 7.5, 50 mM
EDTA) containing Proteinase K (Roche Diagnostics GmbH) from recombinant
Pichia pastoris, PCR Grade with specific activity of 30 U/mg of pro
tein.
Slides
were incubated with Proteinase K (1 or 5 µg/ mL ) during 25, 30, 45 or 60
32
minutes at 37 º C. Proteinase K reaction was stopped by immersing the slide
rack
into Glycine
-
PBS solution (2 mg Glycine/ mL PBS)
for 2 minutes
.
After Glycine-PBS solution, slides were washed in PBS for two minutes.
Then, tissues were submitted to
a
new fixation reaction by dipping slides into
4% p
araformaldehyde in PBS solution for 10 minutes.
Slides were washed again in PBS solution for an additional 2 minutes
and subjected to acetylation treatment. For this, rack was transferred to a fresh
0.5% acetic anhydride solution in 0.1 M t
riethanola
mine pH 8.0 and maintained
for 10 minutes with stirring.
After acetylation reaction slide rack
was
washed in PBS for 2 minutes
and submitted
to tissue
dehydration
treatment
.
2.7.2.3
Deh
ydratation
To perform dehydration treatment, the rack containing the slides was
transferred
sequentially to the following solutions: 0.85% NaCl for 1 minute,
30% ethanol in 0.85% NaCl for 1 minute,
60%
ethanol in 0.85% NaCl for 1
minute,
80% ethanol in 0.85% NaCl for 2 minutes, 95% e
thanol
for 1 minute,
and
100% e
thanol for
1 minute
twice.
Slides were kept
in
the
close
d
box
,
saturated with 100% e
thanol
,
until the
beginning of hybridization step.
2.7.3
Hybridization of the labeled
probe
on
tissue
Labe
led RNA probe of both SBSS1 and SBSS2 genes in the sense and
anti
-
sense
directions
were mixed separately with solution of 50% formamide
to
produce
probe solutions. Then probe solutions were heated at 80 ºC for 2
minutes and quickly transferred
to
and kept
on ice.
Probe solutions were mixed with hybridization buffer to produce f
inal
hybridization solution containing at end concentration: 50% formamide,
20%
Dextran Solution (50% w/v), Denhardt`s Solution (0.2 µg/mL BSA, 0.2
µg/mL
Ficoll 400,1 µg/mL Polyvinylpyrrolidone), 0.5 mg/mL tRNA (from baker`s yeast
–
Roche
Diagnostics GmbH), 0.25 mg/mL Poly(A) (Roche Diagnostics GmbH
),
Salt Solution (300 mM
NaCl
, 10 mM Tris-
HCl
, 5 mM NaH
2
PO
4
, 5 mM Na
2
HPO
4
,
5 mM EDTA, pH
6.8).
33
Final hybridization solution (125 µL/
slide
)
specific for each probe (1
ng/µL final
concentration
) was placed separately on distinct slides. Each slide
was cover
ed
with a piece of Parafi
lm avoiding formation of
bubbles
.
Slides were
placed
in
boxes p
reviously incubated at 55 ºC and saturated with 2x SSC.
Slides containing different probes were incubated
in
separate
boxes at
55 ºC for about
18
hours.
High stringency hybridization conditions were tested to improve the
results.
In order to change stringency condition
,
NaCl concentration in salt
solution was reduced to 150 mM increasing stringency of the final hybridization
solution. F
urthermore
, an additional pre-
hybridization
step
was performed
by
incubating slides with non-probe final hybridization s
olu
tion before the
hybridiza
tion with probes. Finally,
the
incubation temperature of hybridization
was increased to 60 º
C.
2.7.4
Treatment after hybridization
After hybridization slides were washed in 2x SSC Solution at 55 ºC until
removal of Parafilm from slides. Slides without Parafilm were placed on rack
into 0.2 x SSC Solution at 55 ºC for about 20 minutes. The slide rack was
incubated into 0.2x SSC Solution at 55 ºC twice for 40 and 60 minutes.
Then
rack was washed twice
with
NTE solution (500 mM NaCl, 10 mM Tris-HCl pH
8.0, 5 mM EDTA) for 5 minutes at 37 ºC.
RNase A was used to
degrade
probes that were not
hybridized
with
mRNA but were
still
linked
to
tissue. The rack was incubated in NTE Solution
with 20 µg/mL RNase A (Sigma) at 37 ºC for 30 minutes. Then slides were
washed with NTE solution for 5 minutes at 37 ºC twice. After this, Rack was
incubated
in
0.2 SSC at 55 ºC f
or
2 hours.
Following
, slide racks
w
ere
dipped
in
PBS at room temperature for 5 minutes. Sometimes rack was stored in PBS at
4 ºC ove
rnight until detection step.
2.7.5
Immuno
histochemical
detection of DIG probes
All detection steps were performed at room temperature. After PBS
washing, the rack with the slides was
washed
in
b
uffer
I (150 mM NaCl, 100
mM Tris-HCl pH 7.5), for 10 minute
s
in agitation.
Then
rack was incubated
on
35
3. RESULTS AND DISCUSS
ION
3.1 Pr
obe p
roduction
RT
-PCR procedures using
primers
designed
to amplify low conser
ved
region
s between SBSS1 cDNA and SBSS2 cDNA produced successfully
one
cDNA probe fragment for each gene.
The
DNA
probe fragments cloned
in
pCR
II
-TOPO vector are sho
wn
in
Figure 4
, on agarose gel after enzyme restriction reaction.
Figure
4: Gel electrophoresis of the products of restriction enzymes
.
Recombina
nt plasmids containing either SBSS1 probe or SBSS2 probe were
cleaved with
Eco
RI. HL is Hyperladder weight marker. Line 1 and 3 represents
the controls of the restriction reaction without
Eco
RI
(
non
-cleaved plasmids
).
Line 2 - A and 4 - A shows linearized pCRII-TOPO vector. Line 2 - B and 4 -
B
indicate respectively SBSS1 (174 bp) and SBSS2 (200 bp) DNA probe
fragments.
A
HL 1 2
3 4
B
Band
Size
(bp)
4000
1000
200
A
HL 1 2
3 4
B
A
HL 1 2
3 4
B
Band
Size
(bp)
4000
1000
200
36
The SBSS1 probe
fragment
measured
174 bp
in
length
and show
ed
100% of complementariness with SBSS1 cDNA
on
alignment region. The
SBSS1 cDNA from isolated clones of the Sugarbeet Laboratory -
NCSL
-
USDA
is
2482
bp
in
length
. Probe fragment align
ed
with
the
3’ end of
the
cDNA (2294
- 2467 bases in
5’
3’ sense
).
If SBSS1 sequence from Genebank
access
number
X81974
(2563
bp
)
is considered, SBSS1 probe align
ed
on
2120 until
2293 bases in 5’ 3’ sense. Complete sequence of SBSS1 probe fragment is
show
n in
Figure
5
A.
The SBSS2 probe fragment
me
a
sured
200
bp
in
length
an
37
transcribed
and labeled together with
the
true
probe. Because of this, 1
21
nucleotides were added
o9 1287 Tm(,)Tj( )Tj/F7 2048 Tf0.09187 0 4370.09187 3772 13687 Tm99.72 Tz(the )Tj/F7 2048 Tf0.09187 0 47-0.09187 3772 87 Tm3 1287 Tm(o)Tj0.09187 0 0828.09187 3255 1612 Tm(r)Tj0.09187 0 4891.09187 2579 1612 Tm(i)Tj0.09187 0 4 -0.09187 257g 1287 Tm(w)Tj0.09187 0 0 37.09187 2579 1612 Tm(i)Tj0.09187 0 50-0.09187 257n 1287 Tm(t)Tj0.09187 0 0 83.09187 3512 1612 Tm(a)Tj0.09187 0 5289.09187 2276 1612 Tm(d)Tj( )Tj0.09187 0 5420.09187 3930 1287 Tm(p)Tj0.09187 0 5520.09187 3255 1612 Tm(r)Tj0.09187 0 5587.09187 2422 1612 Tm(o)Tj0.09187 0 5691.09187 2572 1287 Tm(b)Tj0.09187 0 -00.09187 2317 1612 Tm(e)Tj0.09187 0 5902.09187 2831 1612 Tm(s)Tj( )Tj0.09187 0 6084.09187 3512 1612 Tm(a)Tj0.09187 0 6190.09187 41250.092 Tm(s)Tj( )Tj0.09187 0 63-0.09187 2831 1617 Tm(o)Tj0.09187 0 0 -5.09187 2317 1612 Tm(e)Tj0.09187 0 -00.09187 257n 1287 Tm(t)Tj0.09187 0 0 -6.09187 2831 1617 Tm(o)Tj0.09187 0 0729.09187 3317 1612 Tm(e)Tj( )Tj0.09187 0 6 -0.09187 2831 1617 Tm(o)Tj0.09187 0 0 -8.09187 2317 1612 Tm(e)Tj0.09187 0 71-0.09187 242q 1287 Tm(a)Tj0.09187 0 0 28.09187 3255 1287 Tm(u)Tj0.09187 0 0 -0.09187 2577 1612 Tm(e)Tj0.09187 0 74-6.09187 283n 1287 Tm(t)Tj0.09187 0 7542.09187 2182 1612 Tm(c)Tj0.09187 0 7635.09187 2317 1612 Tm(e)Tj0.09187 0 7740.09187 2831 1612 Tm(s)Tj( )Tj0.09187 0 7 -0.09187 283a 1287 Tm(i)Tj0.09187 0 0 28.09187 325n 1287 Tm(s)Tj0.09187 0 0 34.09187 3930 1612 Tm(,)Tj( )Tj/F7 2048 Tf0.09187 0 0 -0.09187 3772 10487 Tm -09.88 Tz(21)Tj/F7 2048 Tf100 Tz( )Tj/F7 2048 Tf0.09187 0 0 -0.0936871972 1612 Tm(n)Tj0.09187 0 0 -0.0936871977 1612 Tm(u)Tj0.09187 0 0 -0.0936871972 1612 Tm(c)Tj0.09187 0 0 -0.0936871976 1612 Tm(l)Tj0.09187 0 0 -0.0936871977 1612 Tm(e)Tj0.09187 0 0 -0.0936871972 1612 Tm(o)Tj0.09187 0 0 -0.0936871977 1612 Tm(t)Tj0.09187 0 0 -0.0936871979 1612 Tm(i)Tj0.09187 0 0 -0.0936871971 1612 Tm(d)Tj0.09187 0 0 -0.0936871977 1612 Tm(e)Tj0.09187 0 0 -0.0936871971 1612 Tm(s)Tj( )Tj0.09187 0 0 03.0936871974 1612 Tm(w)Tj0.09187 0 0 40.0936871977 1612 Tm(e)Tj0.09187 0 3244.0936871975 1612 Tm(r)Tj0.09187 0 0 00.0936871977 1617 Tm(d)Tj( )Tj0.09187 0 0 90.0936871973 1287 Tm(a)Tj0.09187 0 0 95.0936871971 1612 Tm(d)Tj0.09187 0 3699.0936871971 1612 Tm(d)Tj0.09187 0 3804.0936871977 1612 Tm(e)Tj0.09187 0 3908.0936871971 1612 Tm(d)Tj( )Tj0.09187 0 0093.0936871977 1612 Tm(t)Tj0.09187 0 -04.0936871972 1612 Tm(d)Tj( )Tj0.09187 0 0328.0936871972 1612 Tm(o)Tj0.09187 0 4433.0936871975 1612 Tm(r)Tj0.09187 0 4495.0936871979 1612 Tm(i)Tj0.09187 0 4538.0936871978 1287 Tm(g)Tj0.09187 0 0642.0936871979 1612 Tm(i)Tj0.09187 0 4684.0936871972 1612 Tm(n)Tj0.09187 0 4788.0936871973 1287 Tm(a)Tj0.09187 0 4893.0936871976 1612 Tm(d)Tj( )Tj0.09187 0 5 -0.0936871970 1287 Tm(p)Tj0.09187 0 5119.0936871975 1612 Tm(r)Tj0.09187 0 0 81.0936871972 1612 Tm(o)Tj0.09187 0 5280.0936871972 1287 Tm(b)Tj0.09187 0 390.0936871977 1612 Tm(e)Tj0.09187 0 5496.0936871971 1612 Tm(s)Tj( )Tj0.09187 0 5668.0936871973 1287 Tm(a)Tj0.09187 0 5773.0936871977 1612 Tm(s)Tj( )Tj0.09187 0 5904.0936871972 1612 Tm(a)Tj0.09187 0 6008.093687197n 1287 Tm(t)Tj0.09187 0 01-0.093687197t 1287 Tm(t)Tj0.09187 0 0165.0936871979 1617 Tm(e)Tj/F7 2048 Tf0.09187 0 0208.093687197- 1617 Tm(e)Tj/F7 2048 Tf0.09187 0 0270.0936871971 161s
38
3.2
RNA dot blot hybridization
The sucrose synthase genes, SBSS1 and SBSS2, are not very close
in
their
genic f
amily
. However, they exhibit 62% nucleotide similarity (
Haagenson
et al., 2006). SBSS1 and SBSS2 exhibit
ed
regions (300 bp) up to 73% identity
but the probes were selected from regions with low identity. Probes
showed
only 38% of identity (data from Align
X
–
Vector NTI).
Despite of low similarity among probes, the specificity of them for each
gene was tested in vitro by RNA dot blot hybridization as
a
guarantee that no
cross hybridization
would take place
i
n
in situ
experiments
(Figure 6)
Fig
ure 6 – RNA dot blot hybridization with chromogenic detection performed
with anti-DiG a
lk
aline phosphatase and NTB/BCIP substrate on filter.
Intersections between arrows and numbers appoint regions where non labeled
mRNAs were blotted. Each membrane has both one dot blotted with SBSS1
mRNA fragments (1) and one dot blotted with SBSS2 mRNA (2). Membranes A,
B, C and D were
hybridized
respectively with SBSS1 RNA anti-sense probes,
SBSS1 RNA sense probes, SBSS2 RNA anti-sense probes and SBSS2 RNA
sense probes. Dark violet-blue stains indicate regions where m
RNA
-
Probe
hybridization
occurred
.
1 2
1 2
A B
1 2
1 2
C
D
1 2
1 2
A B
1 2
1 2
A B
1 2
1 2
C
D
1 2
1 2
C
1 2
1 2
C
D
39
For SBSS1 probe, the hybridization occurred only
between
SBSS1 anti-
sense probe and SBSS1 mRNA fragment (membrane A - 1). No hybridization
was observed
between
SBSS1 anti-sense probe and SBSS2 mRNA
(membrane A - 2). Likewise no hybridization was observed
between
SBSS1
sense probe and either SBSS1 mRNA or SBSS2 mRNA (membrane B - 1 and
2).
For SBSS2 probe, the hybridization occurred only between SBSS2 anti-
sense probe and SBSS2 mRNA (membrane C - 2). No hybridization was
observed between SBSS2 anti-sense probe and SBSS1 mRNA (membrane C -
1). In addition
,
no hybridization was observed
between SBSS2 sense probe and
either SBSS1 mRNA or SBSS2 mRNA (membrane
D –
1 and 2).
3.3
The
empirical art
of
in situ hybridization
The
in situ hybridization experiment involved several steps that need
ed
to be correctly design
ed
and perfor
med
in order to not compromise the quality
of final results. Many
parameters
such as probe quality, plant tissue fixation and
embedding,
prehybridization tissue treatments, hybridization conditions and
hybridization buffer composition, and stringency of po
st
-hybridization washing,
had to be opt
imi
z
ed
to
generate
an
adequate outcome (Borlido et al.,2002; Shu
e
t al., 1999).
Some of t
hese factors
were
used based
on the
consensus
of
many
protocols and were adapted from them. Others parameters such as kind of
fixative and fixation time and proteinase K treatment shows high variability with
the
kind of tissue used which were tested for optimization. In the same manner
h
ybridization conditions were
also tested.
3.3.1
Tissue
f
ixation
Fixation is one of the most critical steps for successful in situ
hybridization. Tissue fixation must give good morphology preservation together
with good staining quality (Ruzin, 1999). The aim of fixation is to kill the cell
rapidly and to immobilize proteins in the same state and location as they were
in
vivo
(Vitha et al., 2000)
.
Fixatives are classified into two groups, cross-
linki
ng fixatives (e.g.,
ethanol, methanol and acetone) and precipitating fixatives (e.g., formaldehyde,
40
paraformaldehyde and glutaraldehyde), according to their action on proteins.
The first group
denature
proteins and transform protoplasm into an artificially
interconnected network. Cross-linking fixatives chemically crosslink with
protoplasmic components (protein and lipids), fixing them in situ. Also there are
mixtures of fixatives that intend to maximize advantages and minimize
disadvantages of different
fi
xatives
(Morel
and Cavalier, 2001).
Three
kinds of fixatives and different times of fixation were tested for 6
week
old
sugarbeet
root tissues.
Par
afor
maldehyde (4%) with 12 hours of fixation
prod
uced better fixation
than other fixatives used, but only for small pieces of tissue.
Tissue
s kept good
structure and morphology. However,
good
staining was not observed with the
experiment
al
conditions performed. This may have occurred due
to
strong
cross
-linking fixation that prevented adequate probe penetration (
Zachgo,
2002).
Histochoice
TM
MB is a non-
hazardous
and
non
-crosslinking fixative, for
molecular biology application with
its formulation
unrevealed by
its
manufacturer
(AMRESCO Inc.)
. It
did not work appropriately and the tissue was not fixed after
12 hour
s of fixation. It was not possible
to section
the
tissue.
FAA
, a mix of formaldehyde, acetic acid and ethanol produced
reasonable morphology of small pieces of tissue after 12 hours of fixation. It
permitted accessibility of probe into the tissue and good staining results.
Fixation during 24 hours improved a little the morphology, but it restrained
probe access demanding stronger proteinase K treatment.
Johansen
(1997) successfully used 3 different fixation procedures (FAA,
4% formaldehyde, 2% paraformaldehyde + 2.5% glutaraldehyde) for plant
tissue fixation, and found that traditional FAA procedure matched the 2 other
fixation methods in terms of structural preservation.
FAA fixation also gave satisfactory results for Urbanczyk-
Wonchniak
(2002), because it met the basic requirements for the protection of nucleic
acids, especially RNA and permeation.
3.3.2
Protease treatment
The deproteinization of the tissue with proteinase K
is
to partially
eliminate proteins, particularly, those associated with nucleic acids, to give
41
better access of the probe to the target complementary sequences (Morel and
Cavalier, 2001).
The optimal protease concentration and digestion time
are
dependent on
factors
, such as type of tissue, fixative and fixation time. They should be
determined
empirically to yield good morphology and strong hybridization signal
(
Silva
-
Valenzuela
et al., 2006)
The best results in relation to equilibrium among signal and morphology
was
found
with 1 µg/
mL proteinase K for 25 minutes
at 37 ºC
to tissue fi
xed with
FAA for 12 hour and with 5 µg/
mL proteinase K for 30 minutes
at 37 ºC
to tissue
fixed with FAA for 24 hours.
3.3.3
Conditions of hybridization
Hybridization conditions are important
for
determining the specificity and
sensitivity of the hybridization signal. Parameters that affect the stability of
hybrids, like melting temperature (T
m
), and kinetics of hybridization, such as
probe concentration and stringency of hybridization, perform a significant role
on success of the hybridization reaction (
W
ilkinson, 1992b)
.
After
the
establishment of hybridization conditions adapted from curren
t
protocols (cited in item 2.7),
the
hybridization reaction was tested in different
condition
s with
high
er
stringency than the last one. The modification of
hybridizat
ion conditions did not improve the signal or alter
the
occasional
background.
Sometimes the signal was lightly reduced.
It is possible that the first hybridization reaction was
performed
under the
limit of stringency. Then the increase of stringency led to less ideal probe
hybridization
conditions.
3.4
In situ
hybridization of
labeled
probe
s
in
sugarbeet
root tissue
The probes were hybridized with 6 week-old beetroot. This stage of
development
was cho
sen
due to the fact that both genes showed high level
of
expression
in beets of this age (
Haagenson
et al., 2006). Then, spatial
expression of both SBSS1 and SBSS2
genes
could be verified on the same
kind of tissue.
Th
e
localization of the transcripts, characterized by dark blue-indigo dye
42
resulted
from
the
anti
-sense probes hybridization
to
tissue,
were
very similar for
both SBSS1 and SBSS2
(F
igure 7
-
14
).
Control of the
reactions was
performed
with
hybridization of sense probes in the same tissue (Figures 7
B,
9
B,
11
B and
13
B
).
The genes showed high expression in the storage parenchyma in inner
layer of tissue among the rings of vascular bundles (
Figure
9A, 10B,
13
A and
14B
).
In this region there are wide areas of mature parenchyma tissue where
the cells are bigger tha
n
in
peripheral zone of the roots.
The p
eripheral zone of
roots include
the
periderm, a thin cork layer five to
eight cells deep with suberized cell walls and lignified middle lamella
(Artschwager, 1926), and concentric rings
intercalated
with vascular and
parenchymal tissue (Klotz, 2005). Color reaction was less intense on peripheral
zone
(Figure 7A and
11
A)
than on inner zone of roots, concentrated on
immature parenchyma tissue of peripherical growth rings. From the external to
ce
43
Figure 7 - Localization of sucrose synthase
transcripts
by in situ hybridization
on peripheral zone of
sugarbeet
roots
.
C
ross
-sections of 6 weeks
old
tap
root
were hybridized with a DIG-
labeled
(A) anti-
sense
SBSS1
RNA probe and (B) a
sense
SBSS1 RNA
probe.
Dark blue
-i
ndigo stain indi
cates positive hybridization
reaction.
Abbreviations: pd, periderm; sc, secondary cortex; cb, secondary
cambium; spc, storage parenchyma.
Bar = 1
00 µm.
pd
sc
cb
spc
pd
sc
cb
spc
pd
sc
cb
spc
pd
sc
cb
spc
pd
sc
cb
spc
pd
sc
cb
spc
A
B
44
Figure
8 - Localization of sucrose synthase
transcripts
by in situ hybridization
on peripheral zone of
sugarbeet
roots
.
C
ross
-sections of 6 weeks
old
taproot
hy
bridized with a DIG-
labeled
antisense
SBSS1
RNA probe
showing
more
details of the growth rings (A) and storage parenchyma (B)
.
Dark blue-
indigo
stain indicates positive hybridization reaction. Abbreviations
:
x
,
xylem;
spc,
storage parenchyma. Bar
= 1
00
µm in
A
and
25 µm in
B.
x
x
spc
A
B
45
Figure 9 -
Localizatio
n of sucrose synthase
transcripts
by in situ hybridization
on inner zone of
sugarbeet
roots
.
C
ross
-sections of 6 weeks
old
taproot
were
hybridized with a DIG
-
labeled
(
A
) antisense
SBSS1
RNA probe and (
B
) a sense
SBSS1 RNA probe. Dark blue-indigo stain indicates positive hybridization
reaction.
Abbreviations
: ph, phloem; x, xylem; cb, secondary cambium; spc,
storage parenchyma. Bar = 100 µm.
cb
ph
cb
x
ph
spc
A
x
x
cb
ph
x
cb
spc
spc
B
ph
cb
ph
cb
x
ph
spc
A
x
cb
ph
cb
x
ph
spc
A
x
x
cb
ph
x
cb
spc
spc
B
ph
x
cb
ph
x
cb
spc
spc
B
ph
46
Figure 10 - Localization of sucrose synthase
transcripts
by in situ hybridization
on
inner
zone of
sugarbeet
roots
.
C
ross
-sections of 6 weeks
old
t
aproot
hy
bridized with a DIG-
labeled
antisense
SBSS1
RNA probe showing more
details of the mature xylem (A) and storage parenchyma (B)
.
Dark blue-
indigo
stain indicates positive hybridization reaction. Abbreviations: x, xylem; spc,
storage parenchyma. Bar
= 25 µm.
x
spc
A
B
x
spc
A
x
spc
A
BB
47
Figure 11 - Localization of sucrose synthase
transcripts
by in situ hybridization
on peripheral zone of
sugarbeet
roots
.
C
ross
-sections of 6 weeks
old
taproot
were hybridized with a DIG-
labeled
(A) antisense
SBSS2
RNA probe and (B) a
sense
SBSS2 RNA
probe.
Dark blue
-
indigo stain indicates positive hybridization
reaction.
Abbreviations: pd, periderm; sc, secondary cortex; cb, secondary
cambium; spc, storage parenchyma. Bar = 100 µm.
pd
sc
cb
spc
Cb
’
Spc
’
A
pd
sc
cb
spc
Cb
’
Spc
’
B
pd
sc
cb
spc
Cb
’
Spc
’
A
pd
sc
cb
spc
Cb
’
Spc
’
A
pd
sc
cb
spc
Cb
’
Spc
’
B
pd
sc
cb
spc
Cb
’
Spc
’
B
48
Figure 12 - Localization of sucrose synthase transcripts by in situ hybridization
on peripheral zone of
sugarbeet
roots. Cross-sections of 6 weeks old
taproot
hybridized with a DIG-
labeled
antisense SBSS2 RNA probe showing more
details of the
immature
growth rings (A) and storage parenchyma (B). Dark
blue
-indigo stain indicates positive hybridization reaction. Abbreviations
:
cb
,
secondary cambium; spc, storage parenchyma. Bar = 50 µm in (A
)
and
10
µm
in
(B).
cb
spc
A
B
cb
spc
A
cb
spc
A
BB
49
Figure 13 - Localization of sucrose synthase transcripts by in situ hybridization
on inner zone of
sugarbeet
roots. Cross-sections of 6 weeks old
taproot
were
hybridized with a DIG
-
labeled
(
A
) antisense
SBSS
2
RNA probe and (
B
) a sense
SBSS
2 RNA probe. Dark blue-indigo stain indicates positive hybridization
reaction
if it do not occur in the control B
.
Abbreviations
: ph, phloem; x, xylem;
cb, secondary cambium; spc, storage parenchyma. Bar = 100 µm.
cb
spc
ph
x
ph
cb
x
spc
A
ph
x
cb
spc
spc
B
cb
spc
ph
x
ph
cb
x
spc
A
cb
spc
ph
x
ph
cb
x
spc
A
ph
x
cb
spc
spc
B
ph
x
cb
spc
spc
B
50
Figure
14
- Localization of sucrose synthase
transcripts
by in situ hybridization
on inner zone of
sugarbeet
roots
.
C
ross
-sections of 6 weeks
old
taproot
hy
bridized with a DIG-
labeled
antisense
SBSS2
RNA probe showing more
details of the mature vascular bundles (A) and storage parenchyma (B)
.
Dark
blue
-indigo stain indicates positive hybridization reaction except on xylem
vessels.
Abbreviations
: x, xylem; spc, storage parenchyma. Bar =
50
µm in A
and 25 µm in B
.
B
spc
x
cb
A
ph
BB
spc
x
cb
A
ph
spc
x
cb
A
ph
51
Several studies showed that the expression of sucrose synthase gene is
spatially and temporally separated within the different compartments of the
plant. Sucrose synthase mRNA were abundant in the surround
ing
cells of the
vascular
bundles in young tomato fruits (Wang et al. 1994), absent in the xylem
of potato microtubers (Vermerris et al., 2001) and phloem-
associate
d in mature
maize leaves (Nolte
and
Koch, 1993). O
therwise
, in young maize tissue, the
transcripts of sucrose synthase accumulated in the xylem, but not in the phloem
(Hanggi
and
Fleming, 2001). Association of sucrose synthase transcripts with
tissues rich in amyloplast also
was
observed on sink organs (Vermerris et al.,
2001, Wang et al.
,
1994).
In root tissues an enhanced level of sucrose synthase mRNA was
detected in the phloem fibers in the differentiated bean root tissues (Blee
and
Anderson, 2002). These authors suggested that sucrose synthase activity in
vascular tissues may promote transport of
photosynthate throug
h these cells
.
In s
ugarcane
culm
, which stock sucrose in the storage parenchyma
tissues, like in
sugarbeet
roots, immunohistochemistry assays showed that
parenchyma and vascular tissue contain
the
sucrose synthase protein in young
and mature internodes (Sc
hafer et al., 2004).
No
similar work with expression of sucrose synthase transcripts was
found
in the literature
for
sugarbeet
roots.
Unlike
from
the
aforementioned
plants, beets are storage root plants
with
supernumerary tissue, unusual
structures originated by
the
development of secondary cambium (Appezzato-
da
-
Glória
and
Hayashi, 2004). This can explain, in part, why the pattern of the
sucrose synthase mRNA localization in root tissues differed from other plants.
Some
reports
of sucrose synthase in the vascular tissue, mainly
in
phloem tissue, were attributed to sink function; by
preventing
accumulation of
sucrose,
the enzyme maintained the unloading process gradient (Rouheir
and
Usuda, 2001; Wittich
and
Vreugdenhil, 1998). Otherwise,
sugarbeet
is one of
the few plants that transport sucrose through the phloem to the vacuole of the
storage cells without
a
sucrose hydrolysis
requirement
for
final
sugar
accumulation (MacFall
and
Johnson, 1994; Leimone et al., 1988). However, a
minor part of sucrose (20
-
30%)
can be hydrolyzed
mainly by sucrose synthase
when sucrose moves from the free space to the vacuole
(Wyse, 1979).
52
The concentration of sucrose synthase mRNA in parenchyma storage
cells observed in this work is an evidence of
the
importance of this tissue n
ot
only in sucrose storage, but in control of sugar distribution to other root tissues.
The high concentration of sucrose
in
sugarbeet storage cells
demand
a
rigorous
control of osmolarity, since tugor can affect cell
function
(Wyse et al., 1986).
One hypothesis is that sucrose after phloem unloading is concentrated and
accumulated in mature storage tissue, and sucrose synthase works on the
regulation between storage and distribution of sugar to other tissues.
In this study an evaluation of spatial distribution of sucrose synthase
transcripts
was performed only in cross section of 6 weeks old tissue. To better
investigate the expression of sucrose synthase genes on different root tissues,
more development stages
must
be studied at different section position
s.
Localization and expression of enzymes often depend on the developmental
stage of plants and their organs (Schafer et al., 2004). The role and tissue
specificity of invertases also
should
be considered.
Ano
ther point to be pondered is the relation between transcript and
protein level. Sucrose synthase mRNA and protein level are not always
temporally synchronized (Wang et al., 1994)
and
regulation of sucrose
synthase
expression is not solely transcriptional. Protein stability and other post-
transcriptiona
l regulatory mechanisms are likely to contribute to sucrose
synthase expression in
sugarbeet
root (
Haagenson
et al., 2006).
53
4.
CONCLUSI
ON
With the goal of better understand
ing
the role of sucrose synthase on
sugar metabolism
of
sugarbeet
roots, labeled RNA probes were produced
from
both SBSS1 and SBSS2 genes that
demonstrated
high specificity for their
respective
transcripts on dot blot assays. The probes were used with an in situ
hybridization protocol adapted to
sugarbeet
roots. The spatial
dist
ribution of the
transcripts of both isoforms of sucrose synthase was evaluated on six weeks
old
sugarbeet
roots and
it
was
observed
that the transcripts of the two genes
have
the same
tissue
localization on
the
evaluated root age. The sucrose
synthase genes were expressed mainly on storage parenchyma and t
ranscripts
were not found on cambial region and vascular bundles. It is still possible that
its concentration in this tissue was sufficiently low to be undetected by
the
used
technique
.
The results gave
da
ta
for
the
role of
the
storage
parenchyma
cell
s
as
center of storage and distribution of sugar for other tissues and the regulatory
role of sucrose synthase in this process. However, more studies
involving
invertases and stages of root development
are nece
ssary
to
better elucidate
the
real role of the two sucrose synthase genes i
n
sugarbeet
tissues
.
54
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60
CHAPTER 2
EFFECT OF THE TEMPERATURE AND PVC FILM COVER ON
FRESH
MASS LOSS AND SUCROSE SYNTHASE ACTIVITY OF STORED
ARRACACHA ROOTS
ABSTRACT
Arracacha is a plant originated from South America that produces a
tuberous
root rich in starch and some vitamins. This crop is cultivated
mainly
by
small producers being an important income resource. However, this crop shows
a long cycle, and the roots have a short postharvest life that depending on the
environment
al conditions
is
restricted to 2 - 3 days. This postharvest restrictions
limit transportation and trade of the roots, being one limitation to the expansion
of the crop. The effect of temperature and PVC cover during storage of
arracacha roots was analyzed by loss of
fre
sh
mass and sucrose synthase
activity. 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7833 6210 Tm0 Tm0.09187 0 0 -0.09187 7786 5236 T00 Tm0.09187 0 0 -0.09187 7331 55609530 Tm0.09187 0 0 -0.09187 7786 523699m0 Tm0.09187 0 0 -0.09187 7833 62180470 Tm0.0y187 0 0 -0.09187 7291 6859 Tm(e Tmm0.0y18 5560 Tm(p)Tj0.09187 0 0 -076Tm0.09187 0 0 -0.09187 2888 52337e7 2359 6854 Tm0 Tm0.0t187 0 0 -0.08 Tm0.09187 0 0 -0.09187 778r5cm0 Tm0.0969169187 5639 2317)Tj0 Tm0.09187 0 0 -9m.0u1a87 0 0 -0.09187 2422 588598877 200.00.0tV(187 0 0 -0.09187 7371 6210 Tmb71 6200.00.0tV(187 0 0 -0.09187 7371 6210 Tmb71 6.0918109187 2422 u187 0 0 -0.09187 2370 5885 Tm-0.09187 7371 6210 Tmb71 6200.00.0t5209187 2422 t187 0 0 -0.09187 2468 4911 T9Tj0 Tm0.09187 0 0 -9m.0u1a87 0 0 -683000 Tm0d0 -00 0 -0.09187 5855 4587 Tm(2867000 Tm0d0 -00 0 -0.09187 2872 6859 T19187 2422 u187 0 0 -0.09187 2370 58830.09187 2422 r187 0 0 -0.09187 3081 6859 T9Tj0 Tm0i187 0 0 -0.09187 3092 4262 T19187 2422 n187 0 0 -0.09187 3185 4262 T59187 2422 g187 0 0 -0.09187 3228 6534 Tm(s)709187 2422 s187 0 0 -0.09187 2223 49135649187 2422 t187 0 0 -0.09187 2468 491361187 0 0 -0.o187 0 0 -0.09187 3708 6210 209187 2422 r187 0 0 -0.09187 3081 6859 Tm-0.09187 7371 6210 Tmb71 6200.00.038879187 2422 g187 0 0 -0.09187 2401 685399)Tj0 Tm0.09180 0 -0.09187 4011 6210 Tm(t)(187 0 0 -0.9187 0 0 -0.09187 2401 6859 T8j0 Tm0i187 0 0 -0.09187 3092 42642709187 2422 m187 0 0 -0.09187 4317 6859 T6Tj0 Tm0.09187 0 0 -9m.0u1a87 0 0 4)Tjj0 Tm0.187 0 0 -0.09187 8536 6534 Tm(e)Tj( )Tj/4 Tmj0 Tm0T0 -0.09187 4606 5236 Tm(e)T79187 2422 h187 0 0 -0.09187 4819 6859 Tmj0 Tm0.09180 0 -0.09187 4011 6210 Tm(5 Tz0 0 0 -0.09187 7371 6210 Tmb71 6.095 Tm7 0 0 -0.o187 0 0 -0.09187 3708 6215F T9187 2422 s187 0 0 -0.09187 2223 49153069187 2422 s187 0 0 -0.09187 4973 5560 Tm(d4T9Tj0 Tm09187 0 0 -0.09187 5416 6534 83000 Tm0f187 0 0 -0.09187 5437 6859 Tm(e714000 Tm0f187 0 0 -0.09187 5655 4587 T69187 2422 r187 0 0 -0.09187 3081 6855829Tj0 Tm0.09187 0 0 -9m.0u1a87 0 0 59349187 2422 s187 0 0 -0.09187 2223 49160T8j0 Tm09187 0 0 -0.09187 6245 4262 Tm(hF T9187 2422 0 -0.100 Tz( )Tj/F7 2048 Tf( )Tj/6-076T87 242299.68 0 -as.09187 4113 5560 Tm100.44 Tz(is )Tj/6 TmT87 242209187 0 9187 0 0 -0.09187 6727 4911 T79187 2422 a187 0 0 -0.09187 2247 62119829187 2422 s187 0 0 -0.09187 4973 5560 Tm(7154187 2422 r187 0 0 -0.09187 3081 685)216Tj0 Tm0.09187 0 0 -9m.0u1a87 0 0 73Tm-0.09187 d187 0 0 -0.09187 7334 6534 T69187 2422 u187 0 0 -0.09187 2370 5887 Tf187 2422 c187 0 0 -0.09187 2888 5237 T5Tj0 Tm0.09187 0 0 -9m.0u1a87 0 0 7730000 Tm0d0 -00 0 -0.09187 5855 4587 Tm(7914000 Tm0b187 0 0 -0.09187 7833 6218019Tj0 Tm0y187 0 0 -0.09187 7291 6859 Tm(e Tm187 2422 u187 0 0 -0.09187 2370 5880 Tm187 2422 s187 0 0 -0.09187 2223 4915 Tmj0 Tm0i187 0 0 -0.09187 3092 4265 Tm187 2422 n187 0 0 -0.09187 3185 426Tm0.87 2422 g187 0 0 -0778r5cm0 Tm0.0969169187 5639 2317)Tj805f0.09185 0 0 -PVC film while uncovered roots de0 -0.100 Tz( )Tj/F7 2048 Tf( )Tj/5019Tj805f0.09183 0 0 -onstrated high mas.0los.0and consequentl0.09187 8443 4587 Tm99.59 Tz(by )Tj/F7 20913Tf0.09187 0 0 -0.09187 4305 5560 Tm1040Tm913Tf0.09187 0 0 -0.09187 2888 5236 Tm913Tf0.0d0 -0.09187 2077 6210 Tm(f)T4913Tf0.0u187 0 0 -0.09187 2401 68593490913Tf0.09187 0 0 -0.09187 2308 6534 43913Tf0.09187 0 0 -0.09187 2888 5236 Tm913Tf0.0d0 -00 0 -0.09187 2547 6859 Tm(.)m(913Tf0.09187 0 0 -0.09187 2789 5885 T1(913Tf0.09187 0 0 -0.09187 2893 5885 T(913Tf0.0s187 0 0 -0.09187 2223 49130T1(913Tf0.09187 0 0 -0.09187 3081 6859 420913Tf0.09187 0 0 -0.09187 3185 4262 4(913Tf0.07371 6210 Tmb71 6200.00.03 Tm913Tf0.0r0 -0.09187 3228 6534 Tm(s)Tm913Tf0.0v0 -0.09187 3341 4587 Tm(h)T8913Tf0.09187 0 0 -0.09187 2888 5233 Tm913Tf0.0s187 0 0 -0.09187 2223 49137T8913Tf0.09187 0 0 -0.09187 3034 4587 Tm(t89m913Tf0.0l187 0 0 -0.09187 2401 685393(913Tf0.0i187 0 0 -0.09187 3092 42629790913Tf0.0f0 -0.09187 4485 4587 Tm(d031913Tf0.09187 0 0 -0.09187 2888 523413 0913Tf0.0.187 0 0 -0.09187 4144 5236 Tm(s)2m913Tf0.0T0 -0.09187 4606 5236 Tm(e43(913Tf0.0h187 0 0 -0.09187 4819 68595420913Tf0.09187 0 0 -0.09187 4606 5236 Tm(e)80913Tf0.0l187 0 0 -0.09187 2401 68587 3(913Tf0.09187 0 0 -0.09187 2893 5884928913Tf0.0s187 0 0 -0.09187 2223 491502m913Tf0.0s187 0 0 -0.09187 4973 5560 Tm(d250(913Tf0.09187 0 0 -0.09187 2893 5885 Tm913Tf0.0f0 -00 0 -0.09187 5347 5560 Tm(2)Tj( )Tj/F7420913Tf0.99. Tf 0 -0.-0.09187 5889 6534 Tm99.59 Tz(sh )Tj/F0950913Tf0.09187 0 0 -0.09187 6181 6534 Tm100251(913Tf0.07371 6210 Tmb71 6200.00.06356913Tf0.0s187 0 0 -0.09187 2223 4916450(913Tf0.09187 0 0 -0.09187 6538 6534 Tm(s)Tj913Tf0.09187 0 0 -0.09187 6727 4911 Tm913Tf0.0a187 0 0 -0.09187 2247 6211920(913Tf0.09187 0 0 -0.09187 6538 6534 Tm(71Tm913Tf0.09187 0 0 -0.09187 7164 5560 53913Tf0.0l187 0 0 -0.09187 2401 68572950913Tf0.0s187 0 0 -0.09187 2223 4910 88(913Tf0.09187 0 0 -0.09187 7537 4911 Tm(s)27913Tf0.0r0 -0.09187 3228 6534 Tm(7 Tm913Tf0.09187 0 0 -0.09187 2888 52377950913Tf0.0d187 0 0 -0.09187 7794 5560 Tm913Tf0.0u187 0 0 -0.09187 2401 68580050913Tf0.09187 0 0 -0.09187 2308 65380Tm913Tf0.09187 0 0 -0.09187 2888 5238203913Tf0.0d0 -00 0 -0.09187 2547 6859 Tm(4 Tm913Tf0.0b187 0 0 -0.09187 8412 4911 Tm913Tf0.0y187 0 0 -0778r5cm0 Tm0.0969169187 5639 2317)T9)Tj0.09187Tf 0 decreasing5560 Tm100.39 Tz(al conditi0 0 -0.09187 2893 5885 m(9)Tj0.0s187 0 0 -0.09187 2223 4913081(9)Tj0.09187 0 0 -0.09187 3081 6859 Tm9)Tj0.0o187 0 0 -0.09187 3708 6210)Tj9)Tj0.0r0 -0.09187 3228 6534 Tm(s301(9)Tj0.0a187 0 0 -0.09187 2566 5233 T6(9)Tj0.0g187 0 0 -0.09187 2401 6853 Tm9)Tj0.09187 0 0 -0.09187 3658 6534 Tm(s711(9)Tj0.09187 0 0 -0.09187 3081 6859s)29)Tj0.09187 .09187 3034 4587 Tm(t86(9)Tj0.0m187 0 0 -0.09187 2401 6534 2m9)Tj0.0p187 0 0 -0.09187 2422 58841289)Tj0.09187 .09187 3034 4587 Tm(42Tm9)Tj0.0r187 0 0 -0.09187 4203 6534 T5(9)Tj0.0a187 0 0 -0.09187 2566 5234400(9)Tj0.09187 0 0 -0.09187 3081 68544529)Tj0.0u187 0 0 -0.09187 4454 5885 T79)Tj0.0r187 0 0 -0.09187 4203 65346209)Tj0.09187 .09187 3034 4587 Tm(47249)Tj0.0,187 0 0 -0.09187 2704 6534 Tm(4871(9)Tj0.0a187 0 0 -0.09187 2566 5234 Tm9)Tj0.09187 0 0 -0.09187 4987 6534 19(9)Tj0.09187 0 0 -0.09187 3081 68550709)Tj0.09187 0 0 -0.09187 3544 52351759)Tj0.0o187 0 0 -0.09187 3708 62152799)Tj0.0u187 0 0 -0.09187 4454 5885384(9)Tj0.0g187 0 0 -0.09187 2401 685 Tm99)Tj0.09187 0 0 -0.09187 5437 6859 Tm(e)889)Tj0.09187 0 0 -0.09187 5742 5885 31(9)Tj0.09187 0 0 -0.09187 3081 6855 Tm9)Tj0.0s187 0 0 -0.09187 5855 4587 Tm(.) 209)Tj0.09187 .09187 3034 4587 Tm(60.099)Tj0.0f187 0 0 -0.09187 6486 4911 Tz9)Tj0.0f187 0 0 -0.09187 6486 4911 809)Tj0.09187 .09187 3034 4587 Tm(6 Tm9)Tj0.0c187 0 0 -0.09187 6161 5885380(9)Tj0.09187 0 0 -0.09187 6268 6210 Tm(C)T69)Tj0.09187 0 0 -0.09187 5742 5886e)89)Tj0.0s187 0 0 -0.09187 5855 4587 Tm(67569)Tj0.0d187 0 0 -0.09187 6727 4911 T29)Tj0.09187 0 0 -0.09187 5742 588690m9)Tj0.0r187 0 0 -0.09187 4203 6535 Tm9)Tj0.09187 .09187 3034 4587 Tm(5 Tm9)Tj0.0c187 0 0 -0.09187 6161 5881 Tm9)Tj0.09187 0 0 -0.09187 3081 6857 Tm9)Tj0.09187 0 0 -0.09187 4987 6537 Tm9)Tj0.0y187 0 0 -0.09187 7291 5885 Tm(o)479)Tj0.0d187 0 0 -0.09187 6727 4917 Tm9)Tj0.09187 .09187 3034 4587 Tm(56T79)Tj0.0p187 0 0 -0.09187 7687 4911 Tm9)Tj0.09187 .09187 3034 4587 Tm(58Tm9)Tj0.0n0 -0.09187 3734 4262 Tm(7 Tm9)Tj0.0d187 0 0 -0.09187 6727 49180759)Tj0.0e187 0 0 -0.09187 2422 58881809
61
Only at 5 ºC the sucrose synthase activity remained high, regardless PVC
cover
ing
. The PVC film modified the atmosphere around the roots decreasing
water loss and associated with low vapor pressure deficit on storage conditions
can prolongate substantially the postharvest life of arracacha roots.
Furthermore, PVC cover reduces the water stress and consequently the
sucrose synthase activity except at 5 ºC, which enzyme seems to be induced by
chilling stress. It is possible that sucrose synthase is also involved on
the
sweetening process possibly associated with chilling injury although more
studies
on
the metabolism of carbohydrate and the involved enzymes as
invertases should be done to confirm this hypothesis.
62
1. INTRODUCTION
1.1
General aspects
The
arracacha
(
Arracacia
xant
horrhiza
Bancroft) also known in Brazil as:
batata
-aipo, batata-fiúza, batata-galinha, batata-salsa, batata-suíça, barão,
baroa, carotole, cenoura amarela, and
mandioquinha
(Santos and Carmo,
1998) belongs to the
Apiaceae
family, the same family of the carrot (Zanin and
Casali, 1984), is part of a group of plants which are rich in carbohydrates
destined for the human nourishment.
The roots are the main economic value product of the
arracacha
,
although
other parts of the plant also have an important nutritional and
medicinal value (Câmara, 1984a). The roots are considered an energetic food
due to its high
starch
content of easy digestibility, being
rich
in calcium,
phosphorus, fibers and vitamins of the C and A complex and, mainly in the B
complex (
niacin
).
Besides, it
shows diuretic properties and
can be characterized
as a
nutraceutic
food, being indicated for children, elders and athletes (Pereira,
2000).
Arracacha has its origin in the South American Ande
an
region
(Venezuela, Colombia, Ecuador, Peru and Bolivia) (Zanin and Casali, 1984),
mainly in the Colombian Andes (Casali and Sediyama, 1997). When the
Spanish conquered that region, the plant was already broadly used by the Incas
(Silva and Norma
nha, 1964).
The
arracacha
introduction did not occur in the European and North
American countries as the potato, for example. This was due to requirement of
mild temperatures during all the cycle and the aspects related to its origin of low
latitude in which the photoperiod is short and the growing season uninterru
pted
.
These conditions sometimes do not happen in those countries where there are
high
temperatures variations between summer and winter, interrupt
ing
the
arracacha
growing period, which is relatively long, making it impossible to
obtain eligible roots
(Ca
rra
s
quilla, 1944; Hodge, 1959; Léon, 1964).
The Nova Friburgo Baron
had
done the introduction of this vegetable in
Brazil around 1900’s from seedlings derived from the Antilleans, therefore
origin
of
the name
baroa
(Casali and Sediyama, 1997). Although the
re are reports that
63
the first seedlings introduced in Brazil came from Colombia and were offered to
the Agricultural Society by the Colombian General Rafael Uribe, in 1907, in his
journey through Brazil (Jaramillo, 1952).
Brazil is the
greatest
world
arrac
acha
producer (Reghin et
al., 2000), with
a cultivation area estimated in about 23
,
000 hectares
,
and a production
of
about
250 tons/year, being 95% of this volume consumed in natura, destining the rest
for the
food
industry (Zoonews, 2007). However, the productivity is still low with
a national a
verage around 8 to 9 t/ha (Bueno, 2004).
This
crop cultivation has expanded itself all over Brazil in regions with
altitudes
lower
than 100 meters, such as Goiás and Tocantins, although its
cultivation is still concentrated in the South-Center of the country where occurs
climatic conditions similar to its original
place
, mainly the areas of elevated
altitudes and mild climate of the Minas Gerais, São Paulo, Paraná, Santa
Catarina and Espírito Santo states. Minas Gerais and Paraná have been the
main producers states (Santos, 1994; Santos et. al., 2000). In the Paraná state
about 3000
small farm
er
families cultivate the
arracacha
(Zoonews, 200
7
).
Because it is a vegetable considered rustic, with a good tolerance to
dise
ases and plagues (Henz, 2002),
demading
little
costs
with
agricultural
inputs
,
the arracacha cultivation has predominated in small areas using the
family labor (Casali and Sediyama, 1997). Besides, the culture adapts well to
the organic cultivation and shows the possibility of various processing ways,
generating a good economic return to the small producers, who have in it an
important mean
of income (Santos, 1997; Bueno, 2004).
However, in spite of the potential that it presents, the
arracacha
is still
lit
tle explored and has a small national production, which corroborates to the
elevated commercialization prices in relation to the other vegetables (Scalon et
al., 2002).
1.2
Culture limitations
Some problems, however, are limiting
the
expansion
of
this culture, such
as: a relatively long cycle of 10 to 12 months (Henz et al., 1991) and the low
capacity of the roots to be preserved after harvest, with an
high
perish ability
and fast deterioration, having an average shelf life of
only
2
- 3
days
(Souza et
64
al
., 2003) and the maximal postharvest life of 6 days when kept without
packaging at room temperature (Thompson, 1980; Avelar Filho, 1989; Scalon et
al., 1998).
The high perish ability
,
characteristic of the
arracacha
roots also occurs
with
other roots (Wenham, 1995) as the cassava that shows a maximum shelf
life of the 2 days (Westby, 2002).
The deterioration of
the
arracacha roots can be increased by superficial
lesions and mechanical damages during the handling from the harvest until the
final destination (Souza et al., 2003), nutritional unbalance, root washing
(Zárate et al., 2001) and postharvest infections (Henz et al., 1991; Henz, 2002).
B
esides
, the physiologic
al
processes that accelerate the senescence, mainly
due to
the
respiration
and transpiration
(Avelar Filho, 1997). According to Avelar
Filho (1989) and Thompson (1980), the
perish
ability of the roots is mainly
related to the high water loss rate.
The search for techniques and/or production practices that are related to
the postharvest factors
aim
ing
the life product extension, is very important
(Zárate et al., 2001
).
Some
practices can be used such as: care
during
harvest and handling,
and the
use of
refrigeration
together with plastic films (Thompson 1980, Câmara
and Medina, 1983; Câmara
,
1984b;
Casali et al.
,
1988; Avelar Filho, 1989).
1.3
Refrigeration
The
use of low temperatures during the storage is considered the most
effective method for the vegetable products preservation (Serrano et al., 1996).
T
he
refrigerated
storage retards many deter
ioration
processes
in peris
hable
vegetal products, such as
ripening
, softening, changes in the texture and color,
undesirable metabolic exchanges, damages caused by microorganisms,
besides controlling and decreasing the
respiration
rates (Bachmann and Earl
es,
2000). Reduction of 10 ºC at temperature induces a decreasing of
2
- 3 times
in
the
respiration rate (Rizvi, 1981). The optimum refrigeration temperature can be
described as that able to retard the senescence and keep the quality without,
cau
sing
damag
e by cold or freezing (Zagory and Kader, 1988).
65
The refrigeration use in the
arracacha
root storage has been tested with
promising results. It is possible to keep the roots with an acceptable
appearance, despite 15% of weight loss, for up to two weeks
at
5 º
C
temperature
(
Câmara
,
1984b
).
However, Avelar Filho (1989) verified that the
medium and big sized
arracacha
roots kept a commercial quality only up to the
eight
-
day of storage at 5
ºC and 78% relative humidity.
In the case of
arracacha
, the refrigeration generates better results for the
post
harvest conservation when it is associated with the plastic film
wrap
(Câmara, 1984b; Casali et al., 1988; Avelar Filho, 1989; Avelar Filho, 1997;
Ribeiro, 2003).
Ribeiro (2003) verified the appearance of
injury
symptoms due to the
chilling
in
arracacha
roots when stored at 5 ºC. However, temperature of 10 ºC
propitiate
d the enlargement of the
postharvest
life without
ca
using symptoms of
damage by
chilling
. According to this author, the prolonged storage at low
temper
atures can also cause the sweetening phenomenon, which is observed
in potatoes
when
stored at low temperatures.
The biochemical alterations, which propitiate the sweetening and change
in flavor resulting from this, led Filgueira (1982) to affirm that the a
rracacha
would
not be good for the
conservation
by using refrigeration
process.
1.4
Chilling injury
Several
tropical and sub-
tropical
plant species origins and some from
temperate
regions
are d
amage
d by temperatures between 0 ºC and 12
o
C. T
he
chilling i
njury
occurs in these species at non freezing temperatures and, in
some cases, way above the freezing temperature. So that this phenomenon is
especially important in the
post
harvest handling and in the vegetal products
stora
ge for which the temperature is the most effective way to extend the
storage life (Kays, 1991).
The exposition of sensitive crops to the chilling injury, at non freezing
temperatures, causes a variety of symptoms that include abnormal maturing,
increase in water loss, “pitting” and superficial discoloration, internal darkening,
tissues rupture, flavor loss, increase in the ethylene and CO
2
production. T
hese
symptoms can occur during the storage or after the re-heating of the product
66
(Cabreira and Saltveit, 1990; Wang, 1993; Lelièvre et al., 1995). The severity of
the damage by
chilling
increases with the length of the storage period, as well
as with the decrease in the storage temperature (Mercer and Smittle, 1992).
Raison and Orr (1990) suggest that the chilling injury would occur in two
stages. One primary stage, in which would occur an increase in the calcium
cytoplasmic levels, a change in the regulatory proteins conformation, and
alterations in the cytoskeleton and a decrease in the
lipidic
membranes fluidity.
In a second
stage
it would occur a tissue rupture due to the cell wall rupture
induced by a
series of reactions due to the increase in the ethylene production.
In
arracacha
, Câmara (1984b)
showed
an inner darkening of the roots
after storage at 5
º
C.
The inner tissues darkening can be a result of the phenolic
compounds oxidation by enzymes, such as the peroxidase and
poliphenoloxidase
, producing kenones or similar components that polymerize
forming macromolecular complexes with amino acids and proteins which lead to
a formation of
pigmentation in the darkened tissues (Beaudry, 1999).
Ribeiro (2003), also studying the
arracacha
storage in low temperatures,
noticed the inner darkening and superficial lesions known as “pitting”.
1.
5
Sweetening
The roots and tubers rich in
starch
s
ub
mitted to low temperatures can
present a sweetening, which consists in an accumulation of the soluble sugars
during the storage due to
the starch
degradati
on (Hertog et al., 1997). In
potato
,
the accumulation of the reducing sugars determines the acceptability for the
processing (Sowokinos, 2001)
.
Then
when the raw and cut tuber is fried in oil at
a high temperature and
in
presence of high content of reducing sugars, there is
a reaction between these sugars and the free amino acids of the potato cells,
known as the Mailard reaction, which causes darkening due to caramel coloring
at
the final product (Lorberth et al., 1998; Beukema and Zaag, 1990). The
maximum content of the reducing sugars
tolerated
in the small tubers
that
mak
es possible obtaining
good
f
ried potatoes
is 0,2% (Melo, 199
9
).
The
arracacha
roots are a very energetic food, rich in
starch
, presenting,
in the 11
th
cycle month, around 83% of dry matter composed by total
carbohydrates, 78%
starch
and 3% sucrose (Câmara,
1984a)
. Similarly, Pereira
67
(2000) affirms that 80% of the total carbohydrates in the
arracacha
correspond
to
starch
and 6% of total sugars.
Due to these characteristics the
arracacha
presents alterations in its
carbohydrates composition during storage at low temperatures (Ribeiro, 2
003)
.
T
herefore,
it can be included among the vegetable products rich in
carbohydrates, and susceptible to sweetening caused by low temperatures as
occurs with
sw
eet potato (Picha, 1987) and
carrot (Phan, 1974).
The metabolic mechanism, responsible for the sweetening induced by
low temperatures, includes many key enzymes involved in the transition of
starch
to sucrose (Chi et al., 2004) and can develop an important role in the
osmoregulation
and
crioprotection
(Guy, 1990).
Initially, it is supposed that the
low temperatures stimulate the increase in
the activity of one or more enzymes of the starch degradation (Krause et al.,
1998). The
starch
phosphorilase would have a predominant role in
starch
mobilization (Davies and Viola, 1992), while the amylases woul
d act helping this
mobilization process (Cottrell et al., 1993). Therefore, the regulation of the
starch
degradation, and over all, the
accumulation
of
reducing sugars
in
potato
would be related to the sucrose cleavage (Chi et al., 2004).
The sucrose can be cleaved either by the invertase or by the sucrose
synthase. In the first case, the sucrose is transformed in glucose and f
ructose in
an irreversible way, while the sucrose synthase uses UDP and sucrose to form
UDP
-glucose and fructose, being this a reversible process (Dennis and
Blakeley, 2000)
In
arracacha
, Ribeiro (2003) observed the reducing sugars accumulation
in roots packed and stored at low temperatures. Besides, a negative correlation
between the non-reducing sugars and activity of the cell wall a
cid invertase,
this
enzyme demonstred to be the most active
enzyme
during the storage at cold
temperatures
. This author proposes that the acid invertase of the cell wall is the
main
sucrolytic
enzyme of the
arracacha
roots. S
he
also
suggests the need for
further studies with other enzymes involved in the process such as sucrose
synthase
and phosphate sucrose synthase.
68
1.6
Objectives
The present work aims to investigate the effect of storage temperature
and the PVC film cover on prolongation of postharvest life of arracacha roots
,
by
analyzing the
fresh
mass loss and the sucrose synthase activity during
storage period.
69
2. MATERIAL
S
AND METHODS
2.1 Harvesting and root preparing
The
arracacha
roots from the ‘Rocha de Viçosa’ clone harvested
between the 9
th
and 11
th
months of the cycle were obtained from producers
from the Ouro Branco r
egion
, Minas Gerais state. The roots were previously
washed and prepared by the producers by immersion in a solution containing
aniline (1.
6 g/L) and Kasugamycin
(400 m
L
/L)
.
The roots were taken to the Postharvest Laboratory at Plant Science
Department at Viçosa Federal University (UFV) and selected by size and
shape.
The deformed
roots by
damaged or
by
any kind of rot were discarded.
The roots were put in a polystyrene tray (24 x 18 x 2 cm) where they were
submitted to the treatments.
2.2 Experiment
The experiment was set in a complete randomized way with 4
re
petitions, being each
composed
by a polystyrene tray containing
two
arracacha
roots.
The experiment setting was done in a 4 x 2 factorial (4 temperatures x 2
“coverings”)
containing on sub-parcels the evaluation times along the storage
period
.
The storage temperatures were 5
º
C, 10
º
C, 15
º
C and room temperature.
To control the tray temperatures containing the roots, they were put in
appropriated chambers with controlled temperature, except for the one at room
temperature, in which the roots were maintained at laboratory bench.
At
each
temperature, part of the trays was wrapped with
a
PVC plastic film (10 µm) from
Conti
nental Brand and the other part was maintained uncovered. The
evaluation times vary according to the “covering” type, being
the
total analyses
time of 20 days. The evaluated parameters were: fresh mass loss, vapor
press
ure
deficit and the activity
of the
s
ucrose synthase
enzyme
.
The data were analyzed by descriptive statistic using the standard error
of the means on enzymatic assays, and analyses of variance and regression to
other assays.
70
2.3
Fresh m
ass l
oss
Th
e
fresh mass loss (FML)
was obtained by we
ighting the tray containing
the roots in an analytical weighing machine during the treatments, being
estimated, in percentage, by the formula below:
F
ML = [(PI
–
PF) x 100]
/PI
In which PI and PF correspond, respectively, to the
root
initial weight a
nd
to the
root
final weight
, respectively.
2.4 Vapor pressure d
eficit
The temperature and
relative
humidity
inside of the chambers and
laboratory
, where the roots were
stored,
were
monitored daily,
at
every hour, by
a portable measurer (WatchDog 150 Data Logger) with a temperature and
relative humidity sensor. The temperature (T) and relative humidity (RH) data
were used to
estimate
the vapor press
ure
deficit (VPD) of the air
,
where the
roots were stored, expressed in kPa, according to the equation proposed by
Jones (1992):
VPD = 0.
61137e
t
* (1
–
RH/100)
in which
“t”
is calculated by the equation:
t = 17.502* (T) / (240.
97 + T)
2.5 Determining of the sucrose
synthase
activity
Pieces
were
extracted from
the
central region of the arracacha roots
(3
cm from the apical superior) cut in small pieces, frozen in liquid nitrogen and
stored at
-
20
ºC. T
wo roots were used in each repetition to obtain the compound
sample.
2.5.1
Extraction
The extraction as well as the enzymatic assays was done according to
the
me
thodology described by Klotz and
Finger (2002), with modifications.
71
Arracacha
samples (1.5 g) were homogenized in 8 mL of extraction
buffer
(50 mM HEPES-NaOH pH 7.2, 5 mM -
mercaptoetanol,
10 mM Na
2
SO
3
and 1 mM MgCl
2
) with an Ultra-TURRAX T25 (IKA - WERKER). Then the
homogenized was filtered in a 10 mL syringe contained 4
cm
3
of cheese cloth
and centrifuged at 17,000 x g for 30 minutes in a centrifuge (Hitachi CR21) with
a R20A2 rotor. The resulting supernatant was dialyzed with more than 20
volumes of dial
yzes
buffer
(10 mM
HEPES
- NaOH pH 7.2, 1 mM -
mercaptoetanol and 1mM MgCl
2
) for 12 hours
under
agitation. The dialyzed
was transferred to microcentrifuge tubes and centrifuged again at 14,000 rpm
for 10 minutes, with the resulting supernatant being separa
ted for the enzymatic
assay
and protein analyses. All the extraction stages were done at 4 ºC or
on
ice.
2.5.2
Sucrose synthase activity
The determination of the sucrose synthase activity in the sucrose
cleavage assay was measured by the incubation of 50 L extract containing
250 mM
sucrose,
2mM UDP and 100mM MES-HCl (pH 6.5) for 30 minutes at
35
ºC. Fructose was quantified by the Nelson method (
Nelson,
1944) as
described below:
The enzymatic reaction was
stopped
with 200 L of the cupric alkaline
reactiv
e of Nelson. Then the tubes were incubated with boiling water for 15
minutes and cooled in cold water, being then added 200 L of the solution
resulting from the mixture of 0.75 M H
2
SO
4
, with the solution
arsenomobilidic
in
a 2:1 proportion.
Following,
60
0 µL
distill
ed
water was added and the
absorba
nce reading was done at 520 nm in a Shimadzu UV-
1601
spectrophotometer.
The enzymatic activity was expressed in fructose
mol h
-1
for
mg protein.
The total protein concentration was determined in the enzymatic extract using
the Bradford method (
Bradford,
1976) and bovine serum albumin (
BSA
) as a
standard
.
72
3
. RESULTS AND DISCUSSION
3
.1
Fresh mass loss
and vapor pressure deficit
The effect of the storage temperature over the mass loss of arracacha
roots was demonstrated for roots wrapped with PVC film (Figure 1). Under all
tested temperatures the loss of fresh mass was linear with the storage period.
At room temperature, roots show the biggest rate of fresh mass loss during
storage period. The rate of fresh mass loss decreased with temperature
reaching the lowest rate at 5 ºC. However, the rate of fresh mass loss did not
decrease proportionally with temperature reduction. While at 5 ºC and 10 ºC the
rate of fresh mass loss was similar, respectively 0.238% and 0
.257%
per
day, at
15 ºC it was about two fold greater (0.5% per day).
0
2
4
6
8
10
12
14
16
18
0 2 4 6 8
10 12 14 16
Figure 1 – Estimation of the fresh mass loss of arracacha roots covered with
PVC films in function of the storage time at temperatures of 5 ºC, 10 ºC, 15 ºC
and at room temperat
ure (RT)
.
The loss of mass at postharvest of fresh vegetables is affected by the
sum of two factors: the loss of water by transpiration and the loss of dry matter
during respiration process (Finger and Vieira, 1997). Both transpiration and
respiration are
affected by temperature (Chitarra and Chitarra, 1990).
-- RT PVC
-- 15 PVC
-- 10 PVC
-- 5 PVC
RT PVC: Y = 1.0757X, r
2
= 0.9953
15 PVC: Y = 0.5060X, r
2
= 0.9999
10 PVC: Y = 0.2573X, r
2
= 0.9971
5 PVC: Y = 0.2383X, r
2
= 0.9951
RT PVC: Y = 1.0757X, r
2
= 0.9953
15 PVC: Y = 0.5060X, r
2
= 0.9999
10 PVC: Y = 0.2573X, r
2
= 0.9971
5 PVC: Y = 0.2383X, r
2
= 0.9951
Fresh mass loss (%)
Storage period (days)
73
In fact, transpiration is consequence of the vapor pressure deficit which
is dependent of the temperature and relative humidity (Almeida, 2005).
To understand the process governing transpiration by the roots, it was
calculated the vapor pressure deficit of the air (VPD) surrounding the PVC
covered roots by mensuration of the temperature and relative humidity inside
chambers and on laboratory where the roots were stored.
The averages of temperature, relative humidity and VPD after 16 days of
storage conditions are shown on Table 1.
Table 1 – Averages of the temperature (T), relative humidity (RH),
temperature
coefficient
(t) and vapor pressure deficit on the surrounding air
(VPD)
inside of
the
storage chambers, and total
fresh
mass loss (FML) of the
arracacha
roots
covered with PVC films after 16 days of storage
.
T (ºC)
RH
(%)
t
VPD (kPa)
ML (%)
5
60
0.34
0.34
3.7
10
76
0.72
0.31
4.1
15
61
1.04
0.66
8.0
23
**
44
1.49
1.51
16.8
**Represent
s
the averag
e temperature of the laboratory room temperature (RT).
The results of VPD explain why the mass loss was not proportional to
temperature variation. It is happened due oscillation in the relative humidity on
store environment that generated divergent VPD.
The average VPD on storage conditions (room temperature, 15 ºC, 10 ºC
and 5 ºC) and total fresh mass loss of the roots after 16 days of storage showed
to be linearly related (Figure 2).
Therefore, in roots covered with PVC the fresh mass loss rate was
pro
portional to VPD. The PVC film
presumably
affected the gases and water
vapor concentration on air in contact with the roots, inhibiting respiration and
controlling water vapor diffusion, and consequently root transpiration.
The PVC, poly(vinyl chloride), is produced by polymerization of vinyl
chloride monomer. A range of PVC films with widely varying properties can be
obtained from the basic polymer (Robertson, 1993). PVC has good gas barrier
property and moderate barrier to moisture vapor (Greengrass, 1993), and it is
among the most commonly types of film used in the preservation and marketing
74
of fresh vegetable products (Riquelme et al., 1994). Furthermore, thin
plasticized PVC film is widely used in supermarkets for the stretch wrapping of
trays containi
ng produce (Robertson, 1993).
r
2
= 0.9962
0
2
4
6
8
10
12
14
16
18
0.0 0.2 0.4 0.6 0.8
1.0 1.2 1.4 1.6
Figure
2 – Relation
among
average values of vapor pressure deficit inside of
storage chambers and accumulated mass lost of
arracacha
roots covered with
PVC films after 16 days of storage.
In the roots stored without PVC film cover, fresh mass loss was affected
mainly by temperature of the storage conditions due the absence of physical
barrier to control gases and water vapor loss.
The effect of the storage temperature during six days of storage is shown
in Figure 3. At room temperature, the roots lost mass strongly in a quadratic
behavior with the storage period. A similar model of mass loss was observed at
15 ºC. However, at 5 ºC and 10 ºC, the fresh mass loss of the roots was linear
with storage period.
The non linear mass loss of roots in function of the storage period, at 15
ºC and room temperature, was probably due high percent of fresh mass loss,
higher than 15% for both treatments. Similar results were obtained with carrots
that reached 40% of fresh mass loss (Caron et al., 2003). This non-
linear
behavior is explained by the mechanism of water loss that is the main
component of mass loss in roots. When the root tissue is turgid, the water is lost
Vapor pressure deficit (kPa)
Fresh mass loss (%)
75
easily by the tissue in consequence of the high VPD
air
-
tissue
. With decrease of
water inside tissue, matric potential decreases due to high concentration of
starch and cell walls components, reducing water potential in tissue. Then water
loss rate decreases proportionality to diminution of VPD
air
-
tissue
(Galindo et al.,
200
4).
0
5
10
15
20
25
30
35
40
45
0 1 2 3 4 5 6
Figure
3 –
Estimation
of
the
fresh
mass loss
of
arracacha
roots
without
PVC
films
cover
in function of the storage
period
at temperatures of 5 ºC, 10 ºC, 15
ºC and at room temperature (RT)
.
As observed with PVC covered roots, the mass loss rate of uncovered
roots was not proportional to storage temperature (Figure 3). The averages of
DPV for the first six storage days for different treatments were calculated (Table
2). The initial VPD was smaller than that observed to the averages from 16
sto
rage days (Table1) due the highest relative humidity in the first days of
storage (Table 2).
Conversely, for the roots covered with PVC, the fresh mass loss was not
governed mainly by VPD and it was not possible to verify a linear relation
between VPD valu
e and fresh mass loss (Figure 4).
-- RT
-- 15
-- 10
-- 5
Fresh mass loss (%)
Storage period (days)
RT: Y
= 10.846X
- 0.749X
2
, R
2
= 0.9988
15 : Y =8.507X – 0.4929X
2
, R
2
= 0.9958
10 : Y = 3.2524X , r
2
= 0.9865
5 : Y = 2.4585X, r
2
= 0.9792
RT: Y
= 10.846X
- 0.749X
2
, R
2
= 0.9988
15 : Y =8.507X – 0.4929X
2
, R
2
= 0.9958
10 : Y = 3.2524X , r
2
= 0.9865
5 : Y = 2.4585X, r
2
= 0.9792
RT: Y
= 10.846X
- 0.749X
2
, R
2
= 0.9988
15 : Y =8.507X – 0.4929X
2
, R
2
= 0.9958
10 : Y = 3.2524X , r
2
= 0.9865
5 : Y = 2.4585X, r
2
= 0.9792
RT: Y
= 10.846X
- 0.749X
2
, R
2
= 0.9988
15 : Y =8.507X – 0.4929X
2
, R
2
= 0.9958
10 : Y = 3.2524X , r
2
= 0.9865
5 : Y = 2.4585X, r
2
= 0.9792
76
Table
2 – Average of temperature (T), relative humidity (RH), temperature
coefficient
(t) and vapor pressure deficit of the surrounding air (VPD) in storage
chambers, and total
fresh
mass loss (FML) of the
arracacha
root
s
without PVC
films cover
after
6
days of storage
.
T (ºC)
RH
(%)
t
VPD (kPa)
F
ML (%)
5
67
0.33
0.28
13.8
10
81
0.72
0.24
18.6
15
67
1.02
0.57
33.5
22
**
50
1.44
1.30
38.4
**Represent
s
the average temperature of the laboratory room temperature (RT).
R
2
= 0.8618
0
5
10
15
20
25
30
35
40
45
0.0 0.2
0.4
0.6 0.8
1.0 1.2
1.4
Figure
4 - Relation among average values of vapor pressure deficit
inside of
storage chambers and accumulated
mass los
s
of
arracacha
roots
without PVC
cover
after
6
days of storage.
It is probable that without the modified atmosphere generated by PVC
film (Kader et al., 1989), variation in the temperature could produce more visible
metabolism alteration with the rise of temperature. Then respiration process
could be acting synergistically with transpiration modifying the linear relation
between VPD and fresh mass loss like the observed in PVC covered roots
(Figure 2). This can be exemplified by comparison among 6 days VPD average
Vapor pressure deficit (kPa)
Fresh mass loss (%)
77
and total mass loss at temperatures of 5
o
C and 10
o
C. A
lthough
VPD
generated at 5 ºC (0.28 kPa) was bigger than at 10 ºC (0.24 kPa) the total
fresh
mass loss of the roots was bigger at 10 ºC (18.6%) than at 5 ºC (13.8%)
demonstrating that other factor above the DPV also affected the
fresh
mass
loss with the raise of temperature.
The final effect of cover treatment (with and without PVC film) in each
temperature condition is shown in Figure 5.
Treatments
0
5
10
15
20
25
30
35
40
45
PVC No PVC
A
a
b
Rrro tmotmm
78
fresh mass loss (Figure 5A). After 8 days of storage at 15 ºC, roots covered with
PVC film lost only 3.9% of the fresh mass in comparison with uncovered roots
that lost more than 40% of fresh mass at end of same period (Figure 5B). After
12 days of storage at 10 ºC uncovered roots lost almost 34% of fresh mass,
while PVC covered roots had 11 fold less of mass loss (Figure 5C). Finally, at 5
ºC treatment after 16 days of storage, roots covered with PVC film
lost 89% less
of fresh mass than uncovered roots (Figure 5D).
The
arracacha roots lose weight rapidly after harvest and as a result of
this become soft and discolored (Thompson, 1980). For Avelar Filho (1989) the
water loss is the main responsible cause for mass loss in arracacha roots on
postharvest period since the respiration rate in these roots is low. The water
loss is preponderant in arracacha roots due the absence of suberification of the
periderm cells (Bustamante, 1994). After harvest, roots maintain a permanent
water loss being
withered
, shrunken, or mummified and then being rejected by
the consumers (Avelar Filho, 1997).
Generally the loss of only 5 - 10% moisture renders a wide range of
products unsalable (Kays, 1991). In a visual evaluation, Avelar Filho (1989)
observed that arracacha roots were useless to consume when reached 15%
mass loss independent of the root size.
Carrot roots stored at about 24 ºC and 65% of relative humidity lost
almost 24% of fresh mass after 4 days of storage (Oliveira et al., 2001). In
arracacha,
Bustamante (1994) observed that roots lost 10 - 21 % of fresh mass
in different clones after 4 storage days at 14 - 19 ºC. Scalon et al. (2002)
verified
losses close
to
19% on roots after 4 storage days at
35
ºC and 80%
relativ
e humidity.
3.2 Activity of sucrose synthase
The effect of storage temperature on sucrose synthase (Susy) activity of
arracacha roots covered with PVC in function of the storage period is shown in
Figure 6. In the first storage day Susy activity showed a slight increase at all
temperatures, except at room temperature. After, the activity of Susy had about
the same behavior for roots stored at room temperature and at 15 ºC, in which
Susy activity decreased until the forth day of storage reaching a stabili
zation
79
with only about 34% of the initial activity. In roots stored at 10 ºC the Susy
activity decreased slowly reaching the same activity level of those above
mentioned after 8 storage days and maintaining the same activity afterwards. In
roots stored at 5 ºC the Susy activity decreased on the second day and was
maintained by about 70% of the initial activity on subsequent storage days.
0
1
2
3
4
5
6
7
8
9
10
11
12
0 2 4 6 8
10 12 14 16 18 20
RT PVC
15 PVC 10 PVC
5 PVC
Figure
6 – Sucrose synthase activity of
arracacha
roots covered with PVC films
in function of the storage time at te
mperatures of 5 ºC, 10 ºC, 15 ºC and
at
room
temperature (RT).
Vertical bars
correspond to the
standard error
s.
Susy activity in roots covered with PVC film at different temperatures
after 16 days of storage were compared with initial activity (before storage) and
indicated a
decrease
of activity at all storage temperature (Figure 7). However,
at room temperature, 15 ºC and 10 ºC the Susy activity was 2.1 - 2.8 µmoles
fructose for hour per mg protein while at 5 º C the activity was 2 fold greater
than the last (5.6 µmoles fructose for h
our per mg protein).
Fructose (µmole h
-1
(mg protein)
-1
)
Storage period (days)
80
0
1
2
3
4
5
6
7
8
9
10
BS
RT PVC
15 PVC 10 PVC
5 PVC
Figure
7 - Sucrose synthase activity of arracacha roots covered with PVC film
after 16 days of storage in function of the storage temperature at 5 ºC, 10 ºC, 15
ºC and room temperature (RT). BS is the control: sucrose synth
ase
activity
on
roots
before storage treatments. Vertical bars correspond to the standard
error
s.
The effect of temperature on Susy activity of roots stored without PVC
during storage time can be observed on Figure 8. After the first storage day
Susy activity had a slight increase at all temperature except at room
temperature. At room temperature Susy activity was maintained about the same
of the initial activity until the third day, showing a decrease on fourth storage
day. At temperature of 15 ºC, 10 ºC and 5 ºC the Susy activity demonstrated
oscillations during storage time, but it was maintained in about 87% of the initial
activity.
The Susy activity in roots without PVC film cover, at different
temperatures after 6 days of storage compared with initial activity (before
storage) indicated a establishment of the initial activity with small reduction
during the storage time, except for roots stored at room temperat
81
0
1
2
3
4
5
6
7
8
9
10
11
12
13
0 1 2 3 4 5 6 7 8
RT
15
10
5
Figure
8 –
Sucrose synthase activity of
arracach
a
roots
without
PVC films
cover
in function of the storage time at temperatures of 5 ºC, 10 ºC, 15 ºC and
room temperature (RT).
Vertical bars
correspond to the
standard error
s.
0
1
2
3
4
5
6
7
8
9
10
BS
RT
15 10
5
Figure
9 - Sucrose synthase activity of
arrac
acha
roots
without PVC
film
s cover
after
6
days of storage in function of the storage temperature
s
at 5 ºC, 10 ºC, 15
ºC and room temperature (RT). BS is the control: sucrose synthase
activity
on
roots
before storage treatments. Vertical bars correspond to the standard
error
s.
Fructose (µmole h
-1
(mg protein)
-1
)
Storage period (days)
Fructose (µmole h
-1
(mg protein)
-1
)
Treatments
82
These results indicate that maximal Susy activity at room temperature
was stimulated by the high water stress condition with VPD of 1.3 kPa (Table
2). The Susy activity was maintained by the continuous water stress until third
stor
age day (Figure 8) when reactions of oxidation and mummification of tissue
with
deterioration of part of the root cortex generated a falling down on Susy
activity.
The temperature was not a preponderant factor on Susy activity since
the reduction of storage temperature did not reduce or increase substantially
the enzyme activity. However, the stress condition caused by water loss seems
to be the determinant factor on Susy activity. Similarly, no variation was found in
Susy activity during storage of sugarbeet roots at 6 ºC, 12 ºC and 21 ºC (Klotz
and Finger, 2004). Furthermore, increase of Susy activity was associated with
dehydratation of sugarbeet roots during the storage period (Sakalo and Tyltu,
1997).
The effect of cover treatment (with and without PVC
film) on Susy activity
inside of each storage temperature in function of the storage time is shown in
Figure 10.
In roots covered with PVC film at room temperature, 15 ºC and 10 ºC, it
was verified reduction of susy activity when compared with uncovered roots at
the same temperature (Figure 10A, B and C). This, again, indicates the
important role of the PVC film in reduction of the water loss. The direct
consequence of this was the decrease of the Susy activity, since root stress
was lessened.
However, at 5 ºC the reduction of the water loss by PVC film did not
affected Susy activity that maintained an activity level next of that observed on
uncovered roots (Figure 10D). This indicates that despite of water stress
decrease by PVC effect, the Susy activity r
emained high.
It is possible that the induction of Susy activity at 5 ºC storage condition
may be related with chilling injury process as previously observed in arracacha
roots in storage at low temperature (Ribeiro et al., 2005 ).
Câmara (1984b) observed chilling injury but only on roots storage at low
temperatures and uncovered with plastic film. It is possible that plastic film
cover acts like a minimizer agent of the chilling injury effects, lessening activity
of oxidative enzymes by reducing water lo
ss and gas changes.
83
Figura
10
- Effect of the “covering” treatment (PVC or without PVC) on activity
of sucrose synthase of arracachas roots in function of storage period at room
temperature (A), 15 ºC (B), 10 ºC (C) an
d 5 ºC (D). Dark blue lines indicate PVC
treatment and red lines indicate no PVC cover. Vertical bars correspond to the
standard error
s.
Sucrose synthase can be still associated to sweetening process that can
occur on sugarbeet roots. According to Avelar Filho (1989), the
arracacha
roots
stored for 20 days at 5 ºC show a mildly sweet flavor. Ribeiro (2003) observed
remarkable
fall in the
starch
content and an increase in the soluble
Fructose (µmole h
-1
(mg protein)
-1
)
0
2
4
6
8
10
12
0 1 2 3 4 5 6
RT PVC
RT
A
Fructose (µmole h
-1
(mg protein)
-1
)
0
2
4
6
8
10
12
14
0 1 2 3 4 5 6 7 8
15 PVC
15
B
0
2
4
6
8
10
12
14
0 2 4 6 8
10
12
10 PVC
10
Fructose (µmole h
-1
(mg protein)
-1
)
C
0
2
4
6
8
10
12
14
0 2 4 6 8
10
12 14
16
5 PVC
5
Fructose (µmole h
-1
(mg protein)
-1
)
D
Storage period (days)
Storage period (days)
Storage period (days)
Storage period (days)
Fructose (µmole h
-1
(mg protein)
-1
)
0
2
4
6
8
10
12
0 1 2 3 4 5 6
RT PVC
RT
A
Fructose (µmole h
-1
(mg protein)
-1
)
0
2
4
6
8
10
12
14
0 1 2 3 4 5 6 7 8
15 PVC
15
B
0
2
4
6
8
10
12
14
0 2 4 6 8
10
12
10 PVC
10
Fructose (µmole h
-1
(mg protein)
-1
)
C
0
2
4
6
8
10
12
14
0 2 4 6 8
10
12 14
16
5 PVC
5
Fructose (µmole h
-1
(mg protein)
-1
)
D
Storage period (days)
Storage period (days)
Storage period (days)
Storage period (days)
84
carbohydrates content along the 21 storage days at 5 ºC and at 10 ºC, in two
clones of
arracacha
.
Sucrose synthase and acid invertases can be induced by cold stress and
are related with sweetening on potato (Chi et al., 2004).
Sucrose synthase activity in roots stored at 5 ºC can be induced by
chilling injury process producing sweetening during root storage. This
hypothesis should be studied on light of chilling injury process and carbohydrate
metabolism at low temperatures in arracacha roots to reveal the true role of
sucrose synthase as
well as other enzymes on these
process
es.
3
.3 Diseases incidence
Despite of antibiotic treatment of roots by the producer, it was observed
incidence of soft rot and some saprophytic fungus attack. Samples of infected
roots were sent to the Fitossanitary Diagnostic Laboratory – UFV to obtain
the
correct identification of pathogens infecting the roots.
The saprophytic fungus growth only on apical pole of the roots, in small
lesions on root periderm or associated with soft rot. The fungus appeared on
restrict areas mainly on roots stored without PVC cover at root temperature, 15
ºC and 10 ºC. More extention of mycelial mass was found on PVC covered
roots at room temperature and at 15 ºC, sometimes associated with soft rot
lesions.
The fungi identified on contaminated samples were
Rhizopus
sp. an
d
Penicillium
sp. Rhizopus negricans was reported as a causal agent of
postharvest diseases on arracacha roots being considered amongst the most
aggressive fungi that infected this crop (Henz, 2002). Infection by this fungus is
characterized for specific soft rot and a dark mycelium covering the root (Lopez
and Henz, 1997). It was not the kind of fungus infection verified during this
experiment.
Soft rots were verified after the second day of storage only on roots
covered with PVC film at room temperature, affecting many roots during the
experiments. Some soft rot were also observed in roots covered with PVC and
stored at 15 ºC, but in smaller proportions than that observed at room
temperature. Soft rot was caused by
Erwinia carotovora
subsp.
carotovora
.
85
So
ft rot in
arracacha
roots usually occurs during summertime, which
coincides
with
warmer temperatures in Southeastern Brazil causing severe
losses (Henz et al.,
200
6). It is the most important disease on arracacha
postharvest causing considerable losses during transport, storage and trade of
the roots. Furthermore, the development of this disease is direct associated with
hot and humid environment (Lopez and Henz, 1997).
86
4. CONCLUSION
The use of PVC film for covering roots during storage was efficient to
prolongate the postharvest life of roots.
Low temperature was important in the preservation of root quality in
postharvest since it was associated with high relative humidity to produce a low
vapor pressure deficit.
Fresh mass loss was significantly reduced when associated with low
vapor pressure deficit and PVC cover.
Sucrose synthase activity was induced by stress conditions of the
environment storage occasioned mainly by water loss, and the activity of this
enzyme decreased with reduction of the
fresh
mass loss. It suffered little
influence of the storage temperature except at 5 ºC in which roots were sensible
at chilling injury.
Sucrose synthase activity seems to be induced by chilling injury process
making part of the sweetening process that occurred w
ith arracacha roots at low
temperatures.
87
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ZOONEWS
– NOTÍCIA.
Mandioquinha
-salsa já tem cartilha de
pad
ronização.
Web address: <http://www.zoonews
.com.br>.
Consulted in:
March,
10
, 200
7.
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