MINISTÉRIO DA EDUCAÇÃO
UNIVERSIDADE FEDERAL DE PELOTAS
FACULDADE DE AGRONOMIA ELISEU MACIEL
PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIA E
TECNOLOGIA DE SEMENTES
TESE
CARACTERIZAÇÃO DE GENÓTIPOS DE ARROZ SUBMETIDOS AOS
ESTRESSES DE FRIO E PROFUNDIDADE DE SEMEADURA
CAROLINE BORGES BEVILACQUA
PELOTAS, 2013
CAROLINE BORGES BEVILACQUA
CARACTERIZAÇÃO DE GENÓTIPOS DE ARROZ SUBMETIDOS AOS
ESTRESSES DE FRIO E PROFUNDIDADE DE SEMEADURA
Tese apresentada à Faculdade de Agronomia “Eliseu
Maciel”, Universidade Federal de Pelotas, sob a
orientação do Prof. Dr. Paulo Dejalma Zimmer,
como parte das exigências do Programa de PósGraduação em Ciência e Tecnología de Sementes,
para obtenção do título de Doutora em Ciências.
Orientador: Prof. Dr. PAULO DEJALMA ZIMMER
Co-orientadora: PhD. NILDA ROMA BURGOS – University of Arkansas (EUA)
PELOTAS, 2013
Dados de catalogação na fonte:
(Gabriela Machado Lopes – CRB: 10/1842)
B571c Bevilacqua, Caroline Borges
Caracterização de genótipos de arroz submetidos aos
estresses de frio e profundidade de semeadura. /
Caroline Borges Bevilacqua; orientador Paulo Dejalma
Zimmer – Pelotas, 2013.
93 f. :il
Tese (Programa de Pós-graduação em Ciência e
Tecnologia de sementes). Faculdade de Agronomia
Eliseu Maciel, Universidade Federal de Pelotas.
Pelotas, 2013.
1. Estádios iniciais; 2. qRT-PCR; 3. Arroz
vermelho; 4. Esterase. I. Zimmer, Paulo Dejalma
(orientador); II. Título.
CDD 633.18
CARACTERIZAÇÃO DE GENÓTIPOS DE ARROZ SUBMETIDOS AOS
ESTRESSES DE FRIO E PROFUNDIDADE DE SEMEADURA
AUTORA: Caroline Borges Bevilacqua, M.Sc.
ORIENTADOR: Prof. Paulo Dejalma Zimmer, Ph.D.
BANCA EXAMINADORA
Prof. PAULO DEJALMA ZIMMER, Dr., UFPel
(Orientador)
Profa LIA REJANE SILVEIRA REINIGER, Dra, UFSM
NACIELE MARINI, Dra, UFPel
Engo Agro GERI EDUARDO MENEGHELLO, Dr., UFPel
ANDREIA DA SILVA ALMEIDA, Dra, UFPel
Prof. LUCIANO CARLOS DA MAIA, Dr., UFPel
Aos meus amores: meus pais Paulo Cezar
Bevilacqua
(in
memorian)
e
Marta
Lúcia
Bevilacqua eminhas irmãs Letícia e Amanda
Borges Bevilacqua
Dedico...
AGRADECIMENTOS
Agradeço a Deus e ao meu pai por terem me guiado.
Aos meus pais, Paulo Cezar Bevilacqua e Marta Lúcia Bevilacqua, por tudo.
As minhas irmãs Letícia e Amanda Bevilacqua e ao meu “irmão”, meu cunhado
Ormuz Neto, pelos conselhos, amor e apoio.
À Universidade Federal de Pelotas e ao meu orientador pela oportunidade de
realização do doutorado.
Ao Dr. Luis Avila por se dispor a entrar em contato e me apresentar para a Dr. Burgos
e assim permitir a realização do meu Doutorado Sanduíche. Além de sempre manter contato e
se preocupar como eu estava nos Estados Unidos.
A Dr. Nilda Roma Burgos por grandiosamente me guiar, orientar, planejar e discutir
os resultados dos experimentos realizados nos Estados Unidos. Além de todo o apoio,
amizade e ensinamentos. Thank you very much Dear Dr. Burgos. I also want to thank all my
coworkers in USA: Hussain, Vijay, Shelpa, Reio, Fernando, Ana Carolina and Jun.
Thanks my sweeties, hard workers and my great friends Dr. Coy Batoy and Dr. Paul
Tseng, I miss you guys, a lot!!! Hope “cya” soon, “dudes”!!
A todos os amigos que fiz nos USA que de alguma forma me ajudaram e me fizeram
bem…Igor mimoso, Bruna, Sergio, Montse, Alejandro, Dimitra, Bia, Fabio, Buri,
Gra...enfim…muitos amigos queridos! E em especial queria agradecer a uma grande amiga
que considero uma irmã de coração Cris Pilon, obrigada por todo apoio, carinho, amizade,
puxões de orelha e gargalhadas...
Hey, Fernando, Lara, Ana, Bea e Cris...nunca vou esquecer aquela madrugada de
“32°F” que vocês me esperaram chegar de viagem só pra me darem “O” abraço que eu
precisava.
Ao Dr. Luciano Maia pelo auxílio nas analises dos experimentos.
Aos Dr. Antonio Costa de Oliveira e Dr. Odir Dellagostin por concederem espaço e
equipamentos indispensáveis à realização dos experimentos.
Aos Dr. Andy Pereira e Dr. Supratim Basu por contribuírem de forma grandiosa desde
o planejamento até a análise dos resultados dos experimentos realizados nos Estados Unidos.
Às colegas Cristiane Brisolara, Daisy e Glacy Silva pela amizade, descontração,
companheirismo, tantos pousos, choros, risadas...Cris, meus estudos para a qualificação sem
tua presença, cafés e explicações seriam bem mais pesados!! Enfim, minha estada em Pelotas
foi mais alegre com vocês!
Aos colegas Carol Terra Borges, Silvana e Eduardo Venske pelo auxílio nos
experimentos.
Ao tio Marco, tio Luiz, tio Beto e tio Salla e às tias Tania, Gilda, Alexia e Bete pelo
super apoio, vocês todos moram no meu coração!
Aos meus avós pela força e exemplo!
Às minhas grandes amigas Babi, Livia e Nati, por me apoiarem tanto e estar comigo
sempre!
Ao pessoal do Laboratório de Sementes e Biotecnologia, por disponibilizar a
realização efetiva dos experimentos.
À Maya Beatriz por ficar ao meu lado durante a escrita da tese.
Ao CNPq pelo auxílio financeiro que possibilitou a realização deste trabalho.
E a todos que, também, contribuíram para que eu concluísse mais essa grande etapa
em minha vida.
RESUMO GERAL
BEVILACQUA, Caroline Borges. Caracterização de genótipos de arroz submetidos aos
estresses de frio e profundidade de semeadura. 2013. 97f. Tese (Doutorado em Ciência e
Tecnologia de Sementes) – Programa de Pós-Graduação em Ciência e Tecnologia de
Sementes, Universidade Federal de Pelotas, Pelotas, RS
O estresse causado pelo frio interfere negativamente na fisiologia, metabolismo, crescimento
e desenvolvimento das plantas e, portanto, limita a produtividade em lavouras de arroz. As
respostas em nível de crescimento em arroz (Oryza sativa L.) submetido a baixas
temperaturas ainda são pouco compreendidas. Um melhor entendimento do mecanismo de
tolerância ao estresse em plantas de arroz pode ajudar na identificação, no germoplasma de
arroz, de plantas com tolerância submetidas à temperatura variável, além de ser útil para
outros estresses abióticos, como diferentes profundidades de semeadura. Para caracterizar
genótipos de arroz, com variação na sensibilidade ao frio, tiveram-se como objetivos:avaliar a
aplicabilidade de diferentes índices de estresse utilizando-se como parâmetro o comprimento
de plântula; classificar acessos de arroz cultivado e vermelho como Japonica ou Indica;
comparar a resposta ao frio de cultivares de arroz tolerante e sensível a esse estresse, com
relação ao acúmulo de massa seca e possíveis alterações no teor de clorofila;categorizá-los
com relação à sensibilidade ao frio e à profundidade de semeadura; e analisar a expressão de
genes que respondem a frio, assim como genes responsivos a submersão, sob condições de
frio e/ou tratamento constituídos por diferentes profundidades de semeadura. Para avaliar o
acúmulo de massa seca e o teor de clorofila, as sementes, após sete dias a 25°C, foram
expostas a 4°C durante 24 h e logo após, foi medida a fotossíntese e,posteriormente, as
plantas ficaram 72 h a 25°C para sua recuperação. Já para os demais experimentos,as
plântulas foram coletadas 7 e/ou 14 dias mantidas a 25°C ou 18/13°C dia/noite e diferentes
profundidades de semeadura (1.5cm, 5cm, 10cm e 15cm); as avaliações da expressão gênica
diferencial foram realizadas com essas amostras coletadas, para 4 diferentes genes induzidos
pelo frio e também em amostras coletadas após exposição a 10°C durante 6, 24 e 96 h a 1.5
cm e 10 cm de profundidade de semeadura.Os resultados indicaram que é possível a
identificação de genótipos superiores para a tolerância a esses estresses abióticos com base em
seus índices de estresse, utilizando como parâmetro o comprimento da parte aérea, devido a
habilidade das plantas tolerar estresses abióticos afetar a morfologia assim como a fisiologia
da planta de arroz. Assim como é possível a utilização do Índice de Tolerância (STI) e da
Média Geométrica (GM) para selecionar genótipos tolerantes ao frio ou profundidade de
semeadura, baseado no comprimento de parte aérea de plântula. As subespécies Japonica e
Indica respondem diferentemente aos estresses abióticos, no entanto, para alguns genes
responsivos a esses estresses, essas subespécies apresentam o mesmo respondem
semelhantemente. Além disso, as análises a nível molecular da tolerância ao frio e a
profundidade de semeadura indicaram a importância das vias ABA-dependente e ABAindependente como vias de transdução do sinal em plantas sob estresse abiótico.
Palavras-chave: estádios iniciais; qRT-PCR; esterase; arroz vermelho.
ABSTRACT GENERAL
BEVILACQUA, Caroline Borges. Characterization of rice genotypes under cold and deep
sowing stresses. 2013. 97f. Tese (Doutorado em Ciência e Tecnologia de Sementes) –
Programa de Pós-Graduação em Ciência e Tecnologia de Sementes, Universidade Federal de
Pelotas, Pelotas, RS
Cold stress adversely modifies their physiology, metabolism plant growth and development,
as well as, it limits crop productivity. The responses of rice (Oryza sativa L.) subjected to low
temperatures are still poorly understood. A better understanding of stress tolerance
mechanism in rice plants will help to develop rice germplasm with improved field level
tolerance under variable temperature and sowing depth conditions. To characterize rice
genotypes with variation in sensitivity to cold, these are the following objectives: to evaluate
the applicability of different Stress Indices using seedling lengthas parameter; classify
accessions cultivated rice and red rice as Indica or Japonica; compare response to rice
cultivars cold-tolerant and cold-sensitive to cold stress according to the dry matter
accumulation and possible changes in chlorophyll content; categorize different genotypes
with regard to sensitivity to cold and to sowing depth stresses and, analyze the expression of
cold-responsive genes, and also genes submergence-responsive. The seeds after seven days at
25°C were exposed at 4°C for 24h and after that, photosynthesis was measured later, the
plants were 72h at 25°C (recovery period) to assess the dry mass and chlorophyll. For the
other experiments, the seedlings were collected 7 and/or 14 days maintained at 25°C or
18/13°C day/night and different sowing depths (1.5cm, 5cm, 10cm and 15cm), differential
gene expression were performed with those seedlings using different genes induced by cold.
To evaluated gene expression using different genes induced by cold and anoxia, samples were
collected after exposure to 10 ° C for 6, 24 and 96 h at 1.5 cm and 10 cm deep sowing. The
results showed that is possible to identify superior genotypes for tolerance to these abiotic
stresses based on the Tolerance Index (STI) and Media Geometric (GM) to select genotypes
tolerant to cold or sowing depth, using as a parameter the seedling shoot length measurement.
Japonica and Indica subspecies respond differently to abiotic stresses, however for some of
these stress-responsive genes, these subspecies responded similarly. Furthermore, the analysis
at the molecular level of cold tolerance and sowing depth indicated the importance of ABAdependent and ABA-independent signal transduction pathways in plants under abiotic stress.
Key words: seedling stages, oRT-PCR, esterase, red rice.
LISTA DE FIGURAS
Página
CAPÍTULO IV..............................................................................................................
Figure 1. Seeding depth tolerance screening of weedy red rice accessions by
measurements of seedling root and shoot lengths (mm), 14 d after
sowing.UFPel/FAEM. 2013 ...........................................................................................
Figure 2. Esterase isoenzyme analysis of red rice ecotype 116 and the cultivars
Diamante and BRS 6 Chui under cold stress (18ºC -10 h/ 13ºC -14 h).UFPel/FAEM.
2013 ................................................................................................................................
Figure 3. Differential gene expression analysis assessed through qRT-PCR in
Ecotype 116, BRS6 Chui and Diamante compared with that of control subjected to
cold stress (Relative fold change, log2 ratio) for each plants. UFPel/FAEM. 2013 ......
Figure 4. Differential Expression analysis of genes: a. ASR1 b: Germin c: JRC 2606
and d: JRC 3709 assessed through qRT-PCR in Ecotype 116, BRS 6 Chui and
Diamante compared with that of control subjected to sowing depth stress (Relative
fold change, log2 ratio) for each plants.UFPel/FAEM. 2013.........................................
CAPÍTULO V ...............................................................................................................
Figure 1. Phenol test of cultivated and weedy red rice accessions for subspecies
determination.UFPel/FAEM. 2013 ................................................................................
Figure 2. Response of selected Oryza genotypes to cold treatment (18C-10 h/13C14h temperature). Shoot lengths were measured (14 days after sowing). Each bar is
the average of 3 replicates, each one with 5 seeds and 2 runs.UFPel/FAEM. 2013 ......
Figure 3. Response of weedy and cultivated rice to seeding depth at 5, 10, and 15 cm
(with depth of 1.5 cm as control. Each bar is the average of 3 data points. Including
the rice genotypes selected (CHI 08-C, GRE08-D01, 1602, PRA08-D02 and Spring).
UFPel/FAEM. 2013 ........................................................................................................
Figure 4. Rice seedling growth at depths of 1.5 cm, 5 cm, 10 cm and 15 cm under
cold stress (18C-10 h/13C-14 h). Cold sensitivity/tolerance shown is relative to the
response of plants exposed to the same depth under cold and normal mean
temperature (25C). Including the rice genotypes selected (CHI 08-C, GRE08-D01,
1602, PRA08-D02 and Spring).UFPel/FAEM. 2013 .....................................................
Figure 5. Gene expression analysis of transcription factors NAM, Dreb2A, MYB and
F-box in PRA 08-D02 and Spring genotypes. Coleoptiles were harvested from 10-dold seedlings. Panel A: Effects of cold (10C) on NAM, Dreb2A, MYB and F-box
gene expression. Coleoptiles were harvested from 10-d-old germinated seedlings of
sensitive (PRA 08-D02) and tolerant (Spring) Japonica accessions. Seedlings were
exposed to cold and depth stress for 6, 24, and 96 hours before RNA extraction.
Panel B: Effects of cold (10C) and depth stress (10 cm) on NAM, Dreb2A, MYB
and F-box gene expression. Coleoptiles were harvested from 10-d-old germinated
seedlings of sensitive (PRA 08-D02) and tolerant (Spring) Japonica accessions.
Seedlings were exposed to cold and depth stress for 6, 24, and 96 hours before RNA
extraction.UFPel/FAEM. 2013 .......................................................................................
Figure 6. Gene expression analysis ofNAM, Dreb2A, MYB and F-box genein CHI
08-C, 1602 and GRE 08-D01genotypes. Coleoptiles were harvested from 10-d-old
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seedlings. Panel A: Effects of cold (10C) on NAM, Dreb2A, MYB and F-box
expression. Coleoptiles were harvested from 10-d-old germinated seedlings of
sensitive (CHI 08-C) and tolerant (1602 and GRE 08-D01) Indica accessions.
Seedlings were exposed to cold and depth stress for 6, 24, and 96 hours before RNA
extraction. Panel B: Effects of cold (10C) and depth stress (10 cm) on NAM,
Dreb2A, MYB and F-box gene expression. Coleoptiles were harvested from 10-d-old
germinated seedlings of sensitive (CHI 08-C) and tolerant (1602 and GRE 08-D01)
Indica accessions. Seedlings were exposed to cold and depth stress for 6, 24, and 96
hours before RNA extraction.UFPel/FAEM. 2013 ........................................................
Figure 7. Gene expression analysis of transcription factors H+ pyrophosphatase, Rab
16, GERMIN and glutamate dehydrogenase in PRA 08-D02 and Springgenotypes.
Coleoptiles were harvested from 10-d-old seedlings. Panel A: Effects of cold (10C)
on H+pyrophosphatase, Rab 16, GERMIN and glutamate dehydrogenase expression.
Coleoptiles were harvested from 10-d-old germinated seedlings of sensitive (PRA
08-D02) and tolerant (Spring) Japonica accessions. Seedlings were exposed to cold
and depth stress for 6, 24, and 96 hours before RNA extraction. Panel B: Effects of
cold (10C) and depth stress (10 cm) on H+pyrophosphatase, Rab 16, GERMIN and
glutamate dehydrogenase gene expression. Coleoptiles were harvested from 10-d-old
germinated seedlings of sensitive (PRA 08-D02) and tolerant (Spring) Japonica
accessions. Seedlings were exposed to cold and depth stress for 6, 24, and 96 hours
before RNA extraction.UFPel/FAEM. 2013 ..................................................................
Figure 8. Gene expression analysis of H+pyrophosphatase, Rab 16, GERMIN and
glutamate de hydrogenase gene in CHI 08-C, 1602 and GRE 08D01genotypes.Coleoptiles were harvested from 10-d-old seedlings. Panel A: Effects
of cold (10C) on H+pyrophosphatase, Rab 16, GERMIN and glutamate
dehydrogenase expression. Coleoptiles were harvested from 10-d-old germinated
seedlings of sensitive (CHI 08-C) and tolerant (1602 and GRE 08-D01) Indica
accessions. Seedlings were exposed to cold and depth stress for 6, 24, and 96 hours
before RNA extraction. Panel B: Effects of cold (10C) and depth stress (10 cm) on
H+pyrophosphatase, Rab 16, GERMIN and glutamate dehydrogenase gene
expression. Coleoptiles were harvested from 10-d-old germinated seedlings of
sensitive (CHI 08-C) and tolerant (1602 and GRE 08-D01) Indica accessions.
Seedlings were exposed to cold and depth stress for 6, 24, and 96 hours before RNA
extraction.UFPel/FAEM. 2013 .......................................................................................
Figure 9. Gene expression analysis of transcription factors ADH1, Expansin 7 and
12, ERF 70 and 68 and alpha-amylase in PRA 08-D02 and Spring genotypes.
Coleoptiles were harvested from 10-d-old seedlings. Panel A: Effects of cold (10C)
on ADH1, Expansin 7 and 12, ERF 70 and 68 and alpha-amylase expression.
Coleoptiles were harvested from 10-d-old germinated seedlings of sensitive (PRA
08-D02) and tolerant (Spring) Japonica accessions. Seedlings were exposed to cold
and depth stress for 6, 24, and 96 hours before RNA extraction. Panel B: Effects of
cold (10C) and depth stress (10 cm) on ADH1, Expansin 7 and 12, ERF 70 and 68
and alpha-amylase gene expression. Coleoptiles were harvested from 10-d-old
germinated seedlings of sensitive (PRA 08-D02) and tolerant (Spring) Japonica
accessions. Seedlings were exposed to cold and depth stress for 6, 24, and 96 hours
before RNA extraction.UFPel/FAEM. 2013 ..................................................................
Figure 10. Gene expression analysis ofADH1, Expansin 7 and 12, ERF 70, ERF 68
and alpha-amylase gene in CHI 08-C, 1602 and GRE 08-D01genotypes. Coleoptiles
were harvested from 10-d-old seedlings. Panel A: Effects of cold (10C) on ADH1,
Expansin 7 and 12, ERF 70, ERF 68 and alpha-amylase expression. Coleoptiles were
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harvested from 10-d-old germinated seedlings of sensitive (CHI 08-C) and tolerant
(1602 and GRE 08-D01) Indica accessions. Seedlings were exposed to cold and
depth stress for 6, 24, and 96 hours before RNA extraction. Panel B: Effects of cold
(10C) and depth stress (10 cm) on ADH1, Expansin 7 and 12, ERF 70, ERF 68 and
alpha-amylase gene expression. Coleoptiles were harvested from 10-d-old
germinated seedlings of sensitive (CHI 08-C) and tolerant (1602 and GRE 08-D01)
Indica accessions. Seedlings were exposed to cold and depth stress for 6, 24, and 96
hours before RNA extraction.UFPel/FAEM. 2013 ........................................................
Figure 11. Gene expression analysis of Sub 1b and APX2 in PRA 08-D02 and Spring
genotypes. Coleoptiles were harvested from 10-d-old seedlings. Panel A: Effects of
cold (10C) on Sub 1b and APX2 expression. Coleoptiles were harvested from 10-dold germinated seedlings of sensitive (PRA 08-D02) and tolerant (Spring) Japonica
accessions. Seedlings were exposed to cold and depth stress for 6, 24, and 96 hours
before RNA extraction. Panel B: Effects of cold (10C) and depth stress (10 cm) on
Sub 1b and APX2 gene expression. Coleoptiles were harvested from 10-d-old
germinated seedlings of sensitive (PRA 08-D02) and tolerant (Spring) Japonica
accessions. Seedlings were exposed to cold and depth stress for 6, 24, and 96 hours
before RNA extraction.UFPel/FAEM. 2013 ..................................................................
Figure 12. Gene expression analysis of Sub 1b and APX2in CHI 08-C, 1602 and
GRE 08-D01genotypes. Coleoptiles were harvested from 10-d-old seedlings. Panel
A: Effects of cold (10C) on Sub 1b and APX2 expression. Coleoptiles were
harvested from 10-d-old germinated seedlings of sensitive (CHI 08-C) and tolerant
(1602 and GRE 08-D01) Indica accessions. Seedlings were exposed to cold and
depth stress for 6, 24, and 96 hours before RNA extraction. Panel B: Effects of cold
(10C) and depth stress (10 cm) on Sub 1b and APX2 gene expression. Coleoptiles
were harvested from 10-d-old germinated seedlings of sensitive (CHI 08-C) and
tolerant (1602 and GRE 08-D01) Indica accessions. Seedlings were exposed to cold
and depth stress for 6, 24, and 96 hours before RNA extraction.UFPel/FAEM. 2013 ..
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LISTA DE TABELAS
Página
CAPÍTULO I ................................................................................................................
Table 1 (a): First count (%), Germination (%) and Cold test of different rice
genotypes. UFPel/FAEM. 2013 .....................................................................................
Table 1(b): Total seedling length (root + shoot) in the different rice genotypes after
cold stress and seed sowing deep stress. UFPel/FAEM. 2013 .......................................
Table 2(a): Averages for seedling shoot length (cm) subjected to stress 14 days after
sowing. UFPel/FAEM. 2013 ..........................................................................................
Table 2(b): Correlation between Indices of Tolerance and Susceptibility and
Geometric Mean for seedling length of the four genotypes. (D: Deep, C: Cold, SI:
Susceptibility Index; TI: Tolerance Index, GM: Geometric Mean; Statistical
Significance was estimated by t test (*) and (**) significant (> 0.05) and (> 0, 01) of
likelihood, respectively).UFPel/FAEM. 2013 ................................................................
26
CAPÍTULO II ...............................................................................................................
Tabela 1. Resposta para massa seca de cultivares de arroz tolerante ou sensível ao
frio submetidas sete dias a 25°C, em seguida, expostas a 4°C (24 h), seguida de 72 h
de recuperação (a 25°C). UFPel/FAEM. 2013 ...............................................................
Tabela 2. Teores de clorofila “a” e “b” e totais (μg.g-1) em plântulas de arroz
tolerantes ou sensíveis ao frio submetidas sete dias a 25C, em seguida, expostas a
frio (4C durante 24 h). UFPel/FAEM. 2013 ...................................................................
37
CAPÍTULO III .............................................................................................................
Tabela I. Informações referentes aos genes e suas sequências de nucleotídeos a serem
utilizados em análises de expressão dos genes em qRT-PCR (Capítulo IV).
UFPel/FAEM. 2013 ........................................................................................................
Tabela II. Informações referentes aos genes e suas sequências de nucleotídeos a
serem utilizados em análises de expressão dos genes em qRT-PCR (Capítulo V).
UFPel/FAEM. 2013 ........................................................................................................
46
CAPÍTULO IV..............................................................................................................
Table 1. Expressed genes in rice (Oryza sativa L.) when subjected to abiotic stress ....
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CAPÍTULO V ...............................................................................................................
Table I. Evaluation of rice tolerance to cold stress.UFPel/FAEM. 2013 .......................
Table II. Screening of rice tolerance to depth stress (5 cm, 10 cm, and 15 cm) ............
Table III. Rice seedling growth at various seeding depths (5 cm, 10 cm and 15 cm)
under cold stress (18C-10h/13C-14h). UFPel/FAEM. 2013 ..........................................
Table IV. Genes analyzed into qRT-PCR to sensitivity and tolerance when subjected
to abiotic stress in (Indica and Japonica) rice genotypes. UFPel/FAEM. 2013 ............
Table V. Genes analyzed into qRT-PCR to sensitivity and tolerance when subjected
to abiotic stress in (Indica and Japonica) rice genotypes. UFPel/FAEM. 2013 ............
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SUMÁRIO
Página
BANCA EXAMINADORA..........................................................................................
2
DEDICATÓRIA ...........................................................................................................
3
AGRADECIMENTOS .................................................................................................
4
RESUMO GERAL .......................................................................................................
6
GENERAL ABSTRACT..............................................................................................
7
LISTA DE FIGURAS...................................................................................................
8
LISTA DE TABELAS ..................................................................................................
11
INTRODUÇÃO GERAL .............................................................................................
13
1. A CULTURA DO ARROZ (Oryza sativa) ................................................................
13
2. DIFERENCIAÇÃO DAS SUBESPÉCIES Indica E Japônica ..................................
14
3. TOLERÂNCIA AO ESTRESSE OCASIONADO PELO FRIO ...............................
14
4. ANÁLISES DA TOLERÂNCIA A NÍVEL MOLECULAR .....................................
16
REFERÊNCIAS BIBLIOGRÁFICAS .......................................................................
21
CAPÍTULO I
Application of Stress Indices for Low Temperature and Deep Sowing Stress:
Screening of Rice Genotypes .........................................................................................
26
CAPÍTULO II
Resposta ao frio em cultivares de arroz com relação ao acúmulo de fitomassa e
possíveis alterações no teor de clorofila .........................................................................
37
CAPÍTULO III
Eficiência de iniciadores relacionados aos genes de tolerância ao estresse em arroz
por baixas temperaturas e anoxia....................................................................................
46
CAPÍTULO IV
Differential gene expression of cold responsive genes in rice and red rice genotypes ..
52
CAPÍTULO V
Screening of different rice genotypes for cold and deep sowing stresses ......................
65
CONSIDERAÇÕES FINAIS .......................................................................................
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INTRODUÇÃO GERAL
1. A CULTURA DO ARROZ (Oryza sativa)
Os dois pools gênicos, do complexo grupo de espécies de arroz (pertencente à
família Poaceae), são compostos pelas espécies cultivadas Oryza sativa L. (arroz asiático) e
Oryza glaberrima Steud. (arroz africano). O processo de domesticação da espécie Oryza
sativa, provavelmente, levou à diferenciação das subespéciesindica e japonicaem resposta as
condições a que foram expostas (MORISHIMA, 2001; GOMES e MAGALHÃES-JÚNIOR,
2004).
O Brasil está entre os 10 países maiores produtores de arroz do mundo, assim como
China, Índia, Indonésia, Bangladesh, Vietnã, Tailândia, Mianmar, Filipinas e Japão, sendo o
maior produtor mundial de arroz, ao se excluírem os países asiáticos (SOSBAI, 2010). No
Brasil, o estado do Rio Grande do Sul (RS) é o principal produtor de arroz irrigado,
apresentando uma área de cultivo estimada em 1.066 milhão de hectares, correspondendo a
66,9% da produção nacional de arroz (CONAB, 2012).
No RS, o período ideal para a semeadura dessa cultura corresponde de 15 de outubro
a 15 de novembro. Nesse período, as normais climatológicas de 30 anos (Tabela 1)
registraram temperaturas em torno de 18,55°C (CONAB, 2011; EMBRAPA/UFPel/INMET,
2011). No entanto, é sabido que temperaturas inferiores a 20°C podem ser prejudiciais ao
desenvolvimento e rendimento da cultura, já que a temperatura ótima para a fase de
germinação do arroz é de 20 a 35°C (YOSHIDA, 1981). Temperaturas abaixo de 20°C podem
ocasionar injúrias, sendo o frio considerado um dos estresses abióticos mais importantes para
o arroz (YOSHIDA, 1981; CRUZ, 2001).
Tabela 1. Normais climatológicas obtidas em análises realizadas durante 30 anos, na região
Sul do Rio Grande do Sul. UFPel/FAEM. 2013.
Variáveis
Jan
Temperatura Média (°C)
23,2 23,0 21,7 18,5 15,1 12,4 12,3 13,4 14,9 17,5 19,6 22,0 17,8
Temperatura Média das Mínimas (°C)
19,1 19,1 17,7 14,4 11,1 8,6
Temperatura Mínima Absoluta (°C)
10,0 9,8
Temperatura Média das Máximas (°C)
28,2 27,9 26,9 24,0 20,8 17,8 17,5 18,6 19,6 22,2 24,6 27,1 22,9
Temperatura Máxima Absoluta (°C)
39,0 36,5 37,4 35,1 31,6 29,4 31,8 33,0 35,6 34,4 39,2 39,6 39,6
Fonte: Embrapa/UFPel/INMET, 2011.
Fev Mar Abr Mai Jun
5,0
2,7
1,2
Jul
8,6
Ago Set
9,5
Out Nov Dez Anual
11,2 13,6 15,3 17,7 13,8
-3,0 -2,7 -1,0 0,2
2,6
6,0
7,9
-3,0
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Apesar dos esforços efetuados, maiores avanços são necessários em programas de
melhoramento de arroz do Rio Grande do Sul quanto à tolerância ao frio. Isso se deve a baixa
disponibilidade de fontes de tolerância, assim como a alta esterilidade presente em híbridos
resultantes do cruzamento indica x japonica e a necessidade de progredir no desenvolvimento
de metodologias de avaliação e seleção de genótipos elite (ROSSO, 2006).
2. DIFERENCIAÇÃO DAS SUBESPÉCIES Indica E Japônica
Diferenças morfológicas podem, algumas vezes, ser utilizadas para distinguir indica
de japonica, como é o caso do formato do grão, sendo que, em geral, as sementes
pertencentes à subespécie japonica possuem grãos curtos, largos e seção transversal
arredondada enquanto que indicase caracteriza por grãos longos, estreitos e levemente planos.
Porém, o método mais utilizado atualmente para distinguir essas subespécies é o Teste de
Fenol em populações de arroz vermelho, o qual foi primariamente utilizado em arroz
cultivado. Esse teste consiste na verificação da mudança da cor (marrom escuro ou preto) da
casca e do grão de amostras de arroz pertencentes, exclusivamente, à subespécie indica como
resultado da atividade da enzima Polifenol Oxidase (GROSS et al., 2009).
Além dessas características que distinguem indica e japonica, as subespécies
segregam para muitos genes e caracteres em associação, de maneira não casualizada
(MORISHIMA, 2001), o que sugere a possiblidade de introdução de novas características,
como fonte de variabilidade, por meio da síntese de híbridos indica x japônica. No entanto, a
esterilidade presente no híbrido resultante desse cruzamento é, ainda, um problema em
programas de melhoramento (LOPES, 2002).
3. TOLERÂNCIA AO ESTRESSE OCASIONADO PELO FRIO
Muitas pesquisa têm sido realizadas com o intuito de identificar e avaliar a tolerância
ao frio em diferentes genótipos, assim como avaliar essa tolerância durante o estádio de
germinação a fim de detectar genótipos tolerantes na subespécie japonica. Essa tolerância foi
demonstrada em trabalhos utilizando-se temperaturas de 13°C e 28°C, no qual foi observado
que as cultivares japonica, “Quilla 64117” e “Diamante”, demonstraram ser mas tolerantes ao
frio, e entre as indica, as cultivares “BR-IRGA 410” e “IRGA 416” (CRUZ e MILACH,
2004). Os caracteres genéticos que levam à tolerância ao frio, ou seja, a fonte de tolerância,
são oriundos dessas cultivares pertencentes à subespécie japonica e que são provenientes de
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países como Chile, Filipinas e Japão. No estudo referido à tolerância foi determinada
mediante a avaliação do comprimento do coleóptilo e pelo recrescimento do mesmo a 28°C
após exposição a 13°C. Em estudos realizados pela Empresa Brasileira de Pesquisa
Agropecuária (EMBRAPA) foram utilizados, como parâmetro de verificação a tolerância ao
frio, de diferentes genótipos, o índice de velocidade de emergência (IVE) (EMBRAPA,
2009).
Estudos de tolerância ao frio em arroz utilizaram temperaturas inferiores a 20°C nas
fases germinativa, vegetativa e reprodutiva (YOSHIDA, 1981; CRUZ, 2000). A temperatura a
ser designada a aplicação do estresse, em condições controladas, pode ser constante ou
alternada (dia e noite). Li e Rutger (1980) utilizaram temperatura constante (18ºC durante 14
dias) para avaliar a estatura de plântulas de arroz. Foram utilizadas temperaturas alternadas,
Martins et al. (2007) avaliaram famílias mutantes quanto à tolerância ao frio, no período
vegetativo, as plântulas mantidas em casa de vegetação à temperatura de 13ºC por 10 dias,
enquanto para avaliação no período reprodutivo utilizou-se estresse de 15ºC por sete dias.
Após a planta ser exposta ao estresse por frio, essa pode se recuperar, ao retornar à
temperatura ideal de cultivo, o que leva a menores percentuais de redução na estatura.
A tolerância ao frio pode ser relativa a um estádio ou fase de desenvolvimento da
planta de arroz (CRUZ e MILACH, 2000). Na fase vegetativa esses danos podem ser
desuniformidade e diminuição na velocidade e porcentagem de germinação, além de redução
na estatura, devido ao retardo no desenvolvimento da planta e amarelecimento das folhas
(SOUZA, 1990; STHAPIT et al., 1995). A tolerância, nessa fase, está sendo buscada a fim de
antecipar a semeadura e evitar que a etapa reprodutiva coincida com a época de início de
temperaturas ainda mais baixas e danosas. Além disso, objetiva favorecer a produtividade, já
que, ao antecipar a semeadura, a fase reprodutiva coincidirá com a época de maior intensidade
de radiação solar (MERTZ et al., 2009). Já na fase reprodutiva, os danos causados pelo frio
são exerção incompleta da panícula e aumento na esterilidade de espiguetas (TERRES, 1991).
Alguns dos fatores que determinam o tempo necessário para a germinação de
sementes e emergência de plântulas são temperatura e profundidade de semeadura
(YOSHIDA, 1981; FERNANDEZ et al., 1985). O desempenho frente à profundidade de
semeadura é considerado outro estresse abiótico que afeta o estande da lavoura, o qual ainda é
muito pouco estudado. Para algumas espécies, essa característica pode ser positiva,
principalmente para o desenvolvimento de bancos de sementes de plantas daninhas no solo,
como por exemplo, o arroz vermelho, o qual apresenta maior longevidade quando as sementes
são depositadas em profundidade comparadas aquelas na superfície (NOLDIN, 1995). Para
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avaliar e selecionar plantas vigorosas seu desempenho pode ser testado em diferentes
profundidades de semeadura, além de o arroz vermelho poder ser utilizado como uma fonte de
variabilidade genética germinação em grandes profundidades (MALONE et al., 2007).
4. ANÁLISES DA TOLERÂNCIA A NÍVEL MOLECULAR
As perturbações morfológicas, bioquímicas e moleculares, decorrentes do estresse
por resfriamento iniciam com a desestabilização das membranas, o que acarreta em sua
disfunção e na alteração dos processos celulares, resultando em modificações (TAIZ &
ZEIGER, 2013). No entanto, as plântulas de arroz, expostas ao estresse pelo frio, podem
apresentar um aumento na síntese de Álcool Desidrogenase, a qual participa do processo de
fermentação alcoólica, e consequente aumento na produção de etanol, o qual pode ajudar na
preservação da fluidez dos lipídeos da membrana (MERTZ et al., 2009). No entanto, o
aumento na produção de etanol, juntamente com o aumento de espécies reativas de oxigênio
ou diminuição na síntese de antioxidantes, pode acarretar no aumento da peroxidação desses
lipídeos insaturados levando à ruptura da membrana plasmática. Essa e outras modificações
dependem de um estímulo extracelular, o qual ativa respostas de defesa.
Esse processo de estimulação, ativação e, portanto, de cascata de sinalização e
transdução de sinal são fundamentais. Os elementos cis-acting, os quais participam dessa
sinalização, são regiões do promotor de um gene que atuam como interruptores moleculares
envolvidos na regulação da transcrição de uma rede gênica dinâmica (STRÄHLE e
RASTEGAR, 2008). Já a proteína denominada “dedo de zinco” (Zinc Finger Protein),
ZFP245, apresenta uma atividade, na região trans, na regulação da tolerância ao frio, como
demonstrado por Huang et al. (2009). Esse acúmulo dá-se pela ativação da síntese de ABA.
Além disso, a super expressão de ZFP245 pode inferir proteção na planta pelo aumento da
atividade antioxidante. Nessas regiões do DNA, os fatores de transcrição (TF) se ligam
fisicamente, influenciando a expressão gênica.
A família de fatores de transcrição CBF (C-repeat binding factor) ou DREB
(dehydration responsive element binding factor) regula a expressão de genes que respondem a
diversos estresses, dentre os quais se destacam o estresse pelo frio, contendo os cis-elementos
denominados CRT/DRE (DUBOUZET et al., 2003). Os fatores de transcrição AREB (ABA responsive elementbinding protein ou TF ABA-dependentes) ou ABFs (binding factors), são
importantes para diversos estresses, inclusive frio, ativando os cis-elementos ABRE (ABA responsive element) assim como a família NAC (petúnia, NAM and Arabidopsis ATAF1,
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ATAF2, e CUC2) (YAMAGUCHI-SHINOZAKI e SHINOZAKI, 2005; HU et al., 2008).
Portanto, existem diversas vias de transdução de sinal, sendo algumas ABA-dependente como
nos genes NAM (determinam a posição do meristema apical caulinar), ABA-independente
(DREBs), ABA-dependentes e independentes como MYBs (Myeloblastosis, família de
numerosos fatores de transcrição), outros pertencentes ao grupo F-box e ao grupo AP2/ERF.
Algumas vezes, essas vias podem não ser específicas, podendo ser combinadas por meio de
uma rede de interações entre diferentes genes, e os quais respondem a diferentes estresses,
seja esses bióticos ou abióticos, o que é definido como “crosstalk” (FUJITA et al., 2006).
Portanto, é de grande relevância o estudo dessa rede de genes responsivos a estresses
mediante análise da sua expressão genica desses, frente a condições adversas.
Para tanto, a técnica de reação da transcriptase reversa, seguida de reação em cadeia
da polimerase, em tempo real (qRT-PCR) faz uso de populações de mRNA expresso, em
determinado tecido e estádio de desenvolvimento da planta. Essa ferramenta permite a
investigação do transcriptoma a partir de genes previamente identificados e com acessos
disponíveis em bancos de dados (PEREIRA, 2011). Além de essa metodologia apresentar
especificidade, sensibilidade e reprodutibilidade na quantificação do produto final
amplificado, essa técnica pode ser utilizada para confirmar informações obtidas através de
análises por microarranjo. Sendo a vantagem do microarranjo é a possibilidade de analisar
milhares de genes ao mesmo tempo e já a qRT-PCR é limitada a poucos genes por corrida
(FREEMAN, 1999; PEREIRA, 2011).
Para que haja um grande número de processos celulares, os quais são necessários à
tolerância ao estresse, é imprescindível um controle combinatório de diferentes fatores de
transcrição que tem sua expressão elevada para efetivar a tolerância. Em trabalho realizado
por Lindlof et al. (2009) foram identificados 1.450 genes relacionados ao estresse do frio em
arroz, os quais foram identificados por meio de análise por microarranjo, o que correspondeu
a um número menor de genes do que aqueles encontrados em Arabdopsis (1753 genes), sob as
mesmas condições experimentais.
Os fatores de transcrição OsDREB1A, OsDREB1B, OsDREB1C, OsDREB1D e
OsDREB2A, ou seja OsDREBs, apresentaram homologia com os fatores de transcrição
(DREBs) em Arabdopsis thaliana, sendo que os dois primeiros (OsDREB1A, OsDREB1B),
em estudos realizados por Dubouzet et al. (2003), tiveram sua expressão induzida sob estresse
por baixa temperatura, mas não por ABA exógeno ou sob estresse por seca. Isso pode
evidenciar maior especificidade de OsDREB1B em resposta a estresse, pois OsDREB1A
também foi induzido por alta salinidade e por ferimentos, o que torna relevante seu potencial
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emprego na produção de transgenia em Liliopsida tolerantes a diferentes estresses abióticos,
já que o arroz é uma espécie modelo (BENNETZEN, 2002). Esses resultados foram possíveis
devido à utilização de Arabdopsis trangênico, ao ser adicionado o gene OsDREB1A, o qual foi
induzido à super expressão a fim de se averiguar a expressão diferencial dos genes alvo de
DREB1A. No entanto, existem algumas diferenças entre OsDREB1A e DREB1A na
especificidade de ligação do TF ao gene alvo, além de diferenças na ação sobre alguns genes
alvo. Essas diferenças foram verificadas por meio de análises de microarranjos, por
hibridização de sondas de cDNA com 7 mil genes, e confirmadas pela utilização de análise de
RNA-gel blot (Northern blot). No estudo mencionado, a exposição ao frio foi temporária
(-6°C por 30h) e considerada congelamento, ocorrendo após 4 semanas de cultivo sob 22°C,
sendo que, após a exposição ao frio, as plantas retornaram ao cultivo à 22°C por mais 5 dias,
como fase de recuperação ao estresse (DUBOUZET et al., 2003). No entanto, em estudos
mais recentes (MATSUKURA et al., 2010) OsDREB2B e OsDREB2A foram induzidos por
estresses abióticos.
Em outro experimento, foi avaliada a expressão de outros três DREBs homólogos à
Arabdopsis (OsDREB1-1, OsDREB4-1 e OsDREB4-2), sendo que após três semanas de
semeadura foi verificado que as plântulas de arroz transferidas para câmara fria (4°C em
intervalos de no máximo 24h). Obtiveram um padrão único de expressão do fator de
transcrição OsDREB1-1 e de OsDREB4-2, porém com diferentes níveis dentre tecidos
analisados (TIAN et al., 2005).
Há relatos de TFs que são expressos constitutivamente e, mesmo assim, apresentam
papel na tolerância ao frio, isso sendo devido à regulação pós-transcricional e ou traducional
(FIGUEIREDO et al., 2010). Em outros casos, a elevação na expressão ocorre tanto em genes
como nos fatores de transcrição. Esse é o caso do gene OsLti6a, que teve sua expressão
elevada em cultivares tolerantes ao frio, além de apresentarem um acréscimo na expressão dos
fatores de transcrição CRT/DRE (MORSY et al., 2005).
As proteínas de ligação ricas em glicina (GRPs/glycine-rich RNA-binding proteins),
também, são sintetizadas sob frio em Arabdopsis, tendo uma função de chaperona. O mesmo
ocorre em arroz, em que esses genes são conhecidos como OsGRPs, mais precisamente
OsGRP1, OsGRP4 e OsGRP6 (KIM et al., 2010).
Diversos outros genes envolvidos na resposta ao frio durante a germinação de arroz
já foram descritos, tais como Cor (Cold-regulated), KIn (cold inducible), ErD (Early
responsive to dehydration), LTI (Low-temperature Induced), RD (Responsive to
Dehydration), Rab16, Expansin 7 e 12, assim como genes que necessitam da sinalização de
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ABA e da presença de espécies reativas de oxigênio (ROS). Igualmente, foram identificadas
proteínas quinases dependentes de cálcio (CDPKs), já que esse elemento é um importante
intermediário na cascata de sinalização, ligando-se, muitas vezes, a calmodulina modificando
o comportamento de quinases (SHINOZAKI e YAMAGUCHI-SHINOZAKI, 2000;
RABBANI et al., 2003; CHINNUSAMY et al., 2006; LASANTHI-KUDAHETTIGE et al.,
2007; WAN et al., 2007; SAIJO et al., 2001; XIANG et al., 2007). Além de genes envolvidos
na resposta ao frio, genes participantes de processos metabólicos da planta são afetados ao
final dessa cascata de transdução de sinal, como por exemplo, os que codificam Glutamato
desidrogenase e Alfa-amilase (RABBANI et al., 2003; LASANTHI-KUDAHETTIGE et al.,
2007).
Um importante Quantitative Locus Trait (QTL) qLTG3-1 foi estudado pela
comparação entre duas cultivares de arroz, outra que não apresenta esse alelo e uma que
apresenta, por meio de análise do nível de expressão sob estresse pelo frio (15°C) por
microarranjo. Esse QTL, na cultivar tolerante ao frio, durante a germinação, teve seus níveis
de expressão aumentados 1 dia antes da germinação e durante o processo germinativo. Outra
diferença observada foi no número de genes com a expressão modificada na comparação das
cultivares contrastantes 1 dia antes da germinação, sendo essa diferença estimada ocorreu em
4.586 genes, dos quais 100 tiveram a expressão diferencial duplicada (FUJINO
&MATSUDA, 2010). Além dos genes já identificados, utilizando microarranjos, foram
descobertos 18 micro RNAs que respondem ao frio em arroz (Lv et al., 2010).
Análises da expressão diferencial dos genes ASR (ABA, Stress and Ripening) foram
realizadas pela utilização de qRT-PCR em arroz sob condições de estresses abióticos pelo frio
e pela posterior averiguação de plantas que sofreram silenciamento gênico. Como resposta ao
frio, os genes ASR1 e ASR4, dos 6 genes da família ASR, apresentaram elevação no número de
seus transcritos (ARENHART et al., 2009). Análises de RNA gel-blot foram utilizadas para
investigar a expressão diferencial em diferentes tecidos de arroz do gene OsARS1, em que
foram detectados altos níveis de expressão na parte aérea tanto na fase vegetativa quanto
reprodutiva sob condições de baixas temperaturas. Já sob condições de temperatura ideal esse
gene foi expresso em todos os tecidos, com exceção dos calos (KIM et al., 2009). Esses
pesquisadores sugerem que esse gene seja regulado pelo FT CBF1, sendo que as amostras de
arroz transformado foram expostas ao frio (4°C por 3, 4, 5, 6, 7, 10 ou 12 dias, precedidas de
10 dias sob 30°C) e após serem submetidas ao frio foram, novamente, submetidas a 30°C por
10 dias.
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Rabbani et al. (2003), após 14 dias da semeadura de Nipponbare (japonica),
utilizando a técnica de microarranjo, verificaram a expressão diferencial de 36 genes
induzidos pelo frio (4°C durante 5, 10 e 24 horas), assim como 43 genes em presença de
ABA, 62 genes em condições de desidratação e 57 genes em estresse de salinidade. Esses
autores ressaltaram a estreita relação que esses genes apresentam, já que 15 desses genes
responderam aos quatro diferentes estresses, assim como alto crosstalk entre frio e salinidade
e, também, entre frio e ABA.
A técnica de microarranjo também permitiu a identificação de 121 genes que
respondem de forma rápida ao frio (10°C durante intervalos de, no máximo, 24 horas) em
arroz subespécie japonica, que são consideradas mais tolerantes a baixas temperaturas
comparativamente à subespécie indica, no período de germinação. Neste estudo, foi proposto
um modelo de regulação no qual participam fatores de transcrição ROS-bZIP1 que
independem de ABA e de CBF/DREB ao contrário do gene GERMIN, que é expresso na
presença de ABA (CHENG et al., 2007). O último que pode servir como parâmetro para
análises, em que se deseja investigar se há aumento na síntese de ABA, assim como a
interferência do ABA na expressão de genes que respondem a condições de baixas
temperaturas.
Em outro estudo também foi investigada a resposta precoce ao estresse causado pelo
frio (10°C) e sua relação com a rota oxidativa, sendo identificada que há sobreposição dos
genes super regulados tanto sob condições de baixa temperatura quanto sob indução do
mecanismo oxidativo, o qual ocorre em quase 60% dos genes averiguados pela técnica de
microarranjo em arroz. O arroz, no estádio V3, apresentou resposta da rota oxidativa mais
rápida que a resposta por grupos mediados por ABA em Nipponbare (YUN et al., 2010).
Além de ser verificada a ação do mecanismo oxidativo mediado por fatores de transcrição
bzip, ERF e R2R3-MYB atuando sobre os respectivos elementos as1/ocs/TGA, GCCbox/JAreeMYB2. Também foi verificado, conforme o proposto por Cheng et al. (2007), que a
resposta do fator de transcrição DREB/CBF independe da resposta oxidativa.
No entanto, há controvérsias sobre a resposta enzimática antioxidante, sendo
sugerido que essa resposta pode ou não estar envolvida à tolerância e/ou à sensitividade ao
estresse pelo frio, assim como também pode estar envolvida na tolerância à semeadura em
grandes profundidades. Porém, foi demonstrada redução da fotoinibição e o dano oxidativo
em cultivares tolerantes quando comparadas às sensíveis ao frio. Além disso, o modo como as
cultivares respondem ao estresse é variável, algumas cultivares expressam constitutivamente
altos níveis de Superóxido Dismutase (SOD) favorecendo a tolerância nas horas iniciais à
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exposição ao estresse (BONNECARRÈRE et al., 2011). Outra enzima que também pode
favorecer a tolerância, por também estar relacionada à ação antioxidante seria a enzima
Ascorbato Peroxidase. Adicionalmente, a tolerância a grandes profundidades de semeadura
pode envolver genes que conferem tolerância a plantas submersas, como é o caso de Sub1b
(LASANTHI-KUDAHETTIGE et al., 2007).
Em pesquisas sobre a expressão ectópica de TERF2 através da transgenia, foi
relatado que esse apresenta uma importante função regulatória na resposta ao estresse por
baixas temperaturas em arroz, aumentando a tolerância ao frio, e, como uma das
consequências, reduz a presença de espécies reativas de oxigênio, estabilizando a membrana.
Além disso, alguns genes regulatórios, como, OsMYB, OsICE e OsCDPK7 tiveram sua
expressão elevada sob o tratamento com frio (TIAN et al., 2011). A família de fatores de
transcrição MYB pode estar relacionada à regulação do metabolismo secundário e à regulação
da formação meristemática (JIN e MARTIN, 1999).
Também pertencente ao grupo MYB, o fator de transcrição MYB3S, além de atuar na
via de sinalização de açúcares atua, de maneira fundamental, na adaptação do arroz ao frio
(SU et al., 2010). Como em arroz transformado geneticamente na presença ou ausência da
função gênica de MYB3S, foi sugerida a onipresença da expressão desse FT. No entanto, sob
estresse pelo frio, houve uma superexpressão de, até, 5 vezes. Foi relevante, nesse estudo, a
descoberta da repressão da transcrição de FT DREB1/CBF por FT MYB3S, evidenciando a
complementaridade de ambos. Esses mesmos autores descrevem a resposta do fator de
transcrição MYB3S como ocorrendo a longo prazo, ou seja, também é fundamental para
quando a planta se mantém sob condições de estresse.
Diante do exposto, o objetivo geral deste trabalho foi identificar e selecionar
genótipos, de arroz cultivado e de arroz vermelho, tolerantes e sensíveis aos estresses
abióticos induzidos por baixas temperaturas e por diferentes profundidades de semeadura.
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CAPÍTULO I
27
Application of Stress Indices for Low Temperature and Deep Sowing Stress
screening of rice genotypes
Caroline Borges Bevilacqua1 (CBB); Daisy Ramirez Monzon2 (DRM); Eduardo
Venske3 (EV); Supratim Basu4 (SB), Paulo Dejalma Zimmer5 (PDZ)
Abstract
Low temperature or cold stress and deep sowing plays a pivotal role in limiting rice
(Oryza sativa L.) productivity in the temperate rice growing regions as well as in
tropical high lands worldwide. A better understanding of stress tolerance mechanism in
rice plants will help to develop rice germplasm with improved field level tolerance
under variable temperature and sowing deep conditions. Using previously developed
stress indices, this study presents results from low temperature and deep sowing
screening of four rice genotypes. A group of 25 seeds per replicate (total of 3 replicates)
was subjected to stress by deep sowing (15 cm) while another group was subjected to
cold stress (13°C-10 h/18°C-14 h), and the control group remained under optimum
conditions (25°C and sowing deep of 1.5 cm). The Geometric Mean (GM), Stress
Tolerance Index (STI) and Stress Susceptibility Index (SSI) were used to evaluate the
genotypic performance under control and stress conditions. The results indicate that it
was possible to identify superior genotypes for tolerance based on their stress indices.
The indices although correlated, were found to be effective for the selection of
genotypes with good yield potential under control and stress treatments and can now be
used for genotypic screening under field conditions.
Keywords: Oryza sativa, temperature response, shoot length.
Running Title: Stress Indices for rice genotypes
INTRODUCTION
Low temperatures have a strong impact on the survival, growth, reproduction
and distribution of plants. The plants are characterized by an inherent level of resistance
to low temperatures, which reduces the metabolic activity. This level of resistance can
vary among individual plants and species. The cold stress is characterized by
physiological perturbations, generally called low-temperature damages (HUDAK &
SALAJ, 1999; YAN et al., 2010; ZHANG et al., 2010). The germination process
followed by fast and uniform seedling emergence is essential for the successful crop
development.
However if the seeds remain under the soil for long time this would impair
germination which will be accompanied by abiotic stresses like low temperature or
biotic stresses like pathogens. Consequently it will decrease the crop yield and increase
the productivity cost.
28
The abiotic stresses caused owing to deep sowing are also an important factor
that plays a major role in impairing plant development. Hence the long term goal for
breeding is the development of germplasm with improved field tolerance under different
stress conditions owing to low temperature (CRUZ e MILACH, 2000) and seed sowing
depths.
Rice is a temperature-sensitive crop; low temperatures and deep sowing
dramatically reduce its production. Good tolerance at the seedling stage is an important
character for stable rice production. Brazil is one of the largest rice producing countries
in the world. However the conditions are not always favorable which makes early
seedlings rotten thereby causing massive seed loss and delayed growth (CRUZ e
MILACH, 2000). Hence it becomes important to develop rice plants that can
circumvent the atrocities of nature. Previous reports have shown the genetics and
physiological responses of plants to temperature stress but yield based on indices are
needed for the evaluation of low temperature and deep sowing stress tolerance for
applied plant breeding programs.
Several yield based stress indices have been developed that may be applicable
for low temperature and deep sowing tolerance. The Geometric Mean (GM) and the
Stress Tolerance Index (STI) (FERNANDEZ, 1993) have been used for comparing
genotypic performance across years or environments. STI was developed to identify
genotypes that perform well under both stress and control conditions. The Stress
Susceptibility Index (SSI) (FISHER e MAURER, 1978) is a ratio of genotypic
performance under stress and non-stress conditions, adjusted for the intensity of each
trial, and have been found to be correlated with yield and canopy temperature in wheat
(RASHID et al., 1999).
These different indices may be applicable to other abiotic stress traits, such as
low temperature or deep sowing stress tolerance. This study presents the low
temperature and deep sowing stress screening of rice plants in Brazil for selection of
cold and depth stress (deep sowing) tolerant rice genotypes with good yield potential
and applicability of several stress indices for genotypic stress under the aforesaid stress
treatments.
29
MATERIAL AND METHODS
Plant Materials
Four rice genotypes were used in this study: “Brilhante” (cold tolerant,
Fagundes et al., 2010), “IRGA 422 CL” (as model rice), “BRS 6 Chuí” (cold sensitive,
Mertz et al., 2009) and one red rice ecotype 116.
Germination test
The experiment was carried out with 100 seeds in each four replicates. The
seeds were set on paper roll previously dampened with sterile double distilled water
using 2.5 times the paper dry mass. After sowing, these rolls were kept inside plastic
bags in growth chamber at 25ºC for 14 days. The evaluations were made on 5 days and
14 days respectively after sowing, according to Brazilian standards (BRASIL, 2009).
Germination Percent (GP) was calculated as described in Scott et al. (1984). The
germination count was calculated as described in Brazilian standards (BRASIL, 2009).
Cold test
The cold test was conducted as described by Vieira e Carvalho (1994) with
some modifications in 4 replicates each with 100 seeds. The seeds were distributed
uniformly on previously dampened filter paper and then these rolls were kept inside
plastic bags in growth chamber for 7 days at 10ºC and these treated seeds were then
kept inside the growth chamber (25ºC) for 7 days. The evaluation was made in a similar
way like Germination test count.
Seed Treatment
The soil was previously autoclaved and then dried in a hot air oven (70ºC). The
experiment was carried out in four replicates, with 25 seeds per genotype under three
different conditions: for cold stress treatment the seeds were sowed at 1.5 cm depth in
cups (200 mL) and kept in growth chamber under alternate temperatures (13°C-10
h/18°C-14 h) while for deep sowing stress, seeds were sowed at 15 cm deep in PVC
tubes and kept in growth chamber at 25 ºC. Seeds sowed at a depth of 1.5 cm in cups
served as control were kept in growth chamber at 25ºC. The measurements of shoot
length and root length were made after 7 days and 14 days respectively. The Stress
Susceptibility Index (SSI): ((1- (Ys/Yp))/(1-(Xs/Xp)), Geometric Mean (GM): ((Ys x
Yp)1/2), Stress Tolerance Index (STI): ((Yp x Ys)/Xp2), were determined using the
30
equations for SSI (Fisher and Maurer 1978), GM, STI and SI (FERNANDEZ, 1993)
respectively.
The data obtained in this study were used in place of the genotypic mean
values for seedling length under stress (Ys) and under control (Yp) variables,
respectively, in the equations for the indices. Xs and Xp are the mean seedling length of
all genotypes per trial under stress and control conditions.
Statistical Analysis
All the experimental data presented in this study are mean from three replicates.
The significance of the values so obtained was evaluated by ANOVA and Scott Knott
test (p≤ 0.05) using SISVAR (FERREIRA, 2011). The values for the indices so
obtained were correlated by Pearson correlation analysis using test t (p ≤0.01 and p≤
0.05) using WinSTAT version 1.0, software package (MACHADO, 2007).
RESULTS AND DISCUSSIONS
Rice (Oryza sativa L.) is a model plant for the genetics and physiology of
cultivated grasses, especially due to its high information content available by synteny
with other cereals (DEVOS & GALE, 1998) and the genetic variability that it has to
characters for importance of seed physiology (SGUAREZI et al., 2003). Furthermore, it
is a major crop species from South of Brazil, which is currently responsible for
approximately 60% of national production. Therefore, rice is an excellent source of
research, either through regional and national economic importance, but also is of great
importance by virtue of the biology of the species itself and other cereals. The ability of
the plants to emerge efficiently and rapidly from the soil or under water is a desirable
quality that needs to be incorporated into rice cultivars (REDOÑA & MACKILL,
1996).
The result presented here puts an insight into the plant vigor which was
assessed by first germination count, germination test and cold test (Table 1a). Among
the four genotypes (“BRS6 Chui”, “Brilhante”, “IRGA 422 CL” and one red rice
ecotype-116) studied here it was observed that “BRS6 Chui” had the best physiological
characteristics demonstrated by germination test and cold test which varied from 9497% followed by “Brilhante” and “IRGA 422 CL” (87-88%) while red rice showed the
less vigor (71%).
31
Table 1(a): First count (%), Germination (%) and Cold test of different rice genotypes.
UFPel/FAEM. 2013.
1st Germination Count (%)
Germination (%)
Cold Treatment
IRGA 422 CL
88b
90b
75c
Ecotype 116
71c
86d
50d
Brilhante
89b
93c
85b
BRS 6 Chuí
96a
97ª
93a
Média
CV (%)
86
2.12 1.5
91.4
6.06
75.6
* Means followed by the same letters differ by Scott-Knott test at 5% level of significance.
The next part of our study focused on the significance of genotype and stresslevel interaction. The effect of stress level and genotypes was considered fixed because
these were the only levels and genotypes of interest. The rice genotypes did not show
significant interaction in shoot length among themselves after 7 days of exposure to
deep sowing stress or cold stress (data not shown). However significant differences
were not observed in the total length (root length + shoot length) among the cultivars
after 7 days of exposure to either of the stresses (Table 1b).
Table 1(b): Total seedling length (root + shoot) in the different rice genotypes after cold
stress and seed deep sowing stress.UFPel/FAEM. 2013.
IRGA 422
Control
7 days
1.1cA
14 days
2.23bcA
Depth Stress
7 days
14 days
0.30aB
0.24aB
Cold Stress
7 days
14 days
0,23aB
0.54bB
Ecotype 116
1.22cA
2.14cA
0.16aB
0.16aB
0,17aB
0.52bB
Brilhante
1.82aA
2.64bA
0.49aB
0.38aC
0,28aB
1.32aB
BRS 6 Chui
1.49bA
3.06aA
0.24aB
0.30aB
0,23aB
0.59bB
Média
CV (%)
26.1 (7 dias)
26 (%)
1.2 (14 dias)
22 (%)
* Means not followed by the same lowercase and uppercase letters in the column and in the row differ by
Scott-Knott test at 5% level of significance, respectively.
Hence in the light of this observation we can suggest that 7 days exposure was
not enough for screening genotypes for cold and deep sowing tolerance at germination
32
stage on the basis of their shoot length. Previously it has been shown that corn
genotypes when exposed to cold stress (10°C) for 7 and 14 days respectively showed a
detrimental effect on seedling development (CRUZ et. al., 2007). Earlier work done
with 494 rice genotypes for screening cold tolerance over a period of four years showed
that Tomoe Mochi, Amber, Diamond Bright, BRS Querência and Japanese Grande
(Japonica group) showed better performance, during two years of review, at 14.7°C in
first year and then third year at 3.3°C to 15.6°C minimum and maximum (FAGUNDES
et al., 2010).
Moreover it was also reported that the cultivars from Japonica subspecies
showed more tolerance to low temperature stress as compared to the Indica subspecies
(CRUZ; MILACH, 1999; MERTZ et al., 2009). Our results showed that Brilhante
showed more shoot length after 14 days of cold stress followed by IRGA 422 CL and
red rice ecotype 116 while BRS6 Chui showed the least shoot length. Although in the
cold test and First germination BRS6 Chui were the best results, comparing with the
results based on shoot length we can conclude that BRS6 Chui is highly sensitive to
cold stress as compared to the other genotypes while Brilhante was cold tolerant.
However when the plants were exposed to deep sowing stress it was observed that all
the plants were highly sensitive as they showed significantly less seedling length (Table
1b). Amidst all the cultivars studied here the red rice ecotype 116 showed better shoot
length as compared to all the other rice genotypes under study which gives us an idea
that this cultivar is moderately tolerant to deep sowing stress which is consistent with
earlier findings from Helpert (1981) who showed that 12% of red rice seeds germinated
at 12 cm deep but at 16 cm germination was only 1%. Earlier when Gealy et al. (2000)
tested the emergence of several ecotypes of weedy red rice in different textures of soil at
a deep of 7.5 cm found that all the ecotypes emerged uniformly. Previously work done
with segregating rice population for analysis of plant vigor showed that red rice ecotype
45 exhibited higher germination at 20 cm deep in the soil (MALONE et al., 2007).
Hence we can conclude that not only abiotic stress caused by low temperature but also
stress mediated by deep sowing is detrimental for rice plant development and
productivity.
The Stress Susceptibility Indices (SSI) and GM for shoot length for 7 days and
14 days of deep sowing stress were correlated negatively which is not desirable.
However for the cold stress (7 days) the GM mean and CSI correlated positively, but on
33
the contrary the GM for deep sowing and cold stress showed a positive correlation
which is inconclusive (Table 2 a and b). This brings us to our earlier conclusion that it is
not possible to screen rice genotypes for cold and deep stress after 7 days.
Table 2(a): Averages for seedling shoot length (cm) subjected to stress 14 days after
sowing.UFPel/FAEM. 2013.
Genotypes
IRGA 422
A. vermelho
Brilhante
BRS 6 Chui
Control
1.95bA
1.98bA
2.25bA
2.74aA
Média
CV(%)
Depth Stress
0.23aB
0.15aB
0.37aC
0.29aB
Cold Stress
0.44bB
0.44bB
1.22aB
0.52bB
1.05
22.19
* Means not followed by the same lowercase and uppercase letters in the column and in the row differ by
Scott-Knott test at 5% level of significance, respectively.
Table 2(b): Correlation between Indices of Tolerance and Susceptibility and Geometric
Mean for seedling length of the four genotypes. (D: Deep, C: Cold, SI: Susceptibility
Index; TI: Tolerance Index, GM: Geometric Mean; Statistical Significance was
estimated by t test (*) and (**) significant (> 0.05) and (> 0, 01) of likelihood,
respectively). UFPel/FAEM. 2013.
Correlation
Correlation (length of seedling)
7 days
7 days
Correlation
Correlation (length of seedling)
14 days
14 days
DSI x Dgm
-0.70**
DSI x Dgm
-0.65**
DSI x Dgm
-0.56* DTI x DSI
- 0.54*
DTI x Dgm
0.99**
DTI x Dgm
0.99**
DTI x Dgm
1.00** CSI x DSI
0.58*
DTI x DSI
-0.75**
DTI x DSI
-0.68**
DTI x DSI
-0.58* DSI x Cgm
-0.55*
Cgm xDgm
0.78**
Cgm xDgm
0.83**
Cgm xDgm
0.67** DTI x Cgm
0.99**
Cgm x DTI
0.71**
Cgm x DTI
0.78**
CSI x Cgm
-0.79**
CSI x Cgm
0.59*
CSI x DSI
0.58*
Cgm x DTI
0.66**
CTI x DSI
0.54*
CTI x CSI
0.98**
Florido et al. (2009) have shown that the index of tolerance can help in
breeding programs to permit the investigation of the performance of that germplasm
under adverse environmental conditions. However the screening results for seedling
length after 14 days under either of the stresses showed a positive correlation between
the Tolerance Indices (STI) and GM, thereby justifying the effectively of the
methodology. Our results are consistent with previous reports on bean genotypes that
showed heat tolerance index and GM proved are the most useful indices for the
34
evaluation of genotypic performance under heat stress and they were highly correlated
(PORCH, 2006). Corroborating this result, Boussen et al. (2010) also found that STI
and GM have high positive correlation, though low correlation was observed between
GM and SI while screening wheat genotypes under drought stress.
The ability of plants to tolerate the harsh effects of abiotic stress plays a critical
role in improving the crop yield. Low temperature or cold stress and deep sowing stress
are two major constraints in rice yield owing to global climactic change. In this
evaluation of screening rice genotypes in Brazil under cold and deep sowing stress it
was observed that STI and GM are the most effective stress indices for the selection of
genotypes with good yield potential under depth sowing stress and low-stress
conditions.
ACKNOWLEDGEMENT
This work was supported by CNPq (National Council for Scientific and
Technological Development – Brazil), CAPES (Coordination of Improvement of
Higher Education Personnel) and FAPERGS (Research Foundation of the State of Rio
Grande do Sul).
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CRUZ, P.R.; MILACH, K.C.S. (2000) Breeding for cold tolerance in irrigated rice.
Ciência Rural 30: 909-917.
CRUZ, H.L.; FERRARI, C.S.; MENEGHELLO, G.E.; KONFLANZ, V.; ZIMMER,
P.D.; VINHOLES, P.S. da S.; CASTRO, M.A. da S. (2007) Evaluation of corn
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FAGUNDES, P.R.R.; MAGALHÃES, J.R. A.M. de; STEINMETZ, S. (2010)
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Grain yield response. Australian Journal of Agricultural Research. 29: 897–907.
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MOYA, C. (2009) Plant heat tolerance screening of ex situ preserved tomato (Solanum
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GUIMARÃES, C.M.; STONE, L.F.; OLIVEIRA, J.P. de; RANGEL, P.H.N.;
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MERTZ, L.M.; HENNING, F.A.; SOARES, R.C.; BALDIGA, R.F.; PESKE, F.B.;
MORAES, D.M. de.(2009) Physiological changes in rice seeds exposed to cold in the
germination phase. Revista brasileira de sementes. 31: 254-262.
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weedy rice.Revista brasileira de sementes.15: 49-54.
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GUO, Z.F. (2010) Comparative study for cold acclimation physiological indicators of
36
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differences in morphological, physiological, and ultrastructural responses of Populus
cathayana to chilling. J. Exp. Bot. 62:1-12.
CAPÍTULO II
*Artigo a ser submetido para a revista Bioscience Journal
Resposta ao frio em cultivares de arroz com relação ao acúmulo de
fitomassa e possíveis alterações no teor de clorofila
Caroline Borges Bevilacqua, Carolina Terra Borges, Paulo Dejalma Zimmer.
Resumo- O estresse por frio durante os estádios iniciais de desenvolvimento do arroz
pode causar desuniformidade e diminuição na velocidade de germinação. Com o
objetivo de comparar a resposta ao frio em cultivares de arroz tolerantes e sensíveis a
esse estresse foram realizadas analises relacionadas ao acúmulo defitomassas seca e
possíveis alterações no teor de clorofila. Para tanto, foram utilizadas cultivares
consideradas tolerantes, sensíveis e moderadamente tolerantes ao frio. As sementes,
após sete dias a 25°C, foram expostas a 4°C durante 24 h e, logo após, foi estimado o
teor de clorofila. Posteriormente, as plantas ficaram durante 72 h a 25°C para
recuperação e, em seguida, foi coletado material para a medição das fitomassas secas. O
período de recuperação, no qual foi determinada a massa seca, especialmente de raiz, é
mais efetivo na discriminação dos genótipos comparativamente ao teor de clorofila.
Palavras-chave: Oryza sativa L, plântula, estresse abiótico.
38
INTRODUÇÃO
O Brasil está entre os 10 maiores produtores de arroz do mundo, sendo o maior
produtor mundial, ao se excluir os países asiáticos (SOSBAI, 2010). O estado do Rio
Grande do Sul é o principal produtor brasileiro de arroz irrigado, apresentando uma área
de cultivo estimada em 1,07 milhão de hectares, correspondendo a 66,9% da produção
nacional (CONAB, 2012).
No Rio Grande do Sul, o período ideal para a semeadura dessa cultura
corresponde de 15 de outubro a 15 de novembro, comas normais climatológicas de 30
anos registrando temperaturas em torno de 18,55°C nesse período. (CONAB, 2011;
Embrapa/UFPel/INMET, 2011). No entanto, sabe-se que temperaturas inferiores a 20°C
podem ser prejudiciais ao desenvolvimento e rendimento da cultura, já que a
temperatura ótima para a fase de germinação do arroz é entre 20°C e 35°C (YOSHIDA,
1981). Temperaturas abaixo de 20°C podem ocasionar injúrias sendo que esse fator é
considerado um dos estresses abióticos mais relevantes para a cultura do arroz. Portanto,
estudos sobre tolerância ao frio em arroz utilizam temperaturas inferiores a 20°C nas
fases germinativa, vegetativa e reprodutiva (YOSHIDA, 1981; CRUZ, 2001).
Os caracteres genéticos que conferem a tolerância ao frio são oriundos de
cultivares provenientes de países como Chile, Filipinas e Japão e pertencentes a
subespécie japonica. No entanto, a maioria das cultivares utilizada no Brasil pertence à
subespécie indica, o que contribui para uma desuniformidade e redução na velocidade
na germinação sob condições de estresse por temperatura (SOUZA, 1990). Essa redução
na velocidade de germinação estando relacionada ao retardo das reações metabólicas
pode acarretar em menor acumulo de massa seca, devido a um possível retardo na
metabolização das matérias de reserva (MERTZ et al., 2004).
As clorofilas são os principais responsáveis pela captação de energia luminosa
que é fundamental para a fotossíntese. Alterações no teor de clorofila podem ser
decorrentes de estresses, o que interfere no desenvolvimento da planta assim como na
produção de biomassa (LIMA et al., 2004; CANCELLIER et al., 2011). Sthapit &
Witcombe (1998) afirmaram que as características fisiológicas, como produção de
clorofila, são controladas geneticamente e devem ser exploradas no melhoramento
genético de arroz irrigado, uma vez que possuem altos valores de herdabilidade.
O período de recuperação de plantas expostas a estresses abióticos pode
contribuir a sua tolerância. Plantas de arroz, após exposição a condições de outro tipo de
39
estresse abiótico, em particular, a seca foram avaliadas quanto à concentração de
pigmentos fotossintetizantes, os resultados observados revelam que essas plantas foram
capazes de combater a fotoinibição causada durante o período de estresse (LOGGINI et
al., 1999).
O objetivo do presente estudo foi comparar a resposta ao frio de cultivares de
arroz tolerantes e sensíveis a esse estresse em relação ao acúmulo de fitomassa e
possíveis alterações no teor de clorofila.
MATERIAL E MÉTODOS
O presente estudo foi conduzido no laboratório de Bio-sementes, Departamento
de Fitotecnia da Universidade Federal de Pelotas (UFPel). As cultivares de arroz
utilizadas no experimento classificadas como tolerantes ao frio foram: Ambar,
Diamante, Oro; enquanto as sensíveis ao frio foram: IRGA 417, BRS PELOTA, BRS
TAIM e BRS SINUELO CL. Também foram incluídas cultivares com média tolerância
ao frio: BRS FRONTEIRA e BRS PAMPA.
Foram semeadas sete sementes de cada cultivar de arroz por copo plástico
(com 200 mL de capacidade), o que foi constituído de três repetições composta por um
copo. Os copos foram preenchidos com vermiculita e mantidas em câmara regulada
durante sete dias a 25°C. Em seguida, as plântulas foram mantidas a 4°C durante 24 h e
logo após, foi medida a fotossíntese. Posteriormente, as plantas foram mantidas por 72 h
a 25°C para sua recuperação, e, em seguida, foi coletado material para realizar as
avaliações de massa seca. Esse tempo de recuperação é necessário para que sejam
obtidas respostas fisiológicas em nível de biomassa, conforme Rabbaniet al. (2003) e
Cruz e Milach (2004).
Determinação dos teores de clorofilas a e b
O material coletado (0,1 gda parte aérea) de cada repetição foi transferido para
tubos falcon com capacidade de 15 mL, os quais foram previamente revestidos com
papel alumínio para evitar a incidência de luz. Nos tubos as amostras foram maceradas
juntamente com 5 mL de Acetona a 80%. (v/v). As amostras foram centrifugadas por 10
min em uma rotação de 5477,5 força g, e, em seguida, o material foi filtrado com
algodão, sendo o volume restante completado a 20 mL com Acetona a 80%. As
absorbâncias foram obtidas em espectrofotômetro (modelo Ultrospec 2000),
40
emcomprimento de onda de 645 nm (para determinação da clorofila b) e 663 nm (para
determinação da clorofila a). Os resultados foram expressos em μg.g-1de fitomassaseca
(MS). Os teores de clorofilas total, ‘a’ e ‘b’ foram calculados por meio de equações
(eq.1, 2 e 3) estabelecidas por Lichtenthaler (1987):
Chl totais = 7,15 (A663) + 18,71 (A645)
(eq. 1)
Chl ‘a’= 12,25 (A663) – 2,79 (A645)
(eq. 2)
Chl ‘b’= 21,50 (A645) – 5,10 (A663)
(eq. 3)
Determinação da Fitomassa após período de recuperação de estresse pelo frio
Após 72 h de recuperação (a 25°C), coletaram-se as plântulas, e separando-se
as partes aéreas e radiculares para a avaliação da fitomassa seca. Em seguida, a parte
aérea e a parte da raiz foram acondicionadas em sacos de papel e colocadas em estufa a
70ºC por 24 h. Logo após, as repetições foram pesadas novamente em balança analíticas
e o valor obtido da soma de cada amostra foi dividido pelo número de plântulas
utilizadas, com os resultados expressos em g plântula-1.
O delineamento experimental utilizado foi o inteiramente casualizado, com três
repetições, em esquema fatorial. As análises de variância foram realizadas aplicando-se
o teste F e quando foi detectado efeito significativo foi realizado o teste Scott-Knott ao
nível de 5% de possibilidade de erro. Os dados foram submetidos à Análise de
Variância, com o auxílio do programa SISVAR (FERREIRA, 2011) e transformados
pela fórmula Raiz quadrada de Y + 1.0 - SQRT (Y + 1.0).
RESULTADOS E DISCUSSAO
Os genótipos Ambar, Diamante e Oro, os quais são enquadrados como
tolerantes ao frio, foram considerados significativamente diferentes e superiores em
relação aos demais genótipos testados para a variável massa seca da raiz (MSR) e massa
seca total (MST) (Tabela 1). Isso pode indicar que durante o processo de recuperação da
planta, após a aplicação do estresse pelo frio, nas cultivares tolerantes foi verificada
maior quantidade de água estava acumulada na Parte Aérea.
Para variável MSR, houve diferença significativa entre as diferentes cultivares,
em que tanto as mediamente quanto as sensíveis ao frio, foram as que tiveram as
menores massas. Esse resultado corrobora com o Wielewick et al., (2002), pois as
41
cultivares de arroz estudadas no estudo referido apresentava um decréscimo no
comprimento radicular quando expostas a temperatura baixa.Durante o estádio de
plântula, o frio pode provocar atraso no desenvolvimento, redução na estatura,
amarelecimento das folhas e redução da matéria seca (MERTZ et al., 2009).
O estresse pelo frio diminui a razão parte aérea/raiz em genótipos tolerantes.
Além disso, o frio diminui a biomassa devido a redução na absorção de água e
nutrientes, assim como afeta a assimilação de CO2 e, portanto, a fotossíntese (AGHAEE
et al., 2011).
Tabela 1. Resposta para massa seca de cultivares de arroz tolerante ou sensível ao frio
submetidas sete dias a 25°C, em seguida, expostas a 4°C (24 h), seguida de 72 h de
recuperação (a 25°C). UFPel/FAEM. 2013.
Cultivar
MSR (g)
MST (g)
Ambar
0,003a
0,010b
Oro
0,004a
0,012a
Diamante
0,003a
0,010b
BRS Fronteira
0,001b
0,006c
BRS Pampa
0,001b
0,008c
IRGA 417
0,001b
0,007c
BRS Pelota
0,001b
0,006c
BRS Taim
0,001b
0,007c
BRS Sinuelo Cl.
0,001b
0,006c
Média
0,002
0,008
CV
0,04
0,06
*Médias seguidas por letras distintas, na coluna, diferem entre si pelo Teste Scott-Knott ao nível de 5% de
significância.
Os teores de clorofila “a” foram mensurados e as cultivares não diferiram
significativamente (média geral= 2,53; cv=25,6), o que sugere que estresse aplicado não
influenciou as cultivares testadas para esse tipo de clorofila. Já para os teores de
clorofila “b” e total ocorreu diferença significativa, no entanto, não foi observado um
padrão diferencial entre cultivares tolerantes, sensíveis e intermediarias, sob as
condições estudadas no estádio inicial de desenvolvimento (Tabela 2). Em outro
trabalho, com diferentes cultivares de arroz as doses de nitrogênio utilizadas
influenciaram os índices de clorofila b e total de clorofila, não apresentando
significativa interação entre os fatores para o índice de clorofila a. O elevado teor de
42
clorofila b pode estar relacionado a condições de baixa luminosidade, característica dos
germinadores utilizados no presente experimento, sugerindo que as cultivares que
conseguem aproveitar melhor a luminosidade apresentaram maiores valores de teor de
clorofila (CANCELLIER et al., 2011). Em um terceiro trabalho com cultivares de arroz
sensíveis ao resfriamento foi verificada a redução no conteúdo de clorofila nas folhas
(AGHAEE et al., 2011).
A redução na concentração de clorofila foi mais proeminentemente observada
durante o período de recuperação ao ser comparado com as avaliações logo após a
aplicação do estresse pelo frio (CRUZ, 2001). O que sugere que o período de
recuperação, no qual foram determinadas a massa seca é mais efetivo na separação dos
genótipos. Além disso, estudos realizados após 14 dias de estresse pelo frio em arroz
(18°C) são mais efetivos para ranqueamento de cultivares tolerantes e sensíveis ao frio
utilizando-se a parte aérea comparando-se com resultados após 7 dias de estresse pelo
frio (BEVILACQUA et al., 2013).
Tabela 2. Teores de clorofila “a” e “b” e totais (μg.g-1) em plântulas de arroz tolerantes
ou sensíveis ao frio submetidas sete dias a 25C, em seguida, expostas a frio (4°C
durante 24 h). UFPel/FAEM. 2013.
Cultivar
Clorofilas totais
Clorofila “b”
Ambar
9,85b
3,83b
Diamante
1,96b
0,57b
Oro
6,15b
1,89b
BRS Fronteira
16,54a
7,94a
BRS Pampa
16,91a
7,63a
9,82b
4,22b
BRS Pelota
15,18a
6,56a
BRS Taim
13,60a
6,60a
8,05b
3,27b
IRGA 417
BRS Sinuelo Cl.
Média
10,372,23
CV
18,44 19,96
* Médias seguidas por letras distintas, na coluna, diferem entre si pelo Teste de Scott-Knott ao nível de
5% de significância.
O estresse pelo frio em plantas pode provocar condições de anaerobiose,
levando ao processo de fermentação alcoólica, e consequente aumento na produção de
etanol e na síntese de álcool desidrogenase. Esse aumento na síntese de etanol pode
43
ajudar na preservação da fluidez dos lipídeos da membrana (MERTZ et al., 2009;
SALTVEIT et al., 2004). No entanto, o aumento na produção de etanol, juntamente com
o aumento de espécies reativas de oxigênio ou diminuição na síntese de antioxidantes,
pode acarretar no aumento da peroxidação dos lipídeos insaturados, presentes nas
membranas e que ajudam na fluidez dessas,sendo esses lipídeos sensíveis à oxidação,
levando a ruptura da membrana plasmática. O rompimento membranar, acarreta a perda
da clorofila e degradação da Rubisco (MARCONDES e GARCIA, 2009). A enzima
álcool desidrogenase produz acetaldeído e NADHo qual pode ser utilizado na cadeia
transportadora de elétrons (LEHNINGER et al., 1995). O que acarreta em alterações na
concentração das clorofilas e fotossíntese de modo geral.
CONCLUSÃO
O período de recuperação das plântulas após estresse pelo frio no qual foi
determinada a massa seca (raiz e total) é efetivo na separação de genótipos de arroz sob
estresse de frio.
Sugerem-se futuras análises da massa seca de parte aérea e teor de clorofila
após um período mais longo de estresse pelo frio.
AGRADECIMENTOS
Agradecimentos ao CNPq (Conselho Nacional de Desenvolvimento Cientifico
e Tecnológico) pelo suporte financeiro.
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fase de germinação. Revista brasileira de sementes, v.31, n.2, Londrina.
SALTVEIT, M.E.; PEISER, G.; RAB, A. Effect of acetaldehyde, arsenite, ethanol, and
heat shock on protein synthesis and chilling sensitivity of cucumber radicles. Plant
Physiology, v.120, p.556-562, 2004.
STREIT, N.M.; CANTERLE, L.P.; CANTO, M.W.; HECKTHEUER, L.H.H.As
clorofilas.Ciência Rural.Santa Maria, v.35, n.3, p.748-755, 2005.
STHAPIT, B.R.; WITCOMBE, J.R. 1998.Inheritance of tolerance to chilling stress in
rice during germination and plumule greening.Crop Science, Madison, v.38, p.660-665.
SOUZA, P.R. 1990. Alguns aspectos da influência do clima temperado sobre a cultura
do arroz irrigado, no sul do Brasil. Lavoura Arrozeira, v.43, p.9-11.
SOSBAI (Sociedade Sul - Brasileira de Arroz Irrigado) 2010. Arroz irrigado:
recomendações técnicas da pesquisa para o Sul do Brasil. 28. Reunião Técnica da
Cultura do Arroz Irrigado. 188 p.
VIANA, M.C.M. Déficit hídrico em genótipos de milho com tolerância diferencial à
seca. 2002. 75 p. Dissertação (Mestrado em Biologia Vegetal) – Universidade Federal
de Minas Gerais, Belo Horizonte.
YOSHIDA, S. 1981. Fundamentals of rice crop science. Los Baños: International
Rice Research Institute.
CAPÍTULO III
Eficiência de iniciadores relacionados aos genes de tolerância ao estresse em arroz
por baixas temperaturas e anoxia
Capítulo com análises preliminares às análises descritas nos Capítulos IV e V
INTRODUÇÃO
O arroz (Oryza sativa L.) está entre os cereais mais consumidos no mundo,
constituindo-se a base da alimentação de 2/3 da população do planeta. Entre os fatores
que podem prejudicar a produtividade desta cultura estão os estresses abióticos como as
baixas temperaturas, especialmente em estágios iniciais de desenvolvimento. A busca
pela tolerância é a principal solução para prejuízos causados pelo frio em arroz
(SERAFIM, 2003).
As respostas biológicas são controladas pela regulação da expressão gênica,
sendo que atualmente existe um grande número de estratégias disponíveis para analisar
a expressão diferencial de genes de interesse. A técnica de PCR quantitativo em Tempo
Real (qRT-PCR) é considerada sensível e precisa (BUSTIN, 2000).
Nessa técnica, a quantidade de produto amplificado é proporcional a
acumulação de fluorescência (HIGUCHI et al., 1993) e a eficiência dessa amplificação
deve ser comparada entre as amostras (FREEMAN et al., 1999). A acurácia desse
método é dependente da análise da curva padrão gerada a partir de diluições do cDNAa
partir da sua concentração inicial conhecida e da qualidade do RNA utilizada para sua
síntese (PEIRSON et al., 2003). Além disso, a metodologia de qRT-PCR que utiliza
SYBR Green é de baixo custo, rápida e confiável em comparação ao emprego de sonda
fluorescente (STANKOVIC e CORFAS, 2003).
Os genes constitutivos que apresentam uma alta estabilidade na expressão
podem ser utilizados como controles, nas reações de qRT-PCR em que os primers
utilizados para a amplificação dos genes a serem investigados devem apresentar uma
eficiência similar àquele utilizados para a amplificação desses genes constitutivos (JAIN
et al., 2006; LIVAK e SCHMITTGEN, 2001). Para tanto, a etapa de desenho dos
primers merece atenção para uma realização de reações de qRT-PCR eficientes.
47
O objetivo desse estudo foi avaliar a eficiência na amplificação de primers
relacionados aos caracteres de tolerância ao frio e/ou anoxia nas fases iniciais do arroz,
a fim de utilizá-los na técnica de qRT-PCR (Capítulos IV e V).
MATERIAL E MÉTODOS
Para o desenho dos primers relacionados aos genes de resposta ao estresse pelo
frio e/ou anoxia no arroz (Tabela 1 e 2), com auxílio do programa Primer 3
(http://frodo.wi.mit.edu/primer3/), observando os seguintes parâmetros:
-temperatura de anelamento (60-65°C);
-tamanho do amplicon (50-150 pb);
-porcentagem de GC (40-60%);
-ausência de dimerização.
Os reagentes utilizados para a realização das amplificações foram: 0,4μl de
DNTPs (5mM), 2 μl de Buffer (10x), 1,2 μl de Cloreto de magnésio (50mM), 2 μl
SYBR Green (1X), Taq Platinum, 5 μl de ROX dye, 1 μl de primer (10 μM) e o 1 μl of
template (cDNA) e 10,95 μl de água. Esse cDNA foi obtido a partir da extração de RNA
de mesocótilo das cultivares utilizadas no experimento em questão, após 2 semanas da
semeadura.
O seguinte programa de amplificação foi utilizado: Estágio 1= 50°C por 2 min;
Estágio 2 = 95°C por 10 min; Estágio 3 = 40 ciclos - 95°C por 15s/60°C por 1 min.
(onde ocorrerá a leitura); Estágio 4 = estágio adicional na qual é efetuada a dissociação
do SYBR - 95°C por 15s/60°C por 1 min./ 95°C por 15s. Os dados foram processados
no software 7500 v.2.0.1.
As amostras foram acondicionadas em placas (96 Well Optic Plates - Applied
Biosystems®) e cobertas com adesivo óptico (Optic Adhesives - Applied Biosystems®).
As reações foram realizadas no termociclador ABI PRISM 7500 Fast (Applied
Biosystems) (para reações referentes ao Capítulo IV) e no termociclador CFX 96 Realtime system, BIO-RAD (para reações referentes ao Capítulo V). Foram utilizadas três
repetições biológicas e três repetições técnicas.
Após o processo de amplificação do produto, que é proporcional à acumulação
de fluorescência, foi analisada a eficiência dos primers (WATSON et al., 1993).
48
Para a obtenção da curva de dissociação (Curva de Melt) foi utilizado o
Programa da Applied Biosystems. O qual apresenta um gráfico de regressão linear do
valor Ct em comparação ao log das diluições de cDNA, apresentando um valor
denominado de slope. Esse valor indica a inclinação da curva padrão gerada pelos
dados, ou seja, log da concentração das amostras x Ct (Threshold Cycle). A seguir, foi
realizada a análise do coeficiente de determinação, gerado a partir da curva padrão, o
qual determina o quanto os valores Ct sobrepõem os pontos da reta obtida pelos valores
log das diluições. A averiguação do comportamento das amostras em quatro pontos de
diluição, foi realizada a partir das curvas de eficiência de amplificação para cada par de
iniciadoresdos genes estudados e para o gene constitutivo (Actina e Ubiquitina,
Capítulos IV e V, respectivamente) foram obtidas a partir de uma série de diluições do
cDNA (1:1; 1:5; 1:25; 1:125).
RESULTADOS E DISCUSSÃO
A Curva de Melt demonstrou que a maioria dos primers obteve apenas um
pico (dados não apresentados), o que indica a provável presença de apenas uma
cópia do fragmento no genoma de arroz, o que representa um bom indício. Foram
utilizadas diluições seriadas do cDNA (1, 1:5, 1:25 e 1:125), sendo a diluição de
1:25 dos cDNAs a escolhida para serem posteriormente ser utilizada para a
quantificação relativa.
Os primers descritos nas Tabelas I e II mostraram-se os mais eficientes, pois
apresentaram seus valores de eficiência mais próximos de 2, para os genes analisados a
fim de serem utilizados para fins de quantificação relativa da expressão gênica. Valores
para eficiência dos primers devem ser o mais próximo possível de 2, com tolerância de
10% para o desvio, pois isso significa que o amplicon duplica sua quantidade durante a
fase geométrica (exponencial) da PCR, ou seja, que a PCR foi 100% eficiente.Os
primers que se destacaram, são os denominados JRC2606 e JRC3709 por apresentaram
os melhores resultados para eficiência, além de aceitáveis valores do coeficiente de
determinação. A qualidade da curva padrão foi aferida a partir do slope da equação de
regressão, utilizado para o cálculo da eficiência de amplificação, assim como do
coeficiente de determinação (R2) (DEPREZ et al., 2002).
49
Tabela I. Informações referentes aos genes e suas sequências de nucleotídeos a serem
utilizados em análises de expressão dos genes em qRT-PCR (Capítulo IV).UFPel/FAEM.
2013.
Gene name
Accession
Reference
Primer sequence
FCAAGATCACGGAGGAGATCGRGCTCTTGTGGTCCTTCTTC
F- TTTTCATGATGCGGAAGTCA
R-TTTTCCCTGTTGAGCACTCC
F- CGGTCACTACCTCCCAACAG
R-GACCGAACATCTCTTGACA
F- CATCTCACTAGCTCGCATCG
R- TGTTCCCTCAAGCACAGTGA
ASR1
AF039573
Arenhart, 2008
CloneJCR2606
BP432999
Rabbani et al., 2003
CloneJCR0937
BP432974
Rabbani et al., 2003
GERMIN
NM_001067692
Cheng et al., 2007
Actin
Pegoraro, 2012
F-CAGCCACACTGTCCCCATCTA
R-AGCAAGGTCGAGACGAAGGA
FONTE: NCBI
Tabela II. Informações referentes aos genes e suas sequências de nucleotídeos a serem
utilizados em análises de expressão dos genes em qRT-PCR (Capítulo V). UFPel/FAEM.
2013.
Name
Accession
Rabbani et
al.(2003)
NAM-F
NAM-R
AK068392
AK108621
Rab 16-R
AK071366
F-box-R
AK072651
Expansin 7-F
Expansin 7-R
Os03g60720
Sub 1b-F
Sub 1b-R
Os03g44290
Alcoholdehydrogenase 2-F
Alcoholdehydrogenase 2-R
Alpha-amylase-R
Os08g36910
Expansin 12-F
Expansin 12-R
Os07g47790
ERF 68-F
ERF 68-R
APX2-R
ERF 70-R
Os03g17690
ACCTGAACCCGTTTATCGTG
AGGGATGTGGTTCGTGCTAC
GTCCATTCACACTCCACACG
AGTGTGGGAGAGGGTGTGAC
AAGGTCATGGTGAAGATCGG
CCTTCTCCCAGACGCTGTAG
AGTTCATGGACTACGACGCC
ATCAAAGCTCCAGAGCTCCA
ACTACATGAGCTTCCTCGGC
AGGTGCCACAAGGAAAGATCTGGT
TCAGCAGGGCTTTGTCACTAGGAA
(Nakano et al,
2006).
Os09g11480
GAGATCAAGTGCGTGAACCA
GACGGCAGCTCGTAGTCTTC
Shigeokaet al.
(2002)
ERF 70-F
CCTGCAACATGAAGCTGAAA
GTGGATGTGCCAACAAAGTG
LasanthuKudahettigeet
al. (2007)
LasanthuKudahettigeet
al. (2007)
Nakano et al,
2006).
Os01g21120
APX2-F
GAAGAAGGGCTTCATGGACA
TCAGTTTCCTTCCGACTGCT
LasanthuKudahettigeet
al. (2007)
LasanthuKudahettigeet
al. (2007)
Fukuda et al.
(2005)
Os11g10510
Alpha-amylase-F
GCTCCTCGTCGACTACATCC
CACCATCACTCGCATTTCAC
Rabbaniet
al.(2003)
F-box-F
GCAAGCCATTCTAGACGACC
CTGGACGATGGACTTCTGCT
Rabbaniet al.
(2003)
Rab 16-F
Primer sequence
GCTCGCCTGAGTCAAAGTTC
Rabbaniet al.
(2003)
Myb 7-F
Myb 7-R
Reference*
GGACGCCACAACGAAGATGAAGAA
TGCACCAGAAGGGAACATGGAAAC
50
(continuação)
Dubouzetet al.
(2003)
Dreb2A-F
Dreb2A-R
AF300971
Glutamatedehydrogenase-R
ATGAAGGTGCTGATGTGCAG
Rabbaniet al.
(2003)
Glutamatedehydrogenase-F
BP432999
Germin-R
Os08g08970
H+ pyrophosphatase-R
D45383
Ubiquitina -R
TTGAGCCTGCCCTCAAGAAG
GGGAGGCCTAACCAACTGAC
Jain et al.(2006)
Ubiquitina -F
CATCTCACTAGCTCGCATCG
TGTTCCCTCAAGCACAGTGA
Rabbaniet al.
(2003)
H+ pyrophosphatase-F
TTTTCATGATGCGGAAGTCA
TTTTCCCTGTTGAGCACTCC
Dubouzetet al
(2003)
Germin-F
TAAGTGGGTGGCTGAGATCC
UBI
CTCACCTACGTCTACAA
GTCAAGGTGTTCAGTTC
FONTE: NCBI
Os demais primers desenhados (dados não mostrados) tanto no que diz respeito
ao valor da eficiência dos iniciadores quanto a sua dissociação após a amplificação
demonstraram uma maior inespecificidade em produzir apenas um amplicon em relação
aos demais testados.
CONCLUSÃO
Os primers testados para reações de qRT-PCR, apresentam diferentes níveis de
eficiência indicando a necessidade de descarte de alguns desses, antes da análise por
qRT-PCR para investigar a tolerância à estresses abióticos em arroz em estádios iniciais
de desenvolvimento sob estresse por frio.
REFERÊNCIAS
BUSTIN S.A. Absolute quantification of mRNA using real-time reverse transcription
polymerase chain reaction assays. Journal of Molecular Endocrinology. 25, p.169–
193. 2000.
DEPREZ, R.H.L. 2002.Sensitivity and accuracy of quantitative real-time polymerase chain
reaction using SYBR green I depends on cDNA synthesis conditions. Analytical
Biochemistry, v.307, p. 63-69.
FREEMAN, D., GARETY, P. A. Worry, worry processes and dimensions of delusions:
An exploratory investigation of a role for anxiety processes in the maintenance of
delusional distress. Behavioral and Cognitive Psychotherapy, 27, p.47–62. 1999.
HIGUCHI, R., FOCKLER, C., DOLLINGER, G, and WATSON, R. Kinetic PCR
analysis: real-time monitoring of DNA amplification reactions. Bio/Technology, 11,
p.1026-1030. 1993.
JAIN, N., R. Koopar and S. Saxena,. Effect of accelerated ageing on seeds of radish
(Raphanus sativus L.).Asian Journal of Plant Sciences, 5, p.461-464.2006.
51
LIVAK, K.J.; SCHMITTGEN, T.D. Analysis of relative gene expression data using
real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods, v.25, p.402408, 2001.
PEIRSON, S.N., BUTLER, J.N., FOSTER, R.G. Experimental validation of novel
andconventional approaches to quantitative real-time PCR data analysis. NucleicAcids
Res 31(14): e73. 2003.
SERAFIM, D. C. S. Mapeamento de QTLs para tolerância ao frio e características
de importância agronômica em arroz. Dissertação (mestrado) em Fitotecnia / Área de
concentração Plantas de Lavoura, Universidade Federal de Santa Maria, Santa Maria,
2003.
WATSON, R. et al. Kinetic PCR Analysis: Real-time Monitoring of DNA
Amplification Reactions. Nature Biotechnology 11, p.1026–1030, 1993.
CAPÍTULO IV
*Artigo a ser submetido para a revista Journal of Plant Physiology
Differential gene expression of cold responsive genes in rice and red rice genotypes
C.B. Bevilacqua, D. Monzon, T. Viana, P.D. Zimmer; S. Basu
Abstract - Plants have strategic adaptations to harsh environments especially after
germination when stress avoidance is no longer possible. Temperature stress occurs at
any ontogenic period during crop production affecting a large array of developmental
processes. Rice seedlings at early planting times are subjected to stress by deep sowing
as well as exposure to low temperatures. Plant species have different tolerance to cold
stress and depth emergence. It has been shown that weedy red rice is more
physiologically adapted to deep sowing than rice cultivars. Likewise, some rice
genotypes are more tolerant to cold stress than others. To achieve a more
comprehensive understanding of the gene expression patterns of rice in response to
depth stress and cold stress, we performed a gene expression analysis using “Diamante”
(cold tolerant), “BRS 6 Chui” (cold sensitive), and an ecotype of weedy red rice. We
then selected the depth-tolerant red rice ecotype 116, and the cultivars BRS6 Chui (cold
sensitive) and Diamante (cold tolerant) for further experiments. Esterase isoenzyme
analysis was done on these plant materials under cold stress (18oC-10h/13oC-14h
temperature), which showed the presence of two unique isoforms in cold sensitive and
ecotype 116. However, all the isoforms were regulated by cold stress. A differential
expression pattern was observed in the transcript accumulation of the genes under study
in all the accessions. These results can help us to study the behavioral responses of
different red rice and rice accessions under deep sowing using these cold inducible
genes as a marker. Moreover we will draw a correlation between cold stress and deep
sowing.
53
INTRODUCTION
The emergence of seedlings influences the yield of crops. Yield increases as
seedling emergence becomes faster and more uniform. The faster and more uniform the
emergence of the seedlings are, the better is the weed control, and there is reduction of
other plant stress (SOLTANI et al. 2006). Abiotic stress is one of the factors that can
decrease the emergence of seedlings. The Abiotic stress signaling is a complex network
that needs multiple information (XIONG et al., 2002).
Rice is a temperature-sensitive crop (CRUZ e MILACH, 2000). Low
temperatures and seed sowing depths are abiotic stresses that can dramatically reduce
yield. Moreover, sowing depth is considered an important abiotic stress that affects crop
productivity. For weedy red rice, this feature can be considered positive, especially for
the development of weed seed banks in soil, which has greater longevity and hence it
adopts a strategy of becoming dormant to persist in the soil (NOLDIN, 1995). However,
weedy red rice is one weed injuring rice fields (SMITH, 1988).
The linkage between rice biomass production and mean minimum temperature
was established. A 10% reduction in grain yield can occur for every 1°C increase in
growing season minimum temperature (PENG et al., 2004). Plants exposed to cold
stress, which includes chilling (< 200C) and freezing (< 00C) temperatures, negatively
affects the growth and development of plants. It can also significantly constraint the
spatial distribution and the yield of crops (CHINNUSAMY et al., 2006).
Many studies have been performed to determine the cold tolerance in different
genotypes. It has been demonstrated that at the stage of germination the subspecies
japonica genotypes have greater tolerance. In Brazil, studies using temperatures of 13
and 28ºC, showed that the japonica cultivars “Quilla 64117” and “Diamante” are more
tolerant to cold. Among the indica genotypes, “BR-IRGA 410” and “IRGA 416”
cultivars were more tolerant (CRUZ e MILACH, 2004).
The genetic traits that lead to cold tolerance (referred to as source of tolerance),
are from cultivars coming from countries such as Chile, Philippines and Japan. In this
case the tolerance was determined by evaluating the coleoptile length and the
regrowth at 28ºC, after exposure to 13ºC. In studies performed by EMBRAPA
(Empresa Brasileira de Pesquisa Agropecuária) the emergence rate index (IVE) was
used as a parameter to verify the tolerance of different genotypes to cold (EMBRAPA,
2009).
54
The isoenzymatic complex esterase is one of more polymorphic complex in
plants (WEEDEN e WENDEL, 1990). It is very useful in rice researches (ENDO E
MORISHMA, 1983) due to their changes in expression pattern under cold stress
(MERTZ et al., 2009).
The expression of the proteins involved with cold tolerance is regulated by
specific transcription factors (TF). TFs are proteins with a DNA domain that binds to
the cis-acting elements present in the promoter of the target gene. The family of
transcription factors CBF (C-repeat binding factor) or DREB (dehydration responsive
element binding factor) regulates the expression of genes that respond to several
stresses, among them the cold stress, containing the cis-element CRT/DRE (Dubouzet et
al., 2003). The transcription factor AREB (ABA - responsive element binding protein)
or ABFs (binding factors) is also important in abiotic stress. It activates the cis-elements
ABRE (ABA - responsive element) as well as the family NAC (NAM, ATAF and NUC)
(YAMAGUCHI-SHINOZAKI & SHINOZAKI, 2005; HU et al., 2008). The zinc finger
proteins show trans regulation of cold tolerance, as demonstrated by Huang et
al. (2009). It is based on the given ZFP245 accumulation by activation of ABA
synthesis. Moreover, further analysis showed that the overexpression of ZFP245 using a
transgenic increases antioxidant activity, thus protecting the plants.
Several other genes involved in cold response during rice germination have
been described such as Cor (Cold-regulated), KIn (cold inducible), ErD (early
responsive
to
dehydration),
LTI
(Low-Temperature
Induced),
and
RD
(Responsive Dehydration). Genes that require the abscisic acid signaling or presence of
reactive oxygen species (ROS), or calcium-dependent protein kinases (CDPKs), or
calcineurin B-like that interacts with protein kinase (OsCIPK03) and others were also
reported (YAMAGUCHI-SHINOZAKI, 2000; CHINNUSAMY et al., 2006; WANet
al., 2007; SAIJO et al., 2001;XIANG et al., 2007).
A large number of cellular processes are involved in the response to stress
tolerance and a large number of genes are required for various abiotic stresses response.
There is an essential combinatorial control of different transcription factors that have
high expression to promote tolerance. In a study conducted by Lindlof et al. (2009)
1450 genes related to cold stress in rice were identified through microarray analysis. It
corresponded to a lower number of genes than those found in the Arabdopsis (1753
genes) under the same experimental conditions.
55
In all these studies, the stress condition is applied in a specific period; on the
other hand, it is relevant to investigate gene expression under conditions of permanent
stress during seedling stage. Moreover studies that will indicate genes that show
crosstalk between the different stresses are actually important to understand the key of
different stress tolerance. It would favor the development of new cultivars showing cold
tolerance.
To achieve a more comprehensive understanding of the gene expression
patterns of rice in response to depth stress and cold stress, we performed a gene
expression analysis using “Diamante” (cold tolerant), “BRS 6 Chui” (cold sensitive),
and an ecotype of weedy red rice. The results obtained here will help us to characterize
the response of weedy red rice towards depth and cold stress. In addition, it will also
provide an insight into the genetic makeup of the weeds. Moreover this will also help us
to get an understanding whether there is any crosstalk between depth stress and cold
stress signaling in rice plants.
MATERIAL AND METHODS
Screening of different weedy red rice genotypes for deep sowing tolerance
The experiment was carried out with 42 red rice accessions (Q1 1A, Q1 19, Q1
13,Q2 14, Q1 32, Q 28, Q1 1A, Q1 2A, Q2 36B, Q1 9, Q1 10A, Q2 33A, Q2 33B, Q1
14A, Q1 18, Q1 10, Q2 6B, Q2 29, Q2 8B oposto, Q2 38B, Q1 22, Q1 5, Q1 4B, Q2
13A,Q2 28A, Q 11A, Q2 32, Q2 10A, Q1 4A, Q2 34b, Q1 28, Q2 16, Q1 36, Q2 13,Q2
14, Q2 27, Q2 2A, Q2 17, Q1 20, Q2 37A, Q2 19 e Q2 ant. 28) with 10 replicates. The
samples were screened for depth tolerance (15cm) based on their ability to germinate
(data not shown). They were evaluated as tolerant or sensitive based on their shoot
length (after 14 days sowed).
Plant Material and Growth Condition
Three rice genotypes were used in this study: “Diamante” (cold tolerant), “BRS
6 Chuí” (cold sensitive) and the weedy red rice accession selected in the Screening of
different weedy red rice genotypes for deep sowing tolerance. Seeds were washed with
water and then were allowed to grow in homogeneous conditions in plastic pots under
vermiculite. For cold stress treatment the plants were kept at 18ºC-14h/13ºC-10h
(during 14 days). For deep sowing stress, seeds were sown, and kept during 14 days, at
a depth of 5, 10 and 15 cm while the seeds sown at a depth of 1.5 cm served as the
control. Plants were washed thoroughly, and the coleoptiles were harvested. Samples of
56
equal fresh weight were frozen in liquid nitrogen. After collection, the plant tissue was
quickly frozen in ultra-freezer (-80ºC) until RNA extraction.
Esterase analysis by NPGE
For esterase isoenzyme analysis, 200 mg of control and stressed (13°C-10
h/18°C-14 h) samples of BRS 6 Chuí (cold sensitive), Diamante (cold tolerant) and
weedy red rice ecotype 116 (depth tolerant) respectively were homogenized in liquid
N2. A total of 400 µl extraction buffer containing solution 0.2(M) Lithium Borate
(pH8.3), 0.2(M) Tris Citrate (pH 8.3) and 0.15% 2-mercaptoethanol was added to the
homogenized samples. The resulting mixture was centrifuged and the supernatant was
collected and analyzed by 7% Native Polyacrylamide gel electrophoresis and stained
according to Scandálios (1969) and Alfenas (1998) with some modifications.
Gene expression analysis by qRT-PCR
The sequences to four cold-responsive genes were obtained in NCBI
(http://www.ncbi.nlm.nih.gov/) (Table 1).To study the expression pattern of these genes
under cold and deep sowing stress treatments from different rice genotypes, quantitative
RT-PCR (qRT-PCR v.2.0.1 7500 Fast (Applied Biosystems) was performed using ROX
(Invitrogen) and SYBR SAFE DNA in DMSO (Invitrogen) for 40 cycles following the
manufacturer’s protocol. Three independent biological replicates of each sample were
used for expression analysis; each sample was normalized using Actin gene (Chapter IV
of this dissertation) and Ubiquitin (Chapter V of this dissertation).
Negative control reactions were also run to confirm the absence of
contaminants.
The
design
the
primers
were
carried
out
by
Primer
3
(http://bioinfo.ut.ee/primer3-0.4.0/). The primer selection was done previously by
Applied BiosystemsTM guide.
The RNA extraction was carried out using 0.1 g from shoot in the seedling
stage by TrizolTM Reagent (InvitrogenTM), followed by DNAse ITM Amplification
Grade (InvitrogenTM).To qualify and quantify the RNA was used electrophoreses and
spectrophotometer and then the cDNA was obtained with 2 µg of RNA
bySuperScriptTM First-Strand System for RT-PCR (Invitrogen TM).
The qPCR was carried out by 7500 Real Time PCR System (Applied
BiosystemsTM). To determine relative fold changes for each sample in each experiment,
57
the Ct (Threshold cycle) value for each gene was normalized to the Ct value for actin
and was calculated relative to a calibrator using the equation 2-ΔΔCt (Livak e Schmittgen,
2001).
To quantify, by real time, was performed 2 μl of the SYBR Green (1X), 1 μl of
each oligonucleotide (10 μM), 1 μl of the first strand cDNA (diluted 1:25, selected
according to results from Chapter 3), 0,4μl of DNTP (5Mm), 2 μl (Buffer 10X), 1,2 μl
of MgCl2 (50Mm), 0,5 μl of ROX dye and 10,95 μl of water.
Table 1. Expressed genes in rice (Oryza sativa L.) when subjected to abiotic stress.
Gene
Accession
Reference*
ASR1
AF039573
Arenhart, 2008
CloneJCR2606
BP432999
CloneJCR0937
BP432974
GERMIN
NM_001067692
Actin
Primer sequences
F- CAAGATCACGGAGGAGATCG
R- GCTCTTGTGGTCCTTCTTC
Rabbani et al., 2003 F- TTTTCATGATGCGGAAGTCA
R-TTTTCCCTGTTGAGCACTCC
Rabbani et al., 2003 F- CGGTCACTACCTCCCAACAG
R-GACCGAACATCTCTTGACA
Cheng et al., 2007
F- CATCTCACTAGCTCGCATCG
R- TGTTCCCTCAAGCACAGTGA
Pegoraro, 2012
F-CAGCCACACTGTCCCCATCTA
R-AGCAAGGTCGAGACGAAGGA
* Based on the literature found in relation to gene function.
RESULTS AND DISCUSSION
Red rice depth screening
The screening for depth tolerance was done with 42 weedy red rice accessions.
The screening was done based on the changes in the shoot and root on emergence length
under depth treatment. To evaluate plants in the germination under depths, weedy red
rice under different depths of sowing can be used a source of genetic variability
(MALONE et al., 2007).
Based on the results shown in Figure 1, the weedy red rice ecotype 116 (Q128) was chosen because the shoot and root length measurement showed good results as
compared in the screening of different weedy red rice genotypes for deep sowing
tolerance.
The sowing depth affects the stand of the crop. For the development of weed
seed banks in soil, the deeply sowing have greater longevity and can become dormant to
persist longer in the soil (NOLDIN, 1995).
58
Figure 1. Seeding depth tolerance screening of weedy red rice accessions by measurements of seedling
root and shoot lengths (mm), 14 d after sowing. UFPel/FAEM. 2013.
Next step was to study the sensitivity of the weedy red rice under low
temperature stress as compared to the cultivars Diamante and BRS6 Chui by Esterase
Isoenzyme Analysis. Our investigation showed that weedy red rice Ecotype 116 and
BRS6 Chui showed the presence of unique isoforms that are down-regulated under cold
stress (Esterase 2 and Esterase 4, respectively). While the other two isoforms are also
down-regulated under cold stress in weedy red rice Ecotype 116 and BRS 6 Chui,
Esterase 1 and Esterase 3, which are present in all the three accessions, are up-regulated
in the cold tolerant cultivar Diamante (Figure 2).
The increment of esterase activity is highly possible that the isoforms 1 and 3
can be related to tolerance, as well as the absence of isoforms 2 and 4. Previous study
indicated 2 alleles for Esterase isoenzyme assay. One of these alleles decreased the
expression in rice seeds exposed to cold stress (MERTZ et al., 2009). This reduction of
intensity of the bands was also observed in Brandão-Júnior et al. (1999) and this
decrease was also in loss of the seeds availability. Majumder et al. (1989) already found
Esterase alleles to sensitivity and tolerance for cold stress in rice.
The Esterase plays a role in the lipid metabolism, which is important for
possible alterations in the membrane lipid phase during cold stress. So this alteration
can contribute to the degenerative processes resulting from the low-temperature stress
59
(PUNEVA e ILIEV, 1995). This is consistent with our observation, so Esterase is a tool
to identify and select cold-tolerant rice genotypes.
Ecotype 116
(Deep sowing-tolerant)
Control
Cold
Diamante
(Cold-tolerant)
Control
Cold
BRS6 Chui
(Cold-sensitive)
Control
Cold
Esterase 1
Esterase 2
Esterase 4
Esterase 3
Figure 2. Esterase isoenzyme analysis of red rice ecotype 116 and the cultivars Diamante and BRS 6 Chui
under cold stress (18ºC-10h/13ºC-14 h). UFPel/FAEM. 2013.
When the plants are exposed to abiotic stresses, the ROS are synthetized during
a normal plant cellular metabolism. However environmental stresses can excessively
increase the ROS production causing progressive damages and/or act as second
messengers involved in the stress tolerance of plants (SHARMA et al., 2012). Germin is
known as oxalate oxidase, which one converts oxalic acid (OA) to CO2 and hydrogen
peroxide (H2O2), this enzyme permit to accumulate that Reactive oxygen species (ROS)
in the apoplast (WOJTASZEK, 1997).
Germin was up-regulated under cold stress as well as with increasing sowing
deep sowing stress for BRS 6 Chui, Diamante and weedy red rice ecotype 116, where
weedy red rice ecotype 116 showed more transcript accumulation as compared to
Diamante and BRS 6 Chui under cold stress (Figures 3 and 4).
Germin is a defense-related protein while ASR 1 is a low temperature induced
chaperone. Consequently, when plants are subjected to abiotic stresses like cold and
deep sowing, transcript accumulation of these genes increases, suggesting their role in
counteracting stress.ASR1 was up-regulated under cold stress as well as in sowing depth
stress in Diamante and BRS 6 Chui and down-regulated for weedy red rice ecotype 116.
ASR1 helps the folding of proteins (TAIZ e ZEIGER, 2009). Previously it has been
reported that ASR1 gene is induced under abiotic stress treatment in rice (Rabbani et al.,
60
2003). Our results also show that this gene has the expression increased in the cultivars
under stress.
On the contrary JRC 3709 (calcineurin B-like protein)was down-regulated
under different stress conditions which is consistent with previous results (Rabbani et
al., 2003). However, this gene was up-regulated for BRS 6 Chui (cold-sensitive) under
depth (15 cm) and cold stress. Rabbani et al. (2003) showed for other also cold sensitive
rice (Oryza sativa var. Nipponbare) exposed to cold (4ºC, after 2; 5; 10 and 24 h). It
showed that probably this cold-sensitive one increased the transcript accumulation of
this enzyme decreasing the successfully energy spend to produce a mechanism of
tolerance (Figures 3 and 4).
JRC 2606 (Glutamate dehydrogenase) was inducible with depth stress for all
the different rice accessions, but it was down-regulated under cold stress for BRS 6
Chui and Diamante. While weedy red rice Ecotype 116 showed up-regulation under
cold stress (Figures 3 and 4).
Glutamate dehydrogenase (GDH) is a hexameric enzyme involved in nitrogen
metabolism (Stewart et al., 1980), which one is encoded by two separate loci of that
gene: gdh1 and gdh2, in Maize and Arabdopsis (MAGALHÃES et al., 1990; TURANO
et al., 1997), while JRC 3709 is a calcineurin B- like protein which plays a role in
regulating the expression of GDH. Due to Calcium is recognized as a universal second
messenger (CARAFOLI, 2005) may be the absence or presence of the calcium binding
domain can regulate differentially the Ca2+.
Based on that, in the present study is suggested the GDH and calcineurin B-like
gene expressions are positively correlated. The cold-tolerant cultivar (Diamante)
showed a balance between the expressions of these two proteins. For the others
genotypes the disbalance was showed. This balance probably is important to the coldtolerance. It also can suggest that is this work was studied only one of the GDH
isozymes, probably the gdh2, because this one is absence in sensitive and in our study
this is down-regulated in the cold-tolerant plants. The cold-sensitive accession, in
Maize, the gdh2 gene product was the only loci detected (Pryor, 1990). Also in Maize
the gdh1 null mutants showed a single GDH isozyme and it was the gdh2 gene product
(MAGALHÃES et al., 1990). This can emphasize the relevance of gdh1 gene product to
the cold-tolerance helping keep the protein synthesis by nitrogen metabolism.
61
So the cold treatment was enough to provide different response. The coldsensitive BRS6 Chui showed repressed JRC 2606 and up-regulated JRC 3709
(calcineurin B-like protein).
Figure 3. Differential gene expression analysis assessed through qRT-PCR in Ecotype 116 (deep sowingtolerant), BRS6 Chui (cold-sensitive) and Diamante (cold-tolerant) compared with that of control
subjected to cold stress (Relative fold change, log2 ratio) for each plants. UFPel/FAEM. 2013.
a
.
b
.
c
.
d
.
Figure 4. Differential Expression analysis of genes: a. ASR1 b: Germin c: JRC 2606 and d: JRC 3709
assessed through qRT-PCR in Ecotype 116, BRS 6 Chui and Diamante compared with that of control
subjected to sowing depth stress (Relative fold change, log2 ratio) for each plants.UFPel/FAEM. 2013.
62
These results can help provide an insight into the cold tolerance and sowing
depth tolerance of different accessions. However a lot of work still needs to be done.
CONCLUSIONS
Esterase isoenzyme analysis showed the presence of two unique isoforms in
cold sensitive and ecotype 116, but all the isoforms were regulated by cold stress.
Esterase is a tool to identify and select cold-tolerant rice genotypes.
A differential expression pattern was observed in the transcript accumulation of
the genes under study in all the accessions.
The genes JRC 2606 e JRC 3709 showed inversely correlated and it can be
used as a molecular marker to cold stress.
The genesASR1 and Germin can be used as a molecular marker for depth
sowing stress.
For future studies is recommended evaluate the response for these gene of
more accessions, under abiotic stress
ACKNOWLEDGMENT
This work was supported by CNPq (National Council for Scientific and
Technological Development – Brazil). I wish to thank Dr. Mariccor Batoy for her
valuable suggestions.
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CAPÍTULO V
*Artigo a ser submetido para a revista Plant Physiology
Screening of different rice genotypes for cold and deep sowing stresses
Caroline Borges Bevilacqua1, SupratimBasu2, Tse-Ming Tseng3, Paulo Dejalma Zimmer1,
Nilda Roma Burgos2, AndyPereira2
1
Universidade Federal de Pelotas, Pelotas, RS, Brazil;
2
University of Arkansas, Fayetteville, Arkansas, USA.
3
Purdue University, West Lafayette, Indiana, USA
ABSTRACT- Cold stress adversely modifies rice physiology, metabolism, plant growth and
development and thus limits crop productivity. Rice (Oryza sativa L.) is one of the important
cereals grown across the world. Although it has been used as a model plant for many years,
the growth responses of rice to cold temperature are still poorly understood. We characterized
a panel of rice genotypes with variation in cold and/or deep sowing sensitivity, with the
following aims: 1) categorize 21 rice accessions as Japonica or Indica by phenol test; 2)
classify them as sowing depth and/or cold-sensitive or –tolerant by evaluating germination
and measuring seedling shoot length after 14 days of incubation at 18/13°C day/night
temperature and at different seedling depths (1.5cm, 5cm, 10cm and 15cm respectively); and
3) analyze the expression of genes under cold and/or sowing depth treatment. We then
selected sensitive and tolerant (3 genotypes from Indica and 2 genotypes from Japonica) rice
subspecies, respectively, to analyze the expression of cold-responsive genes and
submergence-inducible genes by qPCR after exposing the rice plants to 10°C for 6, 24 and 96
h at 1.5 cm and 10 cm sowing depths. These results indicated the importance of ABAdependent and independent pathways for tolerance in both stresses in Japonica and Indica
subspecies. These patterns of gene expression can help to provide an insight into the cold
tolerance and sowing depth tolerance in these different rice subspecies.
66
INTRODUCTION
Temperatures under 20°C cause development and yield injuries, so this abiotic stress
is a relevant problem for rice crop. Many studies during vegetative and reproductive phases
are carried out due to the presence these low temperatures in the field (YOSHIDA, 1981;
CRUZ, 2000).
The genetic traits leading to cold tolerance are from rice cultivated in Chile,
Philippines and Japan, and belong to Japonica subspecies. Nevertheless, the majority of rice
cultivated in Brazil belongs to Indica subspecies, and majority of high yielding cultivars show
little tolerance to low temperature. This contributes to non-uniform germination and
germination is reported to decreases under cold stress (SOUZA, 1990; XU, 2008; NAHAR et
al., 2009). However, the high sterility of the hybrids (Indica x Japonica) shows the
importance of more researches (ROSSO, 2006), as well as studies about the mechanisms
involved in cold stress response. These studies can provide the development of cold-tolerant
cultivars, avoiding the low-temperature damage (LOU et al., 2007).
Cold tolerance, during seedling stage, is necessary for a stable rice production.
According to previous studies the percentage of reduction in coleoptile, at the germination
stage, can identify readily and discriminate the tolerant and sensitive rice genotypes under
abiotic stress (GREGORIO et al., 1997; CRUZ e MILACH, 2004; CRUZ et al., 2006). During
the vegetative phase, rice genotypes were screen out for Salt tolerance indicating the necessity
of molecular markers to improve the rice tolerance identification and selection, encompassing
genotypic and phenotypic studies as well (BHOWMIK et al., 2009).
Different abiotic stress can respond using the same pathway, activating the same
cascades of signal transduction, which leads to crosstalk (RABBANI et al., 2003).This
indicates that stress, like sowing depth, can be evaluated, which can contribute to evaluate the
vigor and the response of less oxygen conditions. Different responses for weedy red rice
under depth sowing were investigated using different depths, these weedy red rice are source
of variability (MALONE et al., 2007).
This study was conducted to achieve objectives to characterize cultivated and weedy
red rice into Indica and Japonica subspecies, and to screen phenotypes for tolerance to low
temperature and/or sowing depth conditions. The accessions selected were analyzed by
expression of cold-responsive genes and submergence-inducible genes using qPCR to
elucidate how rice copes with different abiotic stresses.
67
RESULTS
Rice Subspecies Determination
Twenty-one rice and red rice accessions (Figure 1) were subjected to phenol test to
determine their subspecies grouping. The phenol test showed that 9 accessions were Japonica
and 12 were Indica (Table 1).
Figure 1. Phenol test of cultivated and weedy red rice accessions for subspecies determination. UFPel/FAEM.
2013.
Table I. Rice and red rice genotypesclassified as Japonica or Indica subspecies by phenol test.
UFPel/FAEM. 2013.
Accession code
PRA 08-D02
LIN 08-B3
LIN 08-B5
CHI 08-C
LIN 08-C
GRE 08-DO1
LEE-8-C
CRA 08-D
Law 08-D
210
116
5
135
County
PRAIRIE
LINCOLN
LINCOLN
CHICOT
LINCOLN
GREENE
LEE
CRAIGHEAD
LAWRENCE
Conway
Randolph
Arkansas
Woodruff
Rice ou weedy red rice
weedy red rice
weedy red rice
weedy red rice
weedy red rice
weedy red rice
weedy red rice
weedy red rice
weedy red rice
weedy red rice
weedy red rice
weedy red rice
weedy red rice
weedy red rice
Subspecies
Japonica
Indica
Indica
Indica
Indica
Indica
Indica
Indica
Japonica
Indica
Indica
Indica
Japonica
68
(continuação)
85
44
3011
1200
1602
Spring
Oro
Hayayuki
Lonoke
Desha
Rio Grande do Sul-Brazil
Rio Grande do Sul-Brazil
Rio Grande do Sul-Brazil
USA cultivar
Chile cultivar
Japan cultivar
weedy red rice
weedy red rice
weedy red rice
weedy red rice
weedy red rice
rice
rice
rice
Japonica
Japonica
Japonica
Indica
Indica
Japonica
Japonica
Japonica
Phenotyping for Cold Tolerance
Testing for cold tolerance showed two Indica (GRE 08-D01 and 1602) and one
Japonica (Spring) accessions were tolerant to cold stress (Figure 2; Table II). The others were
sensitive and of these were selected one Indica (CHI 08-C) and one Japonica (PRA 08-D02)
(Table I). Tolerant accessions have an average shoot length reduction of 28-38% relative to
the nonstressed plants compared with approximately 95% in both sensitive accessions choose
Reduction of shoot length (%)
(Figure 2).
Accessions under cold stress
Figure 2. Response of selected Oryza genotypes to cold treatment (18C-10 h/13C-14 h temperature). Shoot lengths
were measured (14 days after sowing). Each bar is the average of 3 replicates, each one with 5 seeds and 2 runs.
UFPel/FAEM. 2013.
69
Table II. Evaluation of rice tolerance to cold stress.UFPel/FAEM. 2013
Shoot length
reduction (mm)
95
31
82
94
95
29
93
92
92
91
95
95
37
Accession
1200
1602
3011
CHI-08C
CRA-08D
GRE-08D01
Havayuki
Lee-8C
LIN08-B3
LIN08-B5
LIN08-C
PRA08-D
SPRING
Tolerance category
Sensitive
Tolerant
X
X
X
X
X
X
X
X
X
X
X
X
X
Prob> F
<.0001*
<.0001*
<.0001*
<.0001*
<.0001*
<.0001*
<.0001*
<.0001*
<.0001*
<.0001*
<.0001*
<.0001*
<.0001*
Rice Tolerance to Seeding Depth
The results for characterization of genotypes with depth tolerance are shown in
Figures 3 and Table III. One Indica weedy rice (GRE 08-D01) and one Japonica cultivar
(Spring) accession are tolerant to depth stress up to 15 cm; the rest are sensitive. The shoot
lengths of tolerant accessions showed less than 50% of reduction.
All accessions did not grow well when planted deep (5, 10 and 15 cm) and subjected
to cold stress (18C-10 h/13C-14 h temperature) (Figure 4) (Table IV). Tolerance to cold stress
was absent when the seed was deep sowed.
Table III. Screening of rice tolerance to depth stress (5 cm, 10 cm, and 15 cm).
Accession
Shoot length reduction (mm)
Prob> F
5 cm depth
10 cm depth
15 cm depth
1200
1602
3011
CHI-08C
CRA-08D
GRE-08D01
Havayuki
Lee-8C
13
-18
0
42
13
9
6
-11
71
50
20
53
7
12
9
0
15
52
-2
53
-8
46
35
0
0.55
0.26
0.0001*
0.007*
0.74
0.0004*
0.33
0.0001*
LIN08-B3
7
16
12
0.83
LIN08-B5
LIN08-C
PRA08-D
SPRING
57
-40
73
26
67
60
83
39
21
29
40
31
0.010*
0.0005*
0.004*
0.1885
Tolerance
Sensitive
Tolerant
X
X
X
X
X
X
X
X
X
X
X
X
X
70
71
72
Table IV. Rice seedling growth at various seeding depths (5 cm, 10 cm and 15 cm) under cold
stress (18C-10 h/13C-14 h). UFPel/FAEM. 2013
Accession
1200
1602
3011
CHI-08C
CRA-08D
GRE-08D01
Havayuki
Lee-8C
LIN08-B3
LIN08-B5
LIN08-C
PRA08-D
SPRING
Shoot length reduction (mm)
5 cm depth
10 cm depth
15 cm depth
88
70
0
75
92
97
90
89
83
64
96
96
87
59
61
61
85
88
90
90
0
75
73
77
76
54
95
79
47
68
98
70
88
0
48
81
85
95
85
Prob> F
0.06
0.64
0.10
0.19
0.06
0.09
0.91
0.0001*
0.01*
0.26
0.63
0.22
0.53
Tolerance
Sensitive
Tolerant
X
X
X
X
X
X
X
X
X
X
X
X
X
Gene Expression
Gene Expression Profiles of Transcription Factors cold responsive
Differential transcript accumulation was observed for the transcription factors
evaluated. These results can help us to study the behavioral responses of different weedy red
rice and rice accessions to cold and deep sowing stress using these cold-inducible genes as
marker. The gene expression patterns can help elucidate how rice copes with cold stress.
Under cold stress: The gene expression analysis of Japonica accessions showed that
the transcript accumulation of NAM in the sensitive accession (PRA 08-D02) was
upregulated under cold stress for up to 24 hours and downregulated at 96 hours. Transcript
accumulation in the cold tolerant accession (Spring) was less than that inPRA08-D02 and was
most expressed at 24 hours of cold exposure (Figure 5, panel A). Upregulation of NAM
occurred faster in the cold-sensitive than in the tolerant accession. Dreb2A and F-BOX were
downregulated in both accessions at all times. MYB was downregulated in the sensitive
accession and upregulated in the tolerant accession, showing the most transcript accumulation
at 24 hours of cold exposure in the cold-tolerant Japonica rice.
Under cold stress and at 10 cm sowing depth: The transcription factor genes
NAM, Dreb2A, and MYB respond similarly in the cold tolerant and cold sensitive accessions.
However, MYB differed from NAM and Dreb2A, after 6 and 96 hours of cold treatment. It is
downregulated in the cold sensitive PRA 08-D02 and upregulated in the cold tolerant Spring
accession (Figure 5, panel B). F-box transcript accumulation decreases with an increase in
time under cold stress for PRA08-D02. Meanwhile, the contrary happened for Spring accessions.
73
Panel A.
Panel B.
Figure 5. Gene expression analysis of transcription factors NAM, Dreb2A, MYB and F-box in
PRA 08-D02 and Spring genotypes. Coleoptiles were harvested from 10-d-old seedlings.
Panel A: Effects of cold (10C) on NAM, Dreb2A, MYB and F-box gene expression.
Coleoptiles were harvested from 10-d-old germinated seedlings of sensitive (PRA 08-D02)
and tolerant (Spring) Japonica accessions. Seedlings were exposed to cold and depth stress
for 6, 24, and 96 hours before RNA extraction. Panel B: Effects of cold (10C) and depth
stress (10 cm) on NAM, Dreb2A, MYB and F-box gene expression. Coleoptiles were
harvested from 10-d-old germinated seedlings of sensitive (PRA 08-D02) and tolerant
(Spring) Japonica accessions. Seedlings were exposed to cold and depth stress for 6, 24, and
96 hours before RNA extraction. UFPel/FAEM. 2013.
Under cold stress: The differential expressions of the transcription factor genes
NAM, Dreb2A, MYB and F-box are more steady responsive in the cold tolerant accession
GRE08-D01 compared to the cold sensitive CHI 08-C.The accession 1602 has some different
form of regulation (Figure 6, panel A).
Under cold stress and at 10 cm sowing depth: The genes NAM, Dreb2A and F-box
showed steady response in the GRE08-D01 accession under the different times of cold and
deep sowing stress. The results for CHI08–C showed that the genes NAM and F-box have
74
higher transcript accumulation compared to Dreb2A and MYB. This means that the
sensitivity to cold is related to ABA-dependence. There was decrease in expression of MYB
gene after 96 hours of cold treatment under deep sowing stress for GRE08-D01 (Figure 6,
panel B).This result is the same for the MYB gene of the said accession under cold stress but
without deep sowing (Figure 6, panel A).
Panel A.
Panel B.
6
24
CHI08-C
96
6
24
(hours)
1602
96
6
24
96
GRE 08-D01
Figure 6. Gene expression analysis of NAM, Dreb2A, MYB and F-box gene in CHI 08-C,
1602 and GRE 08-D01genotypes.Coleoptiles were harvested from 10-d-old seedlings. Panel
A: Effects of cold (10C) on NAM, Dreb2A, MYB and F-box expression. Coleoptiles were
harvested from 10-d-old germinated seedlings of sensitive (CHI 08-C) and tolerant (1602 and
GRE 08-D01) Indica accessions. Seedlings were exposed to cold and depth stress for 6, 24,
and 96 hours before RNA extraction. Panel B: Effects of cold (10C) and depth stress (10
cm) on NAM, Dreb2A, MYB and F-box gene expression. Coleoptiles were harvested from
10-d-old germinated seedlings of sensitive (CHI 08-C) and tolerant (1602 and GRE 08-D01)
Indica accessions. Seedlings were exposed to cold and depth stress for 6, 24, and 96 hours
before RNA extraction. UFPel/FAEM. 2013
75
Gene Expression Profiles of Cytosolic proteins
Under cold stress: There was variation in gene expression of cytosolic proteins
(Figure 6, panel A). The H+pyrophosphatase is only upregulated in the sensitive accession
after 24 and 96 hours under cold. Rab16 was upregulated only in the cold tolerant accession
after 96 hours of exposure to cold stress. GERMIN showed more transcript accumulation
during 24 hours under cold for both accessions. However, this gene is downregulated in
Spring under cold stress for 96 hours. This means that GERMIN is repressed after 96 hours
under cold in the tolerant accession and is downregulated after 6 hours of cold exposure in the
cold sensitive accession. Glutamate dehydrogenase showed a similar response, but the
maximum transcription in the PRA08-D02 was after 96 hours under cold. There is a decrease
in transcription in PRA08-D02 for Spring accessions after 96 hours of cold stress.
Under cold stress and at 10 cm sowing depth: The cytosolic protein H+
pyrophosphatase is only upregulated after 6 hours under cold treatment in the sensitive
accession (Figure 7, panel B). This means that this gene is only upregulated in the sensitive
accession under cold treatment, with and without deep sowing stress, but is early responsive
with deep sowing stress (Figure 7, panel A and B). The genes Rab 16, GERMIN and
Glutamate dehydrogenase are early and steady responsive in Spring (cold and depth tolerant
accession) compared with PRA 08-D02.
Under cold stress: The cytosolic protein genes (Figure 8, panel A) are upregulated in
both GRE 08-D02 and CHI 08-C.However, the amount of transcript accumulation is higher in
the tolerant GRE 08-D01. After 96 hours of cold treatment in the cold sensitive CHI 08-C
accession, only Rab 16 gene is upregulated. The gene glutamate dehydrogenase in the
accession 1602 respond differently compared to the other two accessions CHI 08-Cand
GRE08-D01. The tolerant accession (GRE 08-D02) showed more transcript accumulation
after24 hours of cold stress in the H+pyrophosphatase, Rab 16, GERMIN and glutamate
dehydrogenase genes (Figure 8, panel A).
Under cold stress and at 10 cm sowing depth: The H+pyrophosphatase gene showed a
steady response in GRE08-D01. Gene expression in Rab 16 is increased under cold stress at
different times for the said accession. All three accessions have an increase in gene expression
for GERMIN and glutamate dehydrogenase after 24 hours of cold treatment (Figure 8, panel
B). ADH1 was downregulated in GRE08-D01 and upregulated in CHI08-C and 1602
accessions after 96 hours of cold and deep sowing stress. The differential expression of
76
Expansin 7 decreased in CHI08-C and increased in GRE08-D01 as exposure to cold stress
increases (Figure 8, panel B).
Panel A.
Panel B.
Figure 7. Gene expression analysis of transcription factors H+ pyrophosphatase, Rab 16,
GERMIN and glutamate dehydrogenase in PRA 08-D02 and Spring genotypes. Coleoptiles
were harvested from 10-d-old seedlings. Panel A: Effects of cold (10C) on
H+pyrophosphatase, Rab 16, GERMIN and glutamate dehydrogenase expression. Coleoptiles
were harvested from 10-d-old germinated seedlings of sensitive (PRA 08-D02) and tolerant
(Spring) Japonica accessions. Seedlings were exposed to cold and depth stress for 6, 24, and
96 hours before RNA extraction. Panel B: Effects of cold (10C) and depth stress (10 cm) on
H+pyrophosphatase, Rab 16, GERMIN and glutamate dehydrogenase gene expression.
Coleoptiles were harvested from 10-d-old germinated seedlings of sensitive (PRA 08-D02)
and tolerant (Spring) Japonica accessions. Seedlings were exposed to cold and depth stress
for 6, 24, and 96 hours before RNA extraction.UFPel/FAEM. 2013.
77
Panel A.
Panel B.
Figure 8. Gene expression analysis of H+pyrophosphatase, Rab 16, GERMIN and glutamate
de hydrogenase gene in CHI 08-C, 1602 and GRE 08-D01genotypes.Coleoptiles were
harvested from 10-d-old seedlings. Panel A: Effects of cold (10C) on H+pyrophosphatase,
Rab 16, GERMIN and glutamate dehydrogenase expression. Coleoptiles were harvested from
10-d-old germinated seedlings of sensitive (CHI 08-C) and tolerant (1602 and GRE 08-D01)
Indica accessions. Seedlings were exposed to cold and depth stress for 6, 24, and 96 hours
before RNA extraction. Panel B: Effects of cold (10C) and depth stress (10 cm) on
H+pyrophosphatase, Rab 16, GERMIN and glutamate dehydrogenase gene expression.
Coleoptiles were harvested from 10-d-old germinated seedlings of sensitive (CHI 08-C) and
tolerant (1602 and GRE 08-D01) Indica accessions. Seedlings were exposed to cold and depth
stress for 6, 24, and 96 hours before RNA extraction.UFPel/FAEM. 2013.
Gene Expression Profiles of Transcription Factors with anaerobic responses
Under cold stress: Transcript accumulation observed in genes that are upregulated by
anoxia and submergence (ADH1, Expansin 7 and 12, ERF 70 and 68, and -amylase) for both
sensitive and tolerant accessions showed almost the same pattern of variations (Figure 9,
panel A). Differing by the early response according to the amount of transcript accumulation
for the Expansin 7 and 12 and ERF68 in the sensivite accession.
78
Under cold stress and at 10 cm sowing depth: The transcript accumulation of Expansin
12 is downregulated in PRA08-D02 and upregulated in Spring (Figure 9, panel B).This could
mean that this gene is involved in depth tolerance, because without deep sowing stress (Figure
9, panel A), Expansin 12 gene decrease the transcript accumulation, as the time under cold
treatment increases (Figure 9, panel A and B).
Relative Fold Change
Panel A.
6
24
Pra 08-D02
96
6
(hours)
24
96
Spring
Panel B.
Figure 9. Gene expression analysis of transcription factors ADH1, Expansin 7 and 12, ERF 70
and 68 and alpha-amylase in PRA 08-D02 and Spring genotypes. Coleoptiles were harvested
from 10-d-old seedlings. Panel A: Effects of cold (10C) on ADH1, Expansin 7 and 12, ERF
70 and 68 and alpha-amylase expression. Coleoptiles were harvested from 10-d-old
germinated seedlings of sensitive (PRA 08-D02) and tolerant (Spring) Japonica accessions.
Seedlings were exposed to cold and depth stress for 6, 24, and 96 hours before RNA
extraction. Panel B: Effects of cold (10C) and depth stress (10 cm) on ADH1, Expansin 7
and 12, ERF 70 and 68 and alpha-amylase gene expression. Coleoptiles were harvested from
10-d-old germinated seedlings of sensitive (PRA 08-D02) and tolerant (Spring) Japonica
accessions. Seedlings were exposed to cold and depth stress for 6, 24, and 96 hours before
RNA extraction.UFPel/FAEM. 2013
79
Under cold stress: The tolerant accession (GRE 08-D02) showed more transcript
accumulation after 24 hours of coldtreatment in the gene ERF 70 (Figure 10, panel A).
ADH1, Expansin 7 and 12, ERF 68, and alpha-amylase are downregulated in the tolerant
accession (GRE08- D01) and upregulated in the sensitive (CHI 08-C). After 96 hours of cold
stress, the genes ADH1, Expansin 12 and ERF 68 are also downregulated in GRE 08-D01.
Under cold stress and at 10 cm sowing depth: An increase in gene expression for
Expansin 12 was only observed in GRE 08-D01 The highest expression under deep sowing
was observed at 24 hours of cold stress. ERF 70 just showed a minute amount of transcript in
CHI08-C and ERF 68 was just downregulated in GRE 08-D01. In addition, the maximum
transcript accumulation of alpha-amylase was observed in GRE08-D01 under deep sowing
and after 24 hours of cold stress (Figure 10, panel B).
Gene Expression Profiles of Transcriptions Factors involved in Secondary Metabolism
Under cold stress: Sub1b gene expression declined with time of cold exposure in
both Japonica accessions (Figure 11, panel A). Transcript accumulation ofAPX2 was not
clearly related with time of cold exposure; it was upregulated in the cold-sensitive accession
at 24 and 96 h. The expression of APX2 was lower in the cold-tolerant accession than the
sensitive one at any time interval.
Under cold stress and at 10 cm sowing depth: Sub1b and APX2 genes respond
similarly in both tolerant and sensitive accessions. There is an increase in activity as time
under cold stress and deep of sowing increases. The only difference is that the amount of
transcript accumulation is bigger in Spring than PRA 08-D02 (Figure 11, panel B).
80
Relative Fold Change
Panel A.
6
24
96
6
CHI 08-C
24
(hours)
1602
96
6
24
96
GRE 08-D01
Panel B.
6
24
CHI 08-C
96
6
24
96
(hours)
1602
6
24
96
GRE 08-D01
Figure 10. Gene expression analysis ofADH1, Expansin 7 and 12, ERF 70, ERF 68 and alphaamylase gene in CHI 08-C, 1602 and GRE 08-D01genotypes. Coleoptiles were harvested
from 10-d-old seedlings. Panel A: Effects of cold (10C) on ADH1, Expansin 7 and 12, ERF
70, ERF 68 and alpha-amylase expression. Coleoptiles were harvested from 10-d-old
germinated seedlings of sensitive (CHI 08-C) and tolerant (1602 and GRE 08-D01) Indica
accessions. Seedlings were exposed to cold and depth stress for 6, 24, and 96 hours before
RNA extraction. Panel B: Effects of cold (10C) and depth stress (10 cm) on ADH1,
Expansin 7 and 12, ERF 70, ERF 68 and alpha-amylase gene expression. Coleoptiles were
harvested from 10-d-old germinated seedlings of sensitive (CHI 08-C) and tolerant (1602 and
GRE 08-D01) Indica accessions. Seedlings were exposed to cold and depth stress for 6, 24,
and 96 hours before RNA extraction. UFPel/FAEM. 2013
81
Panel A.
Panel B.
Figure 11.Gene expression analysis of Sub 1b and APX2 in PRA 08-D02 and Spring
genotypes. Coleoptiles were harvested from 10-d-old seedlings. Panel A: Effects of cold
(10C) on Sub 1b and APX2 expression. Coleoptiles were harvested from 10-d-old
germinated seedlings of sensitive (PRA 08-D02) and tolerant (Spring) Japonica accessions.
Seedlings were exposed to cold and depth stress for 6, 24, and 96 hours before RNA
extraction. Panel B: Effects of cold (10C) and depth stress (10 cm) on Sub 1b and APX2
gene expression. Coleoptiles were harvested from 10-d-old germinated seedlings of sensitive
(PRA 08-D02) and tolerant (Spring) Japonica accessions. Seedlings were exposed to cold and
depth stress for 6, 24, and 96 hours before RNA extraction.UFPel/FAEM. 2013
Under cold stress: The Sub1b gene in the accessions 1602 and GRE 08-D01 showed
a decrease in transcript accumulation as time exposure to cold stress increases. However, this
relationship was not observed in the CHI 08-C accession. The transcript accumulation of the
gene Sub1b for CHI08-C increases with time (Figure 12, panel A). Moreover, the
accessions1602 and GRE08-D01 respond very differently for the gene APX2. It is
upregulated in the GRE08-D01 and downregulated in the 1602 accession.
82
Under cold stress and at 10 cm sowing depth: Figure 12, panel B shows that Sub1b
and APX2 genes in CHI08-C have higher amount of transcript accumulation compared to
GRE 08-D01 and 1602 accessions, with an exception observed in GRE08-D01 after 96 hours
of cold stress. This means that Sub1b and APX2, which are genes that are regulated by ROS
detoxification and submergence, are related to deep sowing tolerance. The exception observed
in GRE08-D01 could mean that after 96 hours of cold stress, the accession does not have any
more capacity to tolerate and protect itself from the stress, hence becoming sensitive to it.
Relative Fold Change
Panel A.
6
24
96
6
24
96
(hours)
1602
CHI 08-C
6
24
96
GRE 08-D01
Panel B.
6
24
CHI 08-C
96
6
24
(hours)
1602
96
6
24
96
GRE 08-D01
Figure 12. Gene expression analysis of Sub 1b and APX2in CHI 08-C, 1602 and GRE 08-D01genotypes.
Coleoptiles were harvested from 10-d-old seedlings. Panel A: Effects of cold (10C) on Sub 1b and APX2
expression. Coleoptiles were harvested from 10-d-old germinated seedlings of sensitive (CHI 08-C) and tolerant
(1602 and GRE 08-D01) Indica accessions. Seedlings were exposed to cold and depth stress for 6, 24, and 96
hours before RNA extraction. Panel B: Effects of cold (10C) and depth stress (10 cm) on Sub 1b and APX2
gene expression. Coleoptiles were harvested from 10-d-old germinated seedlings of sensitive (CHI 08-C) and
tolerant (1602 and GRE 08-D01) Indica accessions. Seedlings were exposed to cold and depth stress for 6, 24,
and 96 hours before RNA extraction. UFPel/FAEM. 2013.
83
DISCUSSION
Shoot length reduction can be used to screen out cold or seedling depth tolerant at
seedling stage
In this present study both abiotic stresses were able to decrease the percentage of
shoot length of the accessions evaluated, which was less than 38% in the cold-tolerant
accessions and less than 50% in the seedling depth tolerant accessions. This percentage is
according to Cruz e Milach (2004) scale. Due to cold and seedling depth stresses, the seedling
growth and the development of rice plants can be injured (CHINNUSAMY et al., 2006;
MALONE et al., 2007; NAHAR et al., 2009).
Using the abiotic stresses together did not show any accession tolerance, which
means all accessions were cold and seedling depth sensitive. This may be because the same
pathways were induced, and tolerance depends on the intensity of the stress applied, as well
as, the period under stress (RABBANI et al., 2003).
The stress-inducible genes are not from just one pathway, so it is not following the
same cascade of transduction signaling. In Arabdopsis, ABA-dependent and -independent
signal pathways function in stress response (RABBANI et al., 2003).This is an evidence there
are complex regulatory mechanisms involved (SHINOZAKI e YAMAGUCHI-SHINOSAKI,
2000; ZHU, 2002).
Gene Expression Profiles of Transcription Factors cold responsive
The transcription factors NAM and DREB are ABA-dependent and independent
(respectively) were under cold treatment these ones are down regulated in the cold-tolerant.
Instead of this, the TF MYB, ABA-dependent and independent, was up-regulated under cold
stress in Japonica. DREB2A under cold stress was not inducible, the contrary was observed in
recently studies in rice under abiotic stress-responsive genes (MATSUKURA et al., 2010).
Under cold and seedling depth stresses the TF MYB showed up-regulation, this is an
indicative of the importance of ABA-dependent and independent pathways for tolerance in
both stresses in Japonica subspecies (Figure 5). Tian et al. (2011) also showed the regulatory
gene OsMYB was up-regulated under cold stress, adapting rice for cold-tolerance (SU et al., 2010).
In Indica subspecies there are two tolerant accessions showing differences; probably
due to both of these are weedy red rice. This result can help to emphasize the irreplaceable
pathway for the cold-tolerant accession, as MYB, ABA-dependent and independent pathways,
84
same result showed for Japonica accessions (Figure 9). According to Su et al. (2010) MYB3S
is fundamental when rice plants are kept under cold stress.
Gene Expression Profiles of Cytosolic proteins
Glutamate dehydrogenase (GDH), an enzyme involved in nitrogen metabolism
(STEWART et al., 1980), was showing more transcript accumulation in the cold and seedling
depth tolerant (Figure 7). The strong expression of Rab16, under cold and depth sowing
stresses, suggest the importance of this one for seedling depth tolerance (Figure 6, Panel B).
In Indica subspecies, the GDH was inducible under cold and depth stresses
suggesting a strong alteration in the protein synthesis during cold stress in the tolerant
accessions. This Lea protein (Rab16) in Indica was strongly inducible under depth stresses in
the tolerant one, as well as in Japonica tolerant accession (Figure 7 and 8). That protein plays
a role in the membrane stabilization and in dehydrated conditions LEA proteins protect the
denaturation of proteins (JANSKÁ et al., 2010).
Gene Expression Profiles of Transcription Factors with anaerobic responses
Under cold stress the genes ADH1, Expansins, ERF did not respond strongly
difference that is expected due to these genes are up-regulated in anoxia and submergence
conditions (LASANTHI-KUDAHETTIGE et al., 2007). Under depth sowing stress, Expansin
12 was downregulated and in the sensitive this one was strongly up-regulated (Figure 9). This
result was the same for Lasanthi-Kudahettige et al. (2007), when this gene were more
expressed under anoxia conditions than in air, during anoxic coleoptile elongation phase.
In Indica subspecies, under cold stress the Alpha amylase was up-regulated in the
sensitive accession. The contrary was observed in study hypothesize a role of this enzyme,
under cold stress, protecting the photosystem II (KAPLAN et al., 2006).
ERF 70 showed downregulated in the two depth tolerant accessions and in the
sensitive it was strongly up-regulated (Figure 10), allowing the rice plant preserve energy to
survive (LASANTHI-KUDAHETTIGE et al., 2007). ERF 70 is a transcription factor
regulating processes related to growth and development under environmental stimuli
(NAKANO et al., 2006).
Gene Expression Profiles of Transcriptions Factors involved in Secondary Metabolism
In Japonica subspecies, under cold stress the genes Sub1b and APX2 not respond
strongly differently comparing cold tolerant and sensitive that is expected due to these genes.
85
In Japonica subspecies under depth stress these genes were strongly up-regulated,
showing that anoxia and ROS presence, but these ones are not correlated with seedling depth
tolerance (Figure 11).For Sub1 in Japonica, it was already described as increasing the
flooding tolerance in rice (LASANTHI-KUDAHETTIGE et al., 2007). Maybe the Ascorbate
peroxidase (APX2), under depth, help to keep the anti-oxidative activity under depth.
In Indica subspecies, the tolerant accessions showed less transcript accumulation
comparing with the sensitive for Sub1 under cold and also under depth sowing stress (Figure
12). For anoxia conditions the Sub1 is only slightly regulated. For Sub1 in Japonica, it was
already described as increasing the flooding tolerance (LASANTHI-KUDAHETTIGE et al.,
2007). It can suggest the anoxia conditions injury more Indica than Japonica accession.
According to these authors, the adaptation of rice plants to submergence conditions and,
consequently increases of Sub1 genes cause a decrease of elongation genes. However, ERF 70
was strongly up regulated of ERF 70 in Indica, due to more ethylene production in the
alcoholic fermentation in anoxic conditions.
CONCLUSION
Phenol test identified 9 Japonica and 12 Indica accessions.
Screening of the accessions identified 2 tolerant Indica and 1 Japonica subspecies
and the remaining were cold sensitive and all accessions were seedling depth and cold
sensitive.
Seedling depth identified 1 tolerant Indica and 1 Japonica subspecies and the
remaining were seedling depth sensitive.
Cold stress and seedling depth stresses when applied at the same time prevents the
tolerance.
These results indicated the importance of ABA-dependent and independent pathways
for tolerance in both stresses in Japonica and Indica subspecies; it can help to provide an
insight into the cold tolerance and sowing depth tolerance in this different rice subspecies.
MATERIALS AND METHODS
Plant Material and Growth Condition
In this study were used 21 rice and red rice genotypes (Table V). These accessions
were classified as Japonica or Indica subspecies by phenol test (GROSS et al., 2009).To
overcoming the dormancy the seeds were kept 24h in Sodium Hypochlorite solution (NaOCl,
86
1.5%).Seeds were washed with water, and then were allowed to grow in homogeneous
conditions (growth chamber) in plastic pots under vermiculite.
Table V. Genes analyzed into qRT-PCR to sensitivity and tolerance when subjected to abiotic
stress in (Indica and Japonica) rice genotypes.UFPel/FAEM. 2013
Accession
AK068392
AK108621
AK071366
AK072651
Os03g60720
Os03g44290
Os11g10510
Os08g36910
Os07g47790
Os01g21120
Os03g17690
Os09g11480
AF300971
BP432999
Os08g08970
D45383
UBI
Primer Sequence
Name
GCAAGCCATTCTAGACGACC
NAM-F
GCTCGCCTGAGTCAAAGTTC
NAM-R
GCTCCTCGTCGACTACATCC
Myb 7-F
CTGGACGATGGACTTCTGCT
Myb 7-R
GAAGAAGGGCTTCATGGACA
Rab 16-F
CACCATCACTCGCATTTCAC
Rab 16-R
CCTGCAACATGAAGCTGAAA
F-box-F
TCAGTTTCCTTCCGACTGCT
F-box-R
GAGATCAAGTGCGTGAACCA
Expansin 7-F
ACCTGAACCCGTTTATCGTG
Expansin 7-R
AGGGATGTGGTTCGTGCTAC
Sub 1b-F
GTCCATTCACACTCCACACG
Sub 1b-R
AGTGTGGGAGAGGGTGTGAC
Alcoholdehydrogenase 2-F
GTGGATGTGCCAACAAAGTG
Alcoholdehydrogenase 2-R
AAGGTCATGGTGAAGATCGG
Alpha-amylase-F
CCTTCTCCCAGACGCTGTAG
Alpha-amylase-R
AGTTCATGGACTACGACGCC
Expansin 12-F
ATCAAAGCTCCAGAGCTCCA
Expansin 12-R
ACTACATGAGCTTCCTCGGC
ERF 68-F
GACGGCAGCTCGTAGTCTTC
ERF 68-R
AGGTGCCACAAGGAAAGATCTGGT
APX2-F
TCAGCAGGGCTTTGTCACTAGGAA
APX2-R
GGACGCCACAACGAAGATGAAGAA
ERF 70-F
TGCACCAGAAGGGAACATGGAAAC
ERF 70-R
TAAGTGGGTGGCTGAGATCC
Dreb2A-F
ATGAAGGTGCTGATGTGCAG
Dreb2A-R
TTTTCATGATGCGGAAGTCA
Glutamatedehydrogenase-F
TTTTCCCTGTTGAGCACTCC
Glutamatedehydrogenase-R
CATCTCACTAGCTCGCATCG
Germin-F
TGTTCCCTCAAGCACAGTGA
Germin-R
TTGAGCCTGCCCTCAAGAAG
H+ pyrophosphatase-F
GGGAGGCCTAACCAACTGAC
H+ pyrophosphatase-R
CTCACCTACGTCTACAA
Ubiquitina -F
GTCAAGGTGTTCAGTTC
Ubiquitina -R
Reference*
Rabbani et al.(2003)
Rabbaniet al. (2003)
Rabbaniet al. (2003)
Rabbaniet al.(2003)
Lasanthu-Kudahettigeet
al. (2007)
Lasanthu-Kudahettigeet
al. (2007)
Fukuda et al. (2005)
Lasanthu-Kudahettigeet
al. (2007)
Lasanthu-Kudahettigeet
al. (2007)
Nakano et al, 2006).
Shigeokaet al. (2002)
(Nakano et al, 2006).
Dubouzetet al. (2003)
Rabbaniet al. (2003)
Dubouzetet al (2003)
Rabbaniet al. (2003)
Jain et al.(2006)
87
Screening of different weedy red rice and ricegenotypes for cold and/or deep sowing
tolerance
The accessions were categorized as sowing depth and/or cold-sensitive or -tolerant
by measuring seedling shoot length reduction (Reduction= length control – length under stress
* 100 / length control)(adapted from CRUZ e MILACH, 2004), where coleoptile length is the
average of the 5 seeds evaluated per replication per genotype, after 14 days of incubation at
18/13°C or 25°C (as a control) day/night temperature and at different seedling depths (1.5cm,
5cm, 10cm and 15cm respectively).The reduction was calculated by cold and normal
temperature, as well as for 1.5 cm under only cold treatments (first part of results). For these
two factors at the same time (cold and depth treatments) the reduction was calculated by depth
(5 or 10 or 15 cm) and normal depth (1.5 cm).
Sensitive and tolerant (3 genotypes from Indica and 2 genotypes from Japonica) rice
subspecies were selected to analyze the expression of cold-responsive genes and
submergence-inducible genes by qPCR. These accessions selected with 10 days old were
exposed to 10°C for 6, 24 and 96 h at 1.5 cm and 10 cm seeding depths.
The seedlings were washed thoroughly, the coleoptiles were harvested; samples of
equal fresh weight were frozen in liquid nitrogen. After collection, the plant tissue was
quickly frozen in ultra-freezer (-80C) until RNA extraction (using TrizolTM Reagent
(InvitrogenTM)) and quantification by Nanodrop (ND-1000), verifying the relationship
A260nm/A280nm, the samples that showed only one pic (no contaminant content) and
concentration more than 400 micrograms. The cDNA synthesis (SuperScriptTM First-Strand
System for RT-PCR (Invitrogen
TM)
was carried out with 5000ng of RNA treated with
DNAse(DNAse ITM Amplification Grade (InvitrogenTM),using C 1000 Thermal Cycle.
Gene expression analysis by qPCR
Cold-sensitive and tolerant (IndicaandJaponica) rice genotypes were selected to analyze
the expression of cold-responsive genes. These genes (Table IV) were annotated according to
their functions. We analyzed a) transcription factors including F-box group of TFs, MYB domain
protein, DREBP2, NAM or NAC; b) genes upregulated by submergence: Sub1B, ROS
detoxification: APX2; c) cytosolic proteins including H+pyrophosphatase, Rab16, GERMIN,
glutamate dehydrogenase; d) and genes upregulated by anoxia and submergence including AP2ERF 70, AP2-ERF 68, Expansin 7, Expansin 12, ADH1 and -Amylase.
Gene expression analysis was done by qRT-PCR. Total RNA were isolated from two
Japonica accessions: cold sensitive (PRA 08-D02) and cold tolerant (Spring); and from three
88
Indica accessions: cold sensitive (CHI 08-C) and cold and/or depth tolerant (GRE 08-D01 and
1602).
The expression pattern of these genes under cold and different seedling depths
treatments from different rice genotypes was performed, with 1:25 cDNA concentration, by
quantitative RT-PCR (CFX 96 Real-time system, BIO-RAD). The qPCR was carried
outwith0, 5μlqPCR master mix (Promega), 0.5 μl of each oligonucleotide (10 μM), 2μl of the
first strand cDNA (diluted 1:25), 0,1μl of ROX dye and 1,9 μl of water. It was performed for
40 cycles following the manufacturer’s protocol, and annealing temperature 59°C. Three
independent biological replicates of each sample were used for expression analysis. Negative
control reactions were also run to confirm the absence of contaminants. To determine relative
fold changes for each sample in each experiment, the Ct value for each gene was normalized
to the Ct (Threshold cycle) value for ubiquitin was calculated relative to a calibrator using the
equation 2-ΔΔCt (LIVAK & SCHMITTGEN, 2001).
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CONSIDERAÇOES FINAIS
Os experimentos realizados no presente estudo demonstram que a habilidade das
plantas de arroz em tolerarem estresses abióticos afetam tanto sua morfologia como sua
fisiologia.
Durante a fase vegetativa condições de baixa temperatura afetam o comprimento de
parte aérea de plântulas, sendo esse um parâmetro útil para a seleção de genótipos tolerantes
ao frio aos 14 dias após semeadura (18C/13C).
Atualmente, foi demonstrado que é possível a utilização do Índice de Tolerância
(STI) e da Média Geométrica (GM) para selecionar genótipos tolerantes ao frio ou a
semeaduras mais profundas, baseando-se no comprimento de parte aérea de plântula. Desde
que se considere e análise o vigor das sementes utilizadas.
Em estudos tendo como parâmetros a fitomassa e o teor de clorofila, o período de
recuperação, no qual foi determinado que a massa seca radicular e total são mais efetivo na
discriminação dos genótipos comparando-se com o parâmetro teor de clorofila. Entretanto a
utilização de períodos mais longos de estresse, como nos outros estudos realizados no
presente trabalho seriam efetivos e sendo assim necessitam-se estudos adicionais após um
período mais longo de estresse pelo frio.
Nas análises utilizando-se qRT-PCR, os primers testados nas reações apresentam
diferentes níveis de eficiência indicando a necessidade de descarte de alguns, antes da análise
da expressão gênica diferencial de caracteres de tolerância à estresses abióticos em arroz em
estádios iniciais de desenvolvimento sob estresse por frio.
As subespécies Japonica e Indica respondem diferentemente aos estresses abióticos.
No entanto, para alguns genes responsivos a esses estresses, subespécies respondem
semelhantemente.
Além disso, em nível molecular da tolerância ao frio e a profundidade de semeadura
indicam a importância das vias ABA-dependente e ABA-independente como vias de
transdução do sinal em plantas sob estresse abiótico.