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CÁSSIA ÂNGELA PEDROZO
MORPHOPHYSIOLOGICAL AND MOLECULAR RESPONSES OF SUGARCANE
GENOTYPES TO WATER STRESS
Thesis presented to the Federal
University of Viçosa - Brazil as part of
the demands of the Genetic and Breeding
Program, for obtaining of Doctor
Scientiae title.
VIÇOSA
MINAS GERAIS – BRAZIL
2010
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ACKNOWLEDGEMENTS
First, I would like to acknowledge God for the protection and blessings I have
received through my entire life.
I would like to thank my family members for the support they provided me and in
particular, I must acknowledge my father, Euclydes, and my fiancé, Leandro, for the love and
encouragement.
I would like to acknowledge the advice, guidance and encouragement of my adviser
Dr. Márcio Henrique Pereira Barbosa. Without his help and kindness I would not have
finished this step.
A very special thanks goes out to Dr. John Jifon. Besides guidance, assistance and
technical support, He also provided me motivation and friendship.
I also thank my co-advisers Dr. Jorge da Silva and Dr. Luiz Alexandre Peternelli for
their guidance and suggestions. Finally, I would like to thank the other members of the
committee, Dr. Adriano Nunes Nesi, Dr Felipe Lopes da Silva and Luíz Antônio dos Santos
Dias for the assistance.
I am thankful to Federal University of Viçosa for providing me this opportunity and to
Texas A&M University for the financial assistance.
I wish to thank my friends from Federal University of Viçosa (Jack and Ana Paula)
and from Texas A&M University (Allen, Denise, Jong Won, Juventino, Nora and Lily) for all
the emotional support, entertainment and caring they provided.
Lastly, I would like to thank all of those who supported me in any respect during the
completion of the project.
iii
CONTENTS
TABLE LIST ............................................................................................................................. v
FIGURE LIST ........................................................................................................................... vi
RESUMO ................................................................................................................................. vii
ABSTRACT .............................................................................................................................. ix
GENERAL INTRODUCTION .................................................................................................. 1
SCIENTIFIC PAPER 1.............................................................................................................. 3
RESUMO ................................................................................................................................... 4
ABSTRACT ............................................................................................................................... 5
1. INTRODUCTION ................................................................................................................. 6
2. MATERIALS AND METHODS ........................................................................................... 8
2.1. Planting materials, irrigation treatments, and growing conditions ..................................... 8
2.2. Measurement of morpho-physiological traits ..................................................................... 9
2.3. Experimental design and data analysis ............................................................................. 11
3. RESULTS AND DISCUSSION .......................................................................................... 11
4. CONCLUSIONS.................................................................................................................. 23
5. ACKNOWLEDGEMENTS ................................................................................................. 23
6. REFERENCES .................................................................................................................... 23
SCIENTIFIC PAPER 2............................................................................................................ 31
RESUMO ................................................................................................................................. 32
ABSTRACT ............................................................................................................................. 33
1. INTRODUCTION ............................................................................................................... 34
2. MATERIALS AND METHODS ......................................................................................... 36
2.1. Planting materials, irrigation treatments, and growing conditions ................................... 36
2.2. Sampling procedures ......................................................................................................... 37
2.3. Total RNA extraction, poli (A)+ RNA isolation and cDNA synthesis............................. 37
2.4. cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis ..................... 37
2.5. Isolation and sequencing of transcript-derived fragment (TDF) ...................................... 38
2.6. Analysis of sequences ....................................................................................................... 39
2.7. Validation of cDNA-AFLP experiments by real-time reverse transcription PCR ........... 39
3. RESULTS AND DISCUSSION .......................................................................................... 41
3.1. cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis ..................... 41
3.2. Isolating and characterization of transcript-derived fragment (TDF) ............................... 43
3.3. Validation of cDNA-AFLP experiments by real-time reverse transcription PCR (RT-
PCR) ......................................................................................................................................... 46
3.4. Functional classification of drought-responsive genes ..................................................... 48
iv
4. CONCLUSIONS.................................................................................................................. 50
5. ACKNOWLEDGEMENTS ................................................................................................. 50
6. REFERENCES .................................................................................................................... 50
GENERAL CONCLUSIONS .................................................................................................. 57
v
TABLE LIST
Table 1. Summary of analysis of variance for net photosynthesis rate (P
n
), transpiration rate
(E), stomatal conductance (G
s
), leaf greenness (SPAD), PSII photochemical efficiency
(F
v
/F
m
) leaf relative water content (RWC) of two sugarcane genotypes grown under well-
watered and moderate water stresss conditions and measured at five dates. ........................... 12
Table 2. Summary of analysis of variance for specific leaf area (SLA) and rate among root
and shoot biomass (RS ratio) of two sugarcane genotypes grown under well-watered and
moderate water stress conditions. SLA was measured at five times and RS ratio was
measured at the end of the experiment. ................................................................................... 18
Table 3. Summary of analysis of variance for stalk height (SH), stalk diameter (SD) and stalk
weight (SW) of two sugarcane genotypes grown under well-watered and moderate water
stress conditions. ...................................................................................................................... 21
Table 4. Sequence of adapter strands and primers used for pre-amplification and selective
amplification. ........................................................................................................................... 38
Table 5. Primer sequences of reference gene (RF) and of target TDFs used in the RT-PCR
expression analysis................................................................................................................... 40
Table 6. Differentially expressed transcript derived fragments (TDFs) of two sugarcane
genotypes [drought-tolerant: TSP05-4 (T) and drought-susceptible: TCP02-4589 (S)] under
moderate water stress conditions at two evaluation times [Two (T1) and twelve days after
moderate water stress initiation (T2)]. The TDFs were up-regulated (U) or down-regulated
(D). ........................................................................................................................................... 44
vi
FIGURE LIST
Figure 1. Mean net photosynthesis (P
n
), stomatal conductance (G
s
) and transpiration rate (E)
of two sugarcane genotypes (drought-tolerant: TSP05-4 and drought-susceptible: TCP02-
4589) grown under well-watered (control) and moderate water stress conditions (mean, ±
S.E.). Different letters indicate significant differences between genotypes within each water
supply regime. .......................................................................................................................... 13
Figure 2. Mean net photosynthesis (A), stomatal conductance (B) and transpiration rate (C)
of two sugarcane genotypes (drought-tolerant: TSP05-4 and drought-susceptible: TCP02-
4589) grown under well-watered (control) and moderate water stress conditions, and
measured at five dates (mean, ± S.E.). Different letters indicate significant differences
between water supply regimes within each evaluation time (T0: two days before water stress
initiation, T1, T2 and T3: two, twelve and twenty days after moderate water stress initiation,
and T4: 8 days after re-watering). ............................................................................................ 14
Figure 3. Mean relative water content (RWC), maximum quantum yield of photosystem II
(F
v
/F
m
) and leaf greenness index (SPAD value) of two sugarcane genotypes (drought-
tolerant: TSP05-4 and drought-susceptible: TCP02-4589) grown under well-watered (control)
and moderate water stress conditions (mean, ± S.E.). Different letters indicate significant
differences between water supply regimes (A and B) or between genotypes (C and D). ....... 16
Figure 4. Mean root:shoot ratio (mean, ± S.E.) of two sugarcane genotypes (tolerant and
susceptible) grown under well-watered (control) and moderate water stress conditions.
Different letters indicate significant differences between genotypes. ..................................... 20
Figure 5. Mean stalk height (A) and mean stalk weight (B and C) of two sugarcane
genotypes grown under well-watered (control) and moderate water stress conditions (mean, ±
S.E.). Different letters indicate significant differences between water supply regimes within
genotype (A), between genotypes (B) or between water supply regimes (C). ........................ 22
Figure 6. cDNA-AFLP fingerprints generated from two sugarcane genotypes (T: drought-
tolerant; S: drought-susceptible) under two water regimes (C: control or well-watered
conditions; D: moderate water stress conditions) at three evaluation times (T1: 2 days after
water stress initiation; T2: 12 days after water stress initiation; T3: after 8 days of re-
watering). The primer combination used was TaqI GT x MseI AG. ....................................... 42
Figure 7. Differential expression of three transcript-derived fragments: (A) SugDR01; (B)
SugDR05, and (C) SugDR08 generated from two sugarcane genotypes (T: drought-tolerant;
S: drought-susceptible) under two water regimes (C: control or well-watered conditions; D:
moderate water stress conditions) at two evaluation times (T1: 2 days after water stress
initiation and T2: 12 days after water stress initiation)............................................................ 45
Figure 8. Real time RT-PCR analysis of 8 differentially-expressed transcript derived
fragment (TDFs) of two sugarcane genotypes (drought-tolerant: TSP05-4 and drought-
susceptible: TCP02-4589) under well-watered (C) and moderate water stress conditions (D)
at two evaluation times [two (T1) and twelve days after water stress initiation (T2)]. All data
were normalized to the glyceraldehide-3-phosphate de-hydrogenase (GAPDH) expression
level. Data represent fold change of the gene expression in water-stressed vs. control plants.
Bars indicate the standard deviation obtained of three biological replications. ...................... 47
vii
RESUMO
PEDROZO, Cássia Ângela, D.Sc., Universidade Federal de Viçosa, julho, 2010. Respostas
morfo-fisiológicas e moleculares de genótipos de cana-de-açúcar submetidos ao
estresse hídrico. Orientador: Márcio Henrique Pereira Barbosa. Co-orientadores: Luiz
Alexandre Peternelli e Jorge Alberto da Silva.
O objetivo deste trabalho foi estudar respostas morfo-fisiológicas e moleculares de
dois genótipos de cana-de-açúcar (tolerante à seca: TSP05-4 ou sensível à seca: TCP02-4589)
ao estresse hídrico. Características morfo-fisiológicas foram avaliadas em cinco diferentes
tempos [dois dias antes do início do estresse hídrico (T0), dois (T1), doze (T2) e vinte dias
(T3) após início do estresse hídrico de moderada intensidade, e oito dias após re-irrigação
(T4)]. Características de desenvolvimento foram avaliadas no final do experimento (após T4).
A técnica cDNA-AFLP foi utilizada para identificar genes diferencialmente expressos em T1,
T2 e T4. Tanto sob condições controle (plantas bem irrigadas) quanto sob condições de
estresse hídrico o genótipo tolerante mostrou os maiores valores de indíce de esverdeamento
da folha (SPAD), eficiência quântica máxima do PSII (F
v
/F
m
) e relação biomassa da
raíz/planta inteira. Além disso, sob condições de estresse hídrico, as taxas de fotossíntese,
transpiração e condutância estomática foram significativamente maiores no genótipo
tolerante. O genótipo susceptível apresentou colmos mais altos e mais pesados. No entanto, a
condição de estresse hídrico causou significativa redução na altura deste genótipo, enquanto
que nenhuma alteração foi observada no genótipo tolerante. Um total de 15 fragmentos
derivados de transcritos (TDF) diferencialmente expressos foram caracterizados e 8 desses
foram validados por RT-PCR em tempo real. Três TDFs, os quais mostraram similaridade a
um pentatricopeptideo putativo, à subunidade regulatória CK2β3 da proteina kinase CK2 e ao
transportador glicose-6-fosfato/fosfato 2, foram diferencialmente expressos no genótipo
susceptível. Um TDF similar ao mRNA de uma proteína induzida pela seca foi também
induzido no genótipo tolerante em T2. Tanto as características fisiológicas quanto o padrão
de expressão gênica que foram alterados pelo estresse hídrico foram completamente
revertidos após o período de re-irrigação, demonstrando a plasticidade dos genótipos de cana-
de-açúcar em responder a mudanças nas condições hídricas. Os resultados encontrados neste
estudo demonstram a robustez do genótipo tolerante em responder a condições estresse
hídrico e enfatiza diferenças fisiológicas e moleculares entre os dois genótipos que podem
auxiliar em programas de melhoramento que visam tolerância à seca.
viii
Termos de indexação: estresses abióticos, Saccharum spp, mecanismos fisiológicos, cDNA-
AFLP.
ix
ABSTRACT
PEDROZO, Cássia Ângela, D.Sc., Universidade Federal de Viçosa, July, 2010.
Morphophysiological and molecular responses of sugarcane genotypes to water
stress. Adviser: Márcio Henrique Pereira Barbosa. Co-Advisers: Luiz Alexandre
Peternelli and Jorge Alberto da Silva.
The objective of this work was to study morphophysiological and molecular
responses of two sugarcane genotypes (drought-tolerant: TSP05-4 or drought-susceptible:
TCP02-4589) to water stress. Morphophysiological traits were evaluated at five different
times [two days before water stress initiation (T0), two (T1), twelve (T2) and twenty (T3)
days after moderate water stress initiation, and at eight days after re-watering (T4)]. Growth
traits were evaluated at the end of the experiment (after T4). The cDNA-AFLP technique was
used to identify differentially-expressed genes at T1, T2 and T4. Under control (well-
watered) and water stress conditions, the tolerant genotype showed the highest values of leaf
greenness index (SPAD), maximum quantum yield of PSII (F
v
/F
m
), and root:shoot ratio.
Besides, under water stress conditions, photosynthesis, transpiration and stomatal
conductance were significantly higher in that genotype than in the susceptible genotype. The
susceptible genotype had taller and heavier stalks. However, water stress caused a significant
reduction in stalk length in this genotype, while no differences were observed for the tolerant
genotype. A total of 15 differentially-expressed transcript-derived fragments (TDF) were
characterized and 8 of them were validated by real-time RT-PCR. Three TDFs showing
significant sequence similarities to genes encoding a putative expressed pentatricopeptide, a
protein kinase CK2 regulatory subunity CK2β3, and a glucose-6-phosphate/phosphate
translocator 2 were differentially expressed in the susceptible genotype. One TDF similar to a
drought-inducible protein mRNA was also up-regulated in the tolerant genotype at T2. The
physiological traits and gene expression which were altered by water stress were completely
reversed after re-watering, demonstrating the plasticity of sugarcane genotypes in being able
to respond to changing water conditions. The results found in this study demonstrate the
robustness of the tolerant genotype in response to water stress and highlights physiological
and molecular differences between the two genotypes that could help in sugarcane
improvement programs for stress tolerance.
Indexation terms: abiotic stress, Saccharum spp, physiological mechanisms, cDNA-AFLP.
1
GENERAL INTRODUCTION
Sugarcane (Saccharum spp; Poaceae) is an economically important perennial crop of
tropical origin that is grown in more than 90 countries (FAO, fttp://apps.fao.org) for sugar
production. About 80% of the world‟s sugar (sucrose) supply is from sugarcane, producing
111.8 million tonnes (ton) of sugar for 2002/2003 (Licht; 2003), while the other 20% is
obtained from sugar beet (Beta vulgaris L., Chenopodiaceae). Sugarcane is also a leading
alternative energy feedstock crop both for ethanol and for biomass production.
Sugarcane is widely adapted within the tropical zone (±30
º
of the equator) and its
tolerance to a range of temperatures has allowed cultivation to expand into sub-tropical
regions such as Louisiana, Florida and Texas in the United States of America. For optimum
growth and productivity, sugarcane requires temperatures of above 20ºC and a period of 8 to
24 months to reach maturity, depending on location and agronomic practices. The leading
three sugarcane-producing-countries, Brazil, India, and China, each produced more than 100
Mton of cane sugar in the year 2002/2003. The other major sugarcane-producing countries
include Australia, Mexico, Thailand, Pakistan, USA, South Africa, Colombia, Cuba, and
Philippines.
Even though sugarcane and other C
4
perennial grasses have many adaptive qualities
(such as high productivity, water use efficiency, vegetative propagation, and wide
environmental adaptation), for sugar or bioenergy production, ample water supply and
nutrients are required to assure optimum productivity (Wiedenfeld, 2000; Menossi et al.,
2008; Cha-um and Kirdmanee, 2009). The annual water requirements for optimum yields
have been estimated at about 1500 to 2500 mm (Doorenbos and Pruitt, 1976). Since
sugarcane production is concentrated in many tropical regions where water supply is either
inadequate or irrigation infrastructures are underdeveloped, moisture deficit is a major
limitation to optimal productivity (Inman-Bamber and Smith, 2005; Hemaprabha et al., 2006;
Jangpromma et al., 2007). The major determinants of sugarcane yield (stalk yield and sucrose
content) are highly sensitive to various biotic and abiotic stresses, of which, drought is the
most critical.
A basic understanding of mophophysiological and molecular processes responsible
for high performance under water stress conditions and its interactions is necessary in
sugarcane breeding programs aimed at increasing productivity by enhancing stress tolerance.
Thus, the objective of this study was to characterize and compare morphophysiological and
2
molecular responses of two sugarcane genotypes during moderate water stress exposure and
during a recovery period after re-watering.
3
SCIENTIFIC PAPER 1
MORPHOPHYSIOLOGICAL RESPONSES OF SUGARCANE GENOTYPES TO WATER
STRESS
VIÇOSA
MINAS GERAIS – BRAZIL
2010
4
RESUMO
PEDROZO, Cássia Ângela, D.Sc., Universidade Federal de Viçosa, julho, 2010. Respostas
morfo-fisiológicas de genótipos de cana-de-açúcar submetidos ao estresse hídrico.
Orientador: Márcio Henrique Pereira Barbosa. Co-orientadores: Luiz Alexandre
Peternelli e Jorge Alberto da Silva.
O objetivo deste estudo foi melhor entender a natureza de características morfo-
fisiológicas e de desenvolvimento de dois genótipos de cana-de-açúcar (tolerante à seca:
TSP05-4 ou sensível à seca: TCP02-4589) ao estresse hídrico. Características morfo-
fisiológicas foram avaliadas em cinco diferentes tempos (2 dias antes do início do estresse
hídrico, dois, doze e vinte dias após início do estresse hídrico de intensidade moderada, e oito
dias após re-irrigação). Características de desenvolvimento foram avaliadas no final do
experimento (após o período de re-irrigação). Sob condições controle (plantas bem irrigadas)
e de estresse hídrico, o genótipo tolerante apresentou os maiores valores de indíce de
esverdeamento da folha (SPAD), eficiência quântica máxima do PSII (Fv/Fm) e relação
biomassa raíz/planta inteira (50.83, 0.8085 e 0.6182, respectivamente). Adicionalmente, sob
condições de estresse hídrico, as taxas de fotossíntese (P
n
), condutância estomática (G
s
) e
transpiração (E) foram significativamente superiores no genótipo tolerante (14.61, 133.8 e
3.49 µmol.m
-2
.s
-1
, respectivamente) que no susceptível (11.23, 110.5 e 2.96,
respectivamente). A redução nas taxas de P
n
, G
s
e E devido ao estresse hídrico foi
completemente revertida após o período de re-irrigação, demonstrando a plasticidade dos
genótipos de cana-de-açúcar em responder a mudanças nas condições hídricas e suportam a
hipótese de que o controle fotossintético durante condições de moderado estresse hídrico foi
principalmente estomático. Os colmos do genótipo susceptível apresentaram maior peso e
altura que aqueles do genótipo tolerante. No entanto, as condições de estresse causaram
significativa redução na altura dos colmos daquele primeiro genótipo (de 1.69 m para 1.32 m)
enquanto nenhuma alteração foi observada para o segundo. Os resultados encontrados neste
estudo demonstram a robustez do genótipo tolerante em responder a condições de estresse
hídrico e enfatiza diferenças, principalmente fisiológicas, entre os dois genótipos que podem
auxiliar em programas de melhoramento que visam tolerância à seca.
Termos de indexação: estresse abiótico, Saccharum spp, mecanismos fisiológicos,
melhoramento genético.
5
ABSTRACT
PEDROZO, Cássia Ângela, D.Sc., Universidade Federal de Viçosa, July, 2010.
Morphophysiological responses of sugarcane genotypes to water stress. Adviser:
Márcio Henrique Pereira Barbosa. Co-adviser: Luiz Alexandre Peternelli and Jorge
Alberto da Silva.
The objective of this study was to better understand the nature of
morphophysiological traits in two sugarcane genotypes (tolerant: TSP05-4 or susceptible:
TCP02-4589) to water stress. Physiological and morphological traits were measured at five
different times (two days before water stress initiation, two, twelve and twenty days after
moderate water stress initiation, and at eight days after re-watering) and growth traits were
measured at the end of the experiment (after re-watering). Under control (well-watered) and
simulated water conditions, the tolerant genotype showed the highest values of leaf greenness
index (SPAD), maximum quantum yield of PSII (F
v
/F
m
), and root:shoot ratio (50.83, 0.8085
and 0.6182, respectively). Moreover, under water stress, net photosynthesis (P
n
), stomatal
conductance (G
s
) and transpiration rate (E) were significantly higher in the tolerant genotype
(14.61, 133.8 and 3.49 µmol.m
-2
.s
-1
, respectively). The decrease in P
n
, G
s
and E due to water
stress was completely reversed after re-watering, demonstrating the plasticity of these
genotypes to respond to changing water conditions and supporting the hypothesis that the
control on photosynthesis during moderate water stress was mainly stomatal. Stalks of the
susceptible genotype were taller and heavier than those of the tolerant genotype. However,
water stress caused significant reduction in stalk length of the susceptible genotype (from
1.69 m to 1.32 m), while no differences were observed for the tolerant genotype. These data
demonstrate the robustness of the tolerant genotype in response to water stress and highlights
differences, mainly physiological, between the two genotypes that could help in sugarcane
improvement programs for stress tolerance.
Indexation terms: abiotic stress, Saccharum spp, physiological mechanisms, genetic
improvement.
6
1. INTRODUCTION
Sugarcane (Saccharum spp; Poaceae) is an economically important perennial crop of
tropical origin that is grown in more than 90 countries (FAO, fttp://apps.fao.org) for sugar,
ethanol and biomass production. However, since sugarcane production is concentrated in
many tropical regions where water supply is either inadequate or irrigation infrastructures are
underdeveloped, moisture deficit is a major limitation to optimal productivity (Inman-
Bamber and Smith, 2005; Hemaprabha et al., 2006; Jangpromma et al., 2007). The major
determinants of sugarcane productivity (stalk yield and sucrose content) are highly
susceptible to various biotic and abiotic stresses, of which, drought is the most critical.
Drought stress reduces plant physiological performance and ultimately productivity,
although the magnitude and direction of the response depends on the genotype, duration and
intensity of stress exposure as well as the developmental stage at which stress is applied
(Chaves et al., 2003). Four distinct developmental phases have been identified in sugarcane:
germination, tillering, grand growth and maturity (Van Dillewijn, 1952). The tillering and
early grand growth stages are collectively known as the formative phase, which runs between
60 to 150 days of crop age. Although sugarcane can withstand some degree of water stress
without affecting biomass and sucrose accumulation, formative phase has been identified as a
critical water demand period (Venkataramana et al., 1984; Vasantha et al., 2005). Water
deficit stress during this phase affects growth, morphological, physiological and biochemical
traits, and consequently, cane and sugar yields. In many sugarcane growing areas, the
formative phase coincides with periods of high temperature and water deficit stress.
Therefore, successful culture of sugarcane in areas characterized by inadequate water
availability depends, among other factors, on using appropriate cultural management
practices, such as irrigation, and on growing tolerant varieties. In many production regions of
the world, the high cost of water for irrigation, coupled with poor irrigation infrastructure and
efficiency makes irrigation an expensive and non-sustainable practice. An economic and
sustainable alternative approach would be to plant cultivars that are drought tolerant. There
are currently very few drought tolerant commercial cultivars, however, even though many
sugarcane breeding programs around the world have been selecting for drought tolerance as a
suitable trait for yield improvement.
Drought tolerance is a complex trait that depends on many genes and, thus is
determined by many interactive processes. The polygenic nature of drought tolerance
mechanisms is probably one reason why many conventional selection/breeding approaches
7
have not been fruitful. Moreover, in sugarcane, the high ploidy and large size of the genome
make conventional breeding for this crop more laborious when compared with other crops
(Hogarth, 1987). Hence, yield, sugar content, and disease resistance have received major
attention in sugarcane breeding programs. Nonetheless, some progress in genetic
improvement of sugarcane drought tolerance has been obtained (Ramesh et al., 2000,
Hemaprabha et al., 2004; Vasantha et al., 2005; Hemaprabha et al., 2006; Silva et al., 2008).
Studies have focused on the evaluation of genotypes and families for drought
tolerance-related growth traits such as stomatal behavior, osmotic adjustment, relative water
content, among others (Srivastava et al., 1996; Cha-um and Kirdmanee, 2009; Srivastava et
al., 1997; Ramesh, 2000; Ramesh and Mahadevaswamy, 2000; Hemaprabha et al., 2004).
According to Sleper and Poehlman (2006), combination of genes for frost and drought
hardiness of the S. barberi and S. sinensi with genes for high sugar yield from S. officinarum
should be a major aim in the breeding of sugarcane for marginal environments.
A basic understanding of growth, morphological and physiological processes
responsible for high performance under drought conditions and its interactions are still poor
in sugarcane. This could play a major role in identifying traits for yield improvement and
genetic advance in stress environments. Growth analysis procedures have been utilized to
identify drought-tolerant varieties in certain sugarcane breeding programs (Venkataramana et
al., 1984; Hemaprabha et al., 2004). In this crop, the growth or yield traits that are negatively
impacted by drought stress during the formative phase usually include: stalk height, number
of internodes per stalk, number of millable stalks, stalk diameter, stalk weight and Brix
(Wagih et al., 2003; Hemaprabha et al., 2004; Silva et al., 2008). Among these, stalk height
and stalk weight are generally the most affected traits.
In theory, it is possible to improve productivity either by increasing the
photosynthetic rate, reducing respiration rate, or by increasing allocation of photosynthates to
storage sinks (Zhang et al., 2000). Other drought-responsive physiological traits, such as
stomatal resistance, transpiration, relative water content, chlorophyll content and chlorophyll
a fluorescence traits also influence productivity and constitute useful traits to select drought
tolerant genotypes (Müller et al., 2010; Srivastava et al., 1996). One caveat about these traits
is that even though some of them may be highly correlated with drought tolerance, they may
be of little use during screening because they are too expensive or laborious and time-
consuming.
Morphological responses of plants to drought generally include alterations on leaf
traits (area, shape size, stomatal density, etc), root systems, and mass-area-volume
8
relationships, such as specific leaf area and root-to-shoot ratios (Marron et al., 2002; Marron
et al., 2003; Anyia and Herzog, 2004; Liu and Stützel, 2004; Songsri et al., 2008; Songsri et
al., 2009; Painawadee et al., 2009).
Studies on how plants respond to, and recover from drought stress can reveal
differences in plasticity among genotypes and could be a useful tool for screening for stress
tolerance. Plant physiological/phenotypic plasticity or the ability of the plant to alter its
physiology, morphology and/or behavior in response to a change in the environmental
conditions has not been extensively studied in relation to drought tolerance. Following re-
watering, plants could immediately show a high rate of biological activity, including
photosynthetic capacity and new organ growth, which can be considered as alternative
functional states that may overcompensate for the limitation to plant growth and metabolic
activity due to previous drought (Cai et al., 2004; Montanaro et al., 2007; Efeoglu et al.,
2009; Flexas et al., 2009; Xu et al, 2009). Such overcompensation has also been reported in
sugarcane (Ashton, 1956; Inman-Bamber, 1995). Even after several weeks of severe stress, it
took only few days (3 to 5 days) for leaf extension rates to resume to those rates under normal
conditions (Inman-Bamber, 1995). Genetic variation in physiological/phenotypic plasticity
with respect to drought has not been explored in sugarcane. The objective of this study was to
characterize and compare morphophysiological responses of two sugarcane genotypes during
water stress exposure and during a recovery period after re-watering. The two genotypes used
had previously been classified as drought tolerant and drought susceptible based on yield
performance tests in a field study.
2. MATERIALS AND METHODS
2.1. Planting materials, irrigation treatments, and growing conditions
This study was performed during spring-summer (February-May) 2009 in a ventilated
greenhouse at the Agrilife Research and Extension Center, Texas A&M University, Weslaco
(latitude 26
◦
12‟ N , longitude 97
◦
57‟ W and 18.90 meters of altitude), TX, USA. The two
genotypes of sugarcane selected and evaluated in this study have been previously classified
as drought tolerant (TSP05-4) and drought susceptible (TCP02-4589), based on field yield
trials (Da Silva, personal communication). Among a group of 80 genotypes, TSP05-4 and
TCP02-4589 showed one of the smallest and highest reductions in stalk productivity under
drough stress conditions, respectivelly.
9
Single-node segments containing one lateral bud were germinated in plastics trays
filled with a peat-based substrate (MetroMix MM200, Scotts-Sierra Horticultural Products
Co, Marysville, OH). After two weeks, plantlets were transplanted into 15-L pots containing
MetroMix substrate. Plants were watered at least once per day, and fertilized two times per
week with a complete water-soluble fertilizer (10N-4.4P-8.3K, Peter‟s Corp., St. Louis, Mo.).
The average daily photosynthetic photon flux (PPF) at the canopy level was 15 ± 3.8mol·m
-2
.
Average day/night temperatures were 28.8
± 4.4 / 21.7
± 3.2
°C and average day/night relative
humidity were 48 ± 11 / 68 ± 11%.
Water supply treatments were imposed from 70 to 90 days after planting (DAP) and
after this period the plants were adequately re-watered for eight days. The stress period
coincided with the formative phase, which is the most sensible phase to drought stress in
sugarcane (Venkataramana et al., 1984; Vasantha et al., 2005). Plants were subjected to two
water supply regimes (15% volumetric moisture content, designated “moderate water stress”
and 30% volumetric moisture content, designated as “control” or “well-watered treatment”,
respectively). Volumetric soil moisture content was monitored continuously using soil
moisture sensors (EC5, Decagon Devices, Inc) connected to dataloggers (Em5b, Decagon
Devices, Inc).
2.2. Measurement of morpho-physiological traits
Physiological traits were measured two days before water stress initiation (T
0
), and
then also at two, twelve and, twenty days after stress initiation (T1, T2 and T3, respectively)
and finally at eight days after re-watering (T4). Net photosynthesis rate (P
n
), stomatal
conductance (G
s
), and transpiration (E) were measured using a portable gas exchange system
CIRAS-2 (PPSystems), under ambient temperature, light saturation (1,500 µmol m
-2
s
-1
), and
CO
2
partial pressure of 35 Pa. Leaf chlorophyll a fluorescense measurements were conducted
immediately after P
n
measurements following the procedures of Maxwell and Johnson
(2000), using a pulse amplitude modulation fluorometer (Model OS5-FL, Opti-Sciences,
Tyngsboro, MA, USA). The maximum quantum yield of photosystem II (PSII) was measured
as the dark-adapted F
v
∕F
m
ratio where F
v
is the variable fluorescence (F
m
-F
0
), Fm is the
maximal fluorescence yield following a 1 s saturating pulse at 7500 μmol m
-2
s
-1
, and F
0
is the
minimal fluorescence yield obtained by modulated light at intensity of 0.2 μmol m
-2
s
-1
.
Leaves were dark-adapted for 30 minutes with leaf-clips (FL-DC, Opti-Science) prior to
F
v
∕F
m
measurements. Two measurements were collected per plant.
10
Leaf greenness index (SPAD) measurements were made using a Minolta SPAD-502
chlorophyll meter (Minolta Corp., Ramsey, NJ, USA). The SPAD value is an indicator of the
leaf chlorophyll content (Fanizza et al., 2001; Zaharieva et al., 2001; Anand and Byju, 2008).
Two SPAD readings made on two different leaves were measured per plant. All the above
physiological traits were measured between 10 and 12:00 am, using the 3
rd
or 4
th
leaf from
the top-most visible dewlap of stalk.
Leaf relative water content (RWC: water content relative to the water content of the
tissue at full turgor) was estimated from leaf disks (ten disks per plant). The leaf disks (4,5
mm diameter each) were weighted immediately after collection to determine fresh weight
(FW), and then placed in dislilled water and kept in the dark at room temperature for 24 h
before turgid weight (TW) was recorded. Finally, leaves were dried at 80 ºC for 48 h to
measure the dry weight (DW) (Matin et al., 1989, Silva et al., 2007). RWC trait was
calculated according to the following expression:
RWC (%) = ((FW – DW) / (TW – DW)) x 100
Growth and morphological measurements included stalk height (SH), stalk diameter
(SD), stalk weight (SW), root:shoot ratio (RS ratio) and specific leaf area (SLA) were
collected at the end of the experiment (after T
4
). SLA or the ratio between leaf area and leaf
mass was obtained using the dry weight of the same discs used to measure the RWC trait, by
the following expression:
SLA (cm
2
g
-1
) = (discs leaf area) / DW
SLA has been used to draw conclusions about density and thickness of leaves and the
response of leaf structure to adverse environmental conditions.
Shoot height (SH) was measured from the base of the top-most visible dewlap to the
soil level. Basal stem diameter (SD) was measured with a pair of calipers at 10cm from the
soil level. Single stalk fresh weight was obtained in each pot using a precision scale.
Root:shoot ratio was determined after final harvest by drying root and whole plant tissues at
70ºC for 72 h in an oven and recording dry weights.
11
2.3. Experimental design and data analysis
The physiological investigation experiments were set in a completely randomized
block design with four replications, arranged in a triple factorial 2 x 2 x 5 (two genotypes,
two water treatments and five evaluation times). The growth and morphological investigation
experiments were set in a completely randomized block design with four replications,
arranged in a double factorial 2 x 2 (two genotypes and two water treatments). Analyses of
variance (ANOVA) for each trait were performed to assess the main effects of factors and
interaction between different factors. Genotypes, irrigations treatments and evaluation times
were considered fixed effects. Standard errors were used to detect differences between
treatments means. Statistical tests were considered significant at P ≤ 0.05. All statistical
analyses were performed with SAS statistical package (SAS Institute, Inc.,Cary, NC).
3. RESULTS AND DISCUSSION
Analysis on net photosynthesis (P
n
), transpiration (E) and stomatal conductance (G
s
)
showed significant effects for water supply regime (WR), evaluation time (ET) and the
interactions ET x WR and G x WR (Table 1). Besides, analysis on P
n
showed significant
effects for genotype (G). There were no significant differences in P
n
, G
s
and E between the
genotypes at control (well-watered) treatment, while under water stress treatment tolerant
genotype had values significantly higher for these traits compared to the susceptible genotype
(Fig.1). Water stress reduced Pn, E and Gs rates of the susceptible genotype in 30, 32.70 and
26.53%, respectively. On the other hand, for the tolerant genotype the reductions in these
traits were 6.94, 13.93 and 10%, respectively. Reductions in these physiological traits were
initially non-significant (T0) between the water treatments but with increasing duration of
water stress exposure, differences became more apparent (Fig. 2).
12
Table 1. Summary of analysis of variance for net photosynthesis (P
n
), transpiration rate (E),
stomatal conductance (G
s
), leaf greenness (SPAD), PSII photochemical efficiency (F
v
/F
m
)
and leaf relative water content (RWC) of two sugarcane genotypes (drought-tolerant: TSP05-
4 and drought-susceptible: TCP02-4589) grown under two water supply regimes (control and
moderate water stresss) and evalueted at five times (T0: two days before water stress
initiation, T1, T2 and T3: two, twelve and twenty days after moderate water stress initiation,
and T4: 8 days after re-watering).
SV
DF
P
n
E
G
s
SPAD
F
v
/F
m
RWC
(µmol m
-2
s
-1
)
(µmol m
-2
s
-1
)
(µmol m
-2
s
-1
)
(%)
Mean square
Evaluation time (ET)
4
113.7343*
8.9363*
15906.5125*
109.99*
0.001878*
0.000740
Water regime (WR)
1
186.6605*
10.6142*
28388.1125*
42.490
0.001268*
0.006541*
Genotype (G)
1
40.0445*
0.74110
1058.51250
1214.46*
0.005977*
0.007380
ET x WR
4
67.6343*
2.0989*
5716.3625*
27.550
0.000326
0.001221
ET x G
4
3.61700
0.56800
1400.63750
4.410
0.000238
0.000194
WR x G
1
77.2245*
2.2916*
5136.0125*
0.780
0.000043
0.000798
ET x WR x G
4
8.66830
0.34250
1106.51250
9.420
0.000064
0.000313
Block
3
16.39250
0.29590
1918.64580
75.730
0.000458
0.000168
Error
57
7.41190
0.43810
1135.68970
21.170
0.000180
0.000431
Mean
14.45
3.5853
140.99
46.93
0.7998
91.67
CV (%)
18.84
18.46
23.90
9.80
1.68
2.27
SV: Source of variation; DF: degree of freedom; CV: coefficient of variation; * significant at
p < 0.05.
Studies have shown that stomatal conductance is one of initial short term responses to
water limitation (Jones, 1992) compared to other traits such as leaf expansion, root growth,
gene expression and proteins (Yordanov et al., 2000). A reduction in G
s
caused by stomatal
closure is considered one of the first lines of defense against dehydration damage, and serves
to conserve plant water and maintain high cell and tissue turgor pressure (maintain high leaf
water potential) (Teare et al., 1973; Blum 1974). Stomatal closure and reduction in G
s
caused
by water limitation also limits photosynthetic CO
2
assimilation and transpirational water loss,
as was observed in the present study, suggesting that P
n
was inhibited by gas phase processes
(stomatal limitation). The traits P
n
and G
s
showed a high positive correlation (0.8795, data not
showed).
Differences between plant species and among genotypes of a species in the degree of
stomatal closure and reduction in G
s
, P
n
and E can be indicative of the ability to tolerate mild-
to-moderate water deficit stress. The ability to tolerate low water availability means that the
plant can continue to carry out metabolic processes even during periods of sub-optimal water
13
supply. In the present study, the tolerant genotype was able to maintain significantly higher
rates of P
n
, G
s
and E under water stress conditions (Fig.1).
A
B
C
Figure 1. Mean net photosynthesis (P
n
), stomatal conductance (G
s
) and transpiration rate (E)
of two sugarcane genotypes (drought-tolerant: TSP05-4 and drought-susceptible: TCP02-
4589) grown under two water supply regimes (control and moderate water stress) (mean, ±
S.E.). Different letters indicate significant differences between genotypes within each water
supply regime.
The reductions in P
n
, G
s
and E due to water stress were most severe within 2 days
after water stress initiation (T1). Thereafter, further changes in P
n
and G
s
were not expressive
(Fig. 2). A leveling off in the rate of decline in these traits probably suggest onset of
mechanisms to counteract the effects of water deficit stress, for instance, accumulation of
compatible solutes or adjustments in source-sink allometric relationships such that shoot
water potential is ameliorated. Similar results have also been reported in kidney bean
(Miyashita et al., 2005) and sugarcane leaves (Cha-um and Kirdmanee, 2009; Vu and Allen,
2009).
a
a
a
b
0
5
10
15
20
Control
Water stress
P
n
(µmol.m
-2
.s
-1
)
TSP05-4
TCP02-4589
a
a
a
b
0
40
80
120
160
200
Control
Water stress
G
s
(µmol.m
-2
.s
-1
)
TSP05-4
TCP02-4589
a
a
a
b
0.0
1.0
2.0
3.0
4.0
5.0
Control
Water stress
E (µmol m
-2
s
-1
)
TSP05-4
TCP02-4589
14
A
B
C
Figure 2. Mean net photosynthesis (A), stomatal conductance (B) and transpiration rate (C)
of two sugarcane genotypes (drought-tolerant: TSP05-4 and drought-susceptible: TCP02-
4589) grown under two water supply regimes (control and moderate water stress) and
evaluated at five times (T0: two days before water stress initiation, T1, T2 and T3: two,
twelve and twenty days after moderate water stress initiation, and T4: 8 days after re-
watering) (mean, ± S.E.). Different letters indicate significant differences between water
supply regimes within each evaluation time.
Radiation use efficiency as estimated by PS II chlorophyll a fluorescence (F
v
/F
m
) was
significantly altered by water supply regime, genotype and evaluation time. On average,
F
v
/F
m
was lower in water-stressed plants and in the susceptible genotype (Fig. 3), compared
to control and to tolerant genotype. A decline in F
v
/F
m
ratio under water stress conditions is
indicative of photoinhibition associated with over-reduction of PSII or photoinhibitory
damage (Maxwell and Johnson, 2000; Colom and Vazzana, 2003). Thus, the ability to
maintain high F
v
/F
m
under water stress indicates a high efficiency of radiation use possibly
for photochemistry and carbon assimilation. According to Quiles (2005) the values of F
v
/F
m
in unstressed dark-adapted plants adapted to dark are in the range of 0.75 - 0.80. Zhang et al.
(2000) found that in unstressed sugarcane plants the F
v
/F
m
values were in the range of 0.71 to
0.82.
a
a
a
a
a
a
b
b
b
a
0.0
5.0
10.0
15.0
20.0
25.0
T0
T1
T2
T3
T4
Pn (µmol.m
-2
.s
-1
)
Evaluation time
Control
Water stress
a
a
a
a
a
a
b
b
b
a
50
100
150
200
250
T0
T1
T2
T3
T4
Gs (µmol.m
-2
.s
-1
)
Evaluation time
Control
Water stress
a
a
a
a
a
a
b
b
b
a
1.0
2.5
4.0
5.5
7.0
T0
T1
T2
T3
T4
E (µmol.m-
2
.s
-1
)
Evaluation time
Control
Water stress
15
The leaf greenness index (SPAD) was significantly influenced by genotype and
evaluation time but not by water supply regime (Table 1). SPAD values of the susceptible
genotype were on average 15% lower than those of the tolerant genotype, which is consistent
with their classifications as susceptible and tolerant, respectively (Fig. 3). Similar responses
of SPAD to drought have been reported in the literature (Silva et al., 2007; Cha-um and
Kirdmanee, 2009). SPAD values are indicative of leaf chlorophyll and hence leaf nitrogen
contents (Zaharieva et al., 2001; Fanizza et al., 2001; Anand and Byju, 2008).
Photoinhibitory damage to chlorophyll degradation resulting from water stress-induced
photoinhibition probably accounted for the reduction in leaf greenness indices of the
susceptible genotype. Mechanisms responsible for protection of prevention of chlorophyll
degradation and hence high SPAD values are unclear but probably involve antioxidant
activity of carotenoid pigments. The rates of SPAD and F
v
/F
m
have been considered as rapid
and easy tools to identify drought tolerant genotypes in several species (Fanizza et al., 1991;
Rong-hua et al., 2006; Silva et al., 2007; Arunyanark et al. 2009).
Leaf relative water content (RWC) was significantly altered only by water supply
regime (Table 1), with the control treatment having higher value compared to that of the
water stress treatment (Fig. 3). Even though water supply regime had a significant effect on
RWC, differences between the two water regime treatments only amounted to ~2%. Thus,
RWC values were still high (~91%) compared to results found in other crops such as potato
(Schafleitner et al., 2007) and wheat (Tavakol and Pakniyat, 2007), which have shown drastic
reduction in RWC under water stress. A possible explanation for the lack of differences
found among the genotypes and for the high value of RWC maintained under water stress
may be due the time of the day at which this trait was analyzed. Consistent with observations
of Sarker et al. (1999), RWC measurements tended to be higher in the morning, declined
around midday, and showed some recovery in the afternoon. Thus, the best time to analyze
the RWC was in the afternoon, when evapotranspiration is highest and real differences
among treatments are likely to be revealed. In the present study, RWC samples were
collected at 3:00 pm, when the plants likely have already recovered from drought stress that
had previously occurred. Besides, Painawadee et al. (2009) have found that RWC is an
insensitive indicator of water status in peanut when water deficit was mild.
Relative water content is an easily measured indicator of the current water content in
sampled leaf tissue relative to the water content of the same tissue at full turgor (Matin et al.,
1989). RWC has been used to indicate the plant water status under different water conditions,
and in a diversity of studies (Sarker et al., 1999; Medici et al., 2003; Silva et al., 2007;
16
Bayoumi et al., 2008; Boussadia et al., 2008). While maintenance of high RWC during water
stress is indicative of drought tolerance, the mechanisms for maintaining high RWC are
varied and often confounding (Rampino et al., 2006; Silva et al., 2007). In the present study
there were no significant differences in RWC between the two genotypes studied. This result
indicates that both genotypes may possess the same ability to absorb water from the soil
and/or the same ability to control water loss under moderate stress. Similarly, in crops such as
maize (Efeoglu et al., 2009), common bean (Martínez et al., 2007) and wheat (Tavakol and
Pakniyat, 2007) no differences among genotypes subjected to different water conditions were
detected for RWC.
A
B
C
D
Figure 3. Mean relative water content (RWC), maximum quantum yield of photosystem II
(F
v
/F
m
) and leaf greenness index (SPAD) of two sugarcane genotypes (drought-tolerant:
TSP05-4 and drought-susceptible: TCP02-4589) grown under water supply regimes (control
and moderate water stress) and evaluated at five times (T0: two days before water stress
initiation, T1, T2 and T3: two, twelve and twenty days after moderate water stress initiation,
and T4: 8 days after re-watering) (mean, ± S.E.). Different letters indicate significant
differences between water supply regimes (A and B) or between genotypes (C and D).
a
b
0
20
40
60
80
100
Control
Water stress
RWC (%)
a
b
0.60
0.70
0.80
0.90
Control
Water stress
Fv/Fm
a
b
0
15
30
45
60
TSP05-4
TCP02-4589
SPAD
a
b
0.60
0.70
0.80
0.90
TSP05-4
TCP02-4589
Fv/Fm
17
The non-significant interactions for SPAD, F
v
/F
m
and RWC (Table 1) may indicate
that these traits could be described as constitutive traits, once their expression is independent
of the environment. A constitutive trait is not expected to show high genotype x environment
interaction and could be of advantage as a tool for selection to drought stress (Bayoumi et al.,
2008). The results suggest that F
v
/F
m
could be carried out under any evaluation time,
genotype and water condition under investigation with similar results. This fact was also
observed for SPAD in relation to evaluation time and genotype. This last trait has previously
showed to be a stable trait in peanut genotypes (Arunyanark et al., 2009). The stable
performance of sugarcane genotypes under both water stress and well-watered control for
SPAD and F
v
/F
m
is a flexible tool for breeders to evaluate genotypes for drought tolerance,
once the screening can be carried out without drought conditions.
Plant responses and adaptation to drought stress reflect in changes in photosynthetic
rates. Research on the effect of drought in photosynthesis has been carried out to a less extent
in sugarcane. The inhibition of photosynthesis in water-stressed plants may be attributed to
limited CO
2
diffusion to the leaf intercellular spaces as a consequence of the reduced stomatal
opening (stomatal control) and/or by direct inhibition of biochemical and photochemical
processes (non-stomatal control) imposed by the water deficit (Ashton, 1956; Colom and
Vazzana, 2003; Chaves et al., 2003). Thus, the water stress tolerance was well correlated
with the genotypes capacity to maintain high G
s
and P
n
under water stress and indicate that
these physiological traits were confident to use in early screening for drought tolerance in
sugarcane.
The recovery point upon re-watering following a water shortage is an important and
useful index for the research of recovery processes and for screening of faster-recovery
cultivars. The velocity of recovery after a stress period depends on the species analyzed,
intensity and duration of the stress previously reached, leaf age, light intensity and many
other factors (Flexas et al., 2004; Miyashita et al., 2005; Flexas et al., 2006). The decrease in
P
n
, G
s
and E due the water stress was completely reversed eight days after re-watering
(Fig.2). These results demonstrate the plasticity of the sugarcane genotype to respond to
changing water conditions and support the hypothesis that the basic mechanisms of
photosynthetic biochemistry and photochemistry (non-stomatal control) were not impaired by
water stress and that control on net photosynthesis during water stress was mainly stomatal.
The results on recovery of P
n
, G
s
and E rates found in this study agree with those
reported by Ashton (1956) and Inman-Bamber (1995). In those studies a fast recovery of
sugarcane stressed plants was observed, taking only about few days for photosynthesis and
18
leaf extension rates to return to unstressed conditions. Some 80 to 90 % of normal
photosynthetic activity was recovered within two days in response to irrigation after a series
of high and low soil water content alternating cycles (Ashton, 1956). A fast recovery of
drought effects due the re-watering has also been found in grapevine (Flexas et al., 2009),
maize (Efeoglu et al., 2009), coffee (Cai et al., 2004) and kiwifruit (Montanaro et al., 2007).
Specific leaf area (SLA) and root:shoot ratio (RS) were significantly affected by
evaluation time and genotype, respectively (Table 2). These traits are sensitive to water stress
and have also been correlated with the net photosynthetic capacity (McClendon, 1962, Zhang
et al., 2004). SLA has been used for selecting genotypes for drought tolerance in crops, such
as peanut (Anyia and Herzog, 2004; Songsri et al., 2008; 2009; Painawadee et al., 2009) and
amaranth (Liu and Stützel, 2004). Specific leaf area varies considerably between species and
is a very plastic trait.
Studies have shown that low SLA is associated with slow growth (Poorter and
Remkes, 1990) and this is indicative of low leaf areas available for light interception and
hence photosynthetic carbon assimilation. On the other hand, the inverse of SLA, generally
referred to as specific leaf weight (SLW or leaf mass per unit area) is positively correlate with
leaf thickness and, in some instances, Pn, often through the total number of mesophyll cells
per unit leaf depth (Beadle, 1993). In this regard, a genotype with low SLA may be more
tolerant to water deficit stress since the pathway for water loss is greater and photosynthesis
per unit leaf area is potentially greater. The decrease of SLA under water stress may be
associated with the accumulation of soluble compounds and/or thickening of the cell wall
(Marron et al., 2003). According to Tardieu et al. (1999), a decrease in SLA occurs when
environmental conditions cause a greater depression in growth rate than on photosynthesis
(i.e. the same amount of carbon gain is distributed to a reduced leaf area). However, studies
which emphasize the effects of the water stress in SLA have been contradictory.
Some studies have showed that SLA decrease due to drought (Liu and Stützel, 2004;
Schumacher et al., 2008; Marron et al., 2003; Lal et al., 2009; Painawadee et al., 2009),
while in other ones increased SLA rates were found (Anyia and Herzog, 2004; Aspelmeier
and Leuschner, 2006; Montanaro et al., 2007). Increase in SLA suggests loss in leaf weight in
relation to leaf expansion to compensate for reduced assimilation (Anyia and Herzog, 2004).
The formation of thinner leaves represent less costly leaves. These contradictory results
should be expected in reason of the differences on species, environmental conditions, stress
intensity, stress duration and, plant and leaf age used by different researchers for SLA
determination.
19
Table 2. Summary of analysis of variance for specific leaf area (SLA) and rate among root
and shoot biomass (RS ratio) of two sugarcane genotypes (drought-tolerant: TSP05-4 and
drought-susceptible: TCP02-4589) grown under two water supply regimes (control and
moderate water stress). SLA was evalueted at five times (T0: two days before water stress
initiation, T1, T2 and T3: two, twelve and twenty days after moderate water stress initiation,
and T4: 8 days after re-watering) and RS ratio was measured at the end of the experiment
(after T4).
SV
SLA (cm
2
.g
-1
)
RS ratio
Mean square
Evaluation time (ET)
1437.7424*
-
Water regime (WR)
49.4718
0.0014
Genotype (G)
69.6458
0.2626*
ET x WR
108.8795
-
ET x G
14.9686
-
WR x G
194.0844
0.0031
ET x WR x G
84.6787
-
Block
640.8661
0.0196
Error
80.7958
0.0040
Mean
171.5896
0.4901
CV (%)
5.24
12.94
SV: Source of variation; DF: degree of freedom; CV: coefficient of variation; * significant at
p < 0.05.
In the present study SLA did not differ among genotypes and water supply regimes
(Table 2) showing that the tolerance and the water stress did not affect the leaf mass in
sugarcane. Similar results were found in quinoa plants (González et al., 2009). The lack of
treatment effects on SLA may be due to the early sampling date, which was not enough to
discriminate differences between genotypes and water regimes. Similarly, SPAD values did
not differ in water stressed and non-stressed treatments until two weeks of stress in Vitis
vinifera (Fanizza et al., 1991). Songsri et al. (2009) have also reported that too early or too
late evaluation times were not appropriate to discriminate differences between peanut
genotypes.
The ratio between root and shoot biomass (RS ratio) seems to be governed by a
balance between water absorption by roots and shoot growth. Cell and leaf expansion as well
as stem elongation are more sensitive to water deficit stress than to root growth. In general,
water limitation often results in an increase in RS ratio (Liu and Stützel, 2004). In this study
20
RS ratio was unaffected by water stress (Table 2). This result is in agreement with the finding
of González et al. (2009) who did not observe changes for RS ratio among three treatments in
quinoa plants: drought, water logging and control.
A high RS ratio could reflect an increased capacity of water uptake, thereby
maintaining the shoot in a well-hydrated condition (Blum, 1996). Despite the lack of water
treatments effects, the tolerant genotype had the highest RS ratio (0.62) in both control and
water stress conditions (Fig. 4) and this may have contributed to the higher P
n
, G
s
and E
observations under water stress conditions.
Figure 4. Mean rate among root and shoot
biomass (RS ratio) of two sugarcane genotypes
(drought-tolerant: TSP05-4 and drought-
susceptible: TCP02-4589) grown under two
water supply regimes (control and moderate
water stress) (mean, ± S.E.). Different letters
indicate significant differences between
genotypes.
Sugarcane yield components such as brix, stalk weight, stalk height, stalk diameter
and stalk number are usually affected by drought stress during the formative phase
(Srivastava et al., 1997; Ramesh and Mahadevaswamy, 2000; Hemaprabha et al., 2004;
Vasantha et al., 2005; Silva et al., 2008). Among these, stalk height and stalk weight are
generally the most affected traits. In the present study, stalk height (SH) and stalk weight
(SW) were significantly affected by genotype and water supply regimes (P<0.05). SH was
also affected by the interaction between these two factors (Table 3).
Stalk diameter (SD) was a stable trait across genotypes and water supply regimes.
This result is consistent with studies conducted by Domaingue (1996), Vasantha et al. (2005)
and Silva et al. (2008), who showed that SD was one of the yield component less affected by
a
b
0.0
0.2
0.4
0.6
0.8
TSP05-4
TCP02-4589
RS ratio
21
water shortage. On the other hand, reduction in SH and SW under drought has been observed
by several authors (Domaingue, 1996; Ramesh and Mahadevaswamy, 2000; Hemaprabha et
al., 2004; Vasantha et al., 2005; Cha-um and Kirdmanee, 2009).
Table 3. Summary of analysis of variance for stalk height (SH), stalk diameter (SD) and stalk
weight (SW) of two sugarcane genotypes (drought-tolerant: TSP05-4 and drought-
susceptible: TCP02-4589) grown under two water supply regimes (control and moderate
water stress).
SV
DF
SH (m)
SD (cm)
SW (kg)
Mean square
Genotype (G)
1
0.9409*
0.1444
0.1266*
Water regime (WR)
1
0.2209*
0.0121
0.0990*
G x WR
1
0.0729*
0.064
0.0323
Block
3
0.0052
0.0147
0.0123
Error
9
0.0097
0.0451
0.0164
Mean
1.26
2.63
0.5735
CV (%)
7.81
8.08
22.34
SV: Source of variation; DF: degree of freedom; CV: coefficient of variation; * significant at
p < 0.05.
Among four yield components (SH, SD, stalk number and brix) studied by Silva et al.
(2008), SH was the most affect by unirrigated conditions. In this study, water stress caused a
significant reduction in SH of the susceptible genotype (from 1.69 to 1.32 m), but no
differences were observed for the tolerant genotype (Fig. 5). The reduction in SH in the
susceptible genotype may be caused by suppression of cell division and expansion due to
lower turgor pressure. The mean SH for the susceptible and tolerant genotypes were 1.50 and
1.02 m, respectively.
Water stress reduced SW by 26% in comparison to control treatment and the tolerant
genotype had the smallest single stalk weight (Fig. 5). The diminished weight observed for
tolerant genotype (0.48 kg) is due to the lowest stalk length measured for this genotype (Silva
et al., 2009).
22
A
B
C
Figure 5. Mean stalk height and mean stalk weight of two sugarcane genotypes (drought-
tolerant: TSP05-4 and drought-susceptible: TCP02-4589) grown under two water supply
regimes (control and moderate water stress) (mean, ± S.E.). Different letters indicate
significant differences between water supply regimes within genotype (A), between
genotypes (B) or between water supply regimes (C).
Genotypes which show proportionally less reduction in yield attributes under drought
conditions, could be considered more drought tolerant, but only if the reduction in the
expression of an attribute is associated with a high mean, because it is of little value if the
mean expression of the attribute is too low to satisfy the minimum required criteria
(Domaingue, 1996; Silva et al., 2008). The smaller SH and SW found for the tolerant
genotype suggest that it could have a slower early growth during water stress when compared
to the susceptible genotype. Consequently, the yield superiority cannot be determined solely
on the basis of SH and SW. These traits should be evaluated in plants subjected to a longer
stress period, during the formative phase (60 to 150 days of crop age) to be applied as
drought tolerance indicators. In the present study the plants were submitted to a shorter stress
period (70 to 90 days of crop age), which corresponds to the beginning of the tillering phase.
a
a
a
b
0.0
0.4
0.8
1.2
1.6
2.0
TSP05-4
TCP02-4589
Stalk height (m)
Control
Water stress
b
a
0.0
0.2
0.4
0.6
0.8
TSP05-4
TCP02-4589
Stalk weight (kg)
a
b
0.0
0.2
0.4
0.6
0.8
Control
Water stress
Stalk weight (kg)
23
4. CONCLUSIONS
Physiological traits, especially gas exchange traits, were able to discriminate among
water stress tolerant and susceptible genotypes even at early and moderate stress.
After a re-watering period, the rates of gas exchange traits altered by water stress
returned completely to the rates observed in the control treatment, demonstrating the
plasticity of sugarcane genotypes in responding to water stress.
Morphological traits were not influenced by the imposed water stress.
Growth traits, especially stalk height could be useful to differentiate between tolerant
and susceptible genotypes. However these traits should be evaluated in an advanced water
stress stage during the formative phase to select to drought tolerance and higher productivity
genotypes.
5. ACKNOWLEDGEMENTS
We are grateful to CNPq and Agrilife Research and Extension Center by the financial
assistance and to sugarcane breeding program from Federal University of Viçosa and from
Texas A&M University.
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SCIENTIFIC PAPER 2
DIFFERENTIAL GENE EXPRESSION IN SUGARCANE GENOTYPES IN RESPONSE
TO WATER STRESS
VIÇOSA
MINAS GERAIS – BRAZIL
2010
32
RESUMO
PEDROZO, Cássia Ângela, D.Sc., Universidade Federal de Viçosa, julho, 2010. Expressão
gênica diferencial em genótipos de cana-de-açúcar em resposta ao estresse hídrico.
Orientador: Márcio Henrique Pereira Barbosa. Co-orientadores: Luiz Alexandre
Peternelli e Jorge Alberto da Silva.
A seca é o principal fator limitante à produtividade da cana-de-açúcar em muitas
regiões do mundo e é causada por períodos de estresse hídrico prolongado ou por irrigação
inadequada. Os mecanismos pelos quais as plantas respondem as condições de seca são
variados e ainda pobremente entendidos. A identificação de genes, bem como a
caracterização de suas funções e regulação em resposta à seca é necessária em programas de
melhoramento que visam ao aumento da produtividade por meio da obtenção de cultivares
mais tolerantes. O objetivo deste estudo foi usar a técnica cDNA-AFLP para identificar e
caracterizar genes diferencialmente expressos em dois genótipos de cana-de-açúcar (tolerante
à seca: TSP05-4 ou susceptível à seca: TCP02-4589) ao estresse hídrico. As plantas foram
avaliadas dois (T1) e doze dias após início de estresse hídrico de intensidade moderada (T2),
e oito dias após re-irrigação (T3). Um total de 15 fragmentos derivados de transcritos (TDFs)
diferencialmente expressos foram clonados e caracterizados e, 8 destes foram validados por
RT-PCR em tempo real. Três TDFs mostrando similaridade com genes codificando para um
pentatricopeptídeo putativo, um translocador glicose-6-fosfato/fosfato 2 e para a subunidade
regulatória CK2β3 da proteína kinase CK2 foram diferencialmente expressos no genótipo
susceptível. Os dois primeiros TDFs foram reprimidos enquanto que o último foi induzido.
Por outro lado, um TDF similar a um mRNA de uma proteína induzida pela seca foi também
induzido no genótipo tolerante em T2. A expressão gênica alterada pelo estresse hídrico foi
completamente revertida para todos os fragmentos após o período de re-irrigação
demonstrando, assim, a plasticidade dos genótipos de cana-de-açúcar em responder a
alterações nas condições hídricas. Os resultados encontrados neste estudo demonstram a
robustez do genótipo tolerante em responder a condições de estresse hídrico e enfatiza
diferenças moleculares entre os dois genótipos que podem auxiliar em programas de
melhoramento que visam tolerância à seca.
Termos de indexação: estresse abiótico, cDNA-AFLP, mecanismos moleculares,
melhoramento genético.
33
ABSTRACT
PEDROZO, Cássia Ângela, D.Sc., Universidade Federal de Viçosa, July, 2010. Differential
gene expression in sugarcane genotypes in response to water stress. Adviser: Márcio
Henrique Pereira Barbosa. Co-adviser: Luiz Alexandre Peternelli and Jorge Alberto da
Silva.
Drought is a major factor limiting the productivity of sugarcane in many regions of
the world and is caused by prolonged dry conditions or inadequate irrigation. The
mechanisms by which plants respond to drought are varied and are still poorly understood.
Identifying relevant genes and characterizing their functions and regulation in response to
drought is necessary in crop improvement programs aimed at increasing productivity by
enhancing stress tolerance. The objective of this study was to use the cDNA-AFLP technique
to identify and characterize differentially-expressed genes in two sugarcane genotypes
(tolerant: TSP05-4 or susceptible: TCP02-4589) to drought stress. Plants were evaluated at
two (T1) and twelve days after moderate water stress initiation (T2), and again at eight days
after re-watering (T3). A total of 15 transcript-derived differentially-expressed fragments
(TDF) were cloned and characterized and 8 of them were validated by real-time RT-PCR.
Three TDFs showing sequence similarities to genes encoding a putative expressed
pentatricopeptide, a glucose-6-phosphate/phosphate translocator 2, and a protein kinase CK2
regulatory subunit CK2β3 were differentially expressed in the susceptible genotype. The two
formers TDFs were down-regulated and the last one was up-regulated. On the other hand, one
TDF similar to a drought-inducible protein mRNA was also up-regulated in the tolerant
genotype at T2. The genes that had their expression altered in response to water stress were
completely reversed after re-watering period, thus demonstrating the plasticity of sugarcane
plants in being able to respond to changing water conditions. The results found in this study
demonstrate the robustness of the tolerant genotype in response to water stress and highlights
molecular differences between the two genotypes that could help in sugarcane improvement
programs for stress tolerance.
Indexation terms: abiotic stress, cDNA-AFLP, molecular mechanisms, genetic improvement.
34
1. INTRODUCTION
Modern sugarcane varieties are hybrids derived mostly from hybridization between
Saccharum officinarum L. (2n=80) and Saccharum spontaneum L. (2n=40-128), followed by
a series of backcrosses with Saccharum officinarum, in a process known as „nobilisation‟.
However, other species, such as S.robustum, S. barberi Jesw., and S. sinensi Roxb. have also
been involved to a lesser extent in the development of modern varieties. These varieties are
highly polyploids, heterozygotes, aneuploids, and on average contain 100-130 chromosomes
(Irvine, 1999).
Crop cultivation is negatively affected by abiotic stresses such as drought, high
salinity, cold, flooding, and heat (Grover et al., 2001; Luan, 2002; Wang et al., 2003). These
stresses elicit a variety of responses in plants from alteration of gene expression to changes in
growth and final yield. Among the abiotic stress, drought is the most severe stress limiting
the sugarcane productivity in Brazil and other sugarcane producing countries (Ellis and
Lankford, 1990; Wiedenfeld, 1995; Wiedenfeld, 2000). The level of tolerance or sensitivity
of plants to drought stress is influenced by the time of exposure and severity of stress,
previous exposure to stress, genotype, and developmental age of the plants (Bray, 2002;
Passioura, 2004). In sugarcane, the formative phase (60 to 150 days of crop age) has been
identified as a critical water demand period (Venkataramana et al., 1984; Vasantha et al.,
2005). Water deficit stress during this phase affects growth, morphological and physiological
traits, and consequently, cane and sugar yields.
Drought is a condition of special interest, since increasing water scarcity has been
observed throughout the world. According to Riera et al. (2005), irrigated agriculture
currently accounts for approximately 65% of global fresh water use indicating that the
development varieties tolerant to drought stress can have a potentially huge impact on
productivity in the future. However, drought-tolerance is a polygenic trait and it is difficult to
be improved by traditional breeding (Beever, 2000). Thus, the integration of molecular
approaches with plant breeding and physiology may be advantageous in increasing sugarcane
productivity under water shortage. For molecular breeding, the identification and
characterization of genes regulated by drought stress is essential for understanding the
drought-tolerance mechanisms and developing tolerant genotypes, either for transgenic plant
development or for marker-assisted breeding.
It has been shown that tolerance/sensitivity mechanisms of plants in response to
drought are revealed by changes in the expression level of genes regulated by this adverse
35
condition (Shinozaki and Yamaguchi-Shinozaki, 1997; Shinozaki and Yamaguchi-Shinozaki,
2000; Shinozaki and Yamagushi-Shinozaki, 2007). A variety of drought stress-inducible
genes encodes for proteins which have different functions, such as detoxification, water
channels, transporters, protection factors of macromolecules (LEA proteins), osmolyte
biosynthesis (proline, sugars), transcription factors, protein kinases, phosphatases, ABA
biosynthesis etc (Shinozaki and Yamagushi-Shinozaki, 1997; Shinozaki and Yamaguchi-
Shinozaki, 2000; Wang et al., 2003; Shinozaki and Yamagushi-Shinozaki, 2007). These gene
products are included in three major categories: (1) those involved in signaling cascades and
in transcriptional control; (2) those that function as protectors of membranes and proteins; (3)
those involved in water and ion uptake and transport.
Techniques like suppression subtractive hybridization (SSH), serial analysis of gene
expression (SAGE), macroarray, microarray, differential display reverse transcription-
polymerase chain reaction (DDRT-PCR) and cDNA-amplified fragment length
polymorphism (cDNA-AFLP) are available to monitor gene expression under different biotic
and abiotic stresses. Among them, macroarray and microarray technologies are the most
important and most powerful tools for studying the whole genome transcription (Decorosi et
al., 2005). However, these techniques are relatively expensive and require prior sequence
knowledge of the genes to be investigated, and therefore unable for some laboratories and for
some species.
cDNA-AFLP (Bachem et al., 1996) is a powerful and relatively inexpensive tool for
genome-wide expression analysis, especially when genome sequence information is limited.
Its sensitivity and specificity is compared to those found for the microarray approach (Reijans
et al., 2003). Moreover, cDNA-AFLP enables the identification of new and/or poorly
expressed genes. This technique has been successful to characterize the mechanisms
underlying tolerance to several biotic and abiotic stresses, such as pathogens (Cadle-
Davidson, 2006; Adhikari et al., 2007; LaO et al., 2008), salt (Jayaraman et al., 2008,
Roshandel and Flowers, 2009), cold (Sun et al., 2007; Meng et al., 2008), and heat (Simões-
Araújo et al., 2002). For drought stress there are very few reports on gene expression using
cDNA-AFLP analysis (Yang et al., 2004; Si et al., 2009).
In sugarcane, attempts to reveal the gene expression under drought stress have been
performed using microarray (Rocha et al., 2007) and macroarray (Rodrigues et al., 2009)
analysis. In both these studies, a variety of genes, such as transcription factors, kinases,
phosphatases, transporters, and auxin biosynthesis enzymes were related. Moreover,
Rodrigues et al. (2009) have showed that the number of expressed genes increased with the
36
severity (mild to severe stress) of drought. Despite these positive results, little is known about
molecular mechanisms of drought stress response in sugarcane. Research in this specific
subject may provide new strategies to improve the stress tolerance of sugarcane crop.
The aim of this study was to use the cDNA-AFLP technique to identify and to
characterize differentially expressed genes in leaves of two sugarcane genotypes subjected to
moderate water stress.
2. MATERIALS AND METHODS
2.1. Planting materials, irrigation treatments, and growing conditions
This study was conducted during spring-summer (February-May) 2009 in a ventilated
greenhouse at the Agrilife Research and Extension Center, Texas A&M University, Weslaco
(latitude 26
◦
12‟ N , longitude 97
◦
57‟ W and 18.90 meters of altitude), TX, USA. The two
genotypes of sugarcane selected and evaluated in this study had been previously classified as
drought tolerant (TSP05-4) and drought susceptible (TCP02-4589), based on field yield trials
(Da Silva, personal communication). Among a group of 80 genotypes, TSP05-4 and TCP02-
4589 showed one of the smallest and highest reductions in stalk productivity under drough
stress conditions, respectivelly.
Single-node segments containing one lateral bud were germinated in plastics trays
filled with a peat-based substrate (MetroMix MM200, Scotts-Sierra Horticultural Products
Co, Marysville, OH). After two weeks, plantlets were transplanted into 15-L pots containing
MetroMix substrate. Plants were watered at least once per day, and fertilized two times per
week with a complete water-soluble fertilizer (10N-4.4P-8.3K, Peter‟s Corp., St. Louis, Mo.).
The average daily photosynthetic photon flux (PPF) at the canopy level was 15 ± 3.8mol·m
-2
.
Average day/night temperatures were 28.8
± 4.4
°C / 21.7
± 3.2
°C and average day/night
relative humidity were 48 ± 11% / 68 ± 11%.
Water supply treatments were imposed from 70 to 90 days after planting (DAP) and
after this period the plants were adequately re-watered for eight days. The stress period
coincided with the formative phase, which is the most sensible phase to drought stress in
sugarcane (Venkataramana et al., 1984; Vasantha et al., 2005). Plants were subjected to two
water supply regimes (15% volumetric moisture content, designated “moderate water stress”
and 30% volumetric moisture content, designated as “control” or “well-watered treatment”,
respectively). Volumetric soil moisture content was monitored continuously using soil
37
moisture sensors (EC5, Decagon Devices, Inc) connected to dataloggers (Em5b, Decagon
Devices, Inc).
2.2. Sampling procedures
To study the differential gene expression of two sugarcane genotypes under water
stress, samples of the 3
rd
leaf counted from the top-most visible dewlap of stalk were
collected at three evaluation times: two days (T1) and twelve (T2) days after water stress
initiation, and eight days after re-watering (T3). Leaf tissues of three plants for each treatment
(evaluation date/water regime/genotype) were collected and frozen immediately in liquid
nitrogen and latter stored at -80 ºC prior to analysis.
2.3. Total RNA extraction, poli (A)+ RNA isolation and cDNA synthesis
Approximatelly 100 mg of leaf tissues were frozen with liquid nitrogen and ground to
a fine powder to extract the total RNA using the QIAGEN Rneasy Plant Mini Kit (Qiagen,
Valencia, CA), according to the manufacturer‟s instructions. The total RNA concentration
was measured with a Nanodrop spectrophotometer (NanoDrop ND-1000 UV-Vis
Spectrophotometer, NanoDrop Technologies), while the RNA quality were checked using 1
μg of total RNA by electrophoresis in agarose gel 1% (w/v). Poli (A)
+
RNA was isolated
from 10 μg of total RNA using the MicroPoly(A) Purist
TM
Kit (Ambion), according to the
manufacturer‟s protocol. Single and double stranded-cDNAs were then synthetised from 10
µl of poli (A)
+
RNA, using the SuperScript
TM
Double-Stranded cDNA Synthesis (Invitrogen).
2.4. cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis
The cDNA-AFLP analysis was performed as described by Bachem et al. (1996),
using the AFLP
®
Expression Analysis kit of LI-COR (LI-COR, Lincoln, NE), with minor
modifications. Restriction enzymes TaqI e MseI were used to digest the resulting cDNA and
to generate pre-amplification PCR products. A non-selective pre-amplification was
performed using non-selective adaptor primers without additional nucleotides. The pre-
amplification products were diluted 10-fold before being used in the final selective
amplification. Selective PCRs were performed with a total of 22 primer combinations
obtained by the eight MseI+2 primers and the eight TaqI+2 primers (+2 represents two
38
selective nucleotides: +GA, +GT, +TC, +TG, +CT, +CA, +AG and +AC on both adaptor
primers) provided in the AFLP Expression Analysis Kit. The sequences of adapter strands
and primers used for pre-amplification and selective amplification are summarized in the
Table 4. The TaqI+2 selective primers were labeled with 700 and 800-nm infrared dye (LI-
COR, IRDye 700 and IRDye 800). PCR reactions were performed with Taq DNA
polymerase from Promega (Promega, Madison, WI). Selective PCR products were separated
and visualized by electrophoresis on 6.5% denaturing polyacrylamide in a LI-COR DNA
analyser (model 4300 LI-COR
®
). Eletrophoretic run parameters were: 1500 V, 40 W, 40 mA,
45 ºC, with a 25-min pre-run and 2 h main run.
Table 4. Sequence of adapter strands and primers used for pre-amplification and selective
amplification.
Primer or adapter
Sequence
MseI adapter
5'-GACGATGAGTCCTGAG-3'
3'-TACTCAGGACTCAT-5'
TaqI adapter
5'-CTCGTAGACTGCGTAC-3'
3'-CGGTACGCAGTCT-5'
MseI pre-amplification primer
5'-GACGATGAGTCCTGAGTAA-3'
TaqI pre-amplification primer
5'-GTAGACTGCGTACCGA-3'
MseI amplification primer
5'-GATGAGTCCTGAGTAANN-3'
TaqI amplification primer
5'-GTAGACTGCGTACCGANN-3'
Data images were collected using LI-COR‟s Saga AFLP Analysis software. Gel
images were analyzed visually and the transcript-derived fragments (TDFs) were selected
based on its presence/absence (qualitative variants) between the water regimes. Only the
fragments with reproducible pattern of expression among the three plants sampled were
considered. Use of replicated samples in this study could eliminate false-positive banding
patterns and also allows for the identification of fragments that are water stress-related only.
2.5. Isolation and sequencing of transcript-derived fragment (TDF)
Isolation of TDFs of interest was perfomed according to the AFLP
®
Expression
Analysis kit, mentioned previously. The TDFs were excised from gels and incubated at -20
ºC in Eppendorf tubes containing 40 µl 1X TE buffer. Three freeze-thaw cycles were
39
performed and the solution was centrifuged at 15,000 x g for 20 minutes at 4 ºC. After
centrifugation, 5 µl of the resuspended DNA solution was used for PCR re-amplification
using the same primers and PCR conditions as those in the selective amplification
procedures. To determine the purity and confirm the size of isolated fragments, 10 µl of the
PCR products were run on the 1.5% agarose gel (w/v). After gel visualization, the TDFs were
excised from gel and purified using the Zymoclean
TM
Gel DNA recovery Kit (Zymo
Research). Subsequentely the purified TDFs were cloned into the pGEM
®
-T Easy vector
(Promega Corp., Madison, WI) according to the manufacturer‟s protocol, then used to
transfom Escherichia coli DH5α competent cells.
Recombinant plasmids from six bacterial colonies of each cloned fragment were
isolated using Zyppy
TM
plasmid Miniprep kit (Zymo Research) following the manufacturer‟s
protocol. Before sequencing, the identity of the cloned fragments were verified comparing the
size of the cloned fragment with the size of the fragment in the original polyacrylamide gel.
Purified plasmids containing the insert were sequenced with an automated DNA sequencer
(Applied Biosystem, Inc.) at the DNA Facility, Iowa State University, USA. Sequences not
corresponding to the selective amplification were omitted from further analysis.
2.6. Analysis of sequences
After removal of vector sequence database searches were performed. The nucleotide
as well as translated sequences were analysed for their homology with nucleotide and protein
sequences, respectively, against the data available in the GenBank
(http.//www.ncbi.nlm.nih.gov/BLAST) database using the BLASTx and BLASTn search
tools. Significance of the similarity was based on E-value. Similarities with E-values ≤ 10
-10
were considered significant.
2.7. Validation of cDNA-AFLP experiments by real-time reverse transcription PCR
Real-time reverse transcription PCR (RT-PCR) was used to confirm the differential
expression of 7 TDFs isolated by cDNA-AFLP. The RT-PCR primers for these fragments
were designed using the Primer Express Version 3.0 (Applied Biosystems, Inc). The
following criteria were used for selecting each primer pair: 18 - 25 pb long; melting
temperature of 59 - 61 ºC; 40-60% GC content; amplicon length of 60-150 pb. The primer
40
sequences were searched against the NCBI database using the Blast tool to verify specificity
of the sequences (Table 5).
Single strand cDNA was synthetized from 20 ng of poli (A)
+
RNA using the iScript
TM
cDNA Synthesis Kit (Invitrogen), following the manufacturer‟s instructions. The cDNA of
the three plants sampled in each treatment (genotype/water regime/evaluation time) was
pooled in identical quantities to prepare the RT-PCR reactions. These reactions were
performed with iQ
TM
SYBR
®
Green Supermix (Bio-Rad laboratories, Hercules, CA, USA) in
a BioRad iCycler iQ5 thermocycler (Bio-Rad laboratories, Hercules, CA, USA).
Table 5. Primer sequences of reference gene (RF) and of target TDFs used in the RT-PCR
expression analysis.
Primer name
Forward primer (5'-3')
Reverse primer (5'-3')
RF
GAPDH*
CACGGCCACTGGAAGCA
TCCTC AGGGTTCCTGATGCC
Target TDF
SugF01
CCCTCAAATGCAGGGAACTA
GCCAGCTGTTTTCTGAGACC
SugF02
CCTACGATGACGAGGTCCAT
CCTTTGCTGCAACAATTTCA
SugF05
AGCAACTAACCAACCCATCG
CTTGTTGGAGGGAGATCGAG
SugF06
ATGAGGAAATGGAGCGTGTC
CCATGTGAACCAATCTGTGC
SugF10
AACGCCGAAACTTCTTCTGA
GAGTCGAACTCGGGAACTGA
SugF11
ATCTGGCAGGCGTGAGTTTA
TTCCACTGCTCACTTGCATC
SugF15
TTCTCCAAGAAGGGGATGAA
ATGGAGAGGCAGGCGTAGTA
SugF16
GCAGCAACCGGATATCTCTT
CTGCCTTGGCCTATTTCTTG
PCR was performed for each sample in triplicates. To normalize the gene expression
glyceraldehide-3-phosphate de-hydrogenase (GAPDH) was used as endogenous reference
gene (Iskandar et al., 2004; Table 5). For each RT-PCR reaction, a total of 25 µl was
prepared containing 2 µl of template cDNA, 0.4 µM of each fragment specific primer and
reference gene primers, 8.5 µl water, and 12.5 µl of iQ SYBR Green Supermix. For negative
controls (NTC), cDNA templates synthetized without iScript reverse transcriptase
(Invitrogen) were used. No-template controls were also included to detect any spurious
signals arising from amplification of any DNA contamination or primer dimer formed during
the reaction. The following amplification program was applied: 50 ºC for 2 min, 95 ºC for 10
min followed by 40 cycles of 95 ºC for 15 s and 60 ºC for 1 min.
To ensure optimal PCR efficiencies of TDFs standard curves were generated. Also,
melting curve analyses were performed after the final cycle of amplification to exclude the
occurrence of primer dimers and unspecific PCR products. The relative expression rates of
41
each fragment normalized to the reference gene were calculated using Ct values (threshold
cycle value). The following expression was used to calculate the Ct value (Livak and
Schmittgen, 2001):
Fold change = 2
-Δ(ΔCt)
ΔCt = Ct (target gene) – Ct (reference gene)
Δ(ΔCt) = ΔCt (interess sample) – ΔCt (control sample)
3. RESULTS AND DISCUSSION
3.1. cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis
By using 22 cDNA-AFLP primer combinations, about 1548 TDFs ranging in size
from ~ 50 bp to ~ 500 bp were detected. There were observed few fragments larger than 500
bp but visualization and evaluation of them were very difficult to perform without errors. An
average of 70 TDFs per primer combination was produced. These results are in agreement
with those showed by Yang et al. (2004), Gigliotti et al. (2004), and Polesani et al. (2008).
Figure 6 represents an example of a section of a typical cDNA-AFLP gel, containing banding
patterns of two sugarcane genotypes analyzed under two water supply regimes and at three
evaluation times. The primer combination used in this example was TaqI GT x MseI AG.
Among the total number of TDFs visualized in this study, 30 (about 2% of all TDFs)
were classified as differentially-expressed. A total of 24 differentially expressed TDFs were
down-regulated and 6 were up-regulated. TDFs of interest were classified into three
categories: (1) TDFs that were present only in the tolerant genotype, (2) TDFs present only in
the susceptible genotype, and (3): TDFs that were simultaneously present in both tolerant and
susceptible genotypes. The susceptible genotype had 23 differentially-expressed TDFs, while
the tolerant genotype had only 6 TDFs. Only one down-regulated differentially-expressed
TDF was simultaneously present in both genotypes. Despite the higher number of TDFs
expressed by the susceptible genotype, most of them (19) were down-regulated. Among six
TDFs found exclusively in the tolerant genotype, four were down-regulated and two were up-
regulated. The expression profile exhibited by the susceptible genotype suggests that stress
was detected earlier in the susceptible genotype. These results are in concert with a previous
study by Rodrigues et al. (2009), who observed that the number of differentially expressed
TDFs in sugarcane was increased with water stress severity. In accordance to the results
42
found by these authors, the TDFs differentially expressed in a drought-tolerant cultivar
(SP83-5073) appear as induced or repressed preferentially in severe water stress conditions.
Under moderate drought-stress, SP83-5073 had only one down-regulated TDF. On the other
hand, under the same moderate drought conditions, 36 TDFs were induced and 136 were
repressed in a susceptible cultivar (SP90-1638).
Figure 6. cDNA-AFLP fingerprint generated from two sugarcane genotypes (drought-
tolerant: TSP05-4 and drought-susceptible: TCP02-4589) under two water supply regimes
(control and moderate water stress) evaluated at three times (T1 and T2: two and twelve days
after water stress initiation, respectively ,and T3: eight days after re-watering). The primer
combination used was TaqI GT x MseI AG.
Among other factors, time of exposure and severity of stress is very important for
plant survival under drought conditions (Passioura, 2007). A total of 11 TDFs were
differentially-expressed at T1. 8 TDFs were differentially-expressed only at T2, while 11
43
TDFs were responsive at both evaluation times. These results indicate that some TDFs
overlapped between the two evaluation times, while others are expressed early on or later on
during stress exposure. Only 1 TDF was up-regulated at T1, while 5 TDFs were up-regulated
at T2. On the other hand, 10 TDFs were down-regulated at T1, 3 at T2 and 11 at both
evaluation times. The higher number of down-regulated TDFs compared to those up-
regulated was also observed by Yue et al. (2008) in maize plants exposed to one day and
seven days of water deficit stress treatment.
Following re-watering, plants could immediately show a high rate of biological
activity, including photosynthetic capacity and new organ growth, which can be considered
as alternative functional states that may overcompensate for the limitation to plant growth
and metabolic activity due to previous drought (Xu et al., 2009). In the present study all the
TDFs which were differentially expressed in the susceptible or tolerant genotypes had their
normal expression pattern recovered after re-watering. These results show the plasticity of
sugarcane genotypes in being able to respond rapidly to changing water conditions.
3.2. Isolating and characterization of transcript-derived fragment (TDF)
A total of 15 differentially expressed TDFs was excised from polyacrylamide gels, re-
amplified, cloned and sequenced. The size of these TDFs varied from 84 to 343 bp. The
sequencing failed for 2 TDFs and these fragments were not characterized further. The
remaining sequenced TDFs were renamed as SugDR (sugarcane drought-responsive) and
then compared to nucleotide and protein databases using the BLASTn and BLASTx tools,
respectively. Table 6 describes TDFs along with the measure of similarity (in case of the
TDFs with similarity in the GenBank), regulation (down or up-regulation) under water stress,
and evaluation time when the TDFs were regulated.
44
Table 6. Differentially-expressed transcript derived fragments (TDFs) of two sugarcane genotypes [drought-tolerant: TSP05-4
(T) and drought-susceptible: TCP02-4589 (S)] grown under moderate water stress conditions and evaluated at two times [Two
(T1) and twelve days after moderate water stress initiation (T2)]. The TDFs were up-regulated (U) or down-regulated (D).
TDF name
TDF size
Sequence homology
ID
E-Value
R.P.
G
E.T.
SugDR01*
201
Putative expressed pentatricopeptide, Oriza sativa
ABA99065.2
1.0e
-27
D
S
T1 and T2
SugDR02*
247
Hypothetical protein OsJ-08616, Oriza sativa
EEE57919.1
2.0 e
-40
D
S
T1 and T2
SugDR04
112
No significant similarity
-
-
D
S
T1
SugDR05*
84
22 kDA drought-inducible protein mRNA, Saccharum hybrid cultivar
AY496271.1
3.0 e
-33
U
T
T2
SugDR06*
93
Hypothetical protein OsI-08927, Oriza sativa
EEC74003.1
2.0 e
-07
D
T
T2
SugDR08
264
Protein kinase CK2 regulatory subunit CK2β3, Zea mays
NM001111505.1
2.0 e
-79
U
S
T2
SugDR09
217
No significant similarity
-
1.0 e
-19
U
S
T2
SugDR10*
111
No significant similarity
-
-
D
T
T1
SugDR11*
143
Hipothetical protein LOC100273728, Zea mays
NP001141610.1
1.0 e-
19
D
S
T1 and T2
SugDR14
281
No significant similarity
-
-
D
S
T1 and T2
SugDR15*
282
Glucose-6-phosphate/phosphate translocator 2, Zea mays
NP001147439.1
1.0 e
-21
D
S
T2
SugDR16*
343
Putative tocopherol polyprenyltransferase, Oryza sativa
BAC83059.1
5.0 e
-52
U
S
T2
SugDR19
131
No significant similarity
-
-
D
S and T
T1
ID: Identification on GenBank; RP: regulation pattern; G: genotype; ET: evaluation time
* TDFs selected for RT-PCR analysis
45
Of the total 13 TDFs sequenced, 5 TDFs showed significant similarity to genes with
known or putative function, 3 were similar to hypothetical proteins and 5 did not show
significant similarity to any sequence or protein in the non-redundant database. The identified
and unknown proteins were similar to rice, sugarcane and maize sequences. Approximately
40% of the sequenced TDFs represent yet uncharacterized genes. The characterization of new
genes may provide interesting information to the complex plant response network. The
unclassified genes also represented the largest category in a study previously performed in
sugarcane (Rodrigues et al., 2009). Three differentially expressed TDFs are presented in the
Figure 7.
A
B
C
Figure 7. Three differentially-expressed transcript derived fragment (A: putative expressed
pentatricopeptide, B: 22 kDA drought-inducible protein mRNA and C: protein kinase CK2
regulatory subunit CK2β3) generated from two sugarcane genotypes [drought-tolerant:
TSP05-4 (T) and drought-susceptible: TCP02-4589 (S)] grown under two water supply
regimes (control and moderate water stress) and evaluated at two times (T1: two days after
moderate water stress initiation and T2: twelve days after moderate water stress initiation).
A databank search revealed the relationship of the TDFS SugDR01, SugDR05,
SugDR08, SugDR15, and SugDR16 with other genes involved in environmental stress such
as drought, cold, high salinity, and attack of pathogens. Three of these TDFs showing
significant sequence similarities to genes encoding a putative expressed pentatricopeptide
(SugDR01), a protein kinase CK2 regulatory subunity CK2β3 (SugDR08), and a glucose-6-
phosphate/phosphate translocator 2 (SugDR15) were differentially expressed in the
susceptible genotype. One TDF (SugDR05) similar to a drought-inducible protein mRNA
46
was up-regulated in the tolerant genotype at T2. Finally, one TDF (SugDR16) similar to a
tocopherol polyprenyltransferase gene was down-regulated in both genotypes at T2.
3.3. Validation of cDNA-AFLP experiments by real-time reverse transcription PCR
(RT-PCR)
Real-time reverse transcription PCR (RT-PCR) is one of the most reliable method for
gene expression analysis. Thus, to validate the changes in mRNA abundance detected by
cDNA-AFLP and to quantitatively evaluate the expression level of transcripts in the drought-
tolerant and susceptible genotypes, RT-PCR experiment was performed on five down
regulated and two up-regulated TDFs. Those TDFs selected for RT-PCR analysis are
indicated with an asterisk in the Table 6. The Ct values were normalized using the Ct value of
the GAPDH gene, which was used as a housekeeping gene. No significant variation in the
expression of GAPDH was observed among water treatments (data not shown). Five TDFs
(SugDR01, SugDR02, SugDR05, SugDR11 and SugDR15) were confirmed as either down or
up-regulated in response to water stress. The fragments SugDR10 and SugDR16 did not show
changes in gene expression level among control and water stress treatments.
47
Putative expressed pentatricopeptide -T1
Hypothetical protein OsJ-08616 - T1
22 kDA drought-inducible protein mRNA -
T2
Hypothetical protein OsI-08927 - T2
SugDR10 - No significant similarity - T1
Hipothetical protein LOC100273728 - T1
Glucose-6-phosphate/phosphate translocator 2
-T2
Putative tocopherol polyprenyltransferase -T2
Figure 8. Real time RT-PCR analysis of 8 differentially-expressed transcript derived
fragment generated from two sugarcane genotypes (drought-tolerant: TSP05-4 and drought-
susceptible: TCP02-4589) grown under two water supply regimes (control and moderate
water stress) and evaluated at two times (T1: two days after moderate water stress initiation
and T2: twelve days after moderate water stress initiation). All data were normalized to the
glyceraldehide-3-phosphate de-hydrogenase (GAPDH) expression level. Data represent fold
change of the gene expression in water-stressed vs. control plants. Bars indicate the standard
deviation obtained of three biological replications.
0.00
0.40
0.80
1.20
1.60
TCP02-4589 D
TCP02-4589 C
2
-Δ(ΔCt)
0.00
0.40
0.80
1.20
1.60
TCP02-4589 D
TCP02-4589 C
2
-Δ(ΔCt)
0.00
0.50
1.00
1.50
2.00
2.50
TSP05-4 D
TSP05-4 C
2
-Δ(ΔCt)
0.00
0.40
0.80
1.20
1.60
TSP05-4 D
TSP05-4 C
2
-Δ(ΔCt)
0.00
0.50
1.00
1.50
2.00
2.50
TSP05-4 D
TSP05-4 C
2
-Δ(ΔCt)
0.00
0.30
0.60
0.90
1.20
1.50
TCP02-4589 D
TCP02-4589 C
2
-Δ(ΔCt)
2
-Δ(ΔCt)
0.00
0.30
0.60
0.90
1.20
1.50
TCP02-4589 D
TCP02-4589 C
2
-Δ(ΔCt)
0.00
0.40
0.80
1.20
1.60
2.00
TCP02-4589 D
TCP02-4589 C
2
-Δ(ΔCt)
48
3.4. Functional classification of drought-responsive genes
The pentatricopeptide repeat (PPR) is a protein family capable of specific binding to
both protein and RNA molecules (Lurin et al., 2004; Schmitz-Linneweber and Small, 2008).
Some PPR proteins are involved in plant development (Schmitz-Linneweber and Small,
2008), organelle biogenesis (Lurin et al., 2004), restoring of cytoplasmic male sterilities
(Bentolila et al., 2002; Koizuka et al., 2003), RNA processing and editing in mitochondria
and chloroplasts (Meierhoff et al., 2003; Kotera et al., 2005), and responses to environmental
stresses (Rodrigues et al., 2009). PPR proteins are required for a wide range of different post-
transcriptional processes in plant organelles, and the lack of particular PPR proteins often
leads to phenotypes owing to lack of expression of a specific organelle gene (Schmitz-
Linneweber and Small, 2008). However, there is a little evidence that any of the known PPR
proteins meaningfully regulates expression of organelle proteins under physiological
conditions.
TDFs with similarity to PPR repeat proteins already been found in sugarcane
(Rodrigues et al. (2009), mandarin (Gimeno et al., 2009), and rice (FengHua et al., 2009)
exposed to drought stress conditions. In this study, the PPR like-protein (SugDR01) was
down-regulated at T1 and T2 days after water stress initiation in the susceptible genotype.
Similarly, Rodrigues et al. (2009) have found one TDF (CA128234) which was down-
regulated under moderate water stress conditions in a susceptible genotype and up-regulated
under severe water stress in both tolerant and susceptible genotypes. These authors also found
another TDF (CA120224) which was up-regulated only in the tolerant genotype subjected to
severe water stress conditions. In the present study PPR protein expression was also
suppressed in the susceptible genotype under water stress conditions. An Arabidopsis mutant
in the PPR gene At3g09650, designated as high-chlorophyll-fluorescence (hcf152-1), shows a
defect in photosynthetic-electron transport (petB) and psbH mRNA processing in the
chloroplast, resulting in reduced levels of the cytochrome b6f complex (Meierhoff et al.,
2003). The up-regulation of SugDR01 in the susceptible genotype may be indicative of the
sensitivity of this genotype to water stress conditions.
SugDR05 which was slightly up-regulated at T2 in the tolerant genotype showed
similarity to a drought inducible protein (SoDip22) in Saccharum officinarum (Sugiharto et
al., 2002). Because of the hydrophilic nature of SoDip22, it is plausible that it belongs to the
Asr (abscisic acid-ABA, stress, and ripening induced) protein family and it functions to adapt
to drought stress in the bundle sheath, and the signaling pathway for the induction is, at least
49
in a part, mediated by ABA. Asr genes represent a gene family that is usually induced by a
wide range of stress such as, drought (Maskin et al., 2001; Yang et al., 2005; Philippe et al.,
2010), salt (Yang et al., 2005), ABA (Çakir et al., 2003; Carrari et al., 2004) and pathogen
response (Liu et al., 2010).
Kinase CK2 protein, also known as casein kinase II, is a highly conserved
serine/threonine kinase and it has been classified as a stable tetrameric complex, consisting of
two catalytic subunits (CK2α and CK2α‟) and two regulatory subunits (CK2β1 and CK2β2)
(Pinna, 2002; Litchfield, 2003). However, plant kinase CK2 proteins contain several isoforms
for both catalytic and regulatory subunits, creating the potential for a wide variety of CK2
holoenzyme combinations that may have a role in regulating CK2 activity or substrate
specificity (Riera et al., 2001). The plant CK2 is involved in many different processes such
as, DNA transcription, RNA translation, and cell-cycle regulation.
SugDR08 which was up-regulated at T2 in the susceptible genotype showed similarity
to the regulatory subunit CK2β3. Although several studies have shown that kinase protein
groups are regulated under drought stress conditions (Montalvo-Hernández et al., 2007;
Rocha et al., 2007; Clement et al., 2008; Rodrigues et al., 2009; Mizogushi et al., 2010), few
studies have reported on the regulation of CK2 regulatory subunits under environmental
stress. In sugarcane, Rocha et al. (2007) and Rodrigues et al. (2009) reported that several
kinases are regulated under drought conditions. Rodrigues et al. (2009) have also found two
TDFs similar to serine/threonine kinase-like proteins that were up-regulated under severe
drought conditions. One protein was induced in both tolerant and susceptible sugarcane
cultivars, while two other ones were induced only in the susceptible genotype. They also
found other kinases that were simultaneously up-regulated in the tolerant and susceptible
genotypes. Rocha et al. (2007) have similarly identified ten kinase proteins differentially-
expressed in response to drought.
Changes in osmotic response are associated with changes in the location of cellular
components via transporters and changes in the synthesis of secondary metabolites (Sahin-
Çevic and Moore, 2006; Meng et al., 2008). The induction of transporters enables
osmoprotectants to move to their functional sites. The glucose-6-phosphate/phosphate
translocator (GPT) represents a distinct member of the phosphate translocator protein family
and its proposed physiological function is import glucose 6-phosphate into amyloplasts of
heterotrophic tissues for use as a precursor for starch and fatty acid biosynthesis and a
substrate for the oxidative pentose phosphate pathway (Fischer and Weber, 2002; Hua-wu et
al., 2003). The expression of transcripts similar to GPT was induced by cold (Sahin-Çevic
50
and Moore, 2006; Lee et al., 2010) and heat conditions (Qin et al., 2008) and by combined
drought and heat stress treatments (Rizhsky et al., 2004). However, a TDF (SugDR15)
similar to a glucose-6-phosphate/phosphate translocator was found to be repressed at T2 in
the susceptible genotype. Similarly, in a study achieved by Xue et al. (2008) a significant
reduction in expression was observed for one (TaGPT) of the three chloroplasts GPT genes
analyzed in droughted-wheat plants.
4. CONCLUSIONS
The tolerant and susceptible genotypes had different responses when subjected to
water stress conditions.
Gene response to water stress was quicker in the susceptible genotype, since a higher
number of differentially expressed TDFs were detected in this genotype compared to the
tolerant genotype.
The higher number of up-regulated genes in the susceptible genotype may be an
indicator of its sensitivity to water stress.
The normal gene expression pattern were restored in both genotypes within 8 days
after re-watering, perhaps demonstrating the plasticity of sugarcane plants in being able to
respond rapidly to changing water conditions.
5. ACKNOWLEDGEMENTS
We are grateful to CNPq and Agrilife Research and Extension Center by the financial
assistance and to sugarcane breeding program from Federal University of Viçosa and from
Texas A&M University.
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57
GENERAL CONCLUSIONS
Physiological traits, especially gas exchange traits were more able to
discriminate among drought tolerant and susceptible genotypes even at early and moderate
water stress.
Growth and morphological traits should be evaluated in an advanced water
stress stage during the formative phase to select to drought tolerance and higher productivity
genotypes.
It was possible see that tolerant and susceptible genotypes had different
responses when subjected to water stress conditions and that gene response to drought stress
was quicker in susceptible compared to the tolerant genotype.
After a re-watering period the gas exchange traits and gene expression pattern
returned completely to the rates observed to the control treatment, demonstrating the
plasticity of sugarcane genotypes in responding to changing water conditions.
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