the submergence tolerance regulator sub1a mediates ...the submergence tolerance regulator sub1a...
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The Submergence Tolerance Regulator SUB1A MediatesCrosstalk between Submergence and DroughtTolerance in Rice W OA
Takeshi Fukao, Elaine Yeung, and Julia Bailey-Serres1
Department of Botany and Plant Sciences, Center for Plant Cell Biology, University of California, Riverside, California 92521
Submergence and drought are major constraints to rice (Oryza sativa) production in rain-fed farmlands, both of which can
occur sequentially during a single crop cycle. SUB1A, an ERF transcription factor found in limited rice accessions, dampens
ethylene production and gibberellic acid responsiveness during submergence, economizing carbohydrate reserves and
significantly prolonging endurance. Here, we evaluated the functional role of SUB1A in acclimation to dehydration.
Comparative analysis of genotypes with and without SUB1A revealed that SUB1A enhanced recovery from drought at the
vegetative stage through reduction of leaf water loss and lipid peroxidation and increased expression of genes associated
with acclimation to dehydration. Overexpression of SUB1A augmented ABA responsiveness, thereby activating stress-
inducible gene expression. Paradoxically, vegetative tissue undergoes dehydration upon desubmergence even though the
soil contains sufficient water, indicating that leaf desiccation occurs in the natural progression of a flooding event.
Desubmergence caused the upregulation of gene transcripts associated with acclimation to dehydration, with higher
induction in SUB1A genotypes. SUB1A also restrained accumulation of reactive oxygen species (ROS) in aerial tissue during
drought and desubmergence. Consistently, SUB1A increased the abundance of transcripts encoding ROS scavenging
enzymes, resulting in enhanced tolerance to oxidative stress. Therefore, in addition to providing robust submergence
tolerance, SUB1A improves survival of rapid dehydration following desubmergence and water deficit during drought.
INTRODUCTION
Global climate change influences the frequency and magnitude
of hydrological fluctuations, causing devastating events such as
floods and drought. Both high and low extremes in precipitation
increasingly limit food, fiber, and forest production worldwide
(Easterling et al., 2007). Approximately 30% of the world’s rice
(Oryza sativa) farmlands are at a low elevation and irrigated by
rain (Bailey-Serres et al., 2010). Such rain-fed farming lessens
groundwater depletion, water pollution, and soil salinization,
which are often associated with controlled irrigation systems.
However, rain-fed fields are prone to flooding and drought due to
inadequate water management. Thus, submergence, drought,
and the sequential events (submergence followed by drought
and vice versa) are major constraints to rice production in rain-
fed lowlands. Therefore, improvement of combined tolerance to
submergence and drought would substantially increase rice
productivity while sustaining water resources and soil quality.
Rice is a semiaquatic species that is typically cultivated under
partially flooded conditions. However, flash flooding can cover
the entire plant for prolonged periods, and most rice cultivars die
within 7 d of complete submergence (Xu et al., 2006; Bailey-
Serres et al., 2010). A limited number of rice cultivars overcome
submergence through antithetical growth responses. Deepwater
rice responds to submergence by promoting internode elonga-
tion to outgrow floodwaters. This escape response is regulated
by a polygenic locus that encodes two APETALA2/Ethylene
Response Factor (AP2/ERF) DNA binding proteins, SNORKEL1
(SK1) and SK2 (Hattori et al., 2009). By contrast, submergence-
tolerant lowland rice, including Flood Resistant 13A (FR13A),
restrains elongation growth, economizing carbohydrate reserves
to enable development of new leaves upon desubmergence.
This quiescence response is regulated by another AP2/ERF,
located at the polygenic SUBMERGENCE1 (SUB1) locus (Xu
et al., 2006). The highly submergence-inducible SUB1A gene
that confers this tolerance is absent in all japonica and most
indica accessions. Additionally, the closely related SUB1B and
SUB1C genes are also submergence inducible and invariably
present in rice accessions at the SUB1 locus but are not
associated with submergence tolerance.
During submergence, SUB1A dampens ethylene production
and enhances mRNA and protein accumulation of two negative
regulators of gibberellic acid (GA) signaling, SLENDER RICE1
(SLR1) and SLR1-like 1 (SLRL1), resulting in the suppression of
the energy-consuming escape response (Fukao et al., 2006;
Fukao and Bailey-Serres, 2008). Consistently, global-scale tran-
scriptome analysis revealed that SUB1A regulates the abun-
dance of mRNAs associated with ethylene and GA production
and signaling during submergence (Jung et al., 2010). SUB1A
also coordinates diverse transcription factors, including a subset
1 Address correspondence to [email protected] author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Julia Bailey-Serres([email protected]).WOnline version contains Web-only data.OAOpen Access articles can be viewed online without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.110.080325
The Plant Cell, Vol. 23: 412–427, January 2011, www.plantcell.org ã 2011 American Society of Plant Biologists
of AP2/ERFs at the mRNA accumulation level. A recent survey of
13 rice accessions containing SUB1A found that the degree of
submergence tolerance positively correlates with the expression
level of SUB1A in node and internode regions during submer-
gence (Singh et al., 2010). Although the SKs and SUB1A regulate
opposite growth responses under submerged conditions, all are
group VII ERF subfamily members.
In general, rice is sensitive to drought due to its high water
requirement, but upland and lowland rice varieties range in their
tolerance. Molecular genetic analyses detected a number of
quantitative trait loci that affect the components related to
drought tolerance, including grain production, shoot and root
morphology, and leaf water status (Lanceras et al., 2004; Yue
et al., 2006; Venuprasad et al., 2009). However, no major genes
that regulate these traits have been identified because of the
weak effect and low mapping resolution of the quantitative trait
loci regions. Through genomic and molecular approaches, a
number of transcription factors involved in drought tolerance
have been identified in Arabidopsis thaliana. Among these fac-
tors, members of DEHYDRATION RESPONSIVE ELEMENT
BINDING PROTEIN/C-REPEAT BINDING FACTOR (DREB/CBF),
ABSCISIC ACID RESPONSIVE ELEMENT (ABRE) BINDING
PROTEIN/ABRE BINDING FACTOR (AREB/ABF), NAM ATAF
CUC2 (NAC), and ERF serve functional roles in transcriptional
regulation of acclimation responses and tolerance to drought
(Nakashima et al., 2009; Hirayama andShinozaki, 2010). Drought
stress induces mRNA accumulation of these transcription fac-
tors in an abscisic acid (ABA)-dependent or -independent
manner, regulating expression of their direct target genes that
encode late embryogenesis abundant (LEA) proteins, antioxidant
enzymes, osmolyte biosynthesis enzymes, chaperones, and
transporters. Rice (Os) orthologs of these Arabidopsis transcrip-
tion factors, such as Os DREB1A, Os DREB2B, Os ABF1, and
Os NAC6, induce similar stress-inducible genes, and their ec-
topic expression increases drought tolerance in rice (Dubouzet
et al., 2003; Nakashima et al., 2007; Amir Hossain et al., 2010;
Matsukura et al., 2010), indicating that Arabidopsis and rice
share common transcriptional networks that coordinate drought
response and tolerance mechanisms.
Plants encounter multiple abiotic stresses simultaneously or
sequentially in a natural or agricultural environment. To survive
extreme conditions, plants modulate adaptive responses through
complex signaling pathways, which are integrated at various
levels. ABA plays a crucial role in the adaptive responses to
drought, high salt, and freezing, all of which induce a cellular
osmotic stress. Each stress stimulates accumulation of ABA in
vegetative tissue, resulting in stomatal closure, stress-inducible
gene expression, and metabolic adjustment (Zhu, 2002; Seki
et al., 2007). It is well known that transcription factors such as
DREB1/CBF, DREB2, AREB/ABF, and NAC regulate expres-
sion of genes associated with acclimation to osmotic stress
(Dubouzet et al., 2003; Nakashima et al., 2007; Amir Hossain
et al., 2010; Matsukura et al., 2010). Overexpression of each of
these genes enhances tolerance to multiple stresses, including
drought, salinity, and low temperature in Arabidopsis and rice.
Despite the significance of submergence and drought to rice
production in rain-fed farmlands, the molecular crosstalk be-
tween the two stress responses has not been investigated. Here,
we evaluated the influence of the submergence tolerance reg-
ulator, SUB1A, on acclimation responses to water deficit during
the vegetative growth phase of rice. Comparative analyses
revealed a pivotal role of SUB1A in water relations, detoxification
of reactive oxygen species (ROS), and stress-inducible gene
expression during drought. In addition to conditions where water
is limited, rice experiences dehydration stress following desub-
mergence due to reduced hydraulic conductivity in leaf sheaths
(Setter et al., 2010). Through the SUB1A-mediated responses to
drought, submerged plants overcome this inevitable stress fol-
lowing desubmergence. Thus, SUB1A serves as a convergence
point between submergence and drought response pathways,
allowing rice plants to survive both extremes of precipitation.
RESULTS
SUB1A Confers Drought Tolerance to Rice
Although exposure of rice plants to prolonged submergence
results in severe leaf senescence, genotypes containing SUB1A
are able to recover after desubmergence through formation of
new leaves (Fukao et al., 2006; Fukao and Bailey-Serres, 2008).
This indicates that SUB1A contributes to protection of meriste-
matic cells from submergence and reoxygenation stress. We
considered that the protective role of SUB1A may aid endur-
ance of other abiotic stresses, such as drought. To evaluate the
contribution of SUB1A to water deficit tolerance, a japonica
inbred line, M202, and a near isogenic SUB1 introgression line,
M202(Sub1), were subjected to comparative analysis at physi-
ological and molecular levels. These lines differ in an ;182-kb
interval of chromosome 9 containing the polygenic SUB1 locus
(Xu et al., 2006). M202(Sub1) possesses all three SUB1 genes,
SUB1A,SUB1B, andSUB1C, whereasM202 lacksSUB1A. Both
M202 and M202(Sub1) plants were grown in the same pot for
14 d and uniformly exposed to dehydration stress by withholding
water for 8 d (Figure 1A). Following the drought, pots were placed
in a shallow tray filled with water for complete soil rehydration. In
both genotypes, all of the fully expanded leaves were severely
wilted after 8 d of drought and did not recover with rehydration.
However, there was a significant difference in the establish-
ment of new leaves by the two genotypes after 14 d of recovery;
71.7% of M202(Sub1) plants recommenced leaf development
as compared with only 11.7% of the M202 plants (Figure 1B).
Drought stress reduced the fresh weight of aerial tissue similarly
in the two genotypes, but recovery of M202(Sub1) plants was
distinguished by vigorous leaf formation and fresh weight accu-
mulation (Figure 1C).When leaf relative water content (RWC)was
monitored during 7 d of drought, we found it declined similarly in
the two genotypes for the first 4 d, after which the decline was
significantly less severe in M202(Sub1) leaves (Figure 1D). This
indicates that SUB1A contributes to limiting leaf water loss
during drought. We also investigated two Sub1 introgression
lines in indica backgrounds, IR64(Sub1) and Samba Mahsuri
(Sub1), to verify that the influence ofSUB1A on drought tolerance
was robust (see Supplemental Figure 1 online). The results of
viability tests and freshweightmeasurements in theseSub1 lines
were in accordance with those in M202(Sub1), indicating that
SUB1A Enhances Dehydration Tolerance 413
introgression of SUB1A significantly improves recovery from
prolonged dehydration stress in both japonica and indica culti-
vars with varying drought tolerance.
A variety of environmental stresses, including drought, stim-
ulate the excessive accumulation of ROS, which results in oxi-
dative damage of cellular components, such as lipids, proteins,
and DNA. To determine whether SUB1A influences ROS accu-
mulation in leaves, lipid peroxidation was monitored by mea-
suring a product of the peroxidation events, malondialdehyde
(MDA) (Figure 1E). MDA levels were elevated in response to
drought stress in both genotypes but were significantly sup-
pressed in M202(Sub1) from day 3 to 6. These observations
indicate that SUB1A diminishes the ROS accumulation triggered
by water deficit.
The SUB1 locus encodes up to three ERF transcription fac-
tors, SUB1A, SUB1B, and SUB1C, all of which are induced by
submergence. To obtain the expression profile of SUB1 genes
during drought, we monitored transcripts of these three genes in
Figure 1. SUB1A Confers Drought Tolerance to Rice.
(A) Photos of rice plants treated with drought. Twenty plants of each M202 and M202(Sub1) line were grown in the same pot for 14 d and subjected to
drought treatment for 8 d. After the treatment, plants were recovered under regular watering conditions for 14 d. Bars = 10 cm.
(B) Viability of plants after 8 d of drought treatment and 14 d of recovery. Plants were counted as viable when one or more leaves were produced during
the recovery period. Data represent mean6 SE (n = 203 3 independent biological replicates), and an asterisk indicates a significant difference between
M202 and M202(Sub1) (P < 0.05).
(C) Fresh weight of aerial tissue before and after drought treatment. Data are represented as in (B).
(D) RWC of fully expanded uppermost leaf during drought. Data represent mean6 SE (n = 8), and an asterisk indicates a significant difference between
M202 and M202(Sub1) (*P < 0.05; **P <0.01).
(E) Lipid peroxidation in aerial tissue during drought. The level of MDA, an end product of lipid peroxidation, was monitored in aerial tissue of plants
treated with water deficit. Data represent mean6 SE (n = 3), and an asterisk indicates a significant difference between M202 and M202(Sub1) (P < 0.05).
FW, fresh weight.
(F) Relative mRNA levels of SUB1 genes in aerial tissue during drought. Fourteen-day-old plants were exposed to water deficit for up to 5 d. Aerial tissue
was harvested at the time points specified and analyzed by qRT-PCR. Relative level of each transcript was calculated by comparison to the
nonstressed control [M202(Sub1) at day 0 for SUB1A; M202 at day 0 for SUB1B and SUB1C]. Data represent mean 6 SE from three independent
biological replicates, and an asterisk indicates a significant difference between M202 and M202(Sub1) (P < 0.01).
414 The Plant Cell
aerial tissue of M202 and M202(Sub1) by quantitative RT-PCR
(qRT-PCR) (Figure 1F). Accumulation of SUB1A mRNA was
highly enhanced in response to drought stress. SUB1B and
SUB1C mRNAs were slightly induced by drought, with lower
SUB1CmRNA levels inM202(Sub1). This trend is consistent with
previous reports that SUB1A dampens accumulation of SUB1C
mRNA (Fukao et al., 2006; Xu et al., 2006; Fukao and Bailey-
Serres, 2008).
To evaluate if SUB1A expression also aids in tolerance to
osmotic stress, M202 and M202(Sub1) seedlings were exposed
to constant osmotic stress by use of polyethylene glycol (see
Supplemental Figure 2 online). Osmotic stress (20.6 MPa) con-
siderably reduced the elongation growth of shoots in both
genotypes and roots in M202 (see Supplemental Figures 2A
and 2B online). Inhibition of shoot and root growth was less
severe in M202(Sub1), indicating that SUB1A enhances toler-
ance to osmotic stress. To confirm that osmotic stress induces
expression of SUB1 genes, mRNA levels were monitored in
coleoptiles and roots of seedlings treated with polyethylene
glycol (see Supplemental Figure 2C online). The SUB1A tran-
script was highly elevated 6 h after osmotic stress treatment and
became more abundant at later time points. The rapid accumu-
lation of SUB1A mRNA was observed in both shoots and roots,
but the fold change value was much greater in shoots. Levels of
SUB1B and SUB1C transcripts also increased in response to
osmotic stress. As seen under drought, the accumulation of
SUB1C mRNA was restricted in M202(Sub1) under osmotic
stress.
SUB1A Upregulates Genes Involved in Acclimation
to Drought
Microarray analyses revealed that SUB1A, encoding an ERF
transcription factor, mediates regulation of transcripts encoding
other transcription factors, including AP2/ERF genes, which are
involved in stress response and acclimation (Jung et al., 2010;
Mustroph et al., 2010). To examine the overlap of differentially
regulated genes during submergence and dehydration, publicly
available microarray data sets were analyzed. Direct compar-
ison of significantly induced genes (fold change $2; adjusted
P value # 0.05) demonstrated that 25.5% of submergence-
inducible gene transcripts are also upregulated by dehydration
(seeSupplemental DataSet 1 andSupplemental Figure 3Aonline).
Among 793 genes positively regulated by SUB1A (fold change$
1.5; adjusted P value # 0.05), 226 (28.5%) are upregulated in
response to water deficit (see Supplemental Data Set 2 and
Supplemental Figure 3B online). This suggests that SUB1A
regulates drought-responsive genes, several of which were
monitored at the level of mRNA accumulation over a time course
of water deficit in M202 and M202(Sub1) (Figure 2). DREB1/CBF
encodes group IIIc AP2/ERFs, which regulate expression of
genes associated with tolerance to drought, salinity, and low
temperature in Arabidopsis and rice (Nakashima et al., 2009). It
has been reported that overexpression of rice DREB1A and
DREB1E significantly increases survival of water deficit (Ito et al.,
2006; Chen et al., 2008). We found that drought stress induced
the accumulation of DREB1AmRNA in aerial tissue of M202 and
M202(Sub1), but the level was significantly higher in M202(Sub1)
at days 3 and 4 (Figure 2). The level of DREB1E transcripts
decreased in response to water deficit in both lines, but M202
(Sub1) maintained significantly more transcript during drought
stress. AP37 (ERF3) and AP59 belong to the AP2/ERF groups
VIIIa and IXa, respectively, and constitutive expression of each
enhances recovery from water deficit during vegetative devel-
opment (Oh et al., 2009). Consistently, we found that both were
upregulated by drought stress, with significantly more transcript
in M202(Sub1) (Figure 2).
LEA proteins are a family of highly hydrophilic proteins that
accumulate in maturing seeds and vegetative tissue under
dehydrated conditions, such as drought, salinity, and low tem-
perature (Valliyodan andNguyen, 2006; Hirayama andShinozaki,
2010). It seems that LEAs play a critical role in stabilization of
proteins and membrane structure during cellular dehydration.
Figure 2. SUB1A Enhances mRNA Accumulation of Genes Associated
with Acclimation to Drought.
mRNA levels were measured in leaves of plants subjected to 0, 3, 4, or
5 d of drought and are expressed relative to the level in M202 at day 0 (set
at 1.0). Data represent mean 6 SE from three independent biological
replicates, and an asterisk indicates a significant difference between
M202 and M202(Sub1) (*P < 0.05; **P < 0.01).
SUB1A Enhances Dehydration Tolerance 415
Overexpression of genes encoding LEAs increases survivability,
biomass production, and grain yield in barley (Hordeum vulgare),
wheat (Triticum aestivum), and rice underwater deficit conditions
(Valliyodan and Nguyen, 2006). The rice LEAs, RAB16A (RAB21),
LEA3, and LIP9 (DHN1/DIP1), are strongly induced by drought
and ABA treatment. Constitutive and conditional expression of
LEA3 results in increased spikelet fertility and grain production
during drought (Xiao et al., 2007). SalT is a jacalin-like lectin
protein andwas originally identified as one of themost prominent
proteins induced by high salt in rice roots (Claes et al., 1990).
Drought stress highly induced the accumulation of RAB16B,
LEA3, LIP9, and SalT mRNAs in M202 and M202(Sub1) (Figure
2). Among these genes, the transcripts of RAB16B, LIP9, and
SalT were significantly more abundant in M202(Sub1) under
dehydrated conditions, indicating that SUB1A acts upstream of
these genes.
SUB1A Increases ABA Responsiveness
ABA is a key signaling molecule that coordinates water balance,
expression of stress-inducible genes, and metabolic adjustment
under water deficit conditions (Zhu, 2002; Seki et al., 2007). ABA-
deficient and ABA-insensitive mutants of Arabidopsis exhibit
disturbed water relations and poor acclimation to water deficit
(Koornneef et al., 1998; Zhu, 2002). By contrast, ABA hypersen-
sitive mutants and ABA-overproducing transgenics show re-
stricted water loss and increased tolerance under dehydrated
conditions in Arabidopsis and Nicotiana plumbaginifolia (Pei
et al., 1998; Iuchi et al., 2001; Qin and Zeevaart, 2002). To
determine whether ABA regulates accumulation of SUB1 gene
transcripts, genotypes with and without SUB1A were treated
with two concentrations of ABA (5 or 50 mM) and the aerial tissue
used for RNA analyses. Application of ABA decreased levels of
all three SUB1 transcripts in M202 and M202(Sub1) (Figure 3A).
No significant difference in SUB1C transcript level was observed
between the two genotypes, indicating that the expression of
SUB1A mRNA was not sufficient to repress the accumulation of
SUB1C mRNA in ABA-treated M202(Sub1) plants. The trans-
genic Ubi:SUB1A-3 and nontransformed LG control (O. sativa cv
Liaogeng) were also evaluated. The constitutive accumulation of
SUB1AmRNAwas not altered byABA treatment inUbi:SUB1A-3
(Figure 3B). This suggests that ABA does not influence turnover
of SUB1A mRNA, but it rather restricts SUB1A transcription.
Consistent with results from the M202 and M202(Sub1) geno-
types, ABA downregulated SUB1B and SUB1CmRNA accumu-
lation in the overexpression line and the control. Overexpression
of SUB1A decreased the transcript levels of SUB1B as well as
SUB1C under nontreated conditions.
Although ABA reduces the abundance of theSUB1A transcript
in aerial tissue under nonstress conditions, the transcript is highly
induced by drought and osmotic stress (Figure 1F; see Supple-
mental Figure 2C online). To determine the influence of SUB1A
on ABA responsiveness, seeds of LG and two independent Ubi:
SUB1A lines were incubated in a series of ABA solutions (Figure
3C). Application of 10 mM ABA had little effect on germination
repression in LG seeds but significantly suppressed germination
of the two Ubi:SUB1A lines. Overexpression of SUB1A further
enhanced the inhibition of seed germination at higher ABA
concentrations. ABA responsiveness of seedling shoots was
also evaluated in the three genotypes (Figure 3D). Consistent
with the recalcitrance in seed germination, shoot growth was
more severely restricted by ABA in the two Ubi:SUB1A lines.
These results indicate that SUB1A increases responsiveness to
ABA, in addition to the decrease in GA responsiveness shown
previously (Fukao and Bailey-Serres, 2008).
We reported that seeds of the SUB1A overexpression lines
exhibit a dormancy phenotype (Fukao and Bailey-Serres, 2008).
Application of fluridone, an ABA biosynthesis inhibitor, stimu-
lates germination of dormant seeds of N. plumbaginifolia, Arabi-
dopsis, and red rice (Oryza sativa f. spontanea), emphasizing the
critical role of ABA in seed dormancy (Grappin et al., 2000;
Cadman et al., 2006; Gianinetti and Vernieri, 2007). To evaluate
the significance of ABA biosynthesis in SUB1A-mediated seed
dormancy, we incubated seeds of LG and the two Ubi:SUB1A
transgenics in a series of fluridone solutions (Figure 3E; see
Supplemental Figure 4 online). Application of fluridone slightly
accelerated the rate of seed germination in LG. However, the
inhibitor did not rescue the phenotype of the SUB1A over-
expression lines. It appears that ABA production is not in-
volved in the seed dormancy caused by ectopic expression of
SUB1A.
To discern if SUB1A increases ABA responsiveness at the
gene expression level, we analyzed ABA-regulated genes that
are associated with drought tolerance by qRT-PCR (Figure 4).
Application of ABA decreased the levels of DREB1A, DREB1E,
and AP59 mRNAs in aerial tissue of LG and Ubi:SUB1A-3.
However, these three transcripts constitutively accumulated to
higher levels in the SUB1A overexpression line under mock
conditions, and the higher relative accumulation was maintained
at 5 and 50 mM ABA. AP37 was upregulated by ABA, with
significantly higher induction in the SUB1A overexpression line
under mock and ABA-treated conditions. These results demon-
strate that SUB1A coordinates transcription of other AP2/ERF
transcription factors that are key regulators of dehydration tol-
erance. Representative ABA-responsive genes that have been
well characterized, such asRAB16A, LEA3, LIP9, and SalT, were
analyzed by qRT-PCR (Figure 4). Interestingly, constitutive ex-
pression of SUB1A considerably elevated these ABA-inducible
transcripts under mock conditions and even further with ABA
treatment. These results reveal that SUB1A enhances respon-
siveness to ABA at the mRNA accumulation level, which is in
accordancewith ABA-mediated effects on seed germination and
shoot elongation (Figures 3C and 3D).
SUB1AModerates Leaf Dehydration Triggered
upon Desubmergence
Plants encounter cellular dehydration as a consequence of a
variety of environmental stress, such as drought, high salt, and
low temperature, all of which affect osmotic adjustment and
water balance. Interestingly, we noted that upper leaves of sub-
merged plants were rolled 1 h after desubmergence/reoxygena-
tion following 7 d of submergence (Figure 5A), indicating that
desubmerged plants encounter dehydration stress. Although
this duration of submergence is sublethal to both genotypes
(Fukao et al., 2006), it appeared that desubmerged M202 plants
416 The Plant Cell
were more severely wilted than M202(Sub1) plants. To evaluate
the contribution of SUB1A to leaf water management during the
early recovery period, RWCwasmonitored in the upper leaves of
M202 and M202(Sub1) plants after reoxygenation (Figure 5B).
Upon desubmergence, the RWC decreased rapidly in both
genotypes, but M202(Sub1) significantly moderated this water
loss. Thus, SUB1A functions to maintain leaf water content
following reoxygenation as observed under drought (Figure 1D).
Reoxygenation injury commonly occurs primarily due to the
overproduction of ROS upon sudden reexposure to atmospheric
Figure 3. SUB1A Increases ABA Responsiveness.
(A) and (B) Relative mRNA levels of SUB1 genes following ABA treatment. Developmentally matched plants 14-d-old M202 versus M202(Sub1) (A)
and 14-d-old LG versus 21-d-old Ubi:SUB1A-3 (B) were treated with mock (0.1% [v/v] DMSO) or ABA solution (5 or 50 mM in 0.1% [v/v] DMSO) for
24 h, and aerial tissue was analyzed by qRT-PCR (mock-treated M202 and LG = 1.0). Data represent mean 6 SE from three independent biological
replicates, and an asterisk indicates a significant difference between M202 versus M202(Sub1) or LG versus Ubi:SUB1A-3 (P < 0.05).
(C) and (D) ABA-inhibited seed germination and shoot elongation of LG and two Ubi:SUB1A lines. Seeds were pretreated with 100 mM fluridone at 48C
for 48 h and incubated in a series of ABA solutions (0, 10, 30, or 100 mM) for 4 d (C) or 8 d (D). Relative germination (C) and relative shoot elongation (D)
were calculated by comparison to the nontreated seeds of individual genotypes. Data represent mean 6 SD (n = 20) from three independent biological
replicates, and an asterisk indicates a significant difference between LG and Ubi:SUB1A lines (*P < 0.05; **P < 0.01).
(E) The effect of fluridone on seed germination of LG and Ubi:SUB1A-3. Seeds were incubated on wet filter paper containing 0, 5, 20, or 100 mM
fluridone for 0 to 6 d. Data represent mean 6 SD from three independent biological replicates.
SUB1A Enhances Dehydration Tolerance 417
oxygen (Bailey-Serres and Voesenek, 2008; Blokhina and
Fagerstedt, 2010). To investigate this further, we visualized the
accumulation of superoxide (O22) and hydrogen peroxide (H2O2)
using nitroblue tetrazolium (NBT) and 3,39-diaminobenzidine
tetrahydrochloride (DAB), respectively, in leaves of M202 and
M202(Sub1) immediately following desubmergence (Figure 5C).
Blue and brown coloration was pronounced in M202 leaves but
not in M202(Sub1) leaves. The nonquantitative assay indicates
that SUB1A limits both superoxide and hydrogen peroxide
accumulation that is stimulated by reoxygenation. Lipid perox-
idation, as ameasure of oxidative damage, was also quantified in
the two genotypes during submergence and reoxygenation
(Figure 5D). Submergence did not affect lipid peroxidation in
aerial tissue in either genotype. However, reoxygenation rapidly
increased lipid peroxidation in M202. Consistent with less su-
peroxide and hydrogen peroxide accumulation, lipid peroxida-
tion was significantly repressed in M202(Sub1).
To further understand the interplay between reoxygenation
and SUB1A, wemonitored the alteration inmRNA levels of SUB1
genes in aerial tissue of M202 and M202(Sub1) during reoxy-
genation (Figure 6A). Previously, we showed that all SUB1 genes
are highly induced by submergence, with diverse fold changes in
transcript accumulation (Fukao et al., 2006; Xu et al., 2006). Here,
we found that the elevation inSUB1AmRNAwas;900-fold after
7 d of submergence, whereas that of SUB1B and SUB1Cwas on
the order of 10- to 20-fold. Reoxygenation gradually decreased
accumulation of all three SUB1 mRNAs, but the level of SUB1A
transcript was still abundant in comparison to nonstressed
plants after 4 h of desubmergence (;300-fold). Transcripts of
genes associated with dehydration response/tolerance were
also analyzed in the transition from submergence to reoxygena-
tion (Figure 6B). ALCOHOL DEHYDROGENASE (ADH) genes are
stress responsive, induced by low oxygen, dehydration, and low
temperature (de Bruxelles et al., 1996). We again confirmed that
SUB1A enhances the elevation of ADH1 mRNA during submer-
gence (Fukao et al., 2006) and found that the transcript level
remained high during the time course of reoxygenation in M202
(Sub1). Submergence reduced the steady state levels of
RAB16A and LEA3mRNAs similarly in the two lines. By contrast,
reoxygenation significantly increased accumulation of these
transcripts, particularly in the M202(Sub1) genotype, consistent
with the genotype-specific differences in these mRNAs during
drought treatment (Figure 2).SalTmRNAwas notably induced by
submergence and further elevated after 1 h of reoxygenation,
with higher induction in M202(Sub1). After 2 h of reoxygenation,
the abundance of SalT mRNA decreased similarly in both geno-
types. These results demonstrate that SUB1A modulates accli-
mation responses associated with the dehydration stress that
results from desubmergence.
SUB1A Enhances Oxidative Stress Tolerance
The data presented above indicate that both drought and
reoxygenation trigger accumulation of ROS, which results in
cellular damage by oxidative stress (Figures 1E, 5C, and 5D). We
hypothesized that SUB1A may improve tolerance to oxidative
stress, leading to enhanced acclimation to drought and submer-
gence. To examine this, we incubated M202 and M202(Sub1)
seedlings with methyl viologen (paraquat), which stimulates
formation of ROS within chloroplasts (Figure 7A). Application of
methyl viologen dramatically repressed seedling growth in both
genotypes, but the reduction in shoot fresh weight was more
severe in M202 at all concentrations tested (Figure 7B). Methyl
viologen also reduced chlorophyll content, presumably as a
secondary consequence of oxidative stress (Figure 7C). Notably,
M202(Sub1) restricted the degradation of chlorophyll a and b, as
observed in submerged plants (Fukao et al., 2006). To confirm
that SUB1A moderates the breakdown of chlorophylls by oxi-
dative stress, leaf segments of M202 and M202(Sub1) were
treated with methyl viologen or hydrogen peroxide in the light
(see Supplemental Figures 5A and 5B online). Both chemical
treatments decreased the content of chlorophyll a and b in the
two genotypes, but to a significantly greater extent in M202.
Figure 4. SUB1A Increases ABA Responsiveness at the mRNA Accu-
mulation Level.
Developmentally matched plants 14-d-old LG and 21-d-old Ubi:SUB1A-3
were treated with mock (0.1% [v/v] DMSO) or ABA solution (5 or 50 mM
in 0.1% [v/v] DMSO) for 24 h, and aerial tissue was analyzed by qRT-PCR
(mock-treated LG = 1.0). Data represent mean 6 SE from three inde-
pendent biological replicates, and an asterisk indicates a significant
difference between LG and Ubi:SUB1A-3 (*P < 0.05; **P < 0.01).
418 The Plant Cell
These results confirm that SUB1A can diminish cellular oxidative
damage caused by excessive ROS production.
These observations led us to consider whether oxidative
stress might regulate levels of SUB1 gene transcripts. Indeed,
methyl viologen upregulated the accumulation of SUB1AmRNA
in M202(Sub1), whereas SUB1B and SUB1C mRNAs slightly
decreased in both lines (Figure 7D). The level ofSUB1A induction
was far less than that observed in response to submergence
(Figure 6A). Consistent with SUB1A-mediated repression, the
level of SUB1C transcript was significantly lower in M202(Sub1)
compared with M202.
The abundance of intercellular ROS is tightly regulated through
complex antioxidant systems in diverse subcellular compart-
ments. Among these ROS-scavenging pathways, ascorbate
peroxidase (APX), superoxide dismutase (SOD), and catalase
(CAT) are major enzymes that detoxify superoxide and hydrogen
peroxide under stressed conditions in plants (Mittler et al., 2004).
The rice (japonica) genome has eight annotated genes that
encode APX isoforms, two of which (APX1 and APX2) are
localized at the cytosol (Teixeira et al., 2006). Molecular analysis
of Arabidopsis apx knockout mutants revealed that cytosolic
APX plays a central role in regulation of the ROS scavenging
Figure 5. SUB1A Moderates Dehydration Stress That Is Induced upon Desubmergence.
(A) Photos of rice plants that encounter dehydration after reoxygenation. Fourteen-day-old plants were submerged for 7 d and reoxygenated for 1 h
under aerobic conditions under low light (50 mmol m�2 s�1). Bars = 5 cm.
(B) RWC of the youngest leaves of plants that were submerged and reoxygenated for 0 to 4 h. Data represent mean 6 SE (n = 8), and an asterisk
indicates a significant difference between M202 and M202(Sub1) (P < 0.01).
(C) Accumulation of superoxide anion and hydrogen peroxide in leaf blades upon desubmergence. Fourteen-day-old plants were completely
submerged for 7 d. Immediately following desubmergence, plants were treated with NBT to detect superoxide anion or DAB to detect hydrogen
peroxide in the dark for 3 and 24 h, respectively. Plants grown under aerobic conditions were used as a control. Bar = 2 cm.
(D) Lipid peroxidation of aerial tissue during and after submergence. Fourteen-day-old plants were submerged for 7 d and then reoxygenated for up to 4
h. Aerial tissue was analyzed to determine the level of MDA, an end product of lipid peroxidation. Data represent mean 6 SD from three independent
biological replicates, and an asterisk indicates a significant difference between M202 and M202(Sub1) (P < 0.05). FW, fresh weight.
SUB1A Enhances Dehydration Tolerance 419
systems, which modulates protection from high light and a
combination of drought and heat (Davletova et al., 2005a;
Koussevitzky et al., 2008). Overexpression of rice APX1 and
APX2 in Arabidopsis reduces the accumulation of hydrogen
peroxide, restricts chlorophyll degradation, and enhances sur-
vival under salinity stress (Lu et al., 2007).We determined that the
accumulation of both APX1 and APX2 mRNAs was upregulated
by methyl viologen treatment in M202 and M202(Sub1), but the
induction was more pronounced in the SUB1 genotype (Figure
8). Of the five SOD genes characterized in rice, mitochondrial
SodA1 (mitoochondrial Mn-SOD) and SodB (Fe-SOD) are sig-
nificantly induced by osmotic stress and severe low oxygen,
respectively (Kaminaka et al., 1999; Magneschi and Perata,
2009). We found that SodA1 mRNA was elevated by oxidative
stress, with stronger induction in M202(Sub1), whereas SodB
was not regulated by the stress (Figure 8). Three catalase genes
have been recognized in rice (Iwamoto et al., 2000). We found
that CatA and CatB were oxidative stress inducible and more
prominently induced in M202(Sub1), whereas CatC was down-
regulated by the stress in both genotypes. Based on these
directed transcript studies, in planta ROS detection, and lipid
peroxidation assays (Figures 1E, 5C, 5D, and 8), we suggest that
SUB1A augments the capability of antioxidant systems, thereby
improving tolerance to oxidative stress.
DISCUSSION
The results presented here demonstrate that SUB1A, the sub-
mergence tolerance regulator found in a limited number of rice
accessions, properly coordinates physiological and molecular
responses to cellular water deficit when it occurs independently
or following desubmergence. ABA is a crucial signaling molecule
that affects acclimation responses to both extremes in precip-
itation, drought, and submergence. Drought increases endoge-
nous ABA levels in aerial tissue, modulating leaf water balance
and the expression of genes associated with dehydration toler-
ance (Zhu, 2002; Seki et al., 2007). In the case of deepwater rice,
the decline in ABA augments GA-mediated internode elongation
and ethylene-dependent adventitious root formation during sub-
mergence (Hoffmann-Benning and Kende, 1992; Steffens et al.,
2006). Recently, it was reported that modulation of internal ABA
levels is a key determinant in the rate of underwater petiole
elongation in submergence-tolerant Rumex palustris ecotypes,
allowing escape of leaves from floodwaters (Chen et al., 2010). It
appears that submergence-induced ABA degradation is a pre-
requisite for escape of submergence through rapid shoot elon-
gation. Interestingly, exogenous application of ABA increases
survival of oxygen deprivation in maize (Zea mays) seedlings and
Arabidopsis roots (Hwang and Vantoai, 1991; Ellis et al., 1999).
Thus, it is plausible that ABA may also act as a positive regulator
for submergence tolerance, presumably through restriction of
energy-consuming development and metabolism.
We previously demonstrated that the rapid breakdown of ABA
occurs in a SUB1A-independent manner in aerial tissue of
submerged rice using the same genotypes evaluated in this
study (Fukao and Bailey-Serres, 2008). In accordance with this,
application of fluridone, an ABA biosynthesis inhibitor, did not
rescue the slow germination phenotype of SUB1A overexpres-
sion lines (Figure 3E; see Supplemental Figure 4 online). For
these reasons, it is unlikely that SUB1A regulates the production
or turnover of endogenous ABA. On the other hand, our data
show that SUB1A intensifies responsiveness to ABA, leading to
prevention of leaf water loss and additional induction of stress
responsive genes (Figures 1D, 3C, 3D, and 4).
Acclimation responses to drought are properly coordinated
through integrated regulatory networks that consist of ABA-
dependent and -independent pathways (Nakashima et al., 2009;
Hirayama and Shinozaki, 2010). Besides ABA-regulated genes,
SUB1A enhances mRNA accumulation of representative drought
tolerance regulators, DREB1A, DREB1E, and AP59, which are
Figure 6. SUB1A Further Increases mRNA Accumulation of Genes
Involved in Dehydration Tolerance after Desubmergence.
Relative mRNA levels of SUB1 genes (A) and genes associated with
drought response/tolerance (B). Fourteen-day-old plants were sub-
merged for 7 d and then reoxygenated for up to 4 h. Aerial tissue was
analyzed by qRT-PCR (M202 grown in air = 1.0). Data represent mean6
SE from three independent biological replicates, and an asterisk indicates
a significant difference between M202 and M202(Sub1) (*P < 0.05;
**P < 0.01).
420 The Plant Cell
not induced in response to ABA (Figures 2 and 4). This sug-
gests that SUB1A-mediated dehydration tolerance is mediated
through impacts on both ABA-dependent and -independent
pathways.
ABA and GA antagonistically regulate plant growth, develop-
ment, and stress responses. We previously reported that SUB1A
restricts GA responsiveness through increased accumulation of
the central suppressors for GA signaling, SLR1 and SLRL1,
during submergence (Fukao and Bailey-Serres, 2008). SLR1 is a
DELLA domain-containing protein in the GRAS transcription
factor family. Rice has only one DELLA protein (SLR1), whereas
Arabidopsis possesses five DELLAs that exhibit functional re-
dundancy (Ueguchi-Tanaka et al., 2007).Arabidopsis quadruple-
DELLA mutants display reduced responsiveness to ABA in the
GA-deficient ga1-3 backgrounds (Achard et al., 2006). In ad-
dition, comparative analysis of wild type, ga1-3, and ga1-3
quadruple-DELLA mutants demonstrated that DELLAs activate
expression of genes encoding antioxidant enzymes and diminish
ROS accumulation under high salinity in seedlings (Achard et al.,
2008). The demonstration that SUB1A-contanining genotypes
maintain higher levels of SLR1 and SLRL1 is consistent with their
increased ABA responsiveness and reduced ROS accumulation.
Figure 7. SUB1A Enhances Tolerance to Oxidative Stress.
(A) Photo of rice seedlings treated with an oxidative herbicide. Seven-day-old seedlings were transferred onto 0.53 MS media containing methyl
viologen (0, 1, 5, or 20 mM) and incubated for 14 d (16 h light/8 h dark; light level, 50 mmol m�2 s�1). Bars = 3 cm.
(B) Growth inhibition of aerial tissue by oxidative stress. Relative fresh weight was calculated by comparison to the nontreated seedlings of individual
genotypes. Data represent mean 6 SD (n = 12) from three independent biological replicates, and an asterisk indicates a significant difference between
M202 and M202(Sub1) (*P < 0.05; **P < 0.01).
(C) Chlorophyll (Chl) contents in aerial tissue of plants exposed to oxidative stress. Data represent mean 6 SD from three independent biological
replicates, and an asterisk indicates a significant difference between M202 and M202(Sub1) (*P < 0.05; **P < 0.01). FW, fresh weight.
(D) Relative mRNA levels of SUB1 genes in aerial tissue of plants treated with oxidative stress. Fourteen-day-old plants were exposed to methyl
viologen (0, 1, or 20 mM) for 24 h in the light (100 mmol m�2 s�1), and the aerial tissue was subjected to qRT-PCR analysis. Data represent mean6 SE
from three independent biological replicates, and an asterisk indicates a significant difference between M202 versus M202(Sub1) (*P < 0.05; **P <
0.01).
SUB1A Enhances Dehydration Tolerance 421
The reaeration of aerial tissue is a phase in the natural pro-
gression of a sublethal flooding event. We observed that upper
leaves of M202 and M202(Sub1) plants rapidly rolled and wilted
upon desubmergence (Figure 5A). Accompanying this, repre-
sentative dehydration-responsive genes were rapidly induced in
aerial tissue (Figure 6B). These observations are consistent with
visible symptoms of leaf water deficit observed in rice fields that
had experienced 10 d of complete submergence (Setter et al.,
2010), strongly implicating dehydration stress as a component
of a transient submergence event.
Paradoxically, the desiccation of leaves after desubmergence
occurs even though the soil typically contains sufficient water.
Physiological analysis of the submergence-intolerant rice culti-
var IR42 revealed that leaf water loss upon desubmergence is
restricted by stomatal closure, but water transport to leaf blades
was hampered by low hydraulic conductivity in the leaf sheaths
(Setter et al., 2010). We found that SUB1A moderates desicca-
tion of leaves following desubmergence (Figures 5A and 5B). One
possible explanation is that SUB1A manages water transport
from the root system through regulation of leaf solute potential.
Submergence dramatically promotes consumption of energy
reserves, but SUB1A restricts overall soluble carbohydrate con-
sumption in aerial tissue (Fukao et al., 2006). Preservation of
soluble carbohydrates may be of benefit for maintenance of a
water potential gradient as well as avoidance of carbohydrate
starvation. Future evaluation of metabolite dynamics should
shed additional light on the relevant processes associated with
water relations during submergence and desubmergence.
Both drought and submergence promote rapid accumulation
of ROS, which function as signaling molecules to trigger diverse
acclimation responses to the stresses (Blokhina and Fagerstedt,
2010; Miller et al., 2010). However, excessive accumulation of
ROS results in cellular damage by oxidative stress, mandating
the potential for ROS detoxification to enable survival of water
deficit and submergence. Indeed, several studies successfully
increased tolerance to dehydration through manipulation of
genes encoding ROS detoxification enzymes and their transcrip-
tional regulators in Arabidopsis, rice, and tobacco (Nicotiana
tabacum) (Badawi et al., 2004;Wang et al., 2005; Davletova et al.,
2005b; Mittler et al., 2006; Lu et al., 2007; Wu et al., 2008). In
addition to dehydration tolerance, the ability to manage ROS
detoxification can influence survival of a submergence event.
The specific activity of SOD is more significantly induced upon
reoxygenation in rhizomes of submergence-tolerant Iris pseuda-
corus than intolerant Iris germanica (Monk et al., 1987), which is
negatively correlated with lipid peroxidation (Blokhina et al.,
1999). Similarly, submergence-tolerant FR13A rice, the donor of
SUB1A in this study, restricts lipid peroxidation and chlorophyll
degradation following desubmergence compared with intolerant
IR42 (Ella et al., 2003). The data presented here and previous
analysis of microarray data reveal that SUB1A upregulates
transcripts of genes associated with antioxidant enzymes and
metabolites, thereby dampening oxidative damage during
drought and reoxygenation (Figures 1E, 5C, 5D, and 8) (Jung
et al., 2010; Mustroph et al., 2010). The future characterization of
the direct targets of SUB1A may expose the regulatory mech-
anisms underlying the relevant ROS detoxification network.
We propose a model for SUB1A-mediated responses to a
natural progression of abiotic stresses wrought by a flash-
flooding event (Figure 9). During submergence, SUB1A restricts
ethylene production and GA responsiveness, resulting in the
suppression of shoot elongation and carbohydrate consumption
(Fukao et al., 2006; Fukao and Bailey-Serres, 2008). This quies-
cence response enables the avoidance of carbohydrate starva-
tion, allowing plants to endure submergence for up to 2 weeks.
Maintenance of carbohydrate reserves during submergence
could also supports the rapid production of osmolytes during
desiccation and recommencement of leaf development upon
desubmergence. As floodwaters subside, reoxygenated aerial
tissue encounters an oxidative stress followed by a rapid cellular
dehydration. Under reoxygenation and drought, SUB1A restricts
ROS accumulation, which diminishes oxidative damage. The
induction of SUB1A leads to elevation of ERF mRNAs (e.g.,
DREB1s and AP59), which regulate genes associated with
adaptation to dehydration. Subsequently, the enhancement of
Figure 8. SUB1A Further Increases mRNA Levels of Genes Associated
with ROS Detoxification.
Fourteen-day-old plants were exposed to methyl viologen (0, 1, or 20
mM) for 24 h in the light (100 mmol m�2 s�1), and the aerial tissue was
subjected to qRT-PCR analysis. Data represent mean 6 SE from three
independent biological replicates, and an asterisk indicates a significant
difference between M202 versus M202(Sub1) (*P < 0.05; **P < 0.01).
422 The Plant Cell
ABA responsiveness by SUB1A triggers expression of LEA
mRNAs and dampens leaf water loss. Interestingly, SUB1A
mRNA levels rise in response to submergence, drought, oxida-
tive stress, and ethylene to varying levels (3.5- to 900-fold) but
are downregulated by ABA (Figure 3; Fukao et al., 2006; Fukao
and Bailey-Serres, 2008). ABA might serve as a negative regu-
lator of SUB1A mRNA accumulation to properly manage the
magnitude and duration of the diverse acclimation responses to
submergence, reoxygenation, and dehydration. Although ethyl-
ene upregulates SUB1A mRNA accumulation during submer-
gence, it is not known if ethylene or another signal is responsible
for elevation of this transcript in response to drought. The
relationship between ethylene production and dehydration
stress is species specific, and the role of ethylene in drought
response remains unclear in rice (Acharya and Assmann, 2009).
Further analysis of the SUB1A gene transcript and protein
regulation should aid the characterization of its role as a regulator
of tolerance to multiple abiotic stresses. Altogether, our results
show that SUB1A endows rice with the ability to overcome
prolonged submergence, oxidative damage, and rapid dehydra-
tion that accompanies desubmergence as well as water deficit
caused by drought.
Because submergence and dehydration occur in the natural
progression of flash flooding, it is feasible that tolerances to
extremes in precipitation coexist in other species inhabiting
floodplains and other flood-prone areas. In fact, most tropical
and subtropical floodplains periodically encounter a dry season
and the degree of tolerance to drought is a critical determinant for
species distributions (Miao et al., 2009; Parolin et al., 2010). To
cope with reoxygenation and subsequent water deficit, species
that display quiescence during complete submergence may be
better adapted to dehydration upon desubmergence. Further
analysis of SUB1A target genes and the downstream networks
will aid the elucidation of the molecular mechanisms underlying
water extreme tolerances that contribute to natural variation in
semiaquatic plant species.
METHODS
Plant Materials and Growth Conditions
Rice (Oryza sativa) cv M202, cv IR64, cv Samba Mahsuri, cv Liaogeng
(LG), the Sub1 introgression lines M202(Sub1) and IR64(Sub1), Samba
Mahsuri(Sub1). and SUB1A overexpression lines Ubi:SUB1A-1 and Ubi:
SUB1A-3 (background genotype LG) were analyzed in this study (Fukao
et al., 2006; Xu et al., 2006; Septiningsih et al., 2009). Rice plants were
grown under the conditions described by Fukao et al. (2006). Sterilized
seeds were placed on wet filter paper for 3 d at 258C in the light.
Germinated seeds were transplanted into soil-containing pots (W:L:H,
10 3 10 3 10 cm) and grown in a greenhouse (308C day, 208C night) for
14 d under natural light conditions.
Submergence, Drought, and Chemical Treatments
All submergence, drought, and chemical treatments were replicated in at
least three independent biological experiments. Submergence and ABA
Figure 9. Model of SUB1A-Mediated Response to the Natural Progression of Abiotic Stresses during a Flooding Event in Rice.
SUB1A expression inhibits the increased GA responsiveness during submergence through elevation of the GA signaling repressors, SLR1 and SLRL1.
Negative regulation of GA responses aids in avoidance of carbohydrate starvation, allowing plants to endure submergence. Upon reaeration, leaf
dehydration occurs. SUB1A-mediated enhancement of ROS amelioration limits oxidative damage and chlorophyll degradation during reoxygenation.
Increased ABA responsiveness by SUB1A induces expression of LEA mRNAs and suppresses leaf water loss. SUB1A also upregulates transcripts
encoding ERFs associated with acclimation to drought (e.g., DREB1s and AP59). The SUB1A-mediated responses to water deficit can independently
enhance recovery from submergence or provide protection during drought, both of which induce cellular dehydration.
SUB1A Enhances Dehydration Tolerance 423
treatments were performed following the methods of Fukao et al. (2006)
and Fukao and Bailey-Serres (2008), respectively. Briefly, 14-d-old plants
in soil-containing pots were completely submerged in a plastic tank (W:L:
H, 653 653 95 cm) filled with 90 cm of water in a greenhouse. Following
7 d of submergence, plants were desubmerged at noon on day 7 and
placed in air in the light (50 mmol m22 s21) for up to 4 h of reoxygenation.
The light levels were similar under submerged and recovery conditions.
For ABA treatment, aerial tissue of 14-d-old [M202, M202(Sub1), and LG]
or 21-d-old (Ubi:SUB1A-3) plants was excised at the base of the stemand
immediately placed into 20 mL of mock (0.1% [v/v] DMSO) or ABA
solution (5 or 50 mM in 0.1% DMSO) in a 250-mL glass beaker for 24 h in
the light (50mmol m22 s21). For oxidative stress treatment, aerial tissue of
14-d-old plants was incubated in water or methyl viologen solution (1 or
20 mM) for 24 h in the light (100 mmol m22 s21) as described above. For
osmotic stress treatment, sterilized seeds were grown on wet filter paper
in a Petri dish for 7 d at 258C in the light (100 mmol m22 s21). Seedlings
were placed onto filter paper containing 20% (w/v) polyethylene glycol-
8000 for up to 24 h in the light. For drought treatment, 40 plants (20 plants
per genotype) grown in a 2.5-liter pot for 14 d were exposed to dehydra-
tion stress by withholding water for up to 7 d in a greenhouse (308C day,
208C night). Aerial tissue was harvested at noon on the day specified.
After each treatment, sampled tissue was immediately frozen in liquid
nitrogen and stored in 2808C until use.
RNA Extraction and qRT-PCR
RNA extraction and cDNA synthesis were performed as described by
Fukao and Bailey-Serres (2008). qRT-PCR was performed in a 20-mL
reaction using iQ SYBR Green supermix (Bio-Rad) in a MyiQ real-time
PCR detection system (Bio-Rad). All primers were used at 0.5 mM. Melt-
curve analysis was performed at the end of PCR reactions to validate
amplification specificity. Amplification efficiency was verified following
the method of Schmittgen and Livak (2008). Relative transcript abun-
dance was calculated by the comparative CT method (Livak and
Schmittgen, 2001). ACTIN1 was used as a normalization control. Se-
quences of primer pairs are listed in Supplemental Table 1 online.
Detection and Quantification of ROS
Fourteen-day-old plants were submerged for 7 d as described by Fukao
et al. (2006). Plants grown under normal conditions were used as a
control. To visualize superoxide accumulation, aerial tissue of desub-
merged or nonsubmerged control plants was excised and immediately
placed in a 0.5 mg/mL NBT solution in 10 mM potassium phosphate
buffer, pH 7.6, at 258C for 3 h in the dark. For hydrogen peroxide
detection, excised aerial tissue was treated with 1 mg/mL DAB in 50 mM
Tris acetate buffer, pH 5.0, at 258C for 24 h in the dark. After staining, the
uppermost leaf of each plant was boiled in 95% (v/v) ethanol for 20min to
remove chlorophyll and rehydrated in 40% (v/v) glycerol for 16 h at 258C.
Each experiment was repeated on at least seven different plants, and
representative images are shown.
Lipid Peroxidation
Lipid peroxidation was analyzed with the thiobarbituric acid test, which
determines MDA as an end product of lipid peroxidation (Hodges et al.,
1999). Briefly, 50 mg of aerial tissue was homogenized in 1 mL of 80%
(v/v) ethanol on ice. Following centrifugation at 16,000g for 20 min at 48C,
the supernatant (0.5 mL) was mixed with 0.5 mL of 20% (w/v) trichloro-
acetic acid containing 0.65% (w/v) thiobarbituric acid. The mixture was
incubated at 958C for 30 min and then immediately cooled in an ice bath.
After centrifugation at 10,000g for 10 min, the absorbance of the super-
natant was measured at 532 nm, subtracting the value for nonspecific
absorption at 600 nm. The MDA concentration was calculated from the
extinction coefficient 155 mM21 cm21 (Hodges et al., 1999).
Oxidative Stress Tolerance Analysis
To evaluate whole plant tolerance to oxidative stress, dehulled seeds (25
seeds) were grown in Magenta vessels (Sigma-Aldrich; GA-7) contain-
ing 80 mL 0.53 Murashige and Skoog (MS) media at 238C for 7 d (16 h
light/8 h dark; light level of 50 mmol m22 s21). Seedlings were transferred
onto 0.53MSmedia includingmethyl viologen (Sigma-Aldrich) (0, 1, 5, or
20 mM) and incubated for 14 d under the conditions described above. To
examine oxidative tolerance in leaves, the fully expanded uppermost leaf
of 14-d-old plants was cut into pieces (L 3 W: 8 3 6 mm), and the leaf
segments were floated on a 1 mM methyl viologen or 10 mM H2O2 solu-
tion at 258C in the light (100 mmol m22 s21) for 40 or 24 h, respectively.
This experiment was repeated three times and representative images
are shown. Chlorophyll a and b contents of aerial tissue and leaf pieces
were quantified following the method of Fukao et al. (2006).
Drought Tolerance Analysis
Germinated seeds were transplanted into 2.5-liter soil-containing pots
and grown for 14 d as described above. Drought stress was applied by
withholding water for 8 d in a greenhouse (308C day, 208C night), and
recovery was initiated by placing pots in a shallow tray of water. Plant
viability was evaluated 14 d after initiation of recovery under normal
growth conditions. Plants were scored as viable if one ormore new leaves
appeared during the recovery period. Leaf RWCwasmonitored in the fully
expanded upper leaves (third leaf) of 14-d-old plants during treatments.
Each leaf blade was detached from a plant at noon, and the fresh weight
(FW) was immediately measured. After measurement, leaf blades were
floated on deionized water for 24 h, and rehydrated leaves were re-
weighed to determine turgid weight (TW). Finally, leaf blades were oven
dried at 658C for 3 d, and dry weight (DW) of each leaf was measured.
RWC was calculated using this equation: RWC (%) = (FW – DW)/(TW –
DW) 3 100. For the osmotic stress tolerance test, dehulled sterilized
seeds were grown on 0.53 MS medium equilibrated with polyethylene
glycol-8000 (final concentration: 15% [w/v]) at 238C (16 h light/8 h dark;
light level, 50 mmol m22 s21). After a 10-d incubation, the entire shoot and
root length was recorded.
ABA Response Analysis
Sterilized seeds were soaked in 100 mM fluridone dissolved in 0.1% (v/v)
ethanol for 48 h at 48C. Following the pretreatment, seedswere incubated
on wet filter paper with mock (0.1% [v/v] DMSO) or ABA solutions (10 to
100 mMof + and2ABA in 0.1% [v/v] DMSO) for 8 d at 258C in the light (50
mmol m22 s21). Seeds that formed a shoot or root ($1 mm) were scored
as germinated; shoot length was also recorded. All shoots remained
white for 8 d, indicating fluridone was effective during the entire exper-
imental period. For the fluridone sensitivity test, sterilized seeds were
placed on wet filter paper with mock (0.1% [v/v] ethanol) or fluridone
solutions (5 to 100 mM in 0.1% [v/v] ethanol) for 6 d at 258C in the light (50
mmol m22 s21). Seed germination was scored each day.
Microarray Data Analysis
Publicly available microarray data (Jain et al., 2007; Mustroph et al., 2010)
were reanalyzed by use of the R program (http://cran.at.r-project.org) and
Bioconductor package (http://www.bioconductor.org). Affymetrix CEL
files (accession numbers GSE18930 and GSE6901) were downloaded
from Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/gds). Raw
data were normalized by the GC robust multiarray average (GCRMA)
method. Differential expression analyses [pairwise comparisons of stress
424 The Plant Cell
treatment versus control or M202(Sub1) versus M202 during submer-
gence] were conducted to obtain the signal-to-log ratio by linear models
for microarray data (LIMMA) (Smyth, 2004). The Benjamini and Hochberg
method was applied to adjust P values for multiple experiments.
Accession Numbers
Sequence data from this article can be found in the Michigan State
University Rice Genome Annotation Project database (http://rice.
plantbiology.msu.edu) under the following accession numbers: Actin1
(LOC_Os03g50890), SUB1A (DQ011598b*), SUB1B (LOC_Os09g11480),
SUB1C (LOC_Os09g11460), DREB1A (LOC_Os09g35030), DREB1E
(LOC_Os04g48350), AP37 (LOC_Os01g58420), AP59 (LOC_Os02g43790),
RAB16A (LOC_Os11g26790), LEA3 (LOC_Os05g46480), LIP9 (LOC_
Os02g44870), SalT (LOC_Os01g24710), ADH1 (LOC_Os11g10480), APX1
(LOC_Os03g17690), APX2 (LOC_Os07g49400), SodA1 (LOC_
Os05g25850), SodB (LOC_Os06g05110), CatA (LOC_Os02g02400),
CatB (LOC_Os06g51150), CatC (LOC_Os03g03910). To obtain GenBank
sequences, which are linked to RAP identification numbers, please see
http://rapdb.dna.affrc.go.jp/tools/converter to convert from MSU identi-
fication numbers (LOC_Os00g00000) to Rice Annotation Project identi-
fication numbers (Os00g0000000). * GenBank/EMBL accession number.
Affymetrix CEL files (accession numbers GSE18930 and GSE6901) were
downloaded from Gene Expression Omnibus (http://www.ncbi.nlm.nih.
gov/gds).
Author Contributions
T.F. and J. B.-S. designed the research. T.F. and E.Y. performed the
research. T.F. and J.B.-S. analyzed the data, and T.F. and J.B.-S. wrote
the article.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. SUB1A Enhances Drought Tolerance in
Rice.
Supplemental Figure 2. SUB1A Increases Tolerance to Osmotic
Stress.
Supplemental Figure 3. A Subset of Submergence-Responsive
Genes Is Upregulated by Drought.
Supplemental Figure 4. Fluridone, an ABA Biosynthesis Inhibitor,
Does Not Affect Germination of Ubi:SUB1A-1 Seeds.
Supplemental Figure 5. SUB1A Moderates Chlorophyll Degradation
by Oxidative Stress.
Supplemental Table 1. Primers Used for Quantitative RT-PCR.
Supplemental Data Set 1. Gene Transcripts That Are Upregulated by
Submergence in M202 and Dehydration in IR64.
Supplemental Data Set 2. Gene Transcripts That Are Upregulated by
SUB1A during Submergence and by Dehydration.
ACKNOWLEDGMENTS
We thank Harkamal Walia (University of Nebraska, Lincoln) for valuable
comments and discussion. We also thank Dave Mackill (International
Rice Research Institute, Philippines) for the IR64, IR64(Sub1), Samba
Mahsuri, and Samba Mahsuri(Sub1) lines. This work was supported by a
research grant from the USDA Cooperative State Research, Education,
and Extension Service (2008-35100-042528) to J.B.-S.
Received October 8, 2010; revised December 1, 2010; accepted
December 19, 2010; published January 14, 2010.
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SUB1A Enhances Dehydration Tolerance 427
DOI 10.1105/tpc.110.080325; originally published online January 14, 2011; 2011;23;412-427Plant Cell
Takeshi Fukao, Elaine Yeung and Julia Bailey-SerresDrought Tolerance in Rice
The Submergence Tolerance Regulator SUB1A Mediates Crosstalk between Submergence and
This information is current as of June 16, 2020
Supplemental Data /content/suppl/2010/12/21/tpc.110.080325.DC1.html
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