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 serres@ucr.edu.The 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(serres@ucr.edu).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

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