RESEARCH ARTICLE

PLANT BIOLOGY

Coordinated Responses to Oxygen and

Sugar Deficiency Allow Rice Seedlings to
Tolerate Flooding
Kuo-Wei Lee,1,6 Peng-Wen Chen,2 Chung-An Lu,3 Shu Chen,4 Tuan-Hua David Ho,5 Su-May Yu6*
(Published 6 October 2009; Volume 2 Issue 91 ra61)

Flooding is a widespread natural disaster that leads to oxygen (O2) and energy deficiency in terrestrial
plants, thereby reducing their productivity. Rice is unusually tolerant to flooding, but the underlying
mechanism for this tolerance has remained elusive. Here, we show that protein kinase CIPK15 [calci-
neurin B–like (CBL)–interacting protein kinase] plays a key role in O2-deficiency tolerance in rice.
CIPK15 regulates the plant global energy and stress sensor SnRK1A (Snf1-related protein kinase 1)
and links O2-deficiency signals to the SnRK1-dependent sugar-sensing cascade to regulate sugar and
energy production and to enable rice growth under floodwater. Our studies contribute to understanding

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how rice grows under the conditions of O2 deficiency necessary for growing rice in irrigated lowlands.

INTRODUCTION
Partial or complete submergence in water restricts O2 diffusion to plants
and thereby inhibits their aerobic respiration. Rice (Oryza sativa) has
developed various adaptive mechanisms to survive periods of hypoxia
(limited O2) or anoxia (no O2). These include generating energy in the
absence of O2 through fermentative metabolism (1), developing specific
morphologies (such as aerenchyma in parenchymal tissues) that improve
access to O2 (2), activating gibberellin (GA)- and ethylene-promoted in-
ternode elongation to allow plants to extend above the water surface for
gas exchange (an adaptation found in deepwater rice) (3), and repressing
growth and conserving energy until floodwater recedes (an adaptation
found in lowland rice) (4). These abilities have enabled the global cul-
tivation of rice in a wide range of ecosystems, ranging from rain-fed and
irrigated lowlands to deep water (5).
Submergence-1 locus (Sub1), a major quantitative trait locus that
controls submergence tolerance in flood-prone lowland rice (6), contains
two or three genes with ethylene response factor (ERF) domains that are
transcriptionally induced by submergence (4, 7). Sub1B and Sub1C are
present in the Sub1 locus of many rice varieties, whereas Sub1A is lim-
ited to only a few varieties (7). Sub1A enables mature plants to tolerate
10 to 14 days of complete submergence by stimulating the expression of
the gene encoding alcohol dehydrogenase (Adh), an enzyme necessary
for fermentative metabolism, and repressing GA-mediated induction of
the genes encoding a-amylase and expansin, which are respectively
involved in starch degradation and cell elongation in leaves, thereby pre-
serving energy until floodwaters recede (4, 8). However, in some japonica
rice cultivars that lack Sub1A and whose mature plants are submergence in-
tolerant (7), such as Nipponbare, seed germination and coleoptile elongation
1
Graduate Institute of Life Sciences, National Defense Medical Center,
Neihu, Taipei 114, Taiwan, ROC. 2Institute of Agricultural Biotechnology,
National Chiayi University, Chiayi City 600, Taiwan, ROC. 3Department of
Life Sciences, National Central University, Jhongli City, Taoyuan County 320,
Taiwan, ROC. 4Taiwan Agricultural Research Institute, Wufeng, Taichung
413, Taiwan, ROC. 5Institute of Plant and Microbial Biology, Academia
Sinica, Nankang, Taipei 115, Taiwan, ROC. 6Institute of Molecular Biology,
Academia Sinica, Nankang, Taipei 115, Taiwan, ROC.
*To whom correspondence should be addressed. E-mail: sumay@imb.sinica.
edu.tw
proceed satisfactorily under hypoxic or anoxic conditions (9–11). This indi-
cates that these rice cultivars might use a Sub1A-independent mechanism to
cope with low-O2 stress during germination and coleoptile elongation.
Following imbibition (water adsorption) of rice seeds, sugars in the
embryo are rapidly consumed, leading to sugar depletion and activation
of a-amylase expression in the scutellum between the embryo and en-
dosperm (12, 13). Meanwhile, the embryo synthesizes GAs, which are
released to aleurone cells surrounding the starchy endosperm to activate
the synthesis and secretion of a-amylases and other hydrolases. These
enzymes digest starch and other nutrients stored in the endosperm to
small molecules that are taken up by scutellum and transported to the
embryo to support seedling growth (14). Rice carries out these processes
under conditions of O2 deficiency, thus obtaining fermentable carbohydrates
to provide adenosine 5′-triphosphate (ATP) to the germinating embryo,
whereas other cereal seeds fail to do so and, consequently, do not germi-
nate under comparable conditions (15–18). However, the mechanisms
through which a-amylases are produced in germinating rice seeds under
O2-deficient conditions are largely unknown.
The yeast sucrose nonfermenting 1 (Snf1) protein kinase and its mam-
malian and plant counterparts, adenosine monophosphate (AMP)–activated
protein kinase (AMPK) and Snf1-related protein kinase 1 (SnRK1), re-
spectively, are highly conserved metabolic sensors that monitor cellular
glucose or the ATP/AMP ratio to adjust energy homeostasis under condi-
tions of nutrient deficiency, stress, or both (19–21). An Arabidopsis SnRK1
homolog (KIN10) mediates both hypoxia and glucose starvation re-
sponses of two dark-induced genes (22). In humans, hypoxia-inducible
factor 1a (HIF-1a), which is essential for cancer cell survival under hy-
poxic conditions, is also activated by both sugar starvation and hypoxia
through an AMPK-dependent signaling pathway (23). These findings
suggest that conserved mechanisms regulate crosstalk between sugar
and O2 deficiency–signaling pathways in eukaryotes.
Our earlier studies showed that, in rice, sugar signaling regulates ex-
pression of the gene encoding a-amylase by controlling its transcription
rate and messenger RNA (mRNA) stability (24, 25). Transcriptional reg-
ulation is mediated through a sugar response complex (SRC) in a-amylase
promoters, in which the TA box (TATCCA) is the key regulatory element
(13, 26, 27). The transcription factor MYBS1 interacts with the TA box

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RESEARCH ARTICLE

and stimulates a-amylase promoter activity under conditions of sugar

starvation (28). Protein kinase SnRK1A is an essential upstream positive
regulator of MYBS1 in the response to sugar starvation and plays a key A WT
role in stimulating seed germination and seedling growth in rice (20). Air Water
Calcium signals mediate a multitude of plant responses to various
stimuli, including abiotic stresses, light, pathogens, and hormones, and reg-
ulate a wide range of physiological processes (29, 30). Ca2+ has also been
proposed to act as a physiological transducer of low O2 signaling in plants,
because transient increases in cytosolic Ca2+ concentration can be detected B dry 12 hours 1 day 2 days 3 days 4 days 5 days dry 12 hours 1 day 2 days 3 days 4 days 5 days

soon after flooding of maize roots or anoxic or hypoxic treatment of Ara- CIPK15
bidopsis (31, 32). Furthermore, Ca2+ signaling is required for the activation
of glycolytic enzymes and Adh in maize and Arabidopsis under conditions Amy3
of low O2 (32, 33). However, the pathway that links the sensing of O2 de-
Amy7
ficiency to production of Adh—and the identity of the key regulator(s) of mRNA
this pathway—remains elusive. Amy8
Calcineurin B–like (CBL) proteins are calcium-binding proteins that
interact with the CBL-interacting protein kinases (CIPKs) and propagate Adh2
plant Ca2+ signaling through a complex network (34). CIPKs are a group

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Tubulin
of plant-specific Ser-Thr protein kinases containing a conserved 24–amino
acid motif, called the NAF (Asn-Ala-Phe) domain, which is in the C- MYB S1
terminal nonkinase region and is sufficient and necessary for CIPK inter- Protein
Tubulin
action with CBL (35). The Arabidopsis CBL-CIPK network consists of 10
CBLs and 25 CIPKs (36) that act in combination with one another, and
different pair combinations have been found to participate in signaling C cipk15
pathways related to abiotic stresses, including salt, drought, osmotic, cold,
Air Water
and ABA (abscisic acid) (34, 36).
Here, we show that the protein kinase CIPK15 plays a critical role in the
response to O2 deficiency and acts through the SnRK1A-MYBS1–mediated
sugar signaling pathway to regulate carbohydrate catabolism and fermen-
tation, allowing rice to grow under floodwater.
D dry 12 hours 1 day 2 days 3 days 4 days 5 days dry 12 hours 1 day 2 days 3 days 4 days 5 days

RESULTS
CIPK15 integrates sugar- and O2-deficiency signaling
We observed an intriguing similarity in the sequences of the CIPK family
kinases to those of the Snf1-AMPK family upstream protein kinases (fig.
S1) and hypothesized that CIPKs might act upstream of SnRK1A and be
involved in integrating sugar- and O2-deficiency signaling. We screened
several rice CIPK mutants and found that the CIPK15 knockout mutant
(cipk15) of Nipponbare rice (fig. S2) was hypersensitive to the effect of
anaerobic germination under water. Shoots of the wild type elongated in
air and under water, but growth of true green leaves from coleoptiles and
of roots was delayed under water (Fig. 1A). Two allelic cipk15 knockout
mutants (fig. S2) showed the same hypersensitive phenotype, which was
only apparent in homozygous (–/–) plants; heterozygous (+/–) mutants
showed the wild-type phenotype.
Of eight commonly-studied rice a-amylase genes, aAmy3 is the most
prone to transcriptional activation by both sugar starvation (12, 13, 24) and
anoxia (9). In wild-type seedlings, the abundance of CIPK15, aAmy3, and
Adh2 mRNAs and MYBS1 protein increased under water (Fig. 1B). In air,
cipk15 growth was similar to that of wild-type seedlings, but under water,
cipk15 growth was severely arrested (Fig. 1C). In cipk15 seedlings, accu-
mulation of CIPK15 and aAmy3 mRNAs was abolished and that of Adh2
mRNA was reduced with germination in air, whereas accumulation of
all tested gene mRNAs was abolished and that of MYBS1 protein was
reduced with germination under water (Fig. 1D). The expression of two
other a-amylase genes, aAmy7 and aAmy8, which was similar in air and
under water in wild-type seedlings (Fig. 1B), was reduced in air and abol-
ished under water in cipk15 seedlings (Fig. 1D). The expression of aAmy7
CIPK15
Amy3
Amy7
mRNA
Amy8
Adh2
Tubulin
MYB S1
Protein
Tubulin
Fig. 1. CIPK15 controls seedling development and expression of enzymes
necessary for sugar production and fermentation in rice under water.
Seeds were germinated in air or under water. Thirty seeds were used
for each treatment at each time point. Germinated embryos with shoots,
roots, and endosperms excised were divided into two, with each half used
for extraction of total mRNA and proteins for RT-PCR and Western blot
analyses, respectively. (A) Wild-type (WT) shoots elongated in air or under
water. (B) The abundance of CIPK15, aAmy3, and Adh2 mRNAs and
MYBS1 protein increased in embryos germinated under water, whereas
that of aAmy7 and aAmy8 mRNAs remained constant. (C) cipk15-1 –/–
shoots and roots elongated normally in air but was arrested under water.
(D) In cipk15-1 –/– germinated embryos, aAmy3 mRNA was not apparent;
the abundance of aAmy7, aAmy8, and Adh2 mRNAs was reduced in air
and these mRNAs were not apparent and the abundance of MYBS1 pro-
tein was reduced under water.

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Fig. 2. CIPK15 integrates sugar A WT cipk15 C

Sucrose + – – – – 160 Effector:
and O2-deficiency signaling. (A) +

Luciferase activity (+ 102)

+ Suc 24.4X
Ubi-CIPK15
CIPK15 is necessary for the in- Time (hours) 24 48 24 48 140
– Suc
Lane 1 2 3 4 5 6 Ubi-SnRK1A
crease in SnRK1A and a-amylase 120
17.4X
in response to sugar starvation. CIPK15 100 Reporter:
–/–
WT and cipk15-1 suspension 80 SRC-Luc
10.4X
cells were cultured in medium SnRK1A 10.5X
60 7.8X
containing 90 mM sucrose for 7.8X
8.5X
6.5X
6X
48 hours, and then transferred to 40
3.5X
α Amy3 mRNA 20 1X 0.7X
3X 2.7X
medium without sucrose for 24 1.4X 1.4X

and 48 hours. Cells were col- 0

lected and total mRNA and pro- rRNA CIPK15 – + – + – + – +
SnRK1A – – + + – – + +
teins were extracted for RT-PCR
and Western blot analyses, re- WT cipk15
Tubulin
spectively. (B) CIPK15 is suffi- D
cient for rescuing SnRK1A and Nipponbare (C15ox) Tainung 67 (C15ox)
SnRK1A
a-amylase expression in cipk15. Input IP Input IP
Suspension cells of cipk15-1–/– α -Amylase Protein +S -S +S -S +S -S +S -S

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were transformed with constructs
SnRK1A
containing Ubi::GUS control (CK)
Tubulin
or Ubi::CIPK and cultured with or
without 90 mM sucrose for 24
hours. Total proteins were ex- B
CK CIPK15 CIPK11 CIPK21
tracted for Western blot analysis. + – + – + – + –
Sucrose
(C) CIPK15 and SnRK1A syner- Lane 1 2 3 4 5 6 7 8
gistically enhanced SRC activity SnRK1A
in transfected suspension cells
α−Amylases
[analysis of variance (ANOVA);
–/–
P < 0.01]. WT or cipk15-1 sus- Tubulin
pension cells were cotransfected
with effector and reporter constructs, incubated with (+) or without (−) sucrose Transformed rice suspension cells overexpresssing Ubi::HA-CIPK15 (C15ox)
(Suc), and luciferase activities were assayed. The luciferase activity in WT were cultured in medium with or without 90 mM sucrose for 24 hours. Total
embryos transfected with the reporter only and in the + Suc condition was proteins (Input) were extracted, and the HA-CIPK15-interacting proteins
assigned a value of 1×, and other values were calculated relative to this value. were precipitated with the antibody against HA (IP) and subjected to a
Error bars: SE from three repeats. (D) CIPK15 interacts with SnRK1A in vivo. Western blot analysis with the antibodies against SnRK1A.

and aAmy8 seems to participate in starch degradation in cipk15 in air.
These studies show that CIPK15 regulates a-amylase and Adh production
required for the sugar production and fermentation necessary for seedling
growth under water.
The kinase domain of CIPK15 is homologous to those of SnRK1A,
AMPKa1, and Snf1 and to those of the protein kinases upstream of
AMPKa1 and Snf1 (37, 38), but, of these, only CIPK15 contains the
NAF motif (figs. S3 and S4). To determine whether CIPK15 regulates
SnRK1A, we cultured rice cells in suspension under conditions of su-
crose starvation. Sugar starvation led to an increase in the abundance
of CIPK15 and aAmy3 mRNAs and of SnRK1A and a-amylase proteins
in wild-type but not in cipk15 cells (Fig. 2A). To investigate whether the
lost increase in SnRK1A and a-amylase abundance in cipk15 cells could
be rescued by the introduction of a CIPK15 and other CIPKs, cipk15
suspension cells were transformed with several different CIPKs under
the control of the maize ubiquitin (Ubi) promoter. CIPK11 is closely re-
lated to CIPK15, whereas CIPK21 is more distantly related (fig. S1).
The increase in SnRK1A and a-amylases in sucrose-starved cipk15 cells
was rescued by complementation with CIPK15 and to a lesser extent
with CIPK11, but not with CIPK21 (Fig. 2B).
We used a transient expression assay to delineate the relationship
between CIPK15 and SnRK1A in sugar signaling. In cipk15 cells, trans-
fection with plasmids encoding either CIPK15 or SnRK1A activated tran-
scription from the SRC derived from aAmy3 promoters (Fig. 2C). In both
wild-type and cipk15 cells, cotransfection with plasmids encoding CIPK15
and SnRK1A synergistically activated SRC compared with overexpression
of SnRK1A alone, regardless of sucrose availability (Fig. 2C).
To determine whether CIPK15 interacts with SnRK1A, we performed
coimmunoprecipitation (co-IP) experiments on stably transformed suspen-
sion cells of Nipponbare and Tainung 67 rice bearing influenza hemag-
glutinin (HA)–tagged CIPK15. The HA antibody-precipitated protein
complex containing CIPK15 reacted with SnRK1A antibodies only when
cells were sucrose starved (Fig. 2D). This study suggests that CIPK15 may
interact with SnRK1A in vivo. Together, the above studies show that CIPK15
and SnRK1A connect sensing of the O2-deficiency signal to the flooding
response of seedling growth through the sugar signaling cascade.
CIPK15 is required for growth of rice under
flooded conditions
We investigated the role of CIPK15 in seedling and mature plant growth
under submerged conditions further by germinating seeds in air or under
water with or without sucrose. In air, both wild-type and cipk15 seed-
lings grew normal shoots and roots regardless of sugar availability (Fig.
3A), although shoot elongation in cipk15 seedlings was slightly slower
than in wild-type seedlings (fig. S5A). In submerged seedlings, the re-
tarded shoot and root elongation of cipk15 was rescued by sucrose so that
seedling growth was comparable to that of wild-type seedlings (Fig. 3B
and fig. S5B).

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Fig. 3. CIPK15 controls sugar A C
Air
production necessary for under- WT cipk15
-Suc + Suc
water seedling growth, as well
as mature plant growth under
partially-flooded conditions. (A) Air

In air, neither WT nor cipk15 seed-

WT
ling growth required exogenous
–/–
sucrose. WT and cipk15-1
seeds were germinated in air by
placing on filter papers wetted Water

with water or 90 mM sucrose so-

lution. (B) With anaerobic germi-
nation, WT coleoptile elongation cipk15
did not require exogenous su-
crose but cipk15 coleoptile elon- Water
+ Suc
gation did. WT and cipk15-1–/–
Dry 12 1 2 3 4 5 6 Dry 12 1 2 3 4 5 6
seeds were germinated under hours day days days days days days hours day days days days days days

water or under 90 mM sucrose

B D

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solution. (C) Root growth was Water
80
impaired in WT and arrested in -Suc + Suc Flooded 3 cm above soil

Plant height (cm)

cipk15 ( )
cipk15 under water, but both 60 WT ( )
were rescued by exogenous su-
40
crose. WT and cipk15-1 –/– seeds WT
were germinated on the surface 20
of 5-cm-high 0.3% agarose gel
0
in a 500-ml glass bottle and in- 80
Flooded 10 cm above soil

Plant height (cm)

cubated at 28°C under a 16- WT ( )
hour light/8-hour dark condition 60
7d 8d
for 7 days. Seedlings were di- cipk15 40 cipk15 ( )
vided to three groups and kept
in air submerged in water or sub- 20

merged in 90 mM sucrose so- 0

Dry 12 1 2 3 4 5 6 Dry 12 1 2 3 4 5 6
lution for another 7 days. (D) hours day days days days days days hours day days days days days days
1 2 3 4 5 6 7 8 9 10
The growth rate of wild-type and cipk15 plants in soil was similar when flooded with 3 cm water, Weeks after flooding

but slower in cipk15 than in wild type when flooded with 10 cm water (ANOVA; P < 0.01). Error
E
bars: SE (n = 30). Experimental details are described in the legend of fig. S7. (E) Six weeks after
transplantation to paddy flooded with at least 5 to 10 cm of water, plant growth was impaired in
cipk15-1 –/–.

cipk15 WT

The ability of roots to grow and to deliver inorganic nutrients to
shoots is essential for plant survival (39); thus we paid particular attention
to the effect of the cipk15 mutation on root growth. Paddy rice is typically
grown in flooded soil to limit weed growth, forcing roots to grow under
highly O2-deficient conditions. Seedlings were grown in air for 7 days
and then maintained in air or submerged in water or sucrose solution.
Root growth was similar for wild-type and cipk15 seedlings grown in
air (Fig. 3C and fig. S6A). Shoot growth was also similar for wild-type
and cipk15 seedlings submerged under water, but primary and lateral root
growth was reduced for submerged wild type and arrested in submerged
cipk15 seedlings (Fig. 3C and fig. S6B). Exogenous sucrose increased
root growth of wild-type and cipk15 seedlings by 36 and 44%, respec-
tively (Fig. 3C and fig. S6C). For submerged seedlings, cipk15 roots were
on average 14 to 15% shorter than wild-type roots, regardless of sucrose
availability (fig. S6, compare B with C). These studies further indicate
that CIPK15 controls sugar production required for seedling growth un-
der water. Additionally, root growth is more sensitive than shoot growth
to O2 deficiency.
Nipponbare rice does not contain Sub1A and has thus been regarded
as submergence intolerant (7). To determine whether CIPK15 is essen-
tial for growth of Nipponbare plants under partial flooding (not fully
submerged) conditions, 2-week-old seedlings (13 cm tall) grown in pot
soil were flooded with water to a depth of either 3 cm or 10 cm above
the soil surface (fig. S7A). The growth of wild-type and cipk15 seed-
lings grown under 3 cm of water was similar, and both reached 60 cm
after 10 weeks (Fig. 3D and fig. S7B). When flooded to 10 cm, all wild-
type seedlings continued to grow but 30% of cipk15 seedlings died within
a few days. The growth of wild-type seedlings was rapid and plants
reached 70 cm within 10 weeks, whereas cipk15 seedlings grew slowly,
reaching only 40 cm by 10 weeks (Fig. 3D and fig. S7B). To determine

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RESEARCH ARTICLE
Fig. 4. CIPK15 confers hypoxia A D
Total amylase activity
tolerance on rice through the co-
WT cipk15
ordinated production of sugars Flooding
and Adh through the sugar star- DAI 1 2 3 4 1 2 3 4
vation signaling pathway. (A)
Sugar
CIPK15 increases both total am- CBL/Ca+2 starvation
ylase and a-amylase activity in 7
6 WT total amylases

Unit/mg proteins
germinated seeds under water.
Top, zymogram assay. Bottom, 5 WT -amylase CIPK15
quantitative activity assay. DAI, 4
cipk15 total amylases
days after imbibition. (B) The abun- 3
2 cipk15 -amylase
dance of Adh mRNA was in- SnRK1A
1
creased by sucrose and hypoxia,
0
and CIPK15 was necessary for 0 1 2 3 4
the hypoxia-dependent increase DAI MYBS1
in Adh expression. Seeds were
B
germinated in air for 3 days and WT cipk15
Promoter mRNA
then incubated in air or under wa- Sucrose + — + — + — + —

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ter with or without 90 mM sucrose Hypoxia — — + + — — + + SRE
for 24 hours. Germinated em- Lane 1 2 3 4 5 6 7 8
bryos with shoots, roots, and en- Amylases
dosperms excised were used cipk15
for mRNA extraction for RT-PCR Starch
analysis. (C) In transfected rice Adh1 (Seeds) Photosynthesis
embryos, sucrose (Suc) and hy- (Leaves)
poxia induced Adh2 promoter Adh2
activity. Rice embryos were trans- Sugars
fected with constructs indicated, Tubulin
incubated in the presence (+) or
absence (−) of sucrose (left panel) C Glycolysis
or in air or under hypoxic condi-
Suc Air
Relative luciferase activity

tions (right panel) for 24 hours. 7 + Suc

0.3X Hypoxia 4.2X
Proteins were extracted for lucif- 6 Adh1-Luc Pyruvate
Adh1-Luc Adh2-Luc
erase activity assays (ANOVA; 5 CaMV35S-Luc
Adh2-Luc
P < 0.01). X, fold induction. Error 4 Amy3-Luc Essential Alcohol
bars: SE. (D) Proposed role of 4.6X Energy
3 metabolites dehydrogenases
CIPK15 in flooding adaptation 2.3X 0.5X
2
in rice. SRE, sugar response el- 2.0X
1
ement. Dashed line indicates a Germination
possible pathway. 0
Adh1 Adh2 Amy3 Adh1 Adh2 CaMV35S Seedling & plant growth

whether CIPK15 is required for plant growth in fields, we transplanted 2- and in a CIPK15-dependent manner, because the induction was observed
week-old seedlings to irrigated paddies and showed that, after 6 weeks, in the wild-type but abolished in the cipk15 germinated embryos (Fig.
cipk15 plants were stunted relative to wild-type plants (Fig. 3E). These 4B, compare lane 2 with lane 4 and lane 6 with lane 8). The expression
studies indicate that CIPK15 is required for rice to grow in floodwater. of Adh seems also to be induced by sucrose (Fig. 4B, compare lane 1 with
lane 2 and lane 5 with lane 6); however, there were high background
CIPK15 connects the O2-deficiency response to sugar amounts of Adh mRNA in both wild-type and cipk15 air-germinated
and energy production embryos in the absence of exogenous sucrose (Fig. 4B, lanes 2 and
Next, we investigated the role of CIPK15 in controlling carbohydrate 6). This could be due to transport of sugars from the endosperm, result-
metabolism in germinated rice seeds. The mobilization of starchy en- ing in rise in soluble sugar concentrations in rice embryos 2 days after
dosperms of germinated cereal seeds into sugars requires a number of germination (12).
hydrolytic enzymes (40); however, a-amylase has been considered the To further determine whether transcriptional activity from the Adh
key enzyme capable of hydrolyzing native starch granules in endosperms promoter is up-regulated by sugar and hypoxia, we performed a rice embryo
(40, 41). In the present study, total amylase and a-amylase activities were transient expression assay. We found that both Adh1::Luc and Adh2::Luc
higher in wild-type than cipk15 germinated embryos (Fig. 4A), indicating were transcriptionally activated by sucrose and hypoxia in transfected
CIPK15 regulates all of these hydrolytic enzymes. wild-type embryos (Fig. 4C). Together, these data suggest that, in rice
In wild-type embryos germinated for 3 days, expression of CIPK15 seedlings, the production of sugars and induction of Adh under hypoxic
was induced by sucrose starvation regardless of O2 availability (Fig. 4B), conditions appear to be coordinately regulated by CIPK15. This is phys-
similar to that observed in cultured rice suspension cells (Fig. 2A). In iologically meaningful because soluble sugars are substrates of glycolysis,
contrast, the expression of two Adh genes was induced by hypoxia and pyruvate produced during glycolysis enables Adh to reoxidize NADH

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Table 1. Seedling flooding tolerance is independent of mature plant submergence tolerance in rice varieties.

Relative growth
rate (%)* Amylase Mature plant
Seedling flooding Sub1A
O. sativa cultivar Subspecies activity submergence
phenotype† allele§
Air Water Water + sucrose in seedling‡ phenotype§
M202 japonica 100 47 94 Tolerant High Intolerant Absent
Swarna indica 100 57 81 Tolerant High Intolerant Absent
Nipponbare japonica 100 48 65 Tolerant High Intolerant Absent
Goda Heenati indica 100 43 55 Tolerant High Tolerant Present
Haboganj aman indica 100 7 96 Intolerant Low Intolerant Absent
FR13A indica 100 8 43 Intolerant Low Tolerant Present
Kurkaruppan indica 100 10 42 Intolerant Low Tolerant Present
*
Rice seeds were germinated in air or under water with or without sucrose for 15 days, and the relative growth rate was determined as described in Materials and Meth-
ods. †Seedling flooding phenotype is represented by relative growth rate: tolerant, >40%; intolerant, <10%. ‡Amylase activity determined 4 days after germination in air
or under water. §Mature plant submergence phenotype and presence of Sub1A allele were reported in Xu et al. (7).

(reduced form of nicotinamide adenine dinucleotide) to produce ATP energy consumption. This phenomenon is even more evident in cipk15

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under conditions of reduced O2 (42, 43). Indeed, in maize and Arabi-
dopsis roots, production of key glycolytic enzymes and Adh are induced
by hypoxia, a process that is mediated by Ca2+ (32, 33). The current
work ties all these points together by showing that CIPK15 integrates
sugar- and O2-deficiency signaling processes relevant to germination
and seedling growth under floodwater and in mature plants grown under
partial flooding (Fig. 4D).
The shoots of some submergence-intolerant rice cultivars that lack
Sub1A, including Nipponbare (7), elongate vigorously under water (11, 44),
whereas shoots of some submergence-tolerant rice cultivars that contain
Sub1A (7) elongate well in air but poorly under water (11, 44). Here, we
compared the relative seedling growth rate under water of seven rice cul-
tivars containing or lacking Sub1A. We found that poor seedling develop-
ment under water correlated with lower amylase activities in germinated
embryos but not with Sub1A genotype (Table 1 and fig. S8). This result is
consistent with a similar study using different rice cultivars (44). The seven
rice cultivars used in our studies all contain genes that encode CIPK15 with
almost identical amino acid sequences, indicating that CIPK15 is essential
for survival or flooding tolerance or both. But genes downstream of CIPK15
might have changed through evolution, leading to reduced amylase produc-
tion and therefore reduced germination and seedling tolerance to flooding in
some rice cultivars.
DISCUSSION
Although much remains to be learned about the biochemical and molec-
ular basis of rice germination and seedling growth under conditions of O2
deficiency, the capacity of rice seedlings to produce amylases and Adh is
crucial to these processes. Sugars derived from starch hydrolysis are me-
tabolized through the glycolytic pathway to generate energy and other es-
sential metabolites (such as precursors for amino acids, nucleic acids, and
fatty acids) needed for biosynthesis and growth (Fig. 4D). Fermentation is
the principal pathway of anaerobic carbohydrate metabolism for the gen-
eration of ATP and essential metabolites in plants. Cultured rice cells have
similar ATP concentrations under anoxic and aerobic conditions (45). Con-
sequently, ATP could be initially produced in both wild-type and cipk15
germinated embryos. However, as O2 was depleted, ATP consumption
by wild-type and cipk15 embryos decreased because of slower seedling
growth under water than in air (fig. S9). This indicates that lower rates
of sugar production in hypoxic embryos could limit the supply of essential
metabolites and thereby constrain cell growth, which in turn would reduce
than wild-type seedlings under water.
The characterization of CIPK15 function contributes to our understand-
ing of the mechanisms of O2 deficiency tolerance and sugar sensing in
rice. The synergism between CIPK15 and SnRK1A in activation of the
aAmy3 SRC (Fig. 2C) suggests that CIPK15 may act in parallel or co-
operate with SnRK1A in the sugar starvation signaling pathway. And,
indeed, this possibility cannot be eliminated. However, several lines of evi-
dence suggest that, in rice, CIPK15 acts as the main upstream positive reg-
ulator of SnRK1. First, CIPK15 is necessary (Fig. 2A) and sufficient (Fig.
2B) for accumulation of SnRK1A in response to sugar starvation. Second,
SnRK1A is sufficient to complement the loss of CIPK15 in the cipk15
mutant for activation of aAmy3 SRC (Fig. 2C). Third, knocking down
SnRK1A in transgenic rice to barely detectable amounts virtually abolishes
MYBS1 and aAmy3 expression under conditions of sugar starvation (20).
This suggests that SnRK1A is necessary for increased MYBS1 abundance
and aAmy3 expression and that the direct role of CIPK15 in regulation of
MYBS1 and aAmy3 expression, if any, could be minor. Fourth, SnRK1A is
regulated at the posttranscriptional level (20), which we have now shown
to be regulated by CIPK15 (Fig. 2A). The possibility that CIPK15 may
phosphorylate and activate SnRK1A remains a focus of future studies.
It is still unclear how the O2 deficiency signal is transmitted to CIPK15.
CBL-CIPK pairing has been considered necessary for the transduction of
Ca2+ signals in response to various environmental stresses into regulation
of the expression of downstream genes (46–49). It remains to be deter-
mined which of the 10 CBLs encoded by the rice genome (36) interacts
with CIPK15 in relaying the O2-deficiency signal. It is noteworthy that
CIPK15 itself is transiently activated at the mRNA level by sugar or O2
deficiency (Figs. 1B and 2A), suggesting that other factors could regulate
the expression of CIPK15 in response to either of these two conditions.
Many terrestrial plants have evolved mechanisms to survive transient
hypoxic conditions that are generated by flooding. Transcript profiling by
microarray analyses of anoxic rice coleoptiles and Arabidopsis seedlings
revealed a common approach in their responses to anoxia, increasing the
expression of genes that may provide energy, such as those encoding pro-
teins involved in starch degradation, glycolysis, pyruvate metabolism, and
fermentation (10, 42, 50). However, the mechanism regulating flooding
tolerance in plants is complicated (51). It would be interesting to deter-
mine whether a CIPK15 homolog is present and regulates genes involved
in carbohydrate metabolism in flooding-intolerant plant species.
Here, we show that at least two genes control flooding tolerance in rice.
Sub1A- and CIPK15-based O2-deficiency tolerance complement each other
in protecting rice plants against flooding stress at different developmental

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RESEARCH ARTICLE
stages and under different growth conditions. As shown in Table 1, the
presence of Sub1A correlates with mature plant submergence tolerance,
which is primarily based on growth retardation to outlast the flooding situa-
tion. However, Sub1A does not appear to be related to flooding tolerance
at the germination and seedling stages, when the young plant needs to
quickly establish itself by reaching the surface of the floodwater. The role
of CIPK15 is to regulate a-amylase abundance for sugar production and
Adh abundance for energy production even under O2-limited conditions.
Therefore, the combination of Sub1A- and CIPK15-mediated processes
would provide rice plants two complementary strategies to survive partial
and complete flooding during their entire life cycle.
The solubility of O2 in water is low, with concentrations reduced from
~266 × 10-6 g cm–3 (8.31 mol m–3) in air to less than 8.26 × 10–6 g cm–3
(0.25 mol m–3) in water (52). When O2 concentration declines to = <5%,
plants are subject to O2-deficiency stress (53). Maize mutants lacking
functional Adh1, and thus the ability to carry out fermentation in roots,
succumb more quickly to anoxia than does wild-type maize (39). CIPK15
controls root growth and Adh expression, thereby apparently enabling
seedlings and autotrophic plants, which are capable of carrying out
phytosynthesis, to withstand partial flooding. CIPK15-mediated flooding
tolerance is important for underwater growth of seedlings; this ability
may be particularly critical for roots, which could be constantly
submerged. A similar situation applies to the roots of mature plants that
are partially flooded. These features allow rice to grow in irrigated low-
land systems practiced in nearly 80% of world rice production areas (5).
Rice varieties with higher seedling vigor (13) in standing water that re-
cedes over time are generally preferred for direct water seeding (44), a prac-
tice gradually replacing seedling transplantation in many tropical and
temperate rice-growing areas, because of faster and easier planting, reduced
labor, more efficient water use, and less methane emission (54). Coexpres-
sion of seedling and mature plant flooding tolerance traits exists naturally in
the Goda Heenati cultivar (Table 1), suggesting that both traits could be
incorporated in a breeding program to generate new rice varieties with
greater seedling vigor under floodwater, vigorous growth in irrigated low-
lands, and complete submergence tolerance in flood-prone areas. Similar
approaches might be applied to improve flooding tolerance in other crops.
MATERIALS AND METHODS
Plant materials and cell culture
Rice (O. sativa) cultivar Nipponbare and cultivar Tainung 67 were used in
this study. Rice suspension cell cultures were established as previously de-
scribed (55). Two independent Tos17-tagged Nipponbare mutant lines
(cipk15-1 and cipk15-2) were obtained from the Rice Tos17 Insertion Mu-
tant Library (http://pc7080.abr.affrc.go.jp/~miyao/pub/tos17/index.html.
en). The homozygote and heterozygote mutants and segregated wild-type
lines (fig. S2) were propagated for up to three generations (T3). Seeds of
six rice varieties (Oryza sp.) in addition to Nipponbare and Tainung 67
were obtained from the International Rice Research Institute (IRRI) Germ-
plasm Center (IRGC) (table S1).
Seed germination in air or under water
Rice seeds were sterilized with 1.5% (v/v) sodium hypochlorite and rinsed
with double-distilled water (ddH2O). For germination in air, seeds were
placed on 3M filter papers wetted with water in a 50-ml centrifuge tube.
For germination under water, seeds were placed in a 50-ml centrifuge tube,
autoclaved water was carefully poured into the tube to avoid any air bub-
bles, and tubes were sealed with lids. Seeds were germinated at 28°C under
16 hours light (4802 lux)/8 hours dark conditions. The O2 concentration
www.SCIENCESIGNALING.org
was 0.13 to 0.15 mol m-3 in nonautoclaved water and 0.09 to 0.1 mol m-3
in autoclaved water.
Primers
All primers are listed in table S2.
Semiquantitative RT-PCR
The same plant samples were used for both reverse transcription poly-
merase chain reaction (RT-PCR) and protein gel blot analyses. Total
RNA was extracted from 25 germinated embryos with the TRIZol kit
(Invitrogen). Single-stranded complementary DNA (cDNA) was synthe-
sized from 15 mg of total RNA with SuperScript II reverse transcriptase
(Invitrogen) according to the manufacturer’s instructions. The cDNA
mixture was diluted 10-fold with ddH2O to reduce the amount of cDNA
template for end-point RT-PCR. RT-PCR was performed in a 25-ml reac-
tion mixture containing 5 ml of diluted cDNA solution, 2.5 ml of 10× PCR
buffer, 0.1 mM primers, 0.2 mM deoxynucleotide triphosphate, 6% di-
methyl sulfoxide, and 1.25 U of Taq DNA polymerase (Qiagen). The num-
Downloaded from http://stke.sciencemag.org/ on May 19, 2015
ber of cycles with each primer pair for PCR amplification was adjusted to
be in the linear range.
Transient expression assays
Particle bombardment–mediated transient expression assays were per-
formed as described (13, 20). Rice suspension cells or embryos were co-
bombarded with reporter, effector, and internal control plasmids (Ubi::GUS)
at a ratio of 2:1:0.25. Bombarded suspension cells or embryos were incu-
bated in Murashige & Skoog (MS) medium containing 90 mM sucrose for
3 hours and divided into two halves, each incubated in MS medium con-
taining 90 mM sucrose or 90 mM mannitol. For hypoxia treatment, bom-
barded embryos were incubated in MS medium containing 90 mM sucrose
for 3 hours and divided into two halves, each incubated in air or under
hypoxic conditions (1% O2) with an oxygen-controlled system (Model
101, Prosperous Instrument Co., Ltd.) at 30°C for 24 hours. aAmy8::Luc
and CaMV35S::Luc were used as negative controls in the sucrose and
hypoxia treatment assays, respectively. GUS and luciferase activity as-
says were performed as described (20, 27). All bombardments were re-
peated at least three times.
Antibodies
SnRK1A-specific antibody was produced against synthetic peptides (5′-
SSLAQVTPAETPNSA-3′, amino acid residues 347 to 361) (56) derived
from SnRK1A (20). Polyclonal anti-MYBS1 antibodies against a syn-
thetic peptide (5′-VRRHYEALVEDVAAI-3′, amino acid residues 64 to
79) derived from MYBS1 (28) were raised in rabbits and purified by im-
mobilized peptide affinity chromatography (Biosource). Mouse monoclonal
antibody against a-tubulin (Sigma) and mouse monoclonal antibody–
conjugated beads against HA (AFC-101P, COVANCE) were purchased.
Western blot analysis
Western blot analysis was performed as described (20). Horseradish
peroxidase–conjugated antibody against rabbit immunoglobulin G
(Amersham Biosciences) was used as a secondary antibody. Protein sig-
nals were detected by chemiluminescence with ECL (Amersham Bio-
sciences). Tubulin protein detection was used as an international control.
Plasmids
pAHC18 contains the firefly luciferase (Luc) cDNA fused between the
maize Ubi promoter and the nopaline synthase gene (Nos) terminator (57).
pUGIII contains the Ubi::GUS (27). p3Luc.18 contains the SRC (–186 to
–82 relative to the transcription start site of aAmy3-CaMV35S minimal pro-
6 October 2009 Vol 2 Issue 91 ra61 7
RESEARCH ARTICLE
moter (–46 bp upstream of the transcription site)–Luc fusion gene (27).
pUSnRK1A contains the rice SnRK1A cDNA fused downstream of the
Ubi promoter (20). Coding regions of rice CIPK15, CIPK11, and CIPK21
(with sizes 1302, 1506, and 1491 bp, respectively) were PCR-amplified,
with primers on the basis of their identified genomic DNA sequences (36)
and fused to the Ubi promoter in the pSMY1H binary vector (58) for
rice transformation. There is no intron in the genomic DNA of these
CIPKs. Promoter regions of rice Adh1 and Adh2 (–1 to –1140 and –18 to
–1220 bp upstream from the translation initiation codon ATG, respective-
ly) were PCR-amplified with primers based on sequences in GenBank
(accession nos. AC123521 and AC123515, respectively), and fused to
the Luc cDNA in p35mAluc (27).
Rice stable transformation
Plasmid was introduced into Agrobacterium tumefaciens strain EHA101,
and rice transformation was performed as described (26).
Coimmunoprecipitation analysis
Transformed suspension cells of Nipponbare and Tainung 67 rice carrying
Ubi::HA-CIPK15 construct were cultured in the presence or absence of
sucrose for 24 hours, and total soluble proteins were extracted. In the
co-IP experiment for detection of the in vivo interaction between CIPK15
and SnRK1A, the protein extract containing HA-CIPK15 was immuno-
precipitated with antibody-conjugated beads against HA with slow rotation
at 4°C for 18 hours. After precipitation and washing four times with the
protein extraction buffer, the IP product was eluted three times with 0.1 M
glycine (pH 2.8). The IP product was then concentrated through a Micro-
con filter (YM-30, Millipore) and subjected to Western blot analysis with
antibodies against SnRK1A.
Amylolytic activity assay
Total soluble proteins (TSPs) were extracted from embryos and endo-
sperms as described (20). For in-gel zymogram assay, 10 mg of TSPs were
separated with 10% native polyacrylamide gel electrophoresis at 4°C.
Total amylolytic activity was assayed without heating of protein extracts.
a-Amylase (EC 3.2.1.1) specific activity was assayed by heating protein ex-
tracts containing 3 mM CaCl2 at 70°C for 15 min to inactivate b-amylase,
debranching enzyme, and a-glucosidase (40). Enzyme reactions were car-
ried out in an assay buffer [50 mM sodium acetate (pH 6.0) and 10 mM
CaCl2] containing 1% boiled soluble potato starch and incubated at 37°C
for 1 hour. The amylolytic activity was detected by staining the gel with
0.6% iodine in a potassium-iodine solution.
Liquid assay of amylolytic activities was determined by release of re-
ducing sugars from soluble starch by a modification of a method previous-
ly described (18). For total amylolytic activity, 100 mg of TSPs was assayed
in the same assay buffer described above but containing 2% boiled soluble
potato starch and incubated at 37°C for 1 hour. For a-amylase activity as-
say, 100 mg of TSPs was heat-treated (70°C for 15 min in the presence of 3
mM CaCl2) (40). An aliquot (100 ml) was taken from the assay mixture and
treated with 100 ml of reaction termination buffer (0.1 N NaOH) and 100 ml
of 3,5-dinitrosalycilic acid (DNS) solution (containing 40 mM DNS, 400
mM NaOH, and 1 M K-Na tartrate, heated at 50°C, and filtered through
filter paper) for 5 min at 100°C. After dilution with 600 ml of distilled
water, the absorbance at 540 nm (A540) was measured and reducing power
was evaluated with a standard curve obtained with glucose (0 to 20 mmol).
One enzymatic unit is defined as the amount of enzyme releasing 1 mmol
of glucose in 1 min.
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Determination of seedling growth rate
Heights of seedlings grown in the air or under water were measured daily
for up to 15 days. Slopes of growth curves were determined for each rice
variety with a linear regression equation (Microsoft, Office Excel 2003).
The relative growth rate was calculated by dividing the slope of seedlings
grown under water with that in the air and is presented as a percentage. Ten
seedlings per replicate and a total of three replicates were used for each rice
variety under each treatment.
ATP assay
Rice seeds were germinated in air or under water. Twenty-five isolated
embryos were grounded in liquid N2 and homogenized with 500 ml of
sterile distilled water. The crude extract was centrifuged at 13,000 rpm at
4°C for 30 min. An aliquot of the supernatant was used for luciferin-
luciferase assay with an ATP bioluminescent assay kit FL-AA (Sigma).
The amount of light produced was measured with a luminometer (Lumat
LB 9507, Berthold).
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SUPPLEMENTARY MATERIALS
www.sciencesignaling.org/cgi/content/full/2/91/ra61/DC1
Fig. S1. Dendrogram showing the evolutionary relationship of members of the rice CIPK
family and other Snf1-related protein kinases from rice, mammals and yeast.
Fig. S2. Genotyping identified two allelic Tos17-tagged CIPK homozygous (–/–) and het-
erozygous (+/–) Nipponbare rice mutants.
Fig. S3. Amino acid sequence of CIPK15 closely resembles that of the rice SnRK1A,
mammalian AMPKa1 and yeast Snf1 protein kinases.
Fig. S4. Amino acid sequence of CIPK15 resembles that of protein kinases upstream of
SNF1 and AMPK.
Fig. S5. Sucrose rescues underwater seedling shoot development in the cipk15 mutant.
Fig. S6. CIPK15 controls underwater root development.
Fig. S7. CIPK15 control mature plant growth under partially-flooded conditions.
Fig. S8. Seedling hypoxia tolerance varies among rice varieties.
Fig. S9. ATP is consumed more slowly in WT and cipk15 embryos germinated under wa-
ter than in air.
Table S1. The IRRI Germplasm Center (IRGC) accession number of rice germplasms
used in the present study.
Table S2. Primers used in the present study.
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Submitted 19 March 2009
Accepted 17 September 2009
Final Publication 6 October 2009
10.1126/scisignal.2000333
Citation: K.-W. Lee, P.-W. Chen, C.-A. Lu, S. Chen, T.-H. D. Ho, S.-M. Yu, Coordinated
responses to hypoxia and sugar deficiency through the CIPK15-dependent signaling
pathway allows rice seedlings to tolerate flooding. Sci. Signal. 2, ra61 (2009).
6 October 2009 Vol 2 Issue 91 ra61 9
 Coordinated Responses to Oxygen and Sugar Deficiency Allow
Rice Seedlings to Tolerate Flooding
Kuo-Wei Lee, Peng-Wen Chen, Chung-An Lu, Shu Chen,
Tuan-Hua David Ho and Su-May Yu (October 6, 2009)
Science Signaling 2 (91), ra61. [doi: 10.1126/scisignal.2000333]

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