Regulatory interplay of the Sub1A and CIPK15 pathways in the regulation of α-amylase production in flooded rice plants.

Abstract

Rice (Oryza sativa L.) can successfully germinate and grow even when flooded. Rice varieties possessing the submergence 1A (Sub1A) gene display a distinct flooding-tolerant phenotype, associated with lower carbohydrate consumption and restriction of the fast-elongation phenotype typical of flooding-intolerant rice varieties. Calcineurin B-like interacting protein kinase 15 (CIPK15) was recently indicated as a key regulator of α-amylases under oxygen deprivation, linked to both rice germination and flooding tolerance in adult plants. It is still unknown whether the Sub1A- and CIPK15-mediated pathways act as complementary processes for rice survival under O(2) deprivation. In adult plants Sub1A and CIPK15 may perhaps play an antagonistic role in terms of carbohydrate consumption, with Sub1A acting as a starch degradation repressor and CIPK15 as an activator. In this study, we analysed sugar metabolism in the stem of rice plants under water submergence by selecting cultivars with different traits associated with flooding survival. The relation between the Sub1A and the CIPK15 pathways was investigated. The results show that under O(2) deprivation, the CIPK15 pathway is repressed in the tolerant, Sub1A-containing, FR13A variety. CIPK15 is likely to play a role in the up-regulation of Ramy3D in flooding-intolerant rice varieties that display fast elongation under flooding and that do not possess Sub1A.

Full text

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RESEARCH PAPER
Regulatory interplay of the Sub1A and CIPK15 pathways in the
regulation of a-amylase production in flooded rice plants
N. P. Kudahettige1, C. Pucciariello2, S. Parlanti1, A. Alpi1& P. Perata2
1 Department of Crop Plant Biology, University of Pisa, Pisa, Italy
2 Plantlab, Scuola Superiore Sant’Anna, Pisa, Italy
INTRODUCTION
Flooding is a widespread environmental constraint that has a
dramatic effect on the growth and yield of agronomically
important crops not adapted to a submerged environment
(Perata & Voesenek 2007). It can severely affect food avail-
ability in several regions of South and South-East Asia that
are prone to water submersion during the rainy season. In
the context of crops, all cereals apart from rice (Oryza sativa
L.) are flooding intolerant. The rice plant originates from a
semi-aquatic environment and has evolved specialised func-
tions to adapt to different types of flooding event, either
deepwater or flash (Nagai et al. 2010). Two opposite strate-
gies involving unique adaptations were recently classified as
low oxygen quiescence syndrome (LOQS), where the rice
shoot does not elongate upon submergence but re-grows after
de-submergence, and low oxygen escape syndrome (LOES),
characterised by rapid shoot extension under flooding to
reach the water surface (Bailey-Serres & Voesenek 2008;
Colmer & Voesenek 2009).
The restriction of oxygen (O2) availability due to partial or
complete water submergence inhibits aerobic respiration of
plants, thus leading to an energy deficit (Perata & Alpi 1993).
Rice is characterised by a broad range of metabolic and mor-
phological adaptations to flooding and can successfully ger-
minate and grow even when O2 supply is limited (Alpi &
Beevers 1983; Perata & Alpi 1993). Rice can also germinate
in the absence of O2, thanks to the successful induction of
the enzymes needed to degrade the starch reserves present in
the endosperm (Perata et al. 1992, 1996; Guglielminetti et al.
1995a). Of the enzymes required for starch degradation,
a-amylases are required in order to initiate degradation of
starch granules (Dunn 1974; Sun & Henson 1991). In rice, a-
amylases are encoded by a large multigene family (Huang
et al. 1990),andwhilethe
Ramy1A-encoded isoform plays a major role during aerobic
rice germination, the Ramy3D-encoded isoform is predomi-
nant during anaerobic germination (Loreti et al. 2003a;
Lasanthi-Kudahettige et al. 2007). Ramy3D is not regulated
by GA, whose synthesis requires O2, but is induced by sugar
starvation that is a consequence of the fast anaerobic use of
soluble sugars (Loreti et al. 2003a,b). Ramy3D is therefore
transcriptionally regulated by sugar signaling, through a sugar
response complex (SRC) situated on the promoter region
and activated by the MYBS1 transcription factor under sugar
starvation (Lu et al. 1998; Yu 1999). The calcineurin B-like
interacting protein kinase 15 (CIPK15) has been proposed as
a key regulator of the sensing cascade for successful rice ger-
mination under flooding (Lee et al. 2009). CIPK15 seems to
act through positive regulation of the sucrose non-fermenting
1 related protein kinase (SnRK1A) and MYBS1 to control a-
amylase abundance under O2 deprivation (Lu et al. 2002,
2007; Lee et al. 2009). It has been suggested that the CIPK15
effect goes beyond the seedling stage, thus playing a positive
role in growth of the mature rice plant under partial flooding
(Lee et al. 2009).
gibberellin(GA)-induced
Keywords
a-Amylase; CIPK15; FR13A; Oryza sativa;
Sub1A.
Correspondence
Pierdomenico Perata, Plant Lab, Scuola
Superiore Sant’Anna, Piazza Martiri della
Liberta‘ 33, 56127 Pisa, Italy.
E-mail: p.perata@sssup.it
Editor
T. Elzenga
Received: 10 May 2010; Accepted:
14 October 2010
doi:10.1111/j.1438-8677.2010.00415.x
ABSTRACT
Rice (Oryza sativa L.) can successfully germinate and grow even when flooded. Rice
varieties possessing the submergence 1A (Sub1A) gene display a distinct flooding-
tolerant phenotype, associated with lower carbohydrate consumption and restriction
of the fast-elongation phenotype typical of flooding-intolerant rice varieties. Calci-
neurin B-like interacting protein kinase 15 (CIPK15) was recently indicated as a key
regulator of a-amylases under oxygen deprivation, linked to both rice germination
and flooding tolerance in adult plants. It is still unknown whether the Sub1A- and
CIPK15-mediated pathways act as complementary processes for rice survival under
O2deprivation. In adult plants Sub1A and CIPK15 may perhaps play an antagonis-
tic role in terms of carbohydrate consumption, with Sub1A acting as a starch degra-
dation repressor and CIPK15 as an activator. In this study, we analysed sugar
metabolism in the stem of rice plants under water submergence by selecting culti-
vars with different traits associated with flooding survival. The relation between the
Sub1A and the CIPK15 pathways was investigated. The results show that under O2
deprivation, the CIPK15 pathway is repressed in the tolerant, Sub1A-containing,
FR13A variety. CIPK15 is likely to play a role in the up-regulation of Ramy3D in
flooding-intolerant rice varieties that display fast elongation under flooding and
that do not possess Sub1A.
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However, the survival of established rice plants when sub-
merged is believed to be largely dependent on the presence
of the submergence 1A (Sub1A) ethylene-responsive factor
(ERF) gene, which is limited to a subset of indica varieties
(Xu et al. 2006). Submergence tolerance is in fact strongly
correlated with the presence of the Sub1A-1 allele (Xu et al.
2006), which activates ethanolic fermentation and negatively
regulates carbohydrate catabolism,
dependent starch degradation (Fukao et al. 2006). The
flooding induction of Sub1A results in a quiescence status
that prevents rapid elongation of the submerged plants and
depletion of carbohydrate reserves, thus enabling a fast
recovery after de-submergence (Bailey-Serres & Voesenek
2008).
The physiology of germinating seedlings and established
seedlings is different during complete submergence (Xu et al.
2006; Lee et al. 2009; Magneschi & Perata 2009). Sub1A- and
CIPK15-mediated pathways could complement each other at
different developmental stages and under different growth
conditions (Lee et al. 2009). However, at present it is
unknown whether the Sub1A- and CIPK15-mediated path-
ways act as complementary processes for rice survival under
O2 deprivation. Sub1A is unlikely to play a role during
anoxic germination (Magneschi & Perata 2009) while the
CIPK15 pathway appears to be essential to this growth stage.
In adult plants Sub1A and CIPK15 may perhaps play an
antagonistic role in terms of carbohydrate consumption, with
Sub1A acting as a starch degradation repressor and CIPK15
as an activator.
In this study, we analysed sugar metabolism in stems of
rice plants under water submergence by selecting cultivars
with different traits associated with flooding survival. The
relation between the Sub1A and the CIPK15 pathways was
investigated. The results show that under O2deprivation, the
CIPK15 pathway is repressed in the tolerant Sub1A-contain-
ing FR13A variety (Ellis & Setter 1999; Xu et al. 2006). On
the other hand, CIPK15 is likely to play a role in the up-
regulation of Ramy3D in flooding-intolerant rice varieties
that display fast elongation under flooding and do not pos-
sess Sub1A.
including
a-amylase-
MATERIALS AND METHODS
Plant material and submergence treatment
Seeds of O. sativa varieties FR13A, IR22, Nipponbare,
Lamone and Arborio Precoce (AP) were obtained from our
institute’s farm. Seeds were soaked on sterile water wetted
filter paper in Petri dishes, at 28 ± 2 ?C in dark conditions
for 3–4 days. Pre-germinated healthy seedlings were then
transferred to plastic pots filled with soil and peat (1:1) and
kept in growth chambers for 14 days at 26 ± 2 ?C with a 15-
h photoperiod (light intensity: 50 lmolÆm)2Æs)1). Fourteen-
day-old seedlings (V4 stage, Counce et al. 2000) of all five
varieties were subjected to complete submergence in distilled
water, as described in the legend to Fig. 1.
In the experiment specifically carried out using the FR13A
and AP varieties, the seedlings were germinated as described
above and grown in a sand medium with the following com-
plete nutrient solution: 4.5 mm (CaNO3)2, 0.8 mm MgSO4,
2.6 mm KH2PO4, 9.0 mm KNO3and 0.2 mm K2SO4. Seven-
day-old seedlings (two leaf stage) were subjected to complete
submergence, as described in the legend to Fig. 3.
For the experimentwith an
de-hulled AP and FR13A seeds were sterilised in 70% ethanol
external sugarsource,
A
B
Fig. 1. Growth behaviour of rice varieties under complete submergence
stress. Fourteen-day-old rice seedlings were grown in pots filled with soil
and peat (approximately 10-cm deep) and completely submerged in water,
1 m above the soil surface. Rice plants were grown in a growth chamber
for 17 days after flooding, at 26 ± 2 ?C in a photoperiod of 15-h light. A:
Plant phenotype of rice varieties grown in air and after 14 days of com-
plete submergence. B: Shoot elongation of rice varieties under complete
submergence (empty circles) and after 1 and 3 days of recovery after
de-submergence (filled circles R1and R3). Stem elongation and leaf elon-
gation were taken from the base of the plant to the upper collar and from
there to the edge of the longest leaf, respectively. Data are expressed as
the mean of 30 measurements ± SD.
Regulatory interplay of the Sub1A and CIPK15 pathways
Kudahettige, Pucciariello, Parlanti, Alpi & Perata
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for 1 min, followed by 6% sodium hypochlorite for 15–
20 min. Seeds of each variety were then placed on half-
strength Murashige and Skoog medium, containing 0.8%
agar, in 5-l glass bottles at 26 ± 2 ?C and a 15-h photoperiod
(light intensity: 50 lmolÆm)2Æs)1). The glass bottles and
media where sterilised before use. Seven-day-old seedlings
(V2 stage, Counce et al. 2000) were subjected to complete
submergence, using sterile 50 mm sucrose or 50 mm manni-
tol solutions for 14 days, followed by 7 days of recovery after
de-submergence. The O2 concentration in the media was
measured after 0, 10 and 14 days of submergence with a dis-
solved oxygen meter (Jenway 9071, Jenway, Staffordshire,
UK). Solution contamination was screened on bacterial nutri-
ent agar (at 35 ?C for 24 h) by inoculating plates with media
used to submerge the rice plants.
Screening for Sub1A gene presence
Genomic DNA of FR13A and AP stems was prepared using
GenElute? Plant Genomic DNA Miniprep kit (Sigma-
Aldrich, St Louis, MO, USA), following the manufacturer’s
protocol. The PCR reaction mixture was prepared in 20 ll
total volume using Red Taq Master mix (Invitrogen, Carls-
bad, CA, USA), 0.25 um primers and 100 ng genomic DNA.
PCR was performed using Sub1A genomic specific primers in
accordance with Xu et al. (2006) (Table S1).
RNA extraction, cDNA synthesis and real-time reverse
transcription PCR
Total RNA was extracted from rice stems, subjected to DNase
treatment and reverse-transcribed to produce cDNA, as
previously described (Lasanthi-Kudahettige et al. 2007). Tran-
script abundance was analysed by real-time reverse transcrip-
tion PCR, using TaqMan probes (Table S2) and qPCR
MasterMix Plus for SYBR?green I (Eurogentec, Lie `ge,
Belgium) with specifically designed primers (Table S3). The
relative expression level of each gene was quantified as previ-
ously described (Lasanthi-Kudahettige et al. 2007), using
glyceraldehyde-3-phosphate dehydrogenase (Os08g03290) as
an internal reference.
Semi-quantitative RT-PCR analysis
One microgram of RNA, treated as previously indicated, was
reverse-transcribed using SuperScript?III Reverse Transcrip-
tase (Invitrogen), according to the manufacturer’s protocol.
RT-PCR was performed using GoTaq?Green Master mix
(Promega, Madison, WI, USA) in a reaction volume of 50 ll,
containing 0.2 lm forward and reverse primers and 100 ng
cDNA template. The sequence and conditions of RAmy 3C,
3D, 3E and actin1 (used as control) were as described in
Fukao et al. (2006), RAmy 1A was designed as described in
Hwang et al. (1999) (Table S4). The resulting PCR products
were visualised with ethidium bromide staining on 2% aga-
rose gels after electrophoresis.
Assay of a-amylase activity
a-Amylase activity was determined in rice stems using the
amylopectin azure method, as described in Magneschi et al.
(2009). Total protein content was measured using the Biorad
Protein Assay based with the Bradford (1976) method.
Extraction and determination of soluble sugars and starch
Samples (0.1–0.3 g fresh weight) were extracted as described
by Tobias et al. (1992). Analyses of sucrose, fructose and glu-
cose were carried out as previously described (Guglielminetti
et al. 1995b). The starch-containing pellet was extracted using
10% KOH, the neutralised supernatant was treated with
2.5 units amyloglucosidase (from Rhizopus niger) for 3 h to
release glucose. Starch was quantified on the basis of the glu-
cose units released after amyloglucosidase treatment (Magne-
schi et al. 2009).
RESULTS
Screening of rice cultivars for sensitivity to complete
submergence stress
We analysed the flooding response of five rice varieties
(FR13A, IR22, Lamone, Nipponbare and AP) known to pos-
sess different ability to survive flooding or to elongate when
submerged (Gibbs et al. 2000; Xu et al. 2006; Magneschi et
al. 2009). AP had an unhealthy appearance after 14 days of
complete submergence, and the plants collapsed when
de-submerged. IR22, Lamone and Nipponbare did not
survive beyond 10 days of submergence. Only FR13A, a
Sub1A-harbouring variety (Xu et al. 2006), survived 14 days
of submergence (Fig. 1A). When submerged, AP showed the
highest elongation rates, whereas FR13A had the lowest
(Fig. 1B). Even though IR22 displayed limited shoot elonga-
tion under flooding, comparable to FR13A, it did not survive
flooding, thus suggesting that reduced shoot elongation does
not necessarily lead to submergence tolerance. According to
Gibbs et al. (2000), IR22 possesses insufficient glycolysis
substrate supply to survive anoxia.
The expression of fermentation-related genes was analysed
in rice stems. FR13A had the highest relative expression levels
of alcohol dehydrogenase 2 (ADH2) and pyruvate decarboxy-
lase 1 (PDC1), reaching a maximum after 3 days of flooding
(Fig. 2). All the other varieties failed to accumulate these
transcripts during submergence (Fig. 2). Lamone and AP, but
not FR13A, displayed ADH1 transcript accumulation (Fig. 2).
AP had the highest SUS1 expression after 3 days of complete
submergence, followed by Lamone and Nipponbare after
7 days of flooding (Fig. 2). The expression of SUS1 was
negligible in FR13A and IR22 (Fig. 2).
AP and FR13A had opposite responses to submergence, in
terms of survival, elongation rates and gene expression. These
two varieties were therefore chosen for further analyses.
Differences in shoot elongation were already visible after
3 days of submergence (Fig. 3A). The expression of Sub1A-1
increased rapidly in submerged FR13A seedling stems but
was not expressed in AP (Fig. 3B). PCR analysis of genomic
DNA indicated that this gene is absent in AP (data not
shown). In agreement with the known effects of Sub1A-1
(Fukao et al. 2006; Xu et al. 2006), FR13A showed signifi-
cantly higher PDC1 and ADH2 mRNA accumulation than
AP, while the ADH1 transcript accumulated in both stems of
varieties during submergence (Fig. 3B). Differences in the
Kudahettige, Pucciariello, Parlanti, Alpi & Perata
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expression of ADH genes under O2deprivation are of interest
and suggest that tissue-specific expression of ADH genes
might play a role in the metabolic adaptation of rice plants
to submergence (Xie & Wu 1989).
Surviving complete submergence requires available
carbohydrates
Carbohydrate availability might affect rice ability to survive
submergence. To verify this possibility we submerged FR13A
and AP for 14 days in either mannitol (osmotic control) or
sucrose. Recovery of plants was tested 7 days after de-sub-
mergence. AP submerged in a mannitol solution showed a
high rate of shoot elongation, chlorotic leaves and high mor-
tality (more than 75% of seedlings; Fig. 4). Of the AP seed-
lings, 20% had stunted growth with a low probability of
producing healthy plants after recovery, and only 5% of these
seedlings survived (Fig. 4). FR13A seedlings, on the other
hand, were able to survive when submerged in mannitol
(100% seedling survival; Fig. 4). When seedlings were sub-
merged in sucrose solution, both FR13A and AP seedlings
had less chlorosis, higher stem rigidity and reduced leaf
death. Interestingly, AP also showed 100% survival when sub-
merged in sucrose (Fig. 4), and resumed normal growth after
de-submergence. No differences in elongation due to the car-
bohydrate treatment were noticed for AP (stem length:
22.6 ± 2.34 and 21.27 ± 2.30 cm, n = 20, for sucrose and
Fig. 2. Gene expression patterns of transcripts involved in fermentation
and carbohydrate catabolism in the rice variety stems under complete sub-
mergence stress (empty circles) and after 1 day of recovery after de-sub-
mergence (filled circles R1). The expression level was measured on the
basis that Nipponbare control air = 1. Data are the mean of three repli-
cates ± SD.
A
B
Fig. 3. FR13A and AP under complete submergence stress. Seven-day-old
rice seedlings were grown in pots filled with sand (approximately 5-cm
deep) and completely submerged in water, 1 m above the sand surface.
Rice plants were grown in a growth chamber for 5 days after flooding, at
26 ± 2 ?C in a photoperiod of 15-h light. A: Shoot elongation of rice varie-
ties under complete submergence. Stem elongation and leaf elongation
were taken from the base of the plant to the upper collar and from there
to the edge of the longest leaf, respectively. Data are the means of 30
measurements ± SD. B: FR13A and AP stem gene expression patterns of
Sub1A transcripts and genes involved in fermentation grown in air and in
complete submergence stress. The expression level was measured based
on AP control air at day 0 = 1. Data are the mean of three replicates ± SD.
Regulatory interplay of the Sub1A and CIPK15 pathways
Kudahettige, Pucciariello, Parlanti, Alpi & Perata
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mannitol, respectively). FR13A displayed 100% survival in
both sucrose and mannitol, but showed a significant differ-
ence in elongation between the two treatments (stem length:
12.48 ± 1.28 and 6.90 ± 0.38 cm, n = 20, for sucrose and
mannitol, respectively).
Carbohydrate metabolism in submerged rice plants
The metabolism of carbohydrates is strongly affected by the
presence or absence of the Sub1A gene (Fukao et al. 2006).
AP showed constitutively higher total soluble sugars (TTS)
when compared with FR13A (Fig. 5A). When submerged,
however, the level of TTS dropped significantly only in AP
A
B
Fig. 5. Sugar availability in FR13A and AP stems. A: Total soluble sugars
and starch concentration in stems of rice varieties grown in air and after
3 days of submergence stress. The starch content is expressed as glucose
equivalents. Data are the means of three replicates ± SD. B: a-Amylase
activity in stems of rice varieties grown in air and after 3 days of submer-
gence stress. Data are the means of three replicates ± SD. Different letters
indicate significant differences between treatments (0.05 significance level)
based on an LSD multiple pair-wise comparison test.
Fig. 4. FR13A and AP after 0, 7 and 14 days of complete submergence
(sub) followed by 7 days of recovery (rec), by providing an external sugar
source. Seedlings were grown in in vitro conditions and subjected to com-
plete submersion in 50 mM sucrose or 50 mM mannitol (osmotic control).
The pie charts indicate the viability of plants after 7 days of recovery
(n = 20). Survival criteria correspond to re-growth capability after a recov-
ery period.
Kudahettige, Pucciariello, Parlanti, Alpi & Perata
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(Fig. 5A). Starch content was unaltered in submerged FR13A,
while a slight reduction was observed in AP (Fig. 5A). In this
context, a-amylase activity increased notably in AP during
submergence (Fig. 5A).
In rice, a-amylases are encoded by a multigene family
(Huang et al. 1990). We investigated the expression pattern
of a-amylase genes in rice stems from aerobic and sub-
merged plants. Expression of the RAmy1A gene was unaf-
fected by submergence (data not shown), while an altered
pattern of genes in the RAmy3 sub-family was observed.
Expression of RAmy3B was undetectable in FR13A, while
the low mRNA level of this gene already present in AP
under air was slightly induced in submerged AP at 18:00 h
(Fig. 6). RAmy3C was always expressed in aerobic samples,
but its expression declined under submergence, with the
exception of AP (18:00 h; Fig. 6). RAmy3D displayed clear
submergence-dependent expression (Fig. 6) in both AP and
FR13A, although its induction was higher in AP. RAmy3E
expression was slightly induced in submerged AP, whereas
FR13A did not show any increase in RAmy3E mRNA level
(Fig. 6).
RAmy3D is regulated by a complex, sugar starvation-
dependent pathway (Lu et al. 2002, 2007; Lee et al. 2009).
We therefore analysed the consequences of a starvation-
inducing treatment (submergence in mannitol), compared
with submergence in a sucrose solution, which prevents the
submerged seedlings from starving. Sucrose supply in the
water is likely to increase the CO2 concentration due to
increased respiration and, as a consequence, the submergence
water O2 level might rise due to increased photosynthesis
(Setter et al. 1989; Waters et al. 1989). To test this possibility,
we measured the O2 concentration in the mannitol and
sucrose solutions in which the plants were submerged. We
did not find any significant difference between the treat-
ments, but there was a reduction in the O2 concentration
from 7.5 ± 0.42and 7.7 ± 0.35 mgÆl)1
4.2 ± 0.78 and 4.1 ± 0.82 on 14 day (n = 3), for sucrose and
mannitol,respectively. Sub1A
FR13A was high in mannitol-submerged plants, however its
induction was abolished in the sucrose-submerged plants
(Fig. 7). The Sub1A gene was absent from AP. ADH2 was
induced by submergence in both varieties, although the
response was higher in FR13A, however sucrose reduced its
induction (Fig. 7). Notably, while submergence in mannitol
elicited a marked induction of genes involved in the starva-
on0 day, to
expression insubmerged
tion-dependent RAmy3D pathway (CIPK15, MYBS1) in AP,
this was not observed in FR13A (Fig. 7). Our analysis of
genomic DNA indicated that CIPK15 and MYBS1 are present
in FR13A (data not shown). SnRK1A expression was only
slightly induced by submergence, both in mannitol and
sucrose, in AP but not in FR13A (Fig. 7). Submergence in
sucrose prevented or strongly reduced the induction of the
CIPK15–MYBS1–RAmy3D signalling cascade in AP.
DISCUSSION
The molecular mechanisms that allow rice to germinate, grow
and survive when submerged have remained elusive until
recently, when the discovery of Sub1A, SNORKEL1⁄SNOR-
KEL2 and CIPK15 genes revealed the molecular physiology of
submerged rice (Xu et al. 2006; Hattori et al. 2009; Lee et al.
2009). Different rice groups activate distinct mechanisms to
survive flooding. The indica group of rice comprises several
cultivated lowland rice varieties that overcome submergence
stress by minimal shoot elongation and a drastic reduction in
most metabolic activities, thus activating a ‘quiescence’ strat-
egy. Sub1A-1 has been identified as the key determinant of
submergence tolerance based on the ‘quiescence’ strategy.
This is achieved by controlling the accumulation of tran-
scripts involved in ethanolic fermentation and repression of
genes associated with the carbohydrate catabolism (for a
review see Perata & Voesenek 2007). Rice plants belonging to
the japonica group do not possess the Sub1A-1 and fail to
survive prolonged submergence. Deepwater rice varieties, on
the other hand, are characterised by the ‘escape’ strategy,
based on rapid internode elongation promoted via GA during
flooding (Voesenek & Bailey-Serres 2009), regulated by the
two ERF genes SNORKEL1 and SNORKEL2 (Hattori et al.
2009).
Rice seed germination and early seedling growth under
hypoxia rely instead on the ability to induce a-amylase under
low O2 conditions, thus allowing starch degradation and
sugar availability in submerged seedlings (Perata et al. 1993).
The CIPK15-dependent pathway was recently indentified as a
key regulator of a-amylase abundance under limited O2(Lee
et al. 2009). It is largely unknown whether Sub1A- and
CIPK15-based tolerance mechanisms can co-exist. This would
be illogical, since Sub1A-1 actually represses amylase activity,
while CIPK15 activates the flooding-dependent Ramy3D
gene.
Fig. 6. Differential expression of AP and FR13A rice
stem a-amylase genes under complete submergence
stress analysed using RT-PCR at 08:00 h and 18:00 h,
in air and after 3 days of complete submergence.
Actin1 was used as internal control. Data are from a
representative experiment.
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We analysed the responses of two rice varieties that
differed in their ability to survive submergence because of
presence or absence of the Sub1A gene. FR13A is a submer-
gence-tolerant variety displaying the typical Sub1A response
(limited elongation when submerged). AP, on the other
hand, was selected because of its fast-elongating phenotype
under submergence in the absence of Sub1A (Fig. 1A and B).
When submerged, FR13A showed the highest accumulation
of ADH2 and PDC1 transcripts (Fig. 2). Both ADH and PDC
play important roles in plant adaptation to low O2conditions
(Agarwal & Grover 2006). The high ADH1 expression
observed in Lamone and AP, on the other hand, did not
correlate with survival.
Long-term submersion may cause extensive carbohydrate
consumption leading to energy starvation (Jackson & Ram
2003). The amount of carbohydrate reserves has been posi-
tively correlated with the level of submergence tolerance
(Setter & Laureles 1996; Greenway & Gibbs 2003; Jackson &
Ram 2003; Das et al. 2005). Previous results on submer-
gence-induced rapid internode elongation in deep-water rice
showed the mobilisation of starch from internodes accompa-
nied by enhancement of amylolytic activity (Raskin & Kende
1984).
Although AP had a higher abundance of soluble sugars
when compared to FR13A (Fig. 5A), a significant reduction
in TSS together with a reduction in starch content was
observed after submergence only in AP. A drop in starch and
carbohydrate content was observed by Fukao et al. (2006) in
leaves of the intolerant line M202 when compared to the tol-
erant M202 (Sub1A). The reduced a-amylase and sucrose
synthase (SUS) mRNA accumulation suggests that the preser-
vation of carbohydrate content in Sub1A-containing varieties
is related to the lower activity of starch and sucrose degrad-
ing enzymes (Fukao et al. 2006).
We showed that tolerance to submergence was greatly
enhanced by exogenous sucrose, even in a submergence-
intolerant variety such as AP (Fig. 4). Sucrose improved the
elongation of FR13A, bypassing the suppressive role played
by the presence of the Sub1A gene. Interestingly, while
FR13A displayed clear induction of both Sub1A and ADH2
when submerged in mannitol, a reduction of these mRNAs
was observed when sucrose solution was used to submerge
the seedlings (Fig. 7). These results suggest that there might
be an interaction between the Sub1A–dependent pathway and
sugar signalling.
Sub1A-1 down-regulates a-amylases (Fukao et al. 2006)
and it is also known that regulation of the major anaerobic
amylase (RAmy3D) is independent of hormonal control
(Mitsui & Itoh 1997) but relies on a starvation-sensing mech-
anism (Loreti et al. 2003a). We showed that in FR13A,
Ramy3D is repressed by a mechanism that acts upstream of
CIPK15 and MYBS1, which are both positive regulators of
RAmy3D (Lee et al. 2009). In the case of SnRK1A expression,
a component upstream of MYBS1, we did not observe any
differencesbetweenthe treatments
expected, since the regulation of SnRK1A is at a post-tran-
scriptional level (Lu et al. 2007). The MYBS1 transcription
factor is known to mediate sugar regulation of a-amylase
gene expression (Lu et al. 2002). It has high transactivation
ability to activate TATCCA containing a cis-element, present
in the RAmy3D promoter in two tandem repeats (Lu et al.
1998). Although it would be logical to speculate on the
absence of a starvation signal in FR13A, which, thanks to
Sub1A, retains an adequate level of carbohydrates and pre-
vents activation of the CIPK15⁄MYBS1⁄Ramy3D cascade, this
hypothesis is not entirely satisfactory. In AP, in fact, CIPK15,
SnRK1A, MYBS1 and RAmy3D are expressed, although at
very low levels, even in the sucrose-submerged seedlings
(Fig. 7).
We conclude that sucrose availability modulates the
expression of Sub1A and of its downstream gene ADH2, also
leading to a high elongation rate in FR13A plants when sub-
mergence is not associated with sugar starvation. In addition,
RAmy3D is up-regulated by starvation in AP but not in
(Fig. 7). This was
Fig. 7. Analysis of transcript levels of Sub1A, ADH2, CIPK15, SNRK1,
MYBS1 and RAmy3D genes in stems of both FR13A and AP grown in air
and after 3 days of submergence, with sucrose 50 mM or mannitol
50 mM. The expression level was measured based on AP control air at day
0 = 1. Data are the mean of three replicates ± SD.
Kudahettige, Pucciariello, Parlanti, Alpi & Perata
Regulatory interplay of the Sub1A and CIPK15 pathways
Plant Biology 13 (2011) 611–619 ª 2010 German Botanical Society and The Royal Botanical Society of the Netherlands
617

Page 8

FR13A, suggesting cross-talk between the Sub1A and CIPK15
pathways (Fig. 8). Further work is required to understand
how sugar sensing and signalling interferes with Sub1A-1,
and the possible negative interplay between the Sub1A-1 and
CIPK15 pathways.
ACKNOWLEDGEMENTS
This work was supported by Scuola Superiore Sant’Anna. N.
P. Kudahettige was supported by a PhD fellowship (Biomo-
lecular Sciences, University of Pisa).
SUPPORTING INFORMATION
Additional Supporting Information may be found in the
online version of this article:
Table S1. Sequences of primers used for Sub1A gene pres-
ence screening.
Table S2. Sequences of TaqMan primers and probes used
for quantitative (real-time) PCR analysis.
Table S3. Sequences of SYBR green primers used for quan-
titative (real-time) PCR analysis.
Table S4. Sequences of primers used for semi-quantitative
RT-PCR analysis.
Please note: Wiley-Blackwell are not responsible for the
content or functionality of any supporting materials supplied
by the authors. Any queries (other than missing material)
should be directed to the corresponding author for the article.
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