Received: 28 August 2021 | Revised: 23 November 2021 | Accepted: 9 December 2021

DOI: 10.1111/pce.14250

REVIEW

Finding a breather for Oryza sativa: Understanding hormone

signalling pathways involved in rice plants to submergence
stress

Lavanya Mittal | Sumaira Tayyeba | Alok K. Sinha

National Institute of Plant Genome Research,

New Delhi, India Abstract
During the course of evolution, different ecotypes of rice (Oryza sativa L.) have
Correspondence
Alok K. Sinha, National Institute of Plant evolved distinct strategies to cope with submergence stress. Such contrasting re-
Genome Research, Aruna Asaf Ali Rd, sponses are mediated by plant hormones that are principle regulators of growth,
New Delhi 110067, India.
Email: alok@nipgr.ac.in development and responses to various biotic and abiotic stresses. These hormones
act cooperatively and show extensive crosstalk which is mediated by key regulatory
genes that serve as nodes of molecular communication. The presence or absence of
such genes leads to significant changes in hormone signalling pathways and hence,
governs the type of response that the plant will exhibit. As flooding is one of the
leading causes of crop loss across all the major rice‐producing countries, it is crucial
to deeply understand the molecular nexus governing the response to submergence
to produce flood resilient varieties. This review focuses on the hormonal signalling
pathways that mediate two contrasting responses of the rice plant to submergence
stress namely, rapid internode elongation to escape flood waters and quiescence
response that enables the plant to survive under complete submergence. The sig-
nificance of several key genes such as Sub1A‐1, SLR1, SD1 and SK1/SK2, in defining
the ultimate response to submergence has also been discussed.

KEYWORDS
deepwater rice, eongation response, hormone signalling, lowland rice, quiescence response,
signalling crosstalk, SK1/SK2, SUB1A1, submergence

1 | INTRODUCTION productivity of 0.5–0.8 t h−1 as compared to 3–6 t h−1 in irrigated

Rice is the major food crop for more than half of the world's po-
pulation. 92% of the world's rice is grown in the Asian continent
(Gnanamanickam, 2009). However, monsoon floods that cause
complete submergence of plants are one of the major causes of
crop failure in rice cultivation. South‐ and South‐East Asian
countries are frequently affected by floods every year due to
heavy monsoon rainfall. Among them, India and China, the world's
largest producers of rice, experience floods most frequently. 30%
of Indian rice lands are prone to flash flooding with an average
areas (Bhowmick et al., 2014).
Submergence refers to the condition where the entire aerial part
of the plant is completely submerged underwater. During sub-
mergence, plants experience low oxygen conditions due to impeded
diffusion of gases in water. This is accompanied by a decline in
photosynthesis rates owing to low light penetration (Winkel
et al., 2013). One of the main reasons why rice plants succumb
to submergence is due to exhaustion of carbohydrate reserves
and severe starvation in low light and low oxygen conditions
(Das et al., 2005; Jackson et al., 1987).

Plant Cell Environ. 2022;45:279–295. wileyonlinelibrary.com/journal/pce © 2021 John Wiley & Sons Ltd. | 279
280 | MITTAL ET AL.

Rice has two major ecotypes based on flooding regimes, namely,
rainfed lowland rice which experiences shallow submergence or
partial submergence, and deepwater rice that experiences medium to
deep submergence during the monsoon season. Among these, the
deepwater rice exhibits the potential of rapid internode elongation
upon submergence so that the upper leaves rise above the water
level. In contrast, certain landraces of lowland rice (e.g., FR13A) show
flooding tolerance up to 2 weeks by entering into an energy con-
servation mode (Sarkar et al., 2001; Setter et al., 1987).
Even though the deepwater and the lowland rice have the ability
to survive floods, these landraces are not resistant to all kinds of
flooding regimes (Figure 1). When the water level of the flood is too
high, in spite of all the efforts to elongate, the plants are not able to
emerge above the water level, resulting in the death of plants due to
exhaustion of energy reserves used up in growth (Jackson
et al., 1987). Similarly, lowland rice is only able to tolerate complete
submergence for up to 2 weeks and succumbs to death in case of
prolonged flooding (Figure 1). Therefore, developing varieties toler-
ant to a wide range of flooding regimes would protect the farmers
from crop losses in flood‐prone areas where the amount of rainfall
during monsoons is unpredictable. The preliminary step for devel-
oping such varieties is to gain a deep understanding of the molecular
mechanisms underlying the complex adaptive responses of rice plants
to submergence stress.
Hormones are the central regulators of growth, development and
responses of plants to changing environments. Signalling pathways of
various hormones show crosstalk. Therefore, even a slight change in
hormone levels leads to a change in the expression of thousands of
genes. During the past three decades, studies on the mechanisms of
submergence tolerance in rice have revealed that hormones are the
master regulators of responses of rice plants to submergence stress.
This paper reviews the hormonal signalling pathways that mediate
the elongation and the quiescence response in deepwater and low-
land rice, respectively. It highlights the crosstalk between various
hormones during submergence and how these hormones co-
ordinately mediate distinct responses of the plant to submergence
stress. We give emphasis to the crucial roles of key transcription
factors that are the master regulators to two contrasting strategies

F I G U R E 1 Comparison of phenotype between deepwater and lowland rice in response to two types of flooding regimes. (a) In case of flash
flooding when the water level rises suddenly the deepwater rice plants are not able to emerge above the water level, resulting in the death of
plants when flood recedes due to exhaustion of energy reserves used up in growth. Lowland varieties (harbouring Sub1A‐1) on the other hand
are able to cope up with complete submergence by saving the energy reserves by ceasing interned elongation and shut down sugar metabolism
and grow well when normal conditions are achieved. (b) In the case of long‐term flooding as water levels rise gradually the deepwater rice are
able to elongate the stem internodes to maintain some leaves above the water surface to maintain normal oxygen levels and hence they can
survive. In contrast, lowland rice is only able to tolerate complete submergence for up to 2 weeks and succumbs to death in case of prolonged
flooding
FINDING A BREATHER FOR ORYZA SATIVA
that rice has evolved to cope with different kinds of flooding regimes.
Finally, we discuss the presence of additional unknown mechanisms
and novel QTLs involved in submergence tolerance.
2 | H O R M O NE SI G N A L L I N G P A T H W A Y S
I N V OL V E D I N E S C A P E R E S P O N S E I N
DEEPWATER RICE
During submergence, the deepwater rice varieties have the ability to
elongate at rates that are 25‐fold higher than air grown plants
(Stünzi & Kende, 1989). At such high rates, the plant is quickly able to
attain height and make contact with air. It enables the plant to in-
crease the rate of photosynthesis by the above water leaves and at
the same time secure an oxygen supply for the rest of the plant. This
strategy is known as 'Flood Avoidance' or 'Escape strategy' as the
plant is successful in escaping the severe conditions imposed by
complete submergence. It is a well‐known fact that hormones med-
iate all the growth and stress responses in plants. In the following
paragraphs, we summarize the hormone signalling networks that
mediate the escape response of deepwater rice.
2.1 | Positive feedback regulation between
ethylene and gibberellic acid
Ethylene levels rapidly increase upon submergence, partly due to
accumulation in intercellular spaces owing to reduced gas diffusion
in water and partly, by de‐novo synthesis (Métraux & Kende, 1983).
Its concentration increases 100‐fold in the internodal lacunae within
6 h of submergence (Raskin & Kende, 1984a). Métraux and Kende
(1983) reported that the accumulation of ethylene during sub-
mergence induces internode elongation in deepwater rice. More-
over, it was found that ethylene does not directly regulate the
growth of internodes, but rather mediates internode elongation by
increasing the activity of endogenous gibberellic acids (GAs; Raskin
& Kende, 1984b).
A fourfold increase in the levels of bio‐active GAs is observed
within 3 h of submergence (Hoffmann‐Benning & Kende, 1992).
Ethylene increases levels of endogenous GAs by inducing the ex-
pression of GA metabolic enzymes, GA20ox2 (also called SD1),
GA20ox4 and GA3ox2 that convert GA20 into biologically active GAs
(Choi, 2007). The transcription of the SD1 gene, encoding the GA20
oxidase is activated by direct binding of an ethylene‐responsive
transcription factor OsEIL1a to its promoter upon ethylene accumu-
lation. This phenomenon is restricted only to deepwater rice, as SD1
transcription is not induced by ethylene treatment in non‐deepwater
rice (Kuroha et al., 2018). GA20 oxidases are also under negative
feedback regulation of DELLA proteins, the repressors of the GA
signalling pathway (Fukazawa et al., 2017; Hussain & Peng, 2003;
Vera‐Sirera et al., 2016). Interestingly, SLR1, the only known
DELLA protein in rice is also inhibited by ethylene specifically in
deepwater rice (Fukao & Bailey‐Serres, 2008; Fukazawa et al., 2017).
| 281
These findings imply that the downregulation of SD1 is repressed
in deepwater rice by the ethylene mediated inhibition of the re-
pressor protein, SLR1. Additionally, another ethylene response
transcription factor OsEIL1b directly binds to the promoter of two
ethylene response factors SNORKEL1 and SNORKEL2 (SK1 and
SK2) that directly or indirectly induce GA and trigger the elonga-
tion response (Hattori et al., 2009; Kuroha et al., 2018). The
pathway for GA induction by SK1/SK2 is independent of SD1, and
both these pathways act synergistically to enhance GA accumu-
lation (Kuroha et al., 2018). Interestingly, the functional SK1 and
SK2 genes are only present in deepwater rice and are upregulated
by submergence induced ethylene accumulation. These genes are
either absent or nonfunctional in non‐deepwater rice (Hattori
et al., 2009; Oe et al., 2021). Ethylene also promotes GA accu-
mulation by downregulating the GA biosynthesis inhibitor
OsEATB. It is an ethylene response transcription factor that in-
hibits OsCPS2, a key gene involved in GA biosynthetic pathway (Qi
et al., 2011). Therefore, ethylene increases the levels of en-
dogenous GAs by upregulation of GA the stimulating genes, SD1,
SK1, SK2, GA20ox4 and GA3ox2 and by downregulation of the GA
inhibitors SLR1 and OsEATB (Figure 2).
Likewise, GA also induces ethylene biosynthesis during sub-
mergence resulting in a positive feedback loop between ethylene and
GA. Ethylene is synthesized from methionine via S‐adenosyl‐
methionine (AdoMet) and 1‐Amino‐cyclopropane‐1‐carboxylic acid
(ACC). The enzymes AdoMet synthetase (SAM), ACC synthase (ACS)
and ACC oxidase (ACO) are sequentially involved in this pathway
(Yang & Hoffman, 1984). Induction of ethylene synthesis during
submergence occurs at both, ACS and ACO levels, both of which may
play a rate‐limiting role in this pathway (Rzewuski & Sauter, 2008;
Van Der Straeten et al., 2001; Yang & Hoffman, 1984). Among rice
ACC synthases, OsACS‐5 is induced during submergence (Van Der
Straeten et al., 2001; Zhou et al., 2001). The levels of OsACS‐5 mRNA
increase fivefold upon GA treatment (Van Der Straeten et al., 2001).
Another ACC synthase, OsACS‐1 is slightly induced within 6–12 h of
submergence but there are no reports on its regulation by GA
(Zarembinski & Theologis, 1997). In addition to ACC synthesis, oxi-
dation of ACC to ethylene is also enhanced by GA treatment, sug-
gesting that GA stimulates ACO activity as well (Azuma et al., 1994).
Among rice ACC oxidases, the activity of OsACO1 and OsACO7
significantly increases in the internodes of deepwater rice during
submergence, indicating that GA probably induces either OsACO1 or
OsACO7 or possibly both of them (Mekhedov & Kende, 1996;
Minami et al., 2018; Van Der Straeten et al., 2001). Whether GA
augments ethylene biosynthesis at the level of methionine to Ado-
Met conversion, has not been reported in rice. However, in wheat
aleurones GA is known to induce two AdoMet synthetases SAM1 and
SAM2, by threefold, bringing out the possibility that this phenomenon
might occur in rice also (Mathur et al., 1992). Therefore, it is likely
that GA enhances ethylene biosynthesis at each step of the ethylene
biosynthesis pathway. Talking about ethylene sensing, OsERL1 an
ethylene receptor is upregulated during submergence by GA, sug-
gesting that, apart from increasing ethylene biosynthesis, GA also
282 | MITTAL
increases ethylene sensitivity during submergence (Watanabe
et al., 2004).
In conclusion, both ethylene and GA are induced during sub-
mergence in deepwater rice and these hormones induce biosynthesis
of one another by a positive feedback mechanism (Figure 2).
2.2 | GA is essential for elongation response
One of the primary roles of endogenous GAs in plant development is
stem growth and any defect in GA biosynthesis or signalling path-
ways causes a dwarf phenotype in the plant. Consistent with its role
in growth, the treatment of plants with exogenous GA leads to en-
hanced stem elongation. Although ethylene is induced in both
deepwater and non‐deepwater rice upon submergence, GA is spe-
cifically induced in deepwater rice (Minami et al., 2018). No internode
elongation is observed in submerged deepwater rice plants treated
with a GA inhibitor (Suge, 1985). This implies that an increase in GA
levels is essential for the growth response, and GA is the ultimate
hormone that elicits stem elongation in submerged deepwater rice.
Intercalary meristem (IM) is the primary site for GA action, as it
does not promote elongation of deepwater rice internodes from which
IM has been excised (Sauter et al., 1993). GA first promotes cell
elongation in IM by inducing the expression of genes involved in cell
wall loosening and synthesis (Cho & Kende, 1997, 1998; Métraux &
Kende, 1984; Sauter & Kende, 1992). While the cell is elongating it
simultaneously induces genes (e.g., histones and replication proteins)
ET AL.
F I G U R E 2 Hormone signalling networks
involved in escape response of deepwater rice
during submergence. Submergence induced
ethylene accumulation leads to an increase in
GA and a decrease in ABA levels. GA in turn
induces ethylene biosynthesis resulting in a
positive feedback loop between ethylene and
GA. GA is the final hormone that mediates
internode elongation and carbohydrate
degradation in concert with the low oxygen
and sugar starvation sensing system (escape
response). Furthermore, downregulation of
ABA is essential for GA accumulation and also
leads to an increase in auxin levels which
mediate internode elongation along with GA.
Red/orange colour represents
downregulation/inhibition, whereas black/
blue colour represents upregulation/induction.
Black dashed arrows indicate the presence of
an unknown relationship [Color figure can be
viewed at wileyonlinelibrary.com]
that are involved in DNA replication, which occurs in the S phase of the
cell cycle (van der Knaap & Kende, 1995; Van Der Knaap et al., 1997).
After the cells of IM have sufficiently elongated the hormone induces
cell division at the G2‐M phase checkpoint, thereby initiating mitosis
(Sauter & Kende, 1992).
Cell growth is an energy‐consuming process where 65% of starch
is mobilized within 3 days of submergence, to meet the energy re-
quirements to support rapid internode elongation (Raskin &
Kende, 1984a). Mobilization of starch is accompanied by a 70‐fold
increase in the expression of the starch degrading enzyme, α‐amylase
which is induced by GA (Chen et al., 2006; Das et al., 2005; Lu
et al., 2002; Raskin & Kende, 1984a). The promoter of α‐amylases is
under the control of two MYB transcriptional factors, MYBGA and
MYBS1 (Hong et al., 2012). GAMYB is a transcriptional activator of
GA regulated genes and mediates a variety of GA responses (Aya
et al., 2009, 2011; Hong et al., 2012; Kaneko et al., 2004; Woodger
et al., 2003). MYBS1, on the other hand, activates the expression of
α‐amylases in response to sugar starvation by the formation of Sugar
Response Complex (Lu et al., 1998). It has been found that both these
transcription factors cooperatively function to integrate the sugar
starvation and the GA signalling pathways by co‐nuclear localization
and formation of a stable bi‐partite MYB‐DNA complex at the pro-
moter of starch hydrolases (Hong et al., 2012). Therefore, apart from
induction of the IM, GA mediates carbohydrate degradation in con-
cert with the sugar sensing system to meet the energy requirements
for the elongation response. In addition, GA is also known to mediate
sugar translocation, growth of sink tissues and maintenance of
FINDING A BREATHER FOR ORYZA SATIVA
source‐sink relationships in stressful conditions (Iqbal et al., 2011).
Taken together, we conclude that GA is the ultimate hormone that
executes the 'Escape Strategy' of deepwater rice upon submergence
(Figure 2).
2.3 | Downregulation of ABA is a prerequisite for
elongation response
ABA levels in the internodal region of deepwater rice decrease by
75% during submergence. It is a negative regulator of growth and the
application of exogenous ABA inhibits submergence induced elon-
gation (Hoffmann‐Benning & Kende, 1992). Inhibition of internode
elongation by ABA treatment is reversed on GA application, sug-
gesting that it inhibits growth by decreasing the biosynthesis of GA
(Hoffmann‐Benning & Kende, 1992). Also, time taken in reduction of
ABA levels and the time lag between the onset of elongation re-
sponse implies that decrease in ABA levels precedes submergence
induced growth (Hoffmann‐Benning & Kende, 1992). It is now well
established that ABA is involved in the suppression of GA biosyn-
thetic genes (Liu & Hou, 2018). Thus, the downregulation of ABA is
essential for GA biosynthesis.
Ethylene, which increases 100‐fold during submergence, induces
the expression of CYP707A5 gene, which encodes ABA 8' hydro-
xylase (OsABA8'ox1). This enzyme catabolizes ABA into phaseic
acid, thereby decreasing ABA concentrations in the plant (Saika
et al., 2007; Yang & Choi, 2006). Also, Choi (2007) reported that
ethylene downregulates the expression of key ABA biosynthetic
genes, OsZEP, OsNCED1, OsNCED2 and OsNCED5, specifically in
deepwater rice, upon submergence. On the other hand, in lowland
rice, the downregulation of these ABA biosynthetic genes is mediated
by some other unknown factor and not by ethylene (Saika
et al., 2007). Thus, in deepwater rice, submergence induced ethylene
accumulation decreases ABA levels in two ways, one, by its catabo-
lism and the other through the inhibition of its biosynthesis.
ABA shows antagonistic regulation with ethylene during sub-
mergence. Just like ethylene inhibits ABA during submergence, ABA
also inhibits ethylene by downregulating the ethylene biosynthetic
gene OsACS5 which is induced upto 8 folds during submergence (Van
Der Straeten et al., 2001). Therefore, in addition to GA mediated
positive feedback regulation, the downregulation of ABA further
augments ethylene biosynthesis during submergence. In conclusion,
ABA is downregulated in deepwater rice by submergence‐induced
ethylene accumulation and the decrease in ABA levels is a pre-
requisite for the GA mediated elongation response (Figure 2).
2.4 | Synergistic effect of ethylene and auxin
on GA
Although it is known that auxins are involved in submergence in-
duced internode elongation in deepwater rice, the mechanisms
| 283
behind it are poorly understood (Azuma et al., 1990). Surprisingly,
treatment of submerged rice plants with auxin transport inhibitors
resulted in a threefold decrease in elongation response, suggesting
that auxin signalling has a major role in rapid internode elongation in
deepwater rice (Wu & Yang, 2020). Transcriptomic analysis of sub-
merged rice plants treated with TIBA (2,3,5‐triiodobenzoic acid), an
auxin transport inhibitor, revealed that, during submergence, auxin
increases the expression of genes involved in cell wall modification
and cell division (Wu & Yang, 2020). The hormone probably mediates
elongation by increasing the concentration of bioactive GAs, as auxin
treatment had no effect on internode elongation in presence of a GA
inhibitor (Azuma et al., 1990). According to Ross et al. (2003), auxin
mediates internode elongation by promoting GA biosynthesis and by
preventing the de‐activation of bioactive GAs in pea plants. Also, the
application of auxin transport inhibitors on pea plants significantly
reduced stem elongation along with the reduction in levels of
bioactive GAs (Ross, 1998). Whether the same happens in deepwater
rice, needs to be experimentally verified. Such an experiment would
give more clarity on the mechanism by which auxins and GAs mediate
internode elongation in rice.
Auxin signalling is mediated by TIR1/AFB family proteins which
are auxin receptors. These receptors trigger an auxin response by
posttranslational degradation of the auxin response inhibitors AUX/
IAAs. AUX/IAAs inhibit auxin responses by binding to the promoter
of auxin response transcription factors (ARFs), which ultimately
mediate auxin responses (Quint & Grey, 2006). A microRNA miR393
is involved in auxin homoeostasis, as it negatively regulates auxin
response by targeted degradation of the mRNAs of TIR1/AFBs
(Navarro et al., 2006). Guo et al. (2016) reported that in deepwater
rice, ABA treatment increased miR393 transcript levels and inhibited
coleoptile growth, whereas submergence led to downregulation
of miR393 expression and increase in coleoptile length. The
submergence‐induced decrease in miR393 transcript correlated with
an increased expression of the auxin receptors OsTIR1 and OsAFB2.
Also, it is known that ABA is downregulated during submergence.
Taken together, we suggest a signalling pathway that activates auxin
response during submergence in deepwater rice. Ethylene accumu-
lation decreases the concentration of ABA causing a decrease in
expression of miR393a, a negative regulator of the auxin receptors
OsTIR1 and OsAFB2. This leads to an increase in levels of these auxin
receptors which then target the auxin signalling inhibitors AUX/IAA
bound to the promoters of ARFs. Degradation of AUX/IAA proteins
switches on the expression of ARFs that ultimately mediate auxin
response probably by increasing the accumulation of bioactive GAs
(Figure 2).
Auxin positively regulates ethylene by inducing the ethylene
biosynthetic gene OsACS1 (Zarembinski & Theologis, 1993). There-
fore, in addition to GA, ethylene also shares a positive feedback
regulation with auxin. Thus, we conclude that in deepwater rice,
auxins and ethylene act synergistically in increasing the activity of
GAs which ultimately mediate internode elongation during sub-
mergence (Figure 2).
284 | MITTAL
3 | H O R M O N E SI G N A L L I N G NE T W O R K S
R E G U L A T I N G S U B M E RG E N C E TO L E R A N C E
I N L O W L A N D RI C E
By definition, rice plants that can survive complete submergence up
to 14 days are considered to be submergence tolerant. Such plants
temporarily suspend all their growth‐related activities to minimize
their sugar consumption to use the energy for resuming normal
growth‐related activities after the flood waters recede. The tolerant
phenotype is associated with the ability to restrict stem elongation,
reduce carbohydrate consumption, oxidative damage and chlorophyll
degradation (Fukao et al., 2006; Sarkar et al., 2001). This strategy is
also known as 'Quiescence', a kind of metabolic adaptation, where
the plant goes into an energy‐saving mode. Not all lowland rice
varieties show submergence tolerance. Therefore, the words deep-
water and lowland are the names of rice ecotypes based on habitat
and flooding regimes and are not representative of the tolerance
ability/mechanism of the variety. Since, most of the deepwater
rice varieties show elongation response, for ease in usage, we use the
words elongating rice and deepwater rice synonymously in this re-
view. To further eliminate the confusion, there are certain rice vari-
eties such as Taichung 65, that neither show enhanced elongation,
nor show tolerance due to the lack of the Sub1A‐1 gene. Such
varieties are mostly non‐deepwater with respect to their ecotype
(Fukushima et al., 2020; Minami et al., 2018).
3.1 | A single gene SUB1A‐1 mediates
submergence tolerance
Genetic mapping studies associated the tolerant phenotype to a
single QTL on chromosome 9 named Sub1 (Fukao et al., 2006; Nandi
et al., 1997; Siangliw et al., 2003; Sripongpangkul et al., 2000;
Toojinda et al., 2003; Xu & Mackill, 1996). The Sub1 locus contains
three genes, Sub1A, Sub1B and Sub1C which encode ERFs belonging
to the subfamily of AP2‐like transcription factors (AP2/ERF; Xu
et al., 2006). Out of the three genes, Sub1B and Sub1C are present
invariably in all the varieties harbouring the Sub1 locus, whereas
Sub1A gene is restricted to only some varieties of the indica germ-
plasm that show tolerance to submergence. Genetic analysis revealed
that the populations carrying the Sub1A gene had two alleles,
Sub1A‐1 and Sub1A‐2. The tolerant trait is associated with the strong
upregulation of Sub1A‐1, whereas the Sub1A‐2 allele is poorly in-
duced during submergence, and plants carrying it are intolerant (Xu
et al., 2006). The only difference between the two alleles is the
presence of a proline residue at position 186 in Sub1A‐2 instead of a
serine, in the tolerant allele Sub1A‐1 (Xu et al., 2006). This serine
residue is a MAP kinase target site and it was observed that it is
phosphorylated by MPK3 which leads to the activation of Sub1A‐1 as
a transcriptional regulator, resulting in subsequent upregulation of its
target genes. On the other hand, due to mutation at the MPK3
phosphorylation site, Sub1A‐2 is incapable of activation and there-
fore is unable to induce any tolerance related genes (Singh &
ET AL.
Sinha, 2016). Septiningsih et al. (2008) confirmed that the Sub1A‐1
gene is the primary determinant of submergence tolerance in rice.
They also reported that the level of Sub1A‐1 transcript strongly
correlates with the degree of submergence tolerance as hetero-
zygotes of the Sub1A‐1 allele had lesser mRNA levels and were
significantly less tolerant as compared to the homozygotes (Fukao &
Bailey‐Serres, 2008; Septiningsih et al., 2008). Sub1C on the other
hand has no association with the tolerant phenotype (Septiningsih
et al., 2008).
Both Sub1A‐1 and Sub1C are induced upon submergence or by
ethylene treatment, whereas Sub1B, in spite of being an ERF, is not
responsive to ethylene or submergence (Fukao et al., 2006). Studies
show that Sub1C acts downstream of GA and positively regulates
responses like internode elongation and carbohydrate degradation
(Figure 2; Fukao et al., 2006; Kudahettige et al., 2011). Sub1C acti-
vates α‐amylases in concert with the low oxygen and low sugar
sensing system by the CIPK15‐SnRK1A‐MYBS1 pathway, which is
not discussed in detail, as it is beyond the scope of this review
(Figures 2 and 3; Kudahettige et al., 2011). The transcript levels of
Sub1C are upregulated in deepwater rice and downregulated in the
Sub1A‐1 genotype (Fukao et al., 2006; Xu et al., 2006). This might be
due to the downregulation of GA by Sub1A‐1 (as discussed below).
Therefore, Sub1A‐1 mediates quiescence response, whereas Sub1C
mediates the elongation response to submergence in rice.
In the coming sections, we summarize the hormone signalling
networks that mediate submergence tolerance in lowland rice and
how the presence or absence of a single gene defines the response of
the rice plant to submergence stress.
3.2 | A negative feedback loop between ethylene
and SUB1A‐1
In tolerant varieties, ethylene levels show a transient peak upon the
onset of submergence, following which their levels reduce gradually.
This is in contrast to deepwater rice where ethylene levels are main-
tained throughout submergence (Van Der Straeten et al., 2001). This
initial peak of ethylene is sufficient to induce Sub1A‐1, the levels of
which are subsequently maintained by ethylene independent mechan-
isms (Fukao et al., 2006). Interestingly, Sub1A‐1 is the gene that inhibits,
its own activator, that is, ethylene, by a negative feedback loop that
involves OsERF3. OsERF3 belongs to a group of AP2/ERF transcription
factors that negatively regulate ethylene signalling pathways as well as
ethylene mediated activation of GA (Ohta et al., 2001). OsERF3 is in-
duced during submergence specifically in tolerant rice varieties and in a
Sub1A‐1 dependent manner (Jung et al., 2010).
The decrease in ethylene results in the downregulation of all the
ethylene regulated genes as well as responses, including GA medi-
ated internode elongation and carbohydrate degradation. This implies
that the downregulation of ethylene leads to the subsequent de-
crease in GA levels. In other words, we can say that Sub1A‐1 restricts
growth, by breaking the ethylene‐GA positive feedback loop, which is
the initial driving force for the elongation response (Figure 3).
FINDING A BREATHER FOR ORYZA SATIVA
F I G U R E 3 Hormone signalling networks involved in SUB1A‐
mediated quiescence response in rice during submergence. The
submergence induced ethylene accumulation is restricted in the
presence of the Sub1A‐1 gene which is an ERF. This leads to a
decrease in levels of GA due to the disruption of ethylene‐GA
feedback loop. Sub1A‐1 also induces BRs which further inhibit GA
accumulation and signalling. With the decrease in GA, there is a
decrease in the levels of GA regulated genes that mediate stem
elongation and carbohydrate degradation resulting in restricted
growth and sugar consumption (quiescence response). Apart from
this Sub1A‐1 also increases submergence tolerance by
downregulating the senescence‐associated hormones, JA, SA and
ethylene. Red/orange colour represents downregulation/
inhibition, whereas black/blue colour represents upregulation/
induction. Black dashed arrows indicate the presence of an
unknown relationship [Color figure can be viewed at
wileyonlinelibrary.com]
3.3 | SUB1A‐1 reverses the effect of ethylene
mediated GA accumulation
An increase in GA negatively impacts submergence tolerance in
environments where elongation ability is not required. This is be-
cause GA mediated elongation leads to exhaustion of carbohydrate
reserves resulting in severe starvation and hence, low survival
rate (Das et al., 2005; Setter & Laureles, 1996). In tolerant
| 285
varieties, GA is naturally downregulated by Sub1A‐1 (Fukao &
Bailey‐Serres, 2008). It negatively impacts GA mediated processes
by decreasing GA responsiveness of the tissue. This is done by the
activation of the two GA signalling repressors, SLR1 and SLRL1. As
stated previously, SLR1 is a known DELLA protein, whereas SLRL1,
is distinguished from SLR1 by the lack of DELLA domain (Fukao &
Bailey‐Serres, 2008; Fukazawa et al., 2017). Therefore, in addition
to the disruption of GA‐ethylene feedback regulation, Sub1A‐1
further decreases GA sensitivity by inhibiting the GA signalling
pathway. Another level at which Sub1A‐1 inhibits GA mediated
responses is by the induction of a GA2 oxidase, GA2ox7. GA2
oxidases inhibit GA accumulation by the degradation of bioactive
GAs (Chen, 2018). The activation of GA2ox7 is mediated by a
Sub1A‐1 binding protein called SAB18 (Sub1A binding‐18;
Chen, 2018; Locke et al., 2018). SAB18 is a transcription factor
that is specifically induced in Sub1A‐1 containing genotypes and has
been predicted to function in chromatin remodelling (Chen, 2018).
Surprisingly, SAB18 also interacts with Sub1C, suggesting that this
transcription factor might be an important link, between Sub1A‐1
and Sub1C (Locke et al., 2018). It would be interesting to know the
mechanisms by which SAB18 mediates the crosstalk between two
ERFs having antagonistic functions.
The resulting downregulation of GA would spontaneously lead to
the inhibition of GA mediated responses like carbohydrate degrada-
tion and internode elongation. It is already known that Sub1A‐1 re-
stricts carbohydrate consumption in shoots during submergence
stress (Barding et al., 2012). Also, Fukao et al. (2006) reported a
decrease in the expression of genes involved in carbohydrate cata-
bolism (e.g., α‐amylase and sucrose synthase) and cell elongation (i.e.,
expansins) specifically in tolerant varieties. Taken together, it appears
that in lowland rice, ethylene mediated GA accumulation is reversed
by the presence of Sub1A‐1, which inhibits GA responses at 3 levels,
namely, biosynthesis, catabolism and signalling. As a result, instead of
GA induction, the submergence induced accumulation of ethylene
leads to the downregulation of GA.
3.4 | Synergistic action of Sub1A and
brassinosteroids
Brassinosteroids (BRs) are a relatively recent class of plant hor-
mones that regulate a vast array of genes involved in the growth
and development of plants (Clouse, 2011). They are generally con-
sidered as growth promoters and have been known to promote
stem elongation in a variety of plants (Mandava, 1988). Further-
more, mutants that have defects in BR biosynthesis or signalling
pathways display dwarf phenotypes (Li & Chory, 1999). However, in
rice BRs only induce stem elongation when present in appropriate
amounts (10−6 to 10−8 M) and inhibit growth at high concentrations
(>10−8 M; Jeong et al., 2007). Exogenous treatment with brassino-
lide, an active form of BR, leads to the inhibition of stem elongation
in rice (Schmitz et al., 2013).
286 | MITTAL
During submergence, BRs are specifically upregulated in the toler-
ant rice variety by the Sub1A‐1 gene, whereas no significant change in
its levels is detected in rice lacking Sub1A‐1 (Schmitz et al., 2013). Two
BR biosynthetic genes, DWARF1 (DWF1) and DWARF4 (DWF4) are
upregulated in tolerant rice in a Sub1A‐1 dependent manner (Schmitz
et al., 2013). Also, the BR related genes OsBZR3 and BZS1, are differ-
entially expressed in Sub1A‐1‐containing and intolerant genotypes.
OsBZR3 is the rice ortholog of Arabidopsis BZR1, which is involved in the
BR signalling pathway (Bai et al., 2007), whereas BZS1 gene of Arabi-
dopsis is a known suppressor of the BR signalling pathway (Sun
et al., 2010). BZR1 is known to suppress BZS1 however, whether the
same happens in rice is not known, as functions of both BZR3 and BZS1
have not been well studied in rice. Consistent with the above facts, the
expression of OsBZR3 is upregulated and that of BZS1 is downregulated
by Sub1A‐1 during submergence (Schmitz et al., 2013). Thus in rice, the
Sub1A‐1 gene induces BRs by increasing their biosynthesis, upregulat-
ing the genes involved in their signalling pathway and downregulating
the genes that are suppressors of their signalling.
BRs restrict stem elongation by increasing the expression of two
GA inhibitors, SLR1 and GA2ox7. Both these genes and the me-
chanisms by which they inhibit GA have been discussed above. It
seems that SLR1 and GA2ox7 are important nodes of crosstalk be-
tween the three hormones; ethylene, GA and BRs. In conclusion,
Sub1A‐1 restricts stem elongation and energy consumption by in-
creasing the levels of BRs, which inhibit GA accumulation as well as
responsiveness. BRs also induce the expression of Sub1A‐1 by a
positive feedback loop. This feedback regulation could further aug-
ment Sub1A‐1 and BR responses, that is, the inhibition of GA which is
the ultimate hormone that mediates stem elongation and carbohy-
drate degradation (Figure 3).
3.5 | Downregulation of ABA by unknown factors
ABA is downregulated during submergence in all rice varieties, irre-
spective of their ability to tolerate/avoid floods. But the signalling
pathways involved in its inhibition may differ among lowland and
deepwater rice. As already mentioned earlier the downregulation of
ABA in deepwater rice is mediated by ethylene accumulation.
Whereas, the decrease in ABA levels in tolerant lowland rice is nei-
ther mediated by Sub1A‐1 nor ethylene, but by some other unknown
factors (Fukao & Bailey‐Serres, 2008; Fukao et al., 2006). We pro-
pose that different signalling pathways might be involved in ABA
inhibition in deepwater and lowland rice.
Although signalling may be different, but the mechanism of ABA
downregulation is the same in both varieties. One is by hydroxylation
of ABA into phaseic acid, and secondly, by the inhibition of ABA
biosynthetic genes. Both of these mechanisms are mediated by
ethylene in deepwater rice, whereas in lowland rice these are medi-
ated by some other unknown factor (Fukao & Bailey‐Serres, 2008;
Saika et al., 2007; Yang & Choi, 2006).
ET AL.
A decrease in ABA levels is also a prerequisite for Sub1A‐1
accumulation as treatment with exogenous ABA resulted in a
decrease in Sub1A‐1 expression (Fukao & Bailey‐Serres, 2008;
Fukao et al., 2011). In conclusion, ABA is downregulated in both,
elongating as well as tolerant rice varieties, by same mechanisms,
but by different signalling pathways, and the decrease in ABA
levels is a prerequisite for both the type of flooding responses
(Figures 2 and 3).
3.6 | Sub1A‐1 downregulates the expression of
salicylic acid and jasmonic acid
The plant hormones, ethylene, jasmonic acid (JA) and salicylic acid
(SA) are growth inhibitors and have been implicated to be in-
volved in leaf senescence (Zhang & Zhou, 2013). These hormones
are known to regulate a number of senescence‐associated genes
(SAGs) that mediate processes like starch and protein hydrolysis
and chlorophyll breakdown which are characteristic of senescing
leaves (Morris et al., 2000; Qiu et al., 2015; Reinbothe
et al., 2009). The production of these hormones during biotic and
abiotic stresses leads to the induction of SAGs thereby accel-
erating the process of senescence. It is known that the high
survival rate of the plants tolerant to submergence stress is due to
their ability to reduce stress‐induced chlorophyll degradation
(Fukao et al., 2006; Sarkar et al., 2001). Consistent with this, the
levels of chlorophyll were higher in Sub1A‐1 containing genotype
as compared to the one lacking it, suggesting that Sub1A‐1
somehow restricts chlorophyll degradation and leaf senescence
during submergence (Fukao et al., 2006). It inhibits the expression
of SAGs by inhibiting ethylene accumulation as well as down-
regulating JA and SA mediated responses (Fukao et al., 2012).
JA is also downregulated in deepwater as well as non‐deepwater
rice varieties that lack the Sub1A‐1 gene, suggesting that it is
downregulated during submergence in all rice varieties by
Sub1A‐1 dependent as well as independent pathways (Minami
et al., 2018). One of the known mechanisms by which the en-
dogenous levels of bioactive JAs are reduced is by the induction
of a JA inactive enzyme, CYP94C4 (Kurotani et al., 2015a; Minami
et al., 2018). This gene encodes a cytochrome P450 enzyme that
converts JA‐Ile to its inactive form. The overexpression of
CYP94C2b, a gene from the same family of cytochrome P450
enzymes (CYP94 gene family) leads to enhanced internode elon-
gation in rice (Kurotani et al., 2015b). Coherently, JA is known to
suppress GA mediated internode elongation during submergence,
possibly by the increase in the levels of SLR1 in rice (Minami
et al., 2018; Yang et al., 2012). Moreover, JA also inhibits the
accumulation of Sub1A‐1 transcript (Fukao et al., 2012). Thus we
propose that, like ABA, the downregulation of JA is also a pre-
requisite for both the tolerance and the escape response to
submergence stress in rice.
FINDING A BREATHER FOR ORYZA SATIVA | 287

F I G U R E 4 Comparison of the hormone levels in lowland (SUB1A1) and deepwater rice before and after submergence. In air ethylene, GA,
ABA, auxin, BR, JA and SA are at normal levels which are required for normal growth and development. Upon submergence, there is a change in
the levels of these hormones, but they are regulated differently between the SUB1A1 containing lowland rice, and deepwater rice. In lowland
rice, there is an initial increase in ethylene due to entrapment of the gas in the intercellular spaces (highlighted in red). Ethylene triggers the
expression of SUB1A1 which inhibits ethylene expression by a negative feedback loop (highlighted in red). Additionally, the hormones, GA, ABA,
JA and SA are downregulated whereas BR is upregulated in a SUB1A1‐dependent manner. The downregulation of ethylene and GA majorly
contributes to the inhibition of growth‐related activities in lowland rice and the upregulation of BR leads to the activation of SUB1A1 mediated
quiescence response. On the other hand, in deepwater rice, the levels of the growth stimulating hormones ethylene, GA and auxins increase
many folds leading to enhanced stem elongation, whereas the inhibition of ABA and JA is necessary for the GA‐ and ethylene‐mediated escape
response. Hormones in the green box represent upregulated, and those in the orange box represent downregulated ones [Color figure can
be viewed at wileyonlinelibrary.com]

4 | C ONC LUS I ON
Rice has devised flood avoidance and flood tolerance as the two
strategies to cope with submergence stress. Both these strategies
are based on contrasting physiological mechanisms which are
regulated by a set of plant hormones. The hormones ethylene,
auxin and GA positively regulate the elongation response,
whereas, BRs at high concentrations inhibit it. ABA and JA on the
other hand inhibit both the type of responses to submergence
stress and their levels are kept low by distinct signalling pathways
in deepwater and Sub1A‐1 containing rice. We now know that GA
is the ultimate hormone whose levels define the type of response
of the rice plant to submergence stress. The role of this hormone
becomes pivotal because it directly mediates the two responses
that are critical to the survival of the plant during submergence,
that is, internode elongation and carbohydrate degradation. Both
ethylene and auxin induce GA accumulation as well as sensing,
whereas BRs, ABA and JA inhibit it. If the levels of GA are high in
the internodal tissue, the plant will show rapid internode elon-
gation and massive carbohydrate mobilization. On the other hand,
if the levels of the hormone are low, the plant shows restricted
growth and metabolic activities. A simplified pictorial re-
presentation of roles of different plant growth hormones before
and after submergence in lowland and deepwater rice is depicted
in Figure 4.
The plant hormones have the ability to communicate with each
other and collectively mediate plant responses to specific environ-
mental cues. This communication is established by a set of molecules
that interact among themselves leading to the formation of a complex
network that is tightly regulated. The formation of such a highly
regulated network means that a change in the level of even a single
protein would lead to the change in the dynamics of all the players
involved in the hormonal crosstalk, resulting in distinct phenotypic
responses. One such protein that is the point of crosstalk between
several hormones is, Sub1A‐1 which is specifically involved in sub-
mergence tolerance, having no role in normal growth‐related pro-
cesses (Fukao et al., 2006). It is induced by submergence‐induced
ethylene accumulation and it directly or indirectly regulates the levels
of the hormones: ethylene, BRs, GA, JA and SA. Sub1A‐1 is only
found in some varieties of low land rice and is associated with the
tolerant phenotype (Table 1). Due to its crosstalk with so many
hormones, the presence or absence of Sub1A‐1 entirely defines the
type of response that the plant will exhibit during submergence
(Fukao et al., 2006; Xu et al., 2006; Figure 3).
In deepwater rice, where Sub1A‐1 is absent, submergence cau-
ses the accumulation of ethylene, which induces GAs and auxins and
inhibits ABA. The increase in levels of GA and auxin and decrease in
ABA levels further augments ethylene synthesis by feedback
regulation. The net result of the crosstalk is an increase in the
levels of GA, hence, resulting in an elongation response (Figure 2).
 288
|

TABLE 1 List of genes mediating hormonal crosstalk during submergence

Hormones Induced/ Relationship between

Gene showing crosstalk Function involved Repressed by Target genes identified Effect of Cross talk hormones References

OsEIL1a ERF Ethylene, GA Induced by SD1 (GA Biosynthesis) GA accumulation Synergistic Kuroha et al. (2018)
ethylene

OsEIL1b ERF Ethylene, GA Induced by SK1, SK2 GA accumulation Synergistic Hattori et al. (2009)
ethylene

OsEATB ERF Ethylene, GA Inhibited by OsCPS2 (GA GA accumulation Synergistic Qi et al. (2011)
ethylene Biosynthesis)

OsABA8ox1 Degradation of ABA Ethylene, ABA Induced by – ABA decreases Antagonistic Yang and Choi (2006), Saika
ethylene et al. (2007)

OsZEP OsNCED1 ABA biosynthesis Ethylene, ABA Inhibited by – ABA decrease Antagonistic Choi (2007)
OsNCED2 ethylene
OsNCED5

OsACS5 Ethylene biosynthesis Ethylene, Inhibited by ABA – Ethylene decreases Antagonistic Van der Straetn et al. (2001)
ABA, GA
Induced by GA – Ethylene Synergistic

OsACS1 Ethylene biosynthesis Ethylene, Auxin Induced by auxin – Ethylene accumulation Synergistic Zarembinski and Theologis (1993)

miR393 Degradation of auxin Auxin, ABA induced by ABA OsTIR1/OsAFB2 (auxin Inhibition of auxin Antagonistic Guo et al. (2016), Navarro
receptors receptors) signalling et al. (2006)

OsSLR1 Repressed GA Ethylene, BR, GA Inhibited by – Positive effect on GA Synergistic Fukao and Bailey‐Serres (2008),
signalling ethylene signalling Fukao et al. (2006), Schmitz
et al. (2013)

Induced by BR – Negative effect on GA Antagonistic Schmitz et al. (2013)

signalling

GA2ox7 Degradation of BR, GA Induced by BR – Decrease in GA Antagonistic Chen (2018), Schmitz et al. (2013)
bioactive GA

SUB1A ERF Ethylene, GA, BR Induced by – Decrease in GA Antagonistic Fukao and Bailey‐Serres (2008),
ethylene Fukao et al. (2006)

DWF1, DWF4, BR accumulation and Synergistic Schmitz et al. (2013)

OsBZR3, BZS1 signalling
MITTAL
ET AL.
FINDING A BREATHER FOR ORYZA SATIVA
To manifest this hormone‐mediated elongation response, the deep-
water varieties have evolved many mechanisms at the molecular level
during the course of evolution. Firstly, SD1, the GA biosynthetic gene
is specifically induced by ethylene, only in deepwater rice, and not in
non‐deepwater rice varieties, even though ethylene is induced in
both (Kuroha et al., 2018; Minami et al., 2018). These findings are
suggestive of the existence of an unknown suppressor of ethylene‐
inducible (OsEIL1a mediated) activation of SD1 which was probably
lost from the deepwater rice germplasm during the course of evo-
lution as a consequence of selection (Kuroha et al., 2018). Secondly,
the group VII ERFs, SK1 and SK2 genes that mediate internode
elongation are expressed only in deepwater rice and are either lost or
have a loss of function mutation in non‐deepwater varieties (Hattori
et al., 2009). Their specificity to the elongation response in deep-
water varieties and their high similarity to the SUB1A gene (Hattori
et al., 2009), mark them as important candidates for further in-
vestigation on the associated molecular pathways and mechanisms.
Thirdly, two novel genes ACCELERATOR OF INTERNODE ELONGA-
TION1 (AEC1) and DECELERATOR OF INTERNODE ELONGATION1
(DEC1) have been recently identified, that antagonistically regulated
internode elongation in concert with GA in rice. As the name suggests
ACE1 promotes internode elongation by making the IM cells com-
petent for cell division in presence of GA, whereas the DEC1 is a
suppressor of internode elongation. The full‐length functional allele
of AEC1 is essential for internodal elongation at the vegetative stage
and any mutation in this gene leads to a shorter phenotype. On the
other hand, the GA mediated downregulation of DEC1 promotes
internode elongation irrespective of the presence or absence of a
functional allele of this gene (Nagai et al., 2020). It is believed that the
functional version of ACE1 along with the GA repressed DEC1 are
present in deepwater rice varieties, contributing to the escape
strategy. In parallel, the inactive form of ACE1 and the active form of
DEC1 were selected during the domestication of rice growing in non‐
deepwater conditions (Bailey‐Serres & Voesenek, 2020). It is already
reported that AEC1 is induced both by deepwater conditions and
upon GA treatment (Nagai et al., 2020). To get more insight into their
role in elongation response, it would be interesting to know how
AEC1 and DEC1 are regulated by GA and whether these genes are
associated with ethylene. These results highlight the significance of
genes that serve as a node for crosstalk between plant hormones, in
responses of plants to developmental and environmental cues. Such
genes when subjected to forces of natural selection and domestica-
tion show adaptive radiation and provide flexibility to the plants to
cope with diverse environmental conditions. Mutations or deletions
of these genes lead to the activation of distinct signalling pathways
thereby resulting in different responses (Table 2).
Similarly, in lowland rice, the presence of Sub1A‐1 leads to a
drastic change in the signalling pathways. Firstly, Sub1A‐1 restricts
ethylene synthesis, resulting in a gradual decrease in ethylene levels
with time. Since ethylene is the perpetrator of GA and GA mediated
responses, the inhibition of ethylene leads to the attenuation of the
ethylene—GA feedback loop. The final outcome of ethylene inhibition
is the decrease in GA levels. Secondly, Sub1A‐1 induces BRs, which
| 289
are observed to inhibit stem elongation at concentrations more than
10−8 M. BRs, negatively regulate GA by inducing an enzyme that
degrades bioactive GAs. Furthermore, they inhibit GA responses by
inducing the GA signalling repressor SLR1. The DELLA protein SLR1
is a major point of crosstalk between ethylene, JA, GA and BRs
(Table 1). It is inhibited by ethylene in deepwater rice, whereas, in
lowland rice not only SLR1 is released from ethylene inhibition, but is
further induced by BRs. Both of these mechanisms occur in a
Sub1A‐1 dependent manner. BRs are not induced in genotypes
lacking Sub1A‐1. This differential regulation of BRs significantly
contributes to the differences in hormone signalling networks be-
tween deepwater and lowland rice. In conclusion, the presence of
Sub1A‐1 leads to inhibition of GAs, resulting in restricted growth and
low energy consumption.
What happens to auxins in presence of Sub1A‐1? We know that
the increase in ethylene levels and decrease in ABA concentrations
lead to the upregulation of auxins, in deepwater rice. Auxins favour
the elongation response by increasing the activity of GAs. Due to
their role in stem elongation, one would expect them to be down-
regulated in varieties of lowland rice that show restricted growth. But
we do not know what actually happens to auxins in the presence of
Sub1A‐1. According to the known crosstalk of auxins with ethylene,
ABA and GA, the decrease in ethylene levels should lead to the de-
crease in auxins, whereas the decrease in ABA should induce auxin
accumulation. Owing to the fact that GA levels are low in lowland
rice, we give more weightage to the possibility of auxins being
downregulated in the presence of Sub1A‐1. Adding more to the
complexity, even if auxin levels do not decrease in lowland rice, their
effect on internode elongation might be neutralized by the presence
of BRs. In Arabidopsis, BZR1, directly binds to the promoter of ARF7,
an auxin response factor that mediates auxin responses. The binding
of BZR1 leads to the inhibition of transcription of ARF7 resulting in
repression of auxin responses (Zhou et al., 2013). Even though
OsBZR3, the rice ortholog of BZR1, is induced by Sub1A‐1, we do
not know if the same regulates any ARFs in rice (Bai et al., 2007;
Schmitz et al., 2013). Therefore, it is difficult to predict the changes in
auxin signalling pathways in presence of Sub1A‐1. It would be in-
teresting to know, how the Sub1A‐1 mediated changes in hormone
signal pathways affect auxin signalling.
Since Sub1A‐1 is the master regulator of the quiescence re-
sponse to submergence stress in rice, it becomes imperative to
highlight the molecular aspects of the mechanisms and the pathways
involved. One such striking feature is that the gene (along with SK1
and SK2) belongs to the subgroup VII of ERF transcription factors
that act as oxygen sensors via the N‐end rule pathway (Gibbs
et al., 2011; Licausi et al., 2011). The N‐end rule pathway substrates
contain destabilizing residues in their amino terminal, known as
N‐degron that makes them targets of proteolysis by E3 ubiquitin
ligases (Graciet & Wellmer, 2010). It is believed that these proteins
sense oxygen by oxidation of the tertiary destabilizing cysteine re-
sidue at the second position of the N‐terminal leading to its targeted
proteolysis. Stabilization of these proteins during hypoxic conditions
leads to activation of downstream hypoxia response genes and
290 | MITTAL ET AL.

T A B L E 2 Differential regulation of genes involved in biosynthesis and signalling of various hormones between deepwater and Sub1A‐1
containing rice

Effect on
Hormone Gene Function Hormone Deepwater Sub1A‐1 rice References

Ethylene OsACS5 Biosynthesis Positive Upregulated – Van der Straetn et al. (2001), Zhou
et al. (2001)

OsACS1 Biosynthesis Positive Upregulated – Zarembinski and Theologis (1997)

OsACO1 Biosynthesis Positive Upregulated – Mekhedov and Kende (1996),

Van der Straetn et al. (2001)

OsERL1 Signalling Positive Upregulated – Watanabe et al. (2004)

OsERF3 Signalling Negative – Upregulated Jung et al. (2010), Ohta et al. (2001)

GA SD1 (GA20ox2) Biosynthesis Positive Upregulated – Choi (2007)

GA20ox4 Biosynthesis Positive Upregulated – Choi (2007)

GA3ox1 Biosynthesis Positive Upregulated – Choi (2007)

SLR1 Signalling Negative Downregulated Upregulated Fukao and Bailey‐Serres (2008),

Fukao et al. (2006), Schmitz
et al. (2013)

SLRL1 Signalling Negative Downregulated – Fukao et al. (2006)

SK1 Transcription Positive Upregulated – Hattori et al. (2009)

Factor

SK2 Transcription Positive Upregulated – Hattori et al. (2009)

Factor

OsCPS2 Biosynthesis Positive Upregulated – Qi et al. (2011)

GA2ox7 Biosynthesis Negative – Upregulated Chen (2018), Schmitz et al. (2013)

ABA ABA8ox1 Biosynthesis Negative Upregulated – Yang and Choi (2006),

Saika et al. (2007)

OsZEP Biosynthesis Positive Downregulated – Choi (2007), Saika et al. (2007)

OsNCED1 Biosynthesis Positive Downregulated – Choi (2007), Saika et al. (2007)

OsNCED2 Biosynthesis Positive Downregulated – Choi (2007), Saika et al. (2007)

OsNCED3 Biosynthesis Positive Downregulated – Choi (2007), Saika et al. (2007)

OsNCED5 Biosynthesis Positive Downregulated – Choi (2007), Saika et al. (2007)

Auxin OsTIR1/ Signalling Positive Upregulated – Guo et al. (2016)

OsAFB2

BR OsDWARF1 Biosynthesis Positive Upregulated (slightly) Upregulated Schmitz et al. (2013)

OsDWARF4 Biosynthesis Positive No change Upregulated Schmitz et al. (2013)

OsBZR3 Signalling Positive No change Upregulated Schmitz et al. (2013)

BZS1 Signalling Negative Downregulated Downregulated Schmitz et al. (2013)

(slightly)

therefore enhanced survival of the plant subjected to low‐oxygen
stress. Interestingly, in spite of having the characteristic N‐degron
sequence, Sub1A‐1 is not a substrate of the N‐end rule pathway and
is therefore stable in normoxic conditions also. This is because the
C‐terminal region directly interacts with the N‐terminal region, and
protects it from degradation (Lin et al., 2019). The uncoupling of
Sub1A‐1 from the N‐end rule pathway and its enhanced stability
during normal conditions, suggests that it might have a role in other
plant responses. Consistently, it has been reported that Sub1A‐1
transcript is induced by other abiotic stresses like, prolonged dark-
ness, osmotic and drought stress and enhances recovery from these
stresses (Fukao et al., 2011, 2012). It delays dark‐induced senescence
and dampens damage caused during de‐submergence (Alpuerto
et al., 2016; Fukao et al., 2012). When the plants are submerged they
encounter multiple stresses, including low light, low oxygen and os-
motic stress. Moreover, as floodwaters recede the plants are abruptly
subjected to another set of stresses like, high oxygen, high light and
dehydration. Therefore the high stability of Sub1A‐1 due to escape
FINDING A BREATHER FOR ORYZA SATIVA
from the N‐end rule pathway serves as an evolutionary mechanism
for enhanced flood tolerance at many levels (further justified below).
Given the significance of this gene in submergence tolerance and its
interaction with numerous proteins as reported by Seo et al. (2011),
Sub1A‐1 and its downstream targets have important implications in
studies involving the development of flood‐tolerant varieties. For
example, ERF66 and ERF67 are two group VII ERF transcription
factors that act downstream of Sub1A‐1, but unlike Sub1A‐1 they are
under the regulation of N‐end rule pathway during hypoxia (Lin
et al., 2019). Subsequent analysis revealed that the Sub1A‐1 regu-
lated genes could be divided into two groups, one including genes
whose regulation is dependent on ERF66/67 and the other which
includes genes that are solely regulated by Sub1A‐1 (Lin et al., 2019).
Accordingly, ERF66 and ERF67 individual overexpression lines were
also submergence tolerant. These results suggest that the presence
of ERF66/67 is essential for Sub1A‐1 mediated submergence toler-
ance response making these genes equally important as Sub1A‐1
and additional targets for further molecular studies and breeding
programs.
5 | F UT UR E P R O S PE C TS
Due to the unpredictable nature of monsoon rainfall and climate
change, developing rice varieties that are tolerant to a wide range of
flooding regimes is the need of the hour. To successfully breed such
varieties, it is important to gain a clear picture on the various me-
chanisms of submergence tolerance in rice. Earlier it was believed
that Sub1A‐1 is only expressed during submergence and its role is
limited to submergence tolerance in rice. With further studies, it was
found that the gene is involved in various physiological processes and
enhances tolerance of the rice plant to other abiotic stresses also.
Given the diverse roles of this gene in stress responses and the large
number of Sub1A‐1 interacting proteins predicted by Seo
et al. (2011), investigation of its role in other biotic and abiotic
stresses that have not been reported would help in establishing it as a
candidate gene for producing highly resilient crops. Moreover,
characterization of its interacting proteins would help us understand
the molecular mechanisms underlying Sub1A‐1 mediated responses.
Also, the ERF transcription factors, SK1/SK2 and ERF66/67, have
emerged as key submergence response genes that belong to the
same class of ERF transcription factors but mediate two entirely
different responses of the rice plant to submergence stress. How-
ever, too little is known about the mechanisms and the downstream
targets of these transcription factors. Molecular studies aiming to
find their target genes and their association with various plant hor-
mones would give a clear picture of the submergence tolerance
mechanisms in rice. This would also help us gain an insight into how
rice has evolved different strategies to cope with different kinds of
flooding regimes.
Our current knowledge of submergence tolerance mechanisms in
rice is based on the limited number of genotypes that have been
studied so far. Recent studies suggest that Sub1A‐1 might not be the
| 291
only gene conferring tolerance to plants. Some highly tolerant vari-
eties like Auspachali and Pelbeda and moderately tolerant varieties
like Lathipatni, Katrangi, Gitanjalipatni, Gopalbhog and Dorsal III do
not possess the Sub1A‐1 gene (Samal et al., 2014). Similarly, some
wild rice genotypes belonging to the C genome group lack this gene
and show Sub1A‐1 independent tolerance to submergence stress
(Niroula et al., 2012; Okishio et al., 2015). While studying the allelic
diversity at the Sub1 locus from the gene bank of BRRI (Bangladesh
Rice Research Institute) it was found that the cultivars, DG1‐349,
Kalojoma, DSL‐78‐8, Damsi and Putidepa show greater submergence
tolerance than FR13A, the tolerant check. These cultivars also lacked
the Sub1A‐1 allele, and the Sub1A variant present in these cultivars
showed poor induction during submergence (Iftekharuddaula
et al., 2016). All these results suggest the presence of additional
mechanisms of submergence tolerance in rice that must be in-
vestigated and understood. Also, such genotypes can be used as
potential genetic sources for identifying novel signalling pathways
and genes that mediate submergence tolerance.
Furthermore, the difference in the tolerance ability of FR13A, the
variety from which the Sub1A‐1 gene was discovered, and various
Sub1 introgression lines indicates the presence of additional QTLs
that contribute to the tolerant phenotype (Singh et al., 2014; Xu
et al., 2006). In a similar study, it was observed that the lowland rice
variety Goda Heenati showed lower tolerance in comparison to
FR13A, in spite of the presence of the Sub1A‐1 gene. Transcriptomic
analysis revealed that 2 genes were oppositely regulated between
FR13A and Goda Heenati and 324 genes were expressed only in one
of the two genotypes, whereas their expression remained unchanged
in the other (Xiong et al., 2012). Genetic mapping studies aiming at
QTL mining for submergence tolerance, identified several non‐SUB1
QTLs from diverse rice germplasms, having significant contributions
to the tolerant phenotype (Gonzaga et al., 2016, 2017; Septiningsih
et al., 2012; Singh et al., 2017; Zhang et al., 2017). Identification and
functional characterization of the genes associated with these QTLs
would help us to identify additional physiological mechanisms that
enhance the survival of the plants during submergence. Furthermore,
introgression and pyramiding of such genes in high yielding varieties
would provide food security and benefit to farmers.
As compared to the amount of rice germplasm present in nature,
the genotypes that we have studied till now are only the tip of the
iceberg. Scanning diverse rice genotypes for their submergence tol-
erance abilities would not only help us identify genotypes with high
tolerance ability to a broad range of flooding types but also discover
unique physiological mechanisms by which rice plants adapt to sub-
mergence stress. Such genotypes could be further used to under-
stand the signalling pathways and molecular mechanisms by which
plant hormones mediate these adaptive responses in rice.
ACKNOWLEDGME NT S
LM thanks the Council of Scientific & Industrial Research, Govern-
ment of India while ST thanks the University Grants Commission,
Government of India for fellowships. AKS thanks TATA Innovation
fellowship from the Department of Biotechnology and Sir J.C. Bose
292 | MITTAL
fellowship from the Science and Engineering Research Board,
Government of India. The authors thank Surender Mittal for critical
reading and editing the manuscript.
CO NFL I CT OF INTERES T S
The authors declare that there are no conflict of interests.
D A TA A V A I L A B I L I T Y S T A T E M E N T
This paper did not use any data.
S UM M A R Y ST A T E M E N T
This review focuses on the hormonal signalling pathways that med-
iate two contrasting responses of the rice plant to submergence
stress namely, rapid internode elongation to escape flood waters and
quiescence response that enables the plant to survive under com-
plete submergence.
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