Determination of Adaptive Mechanisms for Flash Flooding Tolerance in Nepalese Cultivated Rice Genepool based on Morpho-physiological and Molecular Analysis
Bishal Gole Tamang,
Raj Kumar Niroula,
Resham Babu Amgai,
Bindeswar Prasad Sah
Surya Kant Ghimire
Flooding is notorious to rice (Oryza sativa L.) cultivation
at any stages threatening the life of >100 millions people in Asia annually.
Compare to other cereals rice has unusual capacity to tolerate both short and
long term flooding attributed to coordinated efforts of several morphological,
anatomical, biochemical and action of adaptive genes. In this study three hundred
and thirteen Nepalese rice landrace collected from diverse geographic regions
were completely submerged for 14 days. Status of Sub1A gene was monitored
in tolerant rice accessions identified here. Diverse rice accession possessed
differential elongation growth response and their survival ability ranged from
0-100%. Some of the rice accessions performed better than the tolerant check;
FR13A. Monitoring of Sub1A gene presence revealed that some of the tolerant
rice accessions are lacking the Sub1A gene and SublA-1 allele.
Four accessions; Bhaatsaar, Kariyaparewa pakha, Sauthari and Karangi possessed
the better survival response than the FR13A and remaining accessions had poorer
response. Among tolerant accessions; Kariyaparewa pakha and Sauthari were lacking
the Sub1A-1 allele though followed the quiescence growth response. Interestingly,
eight rice accessions with Sub1A-1 allele were found to be intolerant
to 14 days of complete submergence. Unlike FR13A all the tolerant and intolerant
rice accessions displayed the bleaching of chlorophyll pigment resulting the
lower SPAD reading. The detailed morpho-physiological and molecular analysis
unveiled that both tolerant and intolerant rice accessions harbored Sub1A
gene and adapt the both quiescence and escape strategies in response to flash
flooding. Beside Sub1A gene, there might be participation of other stress
responsive factors that probably functions in close coordination with Sub1
haplotype. In conclusion, Sub1A-1 alone is not a major contributing
factor to confer submergence tolerance in diverse rice accessions, thus haplotype
based profiling followed by cloning and sequencing are suggested.
to cite this article:
Bishal Gole Tamang, Raj Kumar Niroula, Resham Babu Amgai, Bindeswar Prasad Sah and Surya Kant Ghimire, 2011. Determination of Adaptive Mechanisms for Flash Flooding Tolerance in Nepalese Cultivated Rice Genepool based on Morpho-physiological and Molecular Analysis. Asian Journal of Plant Sciences, 10: 347-356.
Received: August 06, 2011;
Accepted: October 23, 2011;
Published: December 09, 2011
World rice (Oryza sativa L.) production is limited largely by several
forms of climate change directed environmental stresses, of which flooding induced
submergence is a major constraint worldwide (Xu et al.,
2006). Annually about 16% of the world rice areas are unfavorably submerged
and affecting the livelihood of more than 100 millions people (Hossain
and Abedin, 2004). Rice is unusually adapted to semiaquatic environments
because of its well-developed aerenchyma tissues, that facilitate oxygen diffusion
through continuous air spaces from shoot to root and avoid O2 deficiency
in roots. However, complete submergence due to frequent flooding can adversely
affect the plant growth and yield (Fukao et al., 2006;
Xuet al., 2006). In some of the rainfed paddy
areas, the levels of flood water rise progressively during the rice growing
season and can reach up to 7 meters whereas in others, flash flooding can fully
submerge the plants for a few days to weeks (Voesenek and
Bailey-Serres, 2009). Submergence especially limits the oxygen diffusion
rate by 10-4 fold slower than in air resulting anaerobic metabolism
and energy crisis (Bailey-Serres and Voesenek, 2008;
Licausi and Perata, 2009). As a result, most of the
flood-prone areas are planted with five times less yielding landraces that display
a remarkable ability to adapt either in deepwater or in flash flooding conditions
(Voesenek and Bailey-Serres, 2009). These traditional
rice varieties are tolerant to flooding stress due to various adaptive mechanisms
to survive periods of hypoxia or anoxia (Mackill et al.,
1996; Hattori et al., 2009). An adaptation
to submergence involves alterations in molecular, biochemical, physiological,
genetical and anatomical/ morphological attributes in rice plants. These include
energy generation through fermentative metabolism, aerenchyma development in
parenchymal tissues that improves access to O2, activation of ethylene
promoted gibberellin (GA) mediated internode elongation in deepwater rice to
shootup the foliage above the water surface for gas exchange and restricting
growth and conserving precious energy until floodwater recedes in lowland rice
(Bailey-Serres and Voesenek, 2008; Hattori
et al., 2009). These abilities have enabled the rice crop to cultivate
worldwide ranging from rain-fed and irrigated lowlands to deepwater (Khush,
Recent discovery indicated that both quiescence and escape adaptive mechanisms
displayed by different rice accessions are under genetic control (Xu
et al., 2006; Hattori et al., 2009;
Lee et al., 2009) and explicated in terms of the
major Sub1 QTLs and qTIL12 (SK1 and SK2), respectively
(Xu et al., 2006; Hattori
et al., 2009). These QTLs, were identified in two different ecotypes
of rice cultivated in different water regime, encode different subgroup of ERFs
(Ethylene Responsive Factors) genes whose expression is activated by plant hormone
ethylene. Analysis of near-isogenic and transgenic lines confirmed that Sub1A-1
restricts the escape strategy whereas SNORKELs trigger antithetically
through increase GA production and responsiveness and enable the rice plant
to survive in deepwater conditions. Submergence induced ethylene activates Sub1A-1,
limiting ethylene production by feedback mechanism and promoting GA repressors
SLR1 and SLRL1 (Fukao and Bailey-Serres, 2008; Nagai
et al., 2010). Recent findings based on comparative microarray and
metabolite studies also confirmed that Sub1A-1 regulates numerous transcription
factors associated with stress tolerance responses, supporting the past speculation
based on physiological study (Jung et al., 2010).
Likewise mechanisms of seed germination and underwater seedling growth in the
flooded paddy has also recently revealed in which tolerance is governed by CIPK15
gene encoding Calcineurin B-like-interacting protein kinases (Lee
et al., 2009). All three adaptive genes express independently of
each other and are considered crucial to cope the different types of flooding
stress in the rice field. Despite these knowledges on adaptive mechanisms at
the gene and protein level, our understanding of the diversity in mechanisms
by which different rice accessions survive under varying nature of complete
submergence is still to be discovered. Moreover, the potentiality of local Nepalese
rice accessions has not been assessed yet against the flash flooding. Therefore,
this study was undertaken to dissect the morpho-physiological and molecular
mechanism using diverse germplasm representing all Nepalese rice ecosystem.
In this research, we tried to elucidate the underlying diverse mechanisms that
facilitate the rapid recovery of rice seedlings after flash flooding.
MATERIALS AND METHODS
Seeds of 313 rice accessions, collected from different parts of the country
was reterived from National Genetic Resource Center, Nepal Agricultural Research
Council (NARC), comprising landrace from Terai, Hills and Mountains were subjected
to submergence experiment. For the screening of submergence tolerant plants,
seeds of each rice accession were surface sterilized and soaked over night in
Petri dishes containing sterile water wetted filter paper. Petridishes containing
seed were left for 2-3 days in dark with a temperature of 28±2°C
for germination. Approximately 120 pre-germinated caryopses were sown in two
rows in 5 kg plastic tray (0.38x0.27x0.07 m3) supplied with puddled
soil without fertilizers. Each tray consisted of six accessions and performed
the experiment in CRD (Completely Randomized Design) with three replications
(Fig. 1a). Seedlings were allowed to grow for 14 days at 28±2°C
with a 12 h photoperiod. Seedlings when reached the age of 12 days they were
thinned to 50 seedlings/row. Fourteen days old seedlings were completely submerged
in a 3,300 l water tank (2.44x1.22x1.12 m3) for another 14 days (Fig.
1b). Throughout the experiment the water depth was maintained at 1.05 m.
The survival ability was scored after 7 days of desubmergence and survival percent
over universal check (FR13A) was calculated as: Survival % over check = (% survival
of accessions/% survival of check)x100.
Fourty-four accessions were selected based on their survival percentage (>90
%) and quiscence growth during preliminary glasshouse screening. To determine
the molecular mechanism underlying the tolerance in those selected accessions,
presence of Sub1A gene was monitored using six sets of gene specific
primers as described previously (Xu et al., 2006).
To verify the results of preliminary screening and Sub1A monitoring,
the accessions with and without Sub1A but having more than 90% survival
ability were further subjected to air versus submergence experiment following
CRD with three replications, 15 seedlings/replication in 2 kg plastic pots including
three rice varieties; FR13A, Goda Heenati and Kurkaruppan as tolerant and Nipponbarre
(japonica) as intolerant control. Due to the insufficient amount of seeds,
16 rice accessions were excluded from this experiment. Thus, this confirmatory
(pot) experiment only consisted of 28 rice accessions.
||Initial screening of different rice accessions against flash
flooding. (a) 14 d old rice seedlings from different accession prior to
submergence are laid out in CRD experiment inside the water tank, (b) morphological
response of rice seedlings after submergence, (c) differential survival
response of different rice accessions after desubmergence followed by 7
d recovery and (d) differential elongation response showing both quiescence
and escape strategy adapted by different rice genotypes
Height of the seedling and SPAD reading (SPAD 502, Minolta, Japan) were recorded
from five randomly choosen seedlings/replication before and just after desubmergence
to determine the elongation ability and total chlorophyll content. SPAD reading
was recorded from three positions of a leaf. Survival ability of individual
accession was scored after 14 days of submergence followed by 7 days of recovery.
Genomic DNA of rice accessions was prepared using modified CTAB method as described
by Sul and Korban (1996). PCR reaction was conducted
in the 15 μL volume containing 2 μL (100 ng) of genomic DNA, 1.5 μL
(1 μM) of each primer, 7.5 μL of 2x GoTaq Green PCR Master Mix (Promega
Corporation, Madison, WI, USA) and 2.5 μL PCR H2O. The reaction
mixture without template DNA (12.5 μL) was dispensed in each PCR tube.
Finally, the DNA template from respective rice accessions was added in PCR reaction.
PCR mixture was amplified in a MJ Research PTC-100 Programmable Thermal
Controller (MJ Research, Inc, Watertown, MA, USA.) with the following temperature
regimes: initial denaturation for 2 min at 95°C followed by 32 cycles of
95°C for 30 sec, annealing at 56-60°C depending on the primers TM for
1 min, extension at 72°C for 2 min and final extension at 72°C for 7
min followed by holding at 4°C as described by Xu et
al. (2006). Amplified PCR products were separated in 2% analytical grade
agarose gel (Promega Corporation, Madison, WI, USA) using horizontal gel electrophoresis
unit in 1xTAE (0.11% Glacial Acetic acid, 0.5 M EDTA and 0.04M Tris base) buffer
and run at 100v for 1 h. Gels were stained with 0.1 μg mL-1
ethidium bromide (Promega Corporation, Madison, WI, USA) and then visualized
under UV trans illuminator gel documentation system (Wilber Lourmat, Marne-La-Valleen,
France.) using 1 μg guide size DNA ladder (Genetix, Biotech Asia Pvt. Ltd).
Mean and standard deviation for height and chlorophyll contents and percentage
of survival over check were computed using MS EXCEL Software (2007). Monitoring
of Sub1A was done based on the presence/absence of the band in the respective
lane of the accessions.
Three hundred and thirteen Nepalese rice landrace collected from diverse geographic
regions were completely submerged for 14 days.
||Morphological and physiological response of different rice
accessions after 14 days of complete submergence. (a) Survival response
of selected rice accessions (with and without Sub1A gene) after 7
d of recovery. Mean and standard deviation were calculated from three replications
with 15 seedlings/replication and is presented in the graph, (b) The elongation
growth response of rice seedlings under 14 d of complete submergence. Both
quiescence and escape adaptive mechanisms are shown in the graph. Data were
collected as described earlier, (c) Graph showing SPAD reading to determine
the chlorophyll content before and after submergence
Depending upon the genetic makeup, these accessions possessed differential
growth response and survival ability (Fig. 1c-d).
In the initial screening, the submergence tolerance response was varied from
0-100% (Table 1) and some of the accessions were better than
tolerance check; FR13A (Table 1, Fig. 2a,
3). Based on the result of large scale screening, 44 tolerant
rice accessions were selected for further experiment without knowing the molecular
mechanisms behind the tolerance. To know the molecular regulation of the tolerance
in those selected rice accessions, the presence of major genetic regulator;
Sub1A gene was monitored using Sub1A gene and Sub1A-1 allele
specific primer and found that some of the tolerance rice accessions lacking
both Sub1A gene or SublA-1 allele (Table 2,
Fig. 2a). Though 44 rice accessions were selected for Sub1A
presence monitoring, unfortunately high stringent submergence experiment using
all accessions could not be performed due to lack of adequate number of rice
seeds. Therefore, the comparative high throughput screening was restricted to
only 28 accessions. In the repeated experiment some of the rice accessions found
to be tolerant in earlier experiment showed the intolerancy (Fig.
2a). Four accessions; Bhaatsaar, Kariyaparewa pakha, Sauthari and Karangi
possessed the better survival response than the FR13A and rest of the accessions
had poorer response. Among tolerant accession; Kariyaparewa pakha and Sauthari
were lacking the Sub1A-1 allele and also followed the quiescence growth
response (Fig. 3a, b, 2b).
|| List of rice accessions used and their survival ability
||Monitoring of Sub1A gene in 44 selected rice accessions.
Six sets of Sub1A locus specific primer were used as reported earlier
(Xu et al., 2006). (a) DNA amplification
profiling to detect the presence and absence of Sub1A gene using
Sub1-A-7 primer couple, (b) Detection of Sub1A-1 allele in
tolerance and intolerance rice accessions using allele specific primer pair;
Sub1-A-1. The detail information about the primer used are provided
in the Table 3. Number in each lane represents the rice
accessions as 1 = NPGR-3275, 2 = Goda Heenati, 3 = NPGR-3306, 4 = NPGR-3308,
5 = NPGR-3154, 6 = NPGR-3150, 7 = Nipponbarre, 8 = FR13A, 9 = NPGR-3148,
10 = NPGR-3277, 11 = NPGR-3200, 12 = NPGR-3297, 13 = NPGR-3139, 14 = NPGR-3278,
15 = NPGR-3212, 16 = NPGR-3281, 17 = Guthani Saro, 18 = Jharlajhi, 19 =
Rango, 20 = Badhudayan-2, 21 = Mutmud, 22 = Anjana, 23 = Pakhal, 24 = Silhaat,
25 = Belasaro, 26 = Jagad, 27 = Kataush, 28 = Batsar, 29 = Brahambhushi,
30 = Mutmur, 31 = Lalchand, 32 = Jagranthiya, 33 = Wardhar, 34 = Gorra,
35 = Bhatti, 36 = Lajhi, 37 = Raimanuwa, 38 = Karangi=, 39 = Chyaangnya,
40 = Gajargaul , 41 = Kariyaparewa pakha 42 = Nirmoe, 43 = Sauthari, 44
= Bhaatsaar, 45 = Sikichand, 46 = Bhelasaro, 47 = Kurkaruppan and 48 = Bagadi-2
|| Selected tolerant rice accession with their mean survival
ability and status of Sub1A gene
|ND: Not detected due to lack of enough number of rice seed
to repeat the experiment
|| List of primers used for Sub1A monitoring in selected
Nepalese rice accessions
Interestingly, eight rice accessions possessed the presence of Sub1A-1 allele
but did not tolerate 14 days of complete submergence (Table 2,
Fig. 2a). In this experiment, though accessions; NPGR-3148,
-3139 displayed the presence of Sub1A-1allele, they followed the escape
adaptive strategy resulting the very poor survival ability under flash flooding
(Fig. 2b). The chlorophyll retention after submergence was
highest in FR13A than the tolerant accessions identified in this study. Unlike
FR13A all the tolerant and intolerant rice accessions displayed the bleaching
of chlorophyll pigment resulting the lower SPAD reading (Fig.
Flash flooding tolerance response in rice genotypes that have been evaluated
so far harbor Sub1A-1 allele of Sub1A locus and follow an energy
saving quiescence growth mechanism (Xu et al., 2006;
Bailey-Serres and Voesenek, 2008; Singh
et al., 2010). Rice accessions with haplotypes other than Sub1A-1/Sub1C-1
are intolerant to submergence (Singh et al., 2010).
Similarly, deepwater response in rice and hypoxia tolerance in Arabidopsis
are controlled by SK1 and SK2 and RAP2.12, HRE1 and
HRE2 belonging to different members of subgroup VII of the ERF transcription
factor family, respectively (Hattori et al., 2009;
Licausi et al., 2011). Despite these earlier findings
(Fukao et al., 2006; Xu et
al., 2006; Fukao et al., 2011), in the
present study some of the rice accessions such as Kariyaparewa pakha and Sauthari
lacking Sub1A-1 allele displayed the significant level of tolerance.
Interestingly, contrasting response was also observed for rice accessions namely
NPGR 3139, NPGR 3281, Jharlajhi, Raimanuwa, NPGR 3277, NPGR 3148, NPGR 3275
and Batsar. These rice accessions contained Sub1A-1 allele but the recovery
rate was close to nil (Table 2, Fig. 3b).
||Representative photographs showing 7 d of recovery after 14
d of complete submergence in selected rice accessions
The poor survival ability in those genotypes could be explained by extra costing
of energy for rapid stem elongation response (Fig. 2b) which
is to be considered disadvantage for faster regeneration (Perata
and Voesenek, 2007; Colmer and Voesenek, 2009).
Underwater elongation in rice is triggered by ethylene and GA (Fukao
et al., 2006). It is suggested that, in the absence of Sub1A-1,
Sub1C facilitates shoot elongation during drowning, through a GA-dependent
mechanism (Fukao and Bailey-Serres, 2008). Sub1A-1
reverses the ethylene-dependent increase in GA responsiveness and consequent
Sub1C mRNA accumulation. However, based on recombinant genetic studies,
Sub1A seems to be the major determinant of submergence tolerance, as
Sub1C gene expression does not significantly affect the level of tolerance
(Septiningsih et al., 2009).
This study also clearly indicated that the chlorophyll retention capacity after
submergence varied with the overall genetic make up rather than the presence
of Sub1A-1 allele. FR13A was found to be robust interms of chlorophyll
retention capacity and the slow depletion mechanism of chlorophyll could be
plus that helps to replenish the energy crisis through instant photosynthesis
during recovery (Fig. 2c, 4). Positive correlation
between survival and chlorophyll content after submergence was also reported
by Das et al. (2009). However, most tolerant
rice accessions identified in this study such as Bhaatsaar, Karangi and Sauthari
possessed rapid depletion of chlorophyll signifying that chlorophyll retention
capacity of the genotypes is independent of submergence tolerance trait. Compare
to tolerant check, four genotypes viz. Bhaatsaar, Kariyaparewa pakha, Sauthari
and Karangi performed better however, the mechanism of regeneration was quite
dissimilar (Fig. 4). In these accessions most of the older
leaves died soon after the desubmergence but regeneration of new leaves was
abruptly rapid (Fig. 4 and Table 2). Unlike
these accessions, upon desubmergence the older leaves of FR13A showed low level
of senescent and regained its normal growth very soon.
Based on submergence response and monitoring of Sub1A gene among the
28 rice accessions we hypothesized that in the absence of gene regulating elongation
response, the survival ability is independent of Sub1A-1. On the other
hand, in the presence of gene responsible for energy consumption process and
absence of Sub1 haplotype (Sub1A-1/Sub1C-1), Sub1A-1 allelic
form alone is not sufficient to confer the submergence tolerance. To validate
these hypotheses Sub1 haplotypic analysis, transcriptomic and hormonal
profilings are underway.
Following large scale screening of 313 diverse rice accessions we are able to identify four tolerant genotypes. These genotypes were with and without Sub1A-1 allele. From the perspective of rice improvement these germplasm constitute the great assets to broaden the narrow genetic base of submergence tolerance trait. Despite their potential importance, the exact mechanisms of tolerance in those accession are to be unraveled. Therefore, to dissect the underlying hidden mechanisms, further in-depth study using a combined developmental, physiological and omics approaches are suggested.
This research was conducted from the financial support under the Generation
Challenge Program (GCP) of Global Biodiversity Trust (Grant No. 10027). Authors
are grateful to National Genetic Resource Center (NGRC/NARC Nepal) for providing
the seed of the rice accessions included in this study. The laboratory facility
of Biotechnology Unit (NARC-Nepal) and its supportive staffs are also gratefully
acknowledged for their contribution in DNA extraction and data scoring.
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