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1.  Tolerance of anaerobic conditions caused by flooding during germination and early growth in rice (Oryza sativa L.) 
Rice is semi-aquatic, adapted to a wide range of hydrologies, from aerobic soils in uplands to anaerobic and flooded fields in waterlogged lowlands, to even deeply submerged soils in flood-prone areas. Considerable diversity is present in native rice landraces selected by farmers over centuries. Our understanding of the adaptive features of these landraces to native ecosystems has improved considerably over the recent past. In some cases, major genes associated with tolerance have been cloned, such as SUB1A that confers tolerance of complete submergence and SNORKEL genes that control plant elongation to escape deepwater. Modern rice varieties are sensitive to flooding during germination and early growth, a problem commonly encountered in rainfed areas, but few landraces capable of germination under these conditions have recently been identified, enabling research into tolerance mechanisms. Major QTLs were also identified, and are being targeted for molecular breeding and for cloning. Nevertheless, limited progress has been made in identifying regulatory processes for traits that are unique to tolerant genotypes, including faster germination and coleoptile elongation, formation of roots and leaves under hypoxia, ability to catabolize starch into simple sugars for subsequent use in glycolysis and fermentative pathways to generate energy. Here we discuss the state of knowledge on the role of the PDC-ALDH-ACS bypass and the ALDH enzyme as the likely candidates effective in tolerant rice genotypes. Potential involvement of factors such as cytoplasmic pH regulation, phytohormones, reactive oxygen species scavenging and other metabolites is also discussed. Further characterization of contrasting genotypes would help in elucidating the genetic and biochemical regulatory and signaling mechanisms associated with tolerance. This could facilitate breeding rice varieties suitable for direct seeding systems and guide efforts for improving waterlogging tolerance in other crops.
PMCID: PMC3719019  PMID: 23888162
anaerobic germination; alcoholic fermentation; ALDH; pyruvate dehydrogenase bypass; hypoxia; direct seeding; flooding; submergence tolerance
2.  Gas film retention and underwater photosynthesis during field submergence of four contrasting rice genotypes 
Journal of Experimental Botany  2014;65(12):3225-3233.
The flood-tolerant genotype FR13A retains leaf gas films and its capacity for underwater net photosynthesis, whereas gas films are lost faster and photosynthesis declines markedly in sensitive genotypes.
Floods can completely submerge some rice (Oryza sativa L.) fields. Leaves of rice have gas films that aid O2 and CO2 exchange under water. The present study explored the relationship between gas film persistence and underwater net photosynthesis (PN) as influenced by genotype and submergence duration. Four contrasting genotypes (FR13A, IR42, Swarna, and Swarna-Sub1) were submerged for 13 days in the field and leaf gas films, chlorophyll, and the capacity for underwater PN at near ambient and high CO2 were assessed with time of submergence. At high CO2 during the PN assay, all genotypes initially showed high rates of underwater PN, and this rate was not affected by time of submergence in FR13A. This superior photosynthetic performance of FR13A was not evident in Swarna-Sub1 (carrying the SUB1 QTL) and the declines in underwater PN in both Swarna-Sub1 and Swarna were equal to that in IR42. At near ambient CO2 concentration, underwater PN declined in all four genotypes and this corresponded with loss of leaf gas films with time of submergence. FR13A retained leaf gas films moderately longer than the other genotypes, but gas film retention was not linked to SUB1. Diverse rice germplasm should be screened for gas film persistence during submergence, as this trait could potentially increase carbohydrate status and internal aeration owing to increased underwater PN, which contributes to submergence tolerance in rice.
PMCID: PMC4071835  PMID: 24759881
Aerenchyma; flooding stress; leaf gas films; leaf air layer; leaf hydrophobicity; Oryza sativa; submergence tolerance; SUB1; leaf chlorophyll; survival; FR13A; IR42; Swarna; Swarna-Sub1.
3.  Variation in tolerance of rice to long-term stagnant flooding that submerges most of the shoot will aid in breeding tolerant cultivars 
AoB Plants  2014;6:plu055.
Long-term stagnant flooding (SF, 50 cm water depth) is a major problem in rainfed lowland rice. We established a phenotyping protocol and identified tolerant landraces. Genotypes best suited to SF showed moderate elongation of 1.3–2.3 cm d−1 under SF, but semi-dwarf and fast-elongating types are intolerant. Dwarf varieties containing SUB1 are sensitive, but taller varieties with SUB1 are tolerant, suggesting the possibility of combining tolerances to complete submergence and SF. Tolerance of SF is dependent on moderate elongation, high tillering, lesser carbohydrate depletion and higher fertility. Tolerant genotypes identified here will be used for mechanistic studies and in breeding.
Stagnant flooding (SF) is a major problem in rainfed lowlands where floodwater of 25–50 cm stagnates in the field for most of the season. We aimed to establish a system for phenotyping SF tolerance and identifying tolerant germplasm through screening of landraces. A total of 626 rice accessions were evaluated over 3 years under control conditions and two levels of SF. Floodwater was raised to 20 cm at 25 or 30 days after transplanting (DAT). In one trial, the depth was increased subsequently by 5 cm a week and in another (severe stress), it was increased to 40 cm at 37 DAT and to 50 cm at 42 DAT. In both trials, water depth was maintained at 50–60 cm until maturity. In all cases, no plant was completely submerged. Plant height, elongation rate and yield were measured at maturity. Genotypes best suited to SF showed moderate elongation of 1.3–2.3 cm day−1 under SF. In contrast, semi-dwarf and fast-elongating types performed poorly. Subsequent trials using 18 genotypes, including six pairs of near isogenic lines (NILs) with or without SUB1 showed that all SUB1 NILs were sensitive to SF. Five of the other six genotypes contained SUB1 and were SF tolerant, suggesting the possibility of combining tolerances to complete submergence (SUB1) and SF. Stem starch and soluble sugar concentrations were similar under control conditions among the 18 genotypes, but starch was depleted by 37 % under SF, with less depletion in tolerant genotypes. SUB1 NILs contained similar concentrations of starch and sugars under SF. We conclude that survival and yield under SF are dependent on moderate elongation, high tillering, lesser carbohydrate depletion and higher fertility. The tolerant genotypes identified here performed strongly in both wet and dry seasons and will be used to identify tolerance mechanisms and alleles for use in marker-assisted breeding.
PMCID: PMC4196555  PMID: 25202124
Carbohydrates; rice; stagnant flooding; SUB1; tolerance mechanisms.
4.  Physiological analyses of traits associated with tolerance of long-term partial submergence in rice 
AoB Plants  2014;6:plu058.
Long-term stagnant flooding (SF, 50 cm water depth) greatly reduces rice yield. We assessed physiological mechanisms associated with SF tolerance in contrasting rice genotypes. SF reduced yield by 47% due to low biomass caused by reduced light interception and leaf growth above water; and reduced panicle number by 52% because of low tillering. Shoot elongation correlated positively with leaf growth and biomass production, but negatively with stem nonstructural carbohydrates (NSC). Tolerant varieties were either inherently tall or elongate moderately (<2.0 cm d−1) with rising floodwater. Optimum shoot elongation with rising floodwater is therefore a priority for future breeding work.
Floods are major constraints to crop production worldwide. In low-lying, flood-prone areas of the tropics, longer-term partial submergence (stagnant flooding [SF]) greatly reduces rice yield. This study assesses shoot growth and several physiological mechanisms associated with SF tolerance in rice. Five rice genotypes with contrasting responses to SF were evaluated in field ponds. Following transplanting, floodwater was gradually increased at a rate of ∼2 cm day−1 to reach a final depth of 50 cm and then maintained until maturity. Although plants were not fully submerged, the yield was reduced by 47 % across genotypes compared with those grown under control conditions (6.1 vs. 3.3 t ha−1). This reduction was mainly attributed to the reduction in biomass caused by reduced light interception and leaf growth above the water. Stagnant flooding also reduced panicle number per unit area by 52 % because of reduced tillering. Shoot elongation rate kept pace with rising floodwater and correlated positively with leaf growth and biomass production. Conversely, stem non-structural carbohydrate (NSC) concentration correlated negatively with shoot elongation rate, suggesting that fast-elongating genotypes actively consume NSCs to avoid complete submergence. Moderate shoot elongation rate strongly and positively correlated with grain yield under SF; however, elongation at rates >2.0 cm day−1 was associated with reduced harvest index due to a smaller panicle size and increased lodging. Tolerant varieties were found to be either inherently tall or elongate moderately with rising floodwater. Our studies suggest that to improve tolerance of SF an appropriate phenotype should combine both of these traits. Fine-tuning for optimum shoot elongation with rising floodwater is, therefore, a priority for future work.
PMCID: PMC4216431  PMID: 25270231
Flooding stress; rainfed lowland rice; shoot elongation; stagnant flooding; tillering
5.  Differences in responses to flooding by germinating seeds of two contrasting rice cultivars and two species of economically important grass weeds 
AoB Plants  2014;6:plu064.
Barnyard grasses are serious weeds in direct seeded rice. We assessed the effectiveness of using controlled flooding for its control using two rice cultivars and two barnyard grasses contrasting in flood tolerance during germination. Flooding with 100 mm water after seeding suppressed barnyard grasses; but delaying flooding by 2-4 days was ineffective. Flooding increased the activity of alcohol dehydrogenase and pyruvate decarboxylase; the increase was higher in the tolerant rice cultivar but similar in both barnyard grasses. Aldehyde dehydrogenase activity increased only in flood-tolerant types of rice and weeds, but not in flood-sensitive types, implying potential role in tolerance.
Crop productivity is largely affected by abiotic factors such as flooding and by biotic factors such as weeds. Although flooding after direct seeding of rice helps suppress weeds, it also can adversely affects germination and growth of rice, resulting in poor crop establishment. Barnyard grasses (Echinochloa spp.) are among the most widespread weeds affecting rice, especially under direct seeding. The present work aimed to establish effective management options to control these weeds. We assessed the effects of variable depths and time of submergence on germination, seedling growth and carbohydrate metabolism of (i) two cultivars of rice known to differ in their tolerance to flooding during germination and (ii) two barnyard grasses (Echinochloa colona and E. crus-galli) that commonly infest rice fields. Flooding barnyard grasses with 100-mm-deep water immediately after seeding was effective in suppressing germination and growth. Echinochloa colona showed greater reductions in emergence, shoot and root growth than E. crus-galli. Delaying flooding for 2 or 4 days was less injurious to both species. Echinochloa colona was also more susceptible to flooding than the flood-sensitive rice cultivar ‘IR42’. The activity of alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC) in rice seedlings was increased by flooding after sowing but with greater increases in ‘Khao Hlan On’ compared with ‘IR42’. The activity of ADH and PDC was enhanced to a similar extent in both barnyard grasses. Under aerobic conditions, the activity of ADH and PDC in the two barnyard grasses was downregulated, which might contribute to their inherently faster growth compared with rice. Aldehyde dehydrogenase activity was significantly enhanced in flood-tolerant ‘Khao Hlan On’ and E. crus-galli, but did not increase in flood-sensitive E. colona and ‘IR42’, implying a greater ability of the flood-tolerant types to detoxify acetaldehyde generated during anaerobic fermentation. Confirmation of this hypothesis is now being sought.
PMCID: PMC4243074  PMID: 25336336
Alcohol dehydrogenase; aldehyde dehydrogenase; anaerobic germination; barnyard grass; direct-seeded rice; Echinochloa colona; Echinochloa crus-galli; fermentative metabolism; pyruvate decarboxylase; rice weeds.
6.  Physiological basis of tolerance to complete submergence in rice involves genetic factors in addition to the SUB1 gene 
AoB Plants  2014;6:plu060.
Complete submergence reduces survival and yield of modern high-yielding rice cultivars. Tolerant cultivars carrying the SUB1 locus were recently developed. We evaluated survival and growth processes occurring during submergence and recovery that are associated with SUB1 in contrasting genotypes. Sub1 cultivars showed less reduction in shoot biomass and better regulation of non-structural carbohydrates during submergence. They produced new leaves and tillers faster during recovery. FR13A, the source of SUB1 locus showed slower leaf elongation and recovered faster than Sub1 lines, suggesting the possibility of further improvements in submergence tolerance by incorporating additional traits from FR13A or similar landraces.
Recurring floods in Asia cause poor crop establishment. Yields decline drastically when plants are completely submerged for a few days. Traditional rice cultivars predominate because they have acquired moderate tolerance to flooding but they carry the penalty of inherently lower grain yields. In contrast, modern high-yielding varieties are highly susceptible to flooding. Cultivars with tolerance to complete submergence were recently developed in the background of popular varieties by transferring the submergence tolerance gene SUBMERGENCE1 (SUB1) from the highly tolerant Indian landrace FR13A. The present study evaluated three pairs of Sub1 near-isogenic lines (NILs) together with FR13A and two of its submergence-tolerant derivatives under field conditions to assess the survival and growth processes occurring during submergence and recovery that are associated with SUB1. Under control conditions, the NILs showed similar growth and biomass accumulation, indicating that SUB1 had no apparent effects. Submergence substantially decreased biomass accumulation but with greater reduction in the genotypes lacking SUB1, particularly when submergence was prolonged for 17 days. When submerged, the lines lacking SUB1 showed greater elongation and lower or negative biomass accumulation. Sub1 lines maintained higher chlorophyll concentrations during submergence and lost less non-structural carbohydrates (NSC) after submergence. This indicates that the introgression of SUB1 resulted in better regulation of NSC during submergence and that high pre-submergence NSC is not essential for the submergence tolerance conferred by SUB1. During recovery, chlorophyll degradation was faster in genotypes lacking SUB1 and any surviving plants showed poorer and delayed emergence of tillers and leaves. Sub1 lines restored new leaf and tiller production faster. During submergence, FR13A showed not only slower leaf elongation but also accumulated extra biomass and was able to recover faster than Sub1 lines. This suggests the possibility of further improvements in submergence tolerance by incorporating additional traits present in FR13A or other similar landraces.
PMCID: PMC4243076  PMID: 25281725
Flash floods; rainfed lowlands; recovery of submergence; rice; submergence tolerance; SUB1; tillering.
7.  Zn uptake, translocation and grain Zn loading in rice (Oryza sativa L.) genotypes selected for Zn deficiency tolerance and high grain Zn 
Journal of Experimental Botany  2013;64(10):2739-2751.
Zn deficiency is a widespread problem in rice (Oryza sativa L.) grown under flooded conditions, limiting growth and grain Zn accumulation. Genotypes with Zn deficiency tolerance or high grain Zn have been identified in breeding programmes, but little is known about the physiological mechanisms conferring these traits. A protocol was developed for growing rice to maturity in agar nutrient solution (ANS), with optimum Zn-sufficient growth achieved at 1.5 μM ZnSO4.7H2O. The redox potential in ANS showed a decrease from +350 mV to −200 mV, mimicking the reduced conditions of flooded paddy soils. In subsequent experiments, rice genotypes contrasting for Zn deficiency tolerance and grain Zn were grown in ANS with sufficient and deficient Zn to assess differences in root uptake of Zn, root-to-shoot Zn translocation, and in the predominant sources of Zn accumulation in the grain. Zn efficiency of a genotype was highly influenced by root-to-shoot translocation of Zn and total Zn uptake. Translocation of Zn from root to shoot was more limiting at later growth stages than at the vegetative stage. Under Zn-sufficient conditions, continued root uptake during the grain-filling stage was the predominant source of grain Zn loading in rice, whereas, under Zn-deficient conditions, some genotypes demonstrated remobilization of Zn from shoot and root to grain in addition to root uptake. Understanding the mechanisms of grain Zn loading in rice is crucial in selecting high grain Zn donors for target-specific breeding and also to establish fertilizer and water management strategies for achieving high grain Zn.
PMCID: PMC3697949  PMID: 23698631
Agar nutrient solution; grain Zn; grain Zn loading; remobilization; rice; Zn deficiency tolerance; Zn efficiency; Zn translocation; Zn uptake.
8.  Salinity tolerance, Na+ exclusion and allele mining of HKT1;5 in Oryza sativa and O. glaberrima: many sources, many genes, one mechanism? 
BMC Plant Biology  2013;13:32.
Cultivated rice species (Oryza sativa L. and O. glaberrima Steud.) are generally considered among the crop species most sensitive to salt stress. A handful of lines are known to be tolerant, and a small number of these have been used extensively as donors in breeding programs. However, these donors use many of the same genes and physiological mechanisms to confer tolerance. Little information is available on the diversity of mechanisms used by these species to cope with salt stress, and there is a strong need to identify varieties displaying additional physiological and/or genetic mechanisms to confer higher tolerance.
Here we present data on 103 accessions from O. sativa and 12 accessions from O. glaberrima, many of which are identified as salt tolerant for the first time, showing moderate to high tolerance of high salinity. The correlation of salinity-induced senescence (as judged by the Standard Evaluation System for Rice, or SES, score) with whole-plant and leaf blade Na+ concentrations was high across nearly all accessions, and was almost identical in both O. sativa and O. glaberrima. The association of leaf Na+ concentrations with cultivar-groups was very weak, but association with the OsHKT1;5 allele was generally strong. Seven major and three minor alleles of OsHKT1;5 were identified, and their comparisons with the leaf Na+ concentration showed that the Aromatic allele conferred the highest exclusion and the Japonica allele the least. A number of exceptions to this association with the Oryza HKT1;5 allele were identified; these probably indicate the existence of additional highly effective exclusion mechanisms. In addition, two landraces were identified, one from Thailand and the other from Senegal, that show high tissue tolerance.
Significant variation in salinity tolerance exists within both cultivated Oryza species, and this is the first report of significant tolerance in O. glaberrima. The majority of accessions display a strong quantitative relationship between tolerance and leaf blade Na+ concentration, and thus the major tolerance mechanisms found in these species are those contributing to limiting sodium uptake and accumulation in active leaves. However, there appears to be genetic variation for several mechanisms that affect leaf Na+ concentration, and rare cases of accessions displaying different mechanisms also occur. These mechanisms show great promise for improving salt tolerance in rice over that available from current donors.
PMCID: PMC3599985  PMID: 23445750
Allele mining; HKT; Mechanisms of salt tolerance; Rice; Salinity
9.  Molecular Breeding to Improve Salt Tolerance of Rice (Oryza sativa L.) in the Red River Delta of Vietnam 
Rice is a stable food in Vietnam and plays a key role in the economy of the country. However, the production and the cultivating areas are adversely affected from the threats of devastation caused by the rise of sea level. Using marker-assisted backcrossing (MABC) to develop a new salt tolerance rice cultivar is one of the feasible methods to cope with these devastating changes. To improve rice salt tolerance in BT7 cultivar, FL478 was used as a donor parent to introgress the Saltol QTL conferring salt tolerance into BT7. Three backcrosses were conducted and successfully transferred positive alleles of Saltol from FL478 into BT7. The plants numbers IL-30 and IL-32 in BC3F1 population expected recurrent genome recovery of up to 99.2% and 100%, respectively. These selected lines that carried the Saltol alleles were screened in field for their agronomic traits. All improved lines had Saltol allele similar to the donor parent FL478, whereas their agronomic performances were the same as the original BT7. We show here the success of improving rice salt tolerance by MABC and the high efficiency of selection in early generations. In the present study, MABC has accelerated the development of superior qualities in the genetic background of BT7.
PMCID: PMC3544250  PMID: 23326259
10.  Adaptation to flooding during emergence and seedling growth in rice and weeds, and implications for crop establishment 
AoB Plants  2012;2012:pls019.
Direct seeding is replacing transplanting in rice. Early flooding suppresses weeds but selective action is compromised by the sharing of flood-tolerance traits. Understanding adaptive traits in both species is therefore a prerequisite for developing direct seeding systems that control weeds while leaving rice seedlings relatively unharmed.
Background and aims
Direct seeding of rice is being adopted in rainfed and irrigated lowland ecosystems because it reduces labour costs in addition to other benefits. However, early flooding due to uneven fields or rainfall slows down seed germination and hinders crop establishment. Conversely, early flooding helps suppress weeds and reduces the costs of manual weeding and/or dependence on herbicides; however, numerous weed species are adapted to lowlands and present challenges for the use of flooding to control weeds. Advancing knowledge on the mechanisms of tolerance of flooding during germination and early growth in rice and weeds could facilitate the development of improved rice varieties and effective weed management practices for direct-seeded rice.
Principal results
Rice genotypes with a greater ability to germinate and establish in flooded soils were identified, providing opportunities to develop varieties suitable for direct seeding in flooded soils. Tolerance of flooding in these genotypes was mostly attributed to traits associated with better ability to mobilize stored carbohydrates and anaerobic metabolism. Limited studies were undertaken in weeds associated with lowland rice systems. Remaining studies compared rice and weeds and related weed species such as Echinochloa crus-galli and E. colona or compared ecotypes of the same species of Cyperus rotundus adapted to either aerobic or flooded soils.
Tolerant weeds and rice genotypes mostly developed similar adaptive traits that allow them to establish in flooded fields, including the ability to germinate and elongate faster under hypoxia, mobilize stored starch reserves and generate energy through fermentation pathways. Remarkably, some weeds developed additional traits such as larger storage tubers that enlarge further in deeper flooded soils (C. rotundus). Unravelling the mechanisms involved in adaptation to flooding will help design management options that will allow tolerant rice genotypes to adequately establish in flooded soils while simultaneously suppressing weeds.
PMCID: PMC3434364  PMID: 22957137
11.  Seed pre-treatment in rice reduces damage, enhances carbohydrate mobilization and improves emergence and seedling establishment under flooded conditions 
AoB Plants  2011;2011:plr007.
Priming rice seeds (soaking followed by drying) or soaking just before sowing improved emergence from flooded soil, reduced membrane damage from ROS and hastened carbohydrate mobilization. Most benefit was to lines with a superior ability to germinate in flooded soil even when untreated.
Background and aims
Early flooding helps control weeds but reduces seedling establishment in direct-seeded rice (Oryza sativa). When combined with appropriate management practices, the use of genotypes that better tolerate flooding during emergence can enhance crop establishment in flood-prone areas. Management options include seed pre-treatment and we tested the influence of pre-soaking for 24 h prior to sowing or of priming (soaking for 24 or 48 h followed by drying).
The effects on seedling establishment after 21-day flooding of pre-soaking seeds for 24 h before sowing and/or of priming seeds were examined together with physiological responses connected with reactive oxygen scavenging. Seeds of four lines with contrasting abilities to tolerate flooding at the germination stage were compared. Seeds were primed using KCl solutions (48 h) or water (24 h) and pre-soaked using water. Lipid peroxidation and activities of reactive oxygen-scavenging enzymes were measured in seeds before sowing. Carbohydrate mobilization in germinating seeds and seedling growth were also monitored at intervals.
Principal results
Seed pre-treatment by pre-soaking or by priming increased survival of flooding and accelerated and improved seedling establishment, especially in tolerant genotypes. Primed seeds had less lipid peroxidation and higher superoxide dismutase (SOD) and catalase (CAT) activities than non-primed seeds. Amylase activities and starch breakdown were also hastened in primed seeds. Survival after flooding was positively correlated with amylase activity but negatively correlated with the extent of lipid peroxidation.
Pre-soaking and priming improved seedling establishment in flooded soil, enhanced the capacity to scavenge reactive oxygen species in seeds by increasing SOD and CAT activities, and hastened carbohydrate mobilization. Tolerant genotypes responded better to these treatments, emphasizing the effectiveness of combining genetic tolerance with appropriate seed pre-treatment to improve seedling establishment of rice sown in flooded soils.
PMCID: PMC3072768  PMID: 22476478
12.  Morphological and physiological responses of lowland purple nutsedge (Cyperus rotundus L.) to flooding 
AoB Plants  2010;2010:plq010.
Comparing a lowland and an upland ecotype of Cyperus rotundus, the former had greater carbohydrate reserves in tubers, thicker roots and stems with larger air spaces and, under hypoxia, it maintained relatively lower activities of alcohol dehydrogenase and lactate dehydrogenase.
Background and aims
Purple nutsedge (Cyperus rotundus L.) is a major weed of upland crops and vegetables. Recently, a flood-tolerant ecotype evolved as a serious weed in lowland rice. This study attempted to establish the putative growth and physiological features that led to this shift in adaptation.
Tubers of upland C. rotundus (ULCR) and lowland C. rotundus (LLCR) ecotypes were collected from their native habitats and maintained under the respective growth conditions in a greenhouse. Five experiments were conducted to assess the variation between the two ecotypes in germination, growth and tuber morphology when grown in their native or ‘switched’ conditions. Carbohydrate storage and mobilization, and variation in anaerobic respiration under hypoxia were compared.
Principal results
Tubers of LLCR were larger than those of ULCR, with higher carbohydrate content, and larger tubers developed with increasing floodwater depth. Stems of LLCR had larger diameter and proportionally larger air spaces than those of ULCR: a method of aerating submerged plant parts. The LLCR ecotype can also mobilize and use carbohydrate reserves under hypoxia, and it maintained relatively lower and steadier activity of alcohol dehydrogenase (ADH) as a measure of sustained anaerobic respiration. In contrast, ADH activity in ULCR increased faster upon a shift to hypoxia and then sharply decreased, suggesting depletion of available soluble sugar substrates. The LLCR ecotype also maintained lower lactate dehydrogenase activity under flooded conditions, which could reduce chances of cellular acidosis.
These adaptive traits in the LLCR ecotype were expressed constitutively, but some of them, such as tuber growth and aerenchyma development, are enhanced with stress severity. The LLCR ecotype attained numerous adaptive traits that could have evolved as a consequence of natural evolution or repeated management practices, and alternative strategies are necessary because flooding is no longer a feasible management option.
PMCID: PMC3000701  PMID: 22476068
13.  Mechanisms associated with tolerance to flooding during germination and early seedling growth in rice (Oryza sativa) 
Annals of Botany  2008;103(2):197-209.
Background and Aims
Flooding slows seed germination, imposes fatalities and delays seedling establishment in direct-seeded rice. This study describes responses of contrasting rice genotypes subjected to flooding or low oxygen stress during germination and discusses the basis of tolerance shown by certain cultivars.
In one set of experiments, dry seeds were sown in soil and either watered normally or flooded with 10 cm of water. Seedling survival and shoot and root growth were assessed and seed portions of germinating seedlings were assayed for soluble sugars and starch concentrations. The whole germinating seedlings were assayed for amylase and peroxidase activities and for ethylene production. Activities of enzymes associated with anaerobic respiration were examined and gene expression was analysed separately with seeds germinating under different amounts of dissolved oxygen in dilute agar.
Key Results
Flooding during germination reduced survival but to a lesser extent in tolerant genotypes. Starch concentration in germinating seeds decreased while sugar concentration increased under flooding, but more so in tolerant genotypes. Amylase activity correlated positively with elongation (r = 0·85 for shoot and 0·83 for root length) and with plant survival (r = 0·92). Tolerant genotypes had higher amylase activity and higher RAmy3D gene expression. Ethylene was not detected in seeds within 2 d after sowing, but increased thereafter, with a greater increase in tolerant genotypes starting 3 d after sowing. Peroxidase activity was higher in germinating seeds of sensitive genotypes and correlated negatively with survival.
Under low oxygen stress, tolerant genotypes germinate, grow faster and more seedlings survive. They maintain their ability to use stored starch reserves through higher amylase activity and anaerobic respiration, have higher rates of ethylene production and lower peroxidase activity as germinating seeds and as seedlings. Relevance of these traits to tolerance of flooding during germination and early growth is discussed.
PMCID: PMC2707318  PMID: 19001425
Amylase; anoxia; crop establishment; direct-seeded rice; ethylene; flooding; germination; hypoxia; Oryza sativa
14.  Development of submergence-tolerant rice cultivars: the Sub1 locus and beyond 
Annals of Botany  2008;103(2):151-160.
Background and Aims
Submergence is a recurring problem in the rice-producing rainfed lowlands of south and south-east Asia. Developing rice cultivars with tolerance of submergence and with agronomic and quality traits acceptable to farmers is a feasible approach to address this problem. The objectives of this study were to (a) develop mega varieties with Sub1 introgression that are submergence tolerant, (b) assess the performance of Sub1 in different genetic backgrounds, (c) determine the roles of the Sub1A and Sub1C genes in conferring tolerance, and (d) assess the level of tolerance in F1 hybrids heterozygous for the Sub1A-1-tolerant allele.
Tolerant varieties were developed by marker-assisted backcrossing through two or three backcrosses, and their performance was evaluated to determine the effect of Sub1 in different genetic backgrounds. The roles of Sub1A and Sub1C in conferring the tolerant phenotype were further investigated using recombinants identified within the Sub1 gene cluster based on survival and gene expression data.
Key Results
All mega varieties with Sub1 introgression had a significantly higher survival rate than the original parents. An intolerant Sub1C allele combined with the tolerant Sub1A-1 allele did not significantly reduce the level of tolerance, and the Sub1C-1 expression appeared to be independent of the Sub1A allele; however, even when Sub1C-1 expression is completely turned off in the presence of Sub1A-2, plants remained intolerant. Survival rates and Sub1A expression were significantly lower in heterozygotes compared with the homozygous tolerant parent.
Sub1 provided a substantial enhancement in the level of tolerance of all the sensitive mega varieties. Sub1A is confirmed as the primary contributor to tolerance, while Sub1C alleles do not seem important. Lack of dominance of Sub1 suggests that the Sub1A-1 allele should be carried by both parents for developing tolerant rice hybrids.
PMCID: PMC2707316  PMID: 18974101
Oryza sativa; Sub1; marker-assisted backcrossing; mega varieties; submergence tolerance; recombinant; hybrid; abiotic stress
15.  Adaptation to flooding in upland and lowland ecotypes of Cyperus rotundus, a troublesome sedge weed of rice: tuber morphology and carbohydrate metabolism 
Annals of Botany  2008;103(2):295-302.
Background and aims
In recent years, Cyperus rotundus has become a problem weed in lowland rice (Oryza sativa) grown in rotation with vegetables in the Philippines. As the growth of C. rotundus is commonly suppressed by prolonged flooding, the ability of the weed to grow vigorously in flooded as well as upland conditions suggests that adapted ecotypes occur in these rotations. Studies were conducted to elucidate the mechanisms that permit C. rotundus to tolerate flooded soil conditions.
Upland and lowland ecotypes of C. rotundus were compared in terms of growth habit, carbohydrate reserves and metabolism, and activities of enzymes involved in alcoholic fermentation – alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC).
Key Results
The lowland ecotype has much larger tubers than the upland ecotype. Prior to germination, the amylase activity and total non-structural carbohydrate content in the form of soluble sugars were greater in the tubers of lowland plants than in those of upland C. rotundus. At 24 h after germination in hypoxic conditions, PDC and ADH activities in the lowland plants increased, before decreasing at 48 h following germination. In contrast, ADH and PDC activities in the upland plants increased from 24 to 48 h after germination.
Tolerance of lowland C. rotundus of flooding may be attributed to large carbohydrate content and amylase activity, and the ability to maintain high levels of soluble sugars in the tubers during germination and early growth. This is coupled with the modulation of ADH and PDC activities during germination, possibly to control the use of carbohydrate reserves and sustain substrate supply in order to avoid starvation and death of seedlings with prolonged flooding.
PMCID: PMC2707299  PMID: 18515404
Anoxia; ethanol fermentation; flooding tolerance; nutsedge; Cyperus rotundus; Pasteur effect; weed ecology
16.  Comparing genomic expression patterns across plant species reveals highly diverged transcriptional dynamics in response to salt stress 
BMC Genomics  2009;10:398.
Rice and barley are both members of Poaceae (grass family) but have a marked difference in salt tolerance. The molecular mechanism underlying this difference was previously unexplored. This study employs a comparative genomics approach to identify analogous and contrasting gene expression patterns between rice and barley.
A hierarchical clustering approach identified several interesting expression trajectories among rice and barley genotypes. There were no major conserved expression patterns between the two species in response to salt stress. A wheat salt-stress dataset was queried for comparison with rice and barley. Roughly one-third of the salt-stress responses of barley were conserved with wheat while overlap between wheat and rice was minimal. These results demonstrate that, at transcriptome level, rice is strikingly different compared to the more closely related barley and wheat. This apparent lack of analogous transcriptional programs in response to salt stress is further highlighted through close examination of genes associated with root growth and development.
The analysis provides support for the hypothesis that conservation of transcriptional signatures in response to environmental cues depends on the genetic similarity among the genotypes within a species, and on the phylogenetic distance between the species.
PMCID: PMC2739230  PMID: 19706179
17.  Detection and validation of single feature polymorphisms using RNA expression data from a rice genome array 
BMC Plant Biology  2009;9:65.
A large number of genetic variations have been identified in rice. Such variations must in many cases control phenotypic differences in abiotic stress tolerance and other traits. A single feature polymorphism (SFP) is an oligonucleotide array-based polymorphism which can be used for identification of SNPs or insertion/deletions (INDELs) for high throughput genotyping and high density mapping. Here we applied SFP markers to a lingering question about the source of salt tolerance in a particular rice recombinant inbred line (RIL) derived from a salt tolerant and salt sensitive parent.
Expression data obtained by hybridizing RNA to an oligonucleotide array were analyzed using a statistical method called robustified projection pursuit (RPP). By applying the RPP method, a total of 1208 SFP probes were detected between two presumed parental genotypes (Pokkali and IR29) of a RIL population segregating for salt tolerance. We focused on the Saltol region, a major salt tolerance QTL. Analysis of FL478, a salt tolerant RIL, revealed a small (< 1 Mb) region carrying alleles from the presumed salt tolerant parent, flanked by alleles matching the salt sensitive parent IR29. Sequencing of putative SFP-containing amplicons from this region and other positions in the genome yielded a validation rate more than 95%.
Recombinant inbred line FL478 contains a small (< 1 Mb) segment from the salt tolerant parent in the Saltol region. The Affymetrix rice genome array provides a satisfactory platform for high resolution mapping in rice using RNA hybridization and the RPP method of SFP analysis.
PMCID: PMC2697985  PMID: 19480680
18.  Responses of Photosynthesis, Chlorophyll Fluorescence and ROS-Scavenging Systems to Salt Stress During Seedling and Reproductive Stages in Rice 
Annals of Botany  2007;99(6):1161-1173.
Background and Aims
Salinity is a widespread soil problem limiting productivity of cereal crops worldwide. Rice is particularly sensitive to salt stress during the seedling stage, with consequent poor crop establishment, as well as during reproduction where salinity can severely disrupt grain formation and yield. Tolerance at the seedling stage is weakly associated with tolerance during reproduction. Physiological responses to salinity were evaluated for contrasting genotypes, during the seedling and reproductive stages.
Three rice genotypes differing in their tolerance of salinity were evaluated in a set of greenhouse experiments under salt stress during both seedling stage and reproduction.
Key Results
Photosynthetic CO2 fixation, stomatal conductance (gs) and transpiration decreased substantially because of salt stress, but with greater reduction in the sensitive cultivar IR29. The tolerant lines IR651 and IR632 had more responsive stomata that tended to close faster during the first few hours of stress, followed by partial recovery after a brief period of acclimation. However, in the sensitive line, gs continued to decrease for longer duration and with no recovery afterward. Chlorophyll fluorescence measurements revealed that non-photochemical quenching increased, whereas the electron transport rate decreased under salt stress. Salt-tolerant cultivars exhibited much lower lipid peroxidation, maintained elevated levels of reduced ascorbic acid and showed increased activities of the enzymes involved in the reactive oxygen scavenging system during both developmental stages.
Upregulation of the anti-oxidant system appears to play a role in salt tolerance of rice, with tolerant genotypes also maintaining relatively higher photosynthetic function; during both the vegetative and reproductive stages.
PMCID: PMC3243573  PMID: 17428832
Chlorophyll fluorescence; photosynthesis; reactive oxygen species; rice; Oryza sativa; salinity
19.  Array-based genotyping and expression analysis of barley cv. Maythorpe and Golden Promise 
BMC Genomics  2007;8:87.
Golden Promise is a salt-tolerant spring barley closely related to Maythorpe. Salt tolerance in Golden Promise has been attributed to a single mutation at the Ari-e locus (on 5H) resulting from irradiation of Maythorpe. Golden Promise accumulates lower shoot Na+ compared to Maythorpe when growing under saline conditions. This study focused on elucidating the genetic basis and mechanisms involved in this difference.
The level of polymorphism between the two genotypes was explored using the Barley1 GeneChip for single feature polymorphisms (SFPs) and an oligonucleotide pool assay for single nucleotide polymorphisms (SNPs). Polymorphism analyses revealed three haplotype blocks spanning 6.4 cM on chromosome 1H, 23.7 cM on chromosome 4H and 3.0 cM on 5H. The Barley1 GeneChip was used to examine transcript abundance in different tissues and stages during development. Several genes within the polymorphic haplotype blocks were differentially regulated. Additionally, a more global difference in the jasmonic acid pathway regulation was detected between the two genotypes.
The results confirm that Golden Promise and Maythorpe are genetically very closely related but establish that they are not isogenic, as previously reported, due to three polymorphic haplotype blocks. Transcriptome analysis indicates that the response of the two genotypes to salinity stress is quite different. Additionally, the response to salinity stress in the roots and shoot tissue is strikingly different.
PMCID: PMC1851953  PMID: 17394671
20.  Genome-wide transcriptional analysis of salinity stressed japonica and indica rice genotypes during panicle initiation stage 
Plant Molecular Biology  2006;63(5):609-623.
Rice yield is most sensitive to salinity stress imposed during the panicle initiation (PI) stage. In this study, we have focused on physiological and transcriptional responses of four rice genotypes exposed to salinity stress during PI. The genotypes selected included a pair of indicas (IR63731 and IR29) and a pair of japonica (Agami and M103) rice subspecies with contrasting salt tolerance. Physiological characterization showed that tolerant genotypes maintained a much lower shoot Na+ concentration relative to sensitive genotypes under salinity stress. Global gene expression analysis revealed a strikingly large number of genes which are induced by salinity stress in sensitive genotypes, IR29 and M103 relative to tolerant lines. We found 19 probe sets to be commonly induced in all four genotypes. We found several salinity modulated, ion homeostasis related genes from our analysis. We also studied the expression of SKC1, a cation transporter reported by others as a major source of variation in salt tolerance in rice. The transcript abundance of SKC1 did not change in response to salinity stress at PI stage in the shoot tissue of all four genotypes. However, we found the transcript abundance of SKC1 to be significantly higher in tolerant japonica Agami relative to sensitive japonica M103 under control and stressed conditions during PI stage.
Electronic supplementary material
Supplementary material is available in the online version of this article at and is accessible for authorized users.
PMCID: PMC1805040  PMID: 17160619
Rice; Salt stress; Panicle initiation; Microarray

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