OsASR5 enhances drought tolerance through a stomatal closure pathway associated with ABA and H2O2 signalling in rice

Drought is one of the major abiotic stresses that directly implicate plant growth and crop productivity. Although many genes in response to drought stress have been identified, genetic improvement to drought resistance especially in food crops is showing relatively slow progress worldwide. Here, we reported the isolation of abscisic acid, stress and ripening (ASR) genes from upland rice variety, IRAT109 (Oryza sativa L. ssp. japonica), and demonstrated that overexpression of OsASR5 enhanced osmotic tolerance in Escherichia coli and drought tolerance in Arabidopsis and rice by regulating leaf water status under drought stress conditions. Moreover, overexpression of OsASR5 in rice increased endogenous ABA level and showed hypersensitive to exogenous ABA treatment at both germination and postgermination stages. The production of H₂O₂, a second messenger for the induction of stomatal closure in response to ABA, was activated in overexpression plants under drought stress conditions, consequently, increased stomatal closure and decreased stomatal conductance. In contrast, the loss‐of‐function mutant, osasr5, showed sensitivity to drought stress with lower relative water content under drought stress conditions. Further studies demonstrated that OsASR5 functioned as chaperone‐like protein and interacted with stress‐related HSP40 and 2OG‐Fe (II) oxygenase domain containing proteins in yeast and plants. Taken together, we suggest that OsASR5 plays multiple roles in response to drought stress by regulating ABA biosynthesis, promoting stomatal closure, as well as acting as chaperone‐like protein that possibly prevents drought stress‐related proteins from inactivation.     

The putative auxin efflux carrier OsPIN3t is involved in the drought stress response and drought tolerance

The phytohormone auxin plays a critical role in plant growth and development, and its spatial distribution largely depends on the polar localization of the PIN‐FORMED (PIN) auxin efflux carrier family members. In this study, we identify a putative auxin efflux carrier gene in rice, OsPIN3t, which acts in auxin polar transport but is also involved in the drought stress response in rice. We show that OsPIN3t–GFP fusion proteins are localized in plasma membranes, and this subcellular localization changes under 1‐N‐naphthylphthalamic acid (NPA) treatment. The tissue‐specific expression patterns of OsPIN3t were also investigated using a β‐glucuronidase (GUS) reporter, which showed that OsPIN3t was mainly expressed in vascular tissue. The GUS activity in OsPIN3tpro::GUS plants increased by NAA treatment and decreased by NPA treatment. Moreover, knockdown of OsPIN3t caused crown root abnormalities in the seedling stage that could be phenocopied by treatment of wild‐type plants with NPA, which indicated that OsPIN3t is involved in the control of polar auxin transport. Overexpression of OsPIN3t led to improved drought tolerance, and GUS activity significantly increased when OsPIN3tpro::GUS plants were subjected to 20% polyethylene glycol stress. Taken together, these results suggest that OsPIN3t is involved in auxin transport and the drought stress response, which suggests that a polar auxin transport pathway is involved in the regulation of the response to water stress in plants.     

Submergence tolerance of rice – A new glasshouse method for the experimental submergence of plants

A new method is described for evaluation of submergence tolerance of rice ( Oryza sativa L.) plants. Responses of a range of cultivars corresponded with known differences in field performance. The method 1) allows fast and effective determination of submergence tolerance, 2) allows screening of many plants in a small glasshouse area, 3) provides for recovery of superior plants for seed collection, 4) allows manipulation of many environmental variables to mimic the natural submergence environment, and 5) uses simple, inexpensive, readily available equipment. Physiological studies performed with this method gave results similar to those obtained from field studies and showed that submergence tolerance increased in older plants; it decreased with increasing depth, increasing temperature and with high or low light levels. The system is ideal for the rapid evaluation of rice germplasm under controlled conditions and physiological studies on the mechanism of rice submergence tolerance.     

OsPIN5b modulates rice (Oryza sativa) plant architecture and yield by changing auxin homeostasis,transport and distribution

Plant architecture attributes such as tillering, plant height and panicle size are important agronomic traits that determine rice (Oryza sativa) productivity. Here, we report that altered auxin content, transport and distribution affect these traits, and hence rice yield. Overexpression of the auxin efflux carrier‐like gene OsPIN5b causes pleiotropic effects, mainly reducing plant height, leaf and tiller number, shoot and root biomass, seed‐setting rate, panicle length and yield parameters. Conversely, reduced expression of OsPIN5b results in higher tiller number, more vigorous root system, longer panicles and increased yield. We show that OsPIN5b is an endoplasmic reticulum (ER) ‐localized protein that participates in auxin homeostasis, transport and distribution in vivo. This work describes an example of an auxin‐related gene where modulating its expression can simultaneously improve plant architecture and yield potential in rice, and reveals an important effect of hormonal signaling on these traits.     

The key regulator of submergence tolerance,SUB1A,promotes photosynthetic and metabolic recovery from submergence damage in rice leaves

The submergence‐tolerance regulator, SUBMERGENCE1A (SUB1A), of rice (Oryza sativa L.) modulates gene regulation, metabolism and elongation growth during submergence. Its benefits continue during desubmergence through protection from reactive oxygen species and dehydration, but there is limited understanding of SUB1A's role in physiological recovery from the stress. Here, we investigated the contribution of SUB1A to desubmergence recovery using the two near‐isogenic lines, submergence‐sensitive M202 and tolerant M202(Sub1). No visible damage was detected in the two genotypes after 3 d of submergence, but the sublethal stress differentially altered photosynthetic parameters and accumulation of energy reserves. Submergence inhibited photosystem II photochemistry and stimulated breakdown of protein and accumulation of several amino acids in both genotypes at similar levels. Upon desubmergence, however, more rapid return to homeostasis of these factors was observed in M202(Sub1). Submergence considerably restrained non‐photochemical quenching (NPQ) in M202, whereas the value was unaltered in M202(Sub1) during the stress. Upon reaeration, submerged plants encounter sudden exposure to higher light. A greater capability for NPQ‐mediated photoprotection can benefit the rapid recovery of photosynthetic performance and energy reserve metabolism in M202(Sub1). Our findings illuminate the significant role of SUB1A in active physiological recovery upon desubmergence, a component of enhanced tolerance to submergence.     

CLUSTERED PRIMARY BRANCH 1, a new allele of DWARF11, controls panicle architecture and seed size in rice

Panicle architecture and seed size are important agronomic traits that directly determine grain yield in rice (Oryza sativa L.). Although a number of key genes controlling panicle architecture and seed size have been cloned and characterized in recent years, their genetic and molecular mechanisms remain unclear. In this study, we identified a mutant that produced panicles with fascicled primary branching and reduced seeds in size. We isolated the underlying CLUSTERED PRIMARY BRANCH 1 (CPB1) gene, a new allele of DWARF11 (D11) encoding a cytochrome P450 protein involved in brassinosteroid (BR) biosynthesis pathway. Genetic transformation experiments confirmed that a His360Leu amino acid substitution residing in the highly conserved region of CPB1/D11 was responsible for the panicle architecture and seed size changes in the cpb1 mutants. Overexpression of CPB1/D11 under the background of cpb1 mutant not only rescued normal panicle architecture and plant height, but also had a larger leaf angle and seed size than the controls. Furthermore, the CPB1/D11 transgenic plants driven by panicle‐specific promoters can enlarge seed size and enhance grain yield without affecting other favourable agronomic traits. These results demonstrated that the specific mutation in CPB1/D11 influenced development of panicle architecture and seed size, and manipulation of CPB1/D11 expression using the panicle‐specific promoter could be used to increase seed size, leading to grain yield improvement in rice.     

CRD1, an Xpo1 domain protein,regulates miRNA accumulation and crown root development in rice

Crown root (CR) is the main component of the fibrous root system in cereal crops, but the molecular mechanism underlying CR development is still unclear. Here, we isolated the crown root defect 1 (crd1) mutant from ethyl methane sulfonate‐mutated mutant library, which significantly inhibited CR development. The CRD1 was identified through genome resequencing and complementation analysis, which encodes an Xpo1 domain protein: the rice ortholog of Arabidopsis HASTY (HST) and human exportin‐5 (XPO5). CRD1 is ubiquitously expressed, with the highest expression levels in the CR primordium at the stem base. CRD1 is a nucleocytoplasmic protein. The crd1 mutant contains significantly reduced miRNA levels in the cytoplasm and nucleus, suggesting that CRD1 is essential for maintaining normal miRNA levels in plant cells. The altered CR phenotype of crd1 was simulated by target mimicry of miR156, suggesting that this defect is due to the disruption of miR156 regulatory pathways. Our analysis of CRD1, the HST ortholog identified in monocots, expands our understanding of the molecular mechanisms underlying miRNA level and CR development.     

A major locus for submergence tolerance mapped on rice chromosome 9

Submergence stress is a widespread problem in rice-growing environments where drainage is impeded. A few cultivars can tolerate more than 10 days of submergence, but the genes conferring this tolerance have not been identified. We used randon-amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) markers to map submergence tolerance in 169 F₂ plants and the resulting F₃ families of a cross between a tolerant indica rice line, IR40931-26, and a susceptible japonica line, PI543851. IR40931-26 inherited strong submergence tolerance from the unimproved cultivar FR13A. Eight-day old F₃ seedlings were submerged for 14–16 days in 55-cm deep tanks, and tolerance was scored after 7 days recovery on a scale of 1 (tolerant) to 9 (susceptible). The tolerant and susceptible parents scored 1.5 and 8.4, respectively, and the F₃ means ranged from 1.6 to 8.9. Two bulks were formed with DNA from F₂ plants corresponding to the nine most tolerant and the nine most susceptible F₃ families. Of 624 RAPD primers used to screen the bulks, five produced bands associated with either tolerance or susceptibility. These markers were mapped to a region of chromosome 9 by linkage to RFLP markers. A submergence tolerance quantitative trait locus (QTL), here designatedSub1, was located ca. 4 cM from the RFLP marker C1232 and accounted for 69% of the phenotypic variance for the trait.     

The role of Bh4 in parallel evolution of hull colour in domesticated and weedy rice

The two independent domestication events in the genus Oryza that led to African and Asian rice offer an extremely useful system for studying the genetic basis of parallel evolution. This system is also characterized by parallel de‐domestication events, with two genetically distinct weedy rice biotypes in the US derived from the Asian domesticate. One important trait that has been altered by rice domestication and de‐domestication is hull colour. The wild progenitors of the two cultivated rice species have predominantly black‐coloured hulls, as does one of the two U.S. weed biotypes; both cultivated species and one of the US weedy biotypes are characterized by straw‐coloured hulls. Using Black hull 4 (Bh4) as a hull colour candidate gene, we examined DNA sequence variation at this locus to study the parallel evolution of hull colour variation in the domesticated and weedy rice system. We find that independent Bh4‐coding mutations have arisen in African and Asian rice that are correlated with the straw hull phenotype, suggesting that the same gene is responsible for parallel trait evolution. For the U.S. weeds, Bh4 haplotype sequences support current hypotheses on the phylogenetic relationship between the two biotypes and domesticated Asian rice; straw hull weeds are most similar to indica crops, and black hull weeds are most similar to aus crops. Tests for selection indicate that Asian crops and straw hull weeds deviate from neutrality at this gene, suggesting possible selection on Bh4 during both rice domestication and de‐domestication.     

Genomewide discovery of DNA polymorphisms in rice cultivars with contrasting drought and salinity stress response and their functional relevance

Next‐generation sequencing technologies provide opportunities to understand the genetic basis of phenotypic differences, such as abiotic stress response, even in the closely related cultivars via identification of large number of DNA polymorphisms. We performed whole‐genome resequencing of three rice cultivars with contrasting responses to drought and salinity stress (sensitive IR64, drought‐tolerant Nagina 22 and salinity‐tolerant Pokkali). More than 356 million 90‐bp paired‐end reads were generated, which provided about 85% coverage of the rice genome. Applying stringent parameters, we identified a total of 1 784 583 nonredundant single‐nucleotide polymorphisms (SNPs) and 154 275 InDels between reference (Nipponbare) and the three resequenced cultivars. We detected 401 683 and 662 509 SNPs between IR64 and Pokkali, and IR64 and N22 cultivars, respectively. The distribution of DNA polymorphisms was found to be uneven across and within the rice chromosomes. One‐fourth of the SNPs and InDels were detected in genic regions, and about 3.5% of the total SNPs resulted in nonsynonymous changes. Large‐effect SNPs and InDels, which affect the integrity of the encoded protein, were also identified. Further, we identified DNA polymorphisms present in the differentially expressed genes within the known quantitative trait loci. Among these, a total of 548 SNPs in 232 genes, located in the conserved functional domains, were identified. The data presented in this study provide functional markers and promising target genes for salinity and drought tolerance and present a valuable resource for high‐throughput genotyping and molecular breeding for abiotic stress traits in rice.     

Anther culture of an interspecific rice hybrid and selection of fine grain type with submergence tolerance

Improvement of rice with respect to resistance to biotic and abiotic stresses through introgression of alien genetic variation is an important breeding objective of wide hybridization. Anther culture of an interspecific hybrid between Oryza sativa L. cv. pankaj x O. rufipogon Griff. (both of them having "AA" genome) was done to obtain submergence tolerant high yielding recombinant type. Inoculation of anthers in five basal media viz. N6, modified N6, R3, He2 and He5 each one supplemented sith NAA (2 mg l^-1), kinetin (0.5 mg l^-1) and sucrose (5%) showed that the rate of callusing was highest (8.3%) in He2 medium. Calluses initiated in all the above five media when transferred to regeneration medium [MS medium containing NAA (0.5 mg l^-1), kinetin (2 mg l^-1) and sucrose (3%)], percentage of green plant regeneration was also high (13%) in calluses initiated in He2 medium. Androgenic doubled haploid plants, showed 1:1 segregation of the traits for most of the morphological characters studied whereas, different segregating ratios were found in F₂ population. Wider range of variation for different agronomic characters were noted in androgenic population as compared to F₂>. Of the 533 field grown doubled haploid plants, five plants were O. sativa type parental forms with fine grains and submergence tolerant (tested at ten days old seedling stage). Further screening has helped to select only one awnless, fine grain, submergence tolerant line tested at seedling stage and six weeks old plant age. This revised version was published online in June 2006 with corrections to the Cover Date.