Down‐regulation of OsSPX1 caused semi‐male sterility,resulting in reduction of grain yield in rice

OsSPX1, a rice SPX domain gene, involved in the phosphate (Pi)‐sensing mechanism plays an essential role in the Pi‐signalling network through interaction with OsPHR2. In this study, we focused on the potential function of OsSPX1 during rice reproductive phase. Based on investigation of OsSPX1 antisense and sense transgenic rice lines in the paddy fields, we discovered that the down‐regulation of OsSPX1 caused reduction of seed‐setting rate and filled grain number. Through examination of anthers and pollens of the transgenic and wild‐type plants by microscopy, we found that the antisense of OsSPX1 gene led to semi‐male sterility, with lacking of mature pollen grains and phenotypes with a disordered surface of anthers and pollens. We further conducted rice whole‐genome GeneChip analysis to elucidate the possible molecular mechanism underlying why the down‐regulation of OsSPX1 caused deficiencies in anthers and pollens and lower seed‐setting rate in rice. The down‐regulation of OsSPX1 significantly affected expression of genes involved in carbohydrate metabolism and sugar transport, anther development, cell cycle, etc. These genes may be related to pollen fertility and male gametophyte development. Our study demonstrated that down‐regulation of OsSPX1 disrupted rice normal anther and pollen development by affecting carbohydrate metabolism and sugar transport, leading to semi‐male sterility, and ultimately resulted in low seed‐setting rate and grain yield.     

A point mutation in LTT1 enhances cold tolerance at the booting stage in rice

The cold tolerance of rice at the booting stage is a main factor determining sustainability and regional adaptability. However, relatively few cold tolerance genes have been identified that can be effectively used in breeding programmes. Here, we show that a point mutation in the low-temperature tolerance 1 (LTT1) gene improves cold tolerance by maintaining tapetum degradation and pollen development, by activation of systems that metabolize reactive oxygen species (ROS). Cold-induced ROS accumulation is therefore prevented in the anthers of the ltt1 mutants allowing correct development. In contrast, exposure to cold stress dramatically increases ROS accumulation in the wild type anthers, together with the expression of genes encoding proteins associated with programmed cell death and with the accelerated degradation of the tapetum that ultimately leads to pollen abortion. These results demonstrate that appropriate ROS management is critical for the cold tolerance of rice at the booting stage. Hence, the ltt1 mutation can significantly improve the seed setting ability of cold-sensitive rice varieties under low-temperature stress conditions, with little yield penalty under optimal temperature conditions. This study highlights the importance of a valuable genetic resource that may be applied in rice breeding programmes to enhance cold tolerance.     

A novel functional gene associated with cold tolerance at the seedling stage in rice

Identification and cloning of cold‐tolerant genes that can stably express under different cold environments are crucial for molecular rice breeding for cold tolerance. In the previous study, we identified a cold‐tolerant QTL at the seedling stage, qCTS‐9 which could be detected under different cold environments using a recombinant inbred line (RIL) population derived from a cold‐tolerant variety Lijiangxintuanheigu (LTH) and a cold‐sensitive variety Shanhuangzhan 2 (SHZ‐2). In this study, eight candidate genes within the qCTS‐9 interval were identified through integrated analysis of QTL mapping with genomewide differential expression profiling of LTH. The qRT‐PCR assay showed that only Os09g0410300 exhibited different expression patterns between LTH and SHZ‐2 during cold stress, and significantly positive correlation was found between cold induction of Os09g0410300 and seedling cold tolerance in the RI lines. Five SNPs and one InDel in the promoters of Os09g0410300 were detected between LTH and SHZ‐2, and the InDel marker ID410300 designed based on the insertion–deletion polymorphism in the promoter was significantly associated with seedling cold tolerance in RIL population. Further, Os09g0410300 over‐expression plants exhibited enhanced cold tolerance at the seedling stage compared with the wild‐type plants. Thus, our results suggest that Os09g0410300 is the functional gene underlying qCTS‐9. To our knowledge, it is a novel gene contributed to enhance cold tolerance at the seedling stage in rice. Identification of the functional gene underlying qCTS‐9 and development of the gene‐specific marker will facilitate molecular breeding for cold tolerance at the seedling stage in rice through transgenic approach and marker‐assisted selection (MAS).     

Expression of the CCCH‐tandem zinc finger protein gene OsTZF5 under a stress‐inducible promoter mitigates the effect of drought stress on rice grain yield under field conditions

Increasing drought resistance without sacrificing grain yield remains an ongoing challenge in crop improvement. In this study, we report that O ryza s ativa CCCH‐t andem z inc f inger protein 5 (OsTZF5) can confer drought resistance and increase grain yield in transgenic rice plants. Expression of OsTZF5 was induced by abscisic acid, dehydration and cold stress. Upon stress, OsTZF5‐GFP localized to the cytoplasm and cytoplasmic foci. Transgenic rice plants overexpressing OsTZF5 under the constitutive maize ubiquitin promoter exhibited improved survival under drought but also growth retardation. By introducing OsTZF5 behind the stress‐responsive OsNAC6 promoter in two commercial upland cultivars, Curinga and NERICA4, we obtained transgenic plants that showed no growth retardation. Moreover, these plants exhibited significantly increased grain yield compared to non‐transgenic cultivars in different confined field drought environments. Physiological analysis indicated that OsTZF5 promoted both drought tolerance and drought avoidance. Collectively, our results provide strong evidence that OsTZF5 is a useful biotechnological tool to minimize yield losses in rice grown under drought conditions.     

Enhanced salinity tolerance and improved yield properties in Bangladeshi rice Binnatoa through <Emphasis Type="Italic">Agrobacterium-</Emphasis>mediated transformation of <Emphasis Type="Italic">PgNHX1</Emphasis> from <Emphasis Type="Italic">Pennisetum glaucum</Emphasis>

Rice yield is severely affected by high-salt concentration in the vicinity of the plant. In an effort to engineer rice for improved salt tolerance Agrobacterium-mediated transformation of rice cv. Binnatoa was accomplished with the Pennisetum glaucum vacuolar Na⁺/H⁺ antiporter gene (PgNHX1) under the constitutive CaMV35S promoter. For the molecular analysis of putative transgenic plants, PCR and RT-PCR were performed. Transgenic rice plants expressing PgNHX1 showed better physiological status and completed their life cycle by setting flowers and seeds in salt stress, while wild-type plants exhibited rapid chlorosis and growth inhibition. Moreover, transgenic rice plants produced higher grain yields than wild-type plants under salt stress. Assessment of the salinity tolerance of the transgenic plants at seedling and reproductive stages demonstrated the potential of PgNHX1 for imparting enhanced salt tolerance capabilities and improved yield.     

Optimization of TaDREB3 gene expression in transgenic barley using cold‐inducible promoters

Constitutive over‐expression of the TaDREB3 gene in barley improved frost tolerance of transgenic plants at the vegetative stage of plant development, but leads to stunted phenotypes and 3‐ to 6‐week delays in flowering compared to control plants. In this work, two cold‐inducible promoters with contrasting properties, the WRKY71 gene promoter from rice and the Cor39 gene promoter from durum wheat, were applied to optimize expression of TaDREB3. The aim of the work was to increase plant frost tolerance and to decrease or prevent negative developmental phenotypes observed during constitutive expression of TaDREB3. The OsWRKY71 and TdCor39 promoters had low‐to‐moderate basal activity and were activated by cold treatment in leaves, stems and developing spikes of transgenic barley and rice. Expression of the TaDREB3 gene, driven by either of the tested promoters, led to a significant improvement in frost tolerance. The presence of the functional TaDREB3 protein in transgenic plants was confirmed by the detection of strong up‐regulation of cold‐responsive target genes. The OsWRKY71 promoter–driven TaDREB3 provides stronger activation of the same target genes than the TdCor39 promoter. Analysis of the development of transgenic plants in the absence of stress revealed small or no differences in plant characteristics and grain yield compared with wild‐type plants. The WRKY71–TaDREB3 promoter–transgene combination appears to be a promising tool for the enhancement of cold and frost tolerance in crop plants but field evaluation will be needed to confirm that negative development phenotypes have been controlled.     

New insights into the genetic basis of natural chilling and cold shock tolerance in rice by genome‐wide association analysis

In order to understand cold adaptability and explore additional genetic resources for the cold tolerance improvement of rice, we investigated the genetic variation of 529 rice accessions under natural chilling and cold shock stress conditions at the seedling stage using genome‐wide association studies; a total of 132 loci were identified. Among them, 12 loci were common for both chilling and cold shock tolerance, suggesting that rice has a distinct and overlapping genetic response and adaptation to the two stresses. Haplotype analysis of a known gene OsMYB2, which is involved in cold tolerance, revealed indica–japonica differentiation and latitude tendency for the haplotypes of this gene. By checking the subpopulation and geographical distribution of accessions with tolerance or sensitivity under these two stress conditions, we found that the chilling tolerance group, which mainly consisted of japonica accessions, has a wider latitudinal distribution than the chilling sensitivity group. We conclude that the genetic basis of natural chilling stress tolerance in rice is distinct from that of cold shock stress frequently used for low‐temperature treatment in the laboratory and the cold adaptability of rice is associated with the subpopulation and latitudinal distribution.     

Climate‐dependent variation in cold tolerance of weedy rice and rice mediated by OsICE1 promoter methylation

The mechanisms by which weedy rice (Oryza sativa f. spontanea) has adapted to endure low‐temperature stress in northern latitudes remain unresolved. In this study, we assessed cold tolerance of 100 rice varieties and 100 co‐occurring weedy rice populations, which were sampled across a broad range of climates in China. A parallel pattern of latitude‐dependent variation in cold tolerance was detected in cultivated rice and weedy rice. At the molecular level, differential cold tolerance was strongly correlated with relative expression levels of CBF cold response pathway genes and with methylation levels in the promoter region of OsICE1, a regulator of this pathway. Among all methylated cytosine sites of the OsICE1 promoter, levels of CHG and CHH methylation were found to be significantly correlated with cold tolerance among accessions. Furthermore, within many of the collection locales, weedy rice shared identical or near‐identical OsICE1 methylation patterns with co‐occurring cultivated rice. These findings provide new insights on the possible roles that methylation variation in the OsICE1 promoter may play in cold tolerance, and they suggest that weedy rice can rapidly acquire cold tolerance via methylation patterns that are shared with co‐occurring rice cultivars.     

Importance of pre‐anthesis anther sink strength for maintenance of grain number during reproductive stage water stress in wheat

Reproductive stage water stress leads to spikelet sterility in wheat. Whereas drought stress at anthesis affects mainly grain size, stress at the young microspore stage of pollen development is characterized by abortion of pollen development and reduction in grain number. We identified genetic variability for drought tolerance at the reproductive stage. Drought‐tolerant wheat germplasm is able to maintain carbohydrate accumulation in the reproductive organs throughout the stress treatment. Starch depletion in the ovary of drought‐sensitive wheat is reversible upon re‐watering and cross‐pollination experiments indicate that the ovary is more resilient than the anther. The effect on anthers and pollen fertility is irreversible, suggesting that pollen sterility is the main cause of grain loss during drought conditions in wheat. The difference in storage carbohydrate accumulation in drought‐sensitive and drought‐tolerant wheat is correlated with differences in sugar profiles, cell wall invertase gene expression and expression of fructan biosynthesis genes in anther and ovary (sucrose : sucrose 1‐fructosyl‐transferase, 1‐SST; sucrose : fructan 6‐fructosyl‐transferase, 6‐SFT). Our results indicate that the ability to control and maintain sink strength and carbohydrate supply to anthers may be the key to maintaining pollen fertility and grain number in wheat and this mechanism may also provide protection against other abiotic stresses.     

Overexpression of the leucine‐rich receptor‐like kinase gene LRK2 increases drought tolerance and tiller number in rice

Drought represents a key limiting factor of global crop distribution. Receptor‐like kinases play major roles in plant development and defence responses against stresses such as drought. In this study, LRK2, which encodes a leucine‐rich receptor‐like kinase, was cloned and characterized and found to be localized on the plasma membrane in rice. Promoter–GUS analysis revealed strong expression in tiller buds, roots, nodes and anthers. Transgenic plants overexpressing LRK2 exhibited enhanced tolerance to drought stress due to an increased number of lateral roots compared with the wild type at the vegetative stage. Moreover, ectopic expression of LRK2 seedlings resulted in increased tiller development. Yeast two‐hybrid screening and bimolecular fluorescence complementation (BiFC) indicated a possible interaction between LRK2 and elongation factor 1 alpha (OsEF1A) in vitro. These results suggest that LRK2 functions as a positive regulator of the drought stress response and tiller development via increased branch development in rice. These findings will aid our understanding of branch regulation in other grasses and support improvements in rice genetics.     

Physiological and biochemical characterization of NERICA‐L‐44: a novel source of heat tolerance at the vegetative and reproductive stages in rice

The predicted increase in the frequency and magnitude of extreme heat spikes under future climate can reduce rice yields significantly. Rice sensitivity to high temperatures during the reproductive stage is well documented while the same during the vegetative stage is more speculative. Hence, to identify and characterize novel heat‐tolerant donors for both the vegetative and reproductive stages, 71 rice accessions, including approximately 75% New Rice for Africa (NERICAs), were phenotyped across field experiments during summer seasons in Delhi, India, and in a controlled environment study at International Rice Research Institute , Philippines. NERICA‐L‐44 (NL‐44) recorded high seedling survival (52%) and superior growth and greater reproductive success exposed to 42.2°C (sd ± 2.3) under field conditions. NL‐44 and the heat‐tolerant check N22 consistently displayed lower membrane damage and higher antioxidant enzymes activity across leaves and spikelets. NL‐44 recorded 50–60% spikelet fertility, while N22 recorded 67–79% under controlled environment temperature of 38°C (sd ±1.17), although both had about 87% fertility under extremely hot field conditions. N22 and NL‐44, exposed to heat stress (38°C), had similar pollen germination percent and number of pollen tubes reaching the ovary. NL‐44 maintained low hydrogen peroxide production and non‐photochemical quenching (NPQ) with high photosynthesis while N22 avoided photosystem II damage through high NPQ under high‐temperature stress. NL‐44 with its reproductive stage resilience to extreme heat stress, better antioxidant scavenging ability in both vegetative tissue and spikelets and superior yield and grain quality is identified as a novel donor for increasing heat tolerance at both the vegetative and reproductive stages in rice.     

Two NHX‐type transporters from Helianthus tuberosus improve the tolerance of rice to salinity and nutrient deficiency stress

The NHX‐type cation/H⁺ transporters in plants have been shown to mediate Na⁺(K⁺)/H⁺ exchange for salinity tolerance and K⁺ homoeostasis. In this study, we identified and characterized two NHX homologues, HtNHX1 and HtNHX2 from an infertile and salinity tolerant species Helianthus tuberosus (cv. Nanyu No. 1). HtNHX1 and HtNHX2 share identical 5′‐ and 3′‐UTR and coding regions, except for a 342‐bp segment encoding 114 amino acids (L₂₇₂ to Q₃₈₅) which is absent in HtNHX2. Both hydroponics and soil culture experiments showed that the expression of HtNHX1 or HtNHX2 improved the rice tolerance to salinity. Expression of HtNHX2, but not HtNHX1, increased rice grain yield, harvest index, total nutrient uptake under K⁺‐limited salt‐stress or general nutrient deficiency conditions. The results provide a novel insight into NHX function in plant mineral nutrition.     

Mapping quantitative trait loci associated with yield and yield components under reproductive stage salinity stress in rice (Oryza sativa L.)

Salinity tolerance in rice is critical at reproductive stage because it ultimately determines grain yield. An F₂ mapping population derived from a Sadri/FL478 cross was exposed to saline field conditions (6–8 dS m^???1) after the active tillering stage to identify reproductive stage specific QTLs for salinity tolerance. Genetic linkage map was constructed using 123 microsatellite markers on 232 F₂ progenies. Totally 35 QTLs for 11 traits under salinity stress were detected with LOD > 3, out of which 28 QTLs that explained from 5.9 to 30.0% phenotypic variation were found to be significant based on permutation test. Three major QTL clusters were found on chromosomes 2 (RM423–RM174), 4 (RM551–RM518) and 6 (RM20224–RM528) for multiple traits under salinity stress. Both parental lines contributed additively for QTLs identified for the yield components. A majority of the QTLs detected in our study are reported for the first time for reproductive stage salinity stress. Fine-mapping of selected putative QTLs will be the next step to facilitate marker-assisted backcrossing and to detect useful genes for salinity tolerance at the reproductive stage in rice.