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.     

Improving rice tolerance to potassium deficiency by enhancing OsHAK16p:WOX11‐controlled root development

Potassium (K) deficiency in plants confines root growth and decreases root‐to‐shoot ratio, thus limiting root K acquisition in culture medium. A WUSCHEL‐related homeobox (WOX) gene, WOX11, has been reported as an integrator of auxin and cytokinin signalling that regulates root cell proliferation. Here, we report that ectopic expression of WOX11 gene driven by the promoter of OsHAK16 encoding a low‐K‐enhanced K transporter led to an extensive root system and adventitious roots and more effective tiller numbers in rice. The WOX11‐regulated root and shoot phenotypes in the OsHAK16p:WOX11 transgenic lines were supported by K‐deficiency‐enhanced expression of several RR genes encoding type‐A cytokinin‐responsive regulators, PIN genes encoding auxin transporters and Aux/IAA genes. In comparison with WT, the transgenic lines showed increases in root biomass, root activity and K concentrations in the whole plants, and higher soluble sugar concentrations in roots particularly under low K supply condition. The improvement of sugar partitioning to the roots by the expression of OsHAK16p:WOX11 was further indicated by increasing the expression of OsSUT1 and OsSUT4 genes in leaf blades and several OsMSTs genes in roots. Expression of OsHAK16p:WOX11 in the rice grown in moderate K‐deficient soil increased total K uptake by 72% and grain yield by 24%–32%. The results suggest that enlarging root growth and development by the expression of WOX11 in roots could provide a useful option for increasing K acquisition efficiency and cereal crop productivity in low K soil.     

Overexpression of ubiquitin‐like LpHUB1 gene confers drought tolerance in perennial ryegrass

HUB1, also known as Ubl5, is a member of the subfamily of ubiquitin‐like post‐translational modifiers. HUB1 exerts its role by conjugating with protein targets. The function of this protein has not been studied in plants. A HUB1 gene, LpHUB1, was identified from serial analysis of gene expression data and cloned from perennial ryegrass. The expression of this gene was reported previously to be elevated in pastures during the summer and by drought stress in climate‐controlled growth chambers. Here, pasture‐type and turf‐type transgenic perennial ryegrass plants overexpressing LpHUB1 showed improved drought tolerance, as evidenced by improved turf quality, maintenance of turgor and increased growth. Additional analyses revealed that the transgenic plants generally displayed higher relative water content, leaf water potential, and chlorophyll content and increased photosynthetic rate when subjected to drought stress. These results suggest HUB1 may play an important role in the tolerance of perennial ryegrass to abiotic stresses.     

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.     

Biochemical and molecular changes in rice seedlings (Oryza sativa L.) to cope with chromium stress

Chromium (Cr) is very toxic to both humans and plants. This investigation aimed to understand the physiological and molecular responses of rice seedlings to Cr stress. Cr toxicity did not significantly affect morphological features and Cr accumulation in roots and shoots in Pokkali but not in BRRI 51, although there was a reduction in chlorophyll concentration in leaves of both genotypes. These results imply that Pokkali has mechanisms to cope with Cr supplementation. We therefore performed quantitative real‐time PCR on the expression pattern of two chelator genes, OsPCS1 and OsMT1, but there were no significant changes in expression in roots and shoots of Pokkali and BRRI 51 following Cr stress. This suggests that there was no metal sequestration following heavy metal stress in roots of these genotypes. Moreover, no expression of two heavy metal transporter genes, OsHMA3 and OsNRAMP1, was induced after Cr stress in roots and shoots, suggesting that these transporter genes are not induced by Cr stress or might not be involved in Cr uptake in rice. We also performed a targeted study on the effect of Cr on Fe uptake mechanisms. Our studies showed a consistent reduction in Fe uptake, Fe reductase activity and expression of Fe‐related genes (OsFRO1 and OsIRT1) under Cr stress in both roots and leaves of Pokkali. In contrast, these parameters and genes were significantly increased in Cr‐sensitive BRRI 51 under Cr stress. The results confirm that limiting Fe uptake through the down‐regulation of Fe reductase and Fe transporter genes is the main strategy of Cr‐tolerant Pokkali to cope with Cr stress. Finally, increased CAT, POD and GR activity and elevated glutathione and proline synthesis might provide strong antioxidant defence against Cr stress in Pokkali. Taken together, our findings reveal that Cr stress tolerance in rice (Pokkali) is not related to metal sequestration but is associated with reduced Fe transport and increased antioxidant defence.     

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.     

A detailed analysis of the leaf rolling mutant sll2 reveals complex nature in regulation of bulliform cell development in rice (Oryza sativa L.)

Bulliform cells are large, thin‐walled and highly vacuolated cells, and play an important role in controlling leaf rolling in response to drought and high temperature. However, the molecular mechanisms regulating bulliform cell development have not been well documented. Here, we report isolation and characterisation of a rice leaf‐rolling mutant, named shallot‐like 2 (sll2). The sll2 plants exhibit adaxially rolled leaves, starting from the sixth leaf stage, accompanied by increased photosynthesis and reduced plant height and tiller number. Histological analyses showed shrinkage of bulliform cells, resulting in inward‐curved leaves. The mutant is recessive and revertible at a rate of 9%. The leaf rolling is caused by a T‐DNA insertion. Cloning of the insertion using TAIL‐PCR revealed that the T‐DNA was inserted in the promoter region of LOC_Os07 g38664. Unexpectedly, the enhanced expression of LOC_Os07 g38664 by the 35S enhancer in the T‐DNA is not responsible for the leaf rolling phenotype. Further, the enhancer also exerted a long‐distance effect, including up‐regulation of several bulliform cell‐related genes. sll2 suppressed the outward leaf rolling of oul1 in the sll2oul1 double mutant. We conclude that leaf rolling in sll2 could be a result of the combined effect of multi‐genes, implying a complex network in regulation of bulliform cell development.     

An Arabidopsis E3 ligase HUB2 increases histone H2B monoubiquitination and enhances drought tolerance in transgenic cotton

The HUB2 gene encoding histone H2B monoubiquitination E3 ligase is involved in seed dormancy, flowering timing, defence response and salt stress regulation in Arabidopsis thaliana. In this study, we used the cauliflower mosaic virus (CaMV) 35S promoter to drive AtHUB2 overexpression in cotton and found that it can significantly improve the agricultural traits of transgenic cotton plants under drought stress conditions, including increasing the fruit branch number, boll number, and boll‐setting rate and decreasing the boll abscission rate. In addition, survival and soluble sugar, proline and leaf relative water contents were increased in transgenic cotton plants after drought stress treatment. In contrast, RNAi knockdown of GhHUB2 genes reduced the drought resistance of transgenic cotton plants. AtHUB2 overexpression increased the global H2B monoubiquitination (H2Bub1) level through a direct interaction with GhH2B1 and up‐regulated the expression of drought‐related genes in transgenic cotton plants. Furthermore, we found a significant increase in H3K4me3 at the DREB locus in transgenic cotton, although no change in H3K4me3 was identified at the global level. These results demonstrated that AtHUB2 overexpression changed H2Bub1 and H3K4me3 levels at the GhDREB chromatin locus, leading the GhDREB gene to respond quickly to drought stress to improve transgenic cotton drought resistance, but had no influence on transgenic cotton development under normal growth conditions. Our findings also provide a useful route for breeding drought‐resistant transgenic plants.