ABA Inducible Rice Protein Phosphatase 2C Confers ABA Insensitivity and Abiotic Stress Tolerance in Arabidopsis

Arabidopsis PP2C belonging to group A have been extensively worked out and known to negatively regulate ABA signaling. However, rice (Oryza sativa) orthologs of Arabidopsis group A PP2C are scarcely characterized functionally. We have identified a group A PP2C from rice (OsPP108), which is highly inducible under ABA, salt and drought stresses and localized predominantly in the nucleus. Genetic analysis revealed that Arabidopsis plants overexpressing OsPP108 are highly insensitive to ABA and tolerant to high salt and mannitol stresses during seed germination, root growth and overall seedling growth. At adult stage, OsPP108 overexpression leads to high tolerance to salt, mannitol and drought stresses with far better physiological parameters such as water loss, fresh weight, chlorophyll content and photosynthetic potential (Fv/Fm) in transgenic Arabidopsis plants. Expression profile of various stress marker genes in OsPP108 overexpressing plants revealed interplay of ABA dependent and independent pathway for abiotic stress tolerance. Overall, this study has identified a potential rice group A PP2C, which regulates ABA signaling negatively and abiotic stress signaling positively. Transgenic rice plants overexpressing this gene might provide an answer to the problem of low crop yield and productivity during adverse environmental conditions.     

RhEXPA4, a rose expansin gene, modulates leaf growth and confers drought and salt tolerance to Arabidopsis

Drought and high salinity are major environmental conditions limiting plant growth and development. Expansin is a cell-wall-loosening protein known to disrupt hydrogen bonds between xyloglucan and cellulose microfibrils. The expression of expansin increases in plants under various abiotic stresses, and plays an important role in adaptation to these stresses. We aimed to investigate the role of the RhEXPA4, a rose expansin gene, in response to abiotic stresses through its overexpression analysis in Arabidopsis. In transgenic Arabidopsis harboring the Pro _RhEXPA4 ::GUS construct, RhEXPA4 promoter activity was induced by abscisic acid (ABA), drought and salt, particularly in zones of active growth. Transgenic lines with higher RhEXPA4 level developed compact phenotypes with shorter stems, curly leaves and compact inflorescences, while the lines with relatively lower RhEXPA4 expression showed normal phenotypes, similar to the wild type (WT). The germination percentage of transgenic Arabidopsis seeds was higher than that of WT seeds under salt stress and ABA treatments. Transgenic plants showed enhanced tolerance to drought and salt stresses: they displayed higher survival rates after drought, and exhibited more lateral roots and higher content of leaf chlorophyll a under salt stress. Moreover, high-level RhEXPA4 overexpressors have multiple modifications in leaf blade epidermal structure, such as smaller, compact cells, fewer stomata and midvein vascular patterning in leaves, which provides them with more tolerance to abiotic stresses compared to mild overexpressors and the WT. Collectively, our results suggest that RhEXPA4, a cell-wall-loosening protein, confers tolerance to abiotic stresses through modifying cell expansion and plant development in Arabidopsis.     

Transgenic Analysis Reveals 5′ Abbreviated <Emphasis Type="Italic">Os</Emphasis>RGLP2 Promoter(s) as Responsive to Abiotic Stresses

Germins and germin-like proteins are ubiquitous, expressed at various developmental stages and in response to various abiotic and biotic stresses. In this study, to functionally validate the OsRGLP2 promoter, 5′ deletion analysis of the promoter sequences was performed and the deletion fragments fused with the β-glucuronidase (GUS) and green fluorescent protein reporter genes were used for transient expression in tobacco as well as for generating stable transgenic Arabidopsis plants. Very high level of GUS activity was observed in agroinfiltrated tobacco leaves by the construct carrying the P-1063 and P-565 when subjected to abiotic stresses. Histochemical analysis of transgenic Arabidopsis plants revealed expression of reporter gene in root, leaf and stem sections of plants harboring P-1063 and P-565. Real-time qPCR analysis of transiently expressed tobacco leaves and transgenic Arabidopsis plants subjected to several abiotic stresses supported histochemical data and showed that P-565 responded to all the stresses to which the full-length promoter was responsive. The data suggest that P-565 may be a good alternative to full-length promoter region that harbors the necessary cis-elements in providing stable and high level of expression in response to wound, salt and temperature stresses.     

Overexpression of Os2H16 enhances resistance to phytopathogens and tolerance to drought stress in rice

Os2H16, a rice gene of unknown function, has been previously reported to be upregulated in response to infection by Xanthomonas oryzae pv. oryzae. In this study, expression patterns of Os2H16 were analyzed, demonstrating that expression of Os2H16 was dramatically induced by both bacterial and fungal infection as well as by drought stress, but repressed by salt treatment. To further investigate the role of Os2H16 in plant defense responses to abiotic and biotic stresses, transgenic lines of rice were developed. In comparison with wild-type rice, transgenic lines overexpressing Os2H16 show enhanced tolerance to bacterial blight and sheath blight disease, respectively caused by Xanthomonas oryzae pv. oryzae and Rhizoctonia solani. On the contrary, Os2H16 knockdown lines were more susceptible to both pathogens. Consistent with their individual phenotypes, upon inoculation, the expression of defense-related marker genes were elevated in Os2H16 overexpression individuals than in wild-type, while they were significantly reduced in Os2H16 knockdown lines. We also show that Os2H16 overexpression lines display enhanced tolerance to drought stress and elevated induction of drought-related genes, compared to wild-type rice. Os2H16 knockdown lines were more sensitive to drought stress and exhibited reduced induction of drought-related genes. Our study provides the first functional characterization of the rice Os2H16 gene, and suggests that Os2H16 positively modulate plant defense to abiotic and biotic stress.     

A Novel Non-wounding Transient Expression Assay for Cereals Mediated by Agrobacterium tumefaciens

A novel Agrobacterium tumefaciens-mediated transient expression assay (AmTEA) was developed for young plants of different cereal species and the model dicot Arabidopsis thaliana. AmTEA was evaluated using five promoters (six constructs) and two reporter genes, gus and egfp. The constitutive 35S promoter and the promoter of the rice glutaredoxin gene showed gus and egfp expression in the cereals analyzed in the present study. A promoter for the DEAD-box RNA helicase family protein gene from Arabidopsis showed similar expression patterns of reporter genes in stable transgenic lines as well as in transient expression lines of Arabidopsis. Agrobacterium tumefaciens co-cultivation and plant incubation times were optimized using 35S and the rice expressed protein gene promoter (R2-273). The possibility of non-specific expression of the reporter genes was ruled out by using the antibiotic carbenicillin and the comparison of expression of the reporter genes driven by full-length and truncated R2-273 promoters. AmTEA considerably reduced time, space, labor, and cost requirements. Ease of use with stress treatments is another major advantage of this method. AmTEA can be automated and used for large-scale studies to decipher promoter and gene functions with the ultimate goal to enhance the performance of cereal crops against biotic and abiotic stresses.     

The Effect of Silicon on Photosynthesis and Expression of Its Relevant Genes in Rice (Oryza sativa L.) under High-Zinc Stress

The main objectives of this study were to elucidate the roles of silicon (Si) in alleviating the effects of 2 mM zinc (high Zn) stress on photosynthesis and its related gene expression levels in leaves of rice (Oryza sativa L.) grown hydroponically with high-Zn stress. The results showed that photosynthetic parameters, including net photosynthetic rate, transpiration rate, stomatal conductance, intercellular CO₂ concentration, chlorophyll concentration and the chlorophyll fluorescence, were decreased in rice exposed to high-Zn treatment. The leaf chloroplast structure was disordered under high-Zn stress, including uneven swelling, disintegrated and missing thylakoid membranes, and decreased starch granule size and number, which, however, were all counteracted by the addition of 1.5 mM Si. Furthermore, the expression levels of Os08g02630 (PsbY), Os05g48630 (PsaH), Os07g37030 (PetC), Os03g57120 (PetH), Os09g26810 and Os04g38410 decreased in Si-deprived plants under high-Zn stress. Nevertheless, the addition of 1.5 mM Si increased the expression levels of these genes in plants under high-Zn stress at 72 h, and the expression levels were higher in Si-treated plants than in Si-deprived plants. Therefore, we conclude that Si alleviates the Zn-induced damage to photosynthesis in rice. The decline of photosynthesis in Zn-stressed rice was attributed to stomatal limitation, and Si activated and regulated some photosynthesis-related genes in response to high-Zn stress, consequently increasing photosynthesis.     

OsCIPK31, a CBL-interacting protein kinase is involved in germination and seedling growth under abiotic stress conditions in rice plants

Calcineurin B-like protein-interacting protein kinases (CIPKs) are a group of typical Ser/Thr protein kinases that mediate calcium signals. Extensive studies using Arabidopsis plants have demonstrated that many calcium signatures that activate CIPKs originate from abiotic stresses. However, there are few reports on the functional demonstration of CIPKs in other plants, especially in grasses. In this study, we used a loss-of-function mutation to characterize the function of the rice CIPK gene OsCIPK31. Exposure to high concentrations of NaCl or mannitol effected a rapid and transient enhancement of OsCIPK31 expression. These findings were observed only in the light. However, longer exposure to most stresses resulted in downregulation of OsCIPK31 expression in both the presence and absence of light. To determine the physiological roles of OsCIPK31 in rice plants, the sensitivity of oscipk31::Ds, which is a transposon Ds insertion mutant, to abiotic stresses was examined during germination and seedling stages. oscipk31::Ds mutants exhibited hypersensitive phenotypes to ABA, salt, mannitol, and glucose. Compared with wild-type rice plants, mutants exhibited retarded germination and slow seedling growth. In addition, oscipk31::Ds seedlings exhibited enhanced expression of several stress-responsive genes after exposure to these abiotic stresses. However, the expression of ABA metabolic genes and the endogenous levels of ABA were not altered significantly in the oscipk31::Ds mutant. This study demonstrated that rice plants use OsCIPK31 to modulate responses to abiotic stresses during the seed germination and seedling stages and to modulate the expression of stress-responsive genes.     

Comprehensive structural,interaction and expression analysis of CBL and CIPK complement during abiotic stresses and development in rice

Calcium ion is involved in diverse physiological and developmental pathways. One of the important roles of calcium is a signaling messenger, which regulates signal transduction in plants. CBL (calcineurin B-like protein) is one of the calcium sensors that specifically interact with a family of serine–threonine protein kinases designated as CBL-interacting protein kinases (CIPKs). The coordination of these two gene families defines complexity of the signaling networks in several stimulus-response-coupling during various environmental stresses. In Arabidopsis, both of these gene families have been extensively studied. To understand in-depth mechanistic interplay of CBL–CIPK mediated signaling pathways, expression analysis of entire set of CBL and CIPK genes in rice genome under three abiotic stresses (salt, cold and drought) and different developmental stages (3-vegetative stages and 11-reproductive stages) were done using microarray expression data. Interestingly, expression analysis showed that rice CBLs and CIPKs are not only involved in the abiotic stress but their significant role is also speculated in the developmental processes. Chromosomal localization of rice CBL and CIPK genes reveals that only OsCBL7 and OsCBL8 shows tandem duplication among CBLs whereas CIPKs were evolved by many tandem as well as segmental duplications. Duplicated OsCIPK genes showed variable expression pattern indicating the role of gene duplication in the extension and functional diversification of CIPK gene family in rice. Arabidopsis SOS3/CBL4 related genes in rice (OsCBL4, OsCBL5, OsCBL7 and OsCBL8) were employed for interaction studies with rice and Arabidopsis CIPKs. OsCBLs and OsCIPKs are not only found structurally similar but likely to be functionally equivalent to Arabidopsis CBLs and CIPKs genes since SOS3/CBL4 related OsCBLs interact with more or less similarly to rice and Arabidopsis CIPKs and exhibited an interaction pattern comparable with Arabidopsis SOS3/CBL4.     

Transgenic rice plants conferring increased tolerance to rice blast and multiple environmental stresses

We isolated a rice gene (denoted YK1), which showed78 percent amino acid sequence homology to the maize HM1gene. A chimeric gene consisting of a promoter and first intron of maizeubiquitin gene and the cDNA of YK1 was introduced intorice via Agrobacterium mediated transformation. Transgenic riceplants overexpressing this chimeric gene were resistant to rice blast(Magnaporthe grisea) disease, which is one of the mostserious pathogens in rice. Furthermore, the same transgenic plants conferredhigh tolerance to several abiotic stresses such as NaCl, UV-C, submergence, andhydrogen peroxide.     

Homologous expression of γ-glutamylcysteine synthetase increases grain yield and tolerance of transgenic rice plants to environmental stresses

Various environmental stresses induce reactive oxygen species (ROS), causing deleterious effects on plant cells. Glutathione (GSH), a critical antioxidant, is used to combat ROS. GSH is produced by γ-glutamylcysteine synthetase (γ-ECS) and glutathione synthetase (GS). To evaluate the functional roles of the Oryza sativa L. Japonica cv. Ilmi ECS (OsECS) gene, we generated transgenic rice plants overexpressing OsECS under the control of an inducible promoter (Rab21). When grown under saline conditions (100 mM) for 4 weeks, 2-independent transgenic (TGR1 and TGR2) rice plants remained bright green in comparison to control wild-type (WT) rice plants. TGR1 and TGR2 rice plants also showed a higher GSH/GSSG ratio than did WT rice plants in the presence of 100 mM NaCl, which led to enhanced redox homeostasis. TGR1 and TGR2 rice plants also showed lower ion leakage and higher chlorophyll-fluorescence when exposed to 10 μM methyl viologen (MV). Furthermore, the TGR1 and TGR2 rice seeds had approximately 1.5-fold higher germination rates in the presence of 200 mM salt. Under paddy field conditions, OsECS-overexpression in transgenic rice plants increased rice grain yield (TGW) and improved biomass. Overall, our results show that OsECS overexpression in transgenic rice increases tolerance and germination rate in the presence of abiotic stress by improving redox homeostasis via an enhanced GSH pool. Our findings suggest that increases in grain yield by OsECS overexpression could improve crop yields under natural environmental conditions.