The Rice Abscisic Acid-Responsive RING Finger E3 Ligase OsRF1 Targets OsPP2C09 for Degradation and Confers Drought and Salinity Tolerance in Rice

Drought and salinity are major important factors that restrain growth and productivity of rice. In plants, many really interesting new gene (RING) finger proteins have been reported to enhance drought and salt tolerance. However, their mode of action and interacting substrates are largely unknown. Here, we identified a new small RING-H2 type E3 ligase OsRF1, which is involved in the ABA and stress responses of rice. OsRF1 transcripts were highly induced by ABA, salt, or drought treatment. Upregulation of OsRF1 in transgenic rice conferred drought and salt tolerance and increased endogenous ABA levels. Consistent with this, faster transcriptional activation of key ABA biosynthetic genes, ZEP, NCED3, and ABA4, was observed in OsRF1-OE plants compared with wild type in response to drought stress. Yeast two-hybrid assay, BiFC, and co-immunoprecipitation analysis identified clade A PP2C proteins as direct interacting partners with OsRF1. In vitro ubiquitination assay indicated that OsRF1 exhibited E3 ligase activity, and that it targeted OsPP2C09 protein for ubiquitination and degradation. Cell-free degradation assay further showed that the OsPP2C09 protein is more rapidly degraded by ABA in the OsRF1-OE rice than in the wild type. The combined results suggested that OsRF1 is a positive player of stress responses by modulating protein stability of clade A PP2C proteins, negative regulators of ABA signaling.

Overexpression of miR5505 enhanced drought and salt resistance in rice (Orayza sativa)

Rice is one of the most important crops in the world and half of the world population consumes it as their staple food. The abiotic stresses caused by drought, salt and other stresses have severely impacted rice production. MicroRNAs (miRNAs) are a type of small non-coding RNAs which widely reported as gene regulators, suppressing genes expression by degradation mRNA or translation inhibition. Previously, high-throughput sequencing has found a conserved miRNA miR5505 responding to drought stress in Dongxiang wild rice (DXWR). Several other studies also revealed that miR5505 was involved in rice stress responses. We further studied the effect of miRNA in drought and salt tolerance by overexpression it in rice. 2 in 18 successfully transformed transgenic lines with higher miR5505 expression were selected and then drought and salt resistance ability were evaluated. Both transgenic lines showed stronger drought and salt tolerance than wild-type (WT). Putative targets of miR5505 were identified by psRNATarget and several of them were found stress-related. RNA-seq found 1,980 differentially expressed genes (DEGs) in transgenic lines. Among them, 978 genes were down-regulated. Three genes were predicted by psRNATarget and two of them might be stress-related. We also found various environmental stress cis-acting elements in upstream of miR5505 promoter through Software PlantCARE. In all, we improved rice drought and salt tolerance by overexpressing miR5505, and the generated putative targets and cis-acting elements also suggested miR5505 might play important roles in the regulation of drought and salt responses. Keywords: rice, overexpression line , drought and salt stress, miR5505

Establishment of Tissue Culture Regeneration System for Medicago ruthenica L. cv. ‘Zhilixing’

Background: Medicago ruthenica L. ‘Zhilixing’ is a new variety with superior forage and seed yield compared to the wild type. The cold, drought and salt tolerance of Zhlixing are better than those of alfalfa, suggesting that this variety can serve as a high-quality genetic resource for improving the stress resistance of alfalfa. However, because of the lack of tissue culture regeneration system, it is difficult to perform genetic transformation studies on stress resistance genes. This study aimed to establish an efficient tissue culture regeneration system for Zhilixing variety. Methods: Three types of explants were selected and tested on four types of basal media supplemented with different combinations of auxin and cytokinin for callus induction and differentiation, based on orthogonal tests to select the combinations of auxin and cytokinin suitable for callus induction and differentiation. Two-factor combination method was used to formulate a suitable rooting medium. Result: The hypocotyledonary axis was found to be an excellent explant for callus induction on MS medium. The optimum callus induction medium contained thidiazuron (TDZ, 0.5 mg/L), 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg/L) and naphthaleneacetic acid (NAA, 0.5 mg/L) where the callus induction rate was 93.33%. The differentiation medium was supplemented with TDZ (0.75 mg/L), 2,4-D (0.25 mg/L) and 6-benzyladenine (6-BA, 1.5 mg/L) where the differentiation rate was 63.33%. Thidiazuron played the key role in both processes of callus induction and differentiation. Half-strength MS containing 0.1 mg/L of NAA was the most efficient rooting medium.

MicroRNA Member TaMIR5062 in T. Aestivum Involves Plant Drought and Salt Acclimation via Regulating Physiological Processes Associated with Water Retention and ROS Homeostasis

microRNA members negatively regulate target genes via posttranscriptional cleavage or translation repression mechanisms, impacting on plant growth, development, and stress response. In this study, we characterized TaMIR5062, a miRNA member in T. aestivum, in mediating drought and salt responses. TaMIR5062 interacts with six target genes, including two encoding calmodulins, three coding for 4-oxalocrotonate tautomerases, and one for pumilio-family RNA binding domain protein. The TaMIR5062 transcripts were gradually upregulated in plants upon 27-h drought and salt treatments, whose induced expression under stress treatments was restored following the normal recovery condition. Tobacco (N. tabacum) lines transformed with TaMIR5062 modified growth traits under drought and salt treatments; the lines overexpressing miRNA (i.e., Sen 1 and Sen 2) improved growth traits (i.e., biomass, leaf area, and root length) whereas those with knockdown (Anti 1) alleviated growth features compared with wild type. These results suggested the critical role of TaMIR5062 in improving plant drought and salt tolerance. In line with growth traits in stress-challenged lines, improved leaf water retention (i.e., promoted stomata closing, water losing rate, and osmolytes) and ROS-associated parameters (higher SOD, CAT, and POD activities, etc.) were shown in Sen 1 and Sen 2 under stress conditions. Antioxidant enzyme (AE) genes NtMnSOD1, NtCAT, and NtPOD9 encoding SOD, CAT, and POD, respectively, enhanced transcription in Sen 1 and downregulated expression in Anti 1 challenged with drought and salt stress. These results suggested the improved ROS homeostasis mediated by TaMIR5062 associates modified expression of distinct AE genes. Quantities of genes functional into categories “biological process”, “cellular component”, and “molecular function” contribute to TaMIR5062-mediated osmotic stress adaptation by regulating distinct biological pathways (i.e., protein folding) and metabolisms (i.e., photosynthesis and isoprenoid biosynthesis), which impact on plant osmotic-regulation, ROS homeostasis, and stress defensiveness underlying miRNA regulation. TaMIR5062 is a valuable target for molecular breeding of drought-tolerant crop cultivars.

Genome-Wide Analysis of the Soybean Calmodulin-Binding Protein 60 Family and Identification of GmCBP60A-1 Responses to Drought and Salt Stresses

Calmodulin-binding protein 60 (CBP60) members constitute a plant-specific protein family that plays an important role in plant growth and development. In the soybean genome, nineteen CBP60 members were identified and analyzed for their corresponding sequences and structures to explore their functions. Among GmCBP60A-1, which primarily locates in the cytomembrane, was significantly induced by drought and salt stresses. The overexpression of GmCBP60A-1 enhanced drought and salt tolerance in Arabidopsis, which showed better state in the germination of seeds and the root growth of seedlings. In the soybean hairy roots experiment, the overexpression of GmCBP60A-1 increased proline content, lowered water loss rate and malondialdehyde (MDA) content, all of which likely enhanced the drought and salt tolerance of soybean seedlings. Under stress conditions, drought and salt response-related genes showed significant differences in expression in hairy root soybean plants of GmCBP60A-1-overexpressing and hairy root soybean plants of RNAi. The present study identified GmCBP60A-1 as an important gene in response to salt and drought stresses based on the functional analysis of this gene and its potential underlying mechanisms in soybean stress-tolerance.

OsIAA18, an Aux/IAA Transcription Factor Gene, Is Involved in Salt and Drought Tolerance in Rice

Auxin/indoleacetic acid (Aux/IAA) proteins play an important regulatory role in the developmental process of plants and their responses to stresses. A previous study has shown that constitutive expression of OsIAA18, an Aux/IAA transcription factor gene of rice improved salt and osmotic tolerance in transgenic Arabidopsis plants. However, little work is known about the regulatory functions of the OsIAA18 gene in regulating the abiotic stress tolerance of rice. In this study, the OsIAA18 gene was introduced into the rice cultivar, Zhonghua 11 and the OsIAA18 overexpression in rice plants exhibited significantly enhanced salt and drought tolerance compared to the wild type (WT). Moreover, overexpression of OsIAA18 in rice increased endogenous levels of abscisic acid (ABA) and the overexpression of OsIAA18 in rice plants showed hypersensitivity to exogenous ABA treatment at both the germination and postgermination stages compared to WT. Overexpression of OsIAA18 upregulated the genes involved in ABA biosynthesis and signaling pathways, proline biosynthesis pathway, and reactive oxygen species (ROS)-scavenging system in the overexpression of OsIAA18 in rice plants under salt and drought stresses. Proline content, superoxide dismutase (SOD), and peroxidase (POD) activities were significantly increased, whereas malonaldehyde (MDA), hydrogen peroxide (H2O2), and superoxide anion radical (O2–) content were significantly decreased in the transgenic plants under salt and drought stresses. Taken together, we suggest that OsIAA18 plays a positive role in drought and salt tolerance by regulating stress-induced ABA signaling. The OsIAA18 gene has a potential application in genetically modified crops with enhanced tolerance to abiotic stresses.

PLANT GROWTH PROMOTING ACTIVITIES OF RHIZOBACTERIA ISOLATED FROM RHIZOSPHERIC SOILS OF RURAL BANGALORE, INDIA

Soil is a dynamic ecosystem which provides support to plant life. Microorganisms inhabiting the rhizosphere region of soil play a key role in agriculture by promoting the exchange of plant nutrients and reducing the application of chemical fertilizers to a large extent. Engineering of rhizospheric region through exploitation of specific microorganisms leads to higher microbial diversity in the soil which in turn plays a significant role in maintaining the soil health. The present work envisages the isolation, screening and biochemical profiling of potent plant growth promoting rhizobacteria from various rhizospheric soils in and around Bangalore. Sixty isolates from rhizospheric region of fourteen different agricultural soils were screened for plant growth promoting traits such as phosphate solubilization, siderophore production, Ammonia, HCN & Phytohormone production. Twelve isolates that exhibited plant growth promotional traits were further subjected to screening for drought and salt tolerance. Among the twelve isolates, four potential isolates namely Serratia marcescens, Pseudomonas aeruginosa and Acinetobacter pittii were identified based on biochemical methods and 16SrRNA sequencing.

Application of High Voltage Electrical Discharge Treatment to Improve Wheat Germination and Early Growth under Drought and Salinity Conditions

The environmentally friendly, physical method of high voltage electrical discharge (HVED) was developed to improve the drought and salinity tolerance of two wheat genotypes. Unlike other plasma technologies, HVED treatment involves the discharge of electricity in water. In this study, the effect of HVED pretreatment on wheat germination and early vegetative growth under drought (0%, 15%, 20% and 30% PEG) and salinity (0, 90, 160 and 230 mM NaCl) stress conditions was investigated. HVED-exposed seeds showed altered seed surfaces and became more permeable to water uptake, resulting in higher germination percentages, germination index values, and shoot and root growth under the control and all drought and salinity concentrations. Moreover, the electrical conductivity of the water medium increased significantly, indicating HVED-induced reactions of ionization and dissociations of water molecules occurred. In addition, HVED pretreatment in the salt experiment improved the tolerance index values of the shoots and roots. The most pronounced genotypic variations occurred under the highest stress levels (30% PEG or 230 mM NaCl) and varied with the stress intensity and growth stage. The study results indicate that HVED pretreatment has the potential to improve drought and salt tolerance in wheat.