An R2R3-MYB Transcription Factor OsMYBAS1 Promotes Seed Germination under Different Sowing Depths in Transgenic Rice

MYB-type transcription factors play essential regulatory roles in seed germination and the response to seedling establishment stress. This study isolated a rice R2R3-MYB gene, OsMYBAS1, and functionally characterized its role in seed germination by generating transgenic rice plants with the overexpression and knockout of OsMYBAS1. Gene expression analysis suggested that OsMYBAS1 was highly expressed in brown rice and root, respectively. Subcellular localization analysis determined that OsMYBAS1 was localized in the nucleus. No significant differences in seed germination rate were observed among wild-type (WT) and transgenic rice plants at the 0-cm sowing depth. However, when sown at a depth of 4 cm, higher germination rates, root lengths and seedling heights were obtained in OsMYBAS1-overexpressing plants than in WT. Furthermore, the opposite results were recorded between the osmybas1 mutants and WT. Moreover, OsMYBAS1-overexpressing plants significantly enhanced superoxide dismutase (SOD) enzyme activity and suppressed the accumulation of malondialdehyde (MDA) content at the 4-cm sowing depth. These results indicate that the MYB transcription factor OsMYBAS1 may promote rice seed germination and subsequent seedling establishment under deep-sowing conditions. These findings can provide valuable insight into rice seed-quality breeding to facilitate the development of a dry, direct-seeding production system.

Abiotic Stress-Induced Actin-Depolymerizing Factor 3 From Deschampsia antarctica Enhanced Cold Tolerance When Constitutively Expressed in Rice

The Antarctic flowering plant Deschampsia antarctica is highly sensitive to climate change and has shown rapid population increases during regional warming of the Antarctic Peninsula. Several studies have examined the physiological and biochemical changes related to environmental stress tolerance that allow D. antarctica to colonize harsh Antarctic environments; however, the molecular mechanisms of its responses to environmental changes remain poorly understood. To elucidate the survival strategies of D. antarctica in Antarctic environments, we investigated the functions of actin depolymerizing factor (ADF) in this species. We identified eight ADF genes in the transcriptome that were clustered into five subgroups by phylogenetic analysis. DaADF3, which belongs to a monocot-specific clade together with cold-responsive ADF in wheat, showed significant transcriptional induction in response to dehydration and cold, as well as under Antarctic field conditions. Multiple drought and low-temperature responsive elements were identified as possible binding sites of C-repeat-binding factors in the promoter region of DaADF3, indicating a close relationship between DaADF3 transcription control and abiotic stress responses. To investigate the functions of DaADF3 related to abiotic stresses in vivo, we generated transgenic rice plants overexpressing DaADF3. These transgenic plants showed greater tolerance to low-temperature stress than the wild-type in terms of survival rate, leaf chlorophyll content, and electrolyte leakage, accompanied by changes in actin filament organization in the root tips. Together, our results imply that DaADF3 played an important role in the enhancement of cold tolerance in transgenic rice plants and in the adaptation of D. antarctica to its extreme environment.

miR2105 regulates ABA biosynthesis via OsbZIP86-OsNCED3 module to contribute to drought tolerance in rice

Induced abscisic acid (ABA) biosynthesis plays an important role in plant tolerance to abiotic stresses, including drought, cold and salinity. However, regulation pathway of the ABA biosynthesis in response to stresses is unclear. Here, we identified a rice miRNA, osa-miR2105 (miR2105), which plays a crucial role in ABA biosynthesis under drought stress. Analysis of expression, transgenic rice and cleavage site showed that OsbZIP86 is a target gene of miR2105. Subcellular localization and luciferase activity assays showed that OsbZIP86 is a nuclear transcription factor. In vivo and in vitro analyses showed that OsbZIP86 directly binds to the promoter of OsNCED3, and interacts with OsSAPK10, resulting in enhanced-expression of OsNCED3. Transgenic rice plants with knock-down of miR2105 or overexpression of OsbZIP86 showed higher ABA content, more tolerance to drought, a lower rate of water loss, more stomatal closure than wild type rice ZH11 under drought stress. These rice plants showed no penalty with respect to agronomic traits under normal conditions. By contrast, transgenic rice plants with miR2105 overexpression, OsbZIP86 downregulation, or OsbZIP86 knockout displayed less tolerance to drought stress and other phenotypes. Collectively, our results show that a regulatory network of ‘miR2105-OsSAPK10/OsbZIP86-OsNCED3’ control ABA biosynthesis in response to drought stress.One-sentence summary‘miR2105-OsbZIP86-OsNCED3’ module plays crucial role in mediating ABA biosynthesis to contribute to drought tolerance with no penalty with respect to agronomic traits under normal conditions.

Overexpression of OsHMGB707, a High Mobility Group Protein, Enhances Rice Drought Tolerance by Promoting Stress-Related Gene Expression

Drought stress adversely affects crop growth and productivity worldwide. In response, plants have evolved several strategies in which numerous genes are induced to counter stress. High mobility group (HMG) proteins are the second most abundant family of chromosomal proteins. They play a crucial role in gene transcriptional regulation by modulating the chromatin/DNA structure. In this study, we isolated a novel HMG gene, OsHMGB707, one of the candidate genes localized in the quantitative trait loci (QTL) interval of rice drought tolerance, and examined its function on rice stress tolerance. The expression of OsHMGB707 was up-regulated by dehydration and high salt treatment. Its overexpression significantly enhanced drought tolerance in transgenic rice plants, whereas its knockdown through RNA interference (RNAi) did not affect the drought tolerance of the transgenic rice plants. Notably, OsHMGB707-GFP is localized in the cell nucleus, and OsHMGB707 is protein-bound to the synthetic four-way junction DNA. Several genes were up-regulated in OsHMGB707-overexpression (OE) rice lines compared to the wild-type rice varieties. Some of the genes encode stress-related proteins (e.g., DREB transcription factors, heat shock protein 20, and heat shock protein DnaJ). In summary, OsHMGB707 encodes a stress-responsive high mobility group protein and regulates rice drought tolerance by promoting the expression of stress-related genes.

Overexpression of plasma membrane Na+/H+ antiporter OsSOS1 gene improves salt tolerance in transgenic rice plants

Crop productivity is greatly affected by soil salinity; therefore, improvement in salinity tolerance of crops is a major goal in salt-tolerant breeding. The Salt Overly Sensitive (SOS) signal-transduction pathway plays a key role in ion homeostasis and salt tolerance in plants. In plants pumping of Na+ from the root cells is mediated by the plasma membrane Na+/H+ antiporter (SOS1) which plays important role in preventing the accumulation of toxic levels of Na+ in cytosol. In the present study, OsSOS1 (NHX7), gene was overexpressed in rice (var-Vikas) by Agrobacterium mediated In Planta transformation technique. To screen putative T1 plants for salt tolerance, stringent salt screening test was followed and root and shoot growth of transformants were used as selection criterion. Some of the putative transgenics showed significantly higher root growth compared to wild type. To confirm the presence of transgene in putative T1 transgenic plants, PCR based approach was followed using genomic DNA. The result showed that 16 % of the selected seedlings from the stringent salt screening test were PCR positives. Five selected lines were positive for RT-PCR analysis. Physiological studies such as chlorophyll content, membrane permeability, cell viability and sodium /potassium content analysis were also conducted to assess their levels of tolerance. Some of the T1 transformants showed lower percent reduction in chlorophyll content and less membrane leakage, higher cell viability and maintained higher K/Na ratio after NaCl treatment compared to wild type. These results clearly demonstrate that transgenic rice plants overexpressing OsSOS1 have better salt-tolerance. This could be attributed to extrusion of excess Na+ from cytosol into the apoplast and thereby reducing the toxic effects of Na+in the cell.

Overproduction of Chloroplast Glyceraldehyde-3-Phosphate Dehydrogenase Improves Photosynthesis Slightly under Elevated [CO2] Conditions in Rice

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) limits the regeneration of ribulose 1,5-bisphosphate (RuBP) in the Calvin–Benson cycle. However, it does not always limit the rate of CO2 assimilation. In the present study, the effects of overproduction of GAPDH on the rate of CO2 assimilation under elevated [CO2] conditions, where the capacity for RuBP regeneration limits photosynthesis, were examined in transgenic rice (Oryza sativa). GAPDH activity was increased to 3.2- and 4.5-fold of the wild-type levels by co-overexpression of the GAPDH genes, GAPA and GAPB, respectively. In the transgenic rice plants, the rate of CO2 assimilation under elevated [CO2] conditions increased by approximately 10%, whereas that under normal and low [CO2] conditions was not affected. These results indicate that overproduction of GAPDH is effective in improving photosynthesis under elevated [CO2] conditions, although its magnitude is relatively small. By contrast, biomass production of the transgenic rice plants was not greater than that of wild-type plants under elevated [CO2] conditions, although starch content tended to increase marginally.

Rice N-acetylserotonin deacetylase regulates melatonin levels in transgenic rice

A reverse melatonin biosynthetic pathway was recently discovered in plants, by which N-acetylserotonin (NAS) is converted into serotonin by N-acetylserotonin deacetylase (ASDAC) rather than into melatonin by N-acetylserotonin O-methyltransferase (ASMT). In this study, we generated transgenic rice plants in which ASDAC was either suppressed or overexpressed to determine whether ASDAC is functionally involved in melatonin biosynthesis. ASDAC-suppressed rice showed increased levels of NAS, 5-methoxytryptamine (5-MT), and melatonin, whereas ASDAC-overexpressed rice exhibited less melatonin synthesis than observed in the wild type. This finding is strong evidence of the role of ASDAC in melatonin biosynthesis in rice. The increased levels of 5-MT, which is produced either by ASDAC from melatonin or by serotonin O-methyltransferase (SOMT) from serotonin in ASDAC-suppressed rice, was ascribed to enhanced SOMT enzyme activity rather than increased transcripts, such as ASMT or caffeic acid O-methyltransferase (COMT) encoding SOMT activity.