Transgenic creeping bentgrass overexpressing Osa‐miR393a exhibits altered plant development and improved multiple stress tolerance

MicroRNA393 (miR393) has been implicated in plant growth, development and multiple stress responses in annual species such as Arabidopsis and rice. However, the role of miR393 in perennial grasses remains unexplored. Creeping bentgrass (Agrostis stolonifera L.) is an environmentally and economically important C3 cool‐season perennial turfgrass. Understanding how miR393 functions in this representative turf species would allow the development of novel strategies in genetically engineering grass species for improved abiotic stress tolerance. We have generated and characterized transgenic creeping bentgrass plants overexpressing rice pri‐miR393a (Osa‐miR393a). We found that Osa‐miR393a transgenics had fewer, but longer tillers, enhanced drought stress tolerance associated with reduced stomata density and denser cuticles, improved salt stress tolerance associated with increased uptake of potassium and enhanced heat stress tolerance associated with induced expression of small heat‐shock protein in comparison with wild‐type controls. We also identified two targets of miR393, AsAFB2 and AsTIR1, whose expression is repressed in transgenics. Taken together, our results revealed the distinctive roles of miR393/target module in plant development and stress responses between creeping bentgrass and other annual species, suggesting that miR393 would be a promising candidate for generating superior crop cultivars with enhanced multiple stress tolerance, thus contributing to agricultural productivity.     

Differential regulation of microRNAs in response to osmotic,salt and cold stresses in wheat

MicroRNAs (miRNAs) are tiny non-coding regulatory molecules that modulate plant’s gene expression either by cleaving or repressing their mRNA targets. To unravel the plant actions in response to various environmental factors, identification of stress related miRNAs is essential. For understanding the regulatory behaviour of various abiotic stresses and miRNAs in wheat genotype C-306, we examined expression profile of selected conserved miRNAs viz. miR159, miR164, miR168, miR172, miR393, miR397, miR529 and miR1029 tangled in adapting osmotic, salt and cold stresses. The investigation revealed that two miRNAs (miR168, miR397) were down-regulated and miR172 was up-regulated under all the stress conditions. However, miR164 and miR1029 were up-regulated under cold and osmotic stresses in contrast to salt stress. miR529 responded to cold alone and does not change under osmotic and salt stress. miR393 showed up-regulation under osmotic and salt, and down-regulation under cold stress indicating auxin based differential cold response. Variation in expression level of studied miRNAs in presence of target genes delivers a likely elucidation of miRNAs based abiotic stress regulation. In addition, we reported new stress induced miRNAs Ta-miR855 using computational approach. Results revealed first documentation that miR855 is regulated by salinity stress in wheat. These findings indicate that diverse miRNAs were responsive to osmotic, salt and cold stress and could function in wheat response to abiotic stresses.     

GAMYB controls different sets of genes and is differentially regulated by microRNA in aleurone cells and anthers

GAMYB is a component of gibberellin (GA) signaling in cereal aleurone cells, and has an important role in flower development. However, it is unclear how GAMYB function is regulated. We examined the involvement of a microRNA, miR159, in the regulation of GAMYB expression in cereal aleurone cells and flower development. In aleurone cells, no miR159 expression was observed with or without GA treatment, suggesting that miR159 is not involved in the regulation of GAMYB and GAMYB-like genes in this tissue. miR159 was expressed in tissues other than aleurone, and miR159 over-expressors showed similar but more severe phenotypes than the gamyb mutant. GAMYB and GAMYB-like genes are co-expressed with miR159 in anthers, and the mRNA levels for GAMYB and GAMYB-like genes are negatively correlated with miR159 levels during anther development. Thus, OsGAMYB and OsGAMYB-like genes are regulated by miR159 in flowers. A microarray analysis revealed that OsGAMYB and its upstream regulator SLR1 are involved in the regulation of almost all GA-mediated gene expression in rice aleurone cells. Moreover, different sets of genes are regulated by GAMYB in aleurone cells and anthers. GAMYB binds directly to promoter regions of its target genes in anthers as well as aleurone cells. Based on these observations, we suggest that the regulation of GAMYB expression and GAMYB function are different in aleurone cells and flowers in rice.     

Phylogeny and Molecular Evolution of miR820 and miR396 microRNA Families in <Emphasis Type="Italic">Oryza</Emphasis> AA Genomes

The phylogeny and evolution of the microRNA families, miR820 and miR396, was analysed across the AA genomes of the Oryza species, the close relatives of domesticated rice. A highly dynamic evolution of the miR820 family was revealed. The number of copies of MIR820 genes, their chromosomal location and the mature microRNA sequence varied greatly with a total of 16 novel miR820 variants being identified. The phylogeny of pre-MIR820 sequences revealed that MIR820 genes of recently evolved Oryza AA genomes may have derived from sequence divergence of one or a few ancestral genes found in wild Australian perennial rice populations, Taxon B (jpn2)-MIR820 genes. Genomic scale duplication played an important role in the evolution of some of the miR396 family genes in AA genome Oryza species. miR396 family contained a MIR396 gene cluster (MIR396a and MIR396c) which was conserved across the cereal genomes. Nucleotide diversity analysis at these two MIR396 loci revealed that domesticated rice has retained less than 10% of the total diversity present in wild species. In contrast, the nucleotide sequence of four MIR396 loci remained almost conserved across domesticated and wild rices, indicating that they were under extreme functional constraint and may be involved in regulating some fundamental processes which are important both for wild and domesticated rices. Expression analysis demonstrated that miR820 variants were expressed in O. glaberrima O. barthi and O. longistaminata genome. These findings pose new challenges to explain the possible role of miR820 variants identified.     

Overexpression of a wheat Na+/H+ antiporter gene (TaNHX2) enhances tolerance to salt stress in transgenic tomato plants (Solanum lycopersicum L.)

Soil salinity is a major environmental stress limiting plant productivity. Vacuole Na⁺/H⁺ antiporters play important roles for the survival of plants under salt stress conditions. We have developed salt stress tolerant transgenic tomato plants (Solanum lycopersicum cv. PED) by overexpression of the wheat Na⁺/H⁺ antiporter gene TaNHX2 using Agrobacterium tumefaciens strain LBA4404 harbouring a binary vector pBin438 that contains the TaNHX2 gene under the control of double CaMV 35S promoter and npt II as a selectable marker. PCR and Southern blot analysis confirmed that TaNHX2 gene has been integrated and expressed in the T₁ generation transgenic tomato plants. When TaNHX2 expressing plants were exposed to 100 or 150 mM NaCl, they were found to be more tolerant to salt stress compared to wild type plants. Biochemical analyses also showed that transgenic plants have substantial amount of relative water content and chlorophyll content under salt stress conditions compared to wild type plants. The relative water content in transgenic and wild type plants ranged from 68 to 75 % and 46–73 % and chlorophyll content fall in between 1.8 to 2.4 mg/g fw and 1.0 to 2.4 mg/g fw, respectively, in all stress conditions. In the present study, we observed a better germination rate of T₁ transgenic seeds under salt stress conditions compared with wild type plants. Our results indicated that TaNHX2-transgenic tomato plants coped better with salt stress than wild type plants.     

Antioxidative enzymes,calcium, and ABA signaling pathway are required for the stress tolerance of transgenic wheat plant by the ectopic expression of harpin protein fragment Hpa1<Subscript>10–42</Subscript> under heat stress

Genetic engineering for heat stress tolerance can promote crop growth and improve yield. One wheat (Triticum aestivum L.) line Y16 (wild type) and two transgenic plants (Y16-3 and Y16-46) that express Hpa1_10-42, a functional fragment of harpin protein, were used in this study to investigate their possible abiotic stress tolerance under heat stress. Results showed that enhanced thermotolerance was observed in the Y16-3 and Y16-46 lines over the control wheat under stress conditions. However, this increased stress tolerance was significantly abolished by specific inhibitors such as fluridone or sodium tungstate (i.e., arrests abscisic acid (ABA) biosynthesis) and EGTA or La³⁺ (i.e., arrests Ca²⁺ signaling pathway) under heat exposure. By contrast, high activities of antioxidant enzymes such as superoxide dismutase, catalase, and ascorbate peroxidase (but not peroxidase) and low levels of oxidative damage (thiobarbituric acid reactive substance (TBARS) and chlorophyll) were detected in transgenic wheat lines compared with the control plant under stress exposure. However, this significant difference diminished after the addition of these specific inhibitors. Furthermore, a slight increase of H₂O₂ was observed in the transgenic plant, instead of the control, without the addition of chemicals under heat stress. These results suggested that antioxidant enzymes, calcium, and ABA signaling pathways were involved in this Hpa1_10–42-mediated thermotolerance of transgenic wheat plants under stress exposure. Finally, a hypothetical model based on H₂O₂ signaling was proposed to illustrate the possible mechanism of this enhanced heat stress tolerance.