Constitutive expression of a miR319 gene alters plant development and enhances salt and drought tolerance in transgenic creeping bentgrass.

MicroRNA319 (miR319) is one of the first characterized and conserved microRNA families in plants and has been demonstrated to target TCP (for TEOSINTE BRANCHED/CYCLOIDEA/PROLIFERATING CELL FACTORS [PCF]) genes encoding plant-specific transcription factors. MiR319 expression is regulated by environmental stimuli, suggesting its involvement in plant stress response, although experimental evidence is lacking and the underlying mechanism remains elusive. This study investigates the role that miR319 plays in the plant response to abiotic stress using transgenic creeping bentgrass (Agrostis stolonifera) overexpressing a rice (Oryza sativa) miR319 gene, Osa-miR319a. We found that transgenic plants overexpressing Osa-miR319a displayed morphological changes and exhibited enhanced drought and salt tolerance associated with increased leaf wax content and water retention but reduced sodium uptake. Gene expression analysis indicated that at least four putative miR319 target genes, AsPCF5, AsPCF6, AsPCF8, and AsTCP14, and a homolog of the rice NAC domain gene AsNAC60 were down-regulated in transgenic plants. Our results demonstrate that miR319 controls plant responses to drought and salinity stress. The enhanced abiotic stress tolerance in transgenic plants is related to significant down-regulation of miR319 target genes, implying their potential for use in the development of novel molecular strategies to genetically engineer crop species for enhanced resistance to environmental stress.

Overexpression of an Arabidopsis ß-glucosidase gene enhances drought resistance with dwarf phenotype in creeping bentgrass.

UNLABELLED: An Arabidopsis ß-glucosidase, AtBG1 is known to hydrolyze glucose-conjugated, biologically inactive abscisic acid (ABA) to produce active ABA, which increases the level of ABA in plants. Since an increase of ABA in plants confers tolerance against abiotic stress such as drought, we introduced the pCAMBIA3301 vector harboring the AtBG1 gene into creeping bentgrass through Agrobacterium-mediated transformation. After transformation, putative transgenic plants were selected using the BASTA resistance assay at a concentration of 0.8%. Genomic integration of the AtBG1 gene was confirmed by genomic PCR and Southern blot analysis, and gene expression was validated by Northern blot and Western blot analyses. Interestingly, the transgenic bentgrass plants overexpressing AtBG1 had a dwarf phenotype with reduced growth rates when compared to wild-type creeping bentgrass. In addition, the transgenic plants accumulated higher ABA levels and displayed enhanced drought tolerance. These results suggest that the expression of AtBG1 in plants induces the accumulation of higher ABA levels, which results in the formation of dwarf creeping bentgrass and enhances the survival in water-limiting environments. KEY MESSAGE: We used an Arabidopsis ß-glucosidase AtBG1 to engineer a crop with elevated active ABA levels, and developed transgenic creeping bentgrass with enhanced drought tolerance and dwarf phenotype.

Stomatal index responses of Agrostis canina to CO2 and sulphur dioxide: implications for palaeo-[CO2] using the stomatal proxy.

• Stomatal index values of fossil plants are widely used in reconstructing palaeo-[CO(2)]. This depends upon the assumption that the stomatal index is determined by the atmospheric concentration of CO(2) ([CO(2)]). This study investigates whether fumigation with, and resistance to, sulphur dioxide (SO(2)) induces a reduction in the stomatal index that may affect stomatal reconstructions of palaeo-[CO(2)] coinciding with episodes of global-scale volcanism. • Agrostis canina from Mefite di Ansanto, Italy, grow in atmospheres of elevated-[CO(2)], SO(2) and hydrogen sulphide (H(2)S). Mefite A. canina were compared with a control population in a 'common-garden' experiment and a controlled-environment study under elevated-[CO(2)] and SO(2) fumigation. • In A. canina, resistance to toxic volcanic gases is not associated with reduced stomatal index, and fumigation with SO(2) does not cause a decrease in stomatal initiation. The two populations of A. canina analyzed in this study exhibit different stomatal index-[CO(2)] 'responses', with control plants showing a reduction in stomatal index and Mefite plants showing no response. • Stomatal reconstructions of palaeo-[CO(2)] during past episodes of global-scale volcanism probably reflect atmospheric [CO(2)] and not [SO(2)]. The lack of a reduction in the stomatal index in response to elevated [CO(2)] in the Mefite plants, suggests that resistance to toxic gases and/or long-term growth at high [CO(2)] reduces, or negates, sensitivity of the stomatal index-[CO(2)] relationship, or that stomatal index-[CO(2)] in the Mefite plants is attuned to [CO(2)] fluctuations at much higher concentrations.

Turf Grasses.

A reliable and efficient genetic transformation protocol for various turfgrass species and elite cultivars has been achieved using Agrobacterium tumefaciens. We describe a general protocol for the establishment of embryogenic cell cultures, Agrobacterium tumefaciens-mediated transformation, selection, and regeneration of transgenic turfgrass plants. Embryogenic callus is initiated from mature seeds, maintained by visual selection, and infected with an Agrobacterium tumefaciens strain (LBA4404) that contains either an herbicide-resistant bar gene or an antibiotic-resistant hyg gene driven either by a rice ubiquitin or CaMV35S promoter. Stable transformation efficiencies up to 43.3% were achieved. Southern blot and genetic analysis was used to confirm transgene integration in the turfgrass genomes and normal transmission and stable expression of the transgene in the T1 generation. We demonstrate herein that five elite cultivars of bentgrass can be genetically transformed using this single tissue culture media regime. Additionally, we report the successful Agrobacterium-mediated transformation of an elite tall fescue variety using minor variations in the same transformation protocol.