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.

The Arabidopsis vacuolar H + -pyrophosphatase (AVP1), when over-expressed in transgenic (TG) plants, regulates root and shoot development via facilitation of auxin flux, and enhances plant resistance to salt and drought stresses. Here, we report that TG perennial creeping bentgrass plants over-expressing AVP1 exhibited improved resistance to salinity than wild-type (WT) controls. Compared to WT plants, TGs grew well in the presence of 100 mM NaCl, and exhibited higher tolerance and faster recovery from damages from exposure to 200 and 300 mM NaCl. The improved performance of the TG plants was associated with higher relative water content (RWC), higher Na + uptake and lower solute leakage in leaf tissues, and with higher concentrations of Na + , K + , Cl -and total phosphorus in root tissues. Under salt stress, proline content was increased in both WT and TG plants, but more significantly in TGs. Moreover, TG plants exhibited greater biomass production than WT controls under both normal and elevated salinity conditions. When subjected to salt stress, fresh (FW) and dry weights (DW) of both leaves and roots decreased more significantly in WT than in TG plants. Our results demonstrated the great potential of genetic manipulation of vacuolar H + -pyrophosphatase expression in TG perennial species for improvement of plant abiotic stress resistance.

MicroRNA528 (miR528) is a conserved monocot-specific small RNA that has the potential of mediating multiple stress responses. So far, however, experimental functional studies of miR528 are lacking. Here, we report that overexpression of a rice (Oryza sativa) miR528 (Osa-miR528) in transgenic creeping bentgrass (Agrostis stolonifera) alters plant development and improves plant salt stress and nitrogen (N) deficiency tolerance. Morphologically, miR528-overexpressing transgenic plants display shortened internodes, increased tiller number, and upright growth. Improved salt stress resistance is associated with increased water retention, cell membrane integrity, chlorophyll content, capacity for maintaining potassium homeostasis, CATALASE activity, and reduced ASCORBIC ACID OXIDASE (AAO) activity; while enhanced tolerance to N deficiency is associated with increased biomass, total N accumulation and chlorophyll synthesis, nitrite reductase activity, and reduced AAO activity. In addition, AsAAO and COPPER ION BINDING PROTEIN1 are identified as two putative targets of miR528 in creeping bentgrass. Both of them respond to salinity and N starvation and are significantly down-regulated in miR528-overexpressing transgenics. Our data establish a key role that miR528 plays in modulating plant growth and development and in the plant response to salinity and N deficiency and indicate the potential of manipulating miR528 in improving plant abiotic stress resistance.Abiotic stresses, especially drought, salt, and nitrogen (N) deficiency, are limiting factors for plant growth, development, and agricultural productivity. To cope with drought and salt stresses, plants have evolved similar strategies of osmotic adjustment (Munns, 2002;Zhu, 2002). Plants also evolved salinity-specific adjustments against ionic disequilibrium, which encompass excluding salt entry into plants and compartmentalizing ions into vacuoles or old leaves (Munns, 1993;Yeo, 1998). Another worldwide limiting factor for crop yields is N deficiency, which triggers reduced leaf growth rate and photosynthetic rate (Chapin et al., 1988). Due to the sessile nature of plants, abiotic stresses are unavoidable. Therefore, it is critical to develop reliable procedures to genetically modify plants for improved performance under environmental stresses, thereby enhancing agricultural productivity to meet the ever-growing demands in food production.Genetic engineering plays an increasingly important role in agronomic trait modifications in crop species. Currently, many genes encoding functional proteins, transcription factors, and proteins involved in signaling pathways have been identified as abiotic stressresponsive genes (Shinozaki and Yamaguchi-Shinozaki, 2007;Masclaux-Daubresse et al., 2010;Turan et al., 2012). Constitutive expression of some of these genes in transgenic plants has been demonstrated to lead to enhanced salt or drought tolerance (Golldack et al., 2011;Kim et al., 2013;Lu et al., 2013;Li et al., 2014). However, details of the regulatory network in the plant...

In eukaryotic cells IQGAP1 binds to and alters the function of several proteins, including actin, E-cadherin, ␤-catenin, Cdc42, and Rac1. Yeast IQGAP1 homologues have an important role in cytoskeletal organization, suggesting that modulation of the cytoskeleton is a fundamental role of IQGAP1. Phosphorylation is a common mechanism by which cells regulate protein function. Here we demonstrate that endogenous IQGAP1 is highly phosphorylated in MCF-7 human breast epithelial cells. Moreover, incubation of cells with phorbol 12-myristate 13-acetate (PMA) stimulated phosphate incorporation into IQGAP1. By using mass spectrometry, Ser-1443 was identified as the major site phosphorylated on IQGAP1 in intact cells treated with PMA. Ser-1441 was also phosphorylated but to a lesser extent. In vitro analysis with purified proteins documented that IQGAP1 is a substrate for protein kinase C⑀, which catalyzes phosphorylation on Ser-1443. Consistent with these findings, inhibition of cellular protein kinase C via bisindolymaleimide abrogated Ser-1443 phosphorylation in response to PMA. To elucidate the biological sequelae of phosphorylation, Ser-1441 and Ser-1443 were converted either to alanine, to create a nonphosphorylatable construct, or to glutamic acid and aspartic acid, respectively, to generate a phosphomimetic IQGAP1. Although overexpression of wild type IQGAP1 promoted neurite outgrowth in N1E-115 neuroblastoma cells, the nonphosphorylatable IQGAP1 S1441A/S1443A had no effect. In contrast, the S1441E/S1443D mutation markedly enhanced the ability of IQGAP1 to induce neurite outgrowth. Our data disclose that IQGAP1 is phosphorylated at multiple sites in intact cells and that phosphorylation of IQGAP1 will alter its ability to regulate the cytoskeleton of neuronal cells.Initially identified 10 years ago, IQGAP1 has been shown to participate in several fundamental cellular processes (for reviews see Refs 1 and 2). These include cell-cell attachment, ␤-catenin-mediated transcription, cell migration, regulation of actin, microtubule function, the mitogen-activated protein kinase cascade, and Ca 2ϩ /calmodulin signaling (2-4). IQGAP1 is a component of these diverse functions via direct interactions with multiple target proteins that are mediated by a number of protein interaction motifs in IQGAP1. These include the following: a calponin homology domain, responsible for actin binding (5, 6); a WW motif, which is necessary for the association of extracellular signal-regulated kinase 2 (a component of the mitogen-activated protein kinase pathway) (4); a calmodulinbinding IQ domain (5, 7); and a RasGAP-related domain that binds the small GTPases Cdc42 and Rac1 (5, 8). In addition, IQGAP1 binds to and regulates the functions of E-cadherin, ␤-catenin, and CLIP-170 (9 -12).Accumulating evidence reveals an important role for IQGAP1 in cytoskeletal function. The cytoskeleton of eukaryotic cells comprises several elements, including actin, microtubules, and intermediate filaments. Numerous proteins interact with the actin cytoskeleton...