Constitutive Expression of Rice MicroRNA528 Alters Plant Development and Enhances Tolerance to Salinity Stress and Nitrogen Starvation in Creeping Bentgrass

Abstract

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...

Identification of microRNAs in Response to Drought in Common Wild Rice (Oryza rufipogon Griff.) Shoots and Roots

“…With the growth of rice, the expression of miR528 increased in the leaves. It has been reported that overexpression of osa-miR528 in transgenic creeping bentgrass ( Agrostis stolonifera ) changed the plant development and improved the plant tolerance to salt stress and nitrogen (N) deficiency [55]. Morphologically, miR528-overexpressing transgenic plants display shortened internodes, increased tiller numbers, and upright growth [55].…”

“…It has been reported that overexpression of osa-miR528 in transgenic creeping bentgrass ( Agrostis stolonifera ) changed the plant development and improved the plant tolerance to salt stress and nitrogen (N) deficiency [55]. Morphologically, miR528-overexpressing transgenic plants display shortened internodes, increased tiller numbers, and upright growth [55]. Furthermore, overexpression of osa-miR528 in transgenic rice reduced the rice tolerance to Arsenic (As) stress, and the amino acid contents changed and the oxides increased both in the shoots and roots [56].…”

Ectopic expression of a cyanobacterial flavodoxin in creeping bentgrass impacts plant development and confers broad abiotic stress tolerance

“…The plants and the sand were flushed using sufficient water to remove residual nutrients and then natured using modified 19 Murashige and Skoog (MS) solution supplemented with N at different concentrations (0, 0.4, 2, 10 or 40 mM). The preparation of MS solution and N addition was as previously described (Yuan et al, 2015). Three and five weeks after N starvation treatment, the shoots were harvested for further analysis to examine expression of the genes of interest and measure various physiological parameters.…”

“…The nitrite content was calculated from a KNO 2 standard curve. NiR activity was determined following Yuan et al (2015).…”

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

“…All these indicate that miRNAs are master regulators in plant response to various environmental cues and may have great potential in crop genetic engineering for enhanced plant performance under adverse environmental conditions. Indeed, this has been demonstrated for an increasing number of miRNAs, such as miR319, miR528, miR396 and miR408 (Chen et al ., ; Ma et al ., ; Yuan et al ., ; Zhou et al ., ). Recently, a conserved miRNA, miR393 has been identified with the potential of mediating plant response to a variety of biotic and abiotic stresses.…”