Genome-wide analysis of FRF gene family and functional identification of HvFRF9 under drought stress in barley.

FHY3 and its homologous protein FAR1 are the founding members of FRS family. They exhibited diverse and powerful physiological functions during evolution, and participated in the response to multiple abiotic stresses. FRF genes are considered to be truncated FRS family proteins. They competed with FRS for DNA binding sites to regulate gene expression. However, only few studies are available on FRF genes in plants participating in the regulation of abiotic stress. With wide adaptability and high stress-resistance, barley is an excellent candidate for the identification of stress-resistance-related genes. In this study, 22 HvFRFs were detected in barley using bioinformatic analysis from whole genome. According to evolution and conserved motif analysis, the 22 HvFRFs could be divided into subfamilies I and II. Most promoters of subfamily I members contained abscisic acid and methyl jasmonate response elements; however, a large number promoters of subfamily II contained gibberellin and salicylic acid response elements. HvFRF9, one of the members of subfamily II, exhibited a expression advantage in different tissues, and it was most significantly upregulated under drought stress. In-situ PCR revealed that HvFRF9 is mainly expressed in the root epidermal cells, as well as xylem and phloem of roots and leaves, indicating that HvFRF9 may be related to absorption and transportation of water and nutrients. The results of subcellular localization indicated that HvFRF9 was mainly expressed in the nuclei of tobacco epidermal cells and protoplast of arabidopsis. Further, transgenic arabidopsis plants with HvFRF9 overexpression were generated to verify the role of HvFRF9 in drought resistance. Under drought stress, leaf chlorosis and wilting, MDA and O2 - contents were significantly lower, meanwhile, fresh weight, root length, PRO content, and SOD, CAT and POD activities were significantly higher in HvFRF9-overexpressing arabidopsis plants than in wild-type plants. Therefore, overexpression of HvFRF9 could significantly enhance the drought resistance in arabidopsis. These results suggested that HvFRF9 may play a key role in drought resistance in barley by increasing the absorption and transportation of water and the activity of antioxidant enzymes. This study provided a theoretical basis for drought resistance in barley and provided new genes for drought resistance breeding.

Hypoxia preconditioning protects corneal stromal cells against induced apoptosis.

The purpose of this study, was to determine whether hypoxia preconditioning can protect corneal stromal cells from UV stress and cytokine mediated apoptosis. Two models were implemented. First, primary cultured bovine corneal fibroblasts were preconditioned with 0.5-1.5% O2 for 4 hr and stressed with UV-irradiation or stimulation of Fas receptor. Second, bovine eyes were preconditioned with 0.5% O2 for 4 hr and stressed by epithelial scraping to induce anterior keratocyte apoptosis. Cell fate was analyzed at 4 hr after stress using quantitative TUNEL or condensed nuclei assays. Cell apoptotic rates in hypoxia preconditioned groups were significantly lower (50-80%) than that of normoxia control groups. Hypoxia prevented the degradation of the transcription factor HIF-1alpha. CoCl2 (100-200 microM), a chemical inducer of HIF-1alpha, also produced strong protection against UV and Fas induced apoptosis. Moreover, hypoxia preconditioned media protected cells against UV-induced apoptosis. These findings demonstrate that hypoxia preconditioning has a generalized protective effect against stromal fibroblast and keratocyte apoptosis and suggest that HIF-1alpha mediated expression and secretion of protective factors is involved.

TRPC4 knockdown suppresses epidermal growth factor-induced store-operated channel activation and growth in human corneal epithelial cells.

Epidermal growth factor (EGF) in corneal epithelial cells stimulates proliferation by inducing capacitative calcium entry (CCE). However, neither the identity nor the mechanism of activation of the plasma membrane influx pathway that mediates CCE is known. Accordingly, we determined, in human corneal epithelial cells, whether or not (i) CCE is dependent upon stimulation of storeoperated channel (SOC) activity, (ii) the canonical transient receptor potential (TRP) protein isoform TRPC4 is a component of such channels, and (iii) suppression of TRPC4 protein expression decreases EGF-induced stimulation of SOC activity and proliferation. The whole cell patch-clamp technique was used to monitor TRPC4-mediated stimulation of SOC activity following intracellular calcium store depletion and induction of CCE. TRPC4 small interfering RNA transfection suppressed TRPC4 protein expression. Reverse transcription-PCR and Western blot analysis were used to assess knockdown efficiency of mRNA and protein expression. [(3)H]Thymidine incorporation was used to evaluate EGF-in-duced mitogenesis. Ca(2+) transients were measured by single-cell fluorescence imaging. TRPC4 knockdown decreased mRNA and protein expression by 89 and 87%, respectively. In these cells, EGF-induced SOC activation elicited by intracellular calcium store depletion was obviated; 2) EGF-induced CCE fell by 76%; 3) EGF-induced stimulation of SOC activity was eliminated; and 4) EGF-induced increases in proliferation fell by 54%. Thus, TRPC4 is a component of SOC in human corneal epithelial cells whose activation by EGF is requisite for an optimum mitogenic response to this growth factor.