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Phytocystatins (PhyCys) comprise a group of inhibitors for cysteine proteinases in plants. They play a wide range of important roles in regulating endogenous processes and protecting plants against various environmental stresses, but the underlying mechanisms remain largely unknown. Here, we detailed the biological functions of MpCYS4, a member of cystatin genes isolated from Malus prunifolia. This gene was activated under water deficit, heat (40°C), exogenous abscisic acid (ABA), or methyl viologen (MV) (Tan et al., 2014a). At cellular level, MpCYS4 protein was found to be localized in the nucleus, cytoplasm, and plasma membrane of onion epidermal cells. Recombinant MpCYS4 cystatin expressed in Escherichia coli was purified and it exhibited cysteine protease inhibitor activity. Transgenic overexpression of MpCYS4 in Arabidopsis (Arabidopsis thaliana) and apple (Malus domestica) led to ABA hypersensitivity and series of ABA-associated phenotypes, such as enhanced ABA-induced stomatal closing, altered expression of many ABA/stress-responsive genes, and enhanced drought tolerance. Taken together, our results demonstrate that MpCYS4 is involved in ABA-mediated stress signal transduction and confers drought tolerance at least in part by enhancing stomatal closure and up-regulating the transcriptional levels of ABA- and drought-related genes. These findings provide new insights into the molecular mechanisms by which phytocystatins influence plant growth, development, and tolerance to stress.

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Glycine-rich RNA-binding proteins (GRRBPs) are involved in the post-transcriptional regulation of genes. Although they are known to have roles in plant responses to environmental stresses, their functions in diverse species under stress conditions are still unverified. We assessed the biological roles of MpGR-RBP1, a GR-RBP from Malus prunifolia that is up-regulated by salinity, oxidation, or abscisic acid. Under control of the 35S promoter, its ectopic expression in Arabidopsis resulted in accelerated seed germination and seedling growth in two transgenic lines when plants were exposed to high salt or oxidative stress. This gene also contributed to the enhancement of salt tolerance in transgenic Arabidopsis plants. Consistently, the enhanced tolerance was confirmed by the changes of physiological parameters including electrolyte leakage, chlorophyll concentration and malondialdehyde accumulation. The accumulation of reactive oxygen species (ROS) in the transgenics was appreciably decreased under salt stress. In addition, MpGR-RBP1 had an effect on stomatal closure under saline conditions. Taken together, these results demonstrate that MpGR-RBP1 affects the growth and tolerance of salt-stressed Arabidopsis plants. Its functioning may be due, in part, to its influence on ROS accumulation and stomatal behavior, thereby leading to improved salt tolerance.

Cystatins, or phytocystatins, are plant-specific inhibitors of cysteine proteinases that help regulate endogenous processes and protect plants against heat, salinity, cold, water deficits, chilling, and abscisic acid treatment. MpCYS2, a cystatin gene from Malus prunifolia, localized to the nucleus, cytoplasm, and plasma membrane in onion epidermal cells. Controlled by the 35S promoter, its ectopic expression in transgenic Arabidopsis lines caused accelerated seed germination and greater seedling growth when plants were exposed to osmotic or oxidative stress. Expression by this gene was also associated with enhanced drought tolerance in those transgenics. This positive response was manifested by changes measured in electrolyte leakage, the chlorophyll concentration, and malondialdehyde accumulations. Production of reactive oxygen species (ROS) was appreciably decreased in the dehydration-treated transgenic lines. This gene also influenced root hair development under osmotic-stress conditions. Our findings indicate that MpCYS2 affects the growth and tolerance of drought-stressed Arabidopsis plants possibly because of its influence on ROS accumulations and root hair formation.

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Cystatins, or phytocystatins (PhyCys), comprise a family of plant-specific inhibitors of cysteine proteinases. They are thought to help regulate endogenous processes and protect plants against biotic or abiotic stresses, such as heat, salinity, cold, water deficit, chilling, and abscisic acid (ABA) treatment. We isolated and identified a novel cystatin gene from Malus prunifolia, MpCYS5. Its expression was typically induced by salt stress treatment; ectopic expression in Arabidopsis enhanced salt tolerance. Physiological parameters confirmed this phenotype, with the transgenics having remarkably lower electrolyte leakage (EL) values, higher chlorophyll concentrations, and lower levels of malondialdehyde (MDA) upon salt treatment. In addition, the accumulation of reactive oxygen species was markedly regulated by MpCYS5 under stress conditions, as shown by fluctuations in the concentrations of hydrogen peroxide and superoxide radicals and the activities of antioxidant enzymes. We also noted that this gene modulated tunicamycin (TM)-induced endoplasmic reticulum (ER) stress tolerance and functioned in the unfolded protein response (UPR)-signaling pathway in Arabidopsis. This was confirmed by the expression of eight ER stress-responsive genes. All marker genes examined were strongly induced in the wild type, while most of them maintained relatively stable over time in the transgenics. These results demonstrated that ectopic expression of a cystatin gene is associated with salttolerant and TM-tolerant phenotypes. Therefore, the discovery of MpCYS5 from M. prunifolia might establish a molecular link between the ER stress response and salt tolerance in plants.

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