Overexpression of MpCYS4, A Phytocystatin Gene from Malus prunifolia (Willd.) Borkh., Enhances Stomatal Closure to Confer Drought Tolerance in Transgenic Arabidopsis and Apple

Abstract

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.

Genome-Wide Analysis and Cloning of the Apple Stress-Associated Protein Gene Family Reveals MdSAP15, Which Confers Tolerance to Drought and Osmotic Stresses in Transgenic Arabidopsis

“…Relative electrolytic leakage was examined as described by Tan et al [ 56 ]. Chlorophyll concentrations were determined using the protocol of Liang et al [ 57 ], while MDA levels were obtained as described by Wei et al [ 42 ].…”

Overexpression of the class I homeodomain transcription factor TaHDZipI‐5 increases drought and frost tolerance in transgenic wheat

“…Environmental stresses such as frost or drought trigger specific signal transduction pathways, which activate the expression of stress-responsive genes (Braam et al, 1997;Bray, 1997;Hwang et al, 2002;Tena et al, 2001;Zhu, 2016). Gene expression starts from the modulation of transcription by stress-related transcription factors (TFs), which regulate a number of physiological processes under stress, including cuticular wax biosynthesis (Aharoni et al, 2004;Bi et al, 2016Bi et al, , 2017Borisjuk et al, 2014;Seo et al, 2011), stomatal closure (Ren et al, 2010;Tan et al, 2017), reactive oxygen species (ROS) detoxification (Jiang and Deyholos, 2009) and structural alterations in plasma membranes (Pearce, 1999). Manipulation using genes encoding stressrelated TFs offers the possibility to regulate large groups of genes involved in the same physiological processes, and therefore, this intervention draws the attention of plant biotechnologists (Agarwal et al, 2017;Gahlaut et al, 2016;Hrmova and Lopato, 2014).…”

Silencing barley cystatins HvCPI‐2 and HvCPI‐4 specifically modifies leaf responses to drought stress

“…Genetically modified plants have been used to understand how the overexpression of a PhyCys may affect the response of the plant to a specific abiotic stress. For example, enhanced tolerance to drought has been achieved by overexpressing PhyCys in soybean, Arabidopsis , and apple (Prins, van Heerden, Olmos, Kunert, & Foyer, ; Quain et al, ; Tan, Li, Yang, Sun, et al, ; X. Zhang et al, ). However, there is only one report that analyses the response to an abiotic stress in a PhyCys silenced transgenic plant.…”

“…Oryzacystatin‐I‐dependent inhibition of CysProt also enhanced drought tolerance in Glycine max and Arabidopsis through strigolactone‐mediated signalling (Quain et al, ). Very recently, Tan, Li, Yang, Sun, et al () reported that the overexpression of the PhyCys MpCYS4 gene from Malus prunifolia enhanced stomatal closing, up‐regulated the transcriptional levels of abscisic‐acid‐ and drought‐related genes, and conferred drought tolerance in Arabidopsis and apple ( Malus domestica ) transgenic plants. Tan, Li, Yang, Li, et al () also observed delayed leaf senescence in apple MpCYS4‐overexpressing plants, in both the natural and stress‐induced scenarios.…”