A novel Glycine soja cysteine proteinase inhibitor GsCPI14, interacting with the calcium/calmodulin-binding receptor-like kinase GsCBRLK, regulated plant tolerance to alkali stress

Abstract

It has been well demonstrated that cystatins regulated plant stress tolerance through inhibiting the cysteine proteinase activity under environmental stress. However, there was limited information about the role of cystatins in plant alkali stress response, especially in wild soybean. Here, in this study, we focused on the biological characterization of a novel Glycine soja cystatin protein GsCPI14, which interacted with the calcium/calmodulin-binding receptor-like kinase GsCBRLK and positively regulated plant alkali stress tolerance. The protein-protein interaction between GsCBRLK and GsCPI14 was confirmed by using split-ubiquitin based membrane yeast two-hybrid analysis and bimolecular fluorescence complementation assay. Expression of GsCPI14 was greatly induced by salt, ABA and alkali stress in G. soja, and GsCBRLK overexpression (OX) in Glycine max promoted the stress induction of GmCPI14 expression under stress conditions. Furthermore, we found that GsCPI14-eGFP fusion protein localized in the entire Arabidopsis protoplast and onion epidermal cell, and GsCPI14 showed ubiquitous expression in different tissues of G. soja. In addition, we gave evidence that the GST-GsCPI14 fusion protein inhibited the proteolytic activity of papain in vitro. At last, we demonstrated that OX of GsCPI14 in Arabidopsis promoted the seed germination under alkali stress, as evidenced by higher germination rates. GsCPI14 transgenic Arabidopsis seedlings also displayed better growth performance and physiological index under alkali stress. Taken together, results presented in this study demonstrated that the G. soja cysteine proteinase inhibitor GsCPI14 interacted with the calcium/calmodulin-binding receptor-like kinase GsCBRLK and regulated plant tolerance to alkali stress.

Rice bifunctional phytocystatin is a dual modulator of legumain and papain-like proteases

“…Nterm and CtermTemplates, selected by the software to build the OcXII model [including proteins deposited in the protein databank (PDB) (Berman 2000)], during germination (Hong et al 2008). Likewise, phytocystatins can be required for the inhibition of proteolytic activities under stressful conditions including: high soil alkalinity (Sun et al 2014), drought (Quain et al 2014), senescence (Diaz-Mendoza et al 2014), cell death (Belenghi et al 2003), pathogen attack (Carrillo et al 2011b), and phytopathogenic fungi (Martinez et al 2005;Wang et al 2008;Pirovani et al 2010), and a diversity of abiotic stresses (Zhang et al 2008;Hwang et al 2010;.…”

“…For example, legumain proteases are one of the major players in programmed cell death (Hatsugai et al 2015), and consequently, carboxy-extended phytocystatins play important role in this pathway as well. In fact, one homologous phytocystatin appears to be involved in alkali responses in Glycine soja by balancing its target protease activity and regulating programmed cell death induced by papain or other cysteine proteases (Sun et al 2014). Alkalinization is an unfavorable condition for rice germination and growth.…”

“…Some carboxy-extended phytocystatins exhibit antifungal activity by inhibiting mycelium growth (Yang and Yeh 2005;Pirovani et al 2010) and phytopathogenic fungi (Martinez et al 2005;Wang et al 2008), while also providing plant resistance to insects and pathogens (Senthilkumar et al 2010) or inhibiting spore germination and mycelium development in vitro (Carrillo et al 2011a). Chitosan is a fungal elicitor involved in the increased expression of phytocystatin proteins (Agrawal et al 2002;.…”

Overexpression of MpCYS2, a phytocystatin gene from Malus prunifolia (Willd.) Borkh., confers drought tolerance and protects against oxidative stress in Arabidopsis

“…Phytocystatin expression is key to acquiring abiotic stress tolerance (van der Vyver et al 2003;Zhang et al 2008;Benchabane et al 2010;Hwang et al 2010;Wang et al 2012;Sun et al 2014). For example, transformed tobacco plants expressing rice cystatin OC-I are more resistant to the challenges that chilling stress poses to photosynthesis (van der Vyver et al 2003).…”

A Phytocystatin Gene from Malus prunifolia (Willd.) Borkh., MpCYS5, Confers Salt Stress Tolerance and Functions in Endoplasmic Reticulum Stress Response in Arabidopsis

“…The expression of PhyCys is also considered important for the acquisition of abiotic stress tolerance (van der Vyver et al 2003;Zhang et al 2008;Benchabane et al 2010;Hwang et al 2010;Wang et al 2012;Sun et al 2014). For example, transformed tobacco plants expressing the rice cystatin OC-I are more resistant to the negative impacts of chilling stress on photosynthesis (van der Vyver et al 2003).…”