Yanxi Lv1, Jianyi Zhang1, Naveed Ahmad1,2, Hong Yingqi1, Youbao Li1, Nan Wang1, Liu Xiuming3,4,5, Yao Na6, Xiaokun Li7,8. 1. College of Life Sciences, Engineering Research Center of the Chinese Ministry of Education for Bioreactor and Pharmaceutical Development, Jilin Agricultural University, Changchun, 130118, China. 2. Institute of Crop Germplasm Resources (Institute of Biotechnology), Shandong Academy of Agricultural Sciences; Shandong Provincial Key Laboratory of Crop Genetic Improvement, Ecology and Physiology, Jinan, 250100, People's Republic of China. 3. College of Life Sciences, Engineering Research Center of the Chinese Ministry of Education for Bioreactor and Pharmaceutical Development, Jilin Agricultural University, Changchun, 130118, China. xiuming1211@163.com. 4. Institute of Life Sciences, Wenzhou University, Wenzhou, 325035, Zhejiang, China. xiuming1211@163.com. 5. Biomedical Collaborative Innovation Center of Zhejiang Province, Wenzhou University, Wenzhou, 325035, Zhejiang, China. xiuming1211@163.com. 6. College of Life Sciences, Engineering Research Center of the Chinese Ministry of Education for Bioreactor and Pharmaceutical Development, Jilin Agricultural University, Changchun, 130118, China. yaona1103@126.com. 7. Institute of Life Sciences, Wenzhou University, Wenzhou, 325035, Zhejiang, China. xiaokunli@wmu.edu.cn. 8. Biomedical Collaborative Innovation Center of Zhejiang Province, Wenzhou University, Wenzhou, 325035, Zhejiang, China. xiaokunli@wmu.edu.cn.
Abstract
MAIN CONCLUSION: We performed genome-wide and heterologous expression analysis of the safflower cysteine protease family and found that inhibition of CtCP1 expression enhanced plant cold resistance. Cysteine protease (CP) is mainly involved in plant senescence and stress responses. However, the molecular mechanism of endogenous cysteine protease inhibition in plant stress tolerance is yet unknown. Here, we report the discovery and functional characterization of a candidate CP1 gene from safflower. The conserved structural topology of CtCPs revealed important insights into their possible roles in plant growth and stress responses. The qRT-PCR results implied that most of CtCP genes were highly expressed at fading stage suggesting that they are most likely involved in senescence process. The CtCP1 expression was significantly induced at different time points under cold, NaCl, H2O2 and PEG stress, respectively. The in-vitro activity of heterologously expressed CtCP1 protein showed highest protease activity for casein and azocasein substrates. The expression and phenotypic data together with antioxidant activity and physiological indicators revealed that transgenic plants inhibited by CtCP1-anti showed higher tolerance to low temperature than WT and CtCP1-OE plants. Our findings demonstrated the discovery of a new Cysteine protease 1 gene that exerted a detrimental effect on transgenic Arabidopsis under low-temperature stress.
MAIN CONCLUSION: We performed genome-wide and heterologous expression analysis of the safflower cysteine protease family and found that inhibition of CtCP1 expression enhanced plant cold resistance. Cysteine protease (CP) is mainly involved in plant senescence and stress responses. However, the molecular mechanism of endogenous cysteine protease inhibition in plant stress tolerance is yet unknown. Here, we report the discovery and functional characterization of a candidate CP1 gene from safflower. The conserved structural topology of CtCPs revealed important insights into their possible roles in plant growth and stress responses. The qRT-PCR results implied that most of CtCP genes were highly expressed at fading stage suggesting that they are most likely involved in senescence process. The CtCP1 expression was significantly induced at different time points under cold, NaCl, H2O2 and PEG stress, respectively. The in-vitro activity of heterologously expressed CtCP1 protein showed highest protease activity for casein and azocasein substrates. The expression and phenotypic data together with antioxidant activity and physiological indicators revealed that transgenic plants inhibited by CtCP1-anti showed higher tolerance to low temperature than WT and CtCP1-OE plants. Our findings demonstrated the discovery of a new Cysteine protease 1 gene that exerted a detrimental effect on transgenic Arabidopsis under low-temperature stress.