Cold is one of the most important abiotic stresses limiting plant geographical distribution and crop production (Ye et al. 2017). The damage caused by low temperature to plants can be classified as cold injury (low temperatures above 0℃) or freezing injury (temperatures below 0℃). Cold stress can destablize the plant cell membrane, increase the accumulation of reactive oxygen species and disrupt protein structure, all of which negatively impact photosynthesis, metabolism, and growth and development (Chinnusamy et al. 2007; Liu et al. 2018; Yin et al. 2021).
Plants have evolved complex regulatory networks that mediate responses to cold stress to increase survival. These include the rapid deployment of the CBF/DREB (C-repeat binding/dehydration element binding) family of transcription factors, which recognize the C-repeat and DRE cis elements in downstream COLD RESPONSIVE (COR) genes to modulate their expression (Shi et al. 2012). The expression of COR genes, such as COR15A, RD29A, KIN1 and GOLS3, can improve plant tolerance to low temperature (Knight et al. 2004; Thomashow 2010; Park et al. 2015; Guo et al. 2019). In addition, cold stress can induce the expression of abscisic acid (ABA) and jasmonic acid (JA) biosynthetic genes, resulting in the accumulation of ABA and JA, which also regulate genes required for cold response and survival (Knight et al. 2004; Hu et al. 2013; Feng et al. 2016). Allene oxide cyclase (AOC) is one of the key enzymes in jasmonic acid (JA) biosynthesis, which can affect the stereoisomerization and biological activity of jasmonic acid molecules, and plays an important role in plant stress resistance (Sun et al. 2020). Jasmonic acid induces the degradation of JAZ protein and activates the expression of cold resistance-related genes, thus improving plant cold resistance (An et al. 2021). Abiotic stresses such as cold, salt and drought usually increase the content of ABA, which in turn induces stomatal closure and reduces transpiration rate; ABA can also regulate the expression of stress-related genes (Mulholland et al. 2003; Jiang et al. 2014; Li et al. 2019). The rate-limiting enzyme of ABA biosynthesis is 9-cis-epoxycarotenoid dioxygenases (NCEDs). Together, these mechanisms act to enhance the stability of cellular membranes by promoting accumulation of osmolytes such as proline, soluble sugar and soluble protein (Ashraf and Foolad 2007).
Cysteine proteinases, also known as thiol or sulfhydryl proteinases, play various roles in plant growth and development, including senescence and programmed cell death (PCD) (Xu et al. 2003; Paireder et al. 2016; Burke et al. 2020). Senescence can be conditioned by several factors, including water deficit, phytohormones, and pathogen infection (Yang et al. 2003; Munné-Bosch and Alegre 2004; Mercedes et al. 2016). Likewise, PCD can be triggered by a variety of biotic and abiotic stresses (Paireder et al. 2016; Burke et al. 2020). Both abiotic and biotic stress can lead to damage of intracellular proteins, and proteolysis of damaged proteins by cysteine proteinases can minize the potentially negative effects of aberrant protein activity (García-Lorenzo et al. 2006; López-Barón et al. 2017). Abiotic stresses will aggravate the production of reactive oxygen species (ROS), and the excessive accumulation of ROS will lead to oxidative stress, leading to various metabolic disorders and affecting plant growth and development (Bose et al. 2014). Cysteine proteinases are up-regulated under oxidative stress and play an important role in maintaining cell metabolism (Usui et al. 2007). Cysteine proteinases can also regulate the level of reactive oxygen species in cells, thus reducing oxidative damage to membranes and proteins (Hoorn and Renier 2008).
Several studies have revealed that cysteine proteinase genes can be induced by abiotic stress (Jones and Mullet. 1995; Zang et al. 2010; Zheng et al. 2018). For example, in Arabidopsis thaliana (Arabidopsis), Pisum sativum, Nicotiana tabacum and Ipomoea batatas, cysteine proteinase genes are upregulated in response to drought, salt, cold or exogenous hormones such as gibberellin, jasmonic acid and abscisic acid (Koizumi et al. 1993; Jones and Mullet 1995; Beyene 2006; Chen et al. 2010). Zhang et al. exposed wheat seedlings to low temperature, high temperature, salt and drought stresses, and found that wheat TaCP3 was induced by all these conditions, especially drought (Zhang et al. 2019).
Ectopic expression of the cysteine protease SpCP3 from sweet potato in transgenic Arabidopsis enhanced the sensitivity of the plants to drought stress (Chen et al. 2013). Heterologous expression of Salix matsudana SmCP in Arabidopsis improved salt tolerance, and this was associated with increased stability of cell membrane and activity of the antioxidant enzyme system (Zheng et al. 2018). Therefore, plant cysteine proteases play a key role in the defense to abiotic stress.
Vitis amurensis (Amur grape) is native to present-day northeastern China and far-eastern Russia, and is adapted to extremely low winter temperatures. Currently there is wide interest in exploiting genes/alleles conferring cold tolerance in V. amurensis for cultivar improvement of domestic grapes, including the commerically valuable Vitis vinifera. In this study, we identified a cysteine proteinase gene from V. amurensis that is induced in response to cold. We generated transgenic Arabidopsis expressing VaCP17, and analyzed phenotypic, physiological and biochemical indices related to cold stress response, and evaluated expression of cold-resistance related genes. We further identified proteins that could interact with VaCP17 by yeast two-hybrid technology. Together, the results of these experiments lay the foundation for further studies of the molecular mechanisms of VaCP17 in cold resistance of Vitis amurensis.