Abiotic stresses in the environment can disadvantageously affect the normal growth, development and yield of crops. Because of frequent climate abnormalities and inappropriate agricultural management strategies, abiotic stresses have become a major challenge threatening global agricultural production and development. Plant damage from abiotic stresses is mainly caused by the loss of cell homeostasis leading to cell death (Huang et al., 2012; Rengel et al., 2012). In order to maintain the stability of the cell structure and function and survive under adverse conditions, plants have evolved a number of adaptative physiological, biochemical, cellular and molecular responses to abiotic stresses (Bohnert et al., 1995; Browse and Xin, 2001; Chinnusamy et al., 2007). Plants respond to abiotic stresses by regulating the expression of a number of stress-induced genes that may be associated with stress tolerance, transcription regulation or signal transduction (Thomashow 1999; Shinozaki et al., 2003; Nakashima et al., 2009). Transcriptome analysis of four rice genotypes demonstrated that an average of 5975 genes in every genotype, accounting for about 18% of the annotated genes, were differentially expressed under cold stress (Shen et al., 2014). To date, a number of genes have been identified that are associated with mechanisms of abiotic stress defense, and annexin genes are an important category of relevant genes (Clark et al., 2012).
Annexins are an evolutionarily conserved multigene family of Ca2+-dependent phospholipid-binding proteins that occur widely in plants and animals (Rescher and Gerke, 2004; Mortimer et al., 2008; Jami et al., 2012). Sequence analysis has demonstrated that plant annexins harbor motifs or residues related to peroxidase and ATPase/GTPase activity, as well as calcium channel activity(Mortimer et al., 2008), which has also been well demonstrated in subsequent research (Gorecka et al., 2005; Laohavisit et al., 2012, 2013; Richards et al., 2014). A number of annexin genes have been characterized successively in monocot and dicot plants since the first plant annexin protein was isolated successfully in tomato (Calvert et al., 1996; Mortimer et al., 2008; Qiao et al., 2015; Wang et al., 2018). Plant annexins play a role in diverse aspects of plant growth and development, and they are expressed in many tissues from different development stages (Clark et al., 2012). Moreover, previous evidence suggests that annexin genes from a range of plant species are transcriptionally activated in response to abiotic stresses. An initial report suggested that the alfalfa annexin gene (AnnMs2) is activated by drought stress, osmotic stress, and ABA treatment (Kovacs et al., 1998). Subsequent evidence suggests that annexins play an important role in other plant abiotic stress responses. For example, AnnAt1 was found to be associated with drought tolerance in Arabidopsis, with more sensitivity to drought stresses in loss-of-function AnnAt1 mutants and improved drought tolerance in gain-of-function mutants (Konopka-Postupolska et al., 2009). AnnAt1 was also found to interact with AnnAt4, such that AnnAt1 and AnnAt4 regulated salt and drought stress tolerance by interacting with each other in a light-dependent manner (Lee et al., 2004; Huh et al. 2010). Overexpression of the annexin gene AtANN8 enhanced salt and dehydration stress tolerance in Arabidopsis (Yadav et al. 2016). In tomato (Solanum pennellii), the annexin gene SpANN2 was found to be involved in drought and salt stress tolerance, with improved growth in SpANN2-overexpression (OE) lines (Ijaz et al., 2017). The cotton annexin gene GhANN1 was also found to be involved in drought and salt stress tolerance (Zhou et al., 2011; Zhang et al., 2015).
Genome sequencing revealed that there are ten annexin genes in rice (Singh et al., 2014), and the functional roles of several of these genes in responding to abiotic stresses have been characterized. The rice annexin gene OsANN1 (Os02g51750) was found to be associated with heat and drought stress response, with more sensitivity to heat and drought stress in RNA interference plants and improved growth in OsANN1-OE lines (Qiao et al., 2014). Similarly, OsANN3 (Os07g0659600) was also confirmed to be a positive regulator of drought stress tolerance in rice in an ABA-dependent manner (Li et al., 2019). We have recently demonstrated that the rice annexin gene OsAnn3 (Os05g0382600) is relevant for cold stress tolerance, with more sensitivity to cold stress when OsAnn3 was knocked out by CRISPR/Cas9-mediated gene modification (Shen et al., 2017). This study was the first report of an annexin gene involved in cold tolerance in rice, despite the fact that low temperature is a common type of stress in the life cycle of rice. In general, the functional and physiological roles of rice annexin genes in responding to cold stress remain unknown.
In the present study we isolate and characterized a putative annexin protein family gene in rice, designated as OsAnn5 (Os06g0221200) [consistent with the nomenclature of Singh et al (Singh et al., 2014)]. We demonstrate that the expression of OsAnn5 increased following low temperature treatment (4 ~ 6 °C for 4 days). We directly tested the role of OsAnn5 by constructing a series of transgenic rice plants; we used CRISPR/Cas9-mediated genome editing to create an OsAnn5 knock out (KO) line and also constructed OE, OsAnn5pro::GUS and OsAnn5-GFP lines. We found that the OE lines enhanced cold tolerance in rice, whereas the KO lines were more sensitive to cold stress.