4.1 Molecular Mechanism of Response to Cold Stress
Under cold stress, the plant can respond in multiple ways which include the adaptation of leaf and tissue structures, the alteration of cell membrane compositions, the rearrangement of cells, an increase in osmosis maintaining substances (e.g., soluble sugar, proline, betaine), and an increase in the synthesis of antioxidants as a result of enhanced catalytical activity of certain enzymes (e.g., superoxide dismutase, peroxidase, ascorbate reductase). These strategies can help the plant to regain lost components and to balance energy generation, thus enabling it to adapt and survive in cold environments (Viswanathan et al, 2002). At the physiological level, cold stress can induce the perception of cold, signaling and transcriptional regulation (Zhang et al., 2009), which are mediated by specific molecular mechanisms. For this purpose, gene expressions are adjusted via extensive signaling and other regulating mechanisms in order to tune the metabolism and growth of the plant to allow adaptation to low temperatures (Chinnusamy et al., 2004).
4.1.1 Perception of low-temperature signal
The cellular membrane system consists of both the cell membrane and the endomembrane system. The cell membrane, representing the boundary where a cell interacts with the internal environment, is partially permeable to substances, and it maintains the equilibrium of a cell. On the other hand, the endomembrane system is the site where energy production as well as the synthesis, degradation and transfer of substances occur. As far as tolerance to low temperature is concerned, the stability of the cell membrane system is a key factor, as low-temperature stress can reduce the fluidity of the cellular membrane while increasing its permeability and therefore, cause soluble contents in the cells to diffuse out, resulting in metabolic disorders. At low temperatures, the cell membrane first sense the change in temperature, and then trigger appropriate physiological, biochemical and physical changes in its cells. For instance, within a certain low temperature range, fatty acid desaturase located on the cell membrane were found to be activated, causing large quantities of unsaturated fatty acids to be accumulated. This causes the fluidity of the membrane to be reduced, as the membrane lipid is changed from a disordered and liquid crystalline state to an ordered, gelatinous and partially solid one so that the stability of the cellular structure is maintained (Baid, 1994; Wang et al., 1997). In fact, such is the contribution of the fatty acids to cold tolerance that it is considered that the tolerance can be significantly improved by increasing the content of unsaturated fatty acids or the degree of fat unsaturation in plants (Sun et al., 1990; Murelli et al., 1995; Zhou et al., 2001). In Medicago satia and Brassica napus, the rigidification of cell membrane using drugs, induced the expression of COR (cold-responsive) genes, which were shown to be related to cold-acclimatization properties (Sangwan et al., 2001). Furthermore, through a steady drop of temperature, the selectiveness of the cell membrane was found to decline, causing solvents (e.g., K+, sugars and amino acids) to diffuse out. At the same time, ice cores were formed in intracellular spaces, and the plasma gelatinized in a non-reversible way. This caused the cells to freeze, thereby damaging the cell membrane, disturbing the cell metabolism and affecting the overall growth of the plants (Liu et al., 1994).
4.1.2 Transduction of low-temperature signal
After sensing the low-temperature through receptors on the cell membrane, proteins (mainly G-proteins) were phosphorylated by phospholipases, thus activating the Ca2+ channel on the cell membrane and increasing the Ca2+ concentration in cells. In this way, the specific Ca+ signal triggered by the low-temperature are transferred from the external environment to that of the internal cell membrane (Chinnusamy et al., 2004). Currently, Ca2+ is regarded as a second messenger for the transfer of signals related to low-temperature in plants as based on its intracellular concentration, certain Ca2+-dependent protein kinases (CDPK) are phosphorylated or dephosphorylated, hence inducing the expression of genes related to low-temperature. In this context, it has been reported that, when Arabidopsis thaliana L. or Medicago satia were exposed to low-temperatures, Ca2+ stored in the surrounding environment flowed into the cells, causing its intracellular concentration to rise rapidly. This temporary surge of Ca2+ was indispensable for inducing the expression of genes related to cold acclimatization and cold tolerance (Monroy et al., 1995). Furthermore, in Arabidopsis thaliana L., the full expression of some low-temperature-regulated genes such as CRT/DRE (encoding cor 6.6) were also dependent on the increase of Ca2+ levels (Tahtiharju et al., 1997). Similarly, in the case of seedlings of winter wheat and Trititrigia, training the plants for cold acclimatization promoted high activity of Ca2+-ATPase on the cell membrane, so that Ca2+ equilibrium of the cells could be maintained (Jian et al., 2002). Additionally, CDPK was also found to play a role in the signaling of the cold-stress-responsive mechanism while Saijo et al. (2000) found that, under induced cold stress, OsCDPK were overexpressed in transgenic rice, which also displayed better salt- and drought- resistance.
However, in addition to Ca2+, studies have suggested that the plant hormone abscisic acid (ABA) is also associated with low-temperature signaling (Chen et al., 1983). Under normal growth conditions, by applying external ABA and therefore reducing the expression of cold-responsive genes, protein synthesis was facilitated, and cold tolerance was improved in plants (Seki et al., 2002; Li et al., 2003). Studies involving ABA-insensitive mutants (ABI) and ABA-synthesis-deficient mutants (ABA-deficient) of Arabidopsis thaliana L. demonstrated that cold acclimatization and cold tolerance decreased in both mutants. However, when supplemented with external ABA, cold acclimatization properties were recovered in the ABA mutants (Heino et al., 1990; Gilmour et al., 1991; Llorente et al., 2000). Moreover, increasing evidence are suggesting that protein kinases and protein phosphorylases (PP) are also involved in the signaling of low-temperature acclimatization (Zhu et al., 2007).
4.1.3 Low-temperature transcriptional regulation
In response to hormonal stimuli and external environmental stresses, transcriptional regulation controls the intensity of gene expression or regulates gene expression spatiotemporally, through the specific binding of the transcriptional factor proteins to target genes. In this study, the low temperature resulted in drastic changes in the plant transcriptome. As estimated, cold-regulated genes account for 4-20% of the genome in Arabidopsis thaliana L. (Hannah et al., 2005; Lee et al., 2005). When faced with low-temperature stress, the plant senses the cold signal and activate the pathway for downstream signaling. The transductors in the cell wall, the cell membrane and plasma amplify the cold signal in cascade, and transfer it into the nucleus for inducing the expression of cold-regulated genes which will enable the plants to acclimatize to the cold in a short period of time (Yamaguchi-Shinozaki et al., 2006). Owing to the variety of abiotic stress receptors and signaling pathways in plants, cold stress could, in fact, activate a number of different signaling paths, causing various expression results. Meanwhile, different receptors and paths for signal transduction are also present the downstream of the Ca+ and ABA (Yamaguchi-Shinozaki et al., 2006). Collectively, the transduction of stress signals in plants is a complex network, involving interactions between the signal attributed to the multi-gene regulation after environmental stimulation. Several cis-acting elements often locate the promoter of a single cold-regulated gene so that a gene may be regulated by multiple signaling systems rather than being dependent on only a single or one type of signaling molecules. For instance, in the promoters of the genes whose expression were induced by drought, high salinity and low-temperature, RD29A, COR78 and LTI 78, presented the cis-acting elements of ABA response element (ABRE) and dehydration-responsive element (DRE), hence suggesting that the expression of these genes was simultaneously regulated by both ABA-dependent and non-ABA-dependent signaling pathways (Shinozaki et al., 2003).
4.2 Application of RNA-seq in plants
The second generation of RNA-Seq has been widely applied in many aspects of plant research, mainly for studying the differences between developmental stages, tissues and organs, mutants, and plant characteristics under different environments. Gene expression analysis, DEG identification, mining of functional genes, and phylogenetic analysis of these different conditions has been achieved with the help of transcriptome sequencing (Huang et al., 2014). Currently, RNA-seq has revealed the cold-stress-responsive mechanism of dozens of economically important crops including Arabidopsis thaliana L., Oryza sativa L., Brassica napus L., Camellia sinensis, Triticum aestivum L., Nicotiana tabacum, and Sorghum bicolor L.. This approach not only provides insight into the molecular mechanism of cold tolerance and the breeding of cold-tolerant strains, but also provides a technological reference and basic information for the stress-tolerance or avoidance mechanisms in other species (Zhang et al., 2020). As the RNA-Seq technologies continue to upgrade, and the analytical methods improve, it is expected that RNA-Seq would be more extensively and deeply applied in plant biology studies.
Transcriptome analysis has promoted studies on the cold tolerance of tea plants, with the availability of information on a number of cold-related genes. Wang et al. (2013) analyzed the transcriptomes of tea at different stages of cold acclimatization in the wild, and obtained 1,770 DEGs, including those corresponding to cold signal transduction and responsive factors, cell membrane-stabilizing genes and osmosis-responsive genes. Further analyses also demonstrated the crucial role of carbohydrate metabolism and Ca2+ signal transduction in the cold tolerance of tea, with these results providing an important reference for further studies on the cold tolerance of tea plants. In addition, based on transcriptome data, more genes related to cold tolerance in tea were cloned, e.g., the key genes in the betaine synthesis pathway (Cao et al., 2013) and genes encoding glutathione reductase (CsGRs) (Yue et al., 2014). Li et al. (2015) analyzed the mechanisms regulating the biosynthesis of secondary metabolites particularly catechin, caffeine and theanine for different tissues of tea plants via RNA-Seq. Similarly, Wu et al. (2014) demonstrated the molecular mechanism responsible for differences in catechin content of the leaves of different varieties of tea trees via RNA-seq. Wei et al. (2015) also applied this method for elucidating the molecular mechanism of the influence of flavonoid 3'-hydroxylase and flavonoid 3',S'-hydroxylase genes on the ratio of dehydroxylated catechins to trihydroxylated catechins under different shading treatment. Likewise, Li et al. (2012) compared differences between the transcriptomes of Fudingdabai and Xiaoxueya tea breed leaves, to explore the possible molecular genetic mechanism of inducing albino shoots in Xiaoxueya breed.
With the development and application of modern gene technology, researchers have studied the regulation of gene expression for cold tolerance of tea trees. This provided theoretical support for the clarification of cold tolerance mechanism of tea trees and the breeding of cold tolerance varieties. With further research, transcriptomic analysis will be more widely used in the study of tea plants, and its combined analysis with other omics such as proteomics and metabolomics will help to better understand and clarify the physiological and biochemical mechanisms of tea plants.