Although there is no consensus regarding the relationship between the plant tissue water content and cold hardiness, most researchers believe that cold hardiness is negatively associated with increasing water content due to lower water content can decreased osmotic pressure[5, 7, 8, 40]. In this study, an analysis of acclimation revealed that the freezing tolerance of alfalfa was highest in phase 3 because of its decreased water content and freezing conditions (Fig. 2c, d). Additionally, cellular dehydration reportedly decreases metabolic activity, growth, and energy consumption. De-acclimation and regenerative growth are related to tissue rehydration. Compared with tissues with low water contents, tissues with high water contents are more susceptible to mechanical damage caused by extracellular freezing and have a higher ice propagation rate.
Alfalfa survival during a cold winter can be attributed to several causes[13, 23, 31]: 1) decreases in the temperature and photoperiod induce acclimation in alfalfa; 2) a continuously decreasing temperature causes alfalfa to almost stop growing, acquire peak cold hardiness, and survive the midwinter; 3) an increase in the temperature in early spring decreases the cold hardiness of alfalfa and induces regeneration. Moreover, temperature fluctuations may induce re-acclimation and de-acclimation[1, 23]. Plants have evolved various strategies to minimize cellular damage in response to low-temperature stress. Examples include osmotic regulation, synthesis of soluble proteins, changes in cell membrane components, and hormonal regulation.
In the current study, there was no significant difference in the cold hardiness of ZD and WL440 in phase 1, but a significant difference was detected in phase 2, possibly due to the differential metabolism of soluble sugars and amino acids[13, 41]. Soluble sugars are sensitive to temperature changes. An earlier study by Bertrand demonstrated that the soluble sugar content in the root-crown region of alfalfa increases several-fold after only 8 h of a low-temperature treatment. Similarly, several recent studies have confirmed the existence of photoperiod-measuring mechanisms in the plant metabolic network that coordinates seasonal developmental programs to mitigate damages from less predictable stresses[3, 4, 42]. Liu reported that in Arabidopsis, changes in the cellular starch and sucrose contents can regulate the expression of photoperiod-related genes, thereby maintaining the diurnal energy level, which is crucial for plants to adapt to the relatively long and cold nights in winter. Amino acids serve as a nitrogen source for plants, but they are also important for adaptations to low-temperature stress[43, 44]. Amino acids accumulate in the alfalfa root-crown region at low temperatures and specific amino acids can delay protein degradation and maintain the structural stability of the plasma membrane[45–48].
In phase 3, there was a minor difference between the two cultivars, suggesting that sufficient cold acclimation will decrease the differences in the sensitivity to low temperatures among cultivars. Research regarding climate changes due to global warming has generated evidence that temperature fluctuations increase the risk of overwintering failure compared with the effects of an exposure to consistent low temperatures[49, 50].
Temperature increases result in changes in a series of plant metabolic pathways, which lead to the rapid loss of cold hardiness. In the current study, fucose content increased after de-acclimation. This is contrary to earlier findings. As a signaling molecule and protective agent, fucose regulates plant growth and development and the mechanism mediating responses to environmental stimuli. In addition to protecting proteins and cell membranes at low temperatures, fucose also co-regulates the plant heat stress response mechanism with hormones when temperature increases[51–53]. The composition and fluidity of the plasma membrane are key factors influencing plant sensitivity to low temperatures. At low temperatures, the plasma membrane transforms from a relatively fluid structure to a solid gel, in which lipids are tightly packed and highly ordered. The solid gel membrane is highly permeable and is easily ruptured. The decrease in glycerophospholipid metabolic activity during de-acclimation is believed to be related to the loss of cold hardiness. Glycerophosphate lipids, which are phospholipids comprising a phosphate and two fatty acids or fatty alcohols, are the main plasma membrane components. The comparison of different phases in this study revealed decreases in glycerophosphate, glycerophosphocholine, lysophosphatidylcholine (16:0), and lysoPC (15:0) content during de-acclimation. The decrease in unsaturated fatty acid content accelerated the loss of cold hardiness. Unsaturated fatty acids are useful for maintaining plasma membrane fluidity and cellular functions under low-temperature stress conditions, likely because of their cis-double bonds[13, 15, 52]. Miki conducted a proteomics-based analysis of Arabidopsis, which confirmed that changes in amino acid contents in plants are closely related to temperature. During acclimation and de-acclimation, hundreds of proteins are modified, among which transporters are the most reduced. On the basis of an analysis of the circadian clock and energy, Bonnot reported that in response to temperature limitation, organisms selectively translate mRNA to respond to environmental changes. According to a cluster analysis, most of the changes to proteins during acclimation and de-acclimation were reversible. Additionally, the changes during de-acclimation were faster than those during acclimation. In phase 4, tyrosine, succinic acid, and methionine decreased significantly in WL440, but were not significantly changed in ZD, This may help explain the observed differences between these cultivars. These findings were in accordance with the results of a previous study, in which functional amino acids were observed to protect cells from cold damage.
The metabolic pathways that differed between ZD_2 and WL440_2 were mainly related to purine metabolism and plant hormone signal transduction (Fig. 8). Abscisic acid (ABA) is a sesquiterpenoid hormone that affects plant growth and development, while also regulating plant responses to adverse environmental conditions. Furthermore, ABA-dependent signaling is one of the pathways responsive to low-temperature stress[56–58]. An exposure to low temperatures leads to an increase in ABA content. The accumulated ABA binds to receptors, which then interact with PP2C (type 2C protein phosphatase) to inhibit the binding of PP2C to SnRK2 (SNFI-related protein kinase 2). As a result, SnRK2 transcription factors are phosphorylated, which can activate the expression of ABA-responsive genes and increase cold hardiness[59, 60].
The mechanisms underlying alfalfa plant responses to cold conditions are primarily associated with changes in amino acids, lipid molecules, carbohydrates, and their related metabolic pathways. At different stages, various metabolites contribute to alfalfa cold hardiness. For example, alfalfa adapts to low-temperature environments via an increase in the abundance of amino acids (e.g., aspartic acid), fatty acids (e.g., arachidonic acid and norlinolenic acid), glycerophospholipids (e.g., lysophosphatidylcholine and phosphatidyl ethanolamine), and carbohydrates (e.g., glucose, maltose, and raffinose) in the root-crown region during phase 2. However, in phase 3, the contents of most of these metabolites are no longer increased, and may be decreased (with the exception of raffinose). Cystathionine and maltotriose content increase in phase 3. Alfalfa appears to cope with temperature increases mainly through decreases in various amino acid and glycerophospholipid contents, including tyrosine, succinic acid, methionine, glycerophosphate, glycerophosphocholine, lysophosphatidylcholine (16:0), and lysoPC (15:0) (Table 2). These observations, combined with the results of previous studies by Xu[1, 13], highlight the importance of amino acids and glycerophospholipids in both cold acclimation and de-acclimation. Their contents increase during cold acclimation and decrease during de-acclimation. Soluble sugars are mainly involved in increasing the cold hardiness of alfalfa as part of the cold acclimation process.