1. Introduction
Salt stress is one of the important abiotic stress factors that affect the normal growth and development of higher plants and algae [
1]. At present, most of the plant physiological studies on salt stress focus on NaCl, while calcium stress attracts less attention [
2].
The impact of high calcium stress on agricultural production is more and more serious. In the secondary salinization of soil, which is caused by over-irrigation, intensive farming, and industrial pollution, Ca
2+ has accumulated excessively and accounts for over 60% of total cations [
3,
4,
5]. Crop production has been seriously limited by the high calcium stress in greenhouses [
6]. In addition, the high content of calcium ions in karst soil also severely affects the plant community distribution and crop yield [
7,
8,
9,
10]. Understanding the response mechanism to high calcium stress is of great significance. It will contribute to clarifying the damage mechanism of calcium stress in plants and develop salt-tolerant varieties by means of genetic engineering [
2,
11,
12].
When living in a high calcium environment, higher plant cells usually passively absorb massive Ca
2+ [
8,
9]. The Ca
2+ will then combine with PO
43− to form insoluble precipitation in the cytoplasm. This process will not only affect the effective use of phosphorus but also interfere with normal energy metabolism and physiological signal transmission [
8,
9]. The study by Singh and Goswami showed that high calcium stress destroyed the structure of the photosynthetic membrane, thus affecting the photosynthesis and growth rate [
13]. In addition, excessive calcium ions have been proven to cause severe oxidative damage and osmotic stress in plants, which led to reductions in crop yield [
14,
15,
16]. However, up to now, the effect of high calcium stress on algae is unknown.
The small green algae, represented by
Chlamydomonas reinhardtii and
Dunaliella salina, have simple structures and similar cellular metabolic pathways to higher plants. Therefore, they have been the model organisms for studying plant photosynthesis and metabolic regulation [
17]. Compared with the higher plants, small green algae have the advantages of simpler culture conditions, smaller space requirements, and shorter culture periods. They have offered a lot of meaningful information to salt-tolerant research of plants [
18,
19,
20].
Parachlorella kessleri is one of the typical representatives of small green algae. It has diverse habitats and strong adaptability. Some of the strains can even survive in extremely acidic environments [
21]. In the previous study, we found and isolated a strain of
Parachlorella kessleri FACHB-3316 from a high concentration (3.6 g/L) of calcium chloride solution in the laboratory [
22]. These characteristics make the strain a good material for studying plant tolerance to calcium stress.
At present, the genetic regulation mechanism of plant salt tolerance is not completely clear, especially at the gene expression level [
23]. Transcriptomics can reveal the expression of the whole genome under stress, which is of great significance to increase the understanding of the complex regulatory network related to adaptation and tolerance. Thus, transcriptomics has become an important means of plant salt stress research [
24,
25,
26]. Therefore, the method of transcriptome combined with physiological and morphological analysis was used to explore the high CaCl
2 alleviation mechanism of
Parachlorella kessleri FACHB-3316. The purpose of this study is to make up for the deficiency of algal research on calcium stress and provide theoretical data for further explaining the mechanism of plant salt tolerance.
3. Discussion
There are many salt-tolerant plants in nature. They have gradually formed a set of well-developed salt tolerance mechanisms in the long evolutionary process. In the previous study, a green alga that could tolerate high concentrations of CaCl
2 was accidentally found [
22]. In this study, the transcriptome combined with physiological and morphological results revealed the mechanisms of tolerance of this strain to high calcium stress, which were mainly by strengthening photosynthesis, activating antioxidant mechanisms, and regulating osmotic pressure.
Photosynthesis offers the matter and energy for the normal growth and development of plants. Salt stress can accelerate the breakdown of chlorophyll a and chlorophyll b, reduce the activity of PSⅡ and PSⅠ, and destroy the components of the thylakoid membrane [
27,
28]. In this study, with the increase in CaCl
2 concentration, the chlorophyll content of
Parachlorella kessleri FACHB-3316 significantly decreased (
Figure 3 and
Figure 5). This was consistent with the response of cucumber seedlings and tomato seedlings under high Ca(NO
3)
2 stress [
29]. Li et al. (Li Qingyun) revealed that, compared with high sodium salt, the same concentration of calcium salt could cause a greater decline in the chlorophyll content of strawberries [
30]. The possible reason was that high calcium increased the activity of chlorophyllase and loosened the combination of chlorophyll and chloroplast protein, which led to the decomposition and destruction of chlorophyll [
31]. However, unlike NaCl stress, the photosynthetic efficiency of PSⅡ did not decrease under the stress of a high concentration of CaCl
2 (
Figure 2). Transcriptome results revealed that the genes involved in photosynthesis were generally up-regulated under a high calcium environment (
Figure 15,
Figure 16 and
Figure 17). When in the 3.6 g/L CaCl
2, the genes encoding chlorophyll a/b binding protein in the light-harvesting complex (LHC4, LHCB4, and LHCB5) were significantly up-regulated (
p < 0.5). The overexpression of these genes contributed to the increase in light absorption and the reduction in chlorophyll loss. The genes involving the photosynthesis II oxygen-evolving enhancer protein synthesis (
psbO,
psbP), photosystem II 22 kDa protein synthesis (
psbS), and photosystem I subunit synthesis (
psaF,
psaK,
psaL,
psaO) were also significantly up-regulated (
p < 0.5) to maintain the stability of the photosynthetic system. The encoding products of
psbO,
psbP, and
psbS might also participate in the regulation of Ca
2+ and Cl
− [
32,
33]. When living in the 36 g/L CaCl
2, the algae massively up-regulated the photosystem genes participating in the synthesis of most electron transport complexes to accelerate energy absorption (
Figure 15). The phosphatidylglycerol (PG) synthesis also increased by up-regulating the genes encoding CDP-diacylglyerol synthase (EC: 3.1.3.4) and PGP phosphatase (EC: 3.1.3.27), which contributed to the maintenance of electron transport and thylakoid membrane structure [
34]. On the other hand, the genes involved in the C4 cycle and C3 cycle were also significantly up-regulated, which implied an increase in the amount of carbon assimilation. In the C4-dicarboxylic acid cycle, the key gene encoding phosphoenolpyruvate carboxylase (PEP, EC: 4.1.1.31) was up-regulated, which improved the CO
2 fixation efficiency. The carbon cycle was accelerated by upregulated synthesis of malate dehydrogenase (EC: 1.1.1.82) and alanine transaminase (EC: 2.6.1.2). In the reductive pentose phosphate cycle, key genes encoding phosphoglycerate kinase (EC: 2.7.2.3) and NADP
+-glyceraldehyde-3-phosphate dehydrogenase (EC: 1.2.1.13) were upregulated, which increased the production of glyceraldehyde-3P (GAP). The ribulose-1, 5-bisphosphate (RuBP) regeneration cycle was accelerated by upregulated synthesis of a series of enzymes, including fructose-1,6-bisphosphatase I (EC: 3.1.3.11), transketolase (EC: 2.2.1.1), fructose-1,6-bisphosphatase II/sedoheptulose-1,7-bisphosphatase (EC: 3.1.3.11, 3.1.3.37), ribose 5-phosphate isomerase A (EC: 5.3.1.6), and phosphoribulokinase (EC: 2.7.1.19). The up-regulation of carbon fixation pathways indicated that the algae increased the carbon fixation efficiency, which was more conducive to the synthesis of carbon-containing compounds. The products would further synthesize the soluble sugar and starch (
Figure 5) to complete energy utilization and storage. A similar tolerance mechanism of increasing the efficiency of light and carbon reduction reactions was also found in
Dunaliella salina, when dealing with high NaCl stress [
35]. For
Arabidopsis, studies revealed that overexpression of photosynthesis-related genes could enhance salt tolerance [
36]. Luo et al. found that the stability of the photosynthetic rate was the key to adapting to the high-calcium karst soil for
Cyrtogonellum fraxinellum [
37]. Therefore, we speculated that maintaining stable photosynthesis efficiency and energy input was one of the important mechanisms for
Parachlorella kessleri FACHB-3316 to resist high calcium stress.
On the other hand, the reactive oxygen species (ROS) balance of the plant will be broken when subjected to salt stress. Excess ROS accumulation can result in severe oxidative damage to plant cells, and further damage important macromolecular substances, such as DNA, protein, and lipids [
38,
39,
40]. Superoxide dismutase (SOD) can first respond to oxidative damage and rapidly catalyze the conversion of the superoxide anion to hydrogen peroxide and dioxygen [
41]. Therefore, salt-tolerant plants usually increase the SOD activity to survive in saline conditions [
38]. In this experiment, CaCl
2 stress rapidly induced the increase of SOD activity in
P. kessleri FACHB-3316. The degree of increase and duration were proportional to CaCl
2 concentration (
Figure 4). This implied that the SOD could rapidly promote the adaptation of algae cells to the high CaCl
2 environment. The results were similar to the study of calcium stress in cucumber and watermelon [
2,
42]. In addition, transcriptome results revealed that free amino acid (FAA)-synthesized pathways were up-regulated under 3.6 g/L CaCl
2 stress, such as proline, alanine, aspartate, glutamate, and so on. Proline was an important adjustment substance for stress resistance in plants. It could react with excessive oxygen radicals to generate harmless substances for plants so as to eliminate the harm of ROS [
43] and stimulate the activities of catalase, superoxide dismutase, and polyphenol oxidase [
44,
45]. The alanine, aspartate, and glutamate metabolism could also help to maintain redox homeostasis [
46]. In addition, genes related to the selenocompound metabolism pathway were significantly up-regulated in the 3.6 g/L CaCl
2 group (
p < 0.5) for the synthesis of selenoprotein. Trace selenium could not only promote the growth and photosynthesis of algae but also activate the antioxidant defense system to inhibit lipid peroxidation and intracellular ROS formation [
47,
48]. Therefore,
Parachlorella kessleri FACHB-3316 could reduce the oxidative damage of CaCl
2 stress by the synthesis of antioxidant enzymes, free amino acids, and selenides.
Another important mechanism for plants to adapt to salt stress is accumulating osmolytes so as to resist physiological drought, such as soluble sugar, organic acid, and free amino acid [
49,
50]. In this study, when comparing the 3.6 g/L CaCl
2 group to the normal group, the DEGs were largely enriched in various amino acid synthesis pathways (
Table 3), including proline, arginine, alanine, aspartate, glutamate, valine, leucine, and isoleucine (
p < 0.5). Zhen et al. also found that Ca(NO
3)
2 stress induced the increase in aspartate, glutamine, threonine, serine, glutamate, alanine, and proline contents in leaves and roots of melon seedlings, as well as cystine, histidine, and arginine content in roots [
51]. The increasing total free amino acid content was considered a quick response to salt stress in melons [
51]. Liu and Wang found that CaCl
2 promoted the accumulation of free amino acids [
52]. In this way, the plant could increase the concentration of the solution to absorb water and nutrients [
52]. Among free amino acids, proline was considered to be one of the most effective osmolytes [
53,
54]. Xiang et al. found that a high concentration of calcium ions could induce a sharp increase in proline content, and therefore maintain osmotic equilibrium in bryophytes [
55]. A similar effect of osmoregulation was also found in the alanine, aspartate, and glutamate metabolism [
46]. Buayam et al. found that glutamate might provide emergency protection for
Escherichia coli when the damage to osmotic adjustment ability occurred [
56]. Therefore, under the stress of 3.6 g/LCaCl
2,
Parachlorella kessleri FACHB-3316 might resist osmotic stress by the massive synthesis of free amino acids. Under the higher stress of 36 g/L CaCl
2, DEGs were enriched in the glycerol metabolic pathway. Among them, the gene encoding glycerol-3-phosphate dehydrogenase (GPDH, EC: 1.1.1.8, 1.1.5.3), a key regulatory enzyme of glycerol synthesis, was up-regulated. Glycerin was considered the main osmolyte of
Dunaliella salina [
57,
58]. By accumulating glycerol,
Dunaliella salina balanced the osmotic pressure caused by the high salt environment [
59,
60]. Glycerin was also an important component of cell membranes, which could alleviate the influence on membrane permeability by high calcium stress [
61,
62]. In
Dunaliella, glycerol could be synthesized through photosynthesis or degradation of starch metabolic pathways [
63,
64]. In this study, the large amount of GAP produced by up-regulated photosynthesis could be isomerized into dihydroxyacetone phosphate (DHAP). DHAP further generated glycerol-3P by the catalysis of GPDH, and the latter finally generated glycerol [
65]. Therefore, the increases in substrate and enzyme activity might jointly promote the accumulation of glycerol to cope with osmotic stress.