Physiological, Proteomic Analysis, and Calcium-Related Gene Expression Reveal Taxus wallichiana var. mairei Adaptability to Acid Rain Stress Under Various Calcium Levels

As one of the serious environmental problems worldwide, acid rain (AR) has always caused continuous damage to the forestry ecosystem. Studies have shown that AR can leach calcium ions from plants and soil. Calcium (Ca) is also a crucial regulator of the plant stress response, whereas there are few reports on how Ca regulates the response of AR-resistant woody plants to AR stress. In this study, by setting different exogenous Ca levels, we study the physiological and molecular mechanism of Ca in regulating the Taxus wallichiana var. mairei response to AR stress. Our results showed that low Ca level leads to photosynthesis, and antioxidant defense system decreases in T. wallichiana var. mairei leaves; however, these negative effects could be reversed at high Ca level. In addition, proteomic analyses identified 44 differentially expressed proteins in different Ca level treatments of T. wallichiana var. mairei under AR stress. These proteins were classified into seven groups, which include metabolic process, photosynthesis and energy pathway, cell rescue and defense, transcription and translation, protein modification and degradation, signal transduction, etc. Furthermore, the study found that low Ca level leads to an obvious increase of Ca-related gene expression under AR stress in T. wallichiana var. mairei using qRT-PCR analyses and however can be reversed at high Ca level. These findings would enrich and extend the Ca signaling pathways of AR stress in AR-resistant woody plants and are expected to have important theoretical and practical significance in revealing the mechanism of woody plants tolerating AR stress and protecting forestry ecosystem in soil environment under different Ca levels.


INTRODUCTION
As a serious environmental problem, acid rain (AR) affects seriously normal growth and development of forest tree species, which includes balance of leaf nutrient, growth indices, chlorosis, and necrosis in leaves, chlorophyll content, restrict photosynthesis, and the antioxidant enzyme activity (Liu and Diamond, 2005;Larssen et al., 2006;Hu et al., 2014aHu et al., , 2016Hu et al., , 2021Huang et al., 2019;Liu M. et al., 2019;Grennfelt et al., 2020;Zhang et al., 2020). Furthermore, AR can interfere with the material metabolism, transcriptional factors, and secondary metabolites genes, further cause metabolic disorders and inhibit the plant growth and development, and even cause plant death in severe cases (Lee et al., 2006;Hu et al., 2016;Debnath and Ahammed, 2020;Debnath et al., , 2021. In addition, recent studies have found that AR severely affects woody plant vegetation growth through leaching away calcium (Ca) element from plant and soil (Likens et al., 1996;Malakoff, 2010;Liu et al., 2011c). Further studies have also revealed that AR can disturb mineral ion absorption, deplete soil base Ca ion from pools, and limit the Ca uptake in woody plant (Liu et al., 2011c;Hu et al., 2014c;Shu et al., 2019). Many studies have demonstrated that Ca plays important roles in plants responding to environmental stresses, and exogenous Ca level regulates cytosolic Ca concentration and enhances antioxidant defense, thus serving as a source of nutrition and structure, regulatory agent to further modulate signaling functions (Reddy and Reddy, 2004;Hepler, 2005;Juice et al., 2006;Zhang et al., 2021). On the other hand, our previous studies also indicated that exogenous Ca level played a key role in the response process of AR stress in ARsensitive woody plants (Hu et al., 2014c(Hu et al., , 2016. Ma et al. (2021) also found that exogenous Ca enhances antioxidant defense to simulated AR stress in rice. However, the AR-resistant woody plants in response to AR stress at different Ca levels have not been reported in forest tree species.
Taxus wallichiana var. mairei is a well-known gymnosperm with great ornamental and medicinal value (Gao et al., 2007;Cheng et al., 2021;Xiong et al., 2021). As an AR-resistant tree species, T. wallichiana var. mairei is distributed over large areas of southern China, where AR is relatively serious (Gao et al., 2007;Hu et al., 2014a). Some physiological and biochemical changes and growth response to simulated AR have been reported in T. wallichiana var. mairei (Liu K. et al., 2007;Liu et al., 2012). Our previous study also found that T. wallichiana var. mairei is an AR-tolerant species under normal Ca condition (Hu et al., 2014a). However, the molecular mechanisms of AR resistance remain poorly understood in T. wallichiana var. mairei under different Ca conditions.
As responses of different Ca levels to AR stress are very complex processes, and further investigations are needed to clarify the molecular mechanism in AR-resistant species. Although our recent study revealed that there are rescue effects of exogenous Ca against AR stress in AR-sensitive species (Hu et al., 2014c(Hu et al., , 2016, the underlying mechanisms remain unclear in AR-tolerant species at various Ca levels. To better understand the extent to which biological and environmental factors shape AR resistance of T. wallichiana var. mairei at different Ca levels, proteomics and Ca-related gene expression in T. wallichiana var. mairei response to AR stress at different Ca levels is one of powerful ways to identify the molecular mechanism. A recent study has presented a reference genome of T. wallichiana var. mairei, which will provide genetic resources and serve as a platform for identification and decoding of the AR resistance pathway in various Ca conditions in the future (Cheng et al., 2021;Xiong et al., 2021). In this study, we conduct proteomic study to clarify the molecular mechanisms of various Ca levels in T. wallichiana var. mairei under AR treatment. The objective of this study was to characterize the AR-responsive proteins in T. wallichiana var. mairei at different Ca levels, combined with the physiological and gene expression data, and further to establish the molecular metabolism in Ca-mediated AR resistance in AR-resistant woody plants.

Soil Pretreatment
In our experiment, the substrate soil was lateritic soil, and the soil samples were collected from in southern forest areas of China where AR is much too harmful. Ca content in the soil was leached for 6 months of simulated AR, and the specific soil leaching process is referred to Liu J. X. et al. (2007) and Liu et al. (2011c). According to Hu et al. (2014c), Ca content was analyzed using ICP-MS (PerkinElmer Inc., Elan DRC-e, Waltham, MA, United States). Soil nutrients were recovered by a Hoagland nutrient solution, which contains one of the three Ca concentrations (20.0, 2.0, or 0.1 mmol L −1 ), respectively (Liu et al., 2011c). The final soil exchangeable Ca level was high Ca level (107.08 mmol kg −1 ), medium Ca level (19.65 mmol kg −1 ), and low Ca level (1.85 mmol kg −1 ).

Plant Materials and Experimental Procedure
The 6-month-old and size-identical T. wallichiana var. mairei seedlings (the aerial part length was 6.3 ± 0.5 cm) were transplanted into plastic pots. The seedlings were grown in a greenhouse with a light-dark regime of 16/8 h, temperature of 27/21 • C (day/night), relative humidity of 60-70%, and photosynthetically active radiation of 210 µmol m −2 s −1 . After 2 weeks of recovery, the T. wallichiana var. mairei seedlings were sprayed once each day with simulated AR solution (pH 3.0) with medium Ca level as control group and low or high Ca level for treatment group as described by the previous study (Hu et al., 2014c). After 2-month AR treatment, the fresh leaves of T. wallichiana var. mairei seedlings were collected for further experiments, which include physiological and proteomic research and qRT-PCR analysis.

Measurements of Physiological and Growth Indexes
Chlorophyll in T. wallichiana var. mairei leaves was extracted using ice-cold 80% v/v acetone according to the study by Hu et al. (2014c). Net photosynthetic rate (Pn) was performed with a portable photosynthesis system (Li-6400, Li-Cor, Lincoln, NE, United States) as described by Chen et al. (2013). At least eight saplings were randomly selected from the control group or the treatment group for Pn measurements. For Ca element analysis, T. wallichiana var. mairei leaf tissue was dried at 80 • C for 72 h. Ca concentration in the leaves was measured using ICP-MS (PerkinElmer Inc., Elan DRC-e, Waltham, MA, United States), following the study by Hu et al. (2014c).

Assay of Lipid Peroxidation, Reactive Oxygen Species Production, and Antioxidant Enzyme Activity
Soluble protein, proline, H 2 O 2 , and O2 •− content were measured as described by Chen et al. (2013). According to Liu et al. (2011a), lipid peroxidation in leaves was measured by estimating the content of malondialdehyde (MDA) using thiobarbituric acid (TBA) reaction. Superoxide dismutase (SOD) activity, ascorbic peroxidase (APX) activity, peroxidase (POD) activity, and catalase (CAT) activity were measured following the methods of Hu et al. (2014b).
Protein Extraction, Two-Dimensional Electrophoresis, and Data Analysis Protein in T. wallichiana var. mairei leaves was extracted using the method of phenol extraction according to the study of Hu et al. (2014a). Three independent biological repetitions were performed for each treatment. According to the manufacturer's instructions of GE Healthcare Amersham Bioscience, protein concentration of the lysates was measured using a 2-D Quant Kit.
Two-dimensional electrophoresis (2-DE) was conducted according to the study of Hu et al. (2014a). Ettan IPGphor isoelectric focusing system (GE Healthcare Amersham Bioscience, Little Chalfont, United Kingdom) was used for isoelectric focusing (IEF). After IEF, gel strips were equilibrated as described by Hu et al. (2014c). For the second-dimension electrophoresis, it was conducted using a protein apparatus (Bio-Rad), the proteins were separated on 12.5% SDS polyacrylamide gels according to the manufacturer's instructions (Hu et al., 2014c). The 2-DE gels were stained using Coomassie Brilliant Blue R-250, and an image scanner (Uniscan M3600, China) was used for gel images at 600 dots per inch resolution. The 2-D gel images were analyzed using PDQuest software (Version 8.01, Bio-Rad, Hercules, CA, United States). The intensity of protein spots changed more than twofold and passed the Student's t-test (p < 0.05), which were considered for mass spectrometry analysis.

Protein Identification and Protein Classification
Protein digestion and protein identification in T. wallichiana var. mairei was performed according to the study of Hu et al. (2014a). The identified tryptic peptide masses in T. wallichiana var. mairei were searched against the National Center for Biotechnology Information non-redundant (NCBInr) database, and the taxonomy of green plants was selected using the MASCOT interface (Version 2.5; Matrix Science, London, United Kingdom). The following parameters were used for database search: no molecular weight restriction, permitting one missed cleavage, fixed modification of cysteine by carbamidomethylation, oxidation (Met) as a variable modification, the peptide tolerance of 100 ppm, and fragment ion mass tolerance of ±0.3 Da. At least three peptides were matched for protein identification, and the protein scores of MOWSE threshold were set greater than 73 with the NCBInr database (p < 0.05). As described by Hu et al. (2014a), the functions and subcellular localization of identified proteins were searched for the NCBI protein database, 1 UniProt, 2 and published literature.

Hierarchical Cluster Analysis
Hierarchical clustering was performed on density value of differentially expressed proteins according to the study of Hu et al. (2014c). Input value was calculated by dividing volume percentage of each protein spot at the high Ca-AR level and low Ca-AR level by the corresponding protein spot at the medium Ca-AR level. Complete linkage algorithm was enabled, and the results of hierarchical cluster were plotted using TreeView software version 1.1.3.

RNA Extraction and qRT-PCR Analysis
Total RNA from T. wallichiana var. mairei leaves (0.1 g) was extracted using RNA purification reagent (Invitrogen Inc., CA, United States) in liquid nitrogen according to the study of Hu et al. (2014c). The M-MLV reverse transcriptase (TaKaRa, Dalian, China) was used for the synthesis of the first-strand cDNAs as described by the previous study (Hu et al., 2014c). Gene primers were designed for cloning the fragments of Carelated genes in T. wallichiana var. mairei (Supplementary Table 1). The Ca-related gene abundance was analyzed using the Rotor-gene-6000 real-time PCR system (Corbett Research, Mortlake, NSW, Australia) as described by Hu et al. (2014c) with minor modifications. The following temperature program was used for qRT-PCR analysis: 94 • C for 10 min, followed 94 • C for 30 s by 40 cycles, primer annealing at 52-56 • C for 30 s (Supplementary Table 2), and extension at 72 • C for 20 s. The glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) in T. wallichiana var. mairei was used as the internal control for each sample. Three independent biological replicates were performed for each sample.

Statistical Analysis
All data were presented as the mean ± SE of three replicated samples. The statistical significance was analyzed using a univariate analysis of variance (one-way ANOVA; SPSS, version 22.0, Inc., Chicago, IL, United States). Statistical significance was considered at p < 0.05.

Effects of Acid Rain Stress on Physiological Parameters of Taxus wallichiana var. mairei at Different Calcium Levels
To study the responses of woody plant to AR stress at different Ca levels, T. wallichiana var. mairei was treated with simulated AR (pH 3.0) for 2 months. Physiological changes are shown in Figure 1, and Ca content of the leaf, chlorophyll content, and photosynthetic activity (Pn) were also tested and analyzed. After 2-month AR treatment, Ca content and Pn were significantly decreased in T. wallichiana var. mairei leaves at low Ca level; however, high Ca treatment can reverse the decline of the physiological indicators.

Effects of Acid Rain Stress on Antioxidant System Response in Taxus wallichiana var. mairei at Different Calcium Levels
As shown in Figures 2A,B, there was an obvious increase in soluble protein content and proline content in AR-treated T. wallichiana var. mairei at high Ca level. We found that the levels of MDA, H 2 O 2 , and O2 •− were significantly stimulated by low Ca treatment (Figures 2C-E). It found that SOD, APX, POD, and CAT showed a significant increase in T. wallichiana var. mairei leaves at high Ca level (Figures 2F-I).

Protein Profile and Functional Classification in Taxus wallichiana var. mairei Leaves Response to Acid Rain Stress at Different Calcium Levels
To further explore the proteome changes in Pinus massoniana and T. wallichiana var. mairei leaves under AR treatment, 2-DE was performed in this study.
A total of 44 protein spot in T. wallichiana var. mairei leaves response to AR stress with different Ca treatments ( Figure 3A and Table 1). Close-up views of representative different protein spots are shown in Figure 3B. To analyze which biological processes the identified differential proteins are involved in, the identified 44 proteins were analyzed in appropriate functional pathways (Figure 4). The differentially expressed proteins were divided into seven groups based on their biological functions. The largest protein group was sorted to photosynthesis and energy pathway (27.2%), followed by transcription and translation (20.5%), cell rescue and defense (18.2%), signal transduction (11.4%), metabolic process (9.1%), and protein modification and degradation (4.5%) ( Figure 4A). As shown in Figure 4B, the subcellular localization analysis found that 44 differentially expressed proteins were located in the cytoplasm (25.0%), chloroplast (22.7%), mitochondrion (11.4%), membrane (11.4%), nucleus (9.0%), extracellular (4.5%), endoplasmic reticulum (2.3%), and vacuole (2.3%).
In addition, we used a hierarchical cluster analysis to find the regular pattern of different protein expression changes during AR stress in T. wallichiana var. mairei leaves with different Ca treatments. As shown in Figure 5, the differentially expressed proteins are mainly distributed into two branches in T. wallichiana var. mairei. We found that the expression abundance of cell defense-related proteins (spots 21, 22, and 24) showed a downward trend with the increase of Ca concentration under AR stress. Signal transduction-related proteins (spots 3, 37, and 39) showed a significant upregulation trend under low Ca-AR treatment. The expression trend of these proteins was  reversed to downregulated with the increase in Ca concentration. More interestingly, we found that a protein (spot 17, lightharvesting complex II protein Lhcb1) related to photosynthesis process was significantly downregulated in low Ca condition, but turned to upregulated in high Ca condition, which indicates that high Ca treatment helps to improve T. wallichiana var. mairei leaves photosynthesis, which is one of the ways to enhance plant resistance to AR stress. The specific information of the differential proteins is listed in Table 1.

Calcium-Related Gene Expression Analysis
To evaluate the expression abundances of the Ca-related genes at different Ca levels in AR-treated T. wallichiana var. mairei, we further analyze calmodulin gene (CaM1), touch 3 gene (TCH3), calreticulin 3 gene (CRT3), CDPK-related kinase gene (CDPK1), glutamate dehydrogenase 2 gene (GDH2), calcineurin B-like calcium sensor protein 1 gene (CBL1), calnexin 1 gene (CNX1), and respiratory burst oxidase homolog A gene (RbohA). As shown in Figure 7, under AR stress, the expression abundance of most Ca-related genes increased obviously at low Ca level; however, this expression trend can be reversed at high Ca level.

Metabolic Process, Protein Modification, and Degradation-Related Proteins
The environmental stresses severely affect the material metabolism of plants (Liu et al., 2011a;Chen et al., 2014). UDPglucose pyrophosphorylase is the precursor of carbohydrate formation . Our results showed that Ca application reduced AR-induced increases in protein abundance of UDP-glucose pyrophosphorylase (spot 2) in T. wallichiana var. mairei (Table 1). Under AR stress, our previous study indicates that high Ca level enhances the carbohydrate metabolism in Liquidambar formosana (Hu et al., 2016). It has been showed that woody plants against environmental stresses with a complex manner, which results in similar protein expressions (Hu et al., 2014c;. In this study, our finding is consistent with the results from the study of how exogenous Ca affects other woody plants to AR stress. These results showed that Ca application helps in alleviating the adverse effects of AR stress by accelerating the carbohydrate metabolism. On the other hand, Chen et al. (2019) found that UDP-glucose pyrophosphorylase plays an important role in cell wall metabolism and the synthesis of cellulase and hemicellulose. Carbohydrates act as a cytoskeleton and compose the cell wall as the first physical barrier defending against all types of AR stress (Liu et al., 2011b). We speculate that the protecting role of Ca can be explained by the upregulated carbohydrate metabolism-related protein under AR stress. S-adenosylmethionine synthase catalyzes the formation of S-adenosylmethionine from methionine and ATP, which is the critical enzymes in ethylene biosynthetic process (Peleman et al., 1989;Wang et al., 2002). In this study, protein abundances of S-adenosylmethionine synthase (spot 4) increased after low Ca treatment (Figure 5), which indicates that Ca-mediated ethylene biosynthetic process might make effect in AR tolerance in T. wallichiana var. mairei. Additionally, the abundance of S-adenosylmethionine synthase increases in high Ca-AR-treated L. formosana (Hu et al., 2016). These findings indicate that exogenous Ca plays different roles in AR-resistant tree species (T. wallichiana var. mairei) and AR-sensitive tree species (L. formosana and P. massoniana) in response to AR stress. Further study is needed to clarify the different metabolic processes at various Ca levels in AR-tolerance woody plants.
Aspartyl aminopeptidase is likely to play an important role in intracellular protein proteolysis and peptide metabolism (Park et al., 2017). In addition, F-box/kelch-repeat protein is involved in the pathway of protein ubiquitination, which is a part of protein modification. F-box/kelch-repeat protein may mediate the ubiquitination and subsequent proteasomal degradation of target proteins (González-Carranza et al., 2007). In this study, our proteomic analysis has detected increased abundance of probable aspartyl aminopeptidase (spot 6) and F-box/kelch-repeat protein (spot 36) at low Ca level in T. wallichiana var. mairei response to AR stress; however, this expression trend can be reversed at high Ca level ( Table 1). It should be noted that the previous proteome analysis reveals the significantly elevated abundance of  F-box family protein in AR-treated woody plant, which suggests that AR stress affects the biosynthesis and refolding of proteins and exacerbated protein degradation (Chen et al., 2014;Hu et al., 2014a). The findings provide informative clues regarding the protein modification and degradation mechanisms for the negative regulation of exogenous Ca level during T. wallichiana var. mairei response to AR stress. It is reasonable to speculate that AR stress leads to more serious protein degradation in low Ca condition, and high expression of degraded proteins system needs to be activated to maintain the stability of the protein metabolism in T. wallichiana var. mairei; high Ca condition has effective protection mechanism, low expression, or without degradation of protein fragments damaged by AR stress. AR stress can damage the homeostasis of protein metabolism between biosynthesis and degradation (Hu et al., 2014a). Most of these proteins play important roles in linking change of Ca concentration and the subsequent metabolic adaptation to AR stress in woody plants (Hu et al., 2014c(Hu et al., , 2016, but the specific details of the mechanism need to be further studied in the future.

Photosynthesis and Energy Pathway-Related Proteins
Photosynthesis is a key metabolic process of woody plants, which is sensitive to AR stress (Liu et al., 2011a;Chen et al., 2013). In addition, our previous studies found that photosynthesis is greatly affected by simulated AR stress under low Ca level (Hu et al., 2014c(Hu et al., , 2016. In this study, physiological analysis found that the photosynthesis is inhibited with low Ca-AR treatment, but significantly increased with high Ca-AR treatment in T. wallichiana var. mairei leaves ( Figure 1C). Consistent with the results of physiological data, our proteomic data analysis showed that high Ca level can remarkably improve photosynthetic capacity of T. wallichiana var. mairei with AR treatment. For instance, we found that chlorophyll a/b-binding protein (spot 15), ribulose-1,5-bisphosphate carboxylase-oxygenase large subunit (spot 16), light-harvesting complex II protein Lhcb1 (spot 17), and ferredoxin-NADP reductase (spot 18) were decreased in protein expression abundance with low Ca-AR treatment and increased with high Ca-AR treatment in T. wallichiana var. mairei (Table 1). These results indicated that high Ca level could restore the impaired photosynthetic function in AR-treated T. wallichiana var. mairei seedlings. The protective role of exogenous Ca may lie in the increased upregulation of photosynthesis and energy pathway, which enhances AR tolerance in T. wallichiana var. mairei. This inference is supported by our previous studies, in which we demonstrated that exogenous Ca supply improved photosynthesis in woody plants against AR stress (Hu et al., 2014c(Hu et al., , 2016. On the other hand, sufficient ATP is necessary for T. wallichiana var. mairei responding to AR stress in plants. ATP synthase and ATPase are the key enzymes in energy production and conversion (Liu et al., 2011b;Hu et al., 2014a). Previous evidence indicates that enough ATP is necessary for plants in response to environmental stress (Jiang et al., 2007). Our results found that a large number of enzymes were involved in this process that include ATP synthase beta subunit (spots 7, 8), F1-ATP synthase beta subunit (spot 9), and ATP synthase beta chain (spot 10). Our previous studies found that AR-sensitive tree species (L. formosana and P. massoniana) have the increased abundance of energy production-related proteins with high Ca-AR treatment (Hu et al., 2014c(Hu et al., , 2016. In this study, we also find a similar pattern in T. wallichiana var. mairei. Additionally, western blot analysis showed that relative abundance levels of the Rubisco LSU and ATPase were increased with high Ca treatment (Figure 6), and western blot result is consistent with the selected protein of our proteomic data at various Ca levels in AR-treated T. wallichiana var. mairei. Besides, as a membrane-bound protein located in thylakoids, ATP-dependent zinc metalloprotease-related proteins involve in the removal of a damaged D1 protein from PSII in plants (Hu et al., 2014a). Expression abundance of ATP-dependent zinc metalloprotease ThFtsH8 (spot 13) and ATP-dependent zinc metalloprotease FTSH (spot 14) was downregulated in high Ca-AR-treated T. wallichiana var. mairei. This change meant that less damaged protein is produced in high Ca condition, by which we furthermore find that the recovered photosynthesis decline caused by AR stress. Moreover, H + -ATPase plays an important role in the maintenance of ion homeostasis and nutrient uptake in plants (Liu et al., 2011b;Chen et al., 2019). Our study also showed that plasma membrane H + -ATPase (spot 11) was upregulated by high Ca level with AR treatment. Based on these results, high Ca condition may stimulate the resistance of T. wallichiana var. mairei to AR stress by modulating special energy-related pathway. These findings indicate that with high Ca-AR treatment, the mechanisms of energy pathway in ARresistant tree species (T. wallichiana var. mairei) are different from AR-sensitive tree species (L. formosana and P. massoniana). Further investigations are needed to clarify the different energy pathways in AR-tolerance woody plants.

Cell Rescue and Defense-Related Proteins
Acid rain stress has negative effects on plant growth and development, and also the internal functional components. Low  Ca-AR treatment increased the content of MDA and reactive oxygen radicals [reactive oxygen species (ROS), e.g., H 2 O 2 and O2 •− ], which suggests the ROS enrichment in T. wallichiana var. mairei; however, high Ca supplementation can reverse these effects (Figure 2). In addition, we observed that soluble protein content, proline content, SOD, APX, POD, and CAT activity showed a significant increase in T. wallichiana var. mairei leaves with high Ca treatment, which implies that antioxidant defense system was provoked by high Ca supplementation in AR-treated T. wallichiana var. mairei. These changes of physiological and biochemical findings indicated the protective role in exogenous Ca being against AR stress in T. wallichiana var. mairei. To further reveal the different strategies to cope with AR stress at various Ca levels, many cell rescue and defense-related proteins were also identified by proteomic analysis in this study. As stress response proteins and heat shock proteins can be induced by many abiotic stresses (Wang et al., 2004, our study showed that exogenous Ca addition enhanced the abundance of heat shock protein (spot 19) and heat shock protein 70 (spot 20). Additionally, heat shock proteins are also considered as an important part in stabilizing protein folding and play important roles in the cellular stress response . The increased abundance of the heat shock-related proteins in high Ca-AR treatment set indicated the multidimensional role of external Ca supply in the alleviation of AR stress in T. wallichiana var. mairei.
In this study, decreased expressions of thioredoxin-related protein (spot 21) and glutathione S-transferase (spot 24) were also observed at high Ca levels. It was reported that thioredoxin can reduce intramolecular disulfide bridges of target proteins, maintain suppression of apoptosis, and supply reducing equivalents to the antioxidant systems (Tada et al., 2008;Liu et al., 2011b). Under both low and high Ca levels, elevated abundance of thioredoxin superfamily protein was described in L. formosana seedlings in response to AR stress (Hu et al., 2016). In addition, glutathione S-transferases are associated with stress tolerance, which can catalyze the addition of glutathione to electrophilic compounds and resist various cellular damages (Frova, 2003;Aloui et al., 2009). Previous studies reported the enhanced expressions of glutathione S-transferase in Arabidopsis response to AR stress and low Ca-AR-treated P. massoniana seedlings (Hu et al., 2014c(Hu et al., , 2016. As an antioxidative protein, glutathione S-transferase can be strongly induced by biotic and abiotic stresses (Dixon et al., 2002). In this study, downregulation of these proteins indicates that high Ca level has the potential to protect T. wallichiana var. mairei from oxidative stress caused by AR stress, which suggests that AR-resistant tree species has developed different defense strategies against AR stress under high Ca condition.

Transcription and Translation-Related Proteins
Transcription and translation regulate the expression level of stress responsive genes, which are important for plants response to environmental stresses (Jiang et al., 2007;Chen et al., 2014;Hu et al., 2014c). In this study, we have successfully identified nine proteins related to transcription and translation. In plants, maturase K catalyzes intron RNA binding and affects gene expression at the transcriptional level (Ji et al., 2009). The expression level of maturase K is complex in plants response to abiotic stress, which depends on plant species and the type of environmental stresses (Pandey et al., 2008;Chen et al., 2014).
In this study, the abundance of maturase K (spot 27) increases in both low Ca-AR and high Ca-AR-treated T. wallichiana var. mairei, which suggests that expression changes in maturase K may play a part in linking change of Ca level under AR stress. Moreover, Chen et al. (2019) showed that a transcription factor displayed an elevated expression pattern in Arabidopsis of exogenous Ca-alleviated Al toxicity. We also found that exogenous high Ca enhances the abundance of transcription factor JUNGBRUNNEN 1 (spot 32) in T. wallichiana var. mairei. In addition, Hu et al. (2016) found that an increase in ribosomal protein S1 was detected in high Ca-AR-treated woody plant. Consistent with previous research, protein synthesis machinery was also affected by high Ca treatment as increased expression level of ribosomal protein L14 (spot 29) and small subunit ribosomal protein S1 (spot 35) has been detected in T. wallichiana var. mairei under AR stress. Transcription and translation control the expression of stress responsive genes, which play crucial roles in plants in response to various abiotic stresses (Amme et al., 2006). We detected increased abundance of series transcription and translation-related proteins in T. wallichiana var. mairei under high Ca-AR treatment, and the results indicated that Ca-regulated transcription and translation mediate T. wallichiana var. mairei adaptation to AR stress. The complex AR-responsive signaling pathways are associated with stress sensing and activation of defense pathways, which involves in calcium-regulated proteins crosstalk among various transcription factors. Under AR stress, future work on these transcription and translation-related proteins are needed to clarify the specific functions in various Ca conditions.

Signal Transduction-Related Proteins
Under environmental stresses, woody plants initiate multiple signaling pathways through sensing and transducting several stress signals, further regulate the expression level of many functional proteins, and finally exhibit corresponding physiological response to against the abiotic stresses (Hu et al., 2014a;. Membrane steroid-binding protein 1 (MSBP1) can bind steroids and negatively regulates cell elongation and expansion, and also photomorphogenesis and brassinosteroid signaling (Yang et al., 2005;Song et al., 2009;Shi et al., 2011). In this study, we found that the abundance of membrane steroid-binding protein 1 (spot 37) was significantly increased at low Ca level; however, it can reverse the increased abundance of MSBP1 in T. wallichiana var. mairei at high Ca level, which provides informative hints on interactions between Ca and membrane steroid-binding protein. This finding suggested the specific relationship between Ca and steroid-binding protein in the BR signaling pathway. Protein phosphatase 2A plays an important role in plant development and growth through a functional link with the target of rapamycin (TOR) signaling pathway (Ahn et al., 2015). The TOR signaling pathway integrates multiple signals transduction, such as energy, nutrients, growth factors, and environmental conditions, to regulate plant cell metabolism and growth (Wullschleger et al., 2006;Xiong et al., 2013). We found that the expression level of 2A phosphatase-associated protein of 46 kDa (spot 38) was significantly upregulated in T. wallichiana var. mairei with high Ca-AR treatment. On the other hand, it should be noted that the abundance of 2A phosphataseassociated protein of 46 kDa was decreased in P. massoniana under high Ca-AR treatment (Hu et al., 2014c), which suggests that 2A phosphatase-associated protein of 46 kDa could play a specific role in woody plants response to AR stress. These results implied the different expression patterns and coping response strategies between AR-sensitive (P. massoniana) and AR-resistant (T. wallichiana var. mairei) tree species under high Ca condition; however, further studies are needed to elucidate the specific function of the signal transduction-related proteins in AR-stressed tree species.
Moreover, Ca plays an important role in plants coping with a series of environmental stresses (Reddy et al., 2011). In the cytoplasm, free cytosolic Ca 2+ level is a universal second messenger of signal transduction in plants in response to environmental stresses (Kudla et al., 2018). Ca-bindingrelated proteins regulated modulation of intracellular Ca 2+ levels (Hu et al., 2014a(Hu et al., , 2016. In this study, expression level of calcium-binding protein KIC-like (spot 40) showed declines in T. wallichiana var. mairei with high Ca-AR treatment. It has been observed that Ca-binding proteins are regarded as crosstalk key nodes for environmental stress signaling pathways (Hepler, 2005;. These findings suggest that calciumbinding proteins participate in adaptation responses to AR stress in T. wallichiana var. mairei, which provides informative hints on interactions between Ca-associated signal transduction and AR tolerance. In Figure 7, our result provided additional evidence that the expression abundance of eight Ca-related genes (CaM1, TCH3, CRT3, CDPK1, GDH2, CBL1, CNX1, and RbohA) increased obviously at low Ca level, whereas this expression trend can be reversed at high Ca level under AR stress. Plants respond to environmental stresses through altering gene expression and adaptive responses associated with stress sensing and activation of defense pathways, such as expression of stress adaptive proteins, synthesis of oxidative stress protectors, calcium-regulated proteins, and signal pathway (Hasegawa et al., 2000;Hu et al., 2014a). In this study, these findings indicate that Ca-dependent signal transduction could induce upregulated expression of Ca-related genes and further interact with various biological processes to against AR stress in T. wallichiana var. mairei.

CONCLUSION
In this study, physiological and proteomic evidence is carried out to clarify that Ca plays an important role in AR-resistant tree species T. wallichiana var. mairei response to simulated AR treatment. Our data reveal that low Ca treatment activated the cell rescue-and defense-related proteins and Ca-related gene level to respond to AR stress in T. wallichiana var. mairei. These proteins might operate in a dynamic network in the response of AR-resistant tree species under AR stress, and these identified proteins might be useful in revealing insights into the different defense mechanisms of AR-resistant tree species to AR stress. In addition, exogenous Ca supply against AR stress by increasing the abundance of the proteins involved in photosynthesis and energy pathway, translational factors, which suggest that AR-tolerant woody plants can equip themselves better to respond to AR stress by provoking related proteins and gene expression. As shown in Figure 8, we proposed a scheme associated with different defense mechanisms of T. wallichiana var. mairei responding to AR damage at different Ca levels. These results would deepen insights into the adaptation strategies of AR-resistant tree species under AR stress at different Ca levels and furthermore provide theoretical support for the scientific prevention of AR stress.