Calcium content and high calcium adaptation of plants in karst areas of southwestern Hunan, China

Rocky desertification is a major ecological problem of land degradation in karst areas. In these areas, the high soil 10 calcium (Ca) content has become an important environmental factor that can affect the restoration of vegetation. Consequently, the screening of plant species that can adapt to high Ca soil environments is a critical step in vegetation restoration. In this study, three grades of rocky desertification sample areas were selected in karst areas of southwestern Hunan, China (LRD: light rocky desertification; MRD: moderate rocky desertification; and IRD: intense rocky desertification). Each grade of these sample areas had 3 sample plots in different slope positions, each of which had 4 small quadrats (1 in rocky-side areas, 3 in 15 non-rocky-side areas). We measured the Ca content of leaves, branches and roots from 41 plant species, as well as soil total Ca (TCa) and exchangeable Ca (ECa) at depths of 0–15, 15–30 and 30–45 cm in each small quadrat. The results showed that the soil Ca 2+ content in rocky-side areas was significantly higher than that in non-rocky-side areas (p<0.05). The mean soil TCa and ECa content increased gradually along with the grade of rocky desertification, in the order IRD > MRD > LRD. For all plant functional groups, the plant Ca content of aboveground parts was significantly higher than that of the belowground parts 20 (p<0.05). The soil ECa content had significant effects on plant Ca content of the belowground parts but had no significant effects on plant Ca content of the aboveground parts. Of the 41 plant species that were sampled, 17 were found to be dominant (important value >1). The differences in Ca 2+ content between the aboveground and belowground parts of the 17 dominant species were calculated, and their correlations with soil ECa content were analyzed. The results showed that these 17 species


Introduction
Karst is a calcium-rich environment and a unique ecological system. This type of ecosystem is widely distributed, accounting 5 for 12% of the world's total land area (Zeng et al., 2007;Zhou et al., 2009;Luo et al., 2012). Karst landforms in China are mainly distributed in southwestern areas. The Hunan Province of China has been ranked fourth in the severity degree of rocky desertification . Rocky desertification could lead to frequent natural disasters, reduce human survival and development space, threaten local people's production, life and life safety, cause ecological deterioration, reduce arable land resources, aggravate poverty, and affect sustainable economic and social development . In other words, 10 Rocky desertification is an extreme form of land degradation in karst areas, and it has become a major social problem in terms of China's economic and social development (Sheng et al., 2015). Soil with high calcium (Ca) content in rock desertification areas has become one of the most important environmental factors affecting the local plant physiological characteristics and distribution in these areas (Ji et al., 2009). Given the origin of rocky desertification, the main factors that lead to rocky desertification are unreasonable human activities. For example, the cultivation of crops on steep slope can cause vegetation 15 destruction, soil erosion, and then rocky desertification. We should focus on vegetation restoration for the rocky desertification remediation (Wang et al., 2004). Consequently, the screening of plant species that can grow successfully in high-Ca environments in rocky desertification areas is an extremely critical step.
Role of Ca 2+ in plant physiology: over recent decades, progress has been made in identifying the cellular compartments (e.g., endoplasmic reticulum, chloroplasts and mitochondria) that regulate Ca balance and signal transduction in plants (Müller et al., 20 2015). Ca 2+ is an essential nutrients for plant growth and also participate to signal transduction (Poovaiah and Reddy, 1993;Hepler, 2005;Hong-Bo and Ming, 2008;Batistič and Kudla, 2012). And Ca 2+ is a very important signal component in plants responsive to environmental stresses. Ca 2+ signal takes the influential role as a second messenger in hormone signal 3 transduction, particularly in the abscisic acid signal transduction process (Hetherington, et al, 2004). Plants can adapt to high salt, drought and high temperature environments by activating the Ca 2+ signal transduction pathway (Bressan et al., 1998). Ca 2+ is also involved in nutrient cycling coupling process, for example, in the absence of nutrients (such as phosphorus), plants will inhibit the activity of nitrate reductase, thereby inhibiting the absorption of nitrate nitrogen, and ultimately inhibiting the absorption of Ca 2+ (Reuveni et al., 2000). Ca 2+ combines with pectin in the cell walls of plants to form pectin Ca, which is a 5 vital component of the intercellular layer in cell wall, and can buffer the compression between cells without hindering the expansion of cell surface area (Kinzel, 1989). Ca also has the function of maintaining the structure and function of cell membranes, regulating the activity of biological enzymes, and maintaining the anion-cation balance in vacuoles (Marschner, 2011).
Mechanisms of plant defense to high soil Ca 2+ concentrations: Ca 2+ is an essential macronutrient, but low Ca 2+ 10 concentrations must be maintained within the plant cytoplasm to avoid toxicity (Larkindale and Knight, 2002;Borer et al., 2012). The plant cell not only rapidly increases the free Ca 2+ concentration of the cytoplasm to adapt to environmental changes, but also maintains a low Ca concentration to prevent harm caused by high Ca. This fine regulatory mechanism is mainly achieved by Ca 2+ channels (Shang et al., 2003;Hetherington and Brownlee, 2004;Wang et al., 2005). The vacuoles may account for 95% of the plant cell volume and are able to store Ca 2+ within the cell. Thus, empty vacuoles represent an 15 efficient means of Ca storage (Ranjev et al., 1993). Some plants fix excess Ca 2+ by forming calcified deposits in root tissue in order to limit the upward transport of Ca 2+ (Musetti and Favali, 2003). In addition, Ca oxalate crystals in the plant cells play a role in regulating plant Ca content (Ilarslan et al., 2001;Pennisi and McConnell, 2001;Volk et al., 2002), and some plants will form Ca oxalate crystal cells in order to fix excess Ca 2+ (Moore et al., 2002). Furthermore, an active Ca efflux system plays an important role in the adaptation of plants to high-Ca environments (Bose et al., 2011). For example, when the leaves matured, 20 excess Ca 2+ in plants is excreted via stomata on the back of the leaves, thereby maintaining a lower concentration of leaf Ca (Musetti and Favali, 2003). The regulation of internal Ca storage depends predominantly on plasma membrane Ca transport and intracellular Ca storage; collectively these processes can regulate the intracellular Ca 2+ concentration to a lower level (Bowler and Fluhr, 2000). Plants that adapt to high-Ca environments promote excess Ca 2+ flow through the cytoplasm or store 4 Ca 2+ in vacuoles via the cytoplasmic Ca 2+ outflow and influx system (Shang et al., 2003;Hetherington and Brownlee, 2004;Wang et al., 2006). There are Ca 2+ channels, Ca 2+ pump and Ca 2+ /H + reverse conveyor on tonoplast. The former controls Ca 2+ outflow, and the latter two pump cytoplasmic Ca 2+ into vacuole (Wu, 2008). Cytoplasmic Ca 2+ is mainly combined with proteins and other macromolecules. The concentration of free Ca 2+ is generally only 20-200 nmol· L -1 and is stored in cell gaps and organelles such as vacuoles, endoplasmic reticulum, mitochondria and chloroplasts (Wu, 2008). However, excess free 5 Ca 2+ in the cytoplasm combines with phosphate to form a precipitate, which interferes with the physiological processes associated with the phosphorus metabolism, thus hindering normal signal transduction and causing significant detriment to plant growth (White and Broadley, 2003;Hirschi, 2004).
Variation of Ca 2+ content in species and soil: The concentration of free Ca 2+ in vacuoles varies with plant species, cell type and environment, which may also affect the release of Ca 2+ in vacuoles (Peiter, 2011). Some species maintain low calcium 10 content in aboveground part by reducing calcium uptake and transporting from underground part to aboveground part. This type of plant has Nephrolepis auriculata, Parathelypteris glanduligera, Cyrtomium fortunei, Pteris vittata, and so on. In contrast, other plants have a higher demand for calcium. For example, Cayratia japonica and Corchoropsis tomentosa, these plants maintain high calcium content by enhancing calcium uptake and transporting from underground part to aboveground part (Ji et al., 2009). Zhang (2005) studied the growth conditions of Eurycorymbus caraleriel and Rhododendron decorum 15 under different concentrations of Ca 2+ and found that a high Ca 2+ concentration (50 mmol· L -1 ) could promote growth in Eurycorymbus caraleriel but inhibit growth in Rhododendron decorum. Luo et al. (2013) showed that Ca 2+ concentrations affected plant photosynthesis. When the daily net photosynthetic rate of Cyrtogonellum Ching and Diplazium pinfaense Ching reached the highest value, the concentrations of Ca 2+ were 30 mmol· L -1 and 4 mmol· L -1 , respectively. Qi et al. (2013) found that a significant difference in calcium content among Primulina species (P. linearifolia, P. medica, P. swinglei, P. verecunda, 20 P. obtusidentata, P. heterotricha, and so on) from different soil types, and the average Ca content (2,285.6 mg/kg) in Primulina from calcareous soil was higher than the average Ca content of Primulina from both acid soil (1,379.3 mg/kg) and Danxia red soil (1,329.1 mg/kg). The mean soil exchangeable Ca (ECa) was 3.61 g· kg -1 in the Puding, Huajing, Libo and Luodian counties of Guizhou province, which is several times that of non-limestone areas in China (Ji et al., 2009). Wang et al. 5 (2011) found that plant rhizosphere soil total Ca (TCa) content in calcareous soil areas was above 14.0 mg· g -1 .
There are variations in soil Ca content among different areas, and there are differences between calcareous and non-calcareous plants in terms of Ca absorption, transport, storage and other physiological processes. These differences need to be taken into account in order to identify the variety of plants able to adapt to high Ca environments. However, to date, the mechanisms by which plants adapt to high Ca conditions, particularly in karst areas, and the Ca dynamics of plants and soil are 5 not well understood. In this study, we investigated plant Ca content, soil exchangeable Ca (ECa) and total Ca (TCa) contents on the rocky and non-rocky sides of three different grades of rocky desertification areas in southwestern China. Specifically, we hypothesized that the dynamics of Ca content in plants and soil would be significantly affected by the grade of rocky desertification. To test this hypothesis, the following investigations were explored: (i) to measure the soil ECa and TCa contents in rocky-side and non-rocky-side areas; (ii) to investigate and compare the Ca content of aboveground and 10 belowground parts among of plants from different functional groups; and (iii) to reveal correlation between plant Ca content and soil ECa content.

Site description
The study site was located in LijiaPing town in Shaoyang County, Hunan Province, China (latitude 27°0' N; longitude 113°36' 15 E; elevation 400-585 m above sea level), as shown in Fig.1. This region experiences a humid mid-subtropical monsoon climate. Mean annual air temperature is 16.9°C, and maximum and minimum temperatures are 41.0°C and −10.1°C, respectively. Mean annual precipitation is 1399 mm, mostly occurring between April and August, and the frost-free period is 288 days. The study site mainly consists of black and yellow limestone soil, and vegetation is scarce. Groundwater level is low and groundwater storage is poor (see Table. 1). 20

Experimental design and data collection
Rocky desertification was graded by using the sum of four index scores: bedrock exposure rate, vegetation type, vegetation 6 coverage and soil thickness. These index were quantified according to the State Forestry Administration of the People's Republic of China industrial standard 'LY/T 1840-2009' (China, 2009). Three 1 hm 2 sample areas were selected which were representative of the three different grades of rocky desertification: light rocky desertification (LRD); moderate rocky desertification (MRD); and intense rocky desertification (IRD). Within each sample area, we recorded environmental factors which included longitude, latitude, altitude, topography, vegetation type, degree of bare bedrock, and other conditions. The 5 collection of samples in these three sample areas was conducted in October 2016.
Within each of the three sample areas, four (2×2) small quadrats in different slope positions (upper, middle, and lower slope) were set up. In total, we assigned 36 small quadrats (3×4×3) for analysis. The common plant species of the region were gathered using the whole plant harvest method in each small quadrat, as well as shrubs and herbs were collected. Shrubs were divided into three parts: branches, leaves and roots. Herbs were divided into two parts: aboveground and belowground. Plant

Data analysis
All plant species were divided into different functional groups: (1) nitrogen-fixing plant and non-nitrogen-fixing plant groups according to nitrogen-fixing function; (2) dicotyledons and monocotyledons groups according to system development type; (3) C3 and C4 plant groups according to photosynthetic pathway; and (4) deciduous shrubs, evergreen shrubs, annual herbs and perennial herbs according to life form. Biennial herbs were gathered to the 'annual herbs'. Deciduous trees with a 20 height less than 2 m or a ground diameter less than 3 cm were gathered to the 'deciduous shrubs'. Branches and leaves were treated together as the aboveground part, while the belowground part only included roots. We calculated the important values (IV) by the following formula: We carried out two-way analysis of variance (ANOVA) for both species and soil for 17 widespread plants to determine differences in plant Ca content. One-way ANOVA was used to analyze the Ca content of soil and plants between different grades of rocky desertification. Pearson correlation analysis (α = 0.05) was used to analyze the correlation between plant Ca 5 and soil ECa content. All statistical analyses were performed using R 3.3.3 (R Development Core Team, 2017).

The properties of soil in different grades of rocky desertification
The mean TCa content in soil was 2.40 g· kg -1 (range: 0.10-8.09 g· kg −1 ) while the mean ECa content was 1.46 g· kg -1 (range: 0.02-3.92 g· kg -1 ). Differences between different sample locations (non-rocky side and rocky side) were significant (p<0.05) 10 for both TCa and ECa. The mean soil TCa and ECa content were found to be highest in areas of IRD, followed by MRD, then LRD. However, only the mean soil ECa content showed significant differences (p<0.05) across the three different grades of rocky desertification. Regarding the availability of Ca, the average availability of Ca was 59.75%, with the MRD showing the highest value at 72.55%, followed by IRD at 58.98%, and LRD showing the lowest value at 47.72 % (Table. 2).

The Ca content of plants in different grades of rocky desertification areas
A total of 41 plant species were collected from the three different grades of rocky desertification. The mean Ca content of the aboveground parts of these plants was 19.67 g· kg -1 (range: 4.34-40.24 g· kg -1 ). The mean Ca content of the belowground parts 8 was 10.79 g· kg -1 (range: 4.41-33.62 g· kg -1 ). The Ca content of the aboveground parts was significantly higher than that of the belowground parts (p<0.05) throughout the same grades of rocky desertification, but the Ca content of aboveground and belowground parts showed no significant differences (p>0.05) among the three different grades of rocky desertification (Fig.   2).

5
The 41 plant species were identified and divided into different functional groups in the 36 small quadrats. The Ca content of the aboveground parts was significantly higher than that of the belowground parts in each group (p<0.05). Nitrogen-fixing plants (22.48 g· kg -1 ) showed a slightly higher Ca content in the aboveground parts compared to non-nitrogen-fixing plants (19.39 g· kg -1 ; p>0.05), although Ca content in the belowground parts of nitrogen-fixing plants (6.76 g· kg -1 ) was lower than that of non-nitrogen-fixing plants (11.12 g· kg -1 ; p>0.05). For C3 plants, Ca content in the aboveground and belowground parts 10 were 21.08 g· kg -1 and 13.18 g· kg -1 , respectively, and were both significantly higher than that of C4 plants (aboveground: 15.68 g· kg -1 ; belowground: 6.42 g· kg -1 ; p<0.05). In the life form functional groups, shrubs showed a significantly higher in Ca content than herbs in both aboveground and belowground parts (p<0.05), although there were no significant differences (p>0.05) between deciduous and evergreen shrubs (p>0.05). There was no statistical difference in this respect between annual herbs and perennial herbs (p>0.05). The Ca content of dicotyledons in aboveground and belowground parts were 21.39 g· kg -1 15 and 12.19 g· kg -1 , respectively, and were significantly higher than that of monocotyledons (9.63 g· kg -1 and 4.79 g· kg -1 , respectively; p<0.05) (Fig. 3). To monocotyledons and dicotyledons, there were no significant differences in the plant Ca content of the aboveground parts among the different grades of rocky desertification; this was also true for the plant Ca content of the belowground parts. The Ca content of dicotyledons was significantly higher than that of monocotyledons in both aboveground and belowground parts throughout the three grades of rocky desertification (p<0.05) (Fig. 4).

20
Within the total of 41 common plant species, 17 plant species were found in each sample plot and were widespread throughout the southwestern rocky desertification areas of Hunan. These 17 species were calculated their important values (IV) ( Table. 3). Data showed that the differences of Ca content in the aboveground parts of the 17 plant species were highly 9 significant (p<0.01) among species, although these differences were not related to grades of rocky desertification. Differences in the Ca content of the belowground parts were highly significant not only among species, but throughout all the grades of rocky desertification (p<0.01).

Correlation between plant Ca content and soil ECa content
Of these 17 plant species, the Ca content in the aboveground and belowground parts of Sanguisorba officinalis had a

Dynamics of Ca content in plants and soil
The soil Ca content increased with the grade of rocky desertification, which indicates that soil Ca content was affected by the grade of rocky desertification. The mean soil ECa content was 1.46 g· kg -1 in these three different grades of rocky desertification, which was lower than the average ECa content in tobacco-planting soil in Hunan province (3.548 g· kg -1 ) (Xu et al., 2007). The average ECa content in IRD areas was 3.09 g· kg -1 , which was several times higher than the previously reported 10 ECa for non-limestone regions in China (Xu et al., 2007). The range of soil ECa content in the study area is from (LRD) 0.02 to (IRD) 3.92 g· kg -1 , with the maximum and minimum being lower than that of soil on Barro Colorado Island, Panama by Messmer et al. (2014). Tanikawa et al. (2017) revealed that concentrations of TCa and ECa were also low at the deeper horizons in low-acid buffering capacity (ABC) soils, and differences in both soil organic layer depth and soil chemistry could be a reason for the different of Ca accumulation in low-and high-ABC stands. Our research showed that the mean soil TCa 15 and ECa contents were the lowest in LRD areas, and the difference in soil TCa and ECa may be caused by bedrock exposure rate (the main chemical composition: CaCO 3 ) (Ji et al., 2009).
There were no significant differences in plant Ca content among the different grades of rocky desertification either for the aboveground or belowground parts (p>0.05), indicating that the grade of rocky desertification had no obvious effect on the Ca content of the aboveground and belowground parts of the plants. However, the average Ca content of the aboveground parts of 20 plants (19.67 g· kg -1 ) was lower than that of Hunan flue-cured tobacco (21.93 g· kg -1 ) (Xu et al., 2007). The maximum and minimum Ca content of plant aboveground parts were 41.79 g· kg -1 and 2.15 g· kg -1 respectively, and the maximum and 11 minimum Ca content of plant belowground parts were 40.14 g· kg -1 and 0.42 g· kg -1 respectively. The maximum Ca content of plants (41.79 g· kg -1 ) was found in the leaves, which was lower than the Ca content of calcareous plant leaves with the maximum value of 85.13 g· kg -1 detected by Luo et al. (2014). For most plants, the Ca content in the aboveground part was higher than in the belowground part, but for a few plants the Ca content in the aboveground part was lower than in the belowground part (such as Sanguisorba officinalis, Pyracantha fortuneana and Castanea henryi), which was consistent with 5 the findings of Wang et al. (2014).

Correlation between plant Ca content and soil ECa content
Our results showed that most plants had no correlation relationship between soil ECa and plant Ca except for several plants (Sanguisorba officinalis, Dendranthema indicum, Castanea henryi and Themeda japonica) which showed a positive correlation between soil ECa and plant Ca content (Table. 4). Some studies showed that Ca-rich soils caused cells to absorb 10 more Ca than the cells themselves require (White and Broadley, 2003). Additionally, soil ECa content and leaf Ca content (Flue-cured Tobacco) had a significant positive correlation in a pot experiment (Zou et al., 2010). The difference between the findings of these studies and ours may be caused by species factors. The correlation between plant Ca content and soil ECa content reflects what extent soil Ca content influences plant Ca content, and may also reflect how different plants respond to differences in soil ECa content (Ji et al., 2009). The Ca content of Sanguisorba officinalis in the aboveground and 15 belowground parts had a significant positive correlation (p<0.01) with soil ECa content, which indicates that Sanguisorba officinalis was affected greatly by soil ECa content. The Ca content of Dendranthema indicum (p<0.05) and Castanea henryi (p<0.01) in the belowground parts also showed a significant positive correlation (p<0.01) with soil ECa content, indicating that the belowground parts of these species were also greatly affected by soil ECa content. The Ca content of Themeda japonica in the aboveground parts showed a significant positive correlation (p<0.01) with soil ECa content, which indicates 20 that the aboveground parts of Themeda japonica were also greatly affected by soil ECa content.
Two-way ANOVA of species and soil showed that the Ca content of the aboveground parts of the 17 plant species was mainly affected by species factors, while the Ca content of the belowground parts was affected by both species factors and the 12 grade of rocky desertification. This finding is supported by data reported by Ji et al. (2009). The Ca content in the aboveground parts of nitrogen-fixing plants was significantly higher than that of the belowground parts, and this result indicates that nitrogen-fixing plants were the most efficient in Ca upward transport. In contrast, Ji et al. (2009) found that dicotyledons were the most efficient in the upward transport of Ca. However, they used only three types of plants (pteridophytes, dicotyledons, and monocotyledons) omitting nitrogen-fixing plants in their study. We found significant 5 differences (p<0.01) between the aboveground and belowground parts in the Ca content of monocotyledons in our study.
However, Ji et al. (2009) revealed that there were no significant differences between the aboveground and belowground parts in the Ca content of monocotyledons. The main reason for this difference may be the different species. In addition, the Ca content of monocotyledons was lower than that reported for monocotyledons by Ji et al. (2009), highlighting the large difference in ability to absorb soil Ca among monocotyledon species.

High Ca adaptation of plants
The different plant functional groups revealed differences in Ca content (Fig. 3). In some cases, even within the same plant species, there were an inconsistent correlation between Ca content in the aboveground and belowground parts and the soil ECa content. Collectively, these findings show that not all plants adapted to high Ca soil environments in the same way, but rather exhibited a variety of adaptive mechanisms.

15
The aboveground parts of a plant represent the main site of its physiological activity. Thus, the Ca content in the aboveground part reflects the Ca demand of the plant's physiological activity (Grubb and Edwards, 1982). The capacity of those plants that are able to adapt to high Ca soil environments can be reflected by two indicators: (i) the correlation between Ca content in the aboveground parts of the plants and soil ECa content; and (ii) the species differences in terms of the Ca content of the aboveground parts of plants. Thus, based on these two indicators, plants can be placed into the following groups: 20 Ca-indifferent plants, high-Ca plants, and low-Ca plants (Ji et al., 2009). We used this classification method to categorize the 17 plant species that were widely distributed across our study environment, thus providing theoretical guidance for vegetation restoration in rocky desertification areas. In both high-Ca and low-Ca soil environments, the Ca-indifferent plants can survive 13 normally, and their Ca content changes correspondingly with changes in soil ECa content. The physiological activities of high-Ca plants have a higher demand for Ca and may have a strong ability to absorb soil Ca. The physiological activities of low-Ca plants have a lower demand for Ca and can alleviate high Ca stress by inhibiting the absorption of Ca through the root system and its upward transport.
These results are of great significance for vegetation restoration in karst areas. High-Ca plants should be selected 5 preferentially (such as Pyracantha fortuneana, Rhus chinensis, and Loropetalum chinense, Serissa japonica), followed by Ca-indifferent plants (such as Sanguisorba officinalis, Castanea henryi, and Dendranthema indicum). Low-Ca plants should only be used as an alternative species to increase species diversity during the process of ecological restoration. Our findings not only have important significance for guiding solutions to the problem of rocky desertification in China, but also provide species screening ideas for ecosystem restoration in rocky desertification areas in other parts of the world. Rocky 10 desertification is a major ecological problem in karst areas, and further explorations are required to solve this problem. It is necessary to further explore other nutrient elements in soil during vegetation restoration, and long-term positioning observation is crucial for understanding this issue.

Conclusions
Our results indicate that the mean soil TCa and ECa content were highest in areas of IRD, followed by MRD, then LRD. The  The data represent mean ± standard deviation. Different lower-case letters in each column represent significant differences in different sample points within the same grade of rocky desertification. Different upper-case letters in each row represent significant differences between different grades of rocky desertification (p <0.05). "-" indicates that the important value of these species is less than 1.

Fig. 3 Ca content in the aboveground and belowground parts of plants in different functional groups
Different lower-case letters represent significant differences between the Ca content of the aboveground and belowground parts for the same functional groups (p<0.05); different upper-case letters represent significant differences among different 10 functional groups (p<0.05).