Comparisons of plant calcium fraction between two different vegetation zones in semi-arid region

MengQi Li, XingDong He , XiangXiang Yang, YueDan Zhao, YuBao Gao

College of Life Sciences, Nankai University, Tianjin 300071, China

ABSTRACT To explore the characteristics of plant calcium (Ca) fraction, we analyzed 91 plant species in the Ningxia Habahu National Nature Reserve in Yanchi County of Ningxia and 84 plant species in Zhenglan Banner of Inner Mongolia. Results show that, for the two regions, there is no significant difference between Ca fraction for the same growth type, and between water soluble Ca content or between hydrochloric acid soluble Ca content for plants of the same ecosystem and between hydrochloric acid soluble Ca content for plants of the same family. In similar vegetation zones, there is a significant difference among hydrochloric acid soluble Ca content for different growth types in Yanchi County and Zhenglan Banner,which was the highest for annual herbs and the lowest for perennial herbs. There is a significant difference between acetic acid soluble Ca content and between hydrochloric acid soluble Ca content for sandy land and grassland ecosystems in Yanchi County. There is a significant difference among the same Ca fraction of different families in the same region. Thus, the characteristics of plant Ca fraction are results of long-term adaptation to the environment.

Keywords: semi-arid region; plant; calcium fraction; Ningxia Habahu National Nature Reserve; Zhenglan Banner of Inner Mongolia

1 Introduction

Total calcium (Ca) in the plant body accounts for 0.1% to 5.0% of its dry weight. Because of the differences in the proportion of soluble and insoluble forms within a plant, the measurement of total Ca content is recognized as an unreliable predictor of physiologically or nutritional status (Marschner, 1974, 1995;Clark et al., 1987). Ca in a plant is divided into physiologically active and metabolically inactive Ca,the former includes Ca2+, Ca phosphate, Ca protein and Ca pectate, the latter only includes Ca oxalate(Himelrick and Walker, 1982; Clark et al., 1987). In sequential fractionation procedures which are used to determine plant Ca fraction, water soluble Ca consists of Ca2+and some soluble or slightly soluble Ca salts, acetic acid soluble Ca includes Ca carbonate, Ca phosphate and Ca pectinate, while hydrochloric acid soluble Ca consist only of Ca oxalate (Bradfield,1977; Clark et al., 1987).

In plant Ca fractions, Ca2+is the most important cytoplasmic secondary messenger, and participates in the adjustment to resist adversity (Jiang et al., 2005;Zhou and Wang, 2007). Intracellular Ca2+changes in time and space under adverse conditions (Zhang et al., 2007; Zheng and Chen, 2015; Edel et al., 2017).The induced Ca signaling is identified, amplified and transmitted to downstream through Ca binding proteins (Ma et al., 2017), which causes a variety of biochemical reactions in cells to adapt to an adverse environment (Jian and Wang, 2008; Zhang et al., 2009;Zhao et al., 2017). Ca2+is involved in cellular move-ment, regulates cell division, elongation, differentiation as well as formation of polarity, and participates in tropism, photomorphogenesis, defense, stress response and other complex processes (Shao et al.,2008; Tan and Ma, 2017; Wang et al., 2017). On the other hand, there are Ca salt molecules in the plant body. Ca2+combines with pectin to form Ca pectate which is an important component of cell walls. In plant cells, Ca exists in substances such as Ca pectate,calmodulin and Ca phytate. Plant vacuoles contain a large amount of organic acid Ca, such as Ca oxalate,Ca citrate and Ca malate (Himelrick and Walker,1982; Hirschi, 2004). These deposits play an important role in regulating intracellular ionic equilibrium(Li et al., 2003; Prasad and Shivay, 2017). For example, Ca oxalate participates in the regulation of plant Ca ion and detoxification (Volk et al., 2002;Mazen, 2004).

In arid and semi-arid regions, the land undergoes sand desertification (Li et al., 2016; Luo et al., 2016;Liu et al., 2017) and salinization (Yu et al., 2017;Zhang et al., 2017) which is due to little precipitation,strong evaporation, strong winds, and vegetation degradation (He et al., 2016). One possible adaptation to this type of adverse environment is change of the Ca fration in a plant body. Our previous research shows that plant Ca fractions have significant differences between desert and salinization habitats (Ci et al.,2010; Xu et al., 2012; Xue et al., 2013). However, research is lacking in differences in plant Ca fractions in the same climate region. Thus, we analyzed plant Ca fractions in the Ningxia Habahu National Nature Reserve in northern Yanchi County of Ningxia Hui Autonomous Region (vegetation is sand desertified steppe) and the middle north of Zhenglan Banner of Inner Mongolia Autonomous Region (vegetation is typical steppe) with similar semi-arid regions. Our aim is to explain the adversity-resistant mechanism in plant physiology and to promote the restoration of damaged vegetation in arid and semi-arid areas.

2 Materials and methods

2.1 Natural environment

Plant samples were collected from two vegetation zones in semi-arid regions, i.e., the Ningxia Habahu National Nature Reserve in northern Yanchi County of Ningxia Hui Autonomous Region, China and the southeastern part of Sanggendalai in the middle north of Zhenglan Banner of Inner Mongolia Autonomous Region, China. The Reserve belongs to a sand desertified steppe zone, which is located in the transition zone from arid to semi-arid (You et al., 2016), with longitude of 106°53′11″E to 107°38′10″E, latitude of 37°38′36″N to 38°02′00″N, elevation of 1,300 to 1,622 m, average annual temperature of 7.7 °C, average annual precipitation of 296.5 mm, and average annual evaporation of 2,131.7 mm. The soil includes mobile, semi-fixed and fixed aeolian sandy soil with vegetation of mainly Salix psammophila and Artemisia ordosica communities. The main plants are S. psammophila, A. ordosica, A. scoparia, Corispermum deslinatum, Heteropappus altaicus, Cynanchum thesioides, Stipa bungeana, Pennisetum centrasiaticum, Aneurolepidium dasystachys, Poa sinoglauca,Cleistogenes squarrosa, Sophora alopecuroides, Inula salsoloides, Astragalus melilotoides, Thermopussis lanceolata and Hedysarum mongolicum.Zhenglan Banner is a typical steppe zone, with longitude of 115°00′E to 116°43′E, latitude of 41°56′N to 43°07′N, elevation of 1,300 to 1,600 m, average annual temperature of 1.5 °C, annual average precipitation of 370.7 mm and average annual evaporation of 1,931.4 mm. The soil includes chestnut soil, light chestnut soil and fixed aeolian sandy soil with vegetation of mainly S. krylovii, Leymus chinense, Ulmus pumila and Caragana microphylla communities. The main plants are S. krylovii, L. chinensis, U. pumila, C.microphylla, Stellera chamaejasme, Carex korshinskyi, C. squarrosa, Thalictrum Squarrosum, Serratula chinensis, Sanguisorba officinalis, Gentiana dahurica, Potentilla acaulis, Potentilla tanacetifolia,Haplophyllum dauricum, Delphinium grandiflorum and Polygonum divaricatum.

2.2 Sample collection and analyses

In July 2016 and July 2017, 91 plant species were collected from the Ningxia Habahu National Nature Reserve, and 84 plant species from Sanggendalai of Zhenglan Banner. We collected three samples of every plant species in both localities.

Each plant sample was measured three times.Three 0.01 g samples for each repetition were cut into pieces and put into three mortars, respectively.Then, 1 mL of deionized water, 1.05 mol/L dilute acetic acid and 1 mol/L dilute hydrochloric acid were added into three mortars separately, and ground into homogenates. The homogenates were transferred into 7 mL centrifuge tubes, respectively, then, 4 mL of corresponding solution was added and washed into the centrifuge tube. The tubes were centrifuged at 7,000 rpm for 15 min, and the supernatant was the extract. Three conical flasks for each repetition were prepared. For the treatments of deionized water, acetic acid, and hydrochloric acid, 0.16, 0.94 and 0.91 mL of 4 mol/L NaOH and 1.84, 1.06 and 1.09 mL of deionized water as well as 3 mL of the responding extraction solution were added into the responding flask,respectively. Then 3 mL of triethanolamine (1：2 aqueous solution) and 0.08 g of Ca carboxylic acid in-dicator were added into each flask. Every solution was titrated with calibrated EDTA solution, and titration was stopped when the color of the solution changed from mulberry to bright blue. Then the volumes of the titrant were recorded and used to calculate plant Ca fractions.

2.3 Statistical analyses

The SPSS 19.0 software was used for one-way ANOVA and independent sample t-test for the statictical analyses of the plant Ca fractions. Homogeneity test was performed first due to variances of homogeneity in on-way ANOVA. The Duncan test was used to test significant differences in each Ca fraction among different families, growth types and ecosystems in each vegetation zone. Then, independent sample t-test was used to test the differences of each Ca fraction in the same family, growth type and ecosystem of the two vegetation zone. Figures were made by Excel 2016.

3 Results

3.1 Plants with higher and lower calcium content

In the Ningxia Habahu National Nature Reserve,as for water soluble Ca, higher content was found in Dracocephalum mordavica (48.17 g/kg), Tribulus terrestris (35.93 g/kg), Messerschmidia sibirica (33.86 g/kg)and Jurinea mongolica (33.69 g/kg), while lower content was found in Salsola collina (4.65 g/kg), Bassia dasyphylla (5.47 g/kg) and Atriplex centralasiatica(5.66 g/kg). For acetic acid soluble Ca, higher content was found in Nostoc commune (19.56 g/kg), M.sibirica (17.56 g/kg) and Sonchus brachyotus(13.28 g/kg), while lower content was found in Stipa glareosa (0.35 g/kg), Suaeda glauca (1.51 g/kg) and B. dasyphylla (1.98 g/kg). For hydrochloric acid soluble Ca, higher content was found in S. collina(33.05 g/kg), Agriophyllum squarrosum (29.46 g/kg),Chenopodium acuminatum (23.59 g/kg) and Gypsophila davurica (23.10 g/kg), while lower content was found in Psammochola mongolica (0.28 g/kg), S. bungeana (0.37 g/kg), Inula salsoloides (0.66 g/kg) and Astragalus melilotoides (0.67 g/kg).

In Zhenglan Banner, plants with higher water soluble Ca content included Dontostemon dentatus(65.38 g/kg), Plantago asiatica (38.71 g/kg) and Melissitus ruthenica (32.51 g/kg), while lower content included Melandrium apricum (4.64 g/kg), Gentiana dahurica (4.66 g/kg), Chenopodium glaucum(4.66 g/kg) and Stellaria dichotoma (4.82 g/kg). For acetic acid soluble Ca, higher content included Amethystea caerulea (66.91 g/kg), Cannabis sativa(32.51 g/kg), Lappula myosotis (16.29 g/kg) and Cynanchum thesioides (16.04 g/kg), while lower content included Allium senescens (0.74 g/kg), P. asiatica(1.15 g/kg), Saussurea amara (1.51 g/kg), Potentilla bifurca (1.58 g/kg) and S. grandis (1.59 g/kg). For hydrochloric acid soluble Ca, higher content included Amaranthus tricolor (45.51 g/kg), G. dahurica (26.52 g/kg)and S. amara (22.63 g/kg), while lower content included Cleistogenes squarrosa (0.20 g/kg), Heteropappus altaicus (0.38 g/kg), Calamagrostis epigeios(0.40 g/kg), Thermopsis lanceolata (0.41 g/kg) and Ixeris denticulata (0.46 g/kg).

3.2 Plant calcium fractions of Leguminosae,Gramineae, Compositae and Chenopodiaceae

Compositae, Gramineae, Leguminosae and Chenopodiaceae were dominant families in Yanchi County and Zhenglan Banner. For each family of the two regions, there was no significant difference between hydrochloric acid soluble Ca content; for water soluble Ca content and acetic acid soluble Ca content,Gramineae and Chenopodiaceae in Zhenglan Banner was significantly higher than those in Yanchi County,respectively, but no significant difference was observed among other families (Figure 1).

As for different families of the same region, in Yanchi County, water soluble Ca content and acetic acid soluble Ca content of Leguminosae and Compositae were significantly higher than those of Gramineae and Chenopodiaceae, respectively, while hydrochloric acid soluble Ca content of Chenopodiaceae was significantly higher than those of Compositae, Gramineae and Leguminosae (Figure 1). In Zhenglan Banner,water soluble Ca content of Leguminous was significantly higher than those of Compositae, Gramineae,and Chenopodiaceae; acetic acid soluble Ca content of Compositae was significantly higher than that of Gramineae; hydrochloric acid soluble Ca content of Chenopodiaceae was significantly higher than those of Compositae, Gramineae and Leguminosae (Figure 1).

3.3 Plant calcium fraction of different growth types

In each growth type for the two regions, there was no significant differences between water soluble Ca,acetic acid soluble Ca or hydrochloric acid soluble Ca contents (Figure 2).

For different growth types of the same region, in Yanchi County and Zhenglan Banner, there was no significant difference in water soluble Ca and acetic acid soluble Ca among shrub, semi-shrub, perennial herb and annual herb, respectively. For hydrochloric acid soluble Ca all had significant differences in Yanchi County and Zhenglan Banner, which was the highest for annual herb and the lowest for perennial herb (Figure 2).

Figure 1 Comparisons of plant calcium fraction among Leguminosae, Gramineae, Compositae and Chenopodiaceae and between those of Yanchi County of Ningxia and Zhenglan Banner of Inner Mongolia (Capital letter indicates the difference between the same variable of both locations. The lower-case letter indicates the difference among the same variable of different families for each location)

Figure 2 Comparisons of plant calcium fraction among different growth types and between those of Yanchi County of Ningxia and Zhenglan Banner of Inner Mongolia (Capital letter indicates the difference between the same variable for each locality.Lower-case letter indicates the difference among the same variable of different families for each locality)

3.4 Plant calcium fraction of different ecosystems

In each ecosystem for both regions, there was no significance difference between water soluble Ca content or between hydrochloric acid soluble Ca content of sandy land and grassland ecosystems. Acetic acid soluble Ca of grassland plants in Zhenglan Banner was higher than that in Yanchi County, while no significant difference was observed between acetic acid soluble Ca content of the sand land plant for both localities (Figure 3).

For different ecosystems of the same region, in Yanchi County, no significant difference was observed between water soluble Ca content but significant differences was observed between acetic acid soluble Ca content and between hydrochloric acid soluble Ca content of sandy land and grassland plants. In Zhenglan Banner, the amounts were not significantly different between water soluble Ca content, acetic acid soluble Ca and hydrochloric acid soluble Ca of grassland and sandy land plants (Figure 3).

4 Discussion

Changes of plant Ca fraction are sensitive to the ecological environment, such that the more severe drought and salinization, the higher the content of hydrochloric acid soluble Ca (Ca oxalate). Ci et al.(2010) reveals that in the Tengger Desert, perennial herbs has more water soluble Ca, shrubs hold more hydrochloric acid soluble Ca, and grassland plants hold more water soluble Ca; drought-resistance plants possess more Ca oxalate. Xu et al. (2012) implies that in salinized habitats of Tianjin, from tree, shrub, liana to herbs, the concentration of hydrochloric acid soluble Ca decrease gradually, while those of water soluble Ca increases; the concentration of water soluble Ca in herbs are significantly higher than those in tree and shrubs. In this study, we found that there was no significant difference between plant Ca fractions among shrub, subshrub, perennial herb and annual herb for the two vegetation zones (vegetation in Yanchi County is sand desertified steppe, while Zhenglan Banner is typical steppe) in similar semi-arid areas.These results suggest that plant Ca fractions are the result of long-term adaptation to the environment for plants. However, for a single vegetation zone, hydrochloric acid soluble Ca of annual herb was significantly higher than those of other growth types in both localities. We found that hydrochloric acid soluble Ca of C. deslinatum, Ch. acuminatum, K. scoparia var. sieversiana, A. squarrosum and S. collina in annual herb were obviously higher in Yanchi County and in Zhenglan Banner, these plants are psammophyte or halophyte, which accumulate Ca oxalate(hydrochloric acid soluble Ca) in severe environmental conditions. This shows that environmental conditions influence the characteristics of plant Ca fractions, and Ca oxalate is important in plant adversity-resisting.

Figure 3 Comparisons of plant calcium fraction among different ecosystems and between those of Yanchi County of Ningxia and Zhenglan Banner of Inner Mongolia (Capital letter indicates differences between the same variable of both localities.Lower-case letter indicates differences among the same variable of different families of both localities)

In the present study, for the two vegetation zones,there was no significant difference between water soluble Ca contents and hydrochloric acid soluble Ca contents for similar sand land ecosystems or grassland ecosystem in Yanchi County and in Zhenglan Banner. In Zhenglan Banner, no significant difference was found between similar plant Ca fractions of grassland and sandy land ecosystems. In Yanchi County, hydrochloric acid soluble Ca content of plants in sandy land ecosystems was significantly higher than those of plants in grassland ecosystems.Annual precipitation of Yanchi County is 296.5 mm,while that of Zhenglan Banner is 370.7 mm. Thus, the less precipitation the region, the more significant is hydrochloric acid soluble Ca in the sandy land ecosystem. Water soluble Ca and acetic acid soluble Ca are physiologically active Ca. Analysis shows that plants in grasslands of Zhenglan Banner have higher acetic acid soluble Ca, which is a requirement for normal metabolism of plants. In our results, there was no significant difference in hydrochloric acid soluble Ca among plants of Leguminosae, Gramineae, Compositae and Chenopodiaceae both in Yanchi County and in Zhenglan Banner. However, water soluble Ca of Gramineae plants and acetic acid soluble Ca of Chenopodiaceae plants in Zhenglan Banner were higher than those in Yanchi County. In the same semi-arid area, Leguminosae plants had higher water soluble Ca and Chenopodiaceae plants had higher hydrochloric acid soluble Ca for both Yanchi County and Zhenglan Banner. As we know, water soluble Ca and acetic acid soluble Ca are physiologically active Ca, and hydro-chloric acid soluble Ca is physiologically inactive Ca(Ci et al., 2010). Obviously, plants in Zhenglan Banner possess more water soluble Ca and acetic acid soluble Ca due to more rainfall and better natural conditions, while plants in Yanchi County have more hydrochloric acid soluble Ca because of less precipitation and worse natural conditions.

Hydrochloric acid soluble Ca (Ca oxalate) has stronger resistance to stress among plant Ca fractions.Ca oxalate exists mainly in the form of crystals in plants. The formation of Ca oxalate crystals is a kind of Ca regulation mechanism of plants (Volk et al.,2002; Tooulakou et al., 2016). Ca oxalate crystals are widely found in higher plants (Li et al., 2012; Zhu et al., 2014; Islam and Kawasaki, 2015), and deposits in vacuoles of specialized cells are called crystal idioblasts (Yan et al., 2008; Islam and Kawasaki, 2015;Liang and Wang, 2015). On the one hand, crystal idioblasts have strong water retention and absorption capacity, on the other hand, the formation of crystal idioblasts is an accumulation of excess salt and alkali which reduces harmful substances in plants. It is speculated that Ca oxalate crystals are related to the ability of plants to adapt to arid and saline environments.In this study, Ca oxalate concentrations of Chenopodiaceae plants, annual sand plants and halophytes are higher, which is an adaptation and survival strategy of plants to environmental changes.

Thus, water soluble Ca, especially Ca ions, and hydrochloric acid soluble Ca (Ca oxalate), are important to plant stress resistance. Ca ions act primarily as a second messenger which is different with Ca oxalate that enhances the resistance ability. Plants in arid and semi-arid regions tend to accumulate more Ca oxalate to enhance adaptability because of worse survival conditions. Utilizing Ca oxalate to respond to stress is strategically selected by plants.

5 Conclusions

In similar semi-arid regions, each Ca fraction of plants of similar growth types has no significant difference for both vegetation zones of Yanchi County and Zhenglan Banner. This is also true for water soluble Ca and hydrochloric acid soluble Ca for plants of the same ecosystem. For a single vegetation zone, significant differences exist in hydrochloric acid soluble Ca for different growth types, ecosystems or plant families. The characteristics of plant Ca fractions is the result of long-term adaptation and evolution of plants.

Acknowledgment:

This research was supported by the National Key Research and Development Program of China(2016YFC0500706).