Seasonal dynamics of N:P ratio stoichiometry and Ca fraction for four dominant plants in the Alxa Desert

JianTan Guo, XingDong He , HongJuan Jing, YuTing Liang

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

ABSTRACT Desert plants take on unique physiologically adaptive mechanisms in response to an adverse environment. In this study, we determined the concentrations of leaf nitrogen (N), phosphorus (P), and calcium (Ca) fraction for dominant species of Artemisia ordosica, A. frigida, Calligonum mongolicum, and Oxytropis aciphylla in the Alxa Desert and discussed seasonal changes of their leaf N：P ratio and Ca fraction. The results showed that, from May to September, the N：P ratios of A. ordosica and C. mongolicum gradually and significantly increased, while those of A. frigida, and O. aciphylla had an increase trend that was not significant; the physiologically active Ca of A. ordosica and A. frigida increased significantly,while that of C. mongolicum and O. aciphylla decreased significantly. The physiologically inert calcium of C. mongolicum increased extremely significantly, while that of others was not significant. There was a significantly positive correlation between the N：P ratio and physiologically active Ca for A. ordosica, and the N：P ratio was significantly and negatively correlated with physiologically active Ca for O. aciphylla. These findings revealed that the physiological regulation mechanism was different for the plants either in earlier stage or later stage of plant-community succession.

Keywords: the Alxa Desert; N：P ratio; Ca fraction; seasonal dynamic

1 Introduction

In recent years, ecological stoichiometry of the carbon ： nitrogen ： phosphorus (C：N：P) ratio has been a hot topic in ecology research (Niklas, 2006; He and Han, 2010; Martinez-Oró et al., 2017). Ecological stoichiometry has an influence not only on individual development and adaptation to stress (Güsewell et al.,2003; De Senerpont Domis et al., 2014) but also on formation and succession of plant communities(Huang et al., 2012). Compared with the effects of N and P on plants, the plant N：P ratio has a more important role in predicting nutrient limitations(Güsewell and Koerselman, 2002). Up to now, either at home or abroad, the research on ecological stoichiometry has been mostly on large scales, such as global or regional (Güsewell, 2004; Reich and Oleksyn, 2004; Han et al., 2005; Ren et al., 2007; Li et al., 2010; Ren et al., 2012). Few papers have focused on plants in different succession stages, which is very important for elaborating the mechanism of plant-community succession.

Ca fraction includes water-soluble Ca, acetic-acid-soluble Ca, and hydrochloric-acid-soluble Ca. Water-soluble Ca and acetic-acid-soluble Ca are called physiologically active Ca, and hydrochloric-acid-soluble Ca is called physiologically inert Ca (Clark et al.,1987; Ci et al., 2010). Water-soluble Ca is mainly Ca2+, which plays an important role in maintaining stability of the cytoderm and cytomembrane, as well as membrane-bound proteins (Miedema et al., 2001;Hepler, 2005). Ca2+can decrease respiration, work against cell senescence, increase the transfer rate of photosynthetic products, and protect plants from negative effects of metal ions and oxalic acid elimination;and Ca2+has the ability to maintain homeostasis in cells and the development of plants (Reddy, 2001).What's more, Ca2+has been considered to be the second messenger in signal transduction (Poovaiah et al., 1993; Reddy, 2001; Hepler, 2005). Hydrochloric-acid-soluble Ca contains only Ca oxalate,which is of great importance in Ca transformation and resistance to drought stress (Clark et al., 1987). Our previous results indicate a significant difference between the Ca fractions of psammophytes and the xerophytes in the Alxa Desert (Ci et al., 2010). Then,in successional series of plant community, how the Ca fractions of constructive species in different succession stages change still needs to be explored.

In desertified areas, there exist severe land desertification (Li et al., 2016; Luo et al., 2016; Liu et al.,2017) and vegetation degradation (He et al., 2016;Liu et al., 2017; Yu et al., 2017). To deeply study the successional mechanism of plant community helps vegetation restoration of the desertification area. In Alxa Left Banner, over 12 years, we found that there was such a successional series of plant communities：the Artemisia ordosica community was replaced by the A. frigida community and then by the Oxytropis aciphylla community and Calligonum mongolicum community. To reveal the cause and mechanism of succession of the plant communities, in the present study, we determined concentrations of N, P, and Ca fractions for A. ordosica, A. frigida, C. mongolicum,and O. aciphylla in the Alxa Desert; discussed the seasonal dynamics of both their N：P ratios and Ca fractions; and analyzed the relationships between N：P ratios and Ca fractions. Our aim is to facilitate the restoration of damaged vegetation of the desertification area.

2 Materials and methods

2.1 Natural environment

The sample plot is 8 km away from the Alxa Left Banner, on the eastern boundary of the Tengger Desert, located at 38°24′59″N, 105°43′31″E, in an arid area, Inner Mongolia Autonomous Region, China.The altitude of the sample plot is 1,420 m. According to the Alxa League weather station, the average annual temperature is 9.2 °C. The average annual precipitation is 215.2 mm, with rainfall mainly occurring from July to September. The average annual evaporation is 2,349.2 mm. The average annual relative humidity is 39%. The average annual wind speed is 3.1 m/s, and the main wind direction is northwest.The typical soil in this region is a brown calcic soil,and the typical vegetation is semi-desert. Mainly, the plants are A. frigida, A. ordosica, A. sphaerocephala,Bassia dasyphylla, Ceratoides lateens, C. mongolicum, Elaeagnus angustifolia, Euphorbia esula, O. aciphylla, Reaumuria soongorica, Salsola passerina, and Stipa plareosa.

2.2 Sample collection and analyses

We chose A. ordosica, A. frigida, C. mongolicum,and O. aciphylla, which have grown in same sample plot (1km×1km). At 09：00, 12：00, 15：00 and 18：00 on May 26, July 26, and September 26, 2012, a leaf of three plants of each of the four species was collected,then kept in a container filled with liquid nitrogen,carried back to the laboratory, and stored in a refrigerator at -70 °C.

Plant N was determined using the H2SO4-H2O2-Semi-micro-Kjeldahl method (Bao, 2000). Plant P was determined using the H2SO4-H2O2-Mo-Sb-Vc-colormetry method (Bao, 2000). Each treatment was replicated three times. Ca fractions were determined using the sequential fractionation procedure (Bradfield,1977; Clark et al., 1987; Ci et al., 2010), with each treatment being replicated three times.

2.3 Statistical analyses

One-way ANOVA analyses were performed using SPSS 19.0 software. The homogeneity test was performed first; then the least significant difference was used to test the significance of the means of N, P,N：P ratios, and Ca fractions between different plants in different growth seasons if the variances were equal; and Tamhane's T2 multiple comparison test was used if they were not. Then, using the physiologically active Ca concentration as the independent variable and the plant N：P ratio as the dependent variable, we carried out regression analyses in Microsoft Excel 2010, which was tested for their significance using SPSS 19.0 software.

3 Results

3.1 The N and P concentrations and the N:P ratio in the plants

In the growing season from May to September,for the four plants, the average plant N was 16.18-27.72 g/kg; plant P, 2.14-3.39 g/kg; and N：P ratios, 8.06-9.92. The N and P concentrations in A. frigida were higher than those of the other three plants, while those for C. mongolicum were significantly lower than those of the other three plants. The N：P ratios in O. aciphylla were higher than those of the other three plants. From the seasonal variation, the plant N in A. ordosica increased gradually, while decreasing gradually in A. frigida, C. mongolicum, and O. aciphylla. The changes of the plant P in A. ordosica and A. frigida were not obvious and decreased gradually in C. mongolicum and O. aciphylla. The N：P ratios for all four plants increased, significantly for A. ordosica and C. mongolicum but not significantly for A. frigida and O. aciphylla (Figure 1).

Figure 1 Seasonal changes of N, P, and the N：P ratio for four plants in the Alxa Desert

3.2 The Ca fraction in the plants

The physiologically active Ca (water-soluble Ca and acetic-acid-soluble Ca) in A. ordosica and A. frigida was higher in September than in May and July;the physiologically inert Ca (hydrochloric-acid-soluble Ca, Ca oxalate) in A. ordosica was higher in July than in May and did not change significantly over the growing season. The diurnal changes of physiologically active Ca in A. ordosica and A. frigida were larger, and those of physiologically inert Ca were smaller. However, the diurnal variations of physiologically active Ca in C. mongolicum and O. aciphylla were smaller; and those of physiologically inert Ca were larger. From May to September, the physiologically active Ca had a significant increase for A. ordosica and A. frigida and a significant decrease for C. mongolicum and O. aciphylla. Although physiologically inert Ca had a very significant increase for C. mongolicum, those for the other three plants did not change significantly over the growing season(Figure 2).

Figure 2 Seasonal changes of the Ca fraction for four plants in the Alxa Desert

Statistical results showed that the proportions of physiologically active Ca to total Ca content in A. ordosica, A. frigida, and O. aciphylla were 86%, 87%,and 91%, respectively, while the physiologically inert Ca was dominant in C. mongolicum and accounted for 83% of the total Ca. This pattern showed that plants in various stages of the succession had different Ca fractions due to the different species and genetic characteristics.

3.3 The relationships between physiologically active Ca and the N:P ratio in the plants

Regression analysis showed that, with the increase of the physiologically active Ca contents, the N：P ratios significantly increased in A. ordosica(Figure 3a). The regression relationships between physiologically active Ca and the N：P ratios were not significant for A. frigida and C. mongolicum. In O. aciphylla, with the increase of physiologically active Ca, the N：P ratios exponentially reduced significantly (Figure 3d).

4 Discussion

4.1 Characteristics of the plant N:P ratio

Characteristics of the plant N：P ratio are an important indicator by which to judge plant-nutrient limitation (Koerselman and Meuleman, 1996; Aerts and Chapin, 2000; Wu et al., 2010) and plant-growth rate(Elser et al., 2000; Niklas, 2006). Through the study of 5,087 observations of 1,280 species in 452 places all over the world, Reich and Oleksyn (2004) found the average plant N content was 20.1 g/kg; phosphorus, 1.9 g/kg; and the N：P ratio, 13.9. The study of 753 kinds of Chinese terrestrial plants by Han et al. (2005)found the average content of leaf N was 18.6 g/kg;leaf P, 1.21 g/kg; and the N：P ratio, 14.4. Killingbeck and Whitford (1996) studied the leaf N of 78 species and found its average content to be 22.0 g/kg. In the present study, for the whole growing season, the average leaf N for four species in the Alxa Desert was 21.59 g/kg; leaf P, 2.69 g/kg, higher than the average value of the global terrestrial plants and the domestic ones, while the N：P ratio in the Alxa Desert was 8.71,consistent with the findings of Killingbeck and Whitford (1996). It is known that N is the vital component of amino acids, proteins, and nucleic acids in plants; P combines with other organics to form phospholipids,nucleic acids, and coenzymes, and takes on an important role in metabolic processes such as plant photosynthesis, respiration, and synthesis of nucleic acid and membrane lipids (Wang et al., 2007). Because the P content in soil is relatively high in arid and semi-arid areas (Wang et al., 2008), the plant P content also increases (Ren et al., 2007), helping the plants to resist drought stress.

Figure 3 The relationships between the physiologically active Ca and the N：P ratio for four plants in the Alxa Desert

Koerselman and Meuleman (1996) revealed that when the N：P ratio is lower than 14, N mainly influences plant growth; P mainly controls plant growth when the N：P ratio is higher than 16; and plant growth is controlled by both N and P when the N：P ratio is between 14 and 16. In the present study, the N：P ratios for A. ordosica, A. frigida, C. mongolicum, and O. aciphylla were 8.06, 8.74, 8.13, and 9.92, respectively, all lower than 14, which showed that the growth of constructive plants in the Alxa Desert was mainly controlled by N. Meanwhile, this result illustrated that, in the succession series of plant communities in the Alxa Deset, the plants in the earlier stage had the lower N：P ratio, while those in the later stage had the higher.

This study showed that, in the growing season, the N：P ratios increased obviously in A. ordosica and C. mongolicum, while the increase of those in A. frigida and O. aciphylla was not obvious and was higher in autumn. This phenomena was because in the earlier stage of the growing season the synthesis of proteins and nucleic acids required a great deal of N and P and led to the higher concentrations of N and P in the young branches and leaves (Sun and Chen,2001). As plants continued growing, the requirement decreased; and the N and P content in the leaves of the plant decreased (Townsend et al., 2007). Because P content decreased more so than N content, the leaf N：P ratios increased for the four plants in autumn. As shown above, in this study we found in the growing season the average N：P ratios increased gradually for the four plants; this phenomenon was consistent with the growth-rate hypothesis (Sterner and Elser, 2002).

4.2 Characteristics of plant Ca fraction

The total Ca content in leaves increased, along with leaf maturity, in the process of differentiation of pear buds from bud to flower (Peng et al., 1999). The total Ca content in three species of Hedysarum showed an increasing trend from June to September(Yan et al., 2007). Our results were similar to theirs：total Ca content of A. ordosica, A. frigida, and C. mongolicum increased significantly from May to September, with the increase of total Ca content in A. ordosica and A. frigida due to the increase of physiologically active Ca, while that of C. mongolicum resulted from the increase of hydrochloric-acid soluble Ca.

In the present study, the seasonal regularities of physiologically active Ca for the four plants were very evident. The physiologically active Ca in A. ordosica and A. frigida was higher in September than in May and July, while that of C. mongolicum and O. aciphylla was lower in September than in May and July. The physiologically inert Ca was just the opposite in C. mongolicum, and there was no obvious change in O. aciphylla. This finding indicated that physiologically active Ca content in an early successional plant (A. ordosica) and a middle successional plant (A. frigida) was lower in the early period of the growing season and higher in the later period, while the physiologically active Ca in the late successional plants (C. mongolicum and O. aciphylla) was just the opposite. This phenomena illustrated that the physiologically active Ca played a different role in the successional process for the early and late successional plants. The physiologically active Ca content in A. ordosica and A. frigida was lower in the early stages and higher in later stages, with this pattern likely to be because they grew vigorously in the early stage and needed a lot of physiologically active Ca.

4.3 Relationships between plant N:P ratio and physiologically active Ca

In the present study, we demonstrated that, with the increase of physiological Ca content, the plant N：P ratios increased in A. ordosica and decreased in O. aciphylla; and there existed no significant regression relationship for A. frigida and C. mongolicum.This finding indicated that the mechanism of homeostatic regulation in plants situated at earlier or later succession stages was different, which might be due to different soil N and P content at different successional stages.

The mechanism of homeostatic regulation of the N：P ratio is important for sustaining plant life (Sterner and Elser, 2002). A large number of studies have shown that Ca2+reacts with phosphoric acid to precipitate Ca phosphate when the intracellular Ca2+concentration is too high, which can interfere with phosphoric-acid-based energy metabolism (Gong et al.,1990; Zhou and Wang, 2007). When Ca2+is limited,the absorption of exogenous nitrogen in the plant decreases, the transportation of nitrate from underground to aboveground is inhibited, the allocation of nitrate in various organs changes, and the activity of a nitrogen-assimilation enzyme and protein synthetic rate decreases significantly in the aboveground organs(Wang et al., 2000). The research shows that, with the extension of growing season in the Ulan Buh Desert,the Ca content in five shrubs increases. While the P content decreases (Wang et al., 1999), the Ca content increases, but the P content decreases in five shrubs in the Kubuqi Desert from May to October (Yu et al.,2009). Obviously, the plant regulates the rate of the absorption of N and P via the Ca-dependent signaling mechanism, so that the N：P ratio changes. When the concentrations of plant N and P are less than the plant's minimum requirements, it is possible to maintain the normal function of cells in the tissue of the plant by maintaining homeostasis.

5 Conclusions

In the Alxa Desert, leaf N：P ratio and Ca fraction take on obviously seasonal changes for A. ordosica,A. frigida, C. mongolicum and O. aciphylla. From May to July to September, leaf N：P ratios for early stage plant A. ordosica and late stage plant O. aciphylla in September are higher than those in May and in July, but leaf N concentrations significantly increase for A. ordosica and significantly decrease for O. aciphylla, so do the physiologically active Ca. Leaf N：P ratio and physiologically active Ca assume positive correlation relationship for A. ordosica and negative correlation relationship for O. aciphylla. Obviously, there exists a significant difference in physiological mechanism for the early plant and the late plant of succession of plant community.

Acknowledgments:

This work was supported by the National Key R&D Program of China (2016YFC0500706).