Effects of high Ca and Mg stress on plants water use efficiency in a Karst ecosystem

Study area and sample collection

The study area is located in Puding County, Anshun City, Guizhou Province in Southwest China (26°15′ to 26°16′ N, 105°46′ to 105°47′ E) (Fig. 1), a typical Karst area with the dominant dolomite bedrock. Dolomite and limestone from Middle Triassic generated calcareous soil (Han et al., 2020; Zhao et al., 2010) and were further transported and deposited as quaternary soil with high Ca and Mg contents. Subtropical monsoons dominate the climate of the study area, with an annual mean temperature of 15.1 °C and precipitation of 1,400 mm, respectively.

A total of 19 plant samples, including seven types of herbaceous plants and 12 types of woody plants, were collected in this Karst region in June 2016. The herbaceous plants include peanut (Arachis hypogaea Linn.), sunflowers (Helianthus annuus L.), bracken (Pteridium aquilinum), roof iris (Iris tectorum Maxim.), taro (Colocasia esculenta), Indian chrysanthemum (Chrysanthemum indicum), and horseweed (Conyza canadensis). In contrast, the woody plants include Chinese firethorn (Pyracantha fortuneana), bird cherry (Prunus padus L.), oak tree (Quercus fabri), Chinese catalpa (Catalpa ovata), wheel wingnut (Cyclocarya paliurus), prickly wild rose (Rosa acicularis), Japanese camellia (Camellia japonica), dahurian buckthorn (Rhamnus davurica), litsea cubeba (Litsea pungens Hemsl.), lobular buckthorn (Rhamnus parvifolia Bunge), Chinese ilex (Ilex chinensis Sims), and Eucommia bark (Eucommia ulmoides Oliver). The target plants are dominant and relatively abundant in the study area, mainly sampled from the upper canopies and with three to five healthy leaves of similar ages at random of the same plants. The collected leaves were rinsed with deionized water (Cascada™, 18.2 MΩ cm). The tissues were cleaned and then freeze-dried. Before elemental analysis, the dried samples were crushed to a powder and passed through a sieve (<150 μm).

Chemical analysis and calculation of WUE

After grounding into uniformly fine powders, the N contents of leaf samples were determined by an elemental analyzer (Elementar, Langenselbold, Germany) at the Surficial Environment and Hydrological Geochemistry Laboratory of the China University of Geosciences (Beijing). Before analyzing other nutrients, the sample powders were dissolved in 3 mL concentrated HNO₃ for over 48 h inside a high-pressure steel bomb at 190 °C in an oven. The digested samples were then analyzed for P, K, Ca, and Mg contents by Optima 5300DV ICP-OES (PerkinElmer, USA). The N content was measured by a multi-element analyzer (Vario TOC Cube, Elementar, Langenselbold, Germany) (Zeng et al., 2022). The samples were randomly measured again to ensure the recovery percentage from 95% to 105% (Gao et al., 2021; Wang et al., 2021). All plant species and nutrients data were shown in Table S1 in the Supplemental File. The stable carbon isotopic ratio (¹³C/¹²C) was measured using an isotope mass spectrometer (MAT-253;Thermo-Finnigan, San Jose, California, USA) at the Institute of Geochemistry, Chinese Academy of Sciences. The data of δ¹³C were from Liu, Han & Zhang (2020a). The δ¹³C values were described as the following equation:

(1) $${\delta }^{13}C({\mathrm{\%}}_o)=[(R_{sample}-R_{standard})/R_{standard}\times 1000]$$where R_sample and R_standard are the ¹³C/¹²C ratios of the sample and standard of Vienna Pee Dee Belemnite, respectively. The external precision was better than ±0.1‰ after multiple measurements of the standard material. The following formula links Δ¹³C to the ratio of CO₂ in the leaf intercellular space to CO₂ in the atmosphere (C_i/C_a):

(2) $${\mathrm{\Delta }}^{13}C({\mathrm{\%}}_o)=a+(b-a)(C_i/C_a)$$where a (4.4‰) represents the discrimination against ¹³CO₂ during CO₂ diffusion via stomata, and b (27‰) reflects carboxylation discrimination (Farquhar & Richards, 1984; O’Leary, 1981). The atmospheric CO₂ concentration C_a was calculated as the following equation (Feng, 1998):

(3) $$C_a=277.78+1.350exp(0.01572\times (t-1740))$$where t represents the sampling year, which is 2016 in this study. The WUE can be calculated by the relationship between Δ¹³C and C_a using the following equation (Hietz, Wanek & Dünisch, 2005; Osmond, Björkman & Anderson, 1980; Peñuelas, Canadell & Ogaya, 2011):

(4) $WUE=A/g_{H_{2}O}=C_a(b-{\mathrm{\Delta }}^{13}C)/1.6(b-a)$where A is the net photosynthesis, 1.6 is the ratio value of leaf conductance to water vapor (g_H2O) and CO₂ (g_CO2) (Hietz, Wanek & Dünisch, 2005; Peñuelas, Canadell & Ogaya, 2011).

Data analysis

One-way ANOVA with the least significant difference (LSD) test was performed to test the significant differences between herbaceous plants and woody plants of N, P, K, Ca, and Mg contents. Pearson correlation coefficient was used to identify the correlations between WUE and K, Ca, and Mg content. The coefficient of R square and p-value determined the equations of best-fit lines. Statistical analyses were conducted by the IBM SPSS Statistics for Windows, Version 27.0 (IBM, Armonk, NY, USA).

Leaf stoichiometry in herbaceous plants and woody plants

Karst soil is characterized by the scarcity of N and P elements but high Ca and Mg contents (Li et al., 2019). Previous studies reported the soil nutrients of N content (3.1 to 7.3 g·kg⁻¹), P content (0.5 to 1.18 g·kg⁻¹), K content (27.7 g·kg⁻¹), Ca content (3.8 to 34.9 g·kg⁻¹), and Mg content (0.9 to 22.6 g·kg⁻¹) in Puding County (Kuang et al., 2010; Wang et al., 2018). As shown in Figs.2A–2C, herbaceous plants have higher N, P, and K contents than woody plants. Previous studies have reported higher N, P, K, and Mg contents of herbaceous plants compared to woody plants (Borisade, 2020; Tian et al., 2018), which was attributed to the higher growth and turn-over rates and the rapid decomposition of herbaceous plants (Gilliam, 2007; Güsewell & Koerselman, 2002). Our results corroborated the previous findings that herbaceous plants (short life) contained more N and P contents than woody plants (long life) (Güsewell & Koerselman, 2002; Han et al., 2005). However, there were no significant differences in leaf Ca, Mg content between herbaceous plants and woody plants (Figs. 2D and 2E). This phenomenon was probably attributed to the alkaline soil with the enrichment of high Ca and Mg content in the Karst region (Li et al., 2019; Liu et al., 2021), which limited the growth of plants (Carrière et al., 2020). As shown in Fig. 2F, herbaceous plants exhibited a wider WUE range than woody plants. Woody plants are likely to have deep roots and rely on water from deep soil layers, while herbaceous plants rely on water from shallow soil layers (Nie et al., 2014; Nie et al., 2011; Rose, Graham & Parker, 2003; West et al., 2008). Therefore, under the condition of high soil permeability and water shortage in the Karst region (Liu et al., 2021), woody plants should have more steady water-use strategies under drought conditions (Fig. 2F). In other aspects, the various WUE in plants may be related to K content. A possible explanation is that plants collected osmoticum (K⁺) in response to water stress to raise cell osmotic potential, attracting water into cells (Si et al., 2015). Therefore, herbaceous and woody plants have significantly distinctive nutritional needs, and their WUE reveals quite different features.

Nutrient leaking from thin, calcareous soils in the Karst region may decrease the remaining capacity of soil to deliver essential ecosystem services, further affecting carbon sequestration and food production (Ma et al., 2018; Song et al., 2017). Therefore, it is necessary to understand the type of nutrient limitation in plants. Previous study has suggested several nutrient limitations (Olde Venterink et al., 2003): N limitation (N:P < 14.5 and N:K < 2.1), P or P+N limitation (N:P > 14.5 and K:P > 3.4), and K or K+N limitation (N:K > 2.1 and K:P < 3.4). As shown in Fig. 3, almost all herbaceous plants and several woody plants exhibited N limitation, while the other woody plants were P or N+P limited. These results were consistent with the findings that N limited grassland growth, P limited secondary forest growth, and N+P limited shrub growth in Southwest China (Zhang et al., 2015). None of the herbaceous plants and woody plants suffer from K limitation. Therefore, agricultural activities in this Karst area should apply more N fertilizer to crops. However, the limitation of plant nutrients does not rule out the dilution effects due to the specific Karst environment. For example, K, Ca, and Mg contents can influence the NO_3⁻ and NH₄⁺ uptake of plants (Chen et al., 2021) and the antagonistic interaction between K and Ca (Haghshenas, Arshad & Nazarideljou, 2018). Further study should consider using the isotope tracing method to reveal the specific sources of elements and determine the influence of elements on each other.

Water use efficiency in herbaceous plants and woody plants

Drought stress on plants can greatly affect their growth, yield, and grain quality (Seleiman et al., 2021a). Though the precipitation of 1,400 mm is in the study area, water resources in Karst regions are still limited due to shallow soil and high permeability of carbonate rocks, resulting in plants facing drought stress when growing (Liu et al., 2021). The leaf δ¹³C (from −25.3‰ to −30.41‰) of all plants is higher than typical subtropical species with leaf δ¹³C from −31.1‰ to 30.5‰ (Qu et al., 2001), showing plants suffer more drought effect in this area. Under long-term water shortage, plants will employ several strategies in response to drought stress, such as elevating cell osmotic potential or adjusting leaf stomatal conductance (Seleiman et al., 2021a; Si et al., 2015; Vodnik et al., 2019). K plays a critical role in the above functions since K is primarily delivered passively in plant organs through the xylem flow. Regarding leaf cells, guard cells acquire K⁺ and have a higher concentration than nearby cells, and K⁺ outflow causes turgor loss in guard cells (Misra, Reichman & Chen, 2019). Turgor loss in the paired guard cells closes the stomatal gap, which controls water and gases (Melkikh & Sutormina, 2022). Previous studies found a positive relationship between leaf K and WUE (Cao et al., 2009; Zhao et al., 2007), which means high leaf K levels can increase stomatal sensitivity to water stress and lower stomatal conductance (representing higher δ¹³C) (Zhou et al., 2013). However, no significant correlation between leaf K and WUE occurred in the Karst region of this study. On the contrary, WUE showed a strong positive correlation with leaf K:Ca ratio in herbaceous plants (Fig. 4A), implying the effects of Ca on the WUE. Meanwhile, there was a strong correlation between leaf K:Ca raito and leaf Ca content (Fig. 4B), indicating that Ca content mainly controlled the ratio of K and Ca. Similarly, K:Mg ratio showed a strong correlation with WUE and Mg content in the woody plants (Figs. 4C and 4D), indicating the impact of leaf Mg on WUE and the ratio of K and Mg. Under high calcium stress, a large amount of Ca²⁺ influx inhibited the outflow of H⁺ by reducing the activity of plasma membranous H⁺-ATP-ase, which further affected plant stomata, photosynthesis, and transpiration (Sukhova, Akinchits & Sukhov, 2017). Mg plays an essential role in photosynthesis as the central atom of the chlorophyll molecule (Pokharel et al., 2018). Furthermore, the antagonistic effects of Ca and Mg on K in plants have been widely reported (Haghshenas, Arshad & Nazarideljou, 2018; Zhou et al., 2013). Therefore, WUE was affected by the joint influence of leaf Ca, Mg, and K content under high Ca and Mg stress in the Karst region. Additionally, the WUE of herbaceous plants was more sensitive to Ca, whereas the WUE of woody plants was more sensitive to Mg.