Water use efficiency (WUE), defined as the ratio of carbon assimilation to water loss, serves as a fundamental metric for evaluating the resilience of terrestrial ecosystems to climate change (Franks et al., 2015; Li et al., 2023). In terrestrial ecosystem, understanding how plants balance carbon gain against water loss is critical for predicting future carbon sequestration and hydrological feedbacks (Farooq et al., 2019; Migliavacca et al., 2021). Traditional methods for measuring WUE, such as gas exchange measurements, are often limited in temporal and spatial scalability (Franks et al., 2015). The analysis of stable carbon isotope composition (δ¹³C) in plant tissues has emerged as a powerful, integrative tool to quantify intrinsic water use efficiency (iWUE) over the time scale of plant growth, as δ¹³C discrimination during photosynthesis is linked to the interplay between stomatal conductance (g_s) and photosynthetic capacity (A) (Maxwell et al., 2018; Rumman et al., 2018). This dual-isotope approach provides a novel tool to decode plant eco-physiological strategies (Eitelberg et al., 2025). To robustly generalize the patterns and drivers underlying the variation of δ¹³C, investigations spanning extensive natural environmental gradients are essential. Altitudinal transects, which provide dramatic and co-varying gradients in temperature, moisture, and atmospheric pressure over relatively short distances, serve as a unique and powerful “natural laboratory” for exploring how plant functional traits and adaptive strategies respond to multivariate environmental changes (Körner, 2007).

The iWUE of plants is fundamentally shaped by the interaction between stomatal conductance and photosynthetic capacity, both of which are highly sensitive to climatic drivers (Brümmer et al., 2012; Huang et al., 2016). Among these factors, vapor pressure deficit (VPD) is a key driver of stomatal behavior (Yuan et al., 2025). As air dryness increases with elevation rising, the gradient driving water vapor diffusion from leaves to the atmosphere intensifies, potentially leading to stomatal closure to prevent excessive water loss (Mathias and Thomas, 2021; Li et al., 2023). This phenomenon has been observed in various grass species, where elevated VPD conditions during growth periods were found to increase bundle-sheath leakiness and intrinsic water use efficiency (Guerrieri et al., 2019; Hatfield and Dold, 2019). Temperature is another critical climatic factor that controls WUE through its direct effects on metabolic rates and stomatal kinetics (Sage and Kubien, 2007; Koehler et al., 2010). Rising temperatures generally accelerate leaf respiration and transpiration rates, which can reduce WUE if photosynthesis does not keep pace (Hatfield and Dold, 2019; Xu et al., 2023). In mountainous regions, the combined effects of temperature and VPD create a dynamic environment where plants must rapidly adjust their stomatal aperture to maintain optimal hydration while maximizing carbon uptake (Liu et al., 2022; Bai et al., 2025). Nevertheless, the counteracting effects of increasing VPD in response to global warming poses a significant threat to the long-term stability of iWUE in many terrestrial biomes (Zhan et al., 2025). Therefore, understanding how these climatic variables interact to regulate iWUE is key to accurately modeling carbon-water coupling in grassland ecosystems.

Beyond climatic drivers, the internal physiological traits of plants, as reflected by their nutrient’s composition (plant stoichiometry), plays a crucial role in regulating iWUE (Cernusak et al., 2010; Yan et al., 2016). The allocation of nutrients such as nitrogen (N) and phosphorus (P) directly affects the activity of photosynthetic enzymes and the structure of leaf tissues, thereby influencing the iWUE (El-Madany et al., 2021). For instance, nitrogen content is closely linked to the maximum rate of carboxylation (V_cmax) and the capacity for Rubisco regeneration, both of which are key determinants of photosynthetic efficiency and stomatal conductance (Dawson et al., 2022). In grasslands, the nutrients availability often dictates the competitive ability of different functional groups, influencing their dominance along altitudinal gradients and consequently shaping the overall ecosystem iWUE (Bai et al., 2025; Karitter et al., 2025). Moreover, soil properties also constrain iWUE by regulating root water uptake and the hydraulic conductivity of the soil-plant-atmosphere continuum (SPAC) (Orlowski et al., 2023). Soil texture, organic matter content, and water holding capacity determine the availability of soil moisture, which acts as an important limiting factor for stomatal behaviors (Sun et al., 2023). Thus, a comprehensive understanding of iWUE should integrate both the external climatic drivers and the internal physiological constraints imposed by plant stoichiometry and soil properties.

Altitude gradients serve as a natural laboratory, where temperature, precipitation, and VPD vary systematically over short distances, which offers a unique way to study the adaptive strategies of grassland plants (Wu et al., 2015; Midolo et al., 2019). In particular, the study of iWUE along altitudinal gradients provides critical insights into the evolutionary and physiological mechanisms that enable plants to survive in extreme environments. Therefore, integrating altitudinal studies with stable isotope analysis provides a powerful approach to reveal the complex interactions between plant physiology, soil properties, and climate drivers that govern iWUE in terrestrial ecosystems. Thus, our study was aim to address two questions: First, how does the iWUE of different photosynthetic pathways (C₃ vs C₄) vary along an elevation gradient? Second, how do the integrated effects of climatic, plant, and soil factors influence these altitudinal patterns of iWUE in grasslands?

Our study reveals the altitude-adaptive patterns of iWUE between C₃ and C₄ grasses along an elevational transect. While iWUE increased significantly with elevation for C₃ species, it exhibited a slight but significant decrease for C₄ species (Figure 2). This core finding, derived from stable carbon (δ¹³C) and oxygen (δ¹⁸O) isotopes, highlights the distinct eco-physiological strategies and mechanisms that how plants adapt to co-varying environmental gradients. The application of a machine learning framework (XGBoost-SHAP) identified climate variables (VPD, MAT), and plant-soil stoichiometric traits (leaf N:P, leaf C:P, soil C:N) as the key drivers of these adaptive patterns (Figures 3 and 4). Notably, the direction and magnitude of the effects exerted by these drivers differed substantially between the two functional plant types.

The opposing iWUE responses to elevation are rooted in the fundamental physiological differences between C₃ and C₄ photosynthesis pathway. The C₄ pathway, with its CO₂-concentrating mechanism (CCM), confers an inherent advantage in water-use efficiency by enabling high rates of carbon fixation at lower stomatal conductance (g_s) (Ehleringer and Cerling, 2001; Way et al., 2014). This is consistently reflected in our data by the significantly less negative (enriched) leaf δ¹³C values in C₄ grasses along the elevational gradient (Figure 2). The classical model of carbon isotope discrimination (Δ) suggests that leaf δ¹³C is related to the ratio of intercellular to atmospheric CO₂ concentration (c_i/c_a), which is inversely correlated with iWUE (Farquhar et al., 1982). For C₃ plants, a reduction in c_i/c_a directly leads to higher iWUE. This pattern is commonly observed in mountain ecosystems and is often attributed to stomatal limitation under cooler temperatures (Körner, 2007; Maxwell et al., 2018). In contrast, the slight decline in C₄ iWUE with increasing elevation is interesting. It suggests that the C₄ advantage is constrained under the cold conditions of high altitudes (Figure 2). At higher elevations, lower temperatures can inhibit the activity of key C₄ enzymes like PEP carboxylase, which is crucial for initial CO₂ fixation (Carvalho et al., 2024). This reduced enzymatic efficiency weakens the CO₂ concentrating rate, potentially lowering water use efficiency (Carvalho et al., 2024). Meanwhile, cold stress may disrupt the structural integrity of the specialized Kranz anatomy. It can also weaken the efficiency of metabolite shuttling between mesophyll and bundle sheath cells (Sage et al., 2011). These combined biochemical and anatomical constraints explain the observed decline in C₄ plant WUE along elevation gradients (Sage and Kubien, 2007; Way et al., 2014). Consequently, while stomatal conductance may still decrease with elevation (as suggested by rising δ¹³C), the potential parallel decline in photosynthetic rate might be proportionally greater, leading to a net decrease in iWUE. This finding highlights that the higher iWUE of C₄ plants is context-dependent and can be diminished by environmental factors. However, it should be noted that this study used the fixed φ value to calculate the iWUE of C₄ plants. This simplification may introduce a systematic bias in absolute iWUE estimates across the elevation gradient. Nevertheless, the fixed φ value represents a necessary compromise, as deriving species- and site-specific leakiness under field conditions remains unfeasible. Additionally, φ variation in response to environmental drivers (e.g., light and temperature) is often limited to moderate ranges (Kromdijk et al., 2016). Consequently, the relative C₄ iWUE trends and their ecological interpretation are likely robust, though absolute values should be interpreted with appropriate caution.

Our analysis identified VPD as a key climatic driver for both photosynthesis pathways, but with diametrically opposite effects: a strong negative correlation with iWUE in C₃ grasses and a strong positive correlation in C₄ grasses (Figures 3 and 4). This contrast highlights differential stomatal regulation strategies along the elevation rising. For C₃ plants, high VPD primarily triggers stomatal closure to prevent water loss (Wang et al., 2012; Li et al., 2024). However, long-term water stress can lead to significant non-stomatal limitations, such as reduced mesophyll conductance (the ease of CO₂ diffusion to chloroplasts) and impaired biochemical capacity for carboxylation, which further constrain photosynthesis independently of stomatal aperture (Gimeno et al., 2019; Chen et al., 2025). This aligns with observations in some arid systems where iWUE increases or declines under severe drought stress (Cooley et al., 2022; Huang and Zhai, 2023). For C₄ plants, their CO₂-concentrating mechanism (CCM) provides a critical advantage (Zait et al., 2024). It actively elevates CO₂ concentration around the enzyme Rubisco. Therefore, when stomata close in response to high VPD to conserve water, the internal CO₂ reservoir helps sustain photosynthetic rates for longer periods compared to C₃ plants (Márquez et al., 2024). Notably, recent evidence shows that C₄ plants also employ non-stomatal control under VPD stress, which further limits water loss while helping maintain a favorable CO₂ fixation (Gan and Sage, 2024; Márquez et al., 2024). In summary, under high VPD, C₃ plants face a challenge where stomatal closure is rapidly followed by non-stomatal metabolic limitations. In contrast, C₄ plants are more resilient; their CCM buffers the carbon supply against stomatal closure.

The prominent role of plant and soil stoichiometry highlights the tight coupling between plant water-use strategies and nutrient economics (Wright et al., 2004; Du et al., 2021). The positive relationships between iWUE and leaf C:P and N:P ratios in both pathways suggest a coordinated shift towards a more conservative resource-use strategy. High leaf C:P ratios are indicative of greater investment in structural carbon (e.g., cell walls, fibers) or storage compounds relative to metabolically active nutrient pools (e.g., phosphorus) (Xing et al., 2021). This is consistent with the “Leaf Economics Spectrum,” where species characterized by slow growth rates, longer leaf lifespans, and higher tissue density typically exhibit elevated iWUE (Reich, 2014; Luo et al., 2018). The positive effect of N:P ratio further implies that phosphorus limitation, or a shift towards a more higher P-efficient use traits (e.g., thicker leaves, lower g_s) that enhance iWUE (Pinto et al., 2016; Wang and Wen, 2022). P limitation can directly constrain photosynthetic metabolism by limiting ATP synthesis and RuBP regeneration, which drives a coordination between nutrient use and water use strategies (Kumar et al., 2021; Odoom and Ofosu, 2024). When non-stomatal limitations (e.g.,leaf anatomy or biochemistry) intensify due to phosphorus scarcity, plants may strategically reduce stomatal conductance to conserve water—given the limited potential for additional carbon gain (Dijkstra et al., 2016). In terms of nitrogen limitation, C₄ plants generally have higher nitrogen use efficiency compared to C₃ plants (Bai et al., 2025). Under lower N conditions, the priority of C₄ plants tend be to allocate the limited N to maintain the complex CCM unit (e.g., bundle sheath structure) and associated high metabolic rates (Ghannoum et al., 2011). This allocation might come at the cost of optimal stomatal control or leaf hydraulic architecture, potentially reducing iWUE (Togawa and Ueno, 2022). Furthermore, the opposite responses of C₃ and C₄ iWUE to soil C:N ratio is a compelling result (Figures 4 and 5). For C₃ grasses, the positive correlation between iWUE and soil C:N ratio indicates that plants adopt a conservative strategy under low nitrogen availability (high soil C:N) (Figure 4). Specifically, they tend to invest in long-lived tissues with slow gas exchange, which inherently leads to higher iWUE (Figure 4). This divergence underscores that the integration of nutrient and water economies is pathway-specific.

These findings have significant implications for predicting grassland responses to global change. Under a warming climate (increased MAT and VPD), low-elevation C₃ grasses may face intensified environmental pressure due to decreased iWUE. C₄ grasses might expand their advantage in areas where rising VPD is the dominant change, but their performance will be modulated by temperature and nutrient dynamics. Furthermore, atmospheric nitrogen deposition, which reduces soil C:N, may exert differential effects on grassland community composition. Specifically, it could favor C₃ grasses by mitigating the limitation imposed by decreased iWUE or benefit C₄ grasses by alleviating constraints on their iWUE, with the specific outcome dependent on environmental contexts. Future research should aim to mechanistically link the stoichiometric signals to key specific leaf traits (e.g., leaf mass per area, hydraulic conductance) and microbial processes governing nutrient cycling. Integrating these relationships into dynamic vegetation models will improve forecasts of ecosystem carbon-water fluxes in montane grasslands under changing climate.

This study demonstrates a fundamental divergence in how C₃ and C₄ grasses regulate their iWUE along an elevational gradient. We found that iWUE increased with elevation for C₃ species but slightly decreased for C₄ species. These contrasting patterns are driven by the integrated effects of climate and plant-soil stoichiometry, operating through pathway-specific mechanisms. The opposing responses of iWUE to VPD—negative for C₃ and positive for C₄ grasses—and to soil C:N ratio, highlighting distinct strategies in coupling carbon, water, and nutrient economies. The results highlight that the iWUE of C₄ plants is context-dependent, which may be constrained by lower temperatures in high-elevation environments. In contrast, C₃ plants enhance their iWUE under the cooler and nutrient-limited conditions with a conservative resource-use strategies. This research advances our understanding of plant adaptive strategies by integrating stable isotope ecology with stoichiometric theory, revealing that iWUE patterns are not simply climate-driven but emerge from multi-factor interactions specific to photosynthetic pathway. The findings have critical implications for predicting shifts in grassland community structure and ecosystem function under future climate change, particularly where rising VPD, global warming, nitrogen deposition. Integrating these relationships into dynamic vegetation models will improve forecasts of ecosystem carbon-water fluxes in montane grasslands under changing climates.