Greater Biomass Production Under Elevated CO₂ Is Attributed to Physiological Optimality, Trade-Offs in Nutrient Allocation, and Oxidative Defense in Drought-Stressed Mulberry

Abstract

Mulberry (Morus alba L.), a species of significant ecological and economic importance, is widely cultivated for sericulture, soil conservation, and environmental restoration. Despite its remarkable resilience to environmental stresses, the combined impact of elevated CO₂ (eCO₂) and drought stress on aboveground–root–soil interactions remains poorly understood, particularly in the context of global climate change. Here, we investigated the effects of eCO₂ and drought on physiological leaf and root indicators, nutrient absorption and allocation, and soil properties in mulberry seedlings. Mulberry seedlings were grown in environmentally auto-controlled growth chambers under ambient CO₂ (420/470 ppm, day/night) or eCO₂ (710/760 ppm) and well-watered (75–85% soil relative water content, RWC), moderate-drought (55–65% RWC), or severe-drought (35–45% RWC) conditions. Results showed that both above- and below-ground plant biomass production were significantly promoted by eCO₂, particularly by 36% and 15% under severe drought, respectively. This could be attributed to several factors. Firstly, eCO₂ improved leaf photosynthesis by 25–37% and water use efficiency by 104–163% under drought stresses while reducing negative effects of drought on the effective quantum yield of PSII photochemistry and the photochemical quenching coefficient. Secondly, eCO₂ significantly decreased proline accumulation while increasing soluble sugar contents, as well as peroxidase and superoxide dismutase activities, in both leaves and roots under drought stress. Lastly, eCO₂ promoted soil sucrase, urease, and phosphatase activities, as well as plant nitrogen, phosphorus and potassium uptake while facilitating their allocation into roots under drought stress. These findings demonstrate that eCO₂ enhanced the drought tolerance of mulberry plants through improvements in photosystem II efficiency, water use efficiency, antioxidative defense capacity, and nutrient uptake and allocation, providing critical insights for sustainable mulberry plantation management under future climate change scenarios.

1. Introduction

Ambient CO₂ (aCO₂) concentrations have increased from 280 ppm at the beginning of the industrial revolution to the current ~420 ppm, primarily due to anthropogenic activities such as extensive utilization of fossil fuels, deforestation, etc. According to the IPCC (2018), aCO₂ could potentially reach to 550 ppm or higher by the end of this century, with significant implications for global climate patterns and plant responses [1]. Elevated CO₂ (eCO₂) contributes to temperature increases through the greenhouse effect, inducing spatially heterogeneous precipitation frequencies and patterns on a global scale, eventually leading to regional droughts under future climate change [2,3]. Additionally, eCO₂ is commonly practiced in controlled environments such as greenhouses to enhance plant growth and productivity, making it relevant for both natural and agricultural systems. Drought stress typically affects various cellular and physiological processes, such as protein degradation, changes in membrane fluidity, and the inhibition of leaf photosynthesis, ultimately resulting in a decrease in crop yield by 25–30% and deteriorated quality [4,5,6]. However, it is worth noting that the growth-stimulating effect of eCO₂ can partially offset the impacts caused by drought stress, particularly in C₃ species [7,8,9]. Therefore, it is crucial to consider the abovementioned effects when assessing the overall impact of climate change on plant growth.

To comprehend the potential mechanism of agricultural production in response to eCO₂ and drought stress, it is imperative to investigate alterations in the physiological processes and biochemical components of plants that are sensitive to both eCO₂ and drought stress, such as leaf photosynthesis, chlorophyll fluorescence, soluble sugars, proline, and antioxidative ability [9,10,11,12]. Plant photosynthesis is a drought-sensitive process, and water deficit usually leads to a significant decline in leaf photosynthesis [5,10,13]. The decrease in leaf photosynthesis under water deficit may be associated with reduced stomatal conductance [14], nitrogen (N) and chlorophyll contents [15], and 1,5-bisphophate carboxylase/oxygenase (Rubisco) activity [16]. It has been observed that drought stress decreases the effective quantum yield of PSII photochemistry in wheat while also causing increased accumulation of proline and soluble sugar contents in barley and buckwheat [17,18]. Additionally, antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were found to be highly activated in soybean under drought stress in order to adapt and scavenge the free radicals induced by drought stress, accompanied by increased accumulation of malondialdehyde (MDA) [19,20]. Therefore, drought stress limits crop growth and grain yield by reducing aboveground biomass and modifying the shoot-to-root ratio through the development of deeper roots capable of acquiring more water [21].

The increase in atmospheric CO₂ concentration, the primary C source for plants, exerts significant CO₂ fertilization effects on crops [22,23,24] because the Rubisco enzyme in C₃ plants is not saturated at the current atmospheric CO₂ concentration [25,26]; thus, eCO₂ can stimulate leaf photosynthesis and enhance plant growth and grain yield by facilitating C₃ the uptake of more CO₂ molecules by plants [27]. Previous studies have revealed that eCO₂ can mitigate the adverse effects of drought stress on plant growth and physiological functioning by increasing the intercellular CO₂ concentration (Ci), improving water use efficiency (WUE) while reducing transpiration rates [28,29,30,31]. A meta-analysis showed that eCO₂ significantly increases both the net photosynthetic rate (37.2–46.3%) and WUE (84.2–88.9%) [9] while decreasing evapotranspiration by ~10% [32]. The increased WUE under eCO₂ implies that plants may exhibit tolerance to future high CO₂ concentrations under drought conditions [8,33]. Moreover, enhanced photosynthesis under eCO₂ leads to an accumulation of carbohydrates under drought stress, resulting in increased cell osmotic potential and promoting osmotic adjustment [34]. Concurrently, the formation of reactive oxygen species (ROS) was also found to be mitigated under eCO₂ due to increased electron consumption in C fixation processes [7,35]. However, the capacity of the CO₂ fertilization effect to alleviate drought stress may vary not only among different plant species [36,37] but also depending on the levels of water deficit [30,38]. For instance, some studies have reported that eCO₂ can effectively mitigate drought stress by enhancing WUE and photosynthesis in certain species, such as coffee [21] and soybean [19,33]. In contrast, other investigations have revealed that eCO₂ did not significantly improve drought tolerance or even exacerbated water stress in species like wheat and sorghum, particularly under severe drought conditions [39,40]. These inconsistent findings may be attributed to differences in species-specific traits, experimental conditions, and the severity of the applied drought stress. For example, C₄ plants, which have a different photosynthetic pathway, may not benefit as much from eCO₂ as C₃ plants under drought stress [41,42]. Thus, there is currently no consistency regarding the specific physiological mechanisms by which the eCO₂ concentration can affect plant growth and crop yield under drought stress.

In addition to its impact on plant physiology, drought also significantly affects the uptake, accumulation, and distribution of soil nutrients (particularly nitrogen (N), phosphorus (P), and potassium (K)). A recent meta-analysis revealed that drought stress generally decreases nutrient absorption from soil and leads to decreased N and P concentrations in plant tissue [43,44,45]. In contrast, eCO₂ can alter the shoot-to-root ratio by stimulating deeper root growth, thereby enabling better water and nutrients uptake [9,30]. In general, plants under eCO₂ store more C in their above- and below-ground biomass compared to those under aCO₂ [35], leading to changes in their contents of N, P, and K and other nutrients in both plants and soil [46]. Meanwhile, eCO₂ increases fluxes of organic compounds into soil due to a higher rate of plant litterfall and rhizodeposition, resulting in changes in soil organic carbon (SOC) content [47], the availability of soil nutrients [44], composition and activities of the microbial community [48,49], and activities of soil enzymes involved in C and N cycling [47,50]. Such changes in soil are predicted to result in stronger but uncertain feedback at the ecosystem level to eCO₂. However, most studies have explored the individual effects of focusing on either eCO₂ or drought stress on a plant–soil–microbe system, with limited studies addressing their combined effects on plant nutrient distribution and accumulation, soil nutrient availability, and enzyme activity.

Mulberry (Morus alba L.), a rapidly growing multipurpose cash plant, is extensively cultivated in southwest China, accounting for approximately 70% of China’s total plantations (~758,200 hectares) due to its significant economic value. However, this region is currently experiencing reduced precipitation and increased aridity, leading to a scarcity of freshwater resources for agricultural production [51]. These climatic changes pose a significant threat to mulberry cultivation, especially during prolonged drought periods [10,52]. Despite its sensitivity to extreme drought, mulberry exhibits remarkable adaptability to moderate environmental stressors, making it an ideal model for studying climate change adaptation [53,54]. Its high plasticity, deep root system, and efficient water use mechanisms enable it to thrive in arid and semi-arid regions, positioning it as a critical crop for sustainable agriculture in water-limited environments [55,56]. Additionally, mulberry leaves are the primary food source for silkworms, which are integral to the sericulture industry, a major economic driver in many developing regions [57]. The plant’s ability to produce high levels of biomass under moderate stress conditions, coupled with its role in carbon sequestration, further underscores its importance in mitigating climate change impacts [58]. Although studies have reported the morphological, physiological, and biochemical responses of mulberry plants to drought stress [10,52,59,60], most of these studies primarily focused on different mulberry cultivars and/or altered soil–water gradients without considering the potential effect of CO₂. We previously observed a significant 33–40% increase in leaf biomass production of mulberry when the CO₂ concentration was elevated from 410 to 710 ppm [61,62]. Leaf photosynthesis and WUE were enhanced by 550–800 ppm eCO₂ in various mulberry cultivars, including Selection-13, Kanva-2, ‘Qinglong’ mulberry, Gui-sang-you 62, Qiangsang-1, and Nongsang-14, under well-watered conditions [61,62,63,64,65]. However, understanding regarding how eCO₂ affects drought-induced changes in photosynthesis, PSII function, antioxidant defense mechanisms, and nutrient acquisition and partitioning, as well as soil enzyme activities. By understanding how mulberry responds to combined drought and eCO₂ conditions, we can develop strategies to enhance its resilience and productivity, thereby supporting both economic and environmental sustainability. Here, we hypothesized that eCO₂ might have a positive impact on mulberry performance under drought conditions. Specifically, the objectives were to investigate (1) the combined effects of eCO₂ and drought stress on mulberry biomass accumulation; (2) the correlations between drought tolerance, physiological traits, and nutrient acquisition under eCO₂; and (3) the potential mechanisms through which eCO₂ could mitigate negative effects induced by drought stress via alterations in photosynthetic characteristics, antioxidant defense systems, nutrient uptake/distribution, and soil enzyme activities. Therefore, the following physiological traits of mulberry were investigated: leaf gas exchange, PSII efficiency, CAT, POD, SOD, MAD, and contents of proline and soluble sugar at both the leaf and root levels. Meanwhile, the uptake and partitioning of N, P, and K, along with soil enzyme activity. The anticipated findings will contribute to the development of effective water management strategies by elucidating the physiological responses of plants to future drought stress induced by elevated atmospheric CO₂ levels.

2. Materials and Methods

2.1. Growth Chamber Experiments

This experiment was conducted in 12 environmentally controlled plant growth chambers that were located at the National Monitoring Base for Purple Soil Fertility and Fertilizer Efficiency (29°48′ N, 106°24′ E, 266.3 m above the sea level) on the campus of Southwest University, Chongqing, China. The rectangular structure of each chamber measures 1.5 × 1.0 × 2.5 m and is supported by a steel frame suspended 50 cm above a cement floor. The bottom floor of the growth chamber consists of polyvinyl chloride panels, while the four side walls and top roof are lined with by tempered glass (10 mm thick) with transparency allowing approximately 90% of natural sunlight to pass through. Humidity and temperature inside and outside the growth chamber were automatically maintained within ±0.5 °C air temperature and ±5% humidity using electronic control systems. When the humidity inside a chamber was higher than that outside, the inside air was pumped out using pump controlled by a mini-computer and filtered with solid anhydrous calcium chloride. The temperature was automatically maintained at 0.5 °C variation between inside and outside using an air conditioner controlled by a mini-computer. To maintain a targeted CO₂ concentration (±30 ppm) inside each chamber, 90% pure CO₂ gas was injected using a solenoid valve controlled by a mini-computer [42].

The experiment was conducted using a split-plot design consisting two factors (CO₂ and water or drought), with CO₂ as the main factor and drought as the sub-factor. Based on previously observed daytime and night-time atmospheric CO₂ concentrations at the study site, two different CO₂ concentrations were applied: ambient CO₂ (aCO₂, 420 ppm daytime/470 ppm nighttime) for six growth chambers and eCO₂ (710 ppm daytime/760 ppm nighttime) for other six growth chambers. Daytime was defined as 07:00 a.m. to 19:00 p.m., while nighttime was defined 19:00 p.m. to 07:00 a.m. The experimental designs and the procedures are illustrated in Figure 1.

One-year-old, uniformly growing mulberry seedlings (Morus multicaulis Perr. QiangSang-1) were used as experimental materials. Two seedlings were planted in a plastic pot (32 cm in diameter and 20 cm in height). The pots were filled with 11 kg air-dried soil (Eutric Regosol, FAO Soil Classification System). The soil was sieved (2 mm) and had a pH of 6.8, organic carbon content of 7.03 g kg⁻¹, available N content of 31.21 mg kg⁻¹, available P content of 11.47 mg kg⁻¹, and available K content of 127 mg kg⁻¹. Three pots were placed into each growth chamber. Mulberry seedlings were transplanted on 16 April 2021. During the first four months, all of the seedlings were regularly watered two or three times per week with 400 mL of tap water for each pot so that soil relative water content (RWC) was maintained at 75–85%. Meanwhile, 2.2 g N, 0.77 g P₂O₅, and 1.1 g K₂O were applied per pot to meet the plants’ nutrient requirements.

After the four-month experimental period, three pots from each CO₂ chamber were subjected to three different water treatments: well-watered (WW), moderate drought stress (MS), and severe drought stress (SS). Under the well-watered condition, plants were consistently irrigated as described above to maintain an RWC of 75–85%. Plants experiencing moderate drought stress received irrigation twice weekly with 200 mL of water per pot in order to maintain an RWC of 55–65%. Plants exposed to severe stress were irrigated twice weekly with 100 mL of water per pot to maintain an RWC of 35–45%. Soil volumetric water contents were measured using a soil moisture sensor (JXBS-3001, Qingdao, China), then converted to gravimetric water contents. Soil relative water content, which was calculated as the ratio of gravimetric water to saturated moisture content. These three water treatments were performed in each chamber; thus, there were six replicated chambers and six replicate pots for each CO₂ concentration treatment. Additionally, pots were relocated among different chambers once fortnightly to minimize any potential effect of environmental variations across different growth chambers.

2.2. Determination of Photosynthetic Parameters

Measurements of photosynthetic parameters were conducted prior to harvest between 9:00 and 11:00 a.m. on sunny days, specifically on 25, 30 September and 5 October 2021 (data averaged for these three days). The third fully expanded leaf from the top was selected for determination of the net photosynthetic rate (Pn), stomatal conductance (gs), transpiration rate (E), and intercellular CO₂ concentration (Ci) using a Li-6800 portable photosynthesis system (LICOR, Lincoln, NE, USA). Throughout the measurement, the saturated photosynthetically active radiation was maintained at 1600 μmolm⁻² s ⁻¹ using an integrated LED light source (Model 6800-01A, LI-COR Inc.) within the leaf chamber (Model 6800-02A Large Leaf Chamber, LI-COR Inc.). This light intensity was selected based on preliminary light-response curve experiments, which confirmed that it was sufficient to achieve light-saturated photosynthetic rates in the studied mulberry leaves [64]. The LED source provides uniform light distribution and stable intensity, ensuring accurate and reproducible measurements. Meanwhile, the leaf temperature in the leaf chamber was set at 25 ± 1 °C. For plants growing under eCO₂ and aCO₂, the leaf cavity’s CO₂ concentration was maintained at 700 ppm and 420 ppm, respectively. The vapor-pressure deficit at the leaf surface was consistently maintained at 1.2 ± 0.2 kPa. Water use efficiency (WUE) was calculated as the ratio of Pn to E. The intercellular CO₂ and ambient CO₂ concentrations were used to the calculate Ci/Ca ratio.

2.3. Determination of Chlorophyll Fluorescence

The chlorophyll fluorescence was also determined using the Li-6800 fluorescence leaf chamber (LI-COR, Lincoln, USA) connected to an Li-6800 portable photosynthesis system on the same leaves used for photosynthetic leaf gas exchange measurements on 25 and 30 September and 5 October 2021 (data averaged for these three days). To ensure that all photosystem-II (PS-II) reaction centers were open, the leaves were wrapped with tin foil and darkened for 30 min. The following parameters were measured: initial fluorescence yield (F0) and maximum fluorescence yield (Fm) in the dark-adapted stage, minimum fluorescence yield (F0’) and maximum fluorescence (Fm′) in the light-adapted stage, and steady-stage fluorescence yield (Fs). Subsequently, the effective quantum yield of PSII photochemistry (Φ_PSII), maximal quantum yield of PSII photochemistry (Fv/Fm), actual photosynthetic efficiency of PSII under illumination (Fv′/Fm′), photochemical quenching coefficient (qP), non-photochemical quenching coefficient (NPQ), and PSII electron transport rate (ETR) were calculated using the aforementioned measured parameters. The calculation formulas are expressed as follows: Φ_PSII = (Fm′ − Fs)/Fm′, NPQ = (Fm − Fm′)/Fm′, qP = (Fm′ − Fs)/(Fm′ − F0’), and ETR = 0.8 × Φ_PSII × PPFD [66].

2.4. Plant and Soil Sampling

The plants were harvested on 13 October 2021, and the leaves used to measure photosynthetic parameters were collected, immediately frozen in liquid nitrogen, and subsequently stored in a −80 °C freezer for determination of enzyme activity and osmomodulators. The plant tissues were divided into leaves, stems, and roots. The fresh roots were thoroughly washed with tap water, followed by rinsing with deionized water. A portion of the fresh roots was immediately frozen in liquid nitrogen after collection and stored at −80 °C until analysis of enzyme activity and osmomodulators. The remaining portion of the roots, leaves, and stems was dried at 105 °C for 30 min initially, then further dried at 75 °C for ≥ 48 h until reaching a constant weight to determine the biomass and N, P, K, etc. Soil samples from each growth pot were collected after thorough mixing and removal of debris and fine roots and subsequently air-dried (2 mm sieved) to determine their chemical properties and enzyme activities.

2.5. Determination of N, P, and K in Plants and Soils

The dried leaf, stem, and roots of each sample were finely ground into a powder before being subjected to digestion with 98% sulfuric acid and 30% hydrogen peroxide. Tissue N, P, and K concentrations were determined by the trace Kjeldahl method, vanadium molybdate yellow colorimetry, and flame photometry [67], respectively. Soil available N (AN) was measured using the micro-diffusion technique after alkaline hydrolysis. Soil available P (AP) was extracted with 0.5 M NaHCO₃, followed by measurement using the Mo-Sb anti spectrophotometric method. Soil available K (AK) was extracted with 1.0 M ammonium acetate, then determined by flame photometry [67].

2.6. Determination of Osmomodulators

The soluble sugars were determined using anthrone colorimetry [57]. Briefly, 0.5 g of fresh samples was extracted with 10 mL H₂O in boiling water for 20 min. The extraction was mixed with 0.5 mL of anthrone ethyl acetate and 5 mL of sulfuric acid, followed by incubation in boiling water for 10 min. The absorbance was measured spectrophotometrically at a wavelength of 620 nm.

The determination of proline was conducted using the sulfosalicylic acid method [68]. Briefly, 0.5 g of fresh sample was extracted with 5 mL of 3% sulfosalicylic acid in boiling water for 10 min. Then, 2 mL of filtrate was mixed with 2 mL glacial acetic acid and 2 mL of acid ninhydrin. The mixture was then subjected to a boiling water bath for 30 min. After cooling, 4 mL toluene was added into the mixture. The absorbance was measured at a wavelength of 520 nm.

MDA was determined using the thiobarbituric acid (TAB) method [68]. Briefly, 0.5 g of fresh sample was homogenized with 5 mL of 5% TAB solution. Then, 2 mL of the extracted solution was mixed with 2 mL of 0.67% TAB solution and boiled for 15 min. The absorbance of the incubated supernatant was spectrophotometrically measured at wavelengths of 450 nm, 532 nm, and 600 nm.

2.7. Determination of Enzyme Activity in Leaves and Roots

The activity of POD was determined using the guaiacol method and expressed in terms of enzyme units per gram fresh weight (U g⁻¹ FW) [68]. Briefly, 0.5 g of fresh sample was extracted with 10 mL of 0.1 mM phosphate buffer (PBS, pH 6.0), followed by centrifugation at 4000 rpm for 15 min. The supernatant was collected in a 100 mL volumetric flask. Subsequently, the reaction mixture contained 1 mL of enzyme extraction, 2.5 mL guaiacol (0.1 M), and 0.5 mL 30% H₂O₂. The change in absorbance at 470 nm, measured as 1.0 per min, was considered one unit of POD activity.

The activity of SOD was determined using the nitrogen blue tetrazolium (NBT) assay method [68]. Briefly, 0.5 g of fresh sample was extracted with 10 mL of 0.05 mM PBS (pH 7.8), then centrifuged at 10,000 rpm for 10 min. The supernatant was collected in a 100 mL volumetric flask. Subsequently, the reaction mixture contained 0.3 mL of enzyme extraction, 1.5 mL of 0.05 mM PBS, 0.3 mL of 130 mM Met solution, 0.3 mL 750 µM NBT solution, 0.3 mL 100 µM EDTA-Na₂ solution, and 0.3 mL 20 µM riboflavin. The reaction mixture was exposed to fluorescence under 4000 luxes for 20 min; then, the absorbance was measured at a wavelength of 560 nm. The SOD activity was defined as the amount of enzyme required to inhibit 50% of NBT reduction per g fresh weight (U g⁻¹ FW).

The activity of CAT was determined by potassium permanganate titration [57]. Briefly, 0.5 g of fresh sample was extracted with 10 mL of 0.2 mM PBS (pH 7.0), then centrifuged at 4000 rpm for 10 min. The supernatant was collected in a 100 mL volumetric flask. Subsequently, 5 mL of the extraction was added to 2.5 mL of 0.1 M H₂O₂ and placed in a 30 °C water bath for 10 min; then 2.5 mL of 10% sulfuric acid was added, and the mixture was titrated with 0.1 M KMnO₄ until a pink color was achieved, followed by 30 s without color change. CAT activity is expressed as mg of hydrogen peroxide decomposed per minute per g fresh weight of sample.

2.8. Determination of Soil Enzyme Activity

The soil invertase activity was determined using the 3,5-dinitrosalicylic acid colorimetry method [69]. Briefly, 1.0 g of air-dried soil was incubated with a mixture containing 0.2 of ml toluene, 3 mL of 8% (w/v) sucrose, and 1 mL of PBS (pH 5.5) at 37 °C for 24 h. After incubation, 1 mL extraction with 3 mL 3,5-dinitrosalicylic acid was incubated in boiling water for 5 min. Subsequently, the absorbance was measured at a wavelength of 508 nm. Results are expressed as mg of glucose released per g dry soil per h.

The soil urease activity was determined using sodium phenol–sodium hypochlorite colorimetry [69]. Briefly, 1.0 g of air-dried soil was incubated with 0.2 mL of toluene, 2 mL of 10% urea solution, and 4 mL of citrate buffer (pH 6.7) at 37 °C for 24 h. After incubation, the reaction mixture contained 1 mL enzyme extraction, 4 mL sodium phenol solution, and 3 mL sodium hypochlorite solution. The absorbance of the reaction solution was measured at a wavelength of 578 nm. Results are expressed as mg of NH₄⁺–N released per g soil per h.

The neutral phosphatase activity was determined using disodium benzene phosphate colorimetry [69]. Briefly, 1.0 g of air-dried soil was incubated with 0.2 mL of toluene and 5 mL of 0.5% disodium phenyl phosphate solution for 24 h at 37 °C. After incubation, 1 mL extraction with 0.5 mL of citrate buffer (pH 7.0), 1 mL of 8% potassium ferrocyanide (w/v), and 1 mL of 2% 4-aminoantipyrine were thoroughly mixed. Subsequently, the absorbance of the reaction solution was measured at a wavelength of 510 nm. Results are expressed as mg of phenol per g soil per 4 h.

2.9. Statistical Analysis

Statistical analysis was performed using SPSS 19.0 software (SPSS Inc., Chicago, IL, USA). Results (means ± SE, n = 6) between treatments were analyzed by two-way analysis of variance (ANOVA), and significant differences were compared with the Duncan multiple range test at p < 0.05 unless mentioned otherwise. Structural equation modeling (SEM) was employed in the Amos 21.0 software package to investigate potential causative relationships among measured factors (Smallwaters Corporation, Chicago, IL, USA). Figures were generated using Origin Pro 2018 software (OriginLab Corp., Northampton, MA, USA).

3. Results

3.1. Effects of eCO₂ and Drought on Photosynthetic Parameters of Leaves of Mulberry Plants

Compared to well-watered conditions, drought stress significantly decreased the net photosynthetic rates, stomatal conductance, transpiration rates, intercellular CO₂ concentration, and Ci/Ca ratio of mulberry plants, regardless of CO₂ concentrations (Figure 2). However, eCO₂ significantly mitigated these effects. Under eCO₂, the net photosynthetic rates increased by 27%, 25%, and 37% (p = 0.001, Figure 2A), while stomatal conductance decreased by 38%, 41%, and 48% (p = 0.001, Figure 2B) and leaf transpiration rates decreased by 26%, 51%, and 48% (p = 0.001, Figure 2C) under well-watered conditions, moderate drought, and severe drought stress, respectively. As a result, leaf WUE was dramatically enhanced by 96%, 104%, and 163%, respectively (p = 0.001, Figure 2E), under the same conditions. Meanwhile, mulberry plants under eCO₂ exhibited higher Ci values than those grown under aCO₂, regardless of soil water status (Figure 2D). Nevertheless, the Ci/Ca ratio was significantly lower under eCO₂ than under aCO₂ under both well-watered and moderate-drought-stress conditions (Figure 2F). Two-way ANOVA revealed significant interaction effects between eCO₂ and drought on leaf WUE (p = 0.020), Ci (p = 0.004) and Ci/Ca ratio (p = 0.001) but not on the net photosynthetic rate (p = 0.37), stomatal conductance (p = 0.78), or transpiration rate (p = 0.94) (Figure 2).

3.2. Effects of eCO₂ and Drought on Leaf Chlorophyll Fluorescence of Mulberry Plants

Compared to well-watered conditions, drought stress significantly decreased Φ_PSII and ETR under eCO₂ and aCO₂, as well as qP under aCO₂, while increasing NPQ, regardless of CO₂ concentrations (Figure 3). The qP was 21.9% lower in eCO₂ than in aCO₂ under well-watered conditions but 33.3% higher under severe drought (Figure 3C). Similarly, eCO₂ also decreased Φ_PSII by 11.1% under both well-watered and moderate-drought conditions (Figure 3E) and decreased the ETR by 27.0% and 12.9% under well-watered and severe-drought conditions, respectively (Figure 3F). Conversely, eCO₂ significantly increased NPQ by 7.1%, 4.1%, and 13.4% under well-watered, moderate-drought, and severe-drought conditions, respectively (Figure 3D). Neither Fv/Fm nor Fv′/Fm′ was affected by eCO₂ or drought stress (p > 0.05, Figure 3A,B). Two-way ANOVA revealed significant interaction effects between eCO₂ and drought on qP (p = 0.001), NPQ (p < 0.05), Φ_PSII (p < 0.05) and ETR (p = 0.001) but not on Fv/Fm (p > 0.285) or Fv′/Fm′ (p > 0. 5, Figure 3).

3.3. Effects of eCO₂ and Drought on Osmotic Regulators of Mulberry Plants

In comparison to well-watered conditions, leaf MDA concentration was substantially increased by 25.0% and 38.1% under moderate and severe stresses at aCO₂ (p < 0.05, Figure 4A), while no significant changes were observed ateCO₂ (p > 0.05, Figure 4A). Drought stress significantly increased root MDA, irrespective of CO₂ concentrations (p < 0.001, Figure 4D). In contrast, eCO₂ decreased leaf MDA by 12.4% and 13.0% under moderate and severe stresses, respectively, and root MDA by 10.2% and 13.2% under well-watered and severe stress conditions, respectively (p < 0.05, Figure 4D). Similarly, eCO₂ significantly reduced proline concentrations in leaves by 11.2%, 13.3%, and 26.7% and in roots by 9.4%, 21.2%, and 22.0% under well-watered, moderate stress, and severe stress conditions, respectively. However, both leaf and root proline concentrations were obviously increased with declining soil water under aCO₂ (p < 0.05, Figure 4B,E). Conversely, eCO₂ significantly enhanced soluble sugar concentrations in leaves by 27.1%, 15.0%, and 6.7% and in roots by 43.9%, 71.8%, and 62.3% under well-watered, moderate stress, and severe stress conditions, respectively (p < 0.05, Figure 4C,E). Two-way ANOVA results showed significant interaction effects between eCO₂ and drought on leaf soluble sugar (p = 0.03) and leaf malondialdehyde (p = 0.02) but not on root soluble sugar (p = 0.33), root malondialdehyde (p = 0.13), leaf proline (p = 0.23), or root proline (p = 0.23, Figure 4).

3.4. Effects of eCO₂ and Drought on Antioxidant Enzyme Activities in Mulberry Plants

The activities of CAT, POD, and SOD in both leaves and roots consistently increased as soil water declined under both aCO₂ and eCO₂ (Figure 5). Elevated CO₂ significantly increased CAT activity in leaves by 18.3% under well-watered conditions, and in roots by 44.5%, 188.2%, and 100.4% under well-watered, moderate-drought, and severe-drought conditions, respectively (p < 0.05, Figure 5A,D). Similarly, POD activity increased in leaves by 15.5% under severe drought, and in roots by 20.6%, 12.4%, and 10.3% under well-watered, moderate-drought, and severe-drought conditions, respectively (p < 0.05, Figure 5E). SOD activity exhibited a similar pattern, with higher activity under eCO₂ compared to under aCO₂ under well-watered and moderate-drought conditions, but no significant changes were observed under severe-drought (p < 0.05, Figure 5C,F). Two-way ANOVA results showed a significant interaction effect between eCO₂ and drought on CAT activity in leaves (p < 0.001) and roots (p = 0.003), POD activity in roots (p = 0.037), and SOD activity in leaves (p = 0.050) and roots (p < 0.001) but not on POD activity in leaves (p = 0.354) (Figure 5).

3.5. Effects of eCO₂ and Drought on Tissue N, P, and K in Mulberry Plants

Compared to well-watered conditions, drought stress significantly decreased the stem N concentration under both aCO₂ and eCO₂, as well as the root N concentration under aCO₂, but increased the leaf N concentration under aCO₂ (

Table 1. Variations in tissue nitrogen (N), phosphorus (P), and potassium (K) concentrations of mulberry under ambient (aCO₂) and elevated CO₂ (eCO₂) under well-watered, moderate-drought, and severe-drought conditions.

Variable	p-Value			CO2 Treatment	Water Treatment			Moderate Water Deficit Effect (%)	Severe Water Deficit Effect (%)
	CO2	Water	C*W		Well-Watered	Moderate Drought	Severe Drought
Leaf N concentration (mg g−1)	0.001	0.055	0.276	aCO2	34.5 ± 0.6 y,a	35.0 ± 1.1 y,a	38.0 ± 0.1 x,a	+1.3	+10.1
				eCO2	32.4 ± 0.7 x,b	31.4 ± 0.6 x,b	33.0 ± 1.4 x,b	−3.2	+1.8
				CO2 effect (%)	−6.1	−10.1	−13.0
Stem N concentration (mg g−1)	0.042	0.001	0.159	aCO2	16.2 ± 0.4 x,a	13.3 ± 0.5 y,a	13.4 ± 0.8 y,a	−17.9	−17.2
				eCO2	14.5 ± 0.4 x,b	11.3 ± 0.5 y,b	13.11 ± 0.8 x,a	−21.7	−9.6
				CO2 effect (%)	−10.5	−15.9	−2.2
Root N concentration (mg g−1)	0.308	0.526	0.045	aCO2	16.5 ± 0.6 x,a	14.0 ± 0.9 y,a	14.6 ± 0.3 y,a	−15.6	−11.2
				eCO2	13.9 ± 0.5 x,b	15.0 ± 0.9 x,a	14.5 ± 0.3 x,a	+8.3	+4.8
				CO2 effect (%)	−15.9	+7.6	−0.9
Leaf P concentration (mg g−1)	0.007	0.136	0.283	aCO2	1.30 ± 0.06 x,a	1.18 ± 0.04 y,a	1.28 ± 0.04 x,a	−9.1	−0.9
				eCO2	1.08 ± 0.08 y,b	1.10 ± 0.03 y,b	1.20 ± 0.03 x,b	+3.2	+8.2
				CO2 effect (%)	−16.7	−6.6	−5.8
Stem P concentration (mg g−1)	0.012	0.010	0.226	aCO2	0.99 ± 0.04 x,a	0.84 ± 0.02 y,a	0.86 ± 0.04 y,a	−15.1	−13.1
				eCO2	0.85 ± 0.04 x,b	0.72 ± 0.04 y,b	0.85 ± 0.06 x,a	−14.7	−0.8
				CO2 effect (%)	−14.5	−14.2	−0.9
Root P concentration (mg g−1)	0.044	0.039	0.005	aCO2	0.95 ± 0.04 x,a	0.74 ± 0.05 y,a	0.95 ± 0.06 x,a	−22.0	−0.4
				eCO2	0.70 ± 0.03 y,b	0.84 ± 0.06 x,a	0.87 ± 0.02 x,b	+20.8	+26.1
				CO2 effect (%)	−26.6	+12.6	−8.4
Leaf K concentration (mg g−1)	0.294	0.003	0.271	aCO2	20.0 ± 0.4 y,a	19.2 ± 0.8 y,a	21.6 ± 0.3 x,a	−3.7	+8.4
				eCO2	18.8 ± 0.3 z,b	19.7 ± 0.3 y,a	21.15 ± 0. 6 x,a	+4.8	+10.2
				CO2 effect (%)	−6.1	+2.7	−2.7
Stem K concentration (mg g−1)	0.677	0.106	0.785	aCO2	7.6 ± 0.6 x,a	6.0 ± 0.2 y,a	7.2 ± 0.7 x,a	−21.1	−5.6
				eCO2	7.2 ± 0.8 x,a	6.3 ± 0.1 x,a	6.8 ± 0.6 x,a	−12.7	−6.0
				CO2 effect (%)	−5.3	+4.5	−5.8
Root K concentration (mg g−1)	0.802	0.150	0.108	aCO2	8.3 ± 0.6 x,a	6.5 ± 0.7 y,a	8.2 ± 0.1 x,a	−22.2	−1.1
				eCO2	7.5 ± 0.4 x,a	7.3 ± 0.5 x,a	7.6 ± 0.5 x,a	−2.0	+1.8
				CO2 effect (%)	−10.0	13.1	−7.2

Data (means ± SE, n = 6) followed by different letters indicate significant differences between CO₂ treatments for the same water regime (a, b) and among water treatments for the same CO₂ concentration (x, y, z) at p < 0.05. Statistical comparisons (two-way ANOVA) between water regimes and CO₂ treatments, as well as their interaction (water × CO₂), are presented for each variable.

). Under eCO₂, leaf N decreased by 6.1%, 10.1%, and 13.0% (p = 0.001) and stem N decreased by 10.5%, 15.9%, and 2.2% (p = 0.042) under well-watered, moderate-drought, and severe-drought conditions, respectively (Table 1), while root N remained unaffected (p > 0.05, Table 1).

Similarly, P concentrations in leaves, stems, and roots were consistently lower under eCO₂ than under aCO₂ across all water treatments (all p < 0.05, Table 1), except for stem P under severe drought and root P under moderate drought, which showed no significant differences. Compared to the well-watered treatment, moderate drought and severe drought reduced stem P by 15.1% and 13.1%, respectively, under aCO₂, and by 14.7% and 0.8%, respectively, under eCO₂. Conversely, the root P concentration increased significantly under moderate drought and severe drought under eCO₂ but not under aCO₂ (p = 0.044; Table 1).

In addition, only the leaf K concentration exhibited a consistent increase with declining soil water, regardless of CO₂ conditions (p = 0.003; Table 1). In contrast, eCO₂ had no significant effect on K concentrations in leaves, stems, or roots (all p > 0.05, Table 1). Two-way ANOVA revealed significant interactions between eCO₂ and drought for both N and P concentrations in roots (p < 0.05; Table 1).

3.6. Effects of eCO₂ and Drought on Tissue Accumulations and Allocation of N, P, and K in Mulberry Plants

In general, drought stress significantly decreased the accumulation of N, P, and K in the leaves, stems, roots, and total plant under both aCO₂ and eCO₂, except for leaf P accumulation (Figure 6). Severe drought resulted in reductions in N, P, and K accumulation in the total plant of 26.7%, 31.0%, and 24.3% under aCO₂ and of 20.7%, 13.5%, and 14.4% under eCO₂, respectively. Nevertheless, eCO₂ significantly increased N, P, and K accumulation in leaves, stems, roots, and the total plant subjected to well-watered, moderate-drought, and severe-drought conditions. Compared to aCO₂, eCO₂ significantly increased total-plant N accumulation by 13.3%, 18.8%, and 22.6% (p = 0.007, Figure 6D) and total-plant K accumulation by 16.1%, 36.0%, and 31.3% under well-watered, moderate-drought, and severe-drought conditions, respectively (p < 0.001, Figure 6L). Similarly, eCO₂ also increased total-plant P accumulation by 30.7% and 25.9% under moderate drought and severe drought, respectively (p = 0.016), but had no significant effect under well-watered conditions. Two-way ANOVA revealed significant interaction effects between eCO₂ and drought on stem N and stem and root P accumulation (p < 0.05, Figure 6).

Furthermore, severe drought significantly enhanced the allocations of N, P, and K to the leaves but decreased their allocations to the stems and roots of mulberry, regardless of CO₂ concentration. Conversely, eCO₂ decreased N, P, and K allocations to the leaves and stems but increased their allocations to roots under well-watered and severe-drought conditions (Figure 7).

3.7. Effects of eCO₂ and Drought on Soil Nutrients and Soil Enzyme Activities

The response of soil AN, AP, and AK to decreasing soil water status exhibited an increasing trend under both aCO₂ and eCO₂ (Figure 8). Soil AN showed an asymmetric response to CO₂ concentration along the soil water gradient, where it was significantly lower under eCO₂ than under aCO₂ under the well-watered condition but higher under eCO₂ than under aCO₂ under moderate-drought and severe-drought conditions (p = 0.018, Figure 8A). Soil AP and AK were significantly lower under eCO₂ than under aCO₂ along the soil water gradient (p < 0.05, Figure 8B,C).

Moreover, regardless of soil water status, eCO₂ generally significantly increased the activities of soil sucrase and phosphatase, although these enzyme activities obviously decreased with declining soil water content under both aCO₂ and eCO₂ (p < 0.05, Figure 8D,F). In contrast to the observed trends in soil sucrase and phosphatase activities, urease activity exhibited a different pattern: it decreased along the soil water gradient under aCO₂ but slightly increased under moderate-drought conditions under eCO₂ (p < 0.05, Figure 8E). Two-way ANOVA showed significant interaction effects between eCO₂ and drought on soil AN (p = 0.023), AP (p = 0.027), and sucrase (p < 0.001, Figure 8).

3.8. Effects of eCO₂ and Drought on Biomass Production in Mulberry Plants

Compared to the well-watered treatment, drought stress significantly decreased the biomass production in leaves, stems, roots, and total plants, regardless of CO₂ concentration. Moderate drought and severe drought resulted 14.7% and 31.8% reductions in total biomass production, respectively, under aCO₂ and 11.3% and 22.8% reductions, respectively, under eCO₂. Conversely, a very strong effect of CO₂ fertilization on mulberry growth was observed under both well-watered and drought stress conditions, with the effect more pronounced as soil water status declined. Specifically, eCO₂ increased the leaf, stem, root, and total plant biomass production by 26.1%, 20.1%, 20.4%, and 23.1%, respectively, under well-watered conditions; by 32.0%, 29.4%, 18.8%, and 28.0%, respectively, under moderate drought; and by 35.7%, 83.7%, 15.3%, and 39.2% (all p ≤ 0.01), respectively, under severe drought (

Table 2. Variations in plant tissue biomass production of mulberry plants under ambient CO₂ (aCO₂); elevated CO₂ (eCO₂); and well-watered, moderate-drought, and severe-drought conditions.

Variable	p-Value			CO2 Treatment	Water Treatment			Moderate Water Deficit Effect (%)	Severe Water Deficit Effect (%)
	CO2	Water	C*W		Well-Watered	Moderate Drought	Severe Drought
Leaf biomass (g plant−1)	0.002	0.000	0.981	ACO2	37.5 ± 1.8 x,b	32.9 ± 1.0 y,b	28.6 ± 2.9 z,b	−12.4	−23.8
				ECO2	47.3 ± 0.4 x,a	43.4 ± 0.5 y,a	38.8 ± 2.7 z,a	−8.3	−18.0
				CO2 effect (%)	+26.1	+32.0	+35.7
Stem biomass (g plant−1)	0.000	0.000	0.037	ACO2	20.3 ± 0.9 x,b	16.5 ± 0.9 y,b	10.0 ± 0.6 z,b	−18.9	−50.6
				ECO2	24.4 ± 1.4 x,a	21.3 ± 0.8 y,a	18.4 ± 0.9 z,a	−12.6	−24.4
				CO2 effect (%)	+20.1	+29.4	+83.7
Root biomass (g plant−1)	0.000	0.001	0.574	ACO2	19.8 ± 0.9 x,b	16.9 ± 0.8 y,b	14.3 ± 1.2 z,b	−14.8	−27.8
				ECO2	23.9 ± 0.4 x,a	20.1 ± 0.8 y,a	16.5 ± 0.8 z,a	−16.0	−30.9
				CO2 effect (%)	+20.4	+18.8	+15.3
Total biomass (g plant−1)	0.000	0.000	0.876	ACO2	77.7 ± 3.4 x,b	66.2 ± 1.3 y,b	53.0 ± 3.8 z,b	−14.7	−31.8
				ECO2	95.6 ± 2.1 x,a	84.8 ± 1.1 y,a	73.8 ± 14.2 z,a	−11.3	−22.8
				CO2 effect (%)	+23.1	+28.0	+39.2
Root : shoot ratio	0.027	0.164	0.080	ACO2	0.34 ± 0.01 x,a	0.34 ± 0.01 x,a	0.37 ± 0.04 x,a	−0.3	+9.4
				ECO2	0.33 ± 0.01 x,a	0.31 ± 0.02 x,b	0.28 ± 0.02 x,b	−6.8	−13.1
				CO2 effect (%)	−2.9	−9.2	−22.9

Data (means ± SE, n = 6) followed by different letters indicate significant differences between CO₂ treatments for the same water regime (a, b) and among water treatments for the same CO₂ concentration (x, y, z) at p < 0.05. Statistical comparisons (two-way ANOVA) between water regimes and CO₂ treatments, as well as their interaction (water × CO₂), are presented for each variable.

). Additionally, eCO₂ significantly decreased the root/shoot ratio under severe drought. Two-way ANOVA showed a significant interaction effect between eCO₂ and drought on stem biomass production only (p = 0.037, Table 2).

3.9. Multivariate Analyses

To explore the potential mechanisms underlying the impact of eCO₂ on plant response to drought stress, particularly in terms of changes in biomass production, we employed structural equation modeling (SEM) to analyze the relationships between eCO₂ concentration, drought treatments, gas exchange, chlorophyll fluorescence, osmotic regulators, antioxidant enzyme activities, plant nutrient uptake, soil properties, and biomass production. The SEM analysis showed that drought had significant direct, positive effects on osmotic substances and soil nutrients while exerting significant direct, negative effects on photosynthesis, total biomass production, and soil enzyme activities. In contrast, eCO₂ had significant direct, positive effects on photosynthesis, total biomass, and soil enzyme activities and significant direct, negative effects on chlorophyll fluorescence; osmotic substances; and plant N, P, and K (Figure 9).

4. Discussion

Mulberry plants growing in the majority of southwest China frequently experience a moderate drought due to a progressive reduction in soil water availability [10,70,71]. Drought stress has been widely reported to significantly reduce both photosynthesis and biomass accumulation in mulberry plants [72], in agreement with our study findings (Figure 2A, Table 2). In addition, mulberry production faces future challenges due to increases in , drought frequency, and atmospheric CO₂ concentration. This study expands upon previous knowledge regarding how mulberry plants can cope with water scarcity and discusses the dynamics through which eCO₂ mitigates the effects of prolonged drought stress by enhancing CO₂ uptake and biomass production, altering nutrient uptake and partitioning while decreasing oxidative pressure.

4.1. eCO₂ Alleviates Adverse Effects of Drought on Plant Biomass Production and Photosynthesis

Under drought conditions, 710 ppm eCO₂ significantly stimulated the above- and below-ground biomass of mulberry, resulting in a substantial mitigation effect on mulberry productivity (Table 2). This finding agrees with previous studies conducted on coffee plants, Picea abies, Pinus halepensis, and Populus tremula under 700–867 ppm eCO₂ in open-top chambers or free-air CO₂ enrichment experiments [7,30,73,74]. The increase in plant biomass production due to eCO₂ can be primarily attributed to biochemical and photochemical processes such as photosynthesis and respiration [75,76]. Specifically, plants grown under eCO₂ typically exhibit a 30–60% greater leaf photosynthetic rate due to increased Rubisco carboxylation while competitively suppressing plant photorespiration and dark respiration [41,77]. In this study, eCO₂ directly stimulated photosynthesis in mulberry through increased CO₂ uptake (Figure 2A). A considerably greater intercellular CO₂ concentration was observed under eCO₂ (Figure 2D), indicating that the carboxylation rate of Rubisco was constrained by CO₂ diffusion into the carboxylation sites under aCO₂. Consequently, eCO₂ favored the carboxylation reaction of Rubisco over its oxygenation reaction (Figure 5), thereby enhancing carbon assimilation by increasing Ci for Rubisco carboxylation. These results are consistent with findings reported by Kelly et al. (2019) [78] and Fan et al. (2020) [38]. The enhanced photosynthetic rates under eCO₂ directly contribute to higher carbon assimilation, which is a key driver of biomass accumulation and overall productivity in mulberry. This is particularly evident in the significant increase in above- and below-ground biomass observed in our study (Table 2). The greater carbon fixation under eCO₂ provides more resources for growth and development, enabling mulberry to maintain productivity, even under drought stress. Additionally, the increased carbon assimilation not only supports biomass production but also enhances the plant’s ability to cope with drought stress. By maintaining higher photosynthetic rates, mulberry can accumulate more carbohydrates, which are essential for osmotic adjustment and energy metabolism under water-limited conditions [79]. This explains the mitigation of drought stress observed in our study.

Despite the significant impact of eCO₂ on leaf photosynthesis, its effects may be partially mitigated by drought stress. This is evidenced by a greater decline in net photosynthetic rate observed in plants experiencing moderate drought and severe drought compared to well-watered plants (Figure 2A). Such a reduction in the net photosynthetic rate under drought stress could be attributed to both diffusive limitations (such as gs and Ci, as shown in Figure 2B,D) and non-diffusive limitations related to biochemical processes [80]. These observations were supported by decreased leaf N, P, and K levels and their correlations with leaf photosynthesis in mulberry plants (Figure 9). Despite the partial mitigation of photosynthetic rates under drought stress, the overall enhancement of carbon assimilation under eCO₂ still plays a critical role in sustaining mulberry growth and productivity. The ability of mulberry to maintain higher photosynthetic rates under combined eCO₂ and drought conditions highlights its potential as a resilient crop for sustainable agriculture in regions affected by climate change.

However, it is important to critically assess potential limitations and trade-offs associated with long-term exposure to eCO₂. One potential limitation is the acclimation response, where plants may exhibit reduced photosynthetic efficiency over time due to downregulation of Rubisco and other photosynthetic enzymes [62,81]. This acclimation response could diminish the initial benefits of eCO₂ in terms of carbon assimilation and growth, particularly under prolonged exposure [82]. Future studies should investigate whether mulberry exhibits similar acclimation responses under long-term eCO₂ exposure.

4.2. eCO₂ Mitigated the Negative Effects of Drought by Improving Water Use Efficiency

Stomatal responses to eCO₂ exhibit considerable variability and are influenced by environmental conditions. In this study, lower stomatal conductance and transpiration rates were observed under eCO₂ compared to aCO₂ under both well-watered conditions and drought stresses (Figure 2B,C). These findings are consistent with previous studies on various herbaceous and woody species [30,83,84]. The reduction in stomatal conductance under eCO₂ can be an adaptive mechanism for water conservation while maintaining high levels of photosynthesis, which is associated with decreased stomatal density and an altered stomatal aperture [8]. The Ci/Ca ratio reflects the relationship between stomatal conductance and photosynthetic capacity. Our results showed that Ci/Ca values decreased with declining soil water status under aCO₂ but remained unchanged under eCO₂ (Figure 2F), indicating that stomata were sensitive to drought stress under aCO₂ but exhibited reduced sensitivity or physiological adaptation to eCO₂. These results indicate that stomatal conductance is not the limiting factor for the increase in the net photosynthetic rate under eCO₂. Instead, eCO₂ compensates for the decrease in CO₂ caused by stomatal closure during drought stress, thereby alleviating the adverse effects of drought stress on plants [85,86]. Plants typically minimize water loss by reducing their leaf transpiration rate as a key adaptive strategy under short-term drought stress [87]. Our results demonstrate that eCO₂ dramatically improved leaf WUE in mulberry plants subjected to well-watered, moderate-drought, and severe-drought conditions (Figure 2A,C,E). The enhancement in WUE under eCO₂ can be attributed to the combined effects of reduced stomatal conductance and increased photosynthetic efficiency. Elevated CO₂ concentrations reduce the stomatal aperture, thereby minimizing water loss via transpiration while ensuring sufficient CO₂ uptake for photosynthesis. This stomatal regulation is mediated by alterations in guard-cell turgor pressure and the signaling pathways involving abscisic acid and other phytohormones [31]. Furthermore, eCO₂ enhances the carboxylation efficiency of Rubisco, allowing plants to achieve higher photosynthetic rates with lower stomatal conductance [26]. These physiological adaptations enable mulberry to optimize water use and sustain growth under drought stress, highlighting the critical roles of stomatal behavior and photosynthetic efficiency in improving WUE.

4.3. eCO₂ Mitigated the Negative Effects of Drought by Improving Photosystem II Efficiency

Chlorophyll fluorescence was employed to assess the extent of damage to the photosynthetic apparatus—in particular, to photosystem II (PSII). Under drought stress, impairment of PSII reaction centers impedes primary photochemistry, thereby affecting the process of photosynthetic electron transport. In this study, we observed a reduction in PSII efficiency in mulberry plants due to drought stress, as evidenced by decreased Φ_PSII, ETR, and qP values (Figure 3). Importantly, the detrimental effects of severe drought on Φ_PSII, ETR, and qP were more pronounced under aCO₂ compared to under eCO₂ (Φ_PSII, 33% and 25% lower than the control; ETR, 35% and 13% lower; qP, 44% and 4% lower; Figure 3). There was a significant interactive effect between drought stresses and eCO₂ on Φ_PSII, ETR, and qP (p < 0.05, Figure 3), which partially mitigated the damage caused by drought stress. Similar findings have been reported with respect to cucumber [88], coffee plants [30], and wheat [89]. The reduction in PSII efficiency under drought stress directly impacts the photosynthetic electron transport chain, leading to lower energy conversion efficiency and reduced carbon assimilation. This, in turn, limits biomass accumulation and overall productivity in mulberry [90,91]. However, the partial mitigation of PSII efficiency under eCO₂, as indicated by higher ΦPSII, ETR, and qP values, suggests that mulberry can maintain better photochemical efficiency and energy utilization under combined eCO₂ and drought conditions. This enhanced photochemical efficiency likely contributes to the observed increase in biomass and productivity under eCO₂ (Table 2) [41]. Furthermore, a slight decrease in Fv/Fm under eCO₂+SS indicates that mulberry plants exposed to drought conditions may not have undergone photoinhibition. Therefore, mulberry plants activate non-photochemical quenching and reduced drought-induced PSII efficiency under eCO₂ (Figure 3). The activation of NPQ under eCO₂ and drought stress is a protective mechanism that dissipates excess light energy as heat, thereby preventing damage to the photosynthetic apparatus [92]. This mechanism allows mulberry to maintain higher photosynthetic activity and carbon assimilation levels under stress conditions, which are critical for sustaining growth and productivity [93]. The ability of mulberry to optimize energy utilization and protect PSII under combined eCO₂ and drought stress highlights its potential as a resilient crop for cultivation in water-limited environments [94].

4.4. eCO₂ Mitigated the Negative Effects of Drought by Boosting Mulberry’s Defenses

Drought stress significantly reduces the efficiency of the photosynthetic electron transport chain, leading to a decline in photosynthetic efficiency and an increased electron flow towards molecular oxygen and production of superoxide radicals [95]. The accumulation of ROS is detrimental to biomolecules such as nucleic acids, proteins, chloroplast pigments, etc. [96,97]. In response to drought-induced oxidative stress, plants develop a complex antioxidant system to regulate cellular redox status. CAT, POD, and SOD serve as the first line of defense against reactive oxygen species. In this study, the activities of CAT, POD, and SOD were significantly increased in both leaves and roots under drought stress, which is consistent with previous results [10,98]. Furthermore, eCO₂ further improved the activities of CAT, POD, and SOD in leaves and/or roots under drought stress conditions (Figure 5), in line with other studies [12,76]. The impact was more pronounced for SOD under moderate drought and for POD under severe drought, indicating distinct responses in antioxidant metabolism. The mitigation of ROS formation under eCO₂ can be attributed to increased electron consumption in C fixation, reducing the diversion of electrons towards ROS-generating pathways such as photorespiration or the Mehler reaction [99,100]. Enhanced carbon assimilation under eCO₂ provides more resources for the synthesis of antioxidant enzymes and osmoprotectants, thereby mitigating oxidative damage and supporting cellular functions under drought stress. Soluble sugars, acting as osmolyte and metabolite signaling molecules, play a crucial role in modulating plant responses to stress [82]. The elevated concentration of soluble sugars under eCO₂ observed in this study (Figure 4C,D) indicates enhanced C assimilation, which improves turgor pressure through osmotic adjustment. This finding is consistent with a previous study showing that soluble sugars (such as glucose and sugar alcohols) contributed to increased root biomass in Sorghum bicolor and alleviated drought stress under eCO₂ [101]. The accumulation of soluble sugars under eCO₂ not only supports osmotic adjustment but also provides energy for growth and stress responses [79]. Proline serves as both a stress marker and an osmoprotectant, implying that plants may accumulate more proline under drought stress or less proline when experiencing reduced stress levels [102]. Our data demonstrate that eCO₂ decreased proline content in both leaves and roots under drought stress (Figure 4A,B). Hence, the reduced accumulation of proline in plants exposed to eCO₂ suggests that excessive proline buildup is not necessary to withstand drought stress [103]. The reduced proline accumulation under eCO₂ indicates that mulberry experiences less severe drought stress due to improved water use efficiency and antioxidant defense. This allows the plant to allocate more resources towards growth and productivity rather than stress mitigation [104]. These changes in antioxidant metabolites and osmolytes clearly indicate that mulberry plants modify their biochemical system to counteract the detrimental effects of drought stress under eCO₂.

4.5. eCO₂ Mitigated the Negative Effects of Drought by Regulating NPK Partitioning

Drought stress can inhibit plant growth by reducing nutrient uptake and affecting the relocation of N, P, and K in plants [105,106]. A meta-analysis revealed that drought stress results in decreased levels of N and P in plant tissues, as well as reduced nutrient absorption from soil [39]. In this study, the concentrations of N, P, and K in stems and roots were significantly lower under drought stress compared to well-watered conditions under aCO₂, and a greater decrease was observed under moderate drought than under severe drought (Table 2). The impacts of drought on N, P, and K concentrations were more pronounced in roots and stems compared to leaves, indicating that soil drought negatively affected the nutrient uptake capacity of roots and stems. The decline in nutrient levels could be attributed to decreased photosynthesis and transpiration rates under drought conditions, resulting in reduced nutrient uptake (Figure 2 and Figure 8). This coincided with an increase in the allocation of N, P, and K to leaves and a decrease in their allocations to stems and/or roots (Figure 7). However, these findings are inconsistent with studies suggesting drought may accelerate N transfer from green leaves to sink tissues such as roots or grains [107]. The increased allocation of N, P, and K to leaves observed in this study could be a mechanism of drought avoidance through the synthesis and accumulation of osmoprotectants and antioxidant enzymes, which are predominantly found in higher concentrations in leaves than in roots (Figure 5 and Figure 6), as further supported by a significantly positive correlation between plant N, P, and K concentrations and antioxidant enzyme activities (Figure 9).

Fortunately, the detrimental impacts of drought on the concentrations of N, P, and K in stems and roots were mitigated under eCO₂ compared to under aCO₂, and even drought stress increased root P concentrations under eCO₂ (Table 1). Previous studies have reported a decline in plant-tissue N due to CO₂ enrichment [46,61,62,108], which subsequently affects other components involved in N metabolism, such as proline levels [109]. Similar results were observed in this study (Table 1 and Figure 4B,E). Under both well-watered and severe-drought conditions, eCO₂ led to decreases in N and P contents in mulberry tissues, but the reduction was less pronounced under severe drought than under well-watered conditions (Table 1). This phenomenon may be attributed to the nutrient dilution effect, where increased biomass production under eCO₂ leads to lower concentrations of essential nutrients such as N and P in plant tissues [110]. This nutrient dilution could have implications for the nutritional quality of mulberry leaves, which are critical for silkworm feeding and sericulture productivity. Future research should explore strategies to mitigate nutrient dilution effects, such as optimized fertilization practices or genetic improvements in nutrient use efficiency.

Furthermore, eCO₂ induced allocations of N, P, and K to root tissues under drought stresses (Figure 8). This adaptation to eCO₂ facilitates greater allocation to the expanded root system rather than the photosynthetic apparatus of green tissues, optimizing energy costs [111]. Thus, eCO₂ and drought stress may increase nutrient use efficiency by regulating nutrient reallocation [112].

4.6. eCO₂ Mitigated the Negative Effects of Drought by Improving Nutrient Uptake and Stimulating Soil Enzyme Activities

Most studies on eCO₂ and/or drought have primarily focused on plant characteristics, with limited research conducted on the feedback between plant nutrient uptake and soil nutrient supply. It is widely acknowledged that soil nutrient reserves play a crucial role in determining plant growth. However, the impacts of eCO₂ and/or drought on the soil nutrient pool and plant requirements remain unpredictable. Our results demonstrate that plants exhibited reduced uptake of N, P, and K when subjected to decreased soil moisture (Figure 6), resulting in higher contents of available forms of these nutrients in soil (Figure 8). Reduced nutrient uptake during drought may be attributed to factors such as diminished mineralization-mediated nutrient supply, as well as reduced nutrient diffusion and mass flow in soil [113]. Additionally, altered dynamics of root nutrient uptake induced by drought can also contribute to decreased acquisition of essential elements by plants [114]. In contrast, our results indicate that eCO₂ significantly enhanced N, P, and K accumulation in mulberry plants under both well-watered and drought-stressed conditions (Figure 6), leading to decreased levels of AN, AP, and AK in soil, except for AN under drought stress (Figure 8). These findings suggest that eCO₂ ameliorated the adverse impacts of drought by improving nutrient uptake. Consistent with our findings, previous studies have displayed similar effects; for example, 700 ppm eCO₂ upregulated gene expression related to nutrient uptake proteins, enhancing the rates of N and P assimilation [110].

The activities of soil enzymes indicate microbial functions in decomposing organic matter and mobilizing nutrients [48]. In this study, we observed significant decreases in sucrase, urease, and phosphatase activities under drought conditions (Figure 8D–F), consistent with previous studies that have reported negative correlations between soil moisture and enzymatic activities [45,51,115]. The reduction in enzyme activities could also be attributed to the low microbial biomass and physiology [116] or decreased enzyme production and turnover rates [117]. Drought-induced inhibition of plant growth and reductions in rhizodeposition result in decreased substrate availability. However, our study revealed significantly higher activities of soil sucrase, urease, and phosphatase under eCO₂ than under aCO₂ as soil water content declined (Figure 8D–F), indicating a positive correlation between eCO₂ levels and soil enzyme activities (Figure 9). Similar increases in enzyme activities have been reported in herbaceous and woody soils under 550–700 ppm eCO₂ [47,49,118]. A two-year FACE study conducted in a semi-arid grassland at the United States Department of Agriculture’s Agricultural Research Service High Plains Grasslands Research Station demonstrated that 600 ppm eCO₂ had a positive effect on soil C, N, and P cycling by increasing the abundance of microbial functional genes, which were closely correlated with the increase in soil moisture [119]. The increase in soil enzyme activities is directly related to the plant root exudates [120]. Therefore, eCO₂ could accelerate C turnover in the rhizosphere and stimulated soil enzyme activities, thereby influencing the microbial biomass and microbial activity that was not tested in this study, which might ultimately have potential to ameliorate the adverse impacts of drought.

However, the long-term sustainability of eCO₂ effects on mulberry growth and productivity remains uncertain. While eCO₂ can enhance nutrient uptake and soil enzyme activities in the short term, prolonged exposure to elevated CO₂ may alter soil nutrient dynamics and microbial activity, potentially affecting nutrient availability and plant health over time [121]. For example, increased carbon inputs under eCO₂ may accelerate soil organic matter decomposition, leading to changes in nutrient cycling and microbial community composition [122]. Future studies should investigate the long-term impacts of eCO₂ on mulberry growth, nutrient cycling, and ecosystem interactions to fully understand its potential benefits and limitations.

5. Conclusions

We conducted a comprehensive study on the responses of mulberry plants to drought under eCO₂. As shown in Figure 10, eCO₂ promotes mulberry’s photosynthesis and WUE under drought. It also mitigates the adverse effects of drought on ETR and ΦPSII, thereby decreasing damage to photosystems and ROS accumulation in mulberry under drought stress. Moreover, eCO₂ enhanced the tolerance of mulberry to drought stress by increasing activities of antioxidant enzymes such as SOD, POD, and CAT, and the contents of osmolytes such as proline, malondialdehyde, and soluble sugars in both leaves and roots. Furthermore, eCO₂ stimulated the activities soil enzymes under such as soil sucrase, urease, and phosphatase, under drought, improving soil fertility. This stimulation facilitated nutrient uptake of soil N, P, and K, leading to their increased accumulation in mulberry tissues while promoting their allocation to an expanded root system under drought. The enhanced photosynthetic capacity, reduced oxidative stress, and increased nutrient absorption induced by eCO₂ can effectively mitigate the detrimental impact of drought on mulberry growth by augmenting both above- and below-ground biomass production. Our findings provide crucial insights to guide water management strategies in mulberry plantations while increasing leaf growth and productivity through the regulation of N, P, and K nutrition under future conditions of elevated CO₂ and drought.