Photosynthetic Acclimation and Growth Responses to Elevated CO2 Associate with Leaf Nitrogen and Phosphorus Concentrations in Mulberry (Morus multicaulis Perr.)

Mulberry (Morus spp.) is a multipurpose tree that is worldwide planted because of its economic importance. This study was to investigate the likely consequences of anticipated future elevated CO2 (eCO2) on growth, physiology and nutrient uptake of nitrogen (N), phosphorus (P) and potassium (K) in two most widely cultivated mulberry (Morus multicaulis Perr.) varieties, QiangSang-1 and NongSang-14, in southwest China. A pot experiment was conducted in environmentally auto-controlled growth chambers under ambient CO2 (ACO2, 410/460 ppm, daytime/nighttime) and eCO2 (710/760 ppm). eCO2 significantly increased plant height, stem diameter, leaf numbers and biomass production, and decreased chlorophyll concentrations, net photosynthetic rate, stomatal conductance and transpiration rate of these two mulberry varieties. Under eCO2 leaf N and P, and root N, P and K concentrations in both mulberry varieties decreased, while plant total P and K uptake in both varieties were enhanced, and an increased total N uptake in NongSang-4, but not in QiangSang-1. Nutrient dilution and transpiration rate were the main factors driving the reduction of leaf N and P, whereas changes in plant N and P demand had substantial impacts on photosynthetic inhibition. Our results can provide effective nutrient management strategies for a sustainable mulberry production under global atmosphere CO2 rising scenarios.


Introduction
Global atmospheric carbon dioxide (ACO 2 ) concentration is predicted to exceed 700 ppm by the end of this century [1], mostly due to anthropogenic activities. The elevated CO 2 (eCO 2 ) concentration stimulates the photosynthesis especially in C 3 plants, and above-ground biomass accumulation [2]. However, when the synthesis of carbohydrates exceeds the capacity to produce new sinks under eCO 2 , plants would reduce photosynthetic rate in order to balance source-sink, which triggers plant photosynthetic acclimation [3][4][5]. The photosynthetic acclimation during plant growth in response to eCO 2 is often accompanied with reduced in both the amount and activity of ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco), stomatal conductance, leaf nutrient concentrations and chlorophyll content [6][7][8][9]. These effects of eCO 2 have been reported in herbaceous species, such as faba bean [10], cucumber [11], rice [4], tomato [12] and wheat [13]. However, there was less information about the genotypic, inter-or intra-species variations in perennial woody species.
To maintain a high productivity plants need an enhanced supply of essential nutrients such as nitrogen (N), phosphorus (P) and potassium (K) to match their increase in C  2 . Previous studies showed that effects of eCO 2 on N, P and K uptake varied with tree species and experimental conditions [14][15][16]. 550 ppm eCO 2 decreased leaf N concentration during the initial stages of Cajanus cajan, but increased leaf N uptake by 6.5% and 17.5% in vegetative and reproductive stages, respectively [17]. N concentrations in fully expanded leaves declined by 8% under 550 ppm eCO 2 in Eucalyptus tereticornis [18]. In addition, P concentrations in a mature eucalypt woodland [19] and Picea mariana [20] were significantly increased with 550 ppm eCO 2 . In contrast, P concentrations in Populus deltoides [21] and Quercus variabilis [22] were decreased with 660-700 ppm eCO 2 in a pot experiment. Moreover, 700 ppm eCO 2 effects on plant K uptake were speciesspecific, with enhanced K acquisition in Oligostachyum lubricum but not in Phyllostachys edulis [23]. However, 690 ppm eCO 2 had no effect on tissue N, P and K concentrations and uptake rates of N, P and K of Larix kaempferi in a field experiment with open-top chambers [24]. Consequently, contradictions exist when comparing the results of different species and experimental conditions, and the response mechanisms of plants to eCO 2 remain unknown about their uptake of N, P, and K.
Mulberry (Morus spp.) is a fast-growing multipurpose plant and has been widely planted in Asia, Africa, Europe, North and South America [25]. A total of 30 mulberry species and nine varieties are mainly grown in China, India and Japan [26]. Different tissues of mulberry plants are of great interest owing to their nutraceutical values. For instance, mulberry leaves contain an appreciable amount of antioxidants, carbohydrates, fats, fibers, minerals, proteins and vitamins [27], and have been used not only to rear silkworm, cattle, goat and other animals, but also to make tea and for consumption as a vegetable [28]. The root and bark of mulberry are employed as a component of anti-diabetic medicines in Traditional Oriental Medicine [29,30]. Stems of mulberry are used to produce some bioactive molecules and exhibit great antioxidant properties [31]. Given the high economic and medicinal value of mulberry plants, studies on its growth, photosynthesis and nutrient uptake response to global environmental change scenarios are indispensable. Previous studies have demonstrated that 550-800 ppm eCO 2 enhance the growth of different mulberry species, e.g., Selection-13, Kanva-2, 'Qinglong' mulberry and Gui-sang-you 62 [32][33][34][35]. However, it remains unclear how eCO 2 could affect uptake of N, P and K and their allocation in mulberry leaves, which are food source for silkworms.
The objectives of the present study were to quantify (a) the growth and photosynthetic response to eCO 2 of two mulberry (Morus multicaulis Perr.) varieties, QiangSang-1 and NongSang-14, which are widely cultivated in the south of China and (b) the effects of eCO 2 on tissue N, P and K uptake, concentrations and allocation in these mulberry varieties.

Experiment Design
This study was conducted in automatically controlled-environment growth chambers located in the National Monitoring Base for Purple Soil Fertility and Fertilizer Efficiency (29 • 48 N, 106 • 24 E, 266.3 m above sea level) on the campus of Southwest University, Chongqing, China ( Figure S1A,B). Each growth chamber (1.5 × 1.0 × 2.5 m) has a rectangle floor base, which is supported by a steel frame that is hanging 50 cm above the cement ground base ( Figure S1A). The bottom floors of the growth chamber are made up with polyvinyl chloride plates and the four-side walls and top roofs of the chamber are constructed by tempered glasses (10 mm thickness, 90% light transmission rate, Yutao Glass Company, Jiulongpo District, Chongqing, China) ( Figure S1A,B). Detailed information of the automatically controlled-environment facility used in this study has also been described in our previous studies [35,36]. The experiment had a randomized block design with ACO 2 and eCO 2 levels as the main treatment (three chambers or replicates for each treatment) and two plant varieties as the sub-treatment. Two pots per variety were placed in each chamber and thus the three replicated chambers had a total of six replicated pots for each CO 2 treatment. According to the observed daytime and nighttime atmosphere CO 2 concentrations in the study site, we designed CO 2 concentrations (±30 ppm) as followed: Forests 2021, 12, 660 3 of 14 ACO 2 (410 ppm daytime/460 ppm nighttime) and eCO 2 (710 ppm daytime/760 ppm nighttime). Daytime was from 07:00 a.m. to 19:00 p.m. and nighttime was from 19:00 p.m. to 07:00 a.m. Except for the CO 2 concentration, the chambers had similar growth conditions such as fertilization, light, air temperature, and humidity. The similar temperature and humidity between inside and outside the growth chambers were also automatically maintained by the above-mentioned CO 2 auto-controlling facility [35,36]. The sunlight intensity and photosynthetic active radiation (PAR) were supplied by natural sunlight through the growth chamber's four walls and top roof being made up with tempered glasses (90% light transmission rate, see Figure S1A,B).

Plant Materials
One-year-old mulberry seedlings of two varieties (Morus multicaulis Perr. var. Qiang Sang-1 and NongSang-14) with uniform growth status (diameter: 4.0 ± 0.08 mm; height: 25 ± 0.25 cm) were as the experiment materials. Two seedlings per variety were grown in a plastic pot (20 × 32 cm = height × diameter) filled with 11 kg soil (Eutric Regosol, FAO Soil Classification System) from 10 May to 16 September 2020. The soil (pH 6.8) was air-dried and sieved by passing through a 2 mm mesh, which contained 10.56 g kg −1 organic carbon, 0.66 g kg −1 total N, 0.61 g kg −1 total P, 97 mg kg −1 available N, 17 mg kg −1 available P and 197 mg kg −1 available K. Four weeks after growth, 0.5 g N, 0.25 g P and 0.25 g K per pot were applied to meet the nutrient requirement of plant growth. The plants were regularly irrigated to maintain soil moisture at 70-80%. The pots in the chambers were weekly relocated once to minimize differences in growth conditions.

Determination of Plant Growth
The plants were harvested on 16 September 2020 after 129 days of CO 2 exposure. Plant height, stem diameter and leaf numbers were recorded prior to harvest. Plant height was measured from plant base to top of stem using a steel ruler, and the stem diameter was measured using a Vernier caliper. At harvest, plant samples were separated into leaf, stem and root, and then washed with distilled water and dried at 75 • C for 72 h to determine the biomass production.

Determination of Photosynthetic Parameters
The measurements were taken before harvest during the period of 9:00-11:00 a.m. on sunny days of September 8, 11 and 15, 2020 (Data were averaged from these three days). The fifth fully expanded leaf was selected to determine net photosynthetic rate, stomatal conductance, and transpiration rate using a Li-6800 portable photosynthesis system (LI-COR, Lincoln, USA). Throughout the measurements, the saturating photosynthetically active radiation was 1600 µmol m −2 s −1 , leaf temperature in the leaf chamber was set to 25 ± 1 • C. Leaf gas exchange parameters were measured at 410 ppm and 710 ppm CO 2 for plants grown under ACO 2 and eCO 2 , respectively. Water use efficiency was calculated as the ratio of photosynthetic rate and transpiration rate. Meanwhile, leaf chlorophyll a and b concentrations were extracted with 80% (v/v) acetone and measured by spectrophotometry at 663 nm and 645 nm [37].

Plant N, P and K Measurements
The oven-dried leaf, stem and root samples were ground into fine powder and then digested with 98% sulfuric acid and 30% hydrogen peroxide. Concentrations of N, P and K were determined using the micro-Kjeldahl method, vanadium molybdate yellow colorimetric method and flame photometry, respectively [38]. The N, P or K accumulations were multiplied by the N, P or K concentrations with the biomass.

Statistical Analysis
The data were statistically analyzed using the IBM SPSS Statistics 19.0 (SPSS Inc., Chicago, IL, USA). The results were presented as means ± standard error (SE, n = 3). The effects of CO 2 and variety on variables were analyzed using a two-way ANOVA. Significant differences among treatments were compared by the Tukey's Multiple Range Test at p < 0.05.

Plant Growth Traits
The eCO 2 significantly increased the growth of both mulberry varieties (Table 1). The plant height was 8.6% and 16.8% higher for QiangSang and NongSang under eCO 2 than under ACO 2 (p < 0.01), respectively. Similarly, eCO 2 also increased leaf number by 23.0% and 14.3% of QiangSang and NongSang (p < 0.01), respectively. CO 2 enrichment increased stem diameter by 11% in both varieties compared to their respective controls (p < 0.05), whereas the difference between two varieties was not significant (p > 0.05). As a result, leaf, stem, root and total plant biomass production of QiangSang and NongSang grown under eCO 2 were significantly increased by 29.9% and 25.5%, 30.8% and 33.0%, 10.5% and 42.0%, and 22.4% and 33.2% than their ACO 2 counterparts, respectively. However, no significantly different effects on total plant biomass accumulation were found between these two varieties (p > 0.05).

Leaf Photosynthetic Traits
eCO 2 induced significant changes in leaf gas exchange parameters of both mulberry varieties (Figure 1). Decreased net photosynthetic rate under eCO 2 was recorded in both mulberry varieties ( Figure 1A). Net photosynthetic rate decreased from 19.2 to 15.6 µmol m −2 s −1 (19.0%) in QiangSang whereas from 20.0 to 16.6 µmol m −2 s −1 (17.0%) in Nongsang ( Figure 1A). Significant reductions in stomatal conductance of 39.7% and 41.8% (p < 0.001, Figure 1B), or transpiration rate of 44.9% and 47.7% (p < 0.001, Figure 1C) under eCO 2 were recorded for QiangSang and NongSang, respectively. Because of lower transpiration rates, both varieties showed an increase in water use efficiency than their respective controls ( Figure 1D). Meanwhile, concentrations of leaf chlorophyll a, chlorophyll b and chlorophyll a+b were significantly lower under eCO 2 than in ACO 2 for both mulberry varieties, but were significantly greater in NongSang than in QiangSang under both eCO 2 and ACO 2 ( Figure 2). chlorophyll a+b were significantly lower under eCO2 than in ACO2 for both mulberry varieties, but were significantly greater in NongSang than in QiangSang under both eCO2 and ACO2 (Figure 2).

Plant Tissue N, P, and K Concentrations
Concentrations of N and P in both leaves and roots under eCO2 were significantly lower in both mulberry varieties ( Figure 3A,B,G,H), while stem N and P concentrations were not affected by CO2 enrichment ( Figure 3D,E). Significant reductions of leaf and root N concentrations under eCO2 were 20.0% and 21.6% in QiangSang, while 17.6% and (C) transpiration rate; and (D) water use efficiency. Data (means ± SE, n = 3) followed by different letters indicate significant differences (p < 0.05) as revealed by Tukey's test. Statistical comparisons (two-way ANOVA) between varieties and CO 2 treatments as well as their interaction (CO 2 × variety) are presented for each variable. chlorophyll a+b were significantly lower under eCO2 than in ACO2 for both mulberry varieties, but were significantly greater in NongSang than in QiangSang under both eCO2 and ACO2 ( Figure 2).

Plant Tissue N, P, and K Concentrations
Concentrations of N and P in both leaves and roots under eCO2 were significantly lower in both mulberry varieties (Figure 3A,B,G,H), while stem N and P concentrations were not affected by CO2 enrichment (Figure 3D,E). Significant reductions of leaf and root N concentrations under eCO2 were 20.0% and 21.6% in QiangSang, while 17.6% and var. QiangSang and NongSang) grown under ambient CO 2 (ACO 2 , 410/460 ppm, daytime/nighttime) and elevated CO 2 (eCO 2 , 710/760 ppm) levels. Data (means ± SE, n = 3) followed by different letters indicate significant differences (p < 0.05) as revealed by Tukey's test. Statistical comparisons (two-way ANOVA) between varieties and CO 2 treatments as well as their interaction (CO 2 × variety) are presented for each variable.

Plant Tissue N, P, and K Concentrations
Concentrations of N and P in both leaves and roots under eCO 2 were significantly lower in both mulberry varieties (Figure 3A,B,G,H), while stem N and P concentrations were not affected by CO 2 enrichment ( Figure 3D,E). Significant reductions of leaf and root N concentrations under eCO 2 were 20.0% and 21.6% in QiangSang, while 17.6% and 18.0% in NongSang ( Figure 3A,G). An average decrease of leaf and root P concentrations were 9.8% and 26.5% in QiangSang, while 7.3% and 12.2% in NongSang under eCO 2 than under ACO 2 ( Figure 3B,H). Leaf and stem K concentrations were not affected ( Figure 3C,F), but root K concentration (8.8%) were significantly decreased under eCO 2 in both mulberry varieties  Figure 3I). In contrast, stem K concentration was higher in QiangSang than NongSang under both ACO 2 and eCO 2 ( Figure 3F).
18.0% in NongSang ( Figure 3A,G). An average decrease of leaf and root P concentrations were 9.8% and 26.5% in QiangSang, while 7.3% and 12.2% in NongSang under eCO2 than under ACO2 ( Figure 3B,H). Leaf and stem K concentrations were not affected ( Figure  3C,F), but root K concentration (8.8%) were significantly decreased under eCO2 in both mulberry varieties ( Figure 3I). In contrast, stem K concentration was higher in QiangSang than NongSang under both ACO2 and eCO2 ( Figure 3F).

Plant N, P, and K Accumulations
Compared to ACO2, N accumulations in leaves, stems, roots and total plants under eCO2 were significantly respectively increased by 6.7%, 43.7%, 14.5% and 18.8% in NongSang, but not in QiangSang ( Figures 4A-D). Similar trends in P accumulations were observed in both mulberry varieties under eCO2 ( Figure 4E-H). Compared to ACO2, P accumulations in leaves, stems, roots and total plants under eCO2 were significantly en hanced by 18.7%, 31.2%, 8.5% and 19% in QiangSang, and also by 18.8%, 37.1%, 23.9% and 26.0% in NongSang, respectively ( Figures 4E-H). Both mulberry varieties showed significant increases of K accumulations in leaf, stem, root and total plant under eCO than their respective ACO2 counterparts. Meanwhile, More K accumulations in all o these plant tissues were in QiangSang than in NongSang under both ACO2 and eCO ( Figures 4I-L).

Plant N, P, and K Partitioning
On the one hand, eCO2 significantly increased N and P partitioning into the stem in both mulberry varieties (p < 0.05, Figures 5A,B, Table 2), but had no effects on N, P and K partitioning among different plant tissues (p > 0.05, Figure 5, Table 2). On the other hand QiangSang showed higher root N partitioning, leaf P and root P partitioning, whereas lower stem N and P partitioning, compared to NongSang (p < 0.05, Figure 5, Table 2) Although leaf N and K partitioning were not affected by neither CO2 nor variety, while a significant CO2 × variety interaction was observed (p < 0.05, Table 2).

Plant N, P, and K Accumulations
Compared to ACO 2 , N accumulations in leaves, stems, roots and total plants under eCO 2 were significantly respectively increased by 6.7%, 43.7%, 14.5% and 18.8% in NongSang, but not in QiangSang ( Figure 4A-D). Similar trends in P accumulations were observed in both mulberry varieties under eCO 2 ( Figure 4E-H). Compared to ACO 2 , P accumulations in leaves, stems, roots and total plants under eCO 2 were significantly enhanced by 18.7%, 31.2%, 8.5% and 19% in QiangSang, and also by 18.8%, 37.1%, 23.9% and 26.0% in NongSang, respectively ( Figure 4E-H). Both mulberry varieties showed significant increases of K accumulations in leaf, stem, root and total plant under eCO 2 than their respective ACO 2 counterparts. Meanwhile, More K accumulations in all of these plant tissues were in QiangSang than in NongSang under both ACO 2 and eCO 2 ( Figure 4I-L).

Plant N, P, and K Partitioning
On the one hand, eCO 2 significantly increased N and P partitioning into the stem in both mulberry varieties (p < 0.05, Figure 5A,B, Table 2), but had no effects on N, P and K partitioning among different plant tissues (p > 0.05, Figure 5, Table 2). On the other hand, QiangSang showed higher root N partitioning, leaf P and root P partitioning, whereas lower stem N and P partitioning, compared to NongSang (p < 0.05, Figure 5, Table 2). Although leaf N and K partitioning were not affected by neither CO 2 nor variety, while a significant CO 2 × variety interaction was observed (p < 0.05, Table 2).

Relationships between Physiological Parameters and Tissue Nutrient Concentrations
Chlorophyll a+b concentration, net photosynthetic rate and transpiration rate were significantly positively, while leaf biomass production was significantly negatively correlated to leaf N concentrations under both ACO 2 and eCO 2 (R 2 = 0.45-0.81, p < 0.05, Figure 6A,D,G,J).

Relationships between Physiological Parameters and Tissue Nutrient Concentrations
Chlorophyll a+b concentration, net photosynthetic rate and transpiration rate were significantly positively, while leaf biomass production was significantly negatively correlated to leaf N concentrations under both ACO2 and eCO2 (R 2 = 0.45-0.81, p < 0.05, Figure 6A,D,G,J).    Significantly positively correlations were observed between net photosynthetic rate or transpiration rate and leaf P concentrations under eCO 2 only (R 2 = 0.56-0.75, p < 0.05, Figure 6E,H). Meanwhile, under both ACO 2 and eCO 2 , only net photosynthetic rate was significantly positively correlated with leaf K concentration (R 2 = 0.50-0.68, p < 0.05, Figure 6F). In contrast, no relationships under both ACO 2 and eCO 2 were observed between chlorophyll a+b or leaf biomass and leaf P (p = 0.47-0.95, Figure 6B,K), and between chlorophyll a+b, transpiration rate or leaf biomass and leaf K concentration (p = 0.23-0.98, Figure 6C,I,L).

Discussion
Stimulated plant growth and biomass production under eCO2 have been reported in a wide range of tree species [16,[39][40][41]. Biomass accumulation is a complex process that is influenced by various morphological and biochemical adjustments. The increases in biomass due to higher plant height, branch number and leaf area were reported in Artemisia annua [42], Cajanus cajan [17] and Jatropha curcas [43] under 550 ppm eCO2. Similarly, our results showed that mulberry plants displayed morphological changes under 710/760 ppm eCO2, compared to 410/460 ppm ACO2 (Table 1). Both mulberry varieties had higher plant height, and more stem diameter and leaf number under 710/760 ppm eCO2, resulting in an enhanced shoot biomass production (Table 1, Figure S1C). Numerous studies on a variety of plant species have demonstrated that biomass production was generally enhanced under a range of eCO2 than under ACO2 [35,36,44,45]. Previous studies also showed that biomass production was increased by 39% and 44% in Morus alba var.Selection-13 and Kanva-2 under 550 ppm eCO2 for 90 days [33], and by 40% in Morus alba var. Gui-sang-you 62 under 710 ppm eCO2 for 120 days [35]. In the present study, plant biomass production of Morus multicauli var. QiangSang and Nong-Sang were increased by 22% and 33% under 710/760 ppm eCO2 for 129 days, respectively (Table 1). Those results indicated that biomass production of mulberry in response to eCO2 varied with varieties. Such inconsistent responses to 550-710 ppm eCO2 between plant species or varieties were in agreement with previous studies [16,41,46]. For example, the magnitude in enhancing biomass production was greater in Tectona grandis than in Butea monosperma under 550 ppm eCO2, which attributed to a better nutrient use efficiency [39]. Besides, a lower transpiration rate and higher water use efficiency under eCO2 ( Figure 1C,D) could be favorable for plant growth, due to a better water conserva- Figure 7. Relationships between net photosynthetic rate and stomatal conductance (A), and transpiration rate (B); transpiration rate and stomatal conductance (C).Linear regressions were fitted to data for two mulberry varieties (Morus multicaulis Perr. var. QiangSang and NongSang) grown under ambient CO 2 (ACO 2 , 410/460 ppm, daytime/nighttime) and elevated CO 2 (eCO 2 , 710/760 ppm) levels. Open triangles and closed triangles represent data under ACO 2 and eCO 2 , respectively.

Discussion
Stimulated plant growth and biomass production under eCO 2 have been reported in a wide range of tree species [16,[39][40][41]. Biomass accumulation is a complex process that is influenced by various morphological and biochemical adjustments. The increases in biomass due to higher plant height, branch number and leaf area were reported in Artemisia annua [42], Cajanus cajan [17] and Jatropha curcas [43] under 550 ppm eCO 2 . Similarly, our results showed that mulberry plants displayed morphological changes under 710/760 ppm eCO 2 , compared to 410/460 ppm ACO 2 (Table 1). Both mulberry varieties had higher plant height, and more stem diameter and leaf number under 710/760 ppm eCO 2 , resulting in an enhanced shoot biomass production (Table 1, Figure S1C). Numerous studies on a variety of plant species have demonstrated that biomass production was generally enhanced under a range of eCO 2 than under ACO 2 [35,36,44,45]. Previous studies also showed that biomass production was increased by 39% and 44% in Morus alba var.Selection-13 and Kanva-2 under 550 ppm eCO 2 for 90 days [33], and by 40% in Morus alba var. Guisang-you 62 under 710 ppm eCO 2 for 120 days [35]. In the present study, plant biomass production of Morus multicauli var. QiangSang and NongSang were increased by 22% and 33% under 710/760 ppm eCO 2 for 129 days, respectively (Table 1). Those results indicated that biomass production of mulberry in response to eCO 2 varied with varieties. Such inconsistent responses to 550-710 ppm eCO 2 between plant species or varieties were in agreement with previous studies [16,41,46]. For example, the magnitude in enhancing biomass production was greater in Tectona grandis than in Butea monosperma under 550 ppm eCO 2 , which attributed to a better nutrient use efficiency [39]. Besides, a lower transpiration rate and higher water use efficiency under eCO 2 ( Figure 1C,D) could be favorable for plant growth, due to a better water conservation in future eCO 2 scenario [47]. Therefore, eCO 2 may have a fertilizer effect, especially under favorable water and nutrient conditions. The effect of eCO 2 on plant biomass production largely occurs through increased photosynthetic rates [48], and the mechanisms of photosynthetic stimulation by eCO 2 have been reported in many species, such as Artemisia annua [42], Camellia sinensis [49], cassava [50], grapevine [3] and Tabebuia rosea [51]. In mulberry, leaf photosynthetic rates were greatly increased by 27-32% by 550-800 ppm eCO 2 [32][33][34]. This is because under ACO 2 , CO 2 supply is often a limit to growth and eCO 2 accelerates carboxylation processes. However, a down-regulation of leaf photosynthesis was also observed in pot experiments [21,52] or under field conditions [4]. The effect of eCO 2 on growth and photosynthesis was variable throughout plant growth stage and/or time of CO 2 exposure. For example, compared to Glycine max grown under ACO 2 , photosynthetic rates were higher after 8 weeks, while much lower after 12 weeks of exposure to 1,000 ppm eCO 2 [53]. Carbon gain in rice under 695 ppm eCO 2 was increased by 22-79% during the vegetative growth, but decreased to −12-+5% after grain-filling, leading to a 7-22% net increase for the whole season [54]. Net photosynthetic rate of Lycium barbarum displayed a downward trend at 90 and 120 days under 760 ppm eCO 2 [55]. The positive effects of 600 ppm eCO 2 on growth and chlorophyll content were greater in 20 days old than in 40 days old plants, but not on those in 41-65 days old mungbean plants [56]. Similarly, a down-regulation or decline of photosynthetic capacity and chlorophyll concentration for such photosynthetic acclimations was also evidenced for mulberry plants under 710 ppm eCO 2 for 120 days ( Figures 1A and 2). The following mechanisms could explain these decreases in photosynthetic parameters under eCO 2 , as the limited pot space had most likely restricted both the growth and functioning of the root system, leading to a decrease of nutrient uptake and hence a decreased movement of photosynthates to roots. Soil nutrients might also not be sufficient for matching up with CO 2 assimilation or photosynthesis to greater plant biomass production under eCO 2 . We observed that 18-22% and 7-10% of leaf N and P concentrations were decreased, and N and P partitioning into stems were increased in both mulberry varieties under eCO 2 ( Figure 5A,B). Greater positive relationships between net photosynthetic rate and leaf N concentration under ACO 2 than under eCO 2 ( Figure 6D) confirmed that a photosynthetic down-regulation was due to reductions of resource availability [57]. Since RuBisco protein is determined by leaf N allocation [9], the fraction of N allocated to RuBisco would be decreased under eCO 2 [21,54], leading to deficiencies in both amount and activity of Rubisco protein or surplus of C for synthesis of secondary compounds under eCO 2 [58,59]. Moreover, significantly positively greater relationships between leaf N concentration and chlorophyll a+b, net photosynthesis rate or transpiration rate under 410/460 ppm CO 2 than under 710/760 ppm eCO 2 (Figure 6A,D,G), indicating that N limitation under eCO 2 was the cause of photosynthetic acclimation, which was more pronounced in N-deficient plants [60]. Furthermore, eCO 2 and low N supply decreased activities of some antioxidant enzymes and thus increased accumulation of reactive oxygen species [61,62]. These changes in oxidative stress could accelerate the degradation of chlorophyll ( Figure 3) and eventually induce senescence [63], while eCO 2 -induced changes were mainly displayed as a general down-regulation of leaf carbohydrate metabolism [58,59]. Nevertheless, net assimilation rates measured at 350 or at 700 ppm CO 2 were not significantly different, neither [60].
A reduction in mineral concentrations has been frequently reported in wood plants under 550-800 ppm eCO 2 [14,18,64,65]. Similarly, the concentrations of N and P in both mulberry varieties were decreased under 710/760 ppm CO 2 (Figure 3). A significantly negative linear correlation between leaf N concentration and leaf biomass production (R 2 = 0.50-0.55, p < 0.05, Figure 6J) suggested that greater carbohydrate accumulation under eCO 2 had diluted leaf nutrient concentrations. However, plant tissues with different metabolic pathways may exhibit different responses to eCO 2 . Meta-analysis showed that leaf N concentrations were reduced by 14%, which was higher than 9% of N decrease in roots [66]. In this present study, leaf and root N and P concentrations were decreased in both mulberry varieties, whereas stem N, P and K concentrations were not influenced by eCO 2 , indicating that stem as a support structure for plants had less sensitivity to eCO 2 .
Therefore, it seems that the nutrient dilution effect may be plant tissue or organ dependent under eCO 2 . Bloom et al. [67] suggested that eCO 2 inhibited NO 3 − assimilation into organic N-compounds in wheat and Arabidopsis, which plays a major role in the CO 2 acclimation and decline of photosynthesis, so an inhibition of NO 3 − assimilation could be the explanation for the decrease of leaf N concentration in our study (Figure 3A,D,G). In addition, net photosynthetic rate was significantly positive correlated to transpiration rates and stomatal conductance (R 2 = 0.40-0.72, p < 0.05, Figure 7A,B). The reduced stomatal conductance under eCO 2 resulted in a decrease in transpiration rate ( Figures 1B and 7C), thus a declined transpiration-driven mass flow of nutrients from roots to leaves. In the present study, a significantly positive correlation between leaf N or P concentrations and transpiration rate under 710/760 ppm eCO 2 (R 2 = 0.45-0.75, p < 0.05, Figure 6G,H) did give such a piece of substantial evidence. Despite the fact that 700 or 790 ppm eCO 2 decreased foliar K in Coffea [68] and Flindersia brayleyana [69], we found that leaf K concentration was not significant change in both mulberry varieties under 710/760 ppm eCO 2 ( Figure 3C), but the significantly positive correlation between net photosynthetic rate and leaf K concentration under eCO 2 (R 2 = 0.50, p < 0.05, Figure 6F) suggested that high level of leaf K might have accelerated the translocation of products of photosynthesis [70], and an increase of leaf K could alleviate photosynthetic acclimation [11,70].
We found that P and K accumulations in leaves, stems, roots and total plant were increased under 710/760 ppm eCO 2 because of the corresponded biomass increase, while significant increases in N accumulation were only found in NongSang ( Figure 4). These results suggested that mulberry would require more soil nutrients to maintain its constant growth under eCO 2 , so that nutrient dynamics in the soil-plant systems is most likely to be altered under a future climate change scenario. It had been proved that soil available N and P showed a decreasing trend under eCO 2 [39], and an increasing N and P supply would alleviate or mitigate the photosynthetic acclimation [21,60]. Consequently, more fertilizers (especially N and P) would be necessary to minimize the adverse effect of future CO 2 rising for a sustainable mulberry production.

Conclusions
eCO 2 had contrasting effects on tissue N, P and K concentrations, but their total uptakes of both mulberry varieties were enhanced due to the stimulation of growth under 710/760 ppm eCO 2 . Photosynthesis in both mulberry varieties was co-limited by N and P, thus an external N and P application fertilizers is required to match up in parallel with a future increase of atmospheric CO 2 levels for a sustainable enhancement of mulberry plantation.