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Does precipitation mediate the effects of elevated CO2 on plant growth in the grass species Stipa grandis?
Environmental and Experimental Botany 131 (2016) 146–154
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Environmental and Experimental Botany journal homepage: www.elsevier.com/locate/envexpbot
Does precipitation mediate the effects of elevated CO2 on plant growth in the grass species Stipa grandis? Yaohui Shia , Guangsheng Zhoua,* , Yanling Jiangb , Hui Wangb , Zhenzhu Xub a b
State Key Laboratory of Severe Weather, Chinese Academy of Meteorological Sciences, Beijing 100081, China State Key Laboratory of Vegetation and Environmental Change, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China
A R T I C L E I N F O
Article history: Received 10 March 2016 Received in revised form 22 July 2016 Accepted 23 July 2016 Available online 27 July 2016 Keywords: Biomass Nocturnal respiration Elevated CO2 Photosynthetic capacity Precipitation change Stipa grandis
A B S T R A C T
Elevated atmospheric CO2 concentrations and the simultaneous change in precipitation patterns jointly affect plant growth directly or indirectly. The temperate grassland in northern China is primarily located in arid and semi-arid regions and is sensitive to climatic change, particularly to changes in precipitation. Understanding the effects of precipitation on temperate grasslands is essential to the development of countermeasures to cope with the negative effects of climate change. In this study, we aimed to quantify the effects of precipitation and elevated CO2 by exploring the eco-physiological mechanisms of Stipa grandis (a dominant species in typical steppe), which included tests of morphological parameters, photosynthetic capacity, dark respiration (Rn) and biomass accumulation, using open-top chambers (OTCs). S. grandis showed a photosynthetic down-regulation under elevated CO2 due to the decrease in leaf nitrogen (N). Rn was limited by elevated CO2 as a result of the dilution of leaf N, but the effect of precipitation change on Rn was primarily attributed to the change in leaf mass per area (LMA) and nocturnal stomatal conductance (gsn). Precipitation increased the total leaf area primarily by increasing leaf numbers; however, elevated CO2 increased the total leaf area by enlarging the single leaf area. Under conditions of either a serious deficit or an abundance of precipitation, the effect of elevated CO2 on plant biomass was weakened. Thus, Precipitation change mediated the effects of elevated CO2 on S. grandis. ã 2016 Elsevier B.V. All rights reserved.
1. Introduction The atmospheric CO2 concentration has been increasing from the preindustrial level of approximately 280 ppm to the present level of 390 ppm and is expected to exceed 550 ppm under a medium (RCP4.5) scenario in the 21 st century (IPCC, 2013). With the increase of greenhouse gases, the precipitation pattern is projected to change, and extreme precipitation events will increase (IPCC, 2013). An elevated atmospheric CO2 concentration and simultaneous precipitation change affect plant physiology (photosynthesis and respiration) and growth either directly or indirectly (Weltzin et al., 2003; Sun et al., 2009; Ghannoum et al., 2010; Yu et al., 2012; Xu et al., 2013, 2014; van der Kooi et al., 2016). Photosynthesis is commonly stimulated with the (experimental) elevation of atmospheric CO2 concentration. However,
Abbreviations: Ci, intercellular CO2 concentration; Cin, nocturnal intercellular CO2 concentration; gs, stomatal conductance; gsn, nocturnal stomatal conductance; LMA, leaf mass per area; Pn, net photosynthetic rate; WUE, water use efficiency; Rn, nocturnal (dark) respiration. * Corresponding author. E-mail address: [email protected] (G. Zhou). http://dx.doi.org/10.1016/j.envexpbot.2016.07.011 0098-8472/ ã 2016 Elsevier B.V. All rights reserved.
photosynthetic down-regulation with elevated CO2 has also been observed, and the degree of down-regulation depends on the species and the environment (Ainsworth and Long, 2005; Kumar et al., 2014). Plant dark respiration (Rn) is the aerobic respiration in the absence of light, which is affected by the external environment, including moisture and CO2. Whether plant Rn increases, decreases or shows no change in conditions of elevated CO2 has been debated, yet no consensus has emerged on the response mechanism (González-Méler et al., 2004; Ayub et al., 2011; Crous et al., 2012; Tan et al., 2013; Markelz et al., 2014). The response of nocturnal stomatal conductance (gsn) to increasing CO2 concentrations may be different from the daytime response, but the response of gsn is currently unknown. Thus, dark respiration constitutes one of the knowledge gaps in climate change modeling (Atkin et al., 2010; Zeppel et al., 2012; Markelz et al., 2014). Biomass production, as a final result of photosynthesis and respiration, is an indication of the capability of a plant to store carbon in response to environmental change (Kimball et al., 2002; Zhou et al., 2011). The effect of elevated CO2 on plants may depend on the plant metabolism and would be largely influenced by precipitation patterns. Increases in CO2 are expected to enhance biomass production with the stimulation of net photosynthetic CO2 uptake
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in C3 plants (Ainsworth and Long, 2005; Bloom et al., 2010; Zhou et al., 2011; Xu et al., 2013, 2014; van der Kooi et al., 2016). Although this stimulation occurs regardless of water conditions in C3 species, C4 plants benefit from elevated CO2 only under a water-deficit rather than when the water supply is sufficient (Leakey et al., 2006; Morgan et al., 2004; Xu et al., 2013, 2014, 2015; Manderscheid et al., 2014; Zelikova et al., 2015; van der Kooi et al., 2016). Based on the results of Morgan et al. (2011), CO2 enrichment does not significantly increase the aboveground biomass of C3 grasses when precipitation is sufficient precipitation, but the increase is notable under drought. van der Kooi et al. (2016) found that the differences in the responses of C4 crops to the interactive effects of elevated CO2 and change in water status remained when they were pooled per life form, i.e., annuals vs. perennials. Thus, the effects of plant metabolism are likely important in the responses to elevated CO2 and water availability. Quantifying the relationship between precipitation change and the effects of CO2 enrichment on plants is urgently required to determine the integrated responses of plants to environmental changes (Xu et al., 2013; Manderscheid et al., 2014; van der Kooi et al., 2016). Grass-dominated, dry rangelands cover over 30% of the terrestrial surface of the earth and provide most of the forage for the world’s domestic livestock (Morgan et al., 2011). These grasslands are primarily limited by water because they are mainly located in arid and semi-arid regions (Weltzin et al., 2003; Zelikova et al., 2015). Stipa grandis, a C3 perennial bunch grass of the Graminaceae, is a dominant species in the typical steppe in northern China, which is an area that has experienced severe degradation during recent decades and is also sensitive to climate change (Bai et al., 2004; Zhang et al., 2007; Xu et al., 2014; Seddon et al., 2016). S. grandis is widely distributed from the eastern to the middle temperate grasslands of China. The distribution of this grass is primarily affected by precipitation (annual precipitation: 250–350 mm) and temperature (the range of 10 C annual active accumulated temperature is 1800–2100 C). The growing season of S. grandis generally begins in May and ends in August.With high forage value, S. grandis is important for domestic livestock in Inner Mongolia (Bai et al., 2004; Zhang et al., 2007; Xu et al., 2014). Previous studies on S. grandis were primarily concerned with the effect of precipitation change; whereas the interaction between precipitation change and CO2 concentration remains unclear (Yu et al., 2012; Xu et al., 2014; van der Kooi et al., 2016). The elevation of CO2 concentration and change in precipitation will occur simultaneously in the future (IPCC, 2013). In this study, we simulated the interactive effects of elevated CO2 and precipitation change on S. grandis by open-top chambers (OTCs) to (1) quantify the relationship between precipitation and the effects of elevated CO2 on S. grandis; (2) understand the effect of elevated CO2 concentration on photosynthesis of S. grandis; and (3) determine Rn, gsn and their response mechanisms to elevated CO2 and precipitation change. 2. Materials and methods 2.1. Plant materials and experimental design In this study, S. grandis seeds were collected in the autumn of the year before the experiment from the natural grasslands in Xilinhot (44 0800 N, 117 0500 E), Inner Mongolia, China. The soil was also collected from the original grassland in Xilinhot and was placed into plastic pots (10.9 cm in top diameter, 8.5 cm in bottom diameter, and 9.5 cm in height). The soil type belongs to a castanozem, and the soil organic carbon, total nitrogen and available nitrogen concentrations ( SE) were 12.31 0.19 g kg1, 1.45 0.02 g kg1 and 81.61 0.71 mg kg1, respectively. The seeds were sterilized in a 0.5% potassium permanganate solution for
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8 min before sowing. The seedlings were first cultured in a greenhouse (day/night temperatures: 26–28 C/18–20 C; maximum illumination intensity: ca. 1000 mmol m2 s1). Four healthy seedlings with a uniform growth pattern were retained in each pot when the fourth leaf appeared. A total of 60 pots were randomly selected and moved into six OTCs (10 pots in each chamber) on May 23. The hexagonal OTCs were fabricated using an aluminum frame lined with colorless transparent glass and had a length and height of 0.85 m and 1.8 m, respectively. This experiment was designed with two factors: two levels of CO2 concentration (ambient CO2, 390 ppm; elevated CO2, 550 ppm), and five levels of precipitation (30%, 15%, control, +15% and +30%, which were approximately equal to 151, 184, 216, 248 and 281 mm precipitation, respectively). The local average precipitation from June to August during the 30 years from 1978 to 2007 in the seed provenances determined the precipitation control (Supplementary, Table 1). Six OTCs were used for the two CO2 concentration treatments (i.e., three replicates for each CO2 concentration). In each OTC, 10 pots were used for five precipitation treatments, with two replicates (two pots, each with four plants) for each precipitation level; therefore, the total was six replicates (six pots) per treatment. All OTCs were constructed outside the greenhouse. Pure CO2 gas from a cylinder (Chao Hong Ping Gas Co. Ltd, Beijing, China) was supplied for 24 h. The input of CO2 gas was automatically controlled, and an air sample from the middle of the chamber was drawn into a CO2 sensor (eSENSE-D; SenseAir, Delsbo, Sweden) to monitor the concentration change every minute. The monthly precipitation (mm) for each level was converted into an irrigation amount (ml), which was supplied every 3 days. The CO2 enrichment and irrigation began on May 31, 2011. Because of the greenhouse effect of the OTC, an air-exhaust blower was mounted at the base of each OTC to lower the temperature. The air temperature was monitored using thermocouples (HOBO S-TMB-M006; Onset Computer Co., Bourne, MA, USA) installed at a height of 75 cm in and out of the chambers. The monthly mean temperature inside the OTCs was 27.7 C and 26.5 C in July and August, respectively, compared with outside temperatures of 26.9 C and 25.7 C, respectively; the differences were approximately 0.8 C. 2.2. Observation items 2.2.1. Leaf gas exchange parameters The leaf gas exchange parameters were measured using an open gas exchange system (LI-6400F; LI-COR, Inc., Lincoln, NE, USA). The net photosynthetic rate (Pn, mmol CO2 m2 s1), stomatal conductance (gs, mol H2O m2 s1), transpiration rate (E), intercellular CO2 concentration (Ci, mmol H2O m2 s1) and instantaneous water use efficiency (WUE = Pn/E, mmol CO2 mmol1 H2O) were measured for healthy and fully expanded leaves, on sunny days from 9:00 to 11:00 a.m. The measurements were conducted twice, in June and August. The nocturnal stomatal conductance (gsn, mol H2O m2 s1), intercellular CO2 concentration (Cin, mmol H2O m2 s1) and respiration (Rn, mmol CO2 m2 s1) were measured at night from 11:00 p.m. to 1:00 a.m. under a clear sky and calm conditions in August. Three pots were selected (one pot was randomly selected from one OTC) and observed per treatment. S. grandis leaves are linear-lanceolate, and therefore, 5–7 leaves of one plant from each pot were selected to detect the gas exchange parameters simultaneously. 2.2.2. Morphological characteristics, biomass and leaf N At the end of the experiment, three pots were selected from the total six pots of each treatment (one of the two replicates was randomly selected from each OTC) to measure morphological characteristics, biomass and leaf N. The plants of each pot were
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destructively harvested and separated into two parts (aboveground and belowground). The two parts were dried at 65 C to a constant weight and then were weighed to obtain the aboveground and belowground biomass of each pot. Before drying, the plant height, tiller and leaf numbers, and leaf area (cm2) for each pot were measured with a WinFOLIA system for root/leaf analyses (WinRhizo; Régent Instruments, Quebec, Canada). Then, the mean value was calculated for a single plant of each pot. To determine leaf parameters, single leaf area (cm2) = total leaf area/leaf numbers, and leaf mass per area (LMA, mg cm2) = leaf biomass/ leaf area. The leaf N concentration (%) was determined using a
Vario EL III elemental analyzer (Elementar Analysensysteme GmbH, Hanau, Germany). 2.3. Statistical analyses All statistical analyses on the measurements were performed using the SPSS 16.0 statistical software. The data are presented as the mean with standard error (mean SE) for each treatment. The effects of elevated CO2 and precipitation change were analyzed using two-way ANOVAs (p = 0.05). The differences between the means of the elevated CO2 or precipitation treatments were compared using Duncan’s multiple range tests at a 0.05 probability level.
Fig. 1. Effects of elevated CO2 and precipitation change on total leaf area (a), average single leaf area (b), plant height (c), leaf number (d), leaf mass per area (LMA, e) and tiller number (f). Values are the mean SE (n = 3). Different lower case letters indicate significant differences among precipitation treatments for the identical CO2 concentration (p < 0.05); * indicates significant differences between CO2 concentrations for the identical level of precipitation (p < 0.05).
Y. Shi et al. / Environmental and Experimental Botany 131 (2016) 146–154 Table 1 F-values and significance levels (*p < 0.05; **p < 0.01; ***p < 0.001) from two-way ANOVAs for the main effects and interaction of CO2 and precipitation on plant parameters.
Plant height Leaf numbers Tiller numbers Total leaf area Single leaf area LMA Aboveground biomass Belowground biomass Total biomass Leaf N Rn Ci gsn Pn June August gs June August Ci June August WUE June August
CO2 H2O
H2O
CO2
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The elevated CO2 effect under different precipitation conditions was determined by the following equation: Effect of elevated CO2 (%) = (Melevated Mambient)/Mambient Where Melevated is the mean value under elevated CO2; Mambient is the mean value under ambient CO2.
F-values
df
F-values
df
F-values
df
0.291 0.088 0.438 14.00** 16.20** 0.034 2.424 5.388* 5.148* 17.79** 17.67*** 53.89*** 1.503
1 1 1 1 1 1 1 1 1 1 1 1 1
7.391** 19.15*** 14.52*** 39.11*** 11.78*** 10.91*** 29.37*** 16.02*** 31.06*** 6.621** 16.94*** 5.150** 16.91***
4 4 4 4 4 4 4 4 4 4 4 4 4
0.041 0.671 0.578 1.583 3.619* 0.503 0.483 0.419 0.301 1.257 4.450** 0.761 0.147
4 4 4 4 4 4 4 4 4 4 4 4 4
294.0*** 49.90***
1 1
11.02*** 48.03***
4 4
2.074 2.281
4 4
993.3*** 0.029
1 1
418.4*** 31.20***
4 4
94.25*** 3.905*
4 4
The total leaf area, single leaf area, LMA, plant height, tiller and leaf numbers increased significantly with the increase in precipitation at both ambient and elevated levels of CO2 (Fig. 1 and Table 1). The linear regression was significant for the correlation between total leaf area and precipitation (p < 0.01). At the elevated level of CO2, the total leaf area and single leaf area increased, whereas the LMA, plant height, tiller and leaf numbers were not significantly affected (Fig. 1 and Table 1). Additionally, based on the results, the amount of precipitation determined the effects of elevated CO2 on the total leaf area and single leaf area, and with a serious water deficit, the effect would be exacerbated.
303.5*** 130.4***
1 1
17.55*** 11.77***
4 4
1.959 2.260
4 4
3.2. Photosynthetic parameters
248.6*** 0.473
1 1
7.165** 2.877*
4 4
4.295* 11.65***
4 4
3. Results 3.1. Morphological characteristics
In June, Pn, gs and Ci increased significantly with increased precipitation and elevated CO2 (Fig. 2a–c; Table 1). The WUE increased with elevated CO2, but the response was irregular with
Fig. 2. Effects of elevated CO2 and precipitation change on net photosynthetic rate (Pn, a), stomatal conductance (gs, b), intercellular CO2 concentration (Ci, c) and water use efficiency (WUE, d). Values are the mean SE (n = 3).
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precipitation (Fig. 2d). The effects of elevated CO2 varied with the precipitation amount; the maxima occurred at 30% for Pn and Ci, at 15% for gs, and in control conditions for WUE (Fig. 2). In August, the precipitation change produced a significant effect on Pn, with maxima at the +15% level, both under ambient and elevated CO2 conditions (Fig. 2a; Table 1). Both gs and Ci increased significantly as the precipitation increased, under either ambient or elevated levels of CO2 (Fig. 2b and c). The effect of precipitation change on WUE in August was similar to that in June (Fig. 2d). Pn decreased with time under elevated CO2, which indicated a photosynthetic down-regulation phenomenon (Fig. 2a). With elevated CO2, gs decreased under water deficit (at 30% and 15%), but values increased when well watered (+30%; Fig. 2b). Additionally, with elevated CO2, Ci increased significantly under all levels of precipitation, but WUE increased only at 15% and in the control precipitation patterns (Fig. 2c and d). 3.3. Respiration parameters at night and leaf N With increasing precipitation, Rn and gsn increased but Cin decreased significantly under both ambient and elevated CO2 levels (Fig. 3a–c; Table 1). Leaf N gradually decreased with the increment in precipitation and approximated a constant value, which fit well to a logarithmic function (p < 0.01; Fig. 3d). Compared with ambient CO2 conditions, under a CO2 concentration of 550 ppm, Rn changed by +40.0%, 14.4%, 29.0%, 36.5% and 30.5% for the 30%, 15%, control, +15% and +30% precipitation conditions, respectively (Fig. 3a). These results indicated that the effect of elevated CO2 on Rn was dependent on precipitation. With
Fig. 4. Effects of elevated CO2 and precipitation change on biomass accumulation. Values are the mean SE (n = 3). Note: the bars represent total biomass SE. For other notes, see Fig. 1.
Fig. 3. Effects of elevated CO2 and precipitation change on nocturnal respiration (Rn, a), nocturnal intercellular CO2 concentration (Cin, b), nocturnal stomatal conductance (gsn, c) and leaf N concentration (leaf N, e). Values are the mean SE (n = 3).
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elevated CO2, Cin significantly increased and Leaf N decreased, but gsn was not significantly affected (Fig. 3 and Table 1). Additionally, as shown in Fig. 3d, a serious water deficit or an abundance of water limited the effects of elevated CO2 on leaf N. 3.4. Plant biomass accumulation The aboveground biomass, belowground biomass and total biomass increased significantly with increase in precipitation (Fig. 4 and Table 1). The relationships between the total biomass and precipitation for S. grandis were described by significant linear regression functions for both the ambient (R2 = 0.77, p < 0.001) and elevated CO2 conditions (R2 = 0.87, p < 0.01; Fig. 4). Elevated CO2 increased total plant biomass by +1.1%, +11.1%, +13.1%, +11.7% and +9.8% at 151, 184, 216, 248 and 281 mm precipitation, respectively (Fig. 5a). Thus, the stimulation of CO2 enrichment was stronger under moderate precipitation, whereas the effect declined under both a severe water deficit and abundant water. 4. Discussion 4.1. Relationship between effects of elevated CO2 and precipitation condition When light is adequate, Rubisco generally limits leaf photosynthesis under ambient CO2 levels. Elevated CO2 can increase photosynthesis by increasing the carboxylation rate of Rubisco and reducing photorespiration, and an increase in plant biomass under high CO2 is directly proportional to the integral of net photosynthesis (Ainsworth and Long, 2005; Sun et al., 2009; Ghannoum et al., 2010; Zhou et al., 2011; Xu et al., 2015). Plants grow in a multifactor environment, and the effects of elevated CO2 vary with soil moisture, temperature and nitrogen nutrition (Sun et al., 2009; Zheng et al., 2010; Xu et al., 2014, 2015; Reich et al., 2016; van der Kooi et al., 2016). Therefore, the effect of elevated CO2 on plants remains debatable, particularly when combined with temperature and precipitation (Hovenden et al., 2014). With ample water available, elevated CO2 does not stimulate photosynthesis, kernel number, total leaf area, biomass or yield (Leakey et al., 2006). The effects of elevated CO2 under different water regimes on leaf respiration, leaf area and some other eco-physiological characteristics vary during growing periods (Zhou et al., 2011; Crous et al., 2012; Yu et al., 2012; van der Kooi et al., 2016). The root biomass increased proportionally under elevated CO2, which whether be affected by soil moisture is still under debate (Niklaus
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and Körner 2004; Ghannoum et al., 2010; Morgan et al., 2011; Xu et al., 2013; van der Kooi et al., 2016). In this study, we quantified the relationship between precipitation and the effects of elevated CO2 on biomass in an experiment with two concentrations of CO2 and five patterns of precipitation. Our results showed that precipitation mediated the effects of elevated CO2: the “CO2 fertilization” of plant biomass and the dilution of leaf N declined under either a serious water deficit or abundant water (Fig. 5), with serious drought severely restricting the effect of elevated CO2 on leaf area. Elevated CO2 can improve drought tolerance in plants because of a decrease in stomatal conductance, but drought also decreases Pn via down-regulation in photosynthetic capacity (Pmax, Jmax and Vcmax) under severe water shortage (Leakey et al., 2006; Hovenden et al., 2014; Xu et al., 2015; Miranda-Apodaca et al., 2015); thus, elevated CO2 mitigated the degree of change only under moderate drought stress. Summarized by van der Kooi et al. (2016), in mild to moderate drought stress, plants exposed to elevated CO2 show a delay in the effects of drought stress, which is primarily attributed to water savings and increased stomatal closure; however, under severe drought conditions, photosynthesis is fully limited by metabolic inhibition, and the effect of elevated CO2 is absent. Fig. 5 also shows that the stimulation of CO2 enrichment would convert belowground biomass to aboveground biomass with increasing precipitation. The effects of elevated CO2 on Pn, WUE and Rn also varied with the precipitation condition. Precipitation is a critical environmental factor in arid and semi-arid temperate grasslands (Weltzin et al., 2003; Bai et al., 2004; Zhang et al., 2007; Zelikova et al., 2015). In this region, the annual precipitation often varies greatly, indicating that the benefits of increasing CO2 might not be as optimal as expected. Hovenden et al. (2014) reported that the relationship between precipitation and the strength of elevated CO2 effect in grassland is seasonal rather annual; summer rainfall had a positive effect, but autumn and spring rainfall had negative effects on the response to CO2. In this study, we examined the interactive effects of elevated CO2 and precipitation change during the growing period of S. grandis in June and August, and the results were consistent with the findings of Hovenden. In natural ecosystems, plant mixtures coexist with identical or different species, and the response to climate change is species-specific and dependent on the type of competition (Morgan et al., 2004; Hovenden et al., 2014; Miranda-Apodaca et al., 2015 Hovenden et al., 2014). S. grandis, a C3 perennial bunch grass, is a principal species of typical steppe ecosystems in northern China (Zhang et al., 2007). For C3 perennials, elevated CO2 stimulates biomass accumulation under
Fig. 5. Relationship between effects of elevated CO2 and precipitation amount.
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Fig. 6. Relationships of Rn between LMA (a,b), leaf N (c,d) and gsn (e, f). (elevated CO2: a, c, e; precipitation change: b, d, f).
both well-watered and dry conditions (van der Kooi et al., 2016); thus, the enrichment of CO2 would be favorable for S. grandis. The response of forbs to elevated CO2 in well-watered conditions was higher than that of a mixture of grasses, whereas the negative effect of drought was higher in grasses than in forbs because of their lower capacities to acquire water and nutrients (MirandaApodaca et al., 2015). Such differences in species level responses to CO2 and drought may lead to changes in the composition and biodiversity of the S. grandis grasslands under future climate conditions. 4.2. Mechanisms of photosynthetic down-regulation, Rn variability and change of total leaf area In S. grandis, a photosynthetic down-regulation occurred as a result of elevated CO2. The acclimation of photosynthetic capacity can be due to a number of factors, including limited sink capacity,
decreased N content and genetic factors (Ainsworth and Long, 2005; Ainsworth and Rogers, 2007; Leakey et al., 2009; Kumar et al., 2014). In this study, leaf N content decreased under elevated CO2. The decrease in N was caused by the combination of growth dilution and assimilation inhibition (Taub and Wang, 2008; Bloom et al., 2010; Pleijel and Uddling, 2012). Rubisco, a key enzyme to plant photosynthesis, is the most abundant protein in leaves and constitutes approximately 30–50% of the soluble protein of the leaf. The content and activation state of Rubisco decrease as N decreases under high CO2, and then influence photosynthesis (Long et al., 2006; Duval et al., 2012; Kumar et al., 2014). The effects of elevated CO2 on plant respiration at night are mixed and include increases, decreases and no change (González-Méler et al., 2004; Leakey et al., 2009; Tan et al., 2013; Markelz et al., 2014). The positive effects of elevated CO2 on leaf respiration are attributed to greater LMA resulting from the stimulation in photosynthesis and the increase in gsn; the negative effects of elevated CO2 on leaf
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Table 2 Path analysis between total leaf area with single leaf area and leaf number with precipitation change. Independent variable
Correlation with total leaf area
Direct coefficient
Single leaf area Leaf numbers
0.650 0.934
0.374 0.806
respiration are attributed to the reduced demand for energy for protein turnover caused by lower leaf N (González-Méler et al., 2004; Taub and Wang, 2008; Pleijel and Uddling, 2012; Tan et al., 2013; Markelz et al., 2014). Fig. 6 shows the relationships between Rn with the LMA, leaf N and gsn under elevated CO2 and precipitation change. In this study, elevated CO2 decreased leaf N (Fig. 3d) but had no significant effect on LMA (Fig. 1e) and gsn (Fig. 3c); meanwhile, Rn showed significant linear correlation with leaf N concentration (p < 0.01) (Fig. 6c). Consequently, the depression of Rn under elevated CO2 was primarily attributed to the reduction in leaf N. Nevertheless, the stimulating effect of precipitation on Rn was primarily caused by the changes in LMA (Fig. 6b) and gsn (Fig. 6f). The total leaf area and single leaf area increased significantly with increased precipitation and elevated CO2, and the total leaf area is closely correlated with single leaf area and leaf numbers. In this study, the leaf numbers were not significantly affected under elevated CO2; therefore, under elevated CO2, the total leaf area increased because of the enlargement of single leaf area. Ainsworth et al. (2006) found that elevated CO2 prompts early cell division and late-stage cell elongation, thereby increasing single leaf area; the mechanism for this phenomenon is the induction of gene expression for cell proliferation and growth under elevated CO2. For precipitation change, path analysis showed that the effect on total leaf area was due to the changes in leaf numbers and single leaf area but was primarily attributed to the change in leaf numbers (Table 2). Our study showed that the LMA increased with increasing precipitation, whereas LMA did not change significantly under elevated CO2. Combined, these results indicated that precipitation influenced the aboveground biomass with effects on leaf numbers, single leaf area and LMA; however, the effect of elevated CO2 on increasing aboveground biomass occurred only through the enlargement of single leaf area. 5. Conclusions In this experiment, we simulated the effects of elevated CO2 concentration (550 ppm) and varied precipitation (30%, 15%, control, +15% and +30% based on the average monthly precipitation from 1978 to 2007 in locations that support populations of S. grandis) on the morphological characteristics, photosynthetic capacity, dark respiration (Rn) and biomass accumulation of S. grandis. The results showed that (1) a photosynthetic downregulation occurred under elevated CO2 in S. grandis, which was caused by the decrease in leaf N. Elevated CO2 reduced plant Rn resulting from the dilution of leaf N, and precipitation influenced Rn by changing the LMA and gsn; (2) the total leaf area, single leaf area, LMA, plant height, tiller and leaf numbers increased significantly when precipitation increased. With elevated CO2, the total leaf area and single leaf area increased and Leaf N decreased, but the LMA, plant height, tiller and leaf numbers were not affected. Therefore, precipitation influenced aboveground biomass by combined effects on leaf numbers, single leaf area and LMA, whereas elevated CO2 promoted aboveground biomass only by enlarging the single leaf area; (3) the effects of elevated CO2 on the eco-physiological characteristics and biomass depended on
Indirect coefficient Single leaf area
Leaf number
– 0.128
0.276 –
precipitation conditions, and either a serious water deficit or an abundance of water limited the effects of elevated CO2. Author contribution G. Zhou, Y. Jiang, Z. Xu and Y. Shi conceived and designed the experiments. Y. Shi, H. Wang and Y. Jiang performed the experiments. Y. Shi and H. Wang analyzed the data and wrote the manuscript. The authors declare that they have no conflict of interest. Acknowledgments The China Special Fund for Meteorological Research in the Public Interest (Major projects; GYHY201506001-3) and the State Key Development Program of Basic Research (2010CB951300) jointly supported this study. We thank Jian Song, Jingsong Sun, Xiaohong Sun, Yanmin Zhang, Yanhui Hou, Xiaomin Lv and Zhixiang Yang for their support and assistance. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. envexpbot.2016.07.011. References Ainsworth, E.A., Long, S.P., 2005. What have we learned from 15 years of free-air CO2 enrichment (FACE)? A meta-analytic review of the responses of photosynthesis, canopy. New Phytol. 165 (2), 351–371. Ainsworth, E.A., Rogers, A., 2007. The response of photosynthesis and stomatal conductance to rising CO2: mechanisms and environmental interactions. Plant Cell Environ. 30, 258–270. Ainsworth, E.A., Rogers, A., Vodkin, L.O., Walter, A., Schurr, U., 2006. The effects of elevated CO2 concentration on soybean gene expression: an analysis of growing and mature leaves. Plant Physiol. 142, 135–147. Atkin, O.K., Millar, H.A., Turnbull, M.H., 2010. Plant respiration in a changing world. New Phytol. 187, 268–272. Ayub, G., Smith, R.A., Tissue, D.T., Atkin, O.K., 2011. Impacts of drought on leaf respiration in darkness and light in Eucalyptus saligna exposed to industrial-age atmospheric CO2 and growth temperature. New Phytol. 190, 1003–1018. Bai, Y., Han, X., Wu, J., Chen, Z., Li, L., 2004. Ecosystem stability and compensatory effects in the Inner Mongolia grassland. Nature 431, 181–184. Bloom, A.J., Burger, M., Rubio Asensio, J.S., Cousins, A.B., 2010. Carbon dioxide enrichment inhibits nitrate assimilation in wheat and Arabidopsis. Science 328, 899–903. Crous, K.Y., Zaragoza-Castells, J., Ellsworth, D.S., Duursma, R.A., Löw, M., Tissue, D.T., Atkin, O.K., 2012. Light inhibition of leaf respiration in field-grown Eucalyptus saligna in whole-tree chambers under elevated atmospheric CO2 and summer drought. Plant Cell Environ. 35, 966–981. Duval, B.D., Blankinship, J.C., Dijkstra, P., Hungate, B.A., 2012. CO2 effects on plant nutrient concentration depend on plant functional group and available nitrogen: a meta-analysis. Plant Ecol. 213, 505–521. Ghannoum, O., Phillips, N.G., Conroy, J.P., Smith, R.A., Attard, R.D., Woodfield, R., Logan, B.A., Lewis, J.D., Tissue, D.T., 2010. Exposure to preindustrial: current and future atmospheric CO2 and temperature differentially affects growth and photosynthesis in Eucalyptus. Global Change Biol. 16, 303–319. González-Méler, M.A., Taneva, L., Trueman, R.J., 2004. Plant respiration and elevated atmospheric CO2 concentration cellular responses and global significance. Ann. Bot. 94, 647–656. Hovenden, M.J., Newton, P.C.D., Wills, K.E., 2014. Seasonal not annual rainfall determines grassland biomass response to carbon dioxide. Nature 511, 583–586. IPCC, 2013. In: Stocker, T.F., Qin, D., Plattner, G.K., Tignor, M., Allen, S.K., Boschung, J., Nauels, A., Xia, Y., Bex, V., Midgley, P.M. (Eds.), Climate Change 2013: The
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Does precipitation mediate the effects of elevated CO2 on plant growth in the grass species Stipa grandis? Yaohui Shia , Guangsheng Zhoua,* , Yanling Jiangb , Hui Wangb , Zhenzhu Xub a b
State Key Laboratory of Severe Weather, Chinese Academy of Meteorological Sciences, Beijing 100081, China State Key Laboratory of Vegetation and Environmental Change, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China
A R T I C L E I N F O
Article history: Received 10 March 2016 Received in revised form 22 July 2016 Accepted 23 July 2016 Available online 27 July 2016 Keywords: Biomass Nocturnal respiration Elevated CO2 Photosynthetic capacity Precipitation change Stipa grandis
A B S T R A C T
Elevated atmospheric CO2 concentrations and the simultaneous change in precipitation patterns jointly affect plant growth directly or indirectly. The temperate grassland in northern China is primarily located in arid and semi-arid regions and is sensitive to climatic change, particularly to changes in precipitation. Understanding the effects of precipitation on temperate grasslands is essential to the development of countermeasures to cope with the negative effects of climate change. In this study, we aimed to quantify the effects of precipitation and elevated CO2 by exploring the eco-physiological mechanisms of Stipa grandis (a dominant species in typical steppe), which included tests of morphological parameters, photosynthetic capacity, dark respiration (Rn) and biomass accumulation, using open-top chambers (OTCs). S. grandis showed a photosynthetic down-regulation under elevated CO2 due to the decrease in leaf nitrogen (N). Rn was limited by elevated CO2 as a result of the dilution of leaf N, but the effect of precipitation change on Rn was primarily attributed to the change in leaf mass per area (LMA) and nocturnal stomatal conductance (gsn). Precipitation increased the total leaf area primarily by increasing leaf numbers; however, elevated CO2 increased the total leaf area by enlarging the single leaf area. Under conditions of either a serious deficit or an abundance of precipitation, the effect of elevated CO2 on plant biomass was weakened. Thus, Precipitation change mediated the effects of elevated CO2 on S. grandis. ã 2016 Elsevier B.V. All rights reserved.
1. Introduction The atmospheric CO2 concentration has been increasing from the preindustrial level of approximately 280 ppm to the present level of 390 ppm and is expected to exceed 550 ppm under a medium (RCP4.5) scenario in the 21 st century (IPCC, 2013). With the increase of greenhouse gases, the precipitation pattern is projected to change, and extreme precipitation events will increase (IPCC, 2013). An elevated atmospheric CO2 concentration and simultaneous precipitation change affect plant physiology (photosynthesis and respiration) and growth either directly or indirectly (Weltzin et al., 2003; Sun et al., 2009; Ghannoum et al., 2010; Yu et al., 2012; Xu et al., 2013, 2014; van der Kooi et al., 2016). Photosynthesis is commonly stimulated with the (experimental) elevation of atmospheric CO2 concentration. However,
Abbreviations: Ci, intercellular CO2 concentration; Cin, nocturnal intercellular CO2 concentration; gs, stomatal conductance; gsn, nocturnal stomatal conductance; LMA, leaf mass per area; Pn, net photosynthetic rate; WUE, water use efficiency; Rn, nocturnal (dark) respiration. * Corresponding author. E-mail address: [email protected] (G. Zhou). http://dx.doi.org/10.1016/j.envexpbot.2016.07.011 0098-8472/ ã 2016 Elsevier B.V. All rights reserved.
photosynthetic down-regulation with elevated CO2 has also been observed, and the degree of down-regulation depends on the species and the environment (Ainsworth and Long, 2005; Kumar et al., 2014). Plant dark respiration (Rn) is the aerobic respiration in the absence of light, which is affected by the external environment, including moisture and CO2. Whether plant Rn increases, decreases or shows no change in conditions of elevated CO2 has been debated, yet no consensus has emerged on the response mechanism (González-Méler et al., 2004; Ayub et al., 2011; Crous et al., 2012; Tan et al., 2013; Markelz et al., 2014). The response of nocturnal stomatal conductance (gsn) to increasing CO2 concentrations may be different from the daytime response, but the response of gsn is currently unknown. Thus, dark respiration constitutes one of the knowledge gaps in climate change modeling (Atkin et al., 2010; Zeppel et al., 2012; Markelz et al., 2014). Biomass production, as a final result of photosynthesis and respiration, is an indication of the capability of a plant to store carbon in response to environmental change (Kimball et al., 2002; Zhou et al., 2011). The effect of elevated CO2 on plants may depend on the plant metabolism and would be largely influenced by precipitation patterns. Increases in CO2 are expected to enhance biomass production with the stimulation of net photosynthetic CO2 uptake
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in C3 plants (Ainsworth and Long, 2005; Bloom et al., 2010; Zhou et al., 2011; Xu et al., 2013, 2014; van der Kooi et al., 2016). Although this stimulation occurs regardless of water conditions in C3 species, C4 plants benefit from elevated CO2 only under a water-deficit rather than when the water supply is sufficient (Leakey et al., 2006; Morgan et al., 2004; Xu et al., 2013, 2014, 2015; Manderscheid et al., 2014; Zelikova et al., 2015; van der Kooi et al., 2016). Based on the results of Morgan et al. (2011), CO2 enrichment does not significantly increase the aboveground biomass of C3 grasses when precipitation is sufficient precipitation, but the increase is notable under drought. van der Kooi et al. (2016) found that the differences in the responses of C4 crops to the interactive effects of elevated CO2 and change in water status remained when they were pooled per life form, i.e., annuals vs. perennials. Thus, the effects of plant metabolism are likely important in the responses to elevated CO2 and water availability. Quantifying the relationship between precipitation change and the effects of CO2 enrichment on plants is urgently required to determine the integrated responses of plants to environmental changes (Xu et al., 2013; Manderscheid et al., 2014; van der Kooi et al., 2016). Grass-dominated, dry rangelands cover over 30% of the terrestrial surface of the earth and provide most of the forage for the world’s domestic livestock (Morgan et al., 2011). These grasslands are primarily limited by water because they are mainly located in arid and semi-arid regions (Weltzin et al., 2003; Zelikova et al., 2015). Stipa grandis, a C3 perennial bunch grass of the Graminaceae, is a dominant species in the typical steppe in northern China, which is an area that has experienced severe degradation during recent decades and is also sensitive to climate change (Bai et al., 2004; Zhang et al., 2007; Xu et al., 2014; Seddon et al., 2016). S. grandis is widely distributed from the eastern to the middle temperate grasslands of China. The distribution of this grass is primarily affected by precipitation (annual precipitation: 250–350 mm) and temperature (the range of 10 C annual active accumulated temperature is 1800–2100 C). The growing season of S. grandis generally begins in May and ends in August.With high forage value, S. grandis is important for domestic livestock in Inner Mongolia (Bai et al., 2004; Zhang et al., 2007; Xu et al., 2014). Previous studies on S. grandis were primarily concerned with the effect of precipitation change; whereas the interaction between precipitation change and CO2 concentration remains unclear (Yu et al., 2012; Xu et al., 2014; van der Kooi et al., 2016). The elevation of CO2 concentration and change in precipitation will occur simultaneously in the future (IPCC, 2013). In this study, we simulated the interactive effects of elevated CO2 and precipitation change on S. grandis by open-top chambers (OTCs) to (1) quantify the relationship between precipitation and the effects of elevated CO2 on S. grandis; (2) understand the effect of elevated CO2 concentration on photosynthesis of S. grandis; and (3) determine Rn, gsn and their response mechanisms to elevated CO2 and precipitation change. 2. Materials and methods 2.1. Plant materials and experimental design In this study, S. grandis seeds were collected in the autumn of the year before the experiment from the natural grasslands in Xilinhot (44 0800 N, 117 0500 E), Inner Mongolia, China. The soil was also collected from the original grassland in Xilinhot and was placed into plastic pots (10.9 cm in top diameter, 8.5 cm in bottom diameter, and 9.5 cm in height). The soil type belongs to a castanozem, and the soil organic carbon, total nitrogen and available nitrogen concentrations ( SE) were 12.31 0.19 g kg1, 1.45 0.02 g kg1 and 81.61 0.71 mg kg1, respectively. The seeds were sterilized in a 0.5% potassium permanganate solution for
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8 min before sowing. The seedlings were first cultured in a greenhouse (day/night temperatures: 26–28 C/18–20 C; maximum illumination intensity: ca. 1000 mmol m2 s1). Four healthy seedlings with a uniform growth pattern were retained in each pot when the fourth leaf appeared. A total of 60 pots were randomly selected and moved into six OTCs (10 pots in each chamber) on May 23. The hexagonal OTCs were fabricated using an aluminum frame lined with colorless transparent glass and had a length and height of 0.85 m and 1.8 m, respectively. This experiment was designed with two factors: two levels of CO2 concentration (ambient CO2, 390 ppm; elevated CO2, 550 ppm), and five levels of precipitation (30%, 15%, control, +15% and +30%, which were approximately equal to 151, 184, 216, 248 and 281 mm precipitation, respectively). The local average precipitation from June to August during the 30 years from 1978 to 2007 in the seed provenances determined the precipitation control (Supplementary, Table 1). Six OTCs were used for the two CO2 concentration treatments (i.e., three replicates for each CO2 concentration). In each OTC, 10 pots were used for five precipitation treatments, with two replicates (two pots, each with four plants) for each precipitation level; therefore, the total was six replicates (six pots) per treatment. All OTCs were constructed outside the greenhouse. Pure CO2 gas from a cylinder (Chao Hong Ping Gas Co. Ltd, Beijing, China) was supplied for 24 h. The input of CO2 gas was automatically controlled, and an air sample from the middle of the chamber was drawn into a CO2 sensor (eSENSE-D; SenseAir, Delsbo, Sweden) to monitor the concentration change every minute. The monthly precipitation (mm) for each level was converted into an irrigation amount (ml), which was supplied every 3 days. The CO2 enrichment and irrigation began on May 31, 2011. Because of the greenhouse effect of the OTC, an air-exhaust blower was mounted at the base of each OTC to lower the temperature. The air temperature was monitored using thermocouples (HOBO S-TMB-M006; Onset Computer Co., Bourne, MA, USA) installed at a height of 75 cm in and out of the chambers. The monthly mean temperature inside the OTCs was 27.7 C and 26.5 C in July and August, respectively, compared with outside temperatures of 26.9 C and 25.7 C, respectively; the differences were approximately 0.8 C. 2.2. Observation items 2.2.1. Leaf gas exchange parameters The leaf gas exchange parameters were measured using an open gas exchange system (LI-6400F; LI-COR, Inc., Lincoln, NE, USA). The net photosynthetic rate (Pn, mmol CO2 m2 s1), stomatal conductance (gs, mol H2O m2 s1), transpiration rate (E), intercellular CO2 concentration (Ci, mmol H2O m2 s1) and instantaneous water use efficiency (WUE = Pn/E, mmol CO2 mmol1 H2O) were measured for healthy and fully expanded leaves, on sunny days from 9:00 to 11:00 a.m. The measurements were conducted twice, in June and August. The nocturnal stomatal conductance (gsn, mol H2O m2 s1), intercellular CO2 concentration (Cin, mmol H2O m2 s1) and respiration (Rn, mmol CO2 m2 s1) were measured at night from 11:00 p.m. to 1:00 a.m. under a clear sky and calm conditions in August. Three pots were selected (one pot was randomly selected from one OTC) and observed per treatment. S. grandis leaves are linear-lanceolate, and therefore, 5–7 leaves of one plant from each pot were selected to detect the gas exchange parameters simultaneously. 2.2.2. Morphological characteristics, biomass and leaf N At the end of the experiment, three pots were selected from the total six pots of each treatment (one of the two replicates was randomly selected from each OTC) to measure morphological characteristics, biomass and leaf N. The plants of each pot were
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destructively harvested and separated into two parts (aboveground and belowground). The two parts were dried at 65 C to a constant weight and then were weighed to obtain the aboveground and belowground biomass of each pot. Before drying, the plant height, tiller and leaf numbers, and leaf area (cm2) for each pot were measured with a WinFOLIA system for root/leaf analyses (WinRhizo; Régent Instruments, Quebec, Canada). Then, the mean value was calculated for a single plant of each pot. To determine leaf parameters, single leaf area (cm2) = total leaf area/leaf numbers, and leaf mass per area (LMA, mg cm2) = leaf biomass/ leaf area. The leaf N concentration (%) was determined using a
Vario EL III elemental analyzer (Elementar Analysensysteme GmbH, Hanau, Germany). 2.3. Statistical analyses All statistical analyses on the measurements were performed using the SPSS 16.0 statistical software. The data are presented as the mean with standard error (mean SE) for each treatment. The effects of elevated CO2 and precipitation change were analyzed using two-way ANOVAs (p = 0.05). The differences between the means of the elevated CO2 or precipitation treatments were compared using Duncan’s multiple range tests at a 0.05 probability level.
Fig. 1. Effects of elevated CO2 and precipitation change on total leaf area (a), average single leaf area (b), plant height (c), leaf number (d), leaf mass per area (LMA, e) and tiller number (f). Values are the mean SE (n = 3). Different lower case letters indicate significant differences among precipitation treatments for the identical CO2 concentration (p < 0.05); * indicates significant differences between CO2 concentrations for the identical level of precipitation (p < 0.05).
Y. Shi et al. / Environmental and Experimental Botany 131 (2016) 146–154 Table 1 F-values and significance levels (*p < 0.05; **p < 0.01; ***p < 0.001) from two-way ANOVAs for the main effects and interaction of CO2 and precipitation on plant parameters.
Plant height Leaf numbers Tiller numbers Total leaf area Single leaf area LMA Aboveground biomass Belowground biomass Total biomass Leaf N Rn Ci gsn Pn June August gs June August Ci June August WUE June August
CO2 H2O
H2O
CO2
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The elevated CO2 effect under different precipitation conditions was determined by the following equation: Effect of elevated CO2 (%) = (Melevated Mambient)/Mambient Where Melevated is the mean value under elevated CO2; Mambient is the mean value under ambient CO2.
F-values
df
F-values
df
F-values
df
0.291 0.088 0.438 14.00** 16.20** 0.034 2.424 5.388* 5.148* 17.79** 17.67*** 53.89*** 1.503
1 1 1 1 1 1 1 1 1 1 1 1 1
7.391** 19.15*** 14.52*** 39.11*** 11.78*** 10.91*** 29.37*** 16.02*** 31.06*** 6.621** 16.94*** 5.150** 16.91***
4 4 4 4 4 4 4 4 4 4 4 4 4
0.041 0.671 0.578 1.583 3.619* 0.503 0.483 0.419 0.301 1.257 4.450** 0.761 0.147
4 4 4 4 4 4 4 4 4 4 4 4 4
294.0*** 49.90***
1 1
11.02*** 48.03***
4 4
2.074 2.281
4 4
993.3*** 0.029
1 1
418.4*** 31.20***
4 4
94.25*** 3.905*
4 4
The total leaf area, single leaf area, LMA, plant height, tiller and leaf numbers increased significantly with the increase in precipitation at both ambient and elevated levels of CO2 (Fig. 1 and Table 1). The linear regression was significant for the correlation between total leaf area and precipitation (p < 0.01). At the elevated level of CO2, the total leaf area and single leaf area increased, whereas the LMA, plant height, tiller and leaf numbers were not significantly affected (Fig. 1 and Table 1). Additionally, based on the results, the amount of precipitation determined the effects of elevated CO2 on the total leaf area and single leaf area, and with a serious water deficit, the effect would be exacerbated.
303.5*** 130.4***
1 1
17.55*** 11.77***
4 4
1.959 2.260
4 4
3.2. Photosynthetic parameters
248.6*** 0.473
1 1
7.165** 2.877*
4 4
4.295* 11.65***
4 4
3. Results 3.1. Morphological characteristics
In June, Pn, gs and Ci increased significantly with increased precipitation and elevated CO2 (Fig. 2a–c; Table 1). The WUE increased with elevated CO2, but the response was irregular with
Fig. 2. Effects of elevated CO2 and precipitation change on net photosynthetic rate (Pn, a), stomatal conductance (gs, b), intercellular CO2 concentration (Ci, c) and water use efficiency (WUE, d). Values are the mean SE (n = 3).
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precipitation (Fig. 2d). The effects of elevated CO2 varied with the precipitation amount; the maxima occurred at 30% for Pn and Ci, at 15% for gs, and in control conditions for WUE (Fig. 2). In August, the precipitation change produced a significant effect on Pn, with maxima at the +15% level, both under ambient and elevated CO2 conditions (Fig. 2a; Table 1). Both gs and Ci increased significantly as the precipitation increased, under either ambient or elevated levels of CO2 (Fig. 2b and c). The effect of precipitation change on WUE in August was similar to that in June (Fig. 2d). Pn decreased with time under elevated CO2, which indicated a photosynthetic down-regulation phenomenon (Fig. 2a). With elevated CO2, gs decreased under water deficit (at 30% and 15%), but values increased when well watered (+30%; Fig. 2b). Additionally, with elevated CO2, Ci increased significantly under all levels of precipitation, but WUE increased only at 15% and in the control precipitation patterns (Fig. 2c and d). 3.3. Respiration parameters at night and leaf N With increasing precipitation, Rn and gsn increased but Cin decreased significantly under both ambient and elevated CO2 levels (Fig. 3a–c; Table 1). Leaf N gradually decreased with the increment in precipitation and approximated a constant value, which fit well to a logarithmic function (p < 0.01; Fig. 3d). Compared with ambient CO2 conditions, under a CO2 concentration of 550 ppm, Rn changed by +40.0%, 14.4%, 29.0%, 36.5% and 30.5% for the 30%, 15%, control, +15% and +30% precipitation conditions, respectively (Fig. 3a). These results indicated that the effect of elevated CO2 on Rn was dependent on precipitation. With
Fig. 4. Effects of elevated CO2 and precipitation change on biomass accumulation. Values are the mean SE (n = 3). Note: the bars represent total biomass SE. For other notes, see Fig. 1.
Fig. 3. Effects of elevated CO2 and precipitation change on nocturnal respiration (Rn, a), nocturnal intercellular CO2 concentration (Cin, b), nocturnal stomatal conductance (gsn, c) and leaf N concentration (leaf N, e). Values are the mean SE (n = 3).
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elevated CO2, Cin significantly increased and Leaf N decreased, but gsn was not significantly affected (Fig. 3 and Table 1). Additionally, as shown in Fig. 3d, a serious water deficit or an abundance of water limited the effects of elevated CO2 on leaf N. 3.4. Plant biomass accumulation The aboveground biomass, belowground biomass and total biomass increased significantly with increase in precipitation (Fig. 4 and Table 1). The relationships between the total biomass and precipitation for S. grandis were described by significant linear regression functions for both the ambient (R2 = 0.77, p < 0.001) and elevated CO2 conditions (R2 = 0.87, p < 0.01; Fig. 4). Elevated CO2 increased total plant biomass by +1.1%, +11.1%, +13.1%, +11.7% and +9.8% at 151, 184, 216, 248 and 281 mm precipitation, respectively (Fig. 5a). Thus, the stimulation of CO2 enrichment was stronger under moderate precipitation, whereas the effect declined under both a severe water deficit and abundant water. 4. Discussion 4.1. Relationship between effects of elevated CO2 and precipitation condition When light is adequate, Rubisco generally limits leaf photosynthesis under ambient CO2 levels. Elevated CO2 can increase photosynthesis by increasing the carboxylation rate of Rubisco and reducing photorespiration, and an increase in plant biomass under high CO2 is directly proportional to the integral of net photosynthesis (Ainsworth and Long, 2005; Sun et al., 2009; Ghannoum et al., 2010; Zhou et al., 2011; Xu et al., 2015). Plants grow in a multifactor environment, and the effects of elevated CO2 vary with soil moisture, temperature and nitrogen nutrition (Sun et al., 2009; Zheng et al., 2010; Xu et al., 2014, 2015; Reich et al., 2016; van der Kooi et al., 2016). Therefore, the effect of elevated CO2 on plants remains debatable, particularly when combined with temperature and precipitation (Hovenden et al., 2014). With ample water available, elevated CO2 does not stimulate photosynthesis, kernel number, total leaf area, biomass or yield (Leakey et al., 2006). The effects of elevated CO2 under different water regimes on leaf respiration, leaf area and some other eco-physiological characteristics vary during growing periods (Zhou et al., 2011; Crous et al., 2012; Yu et al., 2012; van der Kooi et al., 2016). The root biomass increased proportionally under elevated CO2, which whether be affected by soil moisture is still under debate (Niklaus
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and Körner 2004; Ghannoum et al., 2010; Morgan et al., 2011; Xu et al., 2013; van der Kooi et al., 2016). In this study, we quantified the relationship between precipitation and the effects of elevated CO2 on biomass in an experiment with two concentrations of CO2 and five patterns of precipitation. Our results showed that precipitation mediated the effects of elevated CO2: the “CO2 fertilization” of plant biomass and the dilution of leaf N declined under either a serious water deficit or abundant water (Fig. 5), with serious drought severely restricting the effect of elevated CO2 on leaf area. Elevated CO2 can improve drought tolerance in plants because of a decrease in stomatal conductance, but drought also decreases Pn via down-regulation in photosynthetic capacity (Pmax, Jmax and Vcmax) under severe water shortage (Leakey et al., 2006; Hovenden et al., 2014; Xu et al., 2015; Miranda-Apodaca et al., 2015); thus, elevated CO2 mitigated the degree of change only under moderate drought stress. Summarized by van der Kooi et al. (2016), in mild to moderate drought stress, plants exposed to elevated CO2 show a delay in the effects of drought stress, which is primarily attributed to water savings and increased stomatal closure; however, under severe drought conditions, photosynthesis is fully limited by metabolic inhibition, and the effect of elevated CO2 is absent. Fig. 5 also shows that the stimulation of CO2 enrichment would convert belowground biomass to aboveground biomass with increasing precipitation. The effects of elevated CO2 on Pn, WUE and Rn also varied with the precipitation condition. Precipitation is a critical environmental factor in arid and semi-arid temperate grasslands (Weltzin et al., 2003; Bai et al., 2004; Zhang et al., 2007; Zelikova et al., 2015). In this region, the annual precipitation often varies greatly, indicating that the benefits of increasing CO2 might not be as optimal as expected. Hovenden et al. (2014) reported that the relationship between precipitation and the strength of elevated CO2 effect in grassland is seasonal rather annual; summer rainfall had a positive effect, but autumn and spring rainfall had negative effects on the response to CO2. In this study, we examined the interactive effects of elevated CO2 and precipitation change during the growing period of S. grandis in June and August, and the results were consistent with the findings of Hovenden. In natural ecosystems, plant mixtures coexist with identical or different species, and the response to climate change is species-specific and dependent on the type of competition (Morgan et al., 2004; Hovenden et al., 2014; Miranda-Apodaca et al., 2015 Hovenden et al., 2014). S. grandis, a C3 perennial bunch grass, is a principal species of typical steppe ecosystems in northern China (Zhang et al., 2007). For C3 perennials, elevated CO2 stimulates biomass accumulation under
Fig. 5. Relationship between effects of elevated CO2 and precipitation amount.
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Fig. 6. Relationships of Rn between LMA (a,b), leaf N (c,d) and gsn (e, f). (elevated CO2: a, c, e; precipitation change: b, d, f).
both well-watered and dry conditions (van der Kooi et al., 2016); thus, the enrichment of CO2 would be favorable for S. grandis. The response of forbs to elevated CO2 in well-watered conditions was higher than that of a mixture of grasses, whereas the negative effect of drought was higher in grasses than in forbs because of their lower capacities to acquire water and nutrients (MirandaApodaca et al., 2015). Such differences in species level responses to CO2 and drought may lead to changes in the composition and biodiversity of the S. grandis grasslands under future climate conditions. 4.2. Mechanisms of photosynthetic down-regulation, Rn variability and change of total leaf area In S. grandis, a photosynthetic down-regulation occurred as a result of elevated CO2. The acclimation of photosynthetic capacity can be due to a number of factors, including limited sink capacity,
decreased N content and genetic factors (Ainsworth and Long, 2005; Ainsworth and Rogers, 2007; Leakey et al., 2009; Kumar et al., 2014). In this study, leaf N content decreased under elevated CO2. The decrease in N was caused by the combination of growth dilution and assimilation inhibition (Taub and Wang, 2008; Bloom et al., 2010; Pleijel and Uddling, 2012). Rubisco, a key enzyme to plant photosynthesis, is the most abundant protein in leaves and constitutes approximately 30–50% of the soluble protein of the leaf. The content and activation state of Rubisco decrease as N decreases under high CO2, and then influence photosynthesis (Long et al., 2006; Duval et al., 2012; Kumar et al., 2014). The effects of elevated CO2 on plant respiration at night are mixed and include increases, decreases and no change (González-Méler et al., 2004; Leakey et al., 2009; Tan et al., 2013; Markelz et al., 2014). The positive effects of elevated CO2 on leaf respiration are attributed to greater LMA resulting from the stimulation in photosynthesis and the increase in gsn; the negative effects of elevated CO2 on leaf
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Table 2 Path analysis between total leaf area with single leaf area and leaf number with precipitation change. Independent variable
Correlation with total leaf area
Direct coefficient
Single leaf area Leaf numbers
0.650 0.934
0.374 0.806
respiration are attributed to the reduced demand for energy for protein turnover caused by lower leaf N (González-Méler et al., 2004; Taub and Wang, 2008; Pleijel and Uddling, 2012; Tan et al., 2013; Markelz et al., 2014). Fig. 6 shows the relationships between Rn with the LMA, leaf N and gsn under elevated CO2 and precipitation change. In this study, elevated CO2 decreased leaf N (Fig. 3d) but had no significant effect on LMA (Fig. 1e) and gsn (Fig. 3c); meanwhile, Rn showed significant linear correlation with leaf N concentration (p < 0.01) (Fig. 6c). Consequently, the depression of Rn under elevated CO2 was primarily attributed to the reduction in leaf N. Nevertheless, the stimulating effect of precipitation on Rn was primarily caused by the changes in LMA (Fig. 6b) and gsn (Fig. 6f). The total leaf area and single leaf area increased significantly with increased precipitation and elevated CO2, and the total leaf area is closely correlated with single leaf area and leaf numbers. In this study, the leaf numbers were not significantly affected under elevated CO2; therefore, under elevated CO2, the total leaf area increased because of the enlargement of single leaf area. Ainsworth et al. (2006) found that elevated CO2 prompts early cell division and late-stage cell elongation, thereby increasing single leaf area; the mechanism for this phenomenon is the induction of gene expression for cell proliferation and growth under elevated CO2. For precipitation change, path analysis showed that the effect on total leaf area was due to the changes in leaf numbers and single leaf area but was primarily attributed to the change in leaf numbers (Table 2). Our study showed that the LMA increased with increasing precipitation, whereas LMA did not change significantly under elevated CO2. Combined, these results indicated that precipitation influenced the aboveground biomass with effects on leaf numbers, single leaf area and LMA; however, the effect of elevated CO2 on increasing aboveground biomass occurred only through the enlargement of single leaf area. 5. Conclusions In this experiment, we simulated the effects of elevated CO2 concentration (550 ppm) and varied precipitation (30%, 15%, control, +15% and +30% based on the average monthly precipitation from 1978 to 2007 in locations that support populations of S. grandis) on the morphological characteristics, photosynthetic capacity, dark respiration (Rn) and biomass accumulation of S. grandis. The results showed that (1) a photosynthetic downregulation occurred under elevated CO2 in S. grandis, which was caused by the decrease in leaf N. Elevated CO2 reduced plant Rn resulting from the dilution of leaf N, and precipitation influenced Rn by changing the LMA and gsn; (2) the total leaf area, single leaf area, LMA, plant height, tiller and leaf numbers increased significantly when precipitation increased. With elevated CO2, the total leaf area and single leaf area increased and Leaf N decreased, but the LMA, plant height, tiller and leaf numbers were not affected. Therefore, precipitation influenced aboveground biomass by combined effects on leaf numbers, single leaf area and LMA, whereas elevated CO2 promoted aboveground biomass only by enlarging the single leaf area; (3) the effects of elevated CO2 on the eco-physiological characteristics and biomass depended on
Indirect coefficient Single leaf area
Leaf number
– 0.128
0.276 –
precipitation conditions, and either a serious water deficit or an abundance of water limited the effects of elevated CO2. Author contribution G. Zhou, Y. Jiang, Z. Xu and Y. Shi conceived and designed the experiments. Y. Shi, H. Wang and Y. Jiang performed the experiments. Y. Shi and H. Wang analyzed the data and wrote the manuscript. The authors declare that they have no conflict of interest. Acknowledgments The China Special Fund for Meteorological Research in the Public Interest (Major projects; GYHY201506001-3) and the State Key Development Program of Basic Research (2010CB951300) jointly supported this study. We thank Jian Song, Jingsong Sun, Xiaohong Sun, Yanmin Zhang, Yanhui Hou, Xiaomin Lv and Zhixiang Yang for their support and assistance. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. envexpbot.2016.07.011. References Ainsworth, E.A., Long, S.P., 2005. What have we learned from 15 years of free-air CO2 enrichment (FACE)? A meta-analytic review of the responses of photosynthesis, canopy. New Phytol. 165 (2), 351–371. Ainsworth, E.A., Rogers, A., 2007. The response of photosynthesis and stomatal conductance to rising CO2: mechanisms and environmental interactions. Plant Cell Environ. 30, 258–270. Ainsworth, E.A., Rogers, A., Vodkin, L.O., Walter, A., Schurr, U., 2006. The effects of elevated CO2 concentration on soybean gene expression: an analysis of growing and mature leaves. Plant Physiol. 142, 135–147. Atkin, O.K., Millar, H.A., Turnbull, M.H., 2010. Plant respiration in a changing world. New Phytol. 187, 268–272. Ayub, G., Smith, R.A., Tissue, D.T., Atkin, O.K., 2011. Impacts of drought on leaf respiration in darkness and light in Eucalyptus saligna exposed to industrial-age atmospheric CO2 and growth temperature. New Phytol. 190, 1003–1018. Bai, Y., Han, X., Wu, J., Chen, Z., Li, L., 2004. Ecosystem stability and compensatory effects in the Inner Mongolia grassland. Nature 431, 181–184. Bloom, A.J., Burger, M., Rubio Asensio, J.S., Cousins, A.B., 2010. Carbon dioxide enrichment inhibits nitrate assimilation in wheat and Arabidopsis. Science 328, 899–903. Crous, K.Y., Zaragoza-Castells, J., Ellsworth, D.S., Duursma, R.A., Löw, M., Tissue, D.T., Atkin, O.K., 2012. Light inhibition of leaf respiration in field-grown Eucalyptus saligna in whole-tree chambers under elevated atmospheric CO2 and summer drought. Plant Cell Environ. 35, 966–981. Duval, B.D., Blankinship, J.C., Dijkstra, P., Hungate, B.A., 2012. CO2 effects on plant nutrient concentration depend on plant functional group and available nitrogen: a meta-analysis. Plant Ecol. 213, 505–521. Ghannoum, O., Phillips, N.G., Conroy, J.P., Smith, R.A., Attard, R.D., Woodfield, R., Logan, B.A., Lewis, J.D., Tissue, D.T., 2010. Exposure to preindustrial: current and future atmospheric CO2 and temperature differentially affects growth and photosynthesis in Eucalyptus. Global Change Biol. 16, 303–319. González-Méler, M.A., Taneva, L., Trueman, R.J., 2004. Plant respiration and elevated atmospheric CO2 concentration cellular responses and global significance. Ann. Bot. 94, 647–656. Hovenden, M.J., Newton, P.C.D., Wills, K.E., 2014. Seasonal not annual rainfall determines grassland biomass response to carbon dioxide. Nature 511, 583–586. IPCC, 2013. In: Stocker, T.F., Qin, D., Plattner, G.K., Tignor, M., Allen, S.K., Boschung, J., Nauels, A., Xia, Y., Bex, V., Midgley, P.M. (Eds.), Climate Change 2013: The
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