Effects of Elevated Atmospheric CO2 on Respiratory Rates in Mature Leaves of Two Rice Cultivars Grown at a Free-Air CO2 Enrichment Site and Analyses of the Underlying Mechanisms

Effects of Elevated Atmospheric CO2 on Respiratory Rates in Mature Leaves of Two Rice Cultivars... Abstract Respiratory CO2 efflux and O2 uptake rates in leaves change in response to the growth CO2 concentration ([CO2]). The degrees of change vary depending on the responses of cellular processes such as nitrogen (N) assimilation and accumulation of organic acids to growth [CO2]. However, the underlying mechanisms remain unclear. Here, we examined the respiratory characteristics of mature leaves of two rice varieties with different yield capacities at different growth stages under ambient and elevated [CO2] conditions at a free-air CO2 enrichment site. We also examined the effect of increased water temperature on leaf respiration. We measured the rates of CO2 efflux and O2 uptake, and determined N contents, primary metabolite contents and maximal activities of respiratory enzymes. The leaf CO2 efflux rates decreased in plants grown at elevated [CO2] in both varieties, and were higher in high-yielding Takanari than in Koshihikari. The leaf O2 uptake rates showed little change with respect to growth [CO2] and variety. The increased water temperature did not significantly affect the CO2 efflux and O2 uptake rates. The N and amino acid contents were significantly higher in Takanari than in Koshihikari. The enhanced N assimilation in Takanari may have consumed more respiratory NADH, leading to higher CO2 efflux rates. In Koshihikari, the ratio of tricarboxylic acid (TCA) cycle intermediates changed and maximal activities of enzymes in the TCA cycle decreased at elevated [CO2]. Therefore, the decreased rates of CO2 efflux in Koshihikari may be due to the decreased activities of TCA cycle enzymes at elevated [CO2]. Introduction The atmospheric CO2 concentration ([CO2]) has drastically increased since the industrial revolution. As CO2 is a direct substrate for photosynthesis, the increase in [CO2] will greatly affect photosynthesis and growth in plants. This topic has been discussed in several reviews (Ainsworth and Long 2005, Long et al. 2004, Ainsworth and Rogers 2007, Leakey et al. 2009a). Plants release about half of the fixed carbon (C) via respiration, and plant respiration is closely associated with photosynthesis and growth (Lambers et al. 2005). Therefore, studies on the responses of plant respiration to elevated [CO2] are important to understand the C balance in the whole plant. Many studies have reported that leaf respiration rates of C3 plant species change in response to changes in [CO2] (Drake et al. 1999, Wang and Curtis 2002, Gonzalez-Meler et al. 2004, Leakey et al. 2009b, Haworth et al. 2016). The extent of changes in leaf respiration rates varies depending on the plant species, developmental stage and other environmental factors (Ayub et al. 2011, Griffin et al. 2013, Markelz et al. 2014a, Markelz et al. 2014b, Watanabe et al. 2014, Aranjuelo et al. 2015). In mature leaves of soybean (Glycine max) grown at a free-air CO2 enrichment (FACE) experimental site, the rates of CO2 efflux and O2 uptake on a leaf area basis significantly increased at elevated [CO2] (Leakey et al. 2009b). Rapid increases in the rates of CO2 efflux and O2 uptake were also reported for mature leaves of tomato (Solanum lycopersicum) after transfer from ambient to elevated [CO2] in a growth chamber (Li et al. 2013). In those studies, the increase in leaf respiration rates was thought to be related to the up-regulation of genes involved in the respiratory system. Fukayama et al. (2011) also reported that expression of several respiratory genes was up-regulated under elevated [CO2] in leaves of a japonica variety of rice (Oryza sativa). In contrast, in mature leaves of Arabidopsis thaliana grown under elevated [CO2] in a growth chamber, the responses to elevated [CO2] differed between CO2 efflux and O2 uptake rates (Watanabe et al. 2014). In A. thaliana leaves, the CO2 efflux rate on a leaf fresh weight basis increased under elevated [CO2] at both ends of the light and dark periods, whereas the O2 uptake rate decreased under elevated [CO2] at the end of the light period. The respiration rate in plants is generally limited by substrate availability, enzyme capacity or the ATP consumption rate (Lambers et al. 2005, Noguchi 2005). In most cases, the rates of CO2 efflux and O2 uptake are similar; thus, the respiratory quotient (RQ: ratio of CO2 efflux rate to O2 uptake rate) is around one. However, when NADH produced in both glycolysis and the tricarboxylic acid (TCA) cycle is consumed by various cellular processes such as nitrogen (N) assimilation, the RQ value changes as suggested by the flux-balanced model (Buckley and Adams 2011). Cousins and Bloom (2004) showed that the increased consumption of reducing equivalents via NO3– assimilation decreases the rate of mitochondrial O2 consumption in wheat leaves, which can affect the RQ value. The accumulation and consumption of organic acids also affect the RQ value (Lambers et al. 2005). For example, malate production from glucose leads to an RQ of 0.667. In soybean leaves, the RQ value was reported to be higher at elevated [CO2] than at ambient [CO2] (Leakey et al. 2009b). Therefore, we should pay attention to both CO2 efflux and O2 uptake rates when investigating the mechanisms underlying the respiratory response to elevated [CO2]. At the Tsukuba FACE experimental site in central Japan, growth, photosynthesis and yield have been examined for many rice varieties, and the responses of these parameters to elevated [CO2] have been found to differ among them (Hasegawa et al. 2013, Usui et al. 2014, Ikawa et al. 2018). At this FACE site, the effect of increased water temperature has been examined using a system in which soil water is heated by heating wires under the soil surface (Usui et al. 2016). Not only the warming treatment, but also the interaction between warming and elevated [CO2] were found to affect the yields and grain qualities of rice (Usui et al. 2016). Chen et al. (2014) reported that the mature leaves of a high-yielding indica variety, Takanari, showed consistently higher photosynthesis and stomatal conductance than those of a japonica variety, Koshihikari, under both ambient and enhanced [CO2] conditions. These leaf-level characteristics in Takanari were possibly related to the 21% yield enhancement at elevated [CO2] compared with that at ambient [CO2], whereas the yield enhancement in Koshihikari was only 16% (Hasegawa et al. 2013). The N content in leaves of Takanari was higher than that in Koshihikari (Taylaran et al. 2011), resulting in a greater source capacity of Takanari leaves under both ambient and elevated [CO2] conditions (Chen et al. 2014). Another study at the FACE experimental site showed that the soil-water warming treatment reduced the photosynthetic response to elevated [CO2] at the grain-filling stage (Adachi et al. 2014). In that study, elevated water temperature increased N allocation to the panicles at the grain-filling stage, leading to decreases in leaf N and changes in photosynthetic responses at that stage. The results of these previous studies suggested that leaf photosynthesis in response to elevated [CO2] is highly sensitive to the variety, growth stage and water temperature, and that leaf N status may affect the responses to elevated [CO2]. The respiratory system is closely associated with the photosynthetic system (Noguchi and Yoshida 2008, Vanlerberghe et al. 2016), and leaf respiration rates are correlated with leaf N content (Reich et al. 1996, Tissue et al. 2002, Noguchi and Terashima 2006). Therefore, responses of respiration rates to elevated [CO2] may change depending on leaf N status. Elevated [CO2] affects various primary metabolite contents in leaves. These changes are greatly influenced by leaf N status (Stitt and Krapp 1999, Noguchi et al. 2015). The responses of primary metabolite contents should be tightly related to changes in leaf respiration rates (Araújo et al. 2012, Tcherkez et al. 2012, Tcherkez et al. 2017). However, the physiological mechanisms underlying the changes in leaf respiration rates in response to elevated [CO2] and the roles of various factors in these respiratory changes remain poorly understood. In this study, we examined the following questions. (i) Do the respiratory CO2 efflux and O2 uptake rates in leaves respond differently to elevated [CO2]? (ii) Do these responses change depending on the variety, developmental stage and water tempearture? (iii) Can the difference in the responses to elevated [CO2] be related to leaf N status? To clarify the above questions, we examined respiratory characteristics of mature leaves of two contrasting rice varieties, Koshihikari and Takanari, grown under ambient and elevated [CO2] conditions at the Tsukuba FACE experimental site. We compared their leaf respiration responses under ambient and elevated [CO2] conditions at two water temperatures, and at five different growth stages from the vegetative to the grain-filling stage. We measured the rates of CO2 efflux and O2 uptake, and determined the N and C contents of the leaves. We measured primary metabolites of the respiratory system and determined the maximal activities of respiratory enzymes. Results Effects of elevated CO2 or temperature on CO2 efflux and O2 uptake rates in leaves of plants at different growth stages, and differences in rates between varieties We measured CO2 efflux rates in mature leaves at the panicle initiation stage (early July) and the mid grain-filling stage (mid August) in 2012. We compared the responses of CO2 efflux rates between the two growth [CO2] conditions, the two varieties and the two water temperatures. The CO2 efflux rates on a leaf dry weight basis were significantly lower at elevated [CO2] than at ambient [CO2] at both growth stages (Supplementary Fig. S1a, b). The CO2 efflux rates differed significantly between varieties at both stages, and the 2°C increase in water temperature significantly decreased the CO2 efflux rates at the panicle initiation stage. To confirm these differences in the CO2 efflux rates of leaves detected in 2012, we measured CO2 efflux rates from the tillering stage (late June) to the mid grain-filling stage (mid August) in 2013. In 2013, the CO2 efflux rates were also significantly lower at elevated [CO2] than at ambient [CO2] at all growth stages (Fig. 1a–e). Although the CO2 efflux rate was significantly lower in Takanari than in Koshihikari at the tillering stage, it was significantly higher in Takanari than in Koshihikari from the panicle initiation stage to the heading stage. At the mid grain-filling stage, the difference in CO2 efflux rate between the two varieties was significant at P = 0.0716 (Supplementary Table S1). The responses to elevated [CO2] were similar between the two varieties during all growth stages (P > 0.1 for the interaction between CO2 and variety in Supplementary Table S1). The 2°C increase in water temperature resulted in small changes in the CO2 efflux rates, but a significant difference between the two temperature regimes was observed only at the early growth stage (tillering) in 2013. The CO2 efflux rates on a leaf area basis showed a similar trend to those on a leaf dry weight basis (Supplementary Fig. S2a–e). The CO2 efflux rates on a leaf area basis were significantly lower at elevated [CO2] than at ambient [CO2] at all growth stages. Similar decreases in CO2 efflux rates on a leaf area basis at elevated [CO2] were observed in six herbaceous species that were cultivated at another FACE site (Haworth et al. 2016). The CO2 efflux rates on a leaf area basis differed significantly between the two varieties at all stages except for the panicle initiation stage. At the panicle initiation stage, the difference between the two varieties was significant at P = 0.0577 (Supplementary Table S1). Fig. 1 View largeDownload slide Respiration rates of mature leaves of two rice varieties. CO2 efflux rates on a dry weight basis (a–e), O2 uptake rates on a dry weight basis (f–j) and CO2 efflux rates on a leaf nitrogen basis (k–o) were determined in mature leaves of Koshihikari and Takanari at various developmental stages in 2013. On the x-axis, A and F denote data in ambient and FACE (elevated CO2) plots, respectively. NT and ET denote normal temperature and elevated temperature, respectively. The mean and SEM are shown (n = 4). Statistical results are also shown (*P < 0.05, **P <0.01, ***P < 0.001). CO2, temp and var denote effects of CO2 concentration, temperature and variety, respectively. Fig. 1 View largeDownload slide Respiration rates of mature leaves of two rice varieties. CO2 efflux rates on a dry weight basis (a–e), O2 uptake rates on a dry weight basis (f–j) and CO2 efflux rates on a leaf nitrogen basis (k–o) were determined in mature leaves of Koshihikari and Takanari at various developmental stages in 2013. On the x-axis, A and F denote data in ambient and FACE (elevated CO2) plots, respectively. NT and ET denote normal temperature and elevated temperature, respectively. The mean and SEM are shown (n = 4). Statistical results are also shown (*P < 0.05, **P <0.01, ***P < 0.001). CO2, temp and var denote effects of CO2 concentration, temperature and variety, respectively. To examine differences between the CO2 efflux and O2 uptake rates in terms of the effects of growth [CO2], water temperature and variety, we measured O2 uptake rates from the tillering to the mid grain-filling stages in 2013. In contrast to CO2 efflux rates, O2 uptake rates on a leaf dry weight basis showed little change with respect to growth [CO2], variety and water temperature (Fig. 1f–j). The O2 uptake rates were significantly affected by growth [CO2] and variety at only one growth stage (Supplementary Table S1). The 2°C increase in water temperature resulted in a significant decrease in the O2 uptake rates at three growth stages (tillering, booting and mid grain-filling stages). Since we measured CO2 efflux and O2 uptake rates using different systems and on different days, we could not calculate exact RQ values. The ratios of CO2 efflux to O2 uptake rate were significantly lower at elevated [CO2] than at ambient [CO2] at the three growth stages, and showed similar trends to those of CO2 uptake rates (Supplementary Fig. S2f–j). There were significant differences in the ratio of CO2 efflux to O2 uptake rate between the two varieties, except at the mid grain-filling stage. Effects of elevated CO2 on leaf N and CO2 efflux rate on a leaf N basis, and differences between varieties Leaf mass per area (LMA) increased with rice development, and growth [CO2] significantly increased the LMA at two growth stages (Fig. 2a–e). The LMA significantly differed between the two varieties at the two growth stages (panicle initiation and mid grain-filling). The leaf N content on a leaf dry weight basis was significantly lower at elevated [CO2] than at ambient [CO2] at all growth stages in 2013 (Fig. 2f–j). The N leaf content was significantly higher in Takanari than in Koshihikari, except at the tillering stage in 2013. These trends were similar to those in the CO2 efflux rates on a leaf dry weight basis. Other studies also found that the leaf N content per leaf area was higher in Takanari than in Koshihikari (Taylaran et al. 2011, Chen et al. 2014). Leaf C content on a leaf dry weight basis was significantly higher in Takanari than in Koshihikari at all growth stages, and the growth [CO2] did not affect the leaf C content, except at the tillering stage in 2013 (Fig. 2k–o). The ratio of C to N content (C/N ratio) showed an opposite trend to that of leaf N content (Fig. 2p–u). Growth [CO2] increased the C/N ratio at all growth stages in 2013. The C/N ratio was significantly lower in Takanari than in Koshihikari, except at the tillering stage. The trends in leaf N and C contents in 2012 were similar to those in 2013 (Supplementary Fig. S1c–f). Fig. 2 View largeDownload slide Leaf characteristics of two rice varieties. Leaf mass per area (a–e), nitrogen content (f–j), carbon content (k–o) and ratio of carbon to nitrogen contents (p–u) were determined in mature leaves of Koshihikari and Takanari at different developmental stages in 2013. For other details, see the legend of Fig. 1. Fig. 2 View largeDownload slide Leaf characteristics of two rice varieties. Leaf mass per area (a–e), nitrogen content (f–j), carbon content (k–o) and ratio of carbon to nitrogen contents (p–u) were determined in mature leaves of Koshihikari and Takanari at different developmental stages in 2013. For other details, see the legend of Fig. 1. We calculated the CO2 efflux rate on a leaf N basis from the CO2 efflux rates on a leaf dry weight basis and N contents. In 2013, the differences between the two varieties in the CO2 efflux rate on a leaf N basis were smaller than those in CO2 efflux rate on a leaf dry weight basis (Fig. 1k–o). At the panicle initiation and heading stages, there were no significant differences in the CO2 efflux rate on a leaf N basis between the two varieties. However, there were significant differences between the two [CO2] conditions, except at the mid grain-filling stage. Similar trends were observed in 2012, although there was a significant difference in the CO2 efflux rate on a leaf N basis between the two varieties at the panicle initiation stage (Supplementary Fig. S1g, h). Effects of elevated CO2 on contents of primary metabolites and their relationships to leaf respiratory rates In leaves, contents of primary metabolites are closely related to respiration rates, and elevated [CO2] affects the contents of various primary metabolites (Stitt and Krapp 1999, Misra and Chen 2015, Noguchi et al. 2015). In rice leaves, it has been reported that elevated [CO2] affected primary metabolite contents (Onda et al. 2014). Elevated night temperature affected both O2 uptake rates and primary metabolite contents in rice leaves (Glaubitz et al. 2014, Glaubitz et al. 2015). In this study, we examined whether primary metabolite contents are related to changes in leaf respiration rates in two rice varieties. We measured the contents of various primary metabolites at the booting stage when the CO2 efflux rates were significantly different between two growth [CO2] conditions and between the two varieties (Fig. 1c). Since we had a shortage of booting-stage samples, we determined non-structural carbohydrate contents in samples collected at the heading stage, when the CO2 efflux rates also differed significantly between the two growth [CO2] conditions and between the two varieties (Fig. 1c, d). Among the amino acids measured in this study, glutamate, glutamine, aspartate, glycine and alanine were the most abundant (Fig. 3a–e). The contents of these five amino acids were significantly higher in Takanari leaves than in Koshihikari leaves. Under elevated [CO2], the contents of these major amino acids tended to decrease in Koshihikari leaves, but not in Takanari leaves. The alanine content increased in Takanari leaves at elevated [CO2]. The interaction between CO2 and variety was significant for the contents of glutamate, glycine and alanine (P < 0.01, Supplementary Table S1). Among the amino acids detected at low contents, tyrosine, serine, arginine and γ-aminobutanoic acid (GABA) showed lower contents in Koshihikari leaves at elevated [CO2] than at ambient [CO2], while their contents in Takanari leaves did not differ between ambient and elevated [CO2] conditions (Supplementary Fig. S3f, k, q, s). For these metabolites, the interaction between CO2 and variety was significant (P < 0.05, Supplementary Table S1). Fig. 3 View largeDownload slide Amino acid contents in mature leaves of two rice varieties. Contents of several amino acids, total amino acids and the ratio of glutamine to glutamate (Gln/Glu) were determined in mature leaves of Koshihikari and Takanari at the booting stage in 2013. Total amino acids are the sum of 25 measured amino acids. The unit of amino acids and total amino acids is µmol g FW–1. The mean and SEM are shown (n = 4). For other details, see the legend of Fig. 1. Fig. 3 View largeDownload slide Amino acid contents in mature leaves of two rice varieties. Contents of several amino acids, total amino acids and the ratio of glutamine to glutamate (Gln/Glu) were determined in mature leaves of Koshihikari and Takanari at the booting stage in 2013. Total amino acids are the sum of 25 measured amino acids. The unit of amino acids and total amino acids is µmol g FW–1. The mean and SEM are shown (n = 4). For other details, see the legend of Fig. 1. The branched-chain amino acids valine, leucine and isoleucine accumulated to much higher contents in Takanari leaves than in Koshihikari leaves (Fig. 3f;Supplementary Fig. S3i, j). We calculated total amino acids and TCA cycle organic acids by adding up the amounts of all 25 amino acids and the seven organic acids in the TCA cycle, respectively (Figs. 3g, 4e). The contents of total amino acids and the ratio of total amino acids to TCA cycle organic acids have been reported to increase when amino acid production is up-regulated (Hachiya et al. 2012). Both of these values were significantly higher in Takanari leaves than in Koshihikari leaves (Figs. 3g, 4f). When amino acid production is up-regulated, the ratio of glutamine to glutamate (Gln/Glu) and that of glutamine to 2-oxoglutarate (Gln/2-OG) often increase in leaves (Stitt and Krapp 1999, Noguchi et al. 2015). These ratios were higher in Takanari than in Koshihikari (P = 0.0517 for Gln/Glu in Fig. 3h, P< 0.001 for Gln/2-OG in Fig. 4g). Together, these findings suggested that the production of amino acids was up-regulated in Takanari leaves, and that NADH produced in the TCA cycle may have been consumed in the production of amino acids, leading to the higher rates of CO2 efflux in Takanari leaves than in Koshihikari leaves. Fig. 4 View largeDownload slide TCA cycle organic acid contents in mature leaves of two rice varieties. Contents of several TCA cycle organic acids, ratio of total amino acids to TCA cycle organic acids (total AA/TCA OA), ratio of glutamine to 2-oxoglutarate (Gln/2-OG) and ratio of 2-OG to isocitrate (2-OG/isocitrate) were determined in mature leaves of Koshihikari and Takanari at the booting stage in 2013. Contents of carbohydrates (starch, sucrose and glucose) in mature leaves of two rice cultivars at the heading stage in 2013. TCA cycle organic acids are the sum of seven measured TCA cycle organic acids. The unit of organic acids is µmol g FW–1; the unit of carbohydrates is µmol C g FW–1. The mean and SEM are shown (n = 4). For other details, see the legend of Fig. 1. Fig. 4 View largeDownload slide TCA cycle organic acid contents in mature leaves of two rice varieties. Contents of several TCA cycle organic acids, ratio of total amino acids to TCA cycle organic acids (total AA/TCA OA), ratio of glutamine to 2-oxoglutarate (Gln/2-OG) and ratio of 2-OG to isocitrate (2-OG/isocitrate) were determined in mature leaves of Koshihikari and Takanari at the booting stage in 2013. Contents of carbohydrates (starch, sucrose and glucose) in mature leaves of two rice cultivars at the heading stage in 2013. TCA cycle organic acids are the sum of seven measured TCA cycle organic acids. The unit of organic acids is µmol g FW–1; the unit of carbohydrates is µmol C g FW–1. The mean and SEM are shown (n = 4). For other details, see the legend of Fig. 1. In leaves of both rice varieties, the most abundant organic acids were citrate, isocitrate, 2-OG and malate (Fig. 4a–d). This pattern of organic acid accumulation in rice leaves differed from that in A. thaliana leaves, in which fumarate also accumulated (Watanabe et al. 2014). The content of 2-OG and the ratio of 2-OG to isocitrate (2-OG/isocitrate) were significantly lower at elevated [CO2] than at ambient [CO2] (P < 0.05, Fig. 4c, h), while the isocitrate content tended to be higher at elevated [CO2] (P = 0.0731, Fig. 4b;Supplementary Table S1). The contents of the other organic acids were not higher at elevated [CO2], and the contents of TCA cycle organic acids were comparable between the two [CO2] conditions (Fig. 4e). Therefore, an increase in organic acid production was not responsible for the lower CO2 efflux rate and RQ value in leaves at elevated [CO2]. In leaves of Takanari, where amino acid production may have been up-regulated, the content of TCA cycle organic acids was significantly lower than that in Koshihikari leaves (Fig. 4e); increased production of amino acids in Takanari leaves may have consumed NADH and organic acids in the TCA cycle, leading to lower contents of organic acids than in Koshihikari leaves. In leaves at elevated [CO2], carbohydrate contents often increase (Stitt and Krapp 1999, Noguchi et al. 2015). We quantified starch, sucrose and glucose as non-structural carbohydrates that are consumed as respiratory substrates in leaves. In leaves of both varieties, sucrose accumulated to the highest content, followed by starch and glucose (Fig. 4i–k). All these carbohydrates increased in Takanari leaves at elevated [CO2], whereas only glucose increased in Koshihikari leaves at elevated [CO2]. Sucrose and starch contents in Koshihikari were comparable between the two [CO2] conditions. These data showed that lower contents of carbohydrates were not responsible for the lower rates of CO2 efflux in leaves at elevated [CO2]. In Koshihikari leaves, the contents of metabolites downstream of glycolysis, such as 3-phosphoglycerate (3PGA), phosphoenolpyruvate (PEP) and pyruvate, tended to be low at elevated [CO2] (Supplementary Fig. S4m–o), similar to the results reported for A. thaliana leaves (Watanabe et al. 2014). In contrast, the contents of these metabolites were higher in Takanari leaves, and were not different between the two [CO2] conditions. In Takanari leaves, the content of dihydroxyacetone phosphate (DHAP) was higher at elevated [CO2] than that at ambient [CO2] (Supplementary Fig. S4k). The increase in DHAP content may induce increases in reactive carbonyl contents. In leaves of wheat at elevated [CO2], reactive carbonyl contents increased (Takagi et al. 2014). The contents of glucose 6-phosphate (G6P) and glucose 1-phosphate (G1P) were also significantly higher in Takanari leaves than in Koshihikari leaves (Supplementary Fig. S4e, f). The sum of measured metabolites of glycolysis was greater in Takanari leaves than in Koshihikari leaves (data not shown), in contrast to the pattern observed for TCA cycle organic acids. The photosynthetic rates may be higher in Takanari leaves than in Koshihikari leaves, even at elevated [CO2] (Chen et al. 2014). Therefore, during the daytime, larger amounts of photoassimilates may flow into glycolysis and be consumed in Takanari leaves than in Koshihikari leaves. The contents of intermediates of the Calvin–Benson cycle tended to be lower at elevated [CO2] than at ambient [CO2] in Koshihikari leaves, whereas their contents were comparable between the two [CO2] conditions in Takanari (Supplementary Fig. S4q–t). Effects of elevated CO2 on maximal activities of TCA cycle enzymes The contents of 2-OG and the 2-OG/isocitrate ratio in Fig. 4 suggested that activities of the TCA cycle enzymes may be lower at elevated [CO2] than at ambient [CO2], resulting in lower CO2 efflux rates at elevated [CO2]. Therefore, we determined the maximal activities of TCA cycle enzymes in leaves of the two rice varieties at the booting stage in 2013. In the enzymes of the TCA cycle, maximal activities of citrate synthase and NAD-dependent malate dehydrogenase could not be measured in two rice varieties. We measured maximal activities of aconitase, NAD-dependent isocitrate dehydrogenase (NAD-IDH) and NADP-dependent isocitrate dehydrogenase (NADP-ICDH) in this study. The maximal activity of aconitase was significantly higher in Koshihikari leaves than in Takanari leaves, and it decreased in Koshihikari leaves at elevated [CO2] (Fig. 5a). In Koshihikari leaves, the maximal activities of NADP-ICDH and NAD-IDH tended to be lower at elevated [CO2] than at ambient [CO2]. In Takanari leaves, the maximal activities of NADP-ICDH and NAD-IDH did not differ significantly between the two [CO2] conditions. Fig. 5 View largeDownload slide Maximal activities of three TCA cycle enzymes in mature leaves of two rice varieties. Maximal activities of aconitase, NAD-dependent isocitrate dehydrogenase and NADP-dependent isocitrate dehydogenase were determined in mature leaves of Koshihikari and Takanari at the booting stage in 2013. The mean and SEM are shown (n = 4). For other details, see the legend of Fig. 1. Fig. 5 View largeDownload slide Maximal activities of three TCA cycle enzymes in mature leaves of two rice varieties. Maximal activities of aconitase, NAD-dependent isocitrate dehydrogenase and NADP-dependent isocitrate dehydogenase were determined in mature leaves of Koshihikari and Takanari at the booting stage in 2013. The mean and SEM are shown (n = 4). For other details, see the legend of Fig. 1. Discussion We compared respiratory characteristics in the uppermost fully expanded leaves between two rice varieties cultivated at the FACE experimental site. The O2 uptake rates on a leaf dry weight basis were comparable between the two [CO2] conditions, whereas the CO2 efflux rates on a leaf dry weight basis decreased at elevated [CO2] at all growth stages (Fig. 1). From the panicle initiation stage, the CO2 efflux rates were significantly higher in leaves of Takanari, which showed higher growth and photosynthesis compared with Koshihikari. The 2°C increase in water temperature did not significantly affect the CO2 efflux rate or O2 uptake rate at any growth stage. The N and major amino acid contents were significantly higher in Takanari than in Koshihikari (Figs. 2, 3). Compared with Koshihikari leaves, Takanari leaves with their enhanced N assimilation may consume more respiratory NADH, leading to higher CO2 efflux rates than those in Koshihikari leaves. This hypothesis was supported by the findings that the organic acid contents were lower in Takanari leaves than in Koshihikari leaves, and the CO2 efflux rates on a leaf N basis were comparable between the two varieties (Figs. 1, 4). In Koshihikari leaves, the ratio of TCA cycle intermediates (2-OG/isocitrate) changed and the maximal activities of enzymes in the TCA cycle decreased at elevated [CO2] (Figs. 4, 5). The lower CO2 efflux rates at elevated [CO2] may have been due to the decreased activities of TCA cycle enzymes in Koshihikari leaves, but the activities of TCA cycle enzymes could not explain the decreased CO2 efflux rates at elevated [CO2] in Takanari leaves. Effects of elevated [CO2] on CO2 efflux rate and the related changes in rice leaves In Koshihikari leaves, the decrease in CO2 efflux rates at elevated [CO2] may have been due to the decreased activities of TCA cycle enzymes. In contrast to our study, previous studies have reported that the transcript levels of genes encoding TCA cycle enzymes were up-regulated at elevated [CO2] in mature leaves of rice (Fukayama et al. 2011), soybean (Leakey et al. 2009b) and A. thaliana (Markelz et al. 2014a, Watanabe et al. 2014). So far, the transcript levels of genes encoding TCA cycle enzymes have not been determined in Koshihikari leaves at elevated [CO2]. However, up-regulation of genes encoding respiratory enzymes at elevated [CO2] does not always correlate to increased amounts or activities of their encoded enzymes. In mature leaves of durum wheat (Triticum durum), malate dehydrogenase was the only TCA cycle enzyme that showed increased abundance at elevated [CO2] (Aranjuelo et al. 2015). Quantitative proteomics techniques may clarify which enzymes in the whole respiratory system, including the TCA cycle, increase or decrease at elevated [CO2] (Taylor et al. 2014). At elevated [CO2], a distinct increase in amino acid contents was not observed, but in Takanari leaves, the contents of alanine, asparagine, histidine and tryptophan increased at elevated [CO2] (Fig. 3; Supplementary Fig. S3). Similar increases were also reported in another study using rice leaves at elevated [CO2] (Onda et al. 2014). In tobacco and A. thaliana leaves, contents of major amino acids increased at elevated [CO2] under sufficient N conditions (Geiger et al. 1998, Watanabe et al. 2014). These studies used higher [CO2] at elevated [CO2] conditions (≥700 µmol mol–1) than this study, and thereby different responses may be observed. In this study, since we did not conduct 13 C labeling analysis (metabolic flux analysis; Tcherkez et al. 2012, Sweetlove and Ratcliffe 2011, Sweetlove et al. 2013) , it is difficult to relate the content of each metabolite to CO2 efflux rates directly. However, the decrease in the 2-OG/isocitrate ratio suggests the decrease in the TCA cycle flux and CO2 efflux rate in Koshihikari leaves at elevated [CO2]. Analyses of metabolomic data from some studies suggested that the content of isocitrate increases and the content of 2-OG decreases in A. thaliana leaves at elevated [CO2] (Hachiya et al. 2012, Sato and Yanagisawa 2014). These trends are similar to those observed in this study. In leaves of A. thaliana, the maximal activities of NAD-IDH and/or NADP-ICDH may decrease at elevated [CO2], similar to the case in Koshihikari. Meta-analyses have shown that CO2 efflux rates on a leaf dry weight basis are often lower at elevated [CO2] than at ambient [CO2] (Wang and Curtis 2002, Gonzalez-Meler et al. 2004). Similar changes were observed in two rice varieties in this study. Other studies have also reported significant increases in CO2 efflux rates on a leaf area basis at elevated [CO2] (Leakey et al. 2009b, Li et al. 2013, Markelz et al. 2014a). Since greater LMA at elevated [CO2] can contribute to a greater CO2 efflux rate on a leaf area basis, we should pay attention to the unit of the respiration rate when comparing data. Elevated [CO2] often induces down-regulation of photosynthesis (Makino and Mae 1999, Long et al. 2004). In this FACE site, Rubisco contents decreased in both varieties at elevated [CO2] (Chen et al. 2014). However, the decrease in Rubisco contents may not directly relate to the decrease in respiratory CO2 efflux rates at elevated [CO2]. This is because N and Rubisco contents were tightly correlated (Chen et al. 2014), but relationships between the CO2 efflux rate and the N contents differed between the two [CO2] conditions (Fig. 1). Adachi et al. (2014) reported that the photosynthetic rates under saturated light decreased synergistically in response to elevated [CO2] and water warming treatments in leaves of the japonica variety Akitakomachi at the grain-filling stage. This decrease was due to the decrease in N allocation to the leaves under these conditions. In this study, the N content was significantly decreased by the soil-water warming treatment at the mid grain-filling stage (Fig. 2j), but there was no synergistic effect of warming temperature and elevated [CO2] on the CO2 efflux rates, even at the late growth stage. Since much less N is allocated to the respiratory system than to the photosynthetic system (Makino and Osmond 1991), a small decrease in N content would probably not affect the leaf respiration rates directly. In this study, we examined the uppermost fully expanded leaves of two rice varieties. The effects of elevated [CO2] on CO2 efflux rates of aboveground parts have been investigated in rice plants grown in a growth chamber (Sakai et al. 2001) and at the FACE site (Xu et al. 2006). At the FACE site, the aboveground respiration rates on a dry weight basis were similar between ambient and elevated [CO2] conditions (Xu et al. 2006), whereas, in the growth chamber, the aboveground respiration rates on a dry weight basis differed between the two [CO2] conditions (Sakai et al. 2001). In addition, the aboveground respiration rates at elevated [CO2] were higher at the early growth stage, but lower at the later growth stage. The aboveground part of rice plants consists of leaves with various ages and compositions, which may lead to differences in the responses of the aboveground respiration rate to elevated [CO2]. In A. thaliana, leaf age also strongly affected the response of respiration rates to elevated [CO2] (Markelz et al. 2014b). Effects of elevated [CO2] on O2 uptake rate and the related changes in rice leaves The O2 uptake rates on a leaf dry weight basis were not different between the two [CO2] conditions, except at the panicle initiation stage (Fig. 1f–i). However, other studies have reported contrasting results for different species. In leaves of soybean grown at the FACE site, O2 uptake rates on a leaf area basis were higher at elevated [CO2] than at ambient [CO2] (Leakey et al. 2009b). In leaves of tomato transferred from ambient to elevated [CO2] conditions, the O2 uptake rate on a leaf fresh weight basis increased for a short period (Li et al. 2013). In leaves of A. thaliana, responses of O2 uptake rates to elevated [CO2] changed depending on time. At the end of the light period, the O2 uptake rate on a fresh weight basis was higher at elevated [CO2], while at the end of the night period, the rate was lower at elevated [CO2] than at ambient [CO2] (Watanabe et al. 2014). The O2 uptake rate is generally limited by substrate availability, enzyme activity or the ATP consumption rate (Noguchi 2005). Experiments with additions of substrate or uncoupler indicated that the O2 uptake rate in A. thaliana and tomato leaves is mainly determined by the ATP consumption rate (Li et al. 2013, Watanabe et al. 2014). In the two rice varieties in this study, non-structural carbohydrates accumulated in the leaves at elevated [CO2], but the O2 uptake rates were not higher at elevated [CO2] (Figs. 1, 4). These data suggested that the O2 uptake rates in leaves of the two rice varieties may be determined by ATP consumption rates, as is the case in leaves of A. thaliana and tomato. The plant mitochondrial respiratory chain consists of the cytochrome pathway and the alternative pathway. The cytochrome pathway is associated with H+ translocation, whereas the alternative pathway is catalyzed by the alternative oxidase, which is not coupled to H+ translocation (Vanlerberghe et al. 2016). The in vivo activities of both pathways affect the respiratory ATP production. In A. thaliana leaves, contents of some primary metabolites positively correlated to the in vivo activity of the cytochrome pathway after high-light treatment (Florez-Sarasa et al. 2016). In rice leaves, the correlation between the in vivo flux of the cytochrome pathway and primary metabolite contents may be observed. Differences in leaf respiration between two rice varieties The CO2 efflux rates on a leaf dry weight basis were significantly higher in Takanari leaves than in Koshihikari leaves from the panicle initiation stage (Fig. 1). In a previous study conducted at the same FACE site, photosynthetic rates under saturated light were higher in Takanari leaves than in Koshihikari leaves (Chen et al. 2014). In addition, the physiological parameters related to photosynthetic rates, stomatal conductance, Rubisco carboxylation rate and ribulose bisphosphate (RuBP) regeneration rate were significantly higher in Takanari leaves than in Koshihikari leaves (Chen et al. 2014). The Rubisco, soluble protein and N contents were also significantly higher in Takanari leaves than in Koshihikari leaves. Therefore, the N demand for photosynthetic components should be higher in Takanari leaves. In this study, the content of TCA cycle organic acids was lower and the ratio of total amino acids to TCA cycle organic acids was higher in Takanari leaves than in Koshihikari leaves (Figs. 3, 4), indicating that higher N demand and higher N assimilation may explain the higher rates of CO2 efflux in Takanari leaves than in Koshihikari leaves. In contrast, O2 uptake rates on a leaf dry weight basis did not differ between the two rice varieties, except at the heading stage (Fig. 1f–j). The higher rates of CO2 efflux in Takanari leaves indicate the higher rates of NADH production from the TCA cycle in Takanari leaves. NADH may be oxidized partly by cellular processes other than the respiratory chain in Takanari leaves, and thus NADH oxidation rates by the respiratory chain may be comparable between the two varieties, leading to the similar rates of O2 uptake between the two varieties. Saitoh et al. (2000) compared CO2 efflux rates of whole plants or whole leaves between Takanari and Nipponbare, another japonica variety. In their data, the CO2 efflux rates of whole leaves were comparable between Takanari and Nipponbare. The inconsistency between our data and theirs may be because different parts of the leaves were analyzed. In another study, the canopy top leaves of Takanari had a greater photosynthetic capacity and higher N content than those of Koshihikari, but leaf N per plant was lower in Takanari than in Koshihikari (Muryono et al. 2017). Their study also showed that the vertical gradient of leaf N content was steeper in Takanari than in Koshihikari, which may have contributed to higher productivity and higher photosynthetic and CO2 efflux rates in canopy top leaves of Takanari. The simulated canopy photosynthesis of Takanari was larger than that of Koshihikari (Ikawa et al. 2018). The CO2 efflux rates of whole leaves should be compared between Takanari and Koshihikari to compare the growth and yield of whole plants between the two varieties. Conclusion We compared the respiratory characteristics of mature leaves of two rice varieties grown at the FACE site. The CO2 efflux rates on a leaf dry weight basis decreased at elevated [CO2] at all growth stages. The CO2 efflux rates and the N contents were significantly higher in Takanari leaves than in Koshihikari leaves. In Takanari leaves, the contents of some amino acids were higher and those of some organic acids were lower, suggesting that organic acids may be used for N assimilation in Takanari leaves. This up-regulated N assimilation may support a higher rate of photosynthesis in Takanari. In Koshihikari leaves, the decreased activity of TCA cycle enzymes may have led to the decrease in CO2 efflux rates at elevated [CO2]. In Takanari leaves, the decreased CO2 efflux rates under elevated [CO2] could not be explained by the maximal activities of measured enzymes. The decreased flux of the TCA cycle in Takanari leaves may be caused by other unknown mechanisms under elevated [CO2]. Materials and Methods Site description We conducted the study at the Tsukuba FACE experimental facility in Tsukubamirai, Ibaraki, Japan (3° 8′N, 139°60′E, 10 m a.s.l.). There were four control (ambient [CO2]) plots and four FACE (elevated [CO2]) plots at the site. The average ambient [CO2] at the site across the entire growing season (June–September) and day to day SD was 383 ± 11.2 µmol mol–1 in 2012 and 384 ± 11.4 µmol mol–1 in 2013. The target concentration of the elevated [CO2] treatment was 200 µmol mol–1 above ambient [CO2], and the actual season-long mean [CO2] and day to day SD in the FACE plots was 578 ± 15.7 µmol mol–1 in 2012 and 576 ± 15.5 µmol mol–1 in 2013. In both the ambient and FACE plots, we set up normal temperature and elevated temperature conditions. In elevated temperature conditions, soil and water were heated by 2°C using heating wires under the soil surface between the rows. Previously published papers have provided further details of the experimental site set-up and CO2 control performance (Nakamura et al. 2012), elevated temperature treatment (Adachi et al. 2014, Usui et al. 2016) and soil chemical properties (Hasegawa et al. 2013). Plant materials Two rice (Oryza sativa L.) varieties were used in this study; the japonica variety ‘Koshihikari’ and the indica variety ‘Takanari’. Three-week-old seedlings were transplanted into the experimental plots on May 23–24 in 2012 and on May 22–23 in 2013. Both varieties received equal amounts of fertilizers prior to planting at a rate of 8.00 g m–2 of N, 4.36 g m–2 of P and 8.30 g m–2 of K, as described in Hasegawa et al. (2013). In the plots, Koshihikari achieved 50% heading by August 3 in both years, and Takanari achieved 50% heading by August 9 and 6 in 2012 and 2013, respectively (Chen et al. 2014). We used the uppermost fully expanded leaves for gas exchange measurements and sampling at two different growth stages: July 5–6 (panicle initiation stage) and August 16–17 (mid grain-filling stage) in 2012, and five different growth stages: June 24–27 (tillering stage), July 8–11 (panicle initiation stage), July 22–25 (booting stage), August 5–8 (heading stage) and August 19–22 (mid grain-filling stage), in 2013. At the heading and mid grain-filling stages, we used the flag leaves as the uppermost fully expanded leaves. Gas exchange measurements In 2012 and 2013, measurements of CO2 efflux rates were conducted using a portable photosynthetic gas exchange system (GFS3000, Walz) in the rice paddy from 10:00 to 15:00 h during 2 d at each stage. Measurements were taken on attached leaves (one leaf for each variety per temperature treatment per experimental plot at each growth stage) at a leaf temperature of 30°C, leaf chamber CO2 concentration of 390 (ambient plot) or 590 µmol mol–1 (FACE plot) and leaf chamber H2O concentration of 25,000 µmol mol–1. Since we conducted measurements of CO2 efflux rates using attached leaves in the rice paddy, we could put a limited size of one leaf (<8 cm2) in the chamber. We did not put two different leaves in the chamber in order to obtain more stable data as in Montero et al. (2016), but we waited to obtain constant and stable rates of CO2 efflux before logging. Measurements of O2 uptake rates were conducted using a gas-phase oxygen electrode system (LD2, Hansatech Instruments Ltd.) from 10:00 to 15:00 h during 2 d at each stage in 2013. Leaves were sampled in each plot (one leaf for each variety per temperature treatment per experimental plot at each growth stage), and measured in the cabin at the site. Measurements were taken on detached leaves at a leaf temperature of 30°C and ambient CO2 concentration in the leaf chamber. We measured dark respiration rates of mature leaves in the daytime because CO2 was supplied at the FACE experimental site during daylight hours from sunrise to sunset (Hasegawa et al. 2013). The CO2 efflux or O2 uptake rates were measured using leaves that had been dark adapted for at least 15 min. We checked that the 15 min darkness was enough to obtain constant rates of dark CO2 efflux and O2 uptake in leaves of the two varieties. The mean and the SEM of both rates from four different leaves of four experimental plots were calculated for each variety per temperature treatment per [CO2] treatment at each growth stage. After the gas exchange measurements, samples were kept in the dark and brought to the laboratory on the same day. The leaves were dried for 3 d at 80°C, weighed and the LMA was calculated. Leaf sampling For determinations of carbohydrates, primary metabolites and maximal enzymatic activities, leaves were sampled from 10:00 to 14:00 h (one leaf for each variety per temperature treatment per experimental plot at each growth stage) in 2013. The samples were weighed and immediately frozen in liquid N at each paddy plot. The samples were brought to the laboratory of the university and kept at −80°C until analysis. Determination of carbon and nitrogen contents The C and N contents were quantified as described by Sugiura et al. (2015). Dried leaves were ground using a metalcone with a multibeads shocker (Yasui Kikai), and then the C and N contents of the ground samples were measured with a CN analyzer (Vario micro, Elementar Analyzensysteme GmbH). Determination of carbohydrate content We used leaf samples collected at the heading stage in 2013 to determine the contents of non-structural carbohydrates (starch, glucose and sucrose). Non-structural carbohydrates were quantified as described by Watanabe et al. (2014). Frozen leaves were ground with a multibeads shocker (BS-12 R, Wakenyaku). After adding 1 ml of 80% ethanol, the suspension was incubated at 80°C for 10 min, and then centrifuged at 1,500×g at 4°C for 10 min. The precipitate was used for starch determination. Ethanol was removed from the supernatant by evaporation using a centrifugal concentrator (CC-105, Tomy). Equal volumes of distilled water and chloroform were added to the concentrated supernatant, the mixture was mixed well and then centrifuged at 10,000×g at 4°C for 10 min. The upper aqueous phase was used for determinations of glucose and sucrose. The precipitate was suspended in distilled water and boiled for 60 min at 100°C. An equal volume of amyloglucosidase was added to the boiled suspension and the mixture was incubated for 60 min at 55°C. The mixture was then centrifuged at 10,000×g at 4°C for 10 min, and the upper aqueous phase was used for starch determination. A Glucose C2 Test kit (Wako) was used to analyze the glucose content. Measurement of primary metabolites We used leaf samples collected at the booting stage in 2013 for determination of primary metabolite contents. Metabolites were extracted from leaves as described previously (Watanabe et al. 2010, Onda et al. 2014). Briefly, frozen leaves were ground with a mortar and pestle in liquid N and homogenized in 50% (v/v) methanol (10 µl mg–1 FW) containing 50 µM PIPES and 50 µM methionine sulfone as internal standards. After centrifugation at 21,500×g at 4°C for 5 min, the supernatants were filtered through a 3 kDa cut-off filter (Millipore) at 16,100×g at 4°C for 30 min. Primary metabolites, including amino acids, organic acids and phosphorylated compounds, were separated by capillary electrophoresis–triple quadrupole-mass spectrometry (CE; 7100 Capillary Electrophoresis, MS; 6420 Triple Quad LC/MS, Agilent Technologies), as described previously (Miyagi et al. 2010) with minor modifications. Metabolites were quantified by comparison with 54 compounds at known concentrations (Miyagi et al. 2010, Onda et al. 2014). All capillary electrophoresis mass spectrometry data were processed with Agilent MassHunter software (Agilent Technologies). Measurements of maximal activities of TCA cycle enzymes We used leaf samples collected at the booting stage in 2013 for measurements of enzyme activities. Frozen leaves were ground in liquid N and extracted in buffer [50 mM KH2PO4–K2HPO4 (pH 7.6), 10 mM MgSO4, 1 mM EDTA, 5 mM dithiothreitol, 0.05% (v/v) Triton X-100, and one protease inhibitor cocktail tablet (Complete, Roche Diagnostics GmbH) per 30 ml of buffer]. The mixture was centrifuged at 10,000×g at 4°C for 3 min, and the upper aqueous phase was used for measurements of maximal enzymatic activities. The maximal activities of aconitase, NAD-IDH and NADP-ICDH were determined as described by Noguchi and Terashima (2006). Statistical analysis Data were analyzed with a generalized linear mixed model (GLMM). In this model, the response variable was each datum at each stage, the explanatory variables were CO2, temperature, variety and interactions between these variables, and the random factor was block. When normality was not satisfied based on a Shapiro–Wilks test, we assumed a gamma error distribution with canonical link function, but, if satisfied, we assumed a Gaussian error distribution. All analyses were performed with the statistical software R (R Core Team 2016). The lme4 package (Bates et al. 2015) was used to calculate the GLMM by using maximum likelihood estimation. To determine the effects of fixed factors, we used a Wald test and the χ2 test statistic in the car package (Fox and Weisberg 2011). Statistical significance is noted if P < 0.1, and the results are summarized in Supplementary Table S1. Supplementary Data Supplementary data are available at PCP online. Funding This work was supported by the Ministry of Education, Culture, Sports, Science and Technology, Japan [Grants-in-Aid for Scientific Research on Innovative Areas (21114007 and 24114711)], CREST; the Science and Technology Agency (JST); a Yamaguchi Scholarship; and the Ministry of Agriculture, Forestry and Fisheries, Japan [through the research project ‘Development of Technologies for Mitigation and Adaptation to Climate Change in Agriculture, Forestry and Fisheries’]. 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Effects of Elevated Atmospheric CO2 on Respiratory Rates in Mature Leaves of Two Rice Cultivars Grown at a Free-Air CO2 Enrichment Site and Analyses of the Underlying Mechanisms

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com
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0032-0781
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1471-9053
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10.1093/pcp/pcy017
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Abstract

Abstract Respiratory CO2 efflux and O2 uptake rates in leaves change in response to the growth CO2 concentration ([CO2]). The degrees of change vary depending on the responses of cellular processes such as nitrogen (N) assimilation and accumulation of organic acids to growth [CO2]. However, the underlying mechanisms remain unclear. Here, we examined the respiratory characteristics of mature leaves of two rice varieties with different yield capacities at different growth stages under ambient and elevated [CO2] conditions at a free-air CO2 enrichment site. We also examined the effect of increased water temperature on leaf respiration. We measured the rates of CO2 efflux and O2 uptake, and determined N contents, primary metabolite contents and maximal activities of respiratory enzymes. The leaf CO2 efflux rates decreased in plants grown at elevated [CO2] in both varieties, and were higher in high-yielding Takanari than in Koshihikari. The leaf O2 uptake rates showed little change with respect to growth [CO2] and variety. The increased water temperature did not significantly affect the CO2 efflux and O2 uptake rates. The N and amino acid contents were significantly higher in Takanari than in Koshihikari. The enhanced N assimilation in Takanari may have consumed more respiratory NADH, leading to higher CO2 efflux rates. In Koshihikari, the ratio of tricarboxylic acid (TCA) cycle intermediates changed and maximal activities of enzymes in the TCA cycle decreased at elevated [CO2]. Therefore, the decreased rates of CO2 efflux in Koshihikari may be due to the decreased activities of TCA cycle enzymes at elevated [CO2]. Introduction The atmospheric CO2 concentration ([CO2]) has drastically increased since the industrial revolution. As CO2 is a direct substrate for photosynthesis, the increase in [CO2] will greatly affect photosynthesis and growth in plants. This topic has been discussed in several reviews (Ainsworth and Long 2005, Long et al. 2004, Ainsworth and Rogers 2007, Leakey et al. 2009a). Plants release about half of the fixed carbon (C) via respiration, and plant respiration is closely associated with photosynthesis and growth (Lambers et al. 2005). Therefore, studies on the responses of plant respiration to elevated [CO2] are important to understand the C balance in the whole plant. Many studies have reported that leaf respiration rates of C3 plant species change in response to changes in [CO2] (Drake et al. 1999, Wang and Curtis 2002, Gonzalez-Meler et al. 2004, Leakey et al. 2009b, Haworth et al. 2016). The extent of changes in leaf respiration rates varies depending on the plant species, developmental stage and other environmental factors (Ayub et al. 2011, Griffin et al. 2013, Markelz et al. 2014a, Markelz et al. 2014b, Watanabe et al. 2014, Aranjuelo et al. 2015). In mature leaves of soybean (Glycine max) grown at a free-air CO2 enrichment (FACE) experimental site, the rates of CO2 efflux and O2 uptake on a leaf area basis significantly increased at elevated [CO2] (Leakey et al. 2009b). Rapid increases in the rates of CO2 efflux and O2 uptake were also reported for mature leaves of tomato (Solanum lycopersicum) after transfer from ambient to elevated [CO2] in a growth chamber (Li et al. 2013). In those studies, the increase in leaf respiration rates was thought to be related to the up-regulation of genes involved in the respiratory system. Fukayama et al. (2011) also reported that expression of several respiratory genes was up-regulated under elevated [CO2] in leaves of a japonica variety of rice (Oryza sativa). In contrast, in mature leaves of Arabidopsis thaliana grown under elevated [CO2] in a growth chamber, the responses to elevated [CO2] differed between CO2 efflux and O2 uptake rates (Watanabe et al. 2014). In A. thaliana leaves, the CO2 efflux rate on a leaf fresh weight basis increased under elevated [CO2] at both ends of the light and dark periods, whereas the O2 uptake rate decreased under elevated [CO2] at the end of the light period. The respiration rate in plants is generally limited by substrate availability, enzyme capacity or the ATP consumption rate (Lambers et al. 2005, Noguchi 2005). In most cases, the rates of CO2 efflux and O2 uptake are similar; thus, the respiratory quotient (RQ: ratio of CO2 efflux rate to O2 uptake rate) is around one. However, when NADH produced in both glycolysis and the tricarboxylic acid (TCA) cycle is consumed by various cellular processes such as nitrogen (N) assimilation, the RQ value changes as suggested by the flux-balanced model (Buckley and Adams 2011). Cousins and Bloom (2004) showed that the increased consumption of reducing equivalents via NO3– assimilation decreases the rate of mitochondrial O2 consumption in wheat leaves, which can affect the RQ value. The accumulation and consumption of organic acids also affect the RQ value (Lambers et al. 2005). For example, malate production from glucose leads to an RQ of 0.667. In soybean leaves, the RQ value was reported to be higher at elevated [CO2] than at ambient [CO2] (Leakey et al. 2009b). Therefore, we should pay attention to both CO2 efflux and O2 uptake rates when investigating the mechanisms underlying the respiratory response to elevated [CO2]. At the Tsukuba FACE experimental site in central Japan, growth, photosynthesis and yield have been examined for many rice varieties, and the responses of these parameters to elevated [CO2] have been found to differ among them (Hasegawa et al. 2013, Usui et al. 2014, Ikawa et al. 2018). At this FACE site, the effect of increased water temperature has been examined using a system in which soil water is heated by heating wires under the soil surface (Usui et al. 2016). Not only the warming treatment, but also the interaction between warming and elevated [CO2] were found to affect the yields and grain qualities of rice (Usui et al. 2016). Chen et al. (2014) reported that the mature leaves of a high-yielding indica variety, Takanari, showed consistently higher photosynthesis and stomatal conductance than those of a japonica variety, Koshihikari, under both ambient and enhanced [CO2] conditions. These leaf-level characteristics in Takanari were possibly related to the 21% yield enhancement at elevated [CO2] compared with that at ambient [CO2], whereas the yield enhancement in Koshihikari was only 16% (Hasegawa et al. 2013). The N content in leaves of Takanari was higher than that in Koshihikari (Taylaran et al. 2011), resulting in a greater source capacity of Takanari leaves under both ambient and elevated [CO2] conditions (Chen et al. 2014). Another study at the FACE experimental site showed that the soil-water warming treatment reduced the photosynthetic response to elevated [CO2] at the grain-filling stage (Adachi et al. 2014). In that study, elevated water temperature increased N allocation to the panicles at the grain-filling stage, leading to decreases in leaf N and changes in photosynthetic responses at that stage. The results of these previous studies suggested that leaf photosynthesis in response to elevated [CO2] is highly sensitive to the variety, growth stage and water temperature, and that leaf N status may affect the responses to elevated [CO2]. The respiratory system is closely associated with the photosynthetic system (Noguchi and Yoshida 2008, Vanlerberghe et al. 2016), and leaf respiration rates are correlated with leaf N content (Reich et al. 1996, Tissue et al. 2002, Noguchi and Terashima 2006). Therefore, responses of respiration rates to elevated [CO2] may change depending on leaf N status. Elevated [CO2] affects various primary metabolite contents in leaves. These changes are greatly influenced by leaf N status (Stitt and Krapp 1999, Noguchi et al. 2015). The responses of primary metabolite contents should be tightly related to changes in leaf respiration rates (Araújo et al. 2012, Tcherkez et al. 2012, Tcherkez et al. 2017). However, the physiological mechanisms underlying the changes in leaf respiration rates in response to elevated [CO2] and the roles of various factors in these respiratory changes remain poorly understood. In this study, we examined the following questions. (i) Do the respiratory CO2 efflux and O2 uptake rates in leaves respond differently to elevated [CO2]? (ii) Do these responses change depending on the variety, developmental stage and water tempearture? (iii) Can the difference in the responses to elevated [CO2] be related to leaf N status? To clarify the above questions, we examined respiratory characteristics of mature leaves of two contrasting rice varieties, Koshihikari and Takanari, grown under ambient and elevated [CO2] conditions at the Tsukuba FACE experimental site. We compared their leaf respiration responses under ambient and elevated [CO2] conditions at two water temperatures, and at five different growth stages from the vegetative to the grain-filling stage. We measured the rates of CO2 efflux and O2 uptake, and determined the N and C contents of the leaves. We measured primary metabolites of the respiratory system and determined the maximal activities of respiratory enzymes. Results Effects of elevated CO2 or temperature on CO2 efflux and O2 uptake rates in leaves of plants at different growth stages, and differences in rates between varieties We measured CO2 efflux rates in mature leaves at the panicle initiation stage (early July) and the mid grain-filling stage (mid August) in 2012. We compared the responses of CO2 efflux rates between the two growth [CO2] conditions, the two varieties and the two water temperatures. The CO2 efflux rates on a leaf dry weight basis were significantly lower at elevated [CO2] than at ambient [CO2] at both growth stages (Supplementary Fig. S1a, b). The CO2 efflux rates differed significantly between varieties at both stages, and the 2°C increase in water temperature significantly decreased the CO2 efflux rates at the panicle initiation stage. To confirm these differences in the CO2 efflux rates of leaves detected in 2012, we measured CO2 efflux rates from the tillering stage (late June) to the mid grain-filling stage (mid August) in 2013. In 2013, the CO2 efflux rates were also significantly lower at elevated [CO2] than at ambient [CO2] at all growth stages (Fig. 1a–e). Although the CO2 efflux rate was significantly lower in Takanari than in Koshihikari at the tillering stage, it was significantly higher in Takanari than in Koshihikari from the panicle initiation stage to the heading stage. At the mid grain-filling stage, the difference in CO2 efflux rate between the two varieties was significant at P = 0.0716 (Supplementary Table S1). The responses to elevated [CO2] were similar between the two varieties during all growth stages (P > 0.1 for the interaction between CO2 and variety in Supplementary Table S1). The 2°C increase in water temperature resulted in small changes in the CO2 efflux rates, but a significant difference between the two temperature regimes was observed only at the early growth stage (tillering) in 2013. The CO2 efflux rates on a leaf area basis showed a similar trend to those on a leaf dry weight basis (Supplementary Fig. S2a–e). The CO2 efflux rates on a leaf area basis were significantly lower at elevated [CO2] than at ambient [CO2] at all growth stages. Similar decreases in CO2 efflux rates on a leaf area basis at elevated [CO2] were observed in six herbaceous species that were cultivated at another FACE site (Haworth et al. 2016). The CO2 efflux rates on a leaf area basis differed significantly between the two varieties at all stages except for the panicle initiation stage. At the panicle initiation stage, the difference between the two varieties was significant at P = 0.0577 (Supplementary Table S1). Fig. 1 View largeDownload slide Respiration rates of mature leaves of two rice varieties. CO2 efflux rates on a dry weight basis (a–e), O2 uptake rates on a dry weight basis (f–j) and CO2 efflux rates on a leaf nitrogen basis (k–o) were determined in mature leaves of Koshihikari and Takanari at various developmental stages in 2013. On the x-axis, A and F denote data in ambient and FACE (elevated CO2) plots, respectively. NT and ET denote normal temperature and elevated temperature, respectively. The mean and SEM are shown (n = 4). Statistical results are also shown (*P < 0.05, **P <0.01, ***P < 0.001). CO2, temp and var denote effects of CO2 concentration, temperature and variety, respectively. Fig. 1 View largeDownload slide Respiration rates of mature leaves of two rice varieties. CO2 efflux rates on a dry weight basis (a–e), O2 uptake rates on a dry weight basis (f–j) and CO2 efflux rates on a leaf nitrogen basis (k–o) were determined in mature leaves of Koshihikari and Takanari at various developmental stages in 2013. On the x-axis, A and F denote data in ambient and FACE (elevated CO2) plots, respectively. NT and ET denote normal temperature and elevated temperature, respectively. The mean and SEM are shown (n = 4). Statistical results are also shown (*P < 0.05, **P <0.01, ***P < 0.001). CO2, temp and var denote effects of CO2 concentration, temperature and variety, respectively. To examine differences between the CO2 efflux and O2 uptake rates in terms of the effects of growth [CO2], water temperature and variety, we measured O2 uptake rates from the tillering to the mid grain-filling stages in 2013. In contrast to CO2 efflux rates, O2 uptake rates on a leaf dry weight basis showed little change with respect to growth [CO2], variety and water temperature (Fig. 1f–j). The O2 uptake rates were significantly affected by growth [CO2] and variety at only one growth stage (Supplementary Table S1). The 2°C increase in water temperature resulted in a significant decrease in the O2 uptake rates at three growth stages (tillering, booting and mid grain-filling stages). Since we measured CO2 efflux and O2 uptake rates using different systems and on different days, we could not calculate exact RQ values. The ratios of CO2 efflux to O2 uptake rate were significantly lower at elevated [CO2] than at ambient [CO2] at the three growth stages, and showed similar trends to those of CO2 uptake rates (Supplementary Fig. S2f–j). There were significant differences in the ratio of CO2 efflux to O2 uptake rate between the two varieties, except at the mid grain-filling stage. Effects of elevated CO2 on leaf N and CO2 efflux rate on a leaf N basis, and differences between varieties Leaf mass per area (LMA) increased with rice development, and growth [CO2] significantly increased the LMA at two growth stages (Fig. 2a–e). The LMA significantly differed between the two varieties at the two growth stages (panicle initiation and mid grain-filling). The leaf N content on a leaf dry weight basis was significantly lower at elevated [CO2] than at ambient [CO2] at all growth stages in 2013 (Fig. 2f–j). The N leaf content was significantly higher in Takanari than in Koshihikari, except at the tillering stage in 2013. These trends were similar to those in the CO2 efflux rates on a leaf dry weight basis. Other studies also found that the leaf N content per leaf area was higher in Takanari than in Koshihikari (Taylaran et al. 2011, Chen et al. 2014). Leaf C content on a leaf dry weight basis was significantly higher in Takanari than in Koshihikari at all growth stages, and the growth [CO2] did not affect the leaf C content, except at the tillering stage in 2013 (Fig. 2k–o). The ratio of C to N content (C/N ratio) showed an opposite trend to that of leaf N content (Fig. 2p–u). Growth [CO2] increased the C/N ratio at all growth stages in 2013. The C/N ratio was significantly lower in Takanari than in Koshihikari, except at the tillering stage. The trends in leaf N and C contents in 2012 were similar to those in 2013 (Supplementary Fig. S1c–f). Fig. 2 View largeDownload slide Leaf characteristics of two rice varieties. Leaf mass per area (a–e), nitrogen content (f–j), carbon content (k–o) and ratio of carbon to nitrogen contents (p–u) were determined in mature leaves of Koshihikari and Takanari at different developmental stages in 2013. For other details, see the legend of Fig. 1. Fig. 2 View largeDownload slide Leaf characteristics of two rice varieties. Leaf mass per area (a–e), nitrogen content (f–j), carbon content (k–o) and ratio of carbon to nitrogen contents (p–u) were determined in mature leaves of Koshihikari and Takanari at different developmental stages in 2013. For other details, see the legend of Fig. 1. We calculated the CO2 efflux rate on a leaf N basis from the CO2 efflux rates on a leaf dry weight basis and N contents. In 2013, the differences between the two varieties in the CO2 efflux rate on a leaf N basis were smaller than those in CO2 efflux rate on a leaf dry weight basis (Fig. 1k–o). At the panicle initiation and heading stages, there were no significant differences in the CO2 efflux rate on a leaf N basis between the two varieties. However, there were significant differences between the two [CO2] conditions, except at the mid grain-filling stage. Similar trends were observed in 2012, although there was a significant difference in the CO2 efflux rate on a leaf N basis between the two varieties at the panicle initiation stage (Supplementary Fig. S1g, h). Effects of elevated CO2 on contents of primary metabolites and their relationships to leaf respiratory rates In leaves, contents of primary metabolites are closely related to respiration rates, and elevated [CO2] affects the contents of various primary metabolites (Stitt and Krapp 1999, Misra and Chen 2015, Noguchi et al. 2015). In rice leaves, it has been reported that elevated [CO2] affected primary metabolite contents (Onda et al. 2014). Elevated night temperature affected both O2 uptake rates and primary metabolite contents in rice leaves (Glaubitz et al. 2014, Glaubitz et al. 2015). In this study, we examined whether primary metabolite contents are related to changes in leaf respiration rates in two rice varieties. We measured the contents of various primary metabolites at the booting stage when the CO2 efflux rates were significantly different between two growth [CO2] conditions and between the two varieties (Fig. 1c). Since we had a shortage of booting-stage samples, we determined non-structural carbohydrate contents in samples collected at the heading stage, when the CO2 efflux rates also differed significantly between the two growth [CO2] conditions and between the two varieties (Fig. 1c, d). Among the amino acids measured in this study, glutamate, glutamine, aspartate, glycine and alanine were the most abundant (Fig. 3a–e). The contents of these five amino acids were significantly higher in Takanari leaves than in Koshihikari leaves. Under elevated [CO2], the contents of these major amino acids tended to decrease in Koshihikari leaves, but not in Takanari leaves. The alanine content increased in Takanari leaves at elevated [CO2]. The interaction between CO2 and variety was significant for the contents of glutamate, glycine and alanine (P < 0.01, Supplementary Table S1). Among the amino acids detected at low contents, tyrosine, serine, arginine and γ-aminobutanoic acid (GABA) showed lower contents in Koshihikari leaves at elevated [CO2] than at ambient [CO2], while their contents in Takanari leaves did not differ between ambient and elevated [CO2] conditions (Supplementary Fig. S3f, k, q, s). For these metabolites, the interaction between CO2 and variety was significant (P < 0.05, Supplementary Table S1). Fig. 3 View largeDownload slide Amino acid contents in mature leaves of two rice varieties. Contents of several amino acids, total amino acids and the ratio of glutamine to glutamate (Gln/Glu) were determined in mature leaves of Koshihikari and Takanari at the booting stage in 2013. Total amino acids are the sum of 25 measured amino acids. The unit of amino acids and total amino acids is µmol g FW–1. The mean and SEM are shown (n = 4). For other details, see the legend of Fig. 1. Fig. 3 View largeDownload slide Amino acid contents in mature leaves of two rice varieties. Contents of several amino acids, total amino acids and the ratio of glutamine to glutamate (Gln/Glu) were determined in mature leaves of Koshihikari and Takanari at the booting stage in 2013. Total amino acids are the sum of 25 measured amino acids. The unit of amino acids and total amino acids is µmol g FW–1. The mean and SEM are shown (n = 4). For other details, see the legend of Fig. 1. The branched-chain amino acids valine, leucine and isoleucine accumulated to much higher contents in Takanari leaves than in Koshihikari leaves (Fig. 3f;Supplementary Fig. S3i, j). We calculated total amino acids and TCA cycle organic acids by adding up the amounts of all 25 amino acids and the seven organic acids in the TCA cycle, respectively (Figs. 3g, 4e). The contents of total amino acids and the ratio of total amino acids to TCA cycle organic acids have been reported to increase when amino acid production is up-regulated (Hachiya et al. 2012). Both of these values were significantly higher in Takanari leaves than in Koshihikari leaves (Figs. 3g, 4f). When amino acid production is up-regulated, the ratio of glutamine to glutamate (Gln/Glu) and that of glutamine to 2-oxoglutarate (Gln/2-OG) often increase in leaves (Stitt and Krapp 1999, Noguchi et al. 2015). These ratios were higher in Takanari than in Koshihikari (P = 0.0517 for Gln/Glu in Fig. 3h, P< 0.001 for Gln/2-OG in Fig. 4g). Together, these findings suggested that the production of amino acids was up-regulated in Takanari leaves, and that NADH produced in the TCA cycle may have been consumed in the production of amino acids, leading to the higher rates of CO2 efflux in Takanari leaves than in Koshihikari leaves. Fig. 4 View largeDownload slide TCA cycle organic acid contents in mature leaves of two rice varieties. Contents of several TCA cycle organic acids, ratio of total amino acids to TCA cycle organic acids (total AA/TCA OA), ratio of glutamine to 2-oxoglutarate (Gln/2-OG) and ratio of 2-OG to isocitrate (2-OG/isocitrate) were determined in mature leaves of Koshihikari and Takanari at the booting stage in 2013. Contents of carbohydrates (starch, sucrose and glucose) in mature leaves of two rice cultivars at the heading stage in 2013. TCA cycle organic acids are the sum of seven measured TCA cycle organic acids. The unit of organic acids is µmol g FW–1; the unit of carbohydrates is µmol C g FW–1. The mean and SEM are shown (n = 4). For other details, see the legend of Fig. 1. Fig. 4 View largeDownload slide TCA cycle organic acid contents in mature leaves of two rice varieties. Contents of several TCA cycle organic acids, ratio of total amino acids to TCA cycle organic acids (total AA/TCA OA), ratio of glutamine to 2-oxoglutarate (Gln/2-OG) and ratio of 2-OG to isocitrate (2-OG/isocitrate) were determined in mature leaves of Koshihikari and Takanari at the booting stage in 2013. Contents of carbohydrates (starch, sucrose and glucose) in mature leaves of two rice cultivars at the heading stage in 2013. TCA cycle organic acids are the sum of seven measured TCA cycle organic acids. The unit of organic acids is µmol g FW–1; the unit of carbohydrates is µmol C g FW–1. The mean and SEM are shown (n = 4). For other details, see the legend of Fig. 1. In leaves of both rice varieties, the most abundant organic acids were citrate, isocitrate, 2-OG and malate (Fig. 4a–d). This pattern of organic acid accumulation in rice leaves differed from that in A. thaliana leaves, in which fumarate also accumulated (Watanabe et al. 2014). The content of 2-OG and the ratio of 2-OG to isocitrate (2-OG/isocitrate) were significantly lower at elevated [CO2] than at ambient [CO2] (P < 0.05, Fig. 4c, h), while the isocitrate content tended to be higher at elevated [CO2] (P = 0.0731, Fig. 4b;Supplementary Table S1). The contents of the other organic acids were not higher at elevated [CO2], and the contents of TCA cycle organic acids were comparable between the two [CO2] conditions (Fig. 4e). Therefore, an increase in organic acid production was not responsible for the lower CO2 efflux rate and RQ value in leaves at elevated [CO2]. In leaves of Takanari, where amino acid production may have been up-regulated, the content of TCA cycle organic acids was significantly lower than that in Koshihikari leaves (Fig. 4e); increased production of amino acids in Takanari leaves may have consumed NADH and organic acids in the TCA cycle, leading to lower contents of organic acids than in Koshihikari leaves. In leaves at elevated [CO2], carbohydrate contents often increase (Stitt and Krapp 1999, Noguchi et al. 2015). We quantified starch, sucrose and glucose as non-structural carbohydrates that are consumed as respiratory substrates in leaves. In leaves of both varieties, sucrose accumulated to the highest content, followed by starch and glucose (Fig. 4i–k). All these carbohydrates increased in Takanari leaves at elevated [CO2], whereas only glucose increased in Koshihikari leaves at elevated [CO2]. Sucrose and starch contents in Koshihikari were comparable between the two [CO2] conditions. These data showed that lower contents of carbohydrates were not responsible for the lower rates of CO2 efflux in leaves at elevated [CO2]. In Koshihikari leaves, the contents of metabolites downstream of glycolysis, such as 3-phosphoglycerate (3PGA), phosphoenolpyruvate (PEP) and pyruvate, tended to be low at elevated [CO2] (Supplementary Fig. S4m–o), similar to the results reported for A. thaliana leaves (Watanabe et al. 2014). In contrast, the contents of these metabolites were higher in Takanari leaves, and were not different between the two [CO2] conditions. In Takanari leaves, the content of dihydroxyacetone phosphate (DHAP) was higher at elevated [CO2] than that at ambient [CO2] (Supplementary Fig. S4k). The increase in DHAP content may induce increases in reactive carbonyl contents. In leaves of wheat at elevated [CO2], reactive carbonyl contents increased (Takagi et al. 2014). The contents of glucose 6-phosphate (G6P) and glucose 1-phosphate (G1P) were also significantly higher in Takanari leaves than in Koshihikari leaves (Supplementary Fig. S4e, f). The sum of measured metabolites of glycolysis was greater in Takanari leaves than in Koshihikari leaves (data not shown), in contrast to the pattern observed for TCA cycle organic acids. The photosynthetic rates may be higher in Takanari leaves than in Koshihikari leaves, even at elevated [CO2] (Chen et al. 2014). Therefore, during the daytime, larger amounts of photoassimilates may flow into glycolysis and be consumed in Takanari leaves than in Koshihikari leaves. The contents of intermediates of the Calvin–Benson cycle tended to be lower at elevated [CO2] than at ambient [CO2] in Koshihikari leaves, whereas their contents were comparable between the two [CO2] conditions in Takanari (Supplementary Fig. S4q–t). Effects of elevated CO2 on maximal activities of TCA cycle enzymes The contents of 2-OG and the 2-OG/isocitrate ratio in Fig. 4 suggested that activities of the TCA cycle enzymes may be lower at elevated [CO2] than at ambient [CO2], resulting in lower CO2 efflux rates at elevated [CO2]. Therefore, we determined the maximal activities of TCA cycle enzymes in leaves of the two rice varieties at the booting stage in 2013. In the enzymes of the TCA cycle, maximal activities of citrate synthase and NAD-dependent malate dehydrogenase could not be measured in two rice varieties. We measured maximal activities of aconitase, NAD-dependent isocitrate dehydrogenase (NAD-IDH) and NADP-dependent isocitrate dehydrogenase (NADP-ICDH) in this study. The maximal activity of aconitase was significantly higher in Koshihikari leaves than in Takanari leaves, and it decreased in Koshihikari leaves at elevated [CO2] (Fig. 5a). In Koshihikari leaves, the maximal activities of NADP-ICDH and NAD-IDH tended to be lower at elevated [CO2] than at ambient [CO2]. In Takanari leaves, the maximal activities of NADP-ICDH and NAD-IDH did not differ significantly between the two [CO2] conditions. Fig. 5 View largeDownload slide Maximal activities of three TCA cycle enzymes in mature leaves of two rice varieties. Maximal activities of aconitase, NAD-dependent isocitrate dehydrogenase and NADP-dependent isocitrate dehydogenase were determined in mature leaves of Koshihikari and Takanari at the booting stage in 2013. The mean and SEM are shown (n = 4). For other details, see the legend of Fig. 1. Fig. 5 View largeDownload slide Maximal activities of three TCA cycle enzymes in mature leaves of two rice varieties. Maximal activities of aconitase, NAD-dependent isocitrate dehydrogenase and NADP-dependent isocitrate dehydogenase were determined in mature leaves of Koshihikari and Takanari at the booting stage in 2013. The mean and SEM are shown (n = 4). For other details, see the legend of Fig. 1. Discussion We compared respiratory characteristics in the uppermost fully expanded leaves between two rice varieties cultivated at the FACE experimental site. The O2 uptake rates on a leaf dry weight basis were comparable between the two [CO2] conditions, whereas the CO2 efflux rates on a leaf dry weight basis decreased at elevated [CO2] at all growth stages (Fig. 1). From the panicle initiation stage, the CO2 efflux rates were significantly higher in leaves of Takanari, which showed higher growth and photosynthesis compared with Koshihikari. The 2°C increase in water temperature did not significantly affect the CO2 efflux rate or O2 uptake rate at any growth stage. The N and major amino acid contents were significantly higher in Takanari than in Koshihikari (Figs. 2, 3). Compared with Koshihikari leaves, Takanari leaves with their enhanced N assimilation may consume more respiratory NADH, leading to higher CO2 efflux rates than those in Koshihikari leaves. This hypothesis was supported by the findings that the organic acid contents were lower in Takanari leaves than in Koshihikari leaves, and the CO2 efflux rates on a leaf N basis were comparable between the two varieties (Figs. 1, 4). In Koshihikari leaves, the ratio of TCA cycle intermediates (2-OG/isocitrate) changed and the maximal activities of enzymes in the TCA cycle decreased at elevated [CO2] (Figs. 4, 5). The lower CO2 efflux rates at elevated [CO2] may have been due to the decreased activities of TCA cycle enzymes in Koshihikari leaves, but the activities of TCA cycle enzymes could not explain the decreased CO2 efflux rates at elevated [CO2] in Takanari leaves. Effects of elevated [CO2] on CO2 efflux rate and the related changes in rice leaves In Koshihikari leaves, the decrease in CO2 efflux rates at elevated [CO2] may have been due to the decreased activities of TCA cycle enzymes. In contrast to our study, previous studies have reported that the transcript levels of genes encoding TCA cycle enzymes were up-regulated at elevated [CO2] in mature leaves of rice (Fukayama et al. 2011), soybean (Leakey et al. 2009b) and A. thaliana (Markelz et al. 2014a, Watanabe et al. 2014). So far, the transcript levels of genes encoding TCA cycle enzymes have not been determined in Koshihikari leaves at elevated [CO2]. However, up-regulation of genes encoding respiratory enzymes at elevated [CO2] does not always correlate to increased amounts or activities of their encoded enzymes. In mature leaves of durum wheat (Triticum durum), malate dehydrogenase was the only TCA cycle enzyme that showed increased abundance at elevated [CO2] (Aranjuelo et al. 2015). Quantitative proteomics techniques may clarify which enzymes in the whole respiratory system, including the TCA cycle, increase or decrease at elevated [CO2] (Taylor et al. 2014). At elevated [CO2], a distinct increase in amino acid contents was not observed, but in Takanari leaves, the contents of alanine, asparagine, histidine and tryptophan increased at elevated [CO2] (Fig. 3; Supplementary Fig. S3). Similar increases were also reported in another study using rice leaves at elevated [CO2] (Onda et al. 2014). In tobacco and A. thaliana leaves, contents of major amino acids increased at elevated [CO2] under sufficient N conditions (Geiger et al. 1998, Watanabe et al. 2014). These studies used higher [CO2] at elevated [CO2] conditions (≥700 µmol mol–1) than this study, and thereby different responses may be observed. In this study, since we did not conduct 13 C labeling analysis (metabolic flux analysis; Tcherkez et al. 2012, Sweetlove and Ratcliffe 2011, Sweetlove et al. 2013) , it is difficult to relate the content of each metabolite to CO2 efflux rates directly. However, the decrease in the 2-OG/isocitrate ratio suggests the decrease in the TCA cycle flux and CO2 efflux rate in Koshihikari leaves at elevated [CO2]. Analyses of metabolomic data from some studies suggested that the content of isocitrate increases and the content of 2-OG decreases in A. thaliana leaves at elevated [CO2] (Hachiya et al. 2012, Sato and Yanagisawa 2014). These trends are similar to those observed in this study. In leaves of A. thaliana, the maximal activities of NAD-IDH and/or NADP-ICDH may decrease at elevated [CO2], similar to the case in Koshihikari. Meta-analyses have shown that CO2 efflux rates on a leaf dry weight basis are often lower at elevated [CO2] than at ambient [CO2] (Wang and Curtis 2002, Gonzalez-Meler et al. 2004). Similar changes were observed in two rice varieties in this study. Other studies have also reported significant increases in CO2 efflux rates on a leaf area basis at elevated [CO2] (Leakey et al. 2009b, Li et al. 2013, Markelz et al. 2014a). Since greater LMA at elevated [CO2] can contribute to a greater CO2 efflux rate on a leaf area basis, we should pay attention to the unit of the respiration rate when comparing data. Elevated [CO2] often induces down-regulation of photosynthesis (Makino and Mae 1999, Long et al. 2004). In this FACE site, Rubisco contents decreased in both varieties at elevated [CO2] (Chen et al. 2014). However, the decrease in Rubisco contents may not directly relate to the decrease in respiratory CO2 efflux rates at elevated [CO2]. This is because N and Rubisco contents were tightly correlated (Chen et al. 2014), but relationships between the CO2 efflux rate and the N contents differed between the two [CO2] conditions (Fig. 1). Adachi et al. (2014) reported that the photosynthetic rates under saturated light decreased synergistically in response to elevated [CO2] and water warming treatments in leaves of the japonica variety Akitakomachi at the grain-filling stage. This decrease was due to the decrease in N allocation to the leaves under these conditions. In this study, the N content was significantly decreased by the soil-water warming treatment at the mid grain-filling stage (Fig. 2j), but there was no synergistic effect of warming temperature and elevated [CO2] on the CO2 efflux rates, even at the late growth stage. Since much less N is allocated to the respiratory system than to the photosynthetic system (Makino and Osmond 1991), a small decrease in N content would probably not affect the leaf respiration rates directly. In this study, we examined the uppermost fully expanded leaves of two rice varieties. The effects of elevated [CO2] on CO2 efflux rates of aboveground parts have been investigated in rice plants grown in a growth chamber (Sakai et al. 2001) and at the FACE site (Xu et al. 2006). At the FACE site, the aboveground respiration rates on a dry weight basis were similar between ambient and elevated [CO2] conditions (Xu et al. 2006), whereas, in the growth chamber, the aboveground respiration rates on a dry weight basis differed between the two [CO2] conditions (Sakai et al. 2001). In addition, the aboveground respiration rates at elevated [CO2] were higher at the early growth stage, but lower at the later growth stage. The aboveground part of rice plants consists of leaves with various ages and compositions, which may lead to differences in the responses of the aboveground respiration rate to elevated [CO2]. In A. thaliana, leaf age also strongly affected the response of respiration rates to elevated [CO2] (Markelz et al. 2014b). Effects of elevated [CO2] on O2 uptake rate and the related changes in rice leaves The O2 uptake rates on a leaf dry weight basis were not different between the two [CO2] conditions, except at the panicle initiation stage (Fig. 1f–i). However, other studies have reported contrasting results for different species. In leaves of soybean grown at the FACE site, O2 uptake rates on a leaf area basis were higher at elevated [CO2] than at ambient [CO2] (Leakey et al. 2009b). In leaves of tomato transferred from ambient to elevated [CO2] conditions, the O2 uptake rate on a leaf fresh weight basis increased for a short period (Li et al. 2013). In leaves of A. thaliana, responses of O2 uptake rates to elevated [CO2] changed depending on time. At the end of the light period, the O2 uptake rate on a fresh weight basis was higher at elevated [CO2], while at the end of the night period, the rate was lower at elevated [CO2] than at ambient [CO2] (Watanabe et al. 2014). The O2 uptake rate is generally limited by substrate availability, enzyme activity or the ATP consumption rate (Noguchi 2005). Experiments with additions of substrate or uncoupler indicated that the O2 uptake rate in A. thaliana and tomato leaves is mainly determined by the ATP consumption rate (Li et al. 2013, Watanabe et al. 2014). In the two rice varieties in this study, non-structural carbohydrates accumulated in the leaves at elevated [CO2], but the O2 uptake rates were not higher at elevated [CO2] (Figs. 1, 4). These data suggested that the O2 uptake rates in leaves of the two rice varieties may be determined by ATP consumption rates, as is the case in leaves of A. thaliana and tomato. The plant mitochondrial respiratory chain consists of the cytochrome pathway and the alternative pathway. The cytochrome pathway is associated with H+ translocation, whereas the alternative pathway is catalyzed by the alternative oxidase, which is not coupled to H+ translocation (Vanlerberghe et al. 2016). The in vivo activities of both pathways affect the respiratory ATP production. In A. thaliana leaves, contents of some primary metabolites positively correlated to the in vivo activity of the cytochrome pathway after high-light treatment (Florez-Sarasa et al. 2016). In rice leaves, the correlation between the in vivo flux of the cytochrome pathway and primary metabolite contents may be observed. Differences in leaf respiration between two rice varieties The CO2 efflux rates on a leaf dry weight basis were significantly higher in Takanari leaves than in Koshihikari leaves from the panicle initiation stage (Fig. 1). In a previous study conducted at the same FACE site, photosynthetic rates under saturated light were higher in Takanari leaves than in Koshihikari leaves (Chen et al. 2014). In addition, the physiological parameters related to photosynthetic rates, stomatal conductance, Rubisco carboxylation rate and ribulose bisphosphate (RuBP) regeneration rate were significantly higher in Takanari leaves than in Koshihikari leaves (Chen et al. 2014). The Rubisco, soluble protein and N contents were also significantly higher in Takanari leaves than in Koshihikari leaves. Therefore, the N demand for photosynthetic components should be higher in Takanari leaves. In this study, the content of TCA cycle organic acids was lower and the ratio of total amino acids to TCA cycle organic acids was higher in Takanari leaves than in Koshihikari leaves (Figs. 3, 4), indicating that higher N demand and higher N assimilation may explain the higher rates of CO2 efflux in Takanari leaves than in Koshihikari leaves. In contrast, O2 uptake rates on a leaf dry weight basis did not differ between the two rice varieties, except at the heading stage (Fig. 1f–j). The higher rates of CO2 efflux in Takanari leaves indicate the higher rates of NADH production from the TCA cycle in Takanari leaves. NADH may be oxidized partly by cellular processes other than the respiratory chain in Takanari leaves, and thus NADH oxidation rates by the respiratory chain may be comparable between the two varieties, leading to the similar rates of O2 uptake between the two varieties. Saitoh et al. (2000) compared CO2 efflux rates of whole plants or whole leaves between Takanari and Nipponbare, another japonica variety. In their data, the CO2 efflux rates of whole leaves were comparable between Takanari and Nipponbare. The inconsistency between our data and theirs may be because different parts of the leaves were analyzed. In another study, the canopy top leaves of Takanari had a greater photosynthetic capacity and higher N content than those of Koshihikari, but leaf N per plant was lower in Takanari than in Koshihikari (Muryono et al. 2017). Their study also showed that the vertical gradient of leaf N content was steeper in Takanari than in Koshihikari, which may have contributed to higher productivity and higher photosynthetic and CO2 efflux rates in canopy top leaves of Takanari. The simulated canopy photosynthesis of Takanari was larger than that of Koshihikari (Ikawa et al. 2018). The CO2 efflux rates of whole leaves should be compared between Takanari and Koshihikari to compare the growth and yield of whole plants between the two varieties. Conclusion We compared the respiratory characteristics of mature leaves of two rice varieties grown at the FACE site. The CO2 efflux rates on a leaf dry weight basis decreased at elevated [CO2] at all growth stages. The CO2 efflux rates and the N contents were significantly higher in Takanari leaves than in Koshihikari leaves. In Takanari leaves, the contents of some amino acids were higher and those of some organic acids were lower, suggesting that organic acids may be used for N assimilation in Takanari leaves. This up-regulated N assimilation may support a higher rate of photosynthesis in Takanari. In Koshihikari leaves, the decreased activity of TCA cycle enzymes may have led to the decrease in CO2 efflux rates at elevated [CO2]. In Takanari leaves, the decreased CO2 efflux rates under elevated [CO2] could not be explained by the maximal activities of measured enzymes. The decreased flux of the TCA cycle in Takanari leaves may be caused by other unknown mechanisms under elevated [CO2]. Materials and Methods Site description We conducted the study at the Tsukuba FACE experimental facility in Tsukubamirai, Ibaraki, Japan (3° 8′N, 139°60′E, 10 m a.s.l.). There were four control (ambient [CO2]) plots and four FACE (elevated [CO2]) plots at the site. The average ambient [CO2] at the site across the entire growing season (June–September) and day to day SD was 383 ± 11.2 µmol mol–1 in 2012 and 384 ± 11.4 µmol mol–1 in 2013. The target concentration of the elevated [CO2] treatment was 200 µmol mol–1 above ambient [CO2], and the actual season-long mean [CO2] and day to day SD in the FACE plots was 578 ± 15.7 µmol mol–1 in 2012 and 576 ± 15.5 µmol mol–1 in 2013. In both the ambient and FACE plots, we set up normal temperature and elevated temperature conditions. In elevated temperature conditions, soil and water were heated by 2°C using heating wires under the soil surface between the rows. Previously published papers have provided further details of the experimental site set-up and CO2 control performance (Nakamura et al. 2012), elevated temperature treatment (Adachi et al. 2014, Usui et al. 2016) and soil chemical properties (Hasegawa et al. 2013). Plant materials Two rice (Oryza sativa L.) varieties were used in this study; the japonica variety ‘Koshihikari’ and the indica variety ‘Takanari’. Three-week-old seedlings were transplanted into the experimental plots on May 23–24 in 2012 and on May 22–23 in 2013. Both varieties received equal amounts of fertilizers prior to planting at a rate of 8.00 g m–2 of N, 4.36 g m–2 of P and 8.30 g m–2 of K, as described in Hasegawa et al. (2013). In the plots, Koshihikari achieved 50% heading by August 3 in both years, and Takanari achieved 50% heading by August 9 and 6 in 2012 and 2013, respectively (Chen et al. 2014). We used the uppermost fully expanded leaves for gas exchange measurements and sampling at two different growth stages: July 5–6 (panicle initiation stage) and August 16–17 (mid grain-filling stage) in 2012, and five different growth stages: June 24–27 (tillering stage), July 8–11 (panicle initiation stage), July 22–25 (booting stage), August 5–8 (heading stage) and August 19–22 (mid grain-filling stage), in 2013. At the heading and mid grain-filling stages, we used the flag leaves as the uppermost fully expanded leaves. Gas exchange measurements In 2012 and 2013, measurements of CO2 efflux rates were conducted using a portable photosynthetic gas exchange system (GFS3000, Walz) in the rice paddy from 10:00 to 15:00 h during 2 d at each stage. Measurements were taken on attached leaves (one leaf for each variety per temperature treatment per experimental plot at each growth stage) at a leaf temperature of 30°C, leaf chamber CO2 concentration of 390 (ambient plot) or 590 µmol mol–1 (FACE plot) and leaf chamber H2O concentration of 25,000 µmol mol–1. Since we conducted measurements of CO2 efflux rates using attached leaves in the rice paddy, we could put a limited size of one leaf (<8 cm2) in the chamber. We did not put two different leaves in the chamber in order to obtain more stable data as in Montero et al. (2016), but we waited to obtain constant and stable rates of CO2 efflux before logging. Measurements of O2 uptake rates were conducted using a gas-phase oxygen electrode system (LD2, Hansatech Instruments Ltd.) from 10:00 to 15:00 h during 2 d at each stage in 2013. Leaves were sampled in each plot (one leaf for each variety per temperature treatment per experimental plot at each growth stage), and measured in the cabin at the site. Measurements were taken on detached leaves at a leaf temperature of 30°C and ambient CO2 concentration in the leaf chamber. We measured dark respiration rates of mature leaves in the daytime because CO2 was supplied at the FACE experimental site during daylight hours from sunrise to sunset (Hasegawa et al. 2013). The CO2 efflux or O2 uptake rates were measured using leaves that had been dark adapted for at least 15 min. We checked that the 15 min darkness was enough to obtain constant rates of dark CO2 efflux and O2 uptake in leaves of the two varieties. The mean and the SEM of both rates from four different leaves of four experimental plots were calculated for each variety per temperature treatment per [CO2] treatment at each growth stage. After the gas exchange measurements, samples were kept in the dark and brought to the laboratory on the same day. The leaves were dried for 3 d at 80°C, weighed and the LMA was calculated. Leaf sampling For determinations of carbohydrates, primary metabolites and maximal enzymatic activities, leaves were sampled from 10:00 to 14:00 h (one leaf for each variety per temperature treatment per experimental plot at each growth stage) in 2013. The samples were weighed and immediately frozen in liquid N at each paddy plot. The samples were brought to the laboratory of the university and kept at −80°C until analysis. Determination of carbon and nitrogen contents The C and N contents were quantified as described by Sugiura et al. (2015). Dried leaves were ground using a metalcone with a multibeads shocker (Yasui Kikai), and then the C and N contents of the ground samples were measured with a CN analyzer (Vario micro, Elementar Analyzensysteme GmbH). Determination of carbohydrate content We used leaf samples collected at the heading stage in 2013 to determine the contents of non-structural carbohydrates (starch, glucose and sucrose). Non-structural carbohydrates were quantified as described by Watanabe et al. (2014). Frozen leaves were ground with a multibeads shocker (BS-12 R, Wakenyaku). After adding 1 ml of 80% ethanol, the suspension was incubated at 80°C for 10 min, and then centrifuged at 1,500×g at 4°C for 10 min. The precipitate was used for starch determination. Ethanol was removed from the supernatant by evaporation using a centrifugal concentrator (CC-105, Tomy). Equal volumes of distilled water and chloroform were added to the concentrated supernatant, the mixture was mixed well and then centrifuged at 10,000×g at 4°C for 10 min. The upper aqueous phase was used for determinations of glucose and sucrose. The precipitate was suspended in distilled water and boiled for 60 min at 100°C. An equal volume of amyloglucosidase was added to the boiled suspension and the mixture was incubated for 60 min at 55°C. The mixture was then centrifuged at 10,000×g at 4°C for 10 min, and the upper aqueous phase was used for starch determination. A Glucose C2 Test kit (Wako) was used to analyze the glucose content. Measurement of primary metabolites We used leaf samples collected at the booting stage in 2013 for determination of primary metabolite contents. Metabolites were extracted from leaves as described previously (Watanabe et al. 2010, Onda et al. 2014). Briefly, frozen leaves were ground with a mortar and pestle in liquid N and homogenized in 50% (v/v) methanol (10 µl mg–1 FW) containing 50 µM PIPES and 50 µM methionine sulfone as internal standards. After centrifugation at 21,500×g at 4°C for 5 min, the supernatants were filtered through a 3 kDa cut-off filter (Millipore) at 16,100×g at 4°C for 30 min. Primary metabolites, including amino acids, organic acids and phosphorylated compounds, were separated by capillary electrophoresis–triple quadrupole-mass spectrometry (CE; 7100 Capillary Electrophoresis, MS; 6420 Triple Quad LC/MS, Agilent Technologies), as described previously (Miyagi et al. 2010) with minor modifications. Metabolites were quantified by comparison with 54 compounds at known concentrations (Miyagi et al. 2010, Onda et al. 2014). All capillary electrophoresis mass spectrometry data were processed with Agilent MassHunter software (Agilent Technologies). Measurements of maximal activities of TCA cycle enzymes We used leaf samples collected at the booting stage in 2013 for measurements of enzyme activities. Frozen leaves were ground in liquid N and extracted in buffer [50 mM KH2PO4–K2HPO4 (pH 7.6), 10 mM MgSO4, 1 mM EDTA, 5 mM dithiothreitol, 0.05% (v/v) Triton X-100, and one protease inhibitor cocktail tablet (Complete, Roche Diagnostics GmbH) per 30 ml of buffer]. The mixture was centrifuged at 10,000×g at 4°C for 3 min, and the upper aqueous phase was used for measurements of maximal enzymatic activities. The maximal activities of aconitase, NAD-IDH and NADP-ICDH were determined as described by Noguchi and Terashima (2006). Statistical analysis Data were analyzed with a generalized linear mixed model (GLMM). In this model, the response variable was each datum at each stage, the explanatory variables were CO2, temperature, variety and interactions between these variables, and the random factor was block. When normality was not satisfied based on a Shapiro–Wilks test, we assumed a gamma error distribution with canonical link function, but, if satisfied, we assumed a Gaussian error distribution. All analyses were performed with the statistical software R (R Core Team 2016). The lme4 package (Bates et al. 2015) was used to calculate the GLMM by using maximum likelihood estimation. To determine the effects of fixed factors, we used a Wald test and the χ2 test statistic in the car package (Fox and Weisberg 2011). Statistical significance is noted if P < 0.1, and the results are summarized in Supplementary Table S1. Supplementary Data Supplementary data are available at PCP online. Funding This work was supported by the Ministry of Education, Culture, Sports, Science and Technology, Japan [Grants-in-Aid for Scientific Research on Innovative Areas (21114007 and 24114711)], CREST; the Science and Technology Agency (JST); a Yamaguchi Scholarship; and the Ministry of Agriculture, Forestry and Fisheries, Japan [through the research project ‘Development of Technologies for Mitigation and Adaptation to Climate Change in Agriculture, Forestry and Fisheries’]. 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Plant and Cell PhysiologyOxford University Press

Published: Mar 1, 2018

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