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Nitrogen metabolism correlates with the acclimation of photosynthesis to short-term water stress in rice (Oryza sativa L.)
Plant Physiology and Biochemistry 125 (2018) 52–62
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Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy
Research article
Nitrogen metabolism correlates with the acclimation of photosynthesis to short-term water stress in rice (Oryza sativa L.)
T
Chu Zhonga,b,1, Xiaochuang Caoa,1, Zhigang Baia, Junhua Zhanga, Lianfeng Zhua, Jianliang Huangb, Qianyu Jina,∗ a b
State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006, China Crop Physiology and Production Center, Huazhong Agricultural University, Wuhan 420007, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Soil drying Nitrogen metabolism Photosynthesis Acclimation Rice
Nitrogen metabolism is as sensitive to water stress as photosynthesis, but its role in plant under soil drying is not well understood. We hypothesized that the alterations in N metabolism could be related to the acclimation of photosynthesis to water stress. The features of photosynthesis and N metabolism in a japonica rice ‘Jiayou 5’ and an indica rice ‘Zhongzheyou 1’ were investigated under mild and moderate soil drying with a pot experiment. Soil drying increased non-photochemical quenching (NPQ) and reduced photon quantum efficiency of PSII and CO2 fixation in ‘Zhongzheyou 1’, whereas the effect was much slighter in ‘Jiayou 5’. Nevertheless, the photosynthetic rate of the two cultivars showed no significant difference between control and water stress. Soil drying increased nitrate reducing in leaves of ‘Zhongzheyou 1’, characterized by enhanced nitrate reductase (NR) activity and lowered nitrate content; whereas glutamate dehydrogenase (GDH), glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) were relative slightly affected. ‘Jiayou 5’ plants increased the accumulation of nitrate under soil drying, although its NR activity was increased. In addition, the activities of GDH, GOT and GPT were typically increased under soil drying. Besides, amino acids and soluble sugar were significantly increased under mild and moderate soil drying, respectively. The accumulation of nitrate, amino acid and sugar could serve as osmotica in ‘Jiayou 5’. The results reveal that N metabolism plays diverse roles in the photosynthetic acclimation of rice plants to soil drying.
1. Introduction
required for plant growth and development, as well as a key determinant of plant photosynthetic capacity. N metabolism is tightly linked with carbon (C) metabolism. It has been widely observed that soil water deficit induced reduction in photosynthetic rate was accompanied by the decrease in the activities of N metabolic enzymes such as nitrate reductase (NR), glutamine synthetase (GS), glutamate synthetase (GOGAT), and glutamine dehydrogenase (GDH) (Garg et al., 2001; Xu and Zhou, 2006; Singh et al., 2016). It is proposed that N metabolism plays a significant role in photosynthetic acclimation of plants to water stress (Xu and Zhou, 2006; Zhong et al., 2017). In cereal crops such as wheat (Triticum aestivum L.) and rice (Oryza sativa L.), GS has been viewed as an important metabolic indicator of drought tolerance (Nagy et al., 2013; Singh and Ghosh, 2013). GDH has also been recognized as a stress-response enzyme (Skopelitis et al., 2006). However, water stress had contrasting effects on GDH activity in different species, as remained unaltered in barley (Argandona and Pahlich, 1991), increased
Rice (Oryza sativa L.) consumes about 80% of agricultural irrigation water in Asia, where rice planting area accounts for about 90% of the world (Bouman and Tuong, 2001). Water-saving irrigation regimes have been introduced to rice production to deal with increasing irrigation water scarcity caused by population growth, urban and industry development, and environmental pollution (Bouman and Tuong, 2001; Chaves and Oliveira, 2004; Yang and Zhang, 2006; Price et al., 2013; Wang et al., 2016). However, severe water scarcity inhibits photosynthetic capacity of rice and eventually reduces plant growth and yield (Cao et al., 2017). Improving the adaptability of rice plants to water stress is therefore essential for maintaining the productivity of rice and for rescuing rice yield growth from the threat of increasing water shortage. Nitrogen (N) is a crucial and the largest quantity mineral nutrient
Abbreviations: GDH, glutamate dehydrogenase; GOGAT, glutamate synthetase; GOT, glutamic-oxaloacetic transaminase; GPT, glutamic-pyruvic transaminase; GS, glutamine synthetase; NPQ, non-photochemical quenching; NR, nitrate reductase; RWC, relative water content ∗ Corresponding author. E-mail address: [email protected] (Q. Jin). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.plaphy.2018.01.024 Received 14 November 2017; Received in revised form 18 January 2018; Accepted 23 January 2018 0981-9428/ © 2018 Elsevier Masson SAS. All rights reserved.
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treatment; whereas in moderate soil drying, the guttation was disappeared on day 8 of soil drying treatment. There were 16 pots and 6 plants in each pot, with 4 pots in control and 2 pots each for mild soil drying and moderate soil drying treatments per cultivar. The experiment was arranged in a completely randomized design. Sampling was conducted at the end of each soil drying phase.
in clusterbean (Garg et al., 1998), and decreased in Leymus chinensis (Xu and Zhou, 2006). The roles of N metabolic enzymes in acclimation of plants to water stress have remained need to be verified. The potential roles that N metabolism plays in the acclimation of photosynthesis to water stress could involve in the followings. Firstly, higher N increased the sensitivity of stomata to water stress and maintained better photosynthetic machinery (Otoo et al., 1989). A positive correlation between leaf N content and stomatal conductance (gs) was observed (Xiong et al., 2015a), suggesting N serves as a regulator of stomatal movement. In factor, nitrate (NO3−) participates in the regulation of stomatal opening. Wilkinson et al. (2007) reported that gs increased with increasing NO3− concentration in the rooting substrate even under drying condition. Secondly, N metabolism can consume excessive energy (ATP) and reducing power [NAD(P)H]. It is speculated that the reduction of 1 mol NO3− to NH4+ requires 8–12 mol ATP or 1.3–1.9 mol CO2 assimilated (Bloom et al., 1992). Therefore, N metabolism can partly dissipate the excessive captured light energy to mitigate photoinhibition of photosynthesis induced by water stress (Yi et al., 2014). Thirdly, synthesis of nitrogenous compounds, e.g., amino acid and soluble protein, provides osmotica to defend against water deficit in plant (Ashraf and Foolad, 2007; Cai et al., 2008; Li et al., 2010). Additionally, plants can dissipate excess light energy in the form of heat and increase the accumulation of nonstructural carbohydrates to protect photosynthesis from water stress (Fu et al., 2010; Silva et al., 2015). Nevertheless, how N metabolism in collaboration with these mechanisms in improving plant drought resistance remains unclear. In this study, we examined the photosynthetic and nitrogen metabolic features of two rice (Oryza sativa L.) cultivars ‘Zhongzheyou 1’ (hybrid indica) and ‘Jiayou 5’ (hybrid japonica) in response to different intensities of soil drying. We aimed to test the functional diversities of N metabolism in improving the resistance of rice photosynthesis to soil drying.
2.2. Measurement of gas exchange and chlorophyll fluorescence The youngest fully expanded leaves were used for the measurements of gas exchange and chlorophyll fluorescence simultaneously with LI6400XT portable photosynthesis system (Li-Cor Inc., Lincoln, NE, United States). The measurements were conducted during 08:30 to 12:00 with photosynthetic photon flux density (PPFD) of 1500 μmol m−2·s−1, temperature of 25 °C, and reference CO2 of 400 μmol·mol−1 in the cuvette. The relative humidity is controlled between 70 and 80%. Dark adapted chlorophyll fluorescence and dark respiration rate (Rdark) were determined at night before measurement of gas exchange. Data were recorded after equilibration to a steady-state. The following parameters were calculated: the effective quantum yield of PSII photochemistry [ФPSII=(Fm′-Fs)/Fm′]; the effective quantum yield of CO2 fixation [ФCO2=(Pn-Rdark) × PPFD × αleaf]; the photochemical quenching coefficient [qP=(Fm′-Fs)/(Fm′-F0′)]; the non-photochemical quenching coefficient [NPQ=(Fm-Fm′)/Fm′]; and the total electron transport rate at PSII level [JT = ФPSII × PPFD × 0.5 × αleaf]. The existence of alternative electron sink was evaluated with the ratio of JT/Pn (Silva et al., 2015). The coefficient 0.5 in JT is the fraction of excitation energy distributed to PSII, and αleaf in ФCO2 and JT is the fraction of incoming light absorbed by the leaves. The value of αleaf is assumed to be 0.85, which has been shown to be quite conservative among rice cultivars (Xiong et al., 2015b). 2.3. Xylem exudation collection
2. Materials and methods
Xylem exudation was collected from 18:00 to 8:00. Stems were cut off from 5 cm above the interface of root and stem, and four stems in each plant were covered with known-weight zip-lock bag containing absorbent cotton to capture xylem exudation. Then all the bags were collected and sealed, and the weight was weighed. The difference of the weight of bags was assumed to be the amount of xylem exudation. The density of exudation was assumed to be 1.0 g mL−1. Xylem exudation was stored at −20 °C for further analysis.
2.1. Plant cultivation and experimental treatment Hybrid indica rice cultivar ‘Zhongzheyou 1’ and hybrid japonica rice cultivar ‘Jiayou 5’ were used in this study. These two cultivars are important single-cropping rice cultivars in Zhejiang Province, China. ‘Zhongzheyou 1’ has obtained a widespread promoted application in South China. Seeds were germinated and sown in the seedbed of a rice paddy to grow for 25 days, then the 5-leaf age seedlings were transplanted to pots (45 cm long × 30 cm wide × 30 cm deep) containing 40 kg air-dried paddy soil. The soil fertility was the same as that we used in previous study (Cao et al., 2017), containing 2.65 g kg−1 total N, 36.8 g kg−1 organic matter, 142 mg kg−1 alkali-hydrolysable N, 17 mg kg−1 available P2O5, and 54.1 mg kg−1 available K2O. The pH of soil was 6.9. Phosphorous and potassium fertilizers were applied before transplanting, with the forms of calcium superphosphate (16% P2O5) and potassium chloride (60% K2O), respectively, and at the rates of 0.167 g kg−1 soil and 0.090 g kg−1 soil, respectively. N fertilizer (urea, 46% N) was applied as basal fertilizer and tillering fertilizer at the rates of 0.116 g kg−1 soil and 0.058 g kg−1 soil, respectively. Tillering fertilizer was applied 10 d after transplanting. Water stress was imposed to the plants 30 d after transplanting. There were three treatments in each cultivar. In the control, the plants were well-watered by keeping a 3-cm water layer above soil surface during experiment. Mild soil drying and moderate soil drying were imposed by withholding water application for 5 days and 10 days, respectively, after the disappearance of water layer on the soil surface. Water content of the soil was measured at the end of treatment. Soil water content in mild and moderate soil drying treatment was ∼85% and ∼60% of soil water-retaining capacity, respectively. In mild soil drying, guttation of leaves was occurred at night during soil drying
2.4. Biochemical profiling The youngest fully expanded leaves were sampled and frozen in liquid N2 immediately. Frozen leaves were fine grinded into powder with liquid N2 and stored at −70 °C for biochemical measurements and enzyme assays. Chlorophyll in fresh leaves (0.1 g) was extracted with 25 mL mixture of equivoluminal ethanol and acetone for 24 h at room temperature in dark. The absorbance of the extract was measured at 663 nm, 645 nm, and 470 nm to calculate chlorophyll content (Lichtenthaler, 1987). To measure nitrate (NO3−) and ammonium (NH4+), frozen leaf samples were homogenized with deionized water and centrifuged at 8300 × g and 4 °C for 10 min. The NO3− content in supernatant was quantified colorimetrically at 410 nm as in Cataldo et al. (1975), and KNO3 was used as standard. NH4+ in the supernatant was quantified photometrically as in Zanini (2001) at 630 nm, and (NH4)2SO4 was used as standard. Free amino acid contents in frozen leaves was measured with ninhydrin method according to Yokoyama and Hiramatsu (2003). Samples were homogenized with acetic acid/sodium acetate buffer (pH5.4) and centrifuged at 8300 × g for 10 min at 4 °C. The supernatant was reacted with ninhydrin, and the absorbance was monitored at 580 nm. Amino acid content was calculated by standard curve obtained from L-leucine. 53
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Fig. 1. Xylem secretion rate, and NO3− and free amino acid concentrations in xylem sap of ‘Zhongzheyou 1’ (A, C, and E) and ‘Jiayou 5’ (B, D, and F) under different intensities of soil drying. Five and ten days of soil drying represent mild and moderate soil drying, respectively. Xylem exudation was collected from 18:00 to 8:00. Values are means ± SE (n = 6). The significance of difference between control (well-watered) and water stress is indicated with asterisks by two-tailed Student's t-test: *, p < 0.05; **, p < 0.01.
transaminase (GOT), and glutamic-pyruvic transaminase (GPT). GS activity was determined according to Zhang et al. (1997). The enzyme extract was reacted with hydroxylamine hydrochloride in a 50 mM Tris-HCl buffer (pH7.4, containing 32 mM Mg2+, 7 mM ATP, 8 mM glutamate, 8 mM cysteine, and 0.8 mM EGTA) at 37 °C for 30 min, and the reaction was terminated by adding acidic FeCl3 (0.37 M FeCl3 and 0.2 M TCA in 0.6 M HCl). The protein deposits in the solution was removed by centrifugation at 8300 × g for 10 min. Then the absorbance of supernatant was measured chromometrically at 540 nm, and the activity of GS was expressed indirectly as A540·mg−1 protein·h−1. GOGAT activity was measured with the method described by Loulakakis and Roubelakis-Angelakis (1990). The reaction system consisted of 30 mM α-oxoglutarate, 0.3 mM KCl, 12.5 mM Tris-HCl buffer (pH 7.6), 0.2 mM NADH, 2 mM glutamine and modest enzyme extract. The absorbance at 340 nm was monitored for 300 s, and GOGAT activity was estimated by the oxidation of NADH per mg protein per min. Amination and deamination activities of glutamate dehydrogenase (GDH) were determined as in Loyola-Vargas and De Jimenez (1984). To measure the GDH amination activity, the reaction was carried out in a 100 mM Tris-HCl buffer (pH 8.0) containing 20 mM α-oxoglutarate, 200 mM NH4Cl, 1 mM CaCl2, 0.2 mM NADH, and modest enzyme extract. To measure the GDH deamination activity, the pH value of the buffer was adjusted to 9.2, and the substrates of α-oxoglutarate, NH4Cl and NADH were replaced by 100 mM L-glutamate and 1 mM NAD+. The absorbance at 340 nm was monitored for 300 s. The activities of GDH amination and deamination were estimated by the oxidation of
Proline content in frozen leaves was extracted with 5% sulfosalicylic acid in boiling water bath for 15 min and measure with a rapid method described previously by Bates et al. (1973). Standard curve was obtained from L-proline. To measure soluble sugar and sucrose, the leaf samples were homogenized in deionized water with mortar and pestle at 4 °C. The homogenate was centrifuged at 8300 × g for 10 min at 4 °C. The supernatant was used for soluble sugar and sucrose measurements. Total soluble sugar was measured by the anthrone-sulfuric acid method (Wang et al., 2002). Sucrose was analyzed by resorcinol hydrochloric acid method according to Li et al. (2012). 2.5. Enzyme activities assay Nitrate reductase (NR) in frozen leaf samples (about 100 mg) was extracted with 3 mL 25 mM potassium phosphate buffer (pH 7.5) containing 10 mM L-cysteine and 1 mM EDTA-Na2 (Hageman and Reed, 1980). The samples were homogenized with mortar and pestle, and the homogenates were centrifuged at 8300 × g for 10 min at 4 °C. NR activity in the supernatant was measured colorimetrically at 540 nm based on the reduction of nitrate to nitrite during 30 min at 25 °C. Frozen leaf samples (about 100 mg) were homogenized in 3 mL 50 mM Tris-HCl buffer (pH 8.0, containing 2 mM Mg2+, 2 mM DTT, and 0.4 M sucrose) with mortar and pestle, and the homogenates were centrifuged at 8300 × g for 10 min at 4 °C. The supernatant was used for enzyme assays of glutamine synthetase (GS), glutamate synthetase (GOGAT), glutamate dehydrogenase (GDH), glutamic-oxaloacetic 54
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NADH and reducing of NAD+ per mg protein per min, respectively. Glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) activities were assayed according to Wu et al. (1998). Modest enzyme extract was added to the substrate solution (pH 7.4) containing 2 mM α-oxoglutarate and 200 mM DL-aspartate (GOT) or 200 mM DL-alanine (GPT). The mixture was incubated at 37 °C for 1 h, and the reaction was terminated by adding 2, 4-dinitrophenylhydrazine. After incubating the mixture at 37 °C again for 20 min, 5 mL 0.4 M NaOH was added and the absorbance of the solution was chromometrically measured at 500 nm. The activities of GOT and GPT was expressed as the generating rate of pyruvic acid per mg protein. Soluble protein content in enzyme extract was measured using Bradford method (Bradford, 1976). Bovine serum albumin (BSA) was used as standard.
D). Mild soil drying and moderate soil drying decreased soluble protein content in ‘Zhongzheyou 1’ and ‘Jiayou 5’, respectively (Fig. 5A and B). Soil drying decreased free amino acid content in ‘Zhongzheyou 1’, with significant difference in mild soil drying (Fig. 5C). Free amino acid content in ‘Jiayou 5’ was significantly increased under mild soil drying, but decreased under moderate soil drying (Fig. 5D). Soil drying did not affect proline content in ‘Zhongzheyou 1’, whereas mild soil drying raised proline content remarkably in ‘Jiayou 5’ (Fig. 5E and F). 3.4. Leaf nitrogen metabolic enzymes activities Compared with control, NR activity in both cultivars was significantly higher under soil drying conditions (Fig. 6A and B). GS activity was not significantly affected by soil drying in ‘Zhongzheyou 1’ (Fig. 6C), whereas it was significantly increased under moderate soil drying in ‘Jiayou 5’ (Fig. 6D). Mild soil drying increased GOGAT activity significantly in ‘Zhongzheyou 1’, whereas there was no significant difference in GOGAT activity between control and water stress in ‘Jiayou 5’ (Fig. 6E and F). GDH deamination activity was significantly decreased by moderate soil drying in ‘Zhongzheyou 1’ (Fig. 7A) but significantly increased by mild soil drying in ‘Jiayou 5’ (Fig. 7B). Soil drying did not significantly affect GDH amination activity in ‘Zhongzheyou 1’, whereas mild soil drying and moderate soil drying significantly lowered and raised the GDH amination activity in ‘Jiayou 5’, respectively (Fig. 7C and D). GOT activity in ‘Zhongzheyou 1’ was not significantly affected by soil drying (Fig. 8A), while it was increased remarkably in ‘Jiayou 5’ under both soil drying conditions (Fig. 8B). Moderate soil drying lowered GPT activity in ‘Zhongzheyou 1’ (Fig. 8C). GPT activity responded oppositely to mild soil drying and moderate soil drying in ‘Jiayou 5’, with a significant decrease in the former condition and a significant increase in the latter condition (Fig. 8D).
2.6. Statistical analysis The average values were calculated based on 4 biological replications. The effects of soil drying were evaluated by Student's t-test. Differences were considered statistically significant when p < 0.05. 3. Results 3.1. Xylem secretion rate, and nitrate and amino acid concentrations in xylem sap Mild soil drying (5-d soil drying) had no significant effect on xylem secretion rate of the two rice cultivars, whereas moderate soil drying (10-d soil drying) reduced it extraordinarily in both cultivars (Fig. 1A and B). NO3− concentration in xylem exudation was extremely increased under soil drying in both cultivars, with greater increase in moderate soil drying (Fig. 1C and D). In contrast, free amino acid concentration in xylem exudation was substantially reduced under soil drying conditions, with the exception of ‘Zhongzheyou 1’ under moderate water stress, which was not significantly differ from control (Fig. 1E and F).
4. Discussion Rice is a water-intensive crop, however, a large number of evidence has revealed that moderate soil drying is conducive to rice plant growth and yield formation (Yang and Zhang, 2006, 2010), and can even maintain the rice grain yield as high as traditional flooding irrigation (Belder et al., 2004; Howell et al., 2015). Generally, soil drying reduces the availability of water, and subsequently declines photosynthesis of plant. It has been proposed that photosynthesis is affected in large part by leaf water status (Zhou et al., 2007), decreasing with the decline of leaf relative water content (RWC) (Ding et al., 2014). In this study, photosynthetic rates of the two cultivars were not significantly affected under soil drying conditions. It could be attributed to the stability of leaf RWC. Stomata play a paramount role in regulation of water loss in leaves. In this study, the reduction of stomatal conductance (gs) under moderate soil drying was in accordance with the descent of xylem secretion rate. It is suggested that partial closure of stomata plays a vital role in maintaining leaf RWC when active water absorption of root was constrained under soil drying (Zhong et al., 2017). On the other hand, stomatal closure becomes the major limit of photosynthesis under moderate water stress condition (Chaves and Oliveira, 2004). Mesophyll conductance (gm) determines the CO2 concentration at carboxylation site (Cc), which is as important as Ci for photosynthesis (Niinemets et al., 2009). Additionally, the increased photorespiration under water stress condition can act as an important CO2 concentrating mechanism in mesophyll cells (Peterhansel and Maurino, 2011). Previous study also revealed that gm was less affected by short-term water scarcity (Zhong et al., 2017). Although the stomatal conductance (gs) and intercellular CO2 concentration (Ci) were reduced by water stress, the photosynthetic rate of the both cultivars were not affected (Fig. 2). It is speculated that a slightly affected gm and Cc could partly contribute to this.
3.2. Gas exchange, sugar content, and chlorophyll fluorescence Both leaf relative water content (RWC) and photosynthetic rate (Pn) were not significantly affected under neither mild nor moderate soil drying (Fig. 2A–D). Mild soil drying did not affect stomatal conductance (gs) and intercellular CO2 concentration (Ci) in the two cultivars, whereas moderate soil drying significantly reduced gs and Ci in ‘Zhongzheyou 1’ and Ci in ‘Jiayou 5’ (Fig. 2E–H). Leaf soluble sugar and sucrose contents in ‘Zhongzheyou 1’ were not significantly affected by soil drying, but they were remarkably increased by moderate soil drying in ‘Jiayou 5’ (Fig. 3A–D). There was no significant difference in chlorophyll content between control and water stress treatments in both cultivars (Fig. 3E and F). As shown in Table 1, Fv/Fm was not affected by soil drying, whereas soil drying increased NPQ and JT/Pn in both cultivars, with a significant difference in NPQ in ‘Zhongzheyou 1’. The other chlorophyll fluorescence parameters such as ФPSII, ФCO2, JT, and qP had a tendency of decrease under soil drying conditions in ‘Zhongzheyou 1’, with no significant differences between control and water stress treatments. In ‘Jiayou 5’, these parameters were relatively less affected. 3.3. Leaf nitrogenous compounds The NO3− content in leaves of ‘Zhongzheyou 1’ was not affected under mild soil drying, but significantly decreased under moderate soil drying (Fig. 4A). In contrast, soil drying increased the NO3− content remarkably in ‘Jiayou 5’ (Fig. 4B). NH4+ content in ‘Zhongzheyou 1’ was not significantly affected by soil drying. However, it was significantly decreased by moderate soil drying in ‘Jiayou 5’ (Fig. 4C and 55
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Fig. 2. Leaf relative water content and gas exchange parameters of ‘Zhongzheyou 1’ (A, C, E, and G) and ‘Jiayou 5’ (B, D, F, and H) under different intensities of soil drying. A and B, relative water content (RWC); C and D, net photosynthetic rate (Pn); E and F, stomatal conductance (gs); and G and H, intercellular CO2 concentration (Ci). Five and ten days of soil drying represents mild and moderate soil drying, respectively. Values are means ± SE (n = 4). The significance of difference between control (well-watered) and water stress is indicated as asterisks by two-tailed Student's t-test: *, p < 0.05; **, p < 0.01.
between NPQ and ФCO2 (r = −0.4385, p = 0.012, n = 32), suggesting light energy dissipation is an efficient strategy for ‘Zhongzheyou 1’ to protect photosynthesis when electron flow was dropped in CO2 fixation caused by soil water deficit. Previous studies indicated a negative influence of soil water deficit on plant N metabolism, characterized as reductions in the activities of N metabolism enzymes and the synthesis of nitrogenous compounds (Xu and Zhou, 2006; Pinheiro and Chaves, 2011). Our results, which distinguish from those in previous studies, generally displayed positive responses of the activities of N metabolism enzymes to soil drying, indicating increased N metabolism plays important roles in photosynthetic acclimation in rice when the plants were subjected to water stress. It has been proposed that changes in xylem sap composition influence root to shoot signaling under drought and that NO3− could play a modulatory role in this process (Goodger and Schachtman, 2010). Soil drying ameliorates aeration condition of paddy soil,
The reduced CO2 availability in mesophyll cells due to stomatal closure leads to relative excess of light energy and electron sink, and results in reduction of photochemical efficiency and electron transport (Atkin and Macherel, 2009). In the current study, leaf chlorophyll content was intact under soil drying conditions, indicating the light energy capture is not affected. Therefore, plants have to dissipate excessive excitation energy to attenuate photoinhibition of photosynthesis. Non-photochemical quenching (NPQ) reflects the level of light energy dissipation in the form of heat (Maxwell and Johnson, 2000). In this study, water stress increased NPQ significantly in ‘Zhongzheyou 1’ (Table 1), in which the diffusion of CO2 through stomata was greater limited than in ‘Jiayou 5’. NPQ functions when the electron transport downstream of PSII (e.g., carbon fixation, photorespiration, nitrogen assimilation, etc.) is inhibited (Zhao et al., 2016). In this study, we found a larger reduction of ФCO2 in ‘Zhongzheyou 1’ (13.23%–14.35%) than in ‘Jiayou 5’ (4.29%–8.30%), and a significant negative correlation 56
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Fig. 3. Soluble sugar, sucrose and chlorophyll contents in ‘Zhongzheyou 1’ (A, C and E) and ‘Jiayou 5’ (B, D and F) under different intensities of soil drying. Five and ten days of soil drying represents mild and moderate soil drying, respectively. Values are means ± SE (n = 4). The significance of difference between control (well-watered) and water stress is indicated as asterisks by two-tailed Student's t-test: *, p < 0.05; **, p < 0.01.
increasing the NO3−/NH4+ ratio in soil solution, and subsequently changing xylem sap compositions and the activities of N metabolic enzymes in rice plants (Zhao et al., 2013; Dodd et al., 2015; Hu et al., 2017). Here, we observed a considerable increase in NO3−
concentration but a remarkable decrease in free amino acid concentration in xylem sap of water-stressed plant. The results imply that the responses of N metabolism to water stress could partly due to the changes in xylem sap composition. NO3− is mainly assimilated in
Table 1 Chlorophyll fluorescence parameters of ‘Zhongzheyou 1’ and ‘Jiayou 5’ under different intensities of soil drying. Five and ten days of soil drying represents mild and moderate soil drying, respectively. Values are means ± SE (n = 4). Bold data indicate significant difference from the control (well-watered) by two-tailed Student's t-test at α = 0.05 level. Parameters
Zhongzheyou 1 Fv/Fm ФPSII ФCO2 JT qP NPQ JT/Pn Jiayou 5 Fv/Fm ФPSII ФCO2 JT qP NPQ JT/Pn
5d
10 d
Control
Water stress
Control
Water stress
0.814 ± 0.004 0.354 ± 0.015 0.0209 ± 0.0013 232.39 ± 9.82 0.626 ± 0.020 1.241 ± 0.041 8.76 ± 0.24
0.818 ± 0.001 0.320 ± 0.005 0.0179 ± 0.0005 210.36 ± 3.29 0.591 ± 0.009 1.449 ± 0.036 9.51 ± 0.30
0.815 ± 0.002 0.321 ± 0.011 0.0189 ± 0.0010 211.08 ± 7.19 0.550 ± 0.025 1.183 ± 0.066 8.99 ± 0.18
0.812 ± 0.003 0.298 ± 0.011 0.0164 ± 0.0007 194.97 ± 7.44 0.547 ± 0.024 1.409 ± 0.055 9.68 ± 0.52
0.830 ± 0.002 0.411 ± 0.010 0.0253 ± 0.0006 269.79 ± 6.87 0.671 ± 0.010 1.251 ± 0.036 8.46 ± 0.42
0.825 ± 0.001 0.411 ± 0.011 0.0232 ± 0.0007 269.84 ± 7.08 0.678 ± 0.015 1.326 ± 0.021 9.04 ± 0.41
0.828 ± 0.001 0.333 ± 0.010 0.0163 ± 0.0003 218.41 ± 6.42 0.559 ± 0.014 1.390 ± 0.071 10.95 ± 0.31
0.825 ± 0.001 0.338 ± 0.002 0.0156 ± 0.0011 221.50 ± 1.42 0.575 ± 0.012 1.477 ± 0.109 11.74 ± 0.97
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Fig. 4. Leaf nitrate and ammonium contents in ‘Zhongzheyou 1’ (A and C) and ‘Jiayou 5’ (B and D) under different intensities of soil drying. Five and ten days of soil drying represents mild and moderate soil drying, respectively. Values are means ± SE (n = 4). The significance of difference between control (well-watered) and water stress is indicated as asterisks by two-tailed Student's t-test: *, p < 0.05; **, p < 0.01.
Fig. 5. Soluble protein, free amino acids and proline contents in ‘Zhongzheyou 1’ (A, C, and E) and ‘Jiayou 5’ (B, D, and F) under different intensities of soil drying. Five and ten days of soil drying represents mild and moderate soil drying, respectively. Values are means ± SE (n = 4). The significance of difference between control (well-watered) and water stress is indicated as asterisks by two-tailed Student's t-test: *, p < 0.05; **, p < 0.01.
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Fig. 6. Nitrate reductase (NR), glutamine synthease (GS) and glutamate synthetase (GOGAT) activities in ‘Zhongzheyou 1’ (A, C, and E) and ‘Jiayou 5’ (B, D, and F) under different intensities of soil drying. Five and ten days of soil drying represents mild and moderate soil drying, respectively. Values are means ± SE (n = 4). The significance of difference between control (well-watered) and water stress is indicated as asterisks by two-tailed Student's t-test: *, p < 0.05; **, p < 0.01.
leaves. It should be noted that compared to control, the NO3− transport rate through xylem (μg·h−1) was higher under mild soil drying but lower under moderate soil drying (Fig. 1), which was not completely in line with the NO3− content in leaves (Fig. 4A and B). It is suggested that primary N assimilation in leaves could involves in regulating the photosynthetic acclimation of rice plants to water stress. NR is the rate-limiting enzyme in NO3− assimilation. Numerous studies have reported that water stress reduces NR activity (Foyer et al., 1998; Pandey and Agarwal, 1998; Plaut, 2010). Cornic (2000) proposed that reduction of NR activity can be of some advantage to the plant, because it can rapidly contribute to the maintenance of osmotic pressure in the photosynthetic cells by increasing the NO3− level. Distinct result from previous studies was obtained in this study that soil drying enhanced NR activity in both studied cultivars. NR is a substrate inducible enzyme, and it is regulated by the ratio of NO3−/NH4+ (Oaks et al., 1990). The ratio of NO3−/NH4+ in leaves reflects NO3− uptake and assimilation (Meng et al., 2016). In ‘Zhongzheyou 1’, water stress induced decrease of NO3− in leaves was in line with the increase in NR activity. Additionally, the ratio of NO3−/NH4+ was reduced from 56.11 to 44.82 and from 45.09 to 33.78 when the plants were subjected to mild and moderate soil drying, respectively. It indicates that increasing NO3− reducing is an important mechanism for ‘Zhongzheyou 1’ to cope with soil water stress. In addition, the NO3− transport rate in xylem of ‘Zhongzheyou 1’ was consistent with NO3− content in leaves under moderate soil drying, indicating the coordination between NO3−
uptake and assimilation in improving the acclimation of photosynthesis to water stress. In contrast, even though NO3− uptake was decreased under moderate soil drying condition, the highly accumulation of NO3− and obviously increased NO3−/NH4+ ratio in ‘Jiayou 5’ implies that NO3− reduction was attenuated. Azedo-Silva et al. (2004) reported that the change of NR activity was not agree with NO3− content. The synchronistical increased NO3− content and NR activity in ‘Jiayou 5’ could be due to the compartmentation of NO3− assimilation in leaves (Sechley et al., 1992), for NO3− is mainly stored in vacuoles (Martinoia et al., 2007), isolated from NR which is located in cytoplasm. The different responses of NO3− to soil drying in the two cultivars implies diverse mechanisms that NO3− reducing involves in the acclimation of rice plants to soil drying. Increased NO3− reducing in ‘Zhongzheyou 1’ could play a vital role in dissipation of excessive energy, because NO3− reduction is a highly energy requirement process (Sunil et al., 2013). It requires reduced compounds, such as NADH, NADPH, reduced ferredoxin and ATP produced in mitochondrial oxidative metabolism, photosynthesis, photorespiration, or even the cytosolic reactions, depending on the site where the reaction happens (Sunil et al., 2013). NO3− reducing in leaves could use the excessive energy derived from photosynthetic apparatus, which is more efficient than that takes place in root (Gonzalez-Dugo et al., 2010). The reduced amino acid concentration in xylem sap of water-stressed plants supported this notion. NO3− assimilation is acting as an effective electron sink (evidenced by increase in JT/Pn) alleviating photoinhibition of 59
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Fig. 7. Deamination and amination activities of GDH in ‘Zhongzheyou 1’ (A and C) and ‘Jiayou 5’ (B and D) under different intensities of soil drying. Five and ten days of soil drying represents mild and moderate soil drying, respectively. Values are means ± SE (n = 4). The significance of difference between control (well-watered) and water stress is indicated as asterisks by two-tailed Student's t-test: *, p < 0.05; **, p < 0.01.
affected stomatal conductance (gs) in ‘Jiayou 5’ under moderate soil drying could be partly due to its high NO3− content in leaves. Increasing stomatal aperture is advantageous to gas exchange when leaf water content was not affected under water stress condition. Water stress will cause the accumulation of NH4+ in plant. Avoiding excess NH4+ accumulation in plant tissues is viewed as an important capacity to withstand water stress (Hoai et al., 2003). GS, GOGAT, and GDH are key enzymes in incorporation of NH4+ into amides and amino acids (Lawlor, 2002; Masclaux-Daubresse et al., 2006). In this study, GS
photosynthesis (evidenced by the constant of Fv/Fm and slightly affected JT) under water stress condition (Gonzalez-Dugo et al., 2010; Yi et al., 2014). In addition to as an important nutrient, NO3− plays multiple functions in plant such as regulating plant growth and development and adaptation to fluctuating environments (Linkohr et al., 2002; Wilkinson et al., 2007; Krouk et al., 2010). Besides, NO3− is an important factor regulating stomatal movement (Guo et al., 2003; Wilkinson et al., 2007). Higher NO3− content in guard cells leads to depolarization of guard cells and then stomatal opening. The less
Fig. 8. Glutamic-oxalacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) activities in ‘Zhongzheyou 1’ (A and C) and ‘Jiayou 5’ (B and D) under different intensities of soil drying. Five and ten days of soil drying represents mild and moderate soil drying, respectively. Values are means ± SE (n = 4). The significance of difference between control (well-watered) and water stress is indicated as asterisks by two-tailed Student's t-test: *, p < 0.05; **, p < 0.01.
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Author contributions
and GOGAT activities were increased to a certain extent under soil drying conditions in both cultivars, whereas GDH deamination/amination activity was particularly affected by water stress in ‘Jiayou 5’. It has been well known that GDH is independent of GS-GOGAT in N assimilation (Masclaux-Daubresse et al., 2006). It acts as an anti-stress enzyme in ammonia detoxification and production of Glu for proline synthesis under stress condition (Boussama et al., 1999; Skopelitis et al., 2006). In addition, Glu can be converted into Asp and Ala via glutamicoxalacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT), respectively (King and Waygood, 1968). Asp serves as an additional NH4+ detoxification molecule (Masclaux-Daubresse et al., 2006) and as the precursor for the synthesis of branched-chain amino acids (BCAAs) (Joshi et al., 2010). BCAAs derived from Asp pathway, such as Met, Thr, and Ile provide precursors for a number of plant secondary metabolites (Joshi et al., 2010). For example, ethylene synthesis which plays important roles in plant stress responses is derived from Met. Water stress induced significant increase in GOT activity and the alteration of GPT activity, suggesting that enhanced amino acid metabolism could play a key role in improving the resistance of photosynthesis to soil drying in ‘Jiayou 5’. Increased amino acid synthesis could be advantageous to plant, because amino acids can serve as osmotica to maintain the turgor of leaf under water stress condition (Singh et al., 2016). In this study, a significant increment of free amino acid and proline was observed in mild water stressed plants of ‘Jiayou 5’, indicating N metabolism took part in osmotic adjustment to protect photosynthesis from soil drying. The accumulation of amino acid could be resulted from N assimilation or proteolysis (Wingler et al., 1999). In this study, the increments of free amino acid and proline were more possibly resulted from the amino acid synthesis, for the protein content was constant and the activities of N assimilation enzymes were increased. However, the osmotic adjustment of amino acid and proline was limited under moderate soil drying in ‘Jiayou 5’, as their contents were lowered or not significantly changed. Interestingly, soluble sugar and sucrose were highly accumulated under such condition. Soluble sugars are important osmolyte in water stressed plants (Sánchez et al., 1998). The accumulation of soluble sugar and sucrose could compensate the reduction of soluble protein and free amino acids to maintain the turgor of leaf. It indicates that N and C metabolisms are cooperated in osmoregulation under soil drying condition.
C.Z. and X.C. collected the samples, analyzed the samples, and drafted the manuscript. Z.B. collected the samples and analyzed the samples. L.Z. and J.Z. analyzed the data and revised the manuscript. J.H. and Q.J. conceived and designed this work. All authors read and approved the manuscript. Acknowledgment This work was funded by the Natural Science Foundation of Zhejiang Province (No. LY18C130005, LQ15C130004); the National Key Research and Development Program of China (No. 2017YFD0300100, 2016YFD0101801); and the National Basic Research Program of China (No. 2015CB150502). References Argandona, V., Pahlich, E., 1991. Water stress on proline content and enzyme activities in barley seedlings. Phytochemistry 30, 1093–1094. Ashraf, M., Foolad, M.R., 2007. Roles of glycine betaine and proline in improving plant abiotic stress resistance. Environ. Exp. Bot. 59, 206–216. Atkin, O.K., Macherel, D., 2009. The crucial role of plant mitochondria in orchestrating drought tolerance. Ann. Bot. 103 (4), 581–597. Azedo-Silva, J., Osório, J., Fonseca, F., Correia, M.J., 2004. Effects of soil drying and subsequent re-watering on the activity of nitrate reductase in roots and leaves of Helianthus annuus. Funct. Plant Biol. 31 (6), 611–621. Bates, L.S., Waldren, R.P., Teare, I.D., 1973. Rapid determination of free proline for water-stress studies. Plant Soil 39, 205–207. Belder, P., Bouman, B.A.M., Cabangon, R., Lu, G., Quilang, E.J.P., Li, Y., Spiertz, J.H.J., Tuong, T.P., 2004. Effect of water-saving irrigation on rice yield and water use in typical lowland conditions in Asia. Agric. Water Manag. 65, 193–210. Bloom, A.J., Sukrapanna, S.S., Warner, R.L., 1992. Root respiration associated with ammonium and nitrate absorption and assimilation by barley. Plant Physiol. 99, 1294–1301. Bouman, B.A.M., Tuong, T.P., 2001. Field water management to save water and increase its productivity in irrigated lowland rice. Agric. Water Manag. 49, 11–30. 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5. Conclusion In conclusion, the results reveal that N metabolism is sensitive to soil drying, and that the alterations in N metabolism are of great importance for the acclimation of rice photosynthesis to soil moisture changes. N metabolism plays various roles in improving the acclimation of rice photosynthesis to soil drying in different rice cultivars. In indica rice ‘Zhongzheyou 1’, NO3− reducing was strengthened under soil drying, whereas GDH pathway of N assimilation and Glu transamination through GOT and GPT were relatively slightly affected. NO3− reducing could contribute to alleviating the photoinhibition of photosynthesis with the coordination with NPQ under soil drying, as it is an important energy dissipation pathway in leaves. In japonica rice ‘Jiayou 5’, N assimilation via GDH and transamination through GOP and GPT were strengthened, and the accumulation of NO3− and amino acid was observed, suggesting N metabolism could participate in osmotic adjustment under soil drying conditions. Water stress induced alterations in N metabolism is tightly linked with the changes in chlorophyll fluorescence and carbon metabolism. The results imply that N metabolism could be a key regulatory target for improving the acclimation of photosynthesis to soil drying in rice. Conflicts of interest The authors declare that they have no conflict of interest. 61
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Research article
Nitrogen metabolism correlates with the acclimation of photosynthesis to short-term water stress in rice (Oryza sativa L.)
T
Chu Zhonga,b,1, Xiaochuang Caoa,1, Zhigang Baia, Junhua Zhanga, Lianfeng Zhua, Jianliang Huangb, Qianyu Jina,∗ a b
State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006, China Crop Physiology and Production Center, Huazhong Agricultural University, Wuhan 420007, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Soil drying Nitrogen metabolism Photosynthesis Acclimation Rice
Nitrogen metabolism is as sensitive to water stress as photosynthesis, but its role in plant under soil drying is not well understood. We hypothesized that the alterations in N metabolism could be related to the acclimation of photosynthesis to water stress. The features of photosynthesis and N metabolism in a japonica rice ‘Jiayou 5’ and an indica rice ‘Zhongzheyou 1’ were investigated under mild and moderate soil drying with a pot experiment. Soil drying increased non-photochemical quenching (NPQ) and reduced photon quantum efficiency of PSII and CO2 fixation in ‘Zhongzheyou 1’, whereas the effect was much slighter in ‘Jiayou 5’. Nevertheless, the photosynthetic rate of the two cultivars showed no significant difference between control and water stress. Soil drying increased nitrate reducing in leaves of ‘Zhongzheyou 1’, characterized by enhanced nitrate reductase (NR) activity and lowered nitrate content; whereas glutamate dehydrogenase (GDH), glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) were relative slightly affected. ‘Jiayou 5’ plants increased the accumulation of nitrate under soil drying, although its NR activity was increased. In addition, the activities of GDH, GOT and GPT were typically increased under soil drying. Besides, amino acids and soluble sugar were significantly increased under mild and moderate soil drying, respectively. The accumulation of nitrate, amino acid and sugar could serve as osmotica in ‘Jiayou 5’. The results reveal that N metabolism plays diverse roles in the photosynthetic acclimation of rice plants to soil drying.
1. Introduction
required for plant growth and development, as well as a key determinant of plant photosynthetic capacity. N metabolism is tightly linked with carbon (C) metabolism. It has been widely observed that soil water deficit induced reduction in photosynthetic rate was accompanied by the decrease in the activities of N metabolic enzymes such as nitrate reductase (NR), glutamine synthetase (GS), glutamate synthetase (GOGAT), and glutamine dehydrogenase (GDH) (Garg et al., 2001; Xu and Zhou, 2006; Singh et al., 2016). It is proposed that N metabolism plays a significant role in photosynthetic acclimation of plants to water stress (Xu and Zhou, 2006; Zhong et al., 2017). In cereal crops such as wheat (Triticum aestivum L.) and rice (Oryza sativa L.), GS has been viewed as an important metabolic indicator of drought tolerance (Nagy et al., 2013; Singh and Ghosh, 2013). GDH has also been recognized as a stress-response enzyme (Skopelitis et al., 2006). However, water stress had contrasting effects on GDH activity in different species, as remained unaltered in barley (Argandona and Pahlich, 1991), increased
Rice (Oryza sativa L.) consumes about 80% of agricultural irrigation water in Asia, where rice planting area accounts for about 90% of the world (Bouman and Tuong, 2001). Water-saving irrigation regimes have been introduced to rice production to deal with increasing irrigation water scarcity caused by population growth, urban and industry development, and environmental pollution (Bouman and Tuong, 2001; Chaves and Oliveira, 2004; Yang and Zhang, 2006; Price et al., 2013; Wang et al., 2016). However, severe water scarcity inhibits photosynthetic capacity of rice and eventually reduces plant growth and yield (Cao et al., 2017). Improving the adaptability of rice plants to water stress is therefore essential for maintaining the productivity of rice and for rescuing rice yield growth from the threat of increasing water shortage. Nitrogen (N) is a crucial and the largest quantity mineral nutrient
Abbreviations: GDH, glutamate dehydrogenase; GOGAT, glutamate synthetase; GOT, glutamic-oxaloacetic transaminase; GPT, glutamic-pyruvic transaminase; GS, glutamine synthetase; NPQ, non-photochemical quenching; NR, nitrate reductase; RWC, relative water content ∗ Corresponding author. E-mail address: [email protected] (Q. Jin). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.plaphy.2018.01.024 Received 14 November 2017; Received in revised form 18 January 2018; Accepted 23 January 2018 0981-9428/ © 2018 Elsevier Masson SAS. All rights reserved.
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treatment; whereas in moderate soil drying, the guttation was disappeared on day 8 of soil drying treatment. There were 16 pots and 6 plants in each pot, with 4 pots in control and 2 pots each for mild soil drying and moderate soil drying treatments per cultivar. The experiment was arranged in a completely randomized design. Sampling was conducted at the end of each soil drying phase.
in clusterbean (Garg et al., 1998), and decreased in Leymus chinensis (Xu and Zhou, 2006). The roles of N metabolic enzymes in acclimation of plants to water stress have remained need to be verified. The potential roles that N metabolism plays in the acclimation of photosynthesis to water stress could involve in the followings. Firstly, higher N increased the sensitivity of stomata to water stress and maintained better photosynthetic machinery (Otoo et al., 1989). A positive correlation between leaf N content and stomatal conductance (gs) was observed (Xiong et al., 2015a), suggesting N serves as a regulator of stomatal movement. In factor, nitrate (NO3−) participates in the regulation of stomatal opening. Wilkinson et al. (2007) reported that gs increased with increasing NO3− concentration in the rooting substrate even under drying condition. Secondly, N metabolism can consume excessive energy (ATP) and reducing power [NAD(P)H]. It is speculated that the reduction of 1 mol NO3− to NH4+ requires 8–12 mol ATP or 1.3–1.9 mol CO2 assimilated (Bloom et al., 1992). Therefore, N metabolism can partly dissipate the excessive captured light energy to mitigate photoinhibition of photosynthesis induced by water stress (Yi et al., 2014). Thirdly, synthesis of nitrogenous compounds, e.g., amino acid and soluble protein, provides osmotica to defend against water deficit in plant (Ashraf and Foolad, 2007; Cai et al., 2008; Li et al., 2010). Additionally, plants can dissipate excess light energy in the form of heat and increase the accumulation of nonstructural carbohydrates to protect photosynthesis from water stress (Fu et al., 2010; Silva et al., 2015). Nevertheless, how N metabolism in collaboration with these mechanisms in improving plant drought resistance remains unclear. In this study, we examined the photosynthetic and nitrogen metabolic features of two rice (Oryza sativa L.) cultivars ‘Zhongzheyou 1’ (hybrid indica) and ‘Jiayou 5’ (hybrid japonica) in response to different intensities of soil drying. We aimed to test the functional diversities of N metabolism in improving the resistance of rice photosynthesis to soil drying.
2.2. Measurement of gas exchange and chlorophyll fluorescence The youngest fully expanded leaves were used for the measurements of gas exchange and chlorophyll fluorescence simultaneously with LI6400XT portable photosynthesis system (Li-Cor Inc., Lincoln, NE, United States). The measurements were conducted during 08:30 to 12:00 with photosynthetic photon flux density (PPFD) of 1500 μmol m−2·s−1, temperature of 25 °C, and reference CO2 of 400 μmol·mol−1 in the cuvette. The relative humidity is controlled between 70 and 80%. Dark adapted chlorophyll fluorescence and dark respiration rate (Rdark) were determined at night before measurement of gas exchange. Data were recorded after equilibration to a steady-state. The following parameters were calculated: the effective quantum yield of PSII photochemistry [ФPSII=(Fm′-Fs)/Fm′]; the effective quantum yield of CO2 fixation [ФCO2=(Pn-Rdark) × PPFD × αleaf]; the photochemical quenching coefficient [qP=(Fm′-Fs)/(Fm′-F0′)]; the non-photochemical quenching coefficient [NPQ=(Fm-Fm′)/Fm′]; and the total electron transport rate at PSII level [JT = ФPSII × PPFD × 0.5 × αleaf]. The existence of alternative electron sink was evaluated with the ratio of JT/Pn (Silva et al., 2015). The coefficient 0.5 in JT is the fraction of excitation energy distributed to PSII, and αleaf in ФCO2 and JT is the fraction of incoming light absorbed by the leaves. The value of αleaf is assumed to be 0.85, which has been shown to be quite conservative among rice cultivars (Xiong et al., 2015b). 2.3. Xylem exudation collection
2. Materials and methods
Xylem exudation was collected from 18:00 to 8:00. Stems were cut off from 5 cm above the interface of root and stem, and four stems in each plant were covered with known-weight zip-lock bag containing absorbent cotton to capture xylem exudation. Then all the bags were collected and sealed, and the weight was weighed. The difference of the weight of bags was assumed to be the amount of xylem exudation. The density of exudation was assumed to be 1.0 g mL−1. Xylem exudation was stored at −20 °C for further analysis.
2.1. Plant cultivation and experimental treatment Hybrid indica rice cultivar ‘Zhongzheyou 1’ and hybrid japonica rice cultivar ‘Jiayou 5’ were used in this study. These two cultivars are important single-cropping rice cultivars in Zhejiang Province, China. ‘Zhongzheyou 1’ has obtained a widespread promoted application in South China. Seeds were germinated and sown in the seedbed of a rice paddy to grow for 25 days, then the 5-leaf age seedlings were transplanted to pots (45 cm long × 30 cm wide × 30 cm deep) containing 40 kg air-dried paddy soil. The soil fertility was the same as that we used in previous study (Cao et al., 2017), containing 2.65 g kg−1 total N, 36.8 g kg−1 organic matter, 142 mg kg−1 alkali-hydrolysable N, 17 mg kg−1 available P2O5, and 54.1 mg kg−1 available K2O. The pH of soil was 6.9. Phosphorous and potassium fertilizers were applied before transplanting, with the forms of calcium superphosphate (16% P2O5) and potassium chloride (60% K2O), respectively, and at the rates of 0.167 g kg−1 soil and 0.090 g kg−1 soil, respectively. N fertilizer (urea, 46% N) was applied as basal fertilizer and tillering fertilizer at the rates of 0.116 g kg−1 soil and 0.058 g kg−1 soil, respectively. Tillering fertilizer was applied 10 d after transplanting. Water stress was imposed to the plants 30 d after transplanting. There were three treatments in each cultivar. In the control, the plants were well-watered by keeping a 3-cm water layer above soil surface during experiment. Mild soil drying and moderate soil drying were imposed by withholding water application for 5 days and 10 days, respectively, after the disappearance of water layer on the soil surface. Water content of the soil was measured at the end of treatment. Soil water content in mild and moderate soil drying treatment was ∼85% and ∼60% of soil water-retaining capacity, respectively. In mild soil drying, guttation of leaves was occurred at night during soil drying
2.4. Biochemical profiling The youngest fully expanded leaves were sampled and frozen in liquid N2 immediately. Frozen leaves were fine grinded into powder with liquid N2 and stored at −70 °C for biochemical measurements and enzyme assays. Chlorophyll in fresh leaves (0.1 g) was extracted with 25 mL mixture of equivoluminal ethanol and acetone for 24 h at room temperature in dark. The absorbance of the extract was measured at 663 nm, 645 nm, and 470 nm to calculate chlorophyll content (Lichtenthaler, 1987). To measure nitrate (NO3−) and ammonium (NH4+), frozen leaf samples were homogenized with deionized water and centrifuged at 8300 × g and 4 °C for 10 min. The NO3− content in supernatant was quantified colorimetrically at 410 nm as in Cataldo et al. (1975), and KNO3 was used as standard. NH4+ in the supernatant was quantified photometrically as in Zanini (2001) at 630 nm, and (NH4)2SO4 was used as standard. Free amino acid contents in frozen leaves was measured with ninhydrin method according to Yokoyama and Hiramatsu (2003). Samples were homogenized with acetic acid/sodium acetate buffer (pH5.4) and centrifuged at 8300 × g for 10 min at 4 °C. The supernatant was reacted with ninhydrin, and the absorbance was monitored at 580 nm. Amino acid content was calculated by standard curve obtained from L-leucine. 53
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Fig. 1. Xylem secretion rate, and NO3− and free amino acid concentrations in xylem sap of ‘Zhongzheyou 1’ (A, C, and E) and ‘Jiayou 5’ (B, D, and F) under different intensities of soil drying. Five and ten days of soil drying represent mild and moderate soil drying, respectively. Xylem exudation was collected from 18:00 to 8:00. Values are means ± SE (n = 6). The significance of difference between control (well-watered) and water stress is indicated with asterisks by two-tailed Student's t-test: *, p < 0.05; **, p < 0.01.
transaminase (GOT), and glutamic-pyruvic transaminase (GPT). GS activity was determined according to Zhang et al. (1997). The enzyme extract was reacted with hydroxylamine hydrochloride in a 50 mM Tris-HCl buffer (pH7.4, containing 32 mM Mg2+, 7 mM ATP, 8 mM glutamate, 8 mM cysteine, and 0.8 mM EGTA) at 37 °C for 30 min, and the reaction was terminated by adding acidic FeCl3 (0.37 M FeCl3 and 0.2 M TCA in 0.6 M HCl). The protein deposits in the solution was removed by centrifugation at 8300 × g for 10 min. Then the absorbance of supernatant was measured chromometrically at 540 nm, and the activity of GS was expressed indirectly as A540·mg−1 protein·h−1. GOGAT activity was measured with the method described by Loulakakis and Roubelakis-Angelakis (1990). The reaction system consisted of 30 mM α-oxoglutarate, 0.3 mM KCl, 12.5 mM Tris-HCl buffer (pH 7.6), 0.2 mM NADH, 2 mM glutamine and modest enzyme extract. The absorbance at 340 nm was monitored for 300 s, and GOGAT activity was estimated by the oxidation of NADH per mg protein per min. Amination and deamination activities of glutamate dehydrogenase (GDH) were determined as in Loyola-Vargas and De Jimenez (1984). To measure the GDH amination activity, the reaction was carried out in a 100 mM Tris-HCl buffer (pH 8.0) containing 20 mM α-oxoglutarate, 200 mM NH4Cl, 1 mM CaCl2, 0.2 mM NADH, and modest enzyme extract. To measure the GDH deamination activity, the pH value of the buffer was adjusted to 9.2, and the substrates of α-oxoglutarate, NH4Cl and NADH were replaced by 100 mM L-glutamate and 1 mM NAD+. The absorbance at 340 nm was monitored for 300 s. The activities of GDH amination and deamination were estimated by the oxidation of
Proline content in frozen leaves was extracted with 5% sulfosalicylic acid in boiling water bath for 15 min and measure with a rapid method described previously by Bates et al. (1973). Standard curve was obtained from L-proline. To measure soluble sugar and sucrose, the leaf samples were homogenized in deionized water with mortar and pestle at 4 °C. The homogenate was centrifuged at 8300 × g for 10 min at 4 °C. The supernatant was used for soluble sugar and sucrose measurements. Total soluble sugar was measured by the anthrone-sulfuric acid method (Wang et al., 2002). Sucrose was analyzed by resorcinol hydrochloric acid method according to Li et al. (2012). 2.5. Enzyme activities assay Nitrate reductase (NR) in frozen leaf samples (about 100 mg) was extracted with 3 mL 25 mM potassium phosphate buffer (pH 7.5) containing 10 mM L-cysteine and 1 mM EDTA-Na2 (Hageman and Reed, 1980). The samples were homogenized with mortar and pestle, and the homogenates were centrifuged at 8300 × g for 10 min at 4 °C. NR activity in the supernatant was measured colorimetrically at 540 nm based on the reduction of nitrate to nitrite during 30 min at 25 °C. Frozen leaf samples (about 100 mg) were homogenized in 3 mL 50 mM Tris-HCl buffer (pH 8.0, containing 2 mM Mg2+, 2 mM DTT, and 0.4 M sucrose) with mortar and pestle, and the homogenates were centrifuged at 8300 × g for 10 min at 4 °C. The supernatant was used for enzyme assays of glutamine synthetase (GS), glutamate synthetase (GOGAT), glutamate dehydrogenase (GDH), glutamic-oxaloacetic 54
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NADH and reducing of NAD+ per mg protein per min, respectively. Glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) activities were assayed according to Wu et al. (1998). Modest enzyme extract was added to the substrate solution (pH 7.4) containing 2 mM α-oxoglutarate and 200 mM DL-aspartate (GOT) or 200 mM DL-alanine (GPT). The mixture was incubated at 37 °C for 1 h, and the reaction was terminated by adding 2, 4-dinitrophenylhydrazine. After incubating the mixture at 37 °C again for 20 min, 5 mL 0.4 M NaOH was added and the absorbance of the solution was chromometrically measured at 500 nm. The activities of GOT and GPT was expressed as the generating rate of pyruvic acid per mg protein. Soluble protein content in enzyme extract was measured using Bradford method (Bradford, 1976). Bovine serum albumin (BSA) was used as standard.
D). Mild soil drying and moderate soil drying decreased soluble protein content in ‘Zhongzheyou 1’ and ‘Jiayou 5’, respectively (Fig. 5A and B). Soil drying decreased free amino acid content in ‘Zhongzheyou 1’, with significant difference in mild soil drying (Fig. 5C). Free amino acid content in ‘Jiayou 5’ was significantly increased under mild soil drying, but decreased under moderate soil drying (Fig. 5D). Soil drying did not affect proline content in ‘Zhongzheyou 1’, whereas mild soil drying raised proline content remarkably in ‘Jiayou 5’ (Fig. 5E and F). 3.4. Leaf nitrogen metabolic enzymes activities Compared with control, NR activity in both cultivars was significantly higher under soil drying conditions (Fig. 6A and B). GS activity was not significantly affected by soil drying in ‘Zhongzheyou 1’ (Fig. 6C), whereas it was significantly increased under moderate soil drying in ‘Jiayou 5’ (Fig. 6D). Mild soil drying increased GOGAT activity significantly in ‘Zhongzheyou 1’, whereas there was no significant difference in GOGAT activity between control and water stress in ‘Jiayou 5’ (Fig. 6E and F). GDH deamination activity was significantly decreased by moderate soil drying in ‘Zhongzheyou 1’ (Fig. 7A) but significantly increased by mild soil drying in ‘Jiayou 5’ (Fig. 7B). Soil drying did not significantly affect GDH amination activity in ‘Zhongzheyou 1’, whereas mild soil drying and moderate soil drying significantly lowered and raised the GDH amination activity in ‘Jiayou 5’, respectively (Fig. 7C and D). GOT activity in ‘Zhongzheyou 1’ was not significantly affected by soil drying (Fig. 8A), while it was increased remarkably in ‘Jiayou 5’ under both soil drying conditions (Fig. 8B). Moderate soil drying lowered GPT activity in ‘Zhongzheyou 1’ (Fig. 8C). GPT activity responded oppositely to mild soil drying and moderate soil drying in ‘Jiayou 5’, with a significant decrease in the former condition and a significant increase in the latter condition (Fig. 8D).
2.6. Statistical analysis The average values were calculated based on 4 biological replications. The effects of soil drying were evaluated by Student's t-test. Differences were considered statistically significant when p < 0.05. 3. Results 3.1. Xylem secretion rate, and nitrate and amino acid concentrations in xylem sap Mild soil drying (5-d soil drying) had no significant effect on xylem secretion rate of the two rice cultivars, whereas moderate soil drying (10-d soil drying) reduced it extraordinarily in both cultivars (Fig. 1A and B). NO3− concentration in xylem exudation was extremely increased under soil drying in both cultivars, with greater increase in moderate soil drying (Fig. 1C and D). In contrast, free amino acid concentration in xylem exudation was substantially reduced under soil drying conditions, with the exception of ‘Zhongzheyou 1’ under moderate water stress, which was not significantly differ from control (Fig. 1E and F).
4. Discussion Rice is a water-intensive crop, however, a large number of evidence has revealed that moderate soil drying is conducive to rice plant growth and yield formation (Yang and Zhang, 2006, 2010), and can even maintain the rice grain yield as high as traditional flooding irrigation (Belder et al., 2004; Howell et al., 2015). Generally, soil drying reduces the availability of water, and subsequently declines photosynthesis of plant. It has been proposed that photosynthesis is affected in large part by leaf water status (Zhou et al., 2007), decreasing with the decline of leaf relative water content (RWC) (Ding et al., 2014). In this study, photosynthetic rates of the two cultivars were not significantly affected under soil drying conditions. It could be attributed to the stability of leaf RWC. Stomata play a paramount role in regulation of water loss in leaves. In this study, the reduction of stomatal conductance (gs) under moderate soil drying was in accordance with the descent of xylem secretion rate. It is suggested that partial closure of stomata plays a vital role in maintaining leaf RWC when active water absorption of root was constrained under soil drying (Zhong et al., 2017). On the other hand, stomatal closure becomes the major limit of photosynthesis under moderate water stress condition (Chaves and Oliveira, 2004). Mesophyll conductance (gm) determines the CO2 concentration at carboxylation site (Cc), which is as important as Ci for photosynthesis (Niinemets et al., 2009). Additionally, the increased photorespiration under water stress condition can act as an important CO2 concentrating mechanism in mesophyll cells (Peterhansel and Maurino, 2011). Previous study also revealed that gm was less affected by short-term water scarcity (Zhong et al., 2017). Although the stomatal conductance (gs) and intercellular CO2 concentration (Ci) were reduced by water stress, the photosynthetic rate of the both cultivars were not affected (Fig. 2). It is speculated that a slightly affected gm and Cc could partly contribute to this.
3.2. Gas exchange, sugar content, and chlorophyll fluorescence Both leaf relative water content (RWC) and photosynthetic rate (Pn) were not significantly affected under neither mild nor moderate soil drying (Fig. 2A–D). Mild soil drying did not affect stomatal conductance (gs) and intercellular CO2 concentration (Ci) in the two cultivars, whereas moderate soil drying significantly reduced gs and Ci in ‘Zhongzheyou 1’ and Ci in ‘Jiayou 5’ (Fig. 2E–H). Leaf soluble sugar and sucrose contents in ‘Zhongzheyou 1’ were not significantly affected by soil drying, but they were remarkably increased by moderate soil drying in ‘Jiayou 5’ (Fig. 3A–D). There was no significant difference in chlorophyll content between control and water stress treatments in both cultivars (Fig. 3E and F). As shown in Table 1, Fv/Fm was not affected by soil drying, whereas soil drying increased NPQ and JT/Pn in both cultivars, with a significant difference in NPQ in ‘Zhongzheyou 1’. The other chlorophyll fluorescence parameters such as ФPSII, ФCO2, JT, and qP had a tendency of decrease under soil drying conditions in ‘Zhongzheyou 1’, with no significant differences between control and water stress treatments. In ‘Jiayou 5’, these parameters were relatively less affected. 3.3. Leaf nitrogenous compounds The NO3− content in leaves of ‘Zhongzheyou 1’ was not affected under mild soil drying, but significantly decreased under moderate soil drying (Fig. 4A). In contrast, soil drying increased the NO3− content remarkably in ‘Jiayou 5’ (Fig. 4B). NH4+ content in ‘Zhongzheyou 1’ was not significantly affected by soil drying. However, it was significantly decreased by moderate soil drying in ‘Jiayou 5’ (Fig. 4C and 55
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Fig. 2. Leaf relative water content and gas exchange parameters of ‘Zhongzheyou 1’ (A, C, E, and G) and ‘Jiayou 5’ (B, D, F, and H) under different intensities of soil drying. A and B, relative water content (RWC); C and D, net photosynthetic rate (Pn); E and F, stomatal conductance (gs); and G and H, intercellular CO2 concentration (Ci). Five and ten days of soil drying represents mild and moderate soil drying, respectively. Values are means ± SE (n = 4). The significance of difference between control (well-watered) and water stress is indicated as asterisks by two-tailed Student's t-test: *, p < 0.05; **, p < 0.01.
between NPQ and ФCO2 (r = −0.4385, p = 0.012, n = 32), suggesting light energy dissipation is an efficient strategy for ‘Zhongzheyou 1’ to protect photosynthesis when electron flow was dropped in CO2 fixation caused by soil water deficit. Previous studies indicated a negative influence of soil water deficit on plant N metabolism, characterized as reductions in the activities of N metabolism enzymes and the synthesis of nitrogenous compounds (Xu and Zhou, 2006; Pinheiro and Chaves, 2011). Our results, which distinguish from those in previous studies, generally displayed positive responses of the activities of N metabolism enzymes to soil drying, indicating increased N metabolism plays important roles in photosynthetic acclimation in rice when the plants were subjected to water stress. It has been proposed that changes in xylem sap composition influence root to shoot signaling under drought and that NO3− could play a modulatory role in this process (Goodger and Schachtman, 2010). Soil drying ameliorates aeration condition of paddy soil,
The reduced CO2 availability in mesophyll cells due to stomatal closure leads to relative excess of light energy and electron sink, and results in reduction of photochemical efficiency and electron transport (Atkin and Macherel, 2009). In the current study, leaf chlorophyll content was intact under soil drying conditions, indicating the light energy capture is not affected. Therefore, plants have to dissipate excessive excitation energy to attenuate photoinhibition of photosynthesis. Non-photochemical quenching (NPQ) reflects the level of light energy dissipation in the form of heat (Maxwell and Johnson, 2000). In this study, water stress increased NPQ significantly in ‘Zhongzheyou 1’ (Table 1), in which the diffusion of CO2 through stomata was greater limited than in ‘Jiayou 5’. NPQ functions when the electron transport downstream of PSII (e.g., carbon fixation, photorespiration, nitrogen assimilation, etc.) is inhibited (Zhao et al., 2016). In this study, we found a larger reduction of ФCO2 in ‘Zhongzheyou 1’ (13.23%–14.35%) than in ‘Jiayou 5’ (4.29%–8.30%), and a significant negative correlation 56
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Fig. 3. Soluble sugar, sucrose and chlorophyll contents in ‘Zhongzheyou 1’ (A, C and E) and ‘Jiayou 5’ (B, D and F) under different intensities of soil drying. Five and ten days of soil drying represents mild and moderate soil drying, respectively. Values are means ± SE (n = 4). The significance of difference between control (well-watered) and water stress is indicated as asterisks by two-tailed Student's t-test: *, p < 0.05; **, p < 0.01.
increasing the NO3−/NH4+ ratio in soil solution, and subsequently changing xylem sap compositions and the activities of N metabolic enzymes in rice plants (Zhao et al., 2013; Dodd et al., 2015; Hu et al., 2017). Here, we observed a considerable increase in NO3−
concentration but a remarkable decrease in free amino acid concentration in xylem sap of water-stressed plant. The results imply that the responses of N metabolism to water stress could partly due to the changes in xylem sap composition. NO3− is mainly assimilated in
Table 1 Chlorophyll fluorescence parameters of ‘Zhongzheyou 1’ and ‘Jiayou 5’ under different intensities of soil drying. Five and ten days of soil drying represents mild and moderate soil drying, respectively. Values are means ± SE (n = 4). Bold data indicate significant difference from the control (well-watered) by two-tailed Student's t-test at α = 0.05 level. Parameters
Zhongzheyou 1 Fv/Fm ФPSII ФCO2 JT qP NPQ JT/Pn Jiayou 5 Fv/Fm ФPSII ФCO2 JT qP NPQ JT/Pn
5d
10 d
Control
Water stress
Control
Water stress
0.814 ± 0.004 0.354 ± 0.015 0.0209 ± 0.0013 232.39 ± 9.82 0.626 ± 0.020 1.241 ± 0.041 8.76 ± 0.24
0.818 ± 0.001 0.320 ± 0.005 0.0179 ± 0.0005 210.36 ± 3.29 0.591 ± 0.009 1.449 ± 0.036 9.51 ± 0.30
0.815 ± 0.002 0.321 ± 0.011 0.0189 ± 0.0010 211.08 ± 7.19 0.550 ± 0.025 1.183 ± 0.066 8.99 ± 0.18
0.812 ± 0.003 0.298 ± 0.011 0.0164 ± 0.0007 194.97 ± 7.44 0.547 ± 0.024 1.409 ± 0.055 9.68 ± 0.52
0.830 ± 0.002 0.411 ± 0.010 0.0253 ± 0.0006 269.79 ± 6.87 0.671 ± 0.010 1.251 ± 0.036 8.46 ± 0.42
0.825 ± 0.001 0.411 ± 0.011 0.0232 ± 0.0007 269.84 ± 7.08 0.678 ± 0.015 1.326 ± 0.021 9.04 ± 0.41
0.828 ± 0.001 0.333 ± 0.010 0.0163 ± 0.0003 218.41 ± 6.42 0.559 ± 0.014 1.390 ± 0.071 10.95 ± 0.31
0.825 ± 0.001 0.338 ± 0.002 0.0156 ± 0.0011 221.50 ± 1.42 0.575 ± 0.012 1.477 ± 0.109 11.74 ± 0.97
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Fig. 4. Leaf nitrate and ammonium contents in ‘Zhongzheyou 1’ (A and C) and ‘Jiayou 5’ (B and D) under different intensities of soil drying. Five and ten days of soil drying represents mild and moderate soil drying, respectively. Values are means ± SE (n = 4). The significance of difference between control (well-watered) and water stress is indicated as asterisks by two-tailed Student's t-test: *, p < 0.05; **, p < 0.01.
Fig. 5. Soluble protein, free amino acids and proline contents in ‘Zhongzheyou 1’ (A, C, and E) and ‘Jiayou 5’ (B, D, and F) under different intensities of soil drying. Five and ten days of soil drying represents mild and moderate soil drying, respectively. Values are means ± SE (n = 4). The significance of difference between control (well-watered) and water stress is indicated as asterisks by two-tailed Student's t-test: *, p < 0.05; **, p < 0.01.
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Fig. 6. Nitrate reductase (NR), glutamine synthease (GS) and glutamate synthetase (GOGAT) activities in ‘Zhongzheyou 1’ (A, C, and E) and ‘Jiayou 5’ (B, D, and F) under different intensities of soil drying. Five and ten days of soil drying represents mild and moderate soil drying, respectively. Values are means ± SE (n = 4). The significance of difference between control (well-watered) and water stress is indicated as asterisks by two-tailed Student's t-test: *, p < 0.05; **, p < 0.01.
leaves. It should be noted that compared to control, the NO3− transport rate through xylem (μg·h−1) was higher under mild soil drying but lower under moderate soil drying (Fig. 1), which was not completely in line with the NO3− content in leaves (Fig. 4A and B). It is suggested that primary N assimilation in leaves could involves in regulating the photosynthetic acclimation of rice plants to water stress. NR is the rate-limiting enzyme in NO3− assimilation. Numerous studies have reported that water stress reduces NR activity (Foyer et al., 1998; Pandey and Agarwal, 1998; Plaut, 2010). Cornic (2000) proposed that reduction of NR activity can be of some advantage to the plant, because it can rapidly contribute to the maintenance of osmotic pressure in the photosynthetic cells by increasing the NO3− level. Distinct result from previous studies was obtained in this study that soil drying enhanced NR activity in both studied cultivars. NR is a substrate inducible enzyme, and it is regulated by the ratio of NO3−/NH4+ (Oaks et al., 1990). The ratio of NO3−/NH4+ in leaves reflects NO3− uptake and assimilation (Meng et al., 2016). In ‘Zhongzheyou 1’, water stress induced decrease of NO3− in leaves was in line with the increase in NR activity. Additionally, the ratio of NO3−/NH4+ was reduced from 56.11 to 44.82 and from 45.09 to 33.78 when the plants were subjected to mild and moderate soil drying, respectively. It indicates that increasing NO3− reducing is an important mechanism for ‘Zhongzheyou 1’ to cope with soil water stress. In addition, the NO3− transport rate in xylem of ‘Zhongzheyou 1’ was consistent with NO3− content in leaves under moderate soil drying, indicating the coordination between NO3−
uptake and assimilation in improving the acclimation of photosynthesis to water stress. In contrast, even though NO3− uptake was decreased under moderate soil drying condition, the highly accumulation of NO3− and obviously increased NO3−/NH4+ ratio in ‘Jiayou 5’ implies that NO3− reduction was attenuated. Azedo-Silva et al. (2004) reported that the change of NR activity was not agree with NO3− content. The synchronistical increased NO3− content and NR activity in ‘Jiayou 5’ could be due to the compartmentation of NO3− assimilation in leaves (Sechley et al., 1992), for NO3− is mainly stored in vacuoles (Martinoia et al., 2007), isolated from NR which is located in cytoplasm. The different responses of NO3− to soil drying in the two cultivars implies diverse mechanisms that NO3− reducing involves in the acclimation of rice plants to soil drying. Increased NO3− reducing in ‘Zhongzheyou 1’ could play a vital role in dissipation of excessive energy, because NO3− reduction is a highly energy requirement process (Sunil et al., 2013). It requires reduced compounds, such as NADH, NADPH, reduced ferredoxin and ATP produced in mitochondrial oxidative metabolism, photosynthesis, photorespiration, or even the cytosolic reactions, depending on the site where the reaction happens (Sunil et al., 2013). NO3− reducing in leaves could use the excessive energy derived from photosynthetic apparatus, which is more efficient than that takes place in root (Gonzalez-Dugo et al., 2010). The reduced amino acid concentration in xylem sap of water-stressed plants supported this notion. NO3− assimilation is acting as an effective electron sink (evidenced by increase in JT/Pn) alleviating photoinhibition of 59
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Fig. 7. Deamination and amination activities of GDH in ‘Zhongzheyou 1’ (A and C) and ‘Jiayou 5’ (B and D) under different intensities of soil drying. Five and ten days of soil drying represents mild and moderate soil drying, respectively. Values are means ± SE (n = 4). The significance of difference between control (well-watered) and water stress is indicated as asterisks by two-tailed Student's t-test: *, p < 0.05; **, p < 0.01.
affected stomatal conductance (gs) in ‘Jiayou 5’ under moderate soil drying could be partly due to its high NO3− content in leaves. Increasing stomatal aperture is advantageous to gas exchange when leaf water content was not affected under water stress condition. Water stress will cause the accumulation of NH4+ in plant. Avoiding excess NH4+ accumulation in plant tissues is viewed as an important capacity to withstand water stress (Hoai et al., 2003). GS, GOGAT, and GDH are key enzymes in incorporation of NH4+ into amides and amino acids (Lawlor, 2002; Masclaux-Daubresse et al., 2006). In this study, GS
photosynthesis (evidenced by the constant of Fv/Fm and slightly affected JT) under water stress condition (Gonzalez-Dugo et al., 2010; Yi et al., 2014). In addition to as an important nutrient, NO3− plays multiple functions in plant such as regulating plant growth and development and adaptation to fluctuating environments (Linkohr et al., 2002; Wilkinson et al., 2007; Krouk et al., 2010). Besides, NO3− is an important factor regulating stomatal movement (Guo et al., 2003; Wilkinson et al., 2007). Higher NO3− content in guard cells leads to depolarization of guard cells and then stomatal opening. The less
Fig. 8. Glutamic-oxalacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) activities in ‘Zhongzheyou 1’ (A and C) and ‘Jiayou 5’ (B and D) under different intensities of soil drying. Five and ten days of soil drying represents mild and moderate soil drying, respectively. Values are means ± SE (n = 4). The significance of difference between control (well-watered) and water stress is indicated as asterisks by two-tailed Student's t-test: *, p < 0.05; **, p < 0.01.
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Author contributions
and GOGAT activities were increased to a certain extent under soil drying conditions in both cultivars, whereas GDH deamination/amination activity was particularly affected by water stress in ‘Jiayou 5’. It has been well known that GDH is independent of GS-GOGAT in N assimilation (Masclaux-Daubresse et al., 2006). It acts as an anti-stress enzyme in ammonia detoxification and production of Glu for proline synthesis under stress condition (Boussama et al., 1999; Skopelitis et al., 2006). In addition, Glu can be converted into Asp and Ala via glutamicoxalacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT), respectively (King and Waygood, 1968). Asp serves as an additional NH4+ detoxification molecule (Masclaux-Daubresse et al., 2006) and as the precursor for the synthesis of branched-chain amino acids (BCAAs) (Joshi et al., 2010). BCAAs derived from Asp pathway, such as Met, Thr, and Ile provide precursors for a number of plant secondary metabolites (Joshi et al., 2010). For example, ethylene synthesis which plays important roles in plant stress responses is derived from Met. Water stress induced significant increase in GOT activity and the alteration of GPT activity, suggesting that enhanced amino acid metabolism could play a key role in improving the resistance of photosynthesis to soil drying in ‘Jiayou 5’. Increased amino acid synthesis could be advantageous to plant, because amino acids can serve as osmotica to maintain the turgor of leaf under water stress condition (Singh et al., 2016). In this study, a significant increment of free amino acid and proline was observed in mild water stressed plants of ‘Jiayou 5’, indicating N metabolism took part in osmotic adjustment to protect photosynthesis from soil drying. The accumulation of amino acid could be resulted from N assimilation or proteolysis (Wingler et al., 1999). In this study, the increments of free amino acid and proline were more possibly resulted from the amino acid synthesis, for the protein content was constant and the activities of N assimilation enzymes were increased. However, the osmotic adjustment of amino acid and proline was limited under moderate soil drying in ‘Jiayou 5’, as their contents were lowered or not significantly changed. Interestingly, soluble sugar and sucrose were highly accumulated under such condition. Soluble sugars are important osmolyte in water stressed plants (Sánchez et al., 1998). The accumulation of soluble sugar and sucrose could compensate the reduction of soluble protein and free amino acids to maintain the turgor of leaf. It indicates that N and C metabolisms are cooperated in osmoregulation under soil drying condition.
C.Z. and X.C. collected the samples, analyzed the samples, and drafted the manuscript. Z.B. collected the samples and analyzed the samples. L.Z. and J.Z. analyzed the data and revised the manuscript. J.H. and Q.J. conceived and designed this work. All authors read and approved the manuscript. Acknowledgment This work was funded by the Natural Science Foundation of Zhejiang Province (No. LY18C130005, LQ15C130004); the National Key Research and Development Program of China (No. 2017YFD0300100, 2016YFD0101801); and the National Basic Research Program of China (No. 2015CB150502). References Argandona, V., Pahlich, E., 1991. Water stress on proline content and enzyme activities in barley seedlings. Phytochemistry 30, 1093–1094. Ashraf, M., Foolad, M.R., 2007. Roles of glycine betaine and proline in improving plant abiotic stress resistance. Environ. Exp. Bot. 59, 206–216. Atkin, O.K., Macherel, D., 2009. The crucial role of plant mitochondria in orchestrating drought tolerance. Ann. 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5. Conclusion In conclusion, the results reveal that N metabolism is sensitive to soil drying, and that the alterations in N metabolism are of great importance for the acclimation of rice photosynthesis to soil moisture changes. N metabolism plays various roles in improving the acclimation of rice photosynthesis to soil drying in different rice cultivars. In indica rice ‘Zhongzheyou 1’, NO3− reducing was strengthened under soil drying, whereas GDH pathway of N assimilation and Glu transamination through GOT and GPT were relatively slightly affected. NO3− reducing could contribute to alleviating the photoinhibition of photosynthesis with the coordination with NPQ under soil drying, as it is an important energy dissipation pathway in leaves. In japonica rice ‘Jiayou 5’, N assimilation via GDH and transamination through GOP and GPT were strengthened, and the accumulation of NO3− and amino acid was observed, suggesting N metabolism could participate in osmotic adjustment under soil drying conditions. Water stress induced alterations in N metabolism is tightly linked with the changes in chlorophyll fluorescence and carbon metabolism. The results imply that N metabolism could be a key regulatory target for improving the acclimation of photosynthesis to soil drying in rice. Conflicts of interest The authors declare that they have no conflict of interest. 61
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