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Article

Effects of Nitrogen Deficiency on the Photosynthesis, Chlorophyll a Fluorescence, Antioxidant System, and Sulfur Compounds in Oryza sativa

by
Ling-Hua Chen
1,2,
Ming Xu
2,
Zuxin Cheng
2 and
Lin-Tong Yang
3,*
1
Jinshan College of Fujian Agriculture and Forestry University, Fuzhou 350002, China
2
Engineering Technology Research Center of Fujian Special Crop Breeding and Utilization, College of Agriculture, Fujian Agriculture and Forestry University, Fuzhou 350002, China
3
College of Resources and Environment, Fujian Agriculture and Forestry University, Fuzhou 350002, China
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2024, 25(19), 10409; https://doi.org/10.3390/ijms251910409
Submission received: 30 August 2024 / Revised: 23 September 2024 / Accepted: 25 September 2024 / Published: 27 September 2024
(This article belongs to the Special Issue Changes and Adaptations of the Plant Metabolism under Elemental Nutrient Deficiencies)
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Abstract

:
Decreasing nitrogen (N) supply affected the normal growth of Oryza sativa (O. sativa) seedlings, reducing CO2 assimilation, stomatal conductance (gs), the contents of chlorophylls (Chl) and the ratio of Chl a/Chl b, but increasing the intercellular CO2 concentration. Polyphasic chlorophyll a fluorescence transient and relative fluorescence parameters (JIP test) results indicated that N deficiency increased Fo, but decreased the maximum quantum yield of primary photochemistry (Fv/Fm) and the maximum of the IPphase, implying that N-limiting condition impaired the whole photo electron transport chain from the donor side of photosystem II (PSII) to the end acceptor side of PSI in O. sativa. N deficiency enhanced the activities of the antioxidant enzymes, such as ascorbate peroxidase (APX), guaiacol peroxidase (GuPX), dehydro–ascorbate reductase (DHAR), superoxide dismutase (SOD), glutathione peroxidase (GlPX), glutathione reductase (GR), glutathione S-transferase (GST) and O-acetylserine (thiol) lyase (OASTL), and the contents of antioxidant compounds including reduced glutathione (GSH), total glutathione (GSH+GSSG) and non-protein thiol compounds in O. sativa leaves. In contrast, the enhanced activities of catalase (CAT), DHAR, GR, GST and OASTL, the enhanced ASC–GSH cycle and content of sulfur-containing compounds might provide protective roles against oxidative stress in O. sativa roots under N-limiting conditions. Quantitative real-time PCR (qRT-PCR) analysis indicated that 70% of the enzymes have a consistence between the gene expression pattern and the dynamic of enzyme activity in O. sativa leaves under different N supplies, whereas only 60% of the enzymes have a consistence in O. sativa roots. Our results suggested that the antioxidant system and sulfur metabolism take part in the response of N limiting condition in O. sativa, and this response was different between leaves and roots. Future work should focus on the responsive mechanisms underlying the metabolism of sulfur-containing compounds in O. sativa under nutrient deficient especially N-limiting conditions.

1. Introduction

Nitrogen (N) is one of principal macro–elements for the normal development and fertility of crops [1]. As a necessary component of various bioactive molecules, such as amino acids, proteins, nucleic acids, adenosine triphosphate, vitamins, and chlorophyll (Chl), it constitutes about 2–4% of plant dry weight (DW) [2]. Therefore, N not only plays a key role in miscellaneous biological processes in plants, but also determines the quality and grain yield of cereal crops. Previous statistics indicate that, among the many factors that could determine and increase rice yields, the contribution of chemical N fertilizer is greater than 40% [3]. However, under current agricultural management practices, the average nitrogen use efficiency (NUE) is about 40–50% in the field. The excessive use of N fertilizers causes water and air pollution, resulting in extensive concerns for the environment (e.g., eutrophication and greenhouse effects) and the global economy [4]. Thus, understanding the physiological and molecular mechanisms underlying the adaptive strategy of rice to N-limiting conditions is critical for environmentally friendly and sustainable cultivation.
Moderate N nutrition can ensure the normal biosynthesis of photosynthetic pigments and enhance the ability of light energy capture and conversion, improving photochemical efficiency in plant leaves [3,5]. Measurements of CO2 assimilation and chlorophyll a fluorescence transient are widely adopted methods used to monitor plant growth and development, resistance, and the interactions between a plant and its environment [6]. Studies in the literature indicate that N deficiency decreases the use efficiency of photo-radiation and CO2 assimilation (photosynthesis rate) in many annual crops such as Triticum aestivum [7], Sorghum bicolor [8], Oryza sativa [2,9], Zea mays [10,11], Ipomoea batatas [12], Helianthus annuus [13], and perennial tree including Camellia sinensis [14] and Citrus [15], etc. Although a few studies have attributed the decreased CO2 assimilation in plants under N-limiting conditions to stomatal conductance [8,16], most of the previous works suggest that a decline in the biochemical capacity for carboxylation and damaged photosystem II (PSII), revealed by the decreased activity of Rubisco and the diminished maximal efficiency of PSII photochemistry (Fv/Fm), respectively, are the key constraining factors for photosynthesis in plants under N-limiting conditions. Furthermore, photochemical and non-photochemical quenching can help plants to grow under low-N conditions [16,17].
Due to the decreased light energy utilization efficiency, excessive photo energy inevitably increases the production of reactive oxygen species (ROS) and disrupts the equilibrium of ROS. The strong oxidizing ROS, including superoxide anion radicals (O2–), hydrogen peroxide (H2O2), and hydroxyl radicals (OH) may then attack biological molecules, such as nucleic acids, amino acids, carbohydrates, and lipids, inducing the lipid peroxidation cellular membrane revealed by the increased content of malondialdehyde (MDA) [18]. In plant cells, the ROS-scavenging systems, including the ascorbate–glutathione (ASC–GSH) cycle, recruit the enzymatic antioxidants (e.g., superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), dehydro–ascorbate reductase (DHAR), monodehydro–ascorbate reductase (MDHAR), glutathione reductase (GR), guaiacol peroxidase (GuPX), and glutathione peroxidase (GlPX)), and non-enzymatic antioxidants (e.g., reduced glutathione (GSH), ascorbic acid (Asc), methionine (Met), thioredoxin (TRX), and phytochelatins (PCs)) to counteract oxidative damage in the chloroplast and cytoplasm [19,20]. The observed enhanced antioxidant system has been reported in many plants under N, phosphorus (P), potassium (K), and sulfur (S) stresses [17,21,22,23,24,25,26]. Furthermore, sulfur is the fourth macronutrient for plants, after N, P, and K, and is vital for normal growth and development from multiple aspects. Sulfur is a constituent of the molecule of methionine, cysteine, GSH, vitamins, PCs, chlorophyll, coenzyme A, and S-adenosyl-methionine, which collectively scavenge excessive ROS induced by environmental stresses [27]. Cysteine is the first reduced organic sulfur compound and a central biomolecule in S metabolism in plants; it is synthesized by the reaction of S2− and O-acetyl serine (OAS) catalyzed by O-acetylserine (thiol) lyase (OASTL) [28]. The synthesized cysteine could be further incorporated into S-containing metabolites such as GSH, Met, biotin, coenzyme A, TRX, iron–sulfur-containing proteins (as ferredoxin), adenosine-5′–phosphosulfate, and other non-protein thiol compounds in cells [29]. The increased content of cysteine has been reported in N-starvation tea leaves and Citrus roots [30,31].
Rice is one of the most important staple foods in the world. N stress in rice can be caused by both N deficiency and N excess. Increasing the application of N fertilizer is the main impetus for the grain yield production of rice [32]. Some details about the physiological and molecular mechanisms regarding N-deficiency responses in O. sativa have been investigated [2]. Our previous research showed that N deficiency affected the metabolism of organic acids and N assimilation in O. sativa [1]. However, the details about the effects of N deficiency on the donor side of PSII to the end acceptor side of PSI, and antioxidant system, especially the metabolism of S-containing compounds in O. sativa leaves and roots, were unveiled. The aim of this study is to dissect the effects of N deficiency on the main apparatus of photosynthetic electron transport (PET) in thylakoid, the different responses of the antioxidant system and the gene expression pattern of related enzymes between the leaves and roots of O. sativa seedlings. Our results can be used to further understand the physiological and molecular responses of O. sativa seedlings to N-limiting conditions and for the high-quality cultivation of rice and sustainable development of farmlands.

2. Results

2.1. Effects of N Deficiency on the Growth of Rice Seedlings

Low N and N deficiency apparently inhibited the growth of O. sativa seedlings. The height of the seedlings gradually decreased along with the decreasing supply of N. Furthermore, the chlorosis of O. sativa leaves was observed in the mature leaves of N-deficient seedlings (Figure 1).

2.2. Effects of N Deficiency on Gas Exchange Parameters and Leaf Chlorophylls (Chl) in Rice Seedlings

Both low N and N deficiency dramatically decreased the CO2 assimilation (decreased by 19.7% and 50%, respectively), whereas these two treatments apparently increased the intercellular CO2 concentration in O. sativa leaves (increased by 46.7% and 81.9%, respectively) (Figure 2A,C). N deficiency clearly lowered the stomatal conductance (gs), the contents of chlorophyll a, chlorophyll b, and the ratio of Chl a/Chl b in O. sativa leaves (Figure 2B,D–F). No significant differences in the stomatal conductance, the contents of chlorophyll a, chlorophyll b, and the ratio of Chl a/Chl b were observed between low-N leaves and the control ones (Figure 2B,D–F).

2.3. Effects of N Deficiency on the Parameters of Chlorophyll a Fluorescence in Rice Leaves

The chlorophyll a fluorescence transient (OJIP) showed a typical curve in O. sativa leaves under different N treatments (Figure 3A). Increasing N supply in the nutrient solution gradually enhanced the maximum IP phase of O. sativa leaves (Figure 3B). The dynamics of relative variable fluorescence (Vt) and the differences of normalized low-N and N-deficient transients minus the control transient (ΔVt) showed that the Vt and ΔVt of low-N and N-deficient transients protruded above the control ones (Figure 3C,D). Meanwhile, three positive bands around 300 μs (K-step), 2 ms (J-step) and 30 ms (I-step) were observed in the N-deficient transient, which was more pronounced than the low-N treated samples (Figure 3D). Similarly, a positive band around the immediate vicinity of 120μs (L-step) was observed on the relative variable fluorescence between Fo and F300μs (WK), and the differences in the normalized low-N and N-deficient transients minus the control transient (ΔWK) (Figure 3E,F).
Ten selected primary and calculated fluorescence parameters from different N treated samples are visualized in Figure 4. Compared to the control sample, both low N and N deficiency raised the values of Fo, Vj, ABS/CSo and lowered the value of ETo /CSo, Reo /CSo, and PItotal in O. sativa leaves (Figure 4A,D,F,H–J). Only the N deficiency treatment significantly decreased the values of Fm (decreased by 28.9%), Fv/Fm (decreased by 15.6%), but increased the value of DIo/CSo (increased by 128%), when compared to the controls (Figure 4B,C,G). Elevating the N level apparently increased the value of Vi in O. sativa leaves (Figure 4E).

2.4. Effects of N Deficiency on the Activities of Antioxidant Enzymes in Rice Seedlings

Nitrogen deficiency dramatically increased the activities of leaf APX (Figure 5A; increased by 86.3%), leaf DHAR (Figure 5D; increased by 132%), leaf GlPX (Figure 5F; increased by 195%), leaf GR (Figure 5G; increased by 43%), and leaf GST (Figure 5I; increased by 98.7%), leaf OASTL (Figure 5J; increased by 155%); however, it lowered the activity of leaf CAT (Figure 5C; decreased by 49.6%) in O. sativa seedlings, when compared to the controls. Decreasing the N supply gradually increased the activities of leaf GuPX (Figure 5B) and leaf SOD (Figure 5E), but it did not alter the activity of leaf MDHAR (Figure 5H). Except for increasing the activities of leaf GuPX (Figure 5B) and leaf SOD (Figure 5E), low N did not alter the activities of the abovementioned enzymes in O. sativa leaves when compared to the controls.
Nitrogen deficiency significantly lowered the activities of root APX (Figure 6A; decreased by 69.9%), root GuPX (Figure 6B; decreased by 69.9%), root SOD (Figure 6E; decreased by 84.8%), and root GlPX (Figure 6F; decreased by 48.1%) in O. sativa seedlings, whereas it increased the activities of root CAT (Figure 6C; increased by 318%), root DHAR (Figure 6D; increased by 25.2%), root GR (Figure 6G; increased by 62.8%), and root GST (Figure 6I; increased by 72.7%). Compared to the control, low N and N deficiency decreased the activity of root MDHAR (Figure 6H), but they did not change the activity of root OSALT in O. sativa seedlings (Figure 6J). Furthermore, no significant difference in these enzymes was observed between low-N and control root samples after three weeks of treatment (Figure 6).

2.5. Effects of N Deficiency on the Contents of Antioxidant Compounds, MDA, and the Production of H2O2 in Rice Seedlings

Nitrogen deficiency substantially increased the contents of root Asc (increased by 166%), root Asc+DHA (increased by 124%), leaf GSH (increased by 863%), root GSH (increased by 218%), leaf GSH+GSSG (increased by 79.3%), root GSH+GSSG (increased by 86.6%), and the ratios of leaf Asc/DHA, root Asc/DHA, leaf GSH/GSSG, and root GSH/GSSG in O. sativa (Figure 7B,D–L), but decreased the content of leaf Asc+DHA (Figure 7C). No significant difference was observed in the content of leaf Asc in O. sativa under different N supplies (Figure 7A).
Compared to the 5 mM N treatment, N deficiency significantly raised the contents of leaf non-protein thiol compounds (NPTs) (Figure 8A), root NPTs (Figure 8B) and root cysteine (Figure 8D) in O. sativa. No significant difference in leaf NPTs (Figure 8A), root NPTs (Figure 8B) and root cysteine (Figure 8D) was observed between the 5 mM N and 1 mM N treatments. Different N levels did not change the content of leaf cysteine (Figure 8C). Reducing N levels gradually increased the production of H2O2 in the leaf and root, as well as the content of MDA (Figure 8E–G). N deficiency significantly increased root MDA when compared to both the control and low-N samples (Figure 8H).

2.6. Effects of N Deficiency on the Gene Expression Levels of Antioxidant Related Enzymes in Rice Seedlings

Nitrogen deficiency apparently enhanced the gene expression levels of APX, DHAR, SOD, GlPX, GST and OASTL, whereas it did not change the expression levels of GuPX, CAT, GR, and MDHAR in O. sativa leaves (Figure 9A). Except for increasing the expression level of DHAR, the low-N treatment did not change the expression levels of the other abovementioned genes in O. sativa leaves when compared to the controls (Figure 9A). In O. sativa roots, both low N and N deficiency decreased the expression levels of APX and GuPX but increased the expression level of GlPX. Only N deficiency significantly enhanced the expression levels of GR, GST and OASTL, when compared to those of the controls (Figure 9B). There was no significant difference observed in the expression levels of CAT, DHAR, SOD and MDHAR among the three N treatments (Figure 9B).

3. Discussion

Nitrogen fertilizers are one of the most common fertilizers nowadays; they influence the genetic enhancement of grain yield and play a prominent role in the increment of grain production worldwide [32]. Therefore, understanding the physiological mechanisms underlying plant N nutrition could help to improve crop NUE, as maintaining crop productivity has multiple economic and environmental benefits. Our previous study indicated that low N and N deficiency disturbed the normal metabolism of organic acids and might reduce the carbon skeletons for amino acids assimilation in rice leaves [1]. Decreasing N supply significantly reduced the activities of enzymes related to N assimilation and amino acids metabolism, lowered the contents of total free amino acids (TFAAs) and soluble proteins, eventually hindering the growth of shoots and roots in O. sativa seedlings [1]. Here, we showed that decreasing N supply affected the normal growth of rice seedlings, reducing CO2 assimilation, stomatal conductance, chlorophyll a, chlorophyll b, and the ratio of Chl a/Chl b, but increasing the intercellular CO2 concentration (Figure 1 and Figure 2). The N deficiency induced decreased photosynthesis; related pigments were also reported in wheat [7], tea [14], sorghum [8], rice (cultivar “Zhendao 11” and “Weiyou916”) [2,9], sweet potato [12], sunflower [13], maize [10,11], Citrus [15], grape [33], and so on. Moreover, a decreased Chl a/Chl b, as an indicator of stress response in plants, was also found in N-deficient sunflower [13], copper-stress Citrus [34], and rice seedlings under aluminum treatment and P deficiency [35], indicating that like other abiotic stresses, N deficiency could disrupt the homeostasis of pigment metabolism and thereby lower the photosynthesis rate in O. sativa seedlings. Therefore, in order to confirm whether N deficiency impairs the photosynthetic electron transfer efficiency in the thylakoid membrane, we measured the chlorophyll a transient fluorescence and conducted the JIP test [36,37,38].
Our results showed that both low N and N deficiency displayed a raised Fo, but a descending maximum of the IPphase (Figure 3A,B and Figure 4A), implying that a photo-inhibitory damage to PS II or reaction center (RC) and the electron transfer block at the acceptor side of PSI occurred in O. sativa leaves under N-limiting conditions [37,39,40,41]. Fv/Fm represents the maximum quantum yield of primary photochemistry. The decreased Fv/Fm was only observed in N-deficient O. sativa leaves, which was caused by decreased Fm and increased Fo (Figure 4A–C). This result indicates that the photosynthetic capacity and stability of PSII are lower in O. sativa leaves under N-deficient than those under N -supplying conditions [42]. The K-step, J-step and I-step are correlated with the uncoupling of the oxygen-evolving complex (OEC), accumulation of reduced QA and inhibition of the final reduction of end acceptors, respectively [43]. The positive K-step, J-step and I-step, and the higher value of Vj and Vi, observed in low-N and N-deficient O. sativa leaves indicated that N-limiting conditions could damage the OEC, and impair the whole photosynthetic electron transport chain (PETC) from the PSII donor side up to the reduction in the end acceptor side of PSI, affecting the energy migration properties within the PSs (Figure 3C,D and Figure 4D,E) [15,37,44]. The positive L-step appeared in the relative variable fluorescence between Fo to F300μs (Wk and ΔWk when compared to the control ones), demonstrating that the energy transmission between the independent PSII units is impaired or the PSII units are less grouped [36]. Furthermore, we selected some parameters based on the cross-section (CSo) at t = 0, which showed that leaf samples under low-N or N-deficient conditions absorbed more photo-radiation (ABS/CSo; Figure 4F), but these samples dissipated more energy (DIo/CSo; Figure 4G) and used less energy in electron transport (ETo/CSo; Figure 4H) or caused a reduction in the end acceptor side of PSI (Reo/CSo; Figure 4I), eventually reducing the total performance index of PSs (PItotal; Figure 4J). The observed disruption at the donor side of PSII to the end acceptor side of PSI was also reported in N-deficient and copper-stress Citrus plants [15,34], phosphorus-deficient tea plants [38], N-deficient tomato and maize [6], and N-deficient wheat [7].
Abiotic stresses, including N deficiency, lower light energy utilization efficiency and excess photon energy, inevitably enhancing the production of ROS in the plants. As indicated in the current study, both low N and N deficiency enhanced the production of H2O2 in O. sativa leaves and roots (Figure 8E,F). Therefore, as a responsive strategy, the metabolism of antioxidant compounds and the activities of related enzymes are enhanced in response to adverse conditions. Here, we showed that the activities of ROS-detoxified related enzymes, such as leaf APX, GuPX, DHAR, SOD, GlPX, GR, sulfur-containing compounds, and metabolism-related enzymes, such as GST and OASTL, were enhanced by N deficiency in O. sativa leaves (Figure 5). Accordingly, the ratio of leaf Asc/DHA and leaf GSH/GSSG, and the contents of leaf GSH, total glutathione (GSH + GSSG) and non-protein thiol compounds were also increased by N deficiency in O. sativa leaves (Figure 7E,G,I,K and Figure 8A). The higher ratio of leaf Asc/DHA, leaf GSH/GSSG, and the higher contents of leaf GSH, total glutathione (GSH + GSSG) and non-protein thiol compounds might play positive roles in increasing the activities of leaf APX, DHAR, GlPX, GR, and GST, respectively, in O. sativa under N-deficient conditions.
Although the activity of leaf OASTL was increased by N deficiency, the content of its product, cysteine, was not altered by N deficiency (Figure 5J and Figure 8C). This inconsistency might be due to the consumption of cysteine to synthesize proteins and GSH or due to it acting as a sulfur donor for the biosynthesis of methionine (Met) and some other secondary compounds such as S-adenosylmethionine and S-methylmethionine [45,46]. In contrast, except for increasing the activities of CAT, DHAR, GR, GST and OASTL, N deficiency decreased the activities of the other antioxidant enzymes mentioned above in O. sativa roots (Figure 6). Compared to the leaf, except for increasing the contents of GSH, GSH + GSSG, non-protein thiol compounds and the ratio of GSH/GSSG, N deficiency also increased the contents of Asc, Asc+DHA and cysteine, and the ratio of Asc/DHA in O. sativa roots (Figure 7 and Figure 8B,D). The increased ratios of Asc/DHA and GSH/GSSG in both leave and roots indicates that O. sativa has enhanced its antioxidant system to cope with oxidative stress induced by N deficiency both in the leaves and roots (Figure 7E,F,K,L) [47]. Interestingly, we found that the activity of CAT was decreased by N deficiency, along with the enhanced activity of APX and increased production of H2O2 in N-deficient O. sativa leaves, which might be due to the lower affinity of CAT to H2O2 (Km = 40 mM) than to APX [48]. Similar results were also reported in low-ammonium cultured O. sativa [49], and low-N cultured Solanum lycopersicum [18]. However, the low N-induced decreased activity of CAT needs to be further investigated. We proposed that the enhanced activities of CAT, DHAR, GR, the ASC–GSH cycle and the metabolism of sulfur-containing compounds might play protective roles in O. sativa roots under N-limiting conditions. Enhancing the capacity of the antioxidant enzymes and raising the contents of non-enzymatic antioxidants, such as reduced Asc, GSH, phenolic compounds, flavonoids, and sulfur-containing compounds, etc., is the main adaptive response to oxidative stress in plants [50]. The increased activities of antioxidant enzymes and contents of ascorbic acid and/or glutathione have also been reported in phosphorus-deficient C. sinensis [26], salinity-stress Malus domestica [51], and N-deficient Nicotiana tabaccum [52], O. sativa [17,21,22], Cucumis sativus [23], and Matricaria chamomilla [25]. In order to investigate whether the activities of these enzymes were transcriptionally regulated by their corresponding genes, we carried out the transcription profile of these enzymes related to the antioxidant system and sulfur metabolism using qRT-PCR assay. Our results showed that the expression patterns of most of the genes (70%) except for GuPX, CAT and GR, were consistent with the dynamics of the enzymes related to the antioxidant system and sulfur metabolism under N-limiting conditions in O. sativa leaves (Figure 9A). However, the expression patterns of only 60% of these genes coincided with the dynamics of the enzymes under N-limiting conditions in O. sativa roots (Figure 9B). The inconsistency of some enzymes between the gene expression level and enzyme activity might be attributed to translational regulations, post-translational modifications, and the protein modification or degradation of each enzyme [53]. These results indicate that the activities of most of these enzymes are controlled by the transcriptional regulation of corresponding genes in O. sativa under N-limiting conditions.

4. Materials and Methods

4.1. Plant Material and Treatment

Plant culture and treatment were the same as the methods described in our previous study [1]. Briefly, uniform seeds of the rice variety “Huanghuazhan” (Oryza sativa L. ssp. Indica) were sown in a plastic tray containing natural paddy soil. The seeds were kept moist in a greenhouse under natural light and temperatures. When the height of the rice seedlings were about 15 cm, the seedlings were transplanted to the cultivation bucket containing Hoagland nutrient solutions with the following three different N concentrations: 5 mM NH4NO3 (control); 1 mM NH4NO3 (low N); and 0 mM NH4NO3 (N deficiency). All the rice seedlings were cultivated in a light incubator under a constant temperature of 28 °C. The nutrient solutions were refreshed every two days. The Hoagland nutrient solution contained the following macro-elements except for N (in mM): KH2PO4, 1 mM; MgSO4, 2 mM; and microelements (in μM): H3BO3, 5 μM; CuSO4, 0.5 μM; MnCl2, 2 μM; (NH4)6Mo7O24, 0.065 μM; ZnSO4, 2 μM; FeSO4-EDTA, 20 μM; and Na2SiO3, 0.1 μM. Three weeks later, rice seedlings were separated into shoots and roots and wrapped with aluminum foil. All the samples were immediately frozen in liquid nitrogen and stored at −80 °C until assayed. The experimental conditions for all the seedlings were the same except for the N levels in the nutrient solution.

4.2. Measurement of Leaf Gas Exchange, Chlorophyll a Transient Fluorescence (OJIP) and the Contents of Chlorophyll in Rice Leaves

After the end of the different N treatments, leaf gas exchange was monitored with a CIARS-II portable photosynthesis system (PP systems, Amesbury, MA, USA) between 10:00 am and 11:30 am on a clear day. The CO2 concentration was supplied by a CO2 cylinder and controlled at ~380 µmol mol–1. The controlled light intensity was 1000 µmol m−2 s−1. During the measurements, the leaf temperature and vapor pressure deficit (VPD) were 29.6 ± 0.92 °C and 1.6 ± 0.12 kPa, respectively. The chlorophyll a transient fluorescence was measured using the portable Handy PEA (Hansatech Instruments, Norfolk, UK) after dark adaptation for 90 minutes. Relative OJIP transient parameters were calculated according to the JIP test [37]. Leaf chlorophylls were extracted with 80% (v/v) acetone in the dark and the absorbance of the extraction was measured at 470 nm, 645 nm, and 663 nm, respectively [54].

4.3. Extraction and Measurement of Antioxidant Metabolites and Malondialdehyde (MDA)

Reduced ascorbate (Asc) and dehydroascorbate (DHA) were extracted with ice-cold 6% (v/v) HClO4 and measured according to the method reported by Longan [37]. Reduced glutathione (GSH) and oxidized glutathione (GSSG) were extracted with ice-cold 5% (w/v) trichloroacetic acid (TCA) and determined according to the protocol described by Chen and Cheng [33]. MDA in the leaf and root of O. sativa was extracted with 80% (v/v) ethanol and represented as the thiobarbituric acid reactive substances (TBARS) [55].

4.4. Extraction and Measurement of Non-Protein Thiol Compounds (NPTs)

The content of NPTs was assayed according to the method described by Dixit et al. [56]. Briefly, about 50 mg of leaf or root samples were ground with 1.5 mL extraction buffer (pH = 8.0) containing 150 mM Tris–HCl and 5.5 mM EDTA. After being centrifuged at 12,000× g for 10 minutes at 4 °C, the supernatant was used to measure the content of NPTs in a 1 mL reaction solution containing 150 mM Tris–HCl (pH = 8.0), 5.5 mM EDTA, 1.2 mM DTNB. The reaction solution was allowed to stand at room temperature for 10 minutes and then centrifuged at 5000× g for 10 minutes at 4 °C. The obtained supernatant was used to monitor the absorbance at 412 nm. The contents of cysteine were extracted and measured according to the method previously described by Zhang et al. [57].

4.5. Extraction and Measurement of Antioxidant Enzymes

SOD, CAT, APX, GuPX, GlPX, DHAR, MDHAR, GR, GST and OASTL were extracted with buffer solution containing KH2PO4-KOH (50 mM, pH = 7.5), EDTA (0.1 mM), Triton X-100 (0.3%, w/v), and insoluble polyvinylpolypyrrolidone (4%, w/v) [55].
The activity of SOD was measured according to a previously described method [58]. The activities of APX, CAT, GuPX, DHAR, MDHAR and GR were assayed according to Chen et al. [59]. The activities of GlPX and GST were assayed according to Cai et al. [55]. The activity of OASTL was assayed by the method described by Warrilow and Hawkesford [60].

4.6. Quantitative Real-Time PCR (qRT-PCR) Analysis

Total RNA was extracted from O. sativa leaves and roots using a RNAprep Pure Plant Plus Kit (Tiangen, Beijing, China). After the integrity and concentration of total RNA were assessed, total RNA was reversely transcribed into single-strand cDNA using a MMLV transcriptase. The gene special primer pairs were designed by PrimerPremier software (Version 5.0) using the mRNA sequences of O. sativa genome data deposited in NCBI (https://www.ncbi.nlm.nih.gov/assembly/GCF_001433935.1, accessed on 15 July 2024) (Table S1). qRT-qPCR was carried out using iQ SYBR green super-mix kit with a CFX96 Real-time System (Bio–Rad, Hercules, CA, USA). The PCR procedure was as follows: 95 °C, 2 min; 40 cycles of 95 °C, 10 s, 60 °C, 10 s, 72 °C, 30 s. The relative expression level of each gene was calculated by the 2−ΔΔCt method [61]. Ubiquitin (accession number: LOC107277317) was used as the internal control gene and the control samples were set as the reference samples. Three biological replicates per sample were analyzed for the expression levels.

4.7. Experiment Design and Statistical Analysis

Three culture pots were set for each N treatment, and each culture pot contained 24 O. sativa seedlings. The experimental data were processed by Microsoft Excel 2010, analyzed by SPSS 19.0, and visualized by Sigmaplot 10.0. Differences between means were separated by a least significant difference (LSD) test at p-value < 0.05.

5. Conclusions

Decreasing the N supply affected the normal growth of O. sativa seedlings, reducing CO2 assimilation, stomatal conductance, the contents of chlorophylls, and the ratio of Chl a/Chl b, but increasing the intercellular CO2 concentration. The JIP test results indicated that N deficiency increased Fo but decreased the Fv/Fm and the maximum of the IPphase, implying that N-limiting conditions impaired the whole photo electron transport chain from the donor side of PSII to the end acceptor side of PSI in O. sativa. N deficiency enhanced the activities of antioxidant enzymes, such as APX, GuPX, DHAR, SOD, GuPX, GR, GST and OASTL, and the contents of antioxidant compounds, including GSH, GSH + GSSG, and non-protein thiol compounds in O. sativa leaves. In contrast, the increased activities of CAT, DHAR, GR, GST and OASTL, the enhanced ASC–GSH cycle, and the content of sulfur-containing compounds might provide protective roles against oxidative stress in O. sativa roots under N-limiting conditions. When the results of the qRT-PCR analysis and the measurement of enzyme activity were combined, it was found that the number of enzymes was larger in O. sativa leaves than in roots; however, the dynamics of enzyme activity were consistent with their gene expression patterns under N–limiting conditions.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms251910409/s1.

Author Contributions

Conceptualization, L.-H.C. and L.-T.Y.; software, Z.C.; methodology, M.X.; investigation, L.-H.C.; writing—original draft, L.-H.C.; writing—review and editing, L.-T.Y.; funding acquisition, Z.C. and M.X. All authors have read and agreed to the published version of the manuscript.

Funding

This study was financially supported by the Guiding Project of Scientific Technology Plan of Fujian province, Fujian, China (2024N0004, 2023N0005 and 2022N0005).

Institutional Review Board Statement

Not applicable for studies not involving humans or animals.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data will be made available on request.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Effects of different nitrogen treatments on the growth of O. sativa seedlings. Rice seedlings were transplanted to the Hoagland nutrient solutions containing 5 mM NH4NO3 (control), 1 mM NH4NO3 (low N), and 0 mM NH4NO3 (N deficiency), respectively. Seedlings were cultured under a 14 h light/10 h dark regime with a photo radiation of 150 μmol m−2 s−1 and a relative humidity of 68% at 28 °C. Three weeks later, rice seedlings were separated into shoots and roots and wrapped with aluminum foil.
Figure 1. Effects of different nitrogen treatments on the growth of O. sativa seedlings. Rice seedlings were transplanted to the Hoagland nutrient solutions containing 5 mM NH4NO3 (control), 1 mM NH4NO3 (low N), and 0 mM NH4NO3 (N deficiency), respectively. Seedlings were cultured under a 14 h light/10 h dark regime with a photo radiation of 150 μmol m−2 s−1 and a relative humidity of 68% at 28 °C. Three weeks later, rice seedlings were separated into shoots and roots and wrapped with aluminum foil.
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Figure 2. Effects of different nitrogen treatments on CO2 assimilation rate (A); stomatal conductance ((B) n = 6); internal CO2 concentration ((C) n = 6); chlorophyll a (Chl a, (D) n = 3), Chl b ((E) n = 3) and the ratio of Chl a to Chl b (Chl a/Chl b (F) n = 3) in O. sativa seedlings. Bars represent means ± SD (n = 6). Different letters above the bars represent a significant difference at p < 0.05.
Figure 2. Effects of different nitrogen treatments on CO2 assimilation rate (A); stomatal conductance ((B) n = 6); internal CO2 concentration ((C) n = 6); chlorophyll a (Chl a, (D) n = 3), Chl b ((E) n = 3) and the ratio of Chl a to Chl b (Chl a/Chl b (F) n = 3) in O. sativa seedlings. Bars represent means ± SD (n = 6). Different letters above the bars represent a significant difference at p < 0.05.
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Figure 3. Effects of different nitrogen treatments on the average Chl a fluorescence transient (A); IP phase: (Ft − FI)/(FI − Fo) in dark-adapted O. sativa leaves (B); the relatively variable fluorescence between Fo to Fm: Vt = (Ft − Fo)/(Fm − Fo) (C); the differences in Vt between low-N and N-deficient samples and the control samples (5 mM N) (D); the relatively variable fluorescence between Fo to F300μs: Wk = (Ft − Fo)/(F300μs − Fo) (E); and the differences in Wk between low-N and N-deficient samples and the control samples (5 mM N) (F). Values are represented by the means of six biological replicates.
Figure 3. Effects of different nitrogen treatments on the average Chl a fluorescence transient (A); IP phase: (Ft − FI)/(FI − Fo) in dark-adapted O. sativa leaves (B); the relatively variable fluorescence between Fo to Fm: Vt = (Ft − Fo)/(Fm − Fo) (C); the differences in Vt between low-N and N-deficient samples and the control samples (5 mM N) (D); the relatively variable fluorescence between Fo to F300μs: Wk = (Ft − Fo)/(F300μs − Fo) (E); and the differences in Wk between low-N and N-deficient samples and the control samples (5 mM N) (F). Values are represented by the means of six biological replicates.
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Figure 4. Effects of different nitrogen treatments on the primary fluorescence: Fo (A); maximum fluorescence: Fm (B); maximum quantum yield of primary photochemistry: Fm/Fo (C); variable fluorescence at t = 2 ms: Vj (D); variable fluorescence at t = 30 ms: Vi (E); absorbed photo radiation based on the cross section: ABS/CSo (F); dissipated energy based on the cross section: DIo/CSo (G); energy used in the electron transport: ETo/CSo (H); energy used in the reduction of end acceptor side of PSI: Reo/CSo (I); and the total performance index of PSs: PItotal (J) in dark-adapted O. sativa leaves. Bars represent means ± SD (n = 6). Different letters above the bars represent a significant difference at p < 0.05.
Figure 4. Effects of different nitrogen treatments on the primary fluorescence: Fo (A); maximum fluorescence: Fm (B); maximum quantum yield of primary photochemistry: Fm/Fo (C); variable fluorescence at t = 2 ms: Vj (D); variable fluorescence at t = 30 ms: Vi (E); absorbed photo radiation based on the cross section: ABS/CSo (F); dissipated energy based on the cross section: DIo/CSo (G); energy used in the electron transport: ETo/CSo (H); energy used in the reduction of end acceptor side of PSI: Reo/CSo (I); and the total performance index of PSs: PItotal (J) in dark-adapted O. sativa leaves. Bars represent means ± SD (n = 6). Different letters above the bars represent a significant difference at p < 0.05.
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Figure 5. Effects of different nitrogen treatments on the activities of antioxidant enzymes (A–H); and sulfur metabolism related enzymes (I,J) in O. sativa leaves. Bars represent means ± SD (n = 3). Different letters above the bars represent a significant difference at p < 0.05.
Figure 5. Effects of different nitrogen treatments on the activities of antioxidant enzymes (A–H); and sulfur metabolism related enzymes (I,J) in O. sativa leaves. Bars represent means ± SD (n = 3). Different letters above the bars represent a significant difference at p < 0.05.
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Figure 6. Effects of different nitrogen treatments on the activities of antioxidant enzymes (A–H); and sulfur metabolism related enzymes (I,J) in O. sativa roots. Bars represent means ± SD (n = 3). Different letters above the bars represent a significant difference at p < 0.05.
Figure 6. Effects of different nitrogen treatments on the activities of antioxidant enzymes (A–H); and sulfur metabolism related enzymes (I,J) in O. sativa roots. Bars represent means ± SD (n = 3). Different letters above the bars represent a significant difference at p < 0.05.
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Figure 7. Effects of different nitrogen treatments on the contents of antioxidant compounds in the leaves (A,C,E,G,I,K); and roots (B,D,F,H,J,L) of rice. Bars represent means ± SD (n = 3). Different letters above the bars represent a significant difference at p < 0.05.
Figure 7. Effects of different nitrogen treatments on the contents of antioxidant compounds in the leaves (A,C,E,G,I,K); and roots (B,D,F,H,J,L) of rice. Bars represent means ± SD (n = 3). Different letters above the bars represent a significant difference at p < 0.05.
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Figure 8. Effects of different nitrogen treatments on the contents of non-protein thiol compounds (A,B); cysteine (C,D); MDA (E,F); and the production of H2O2 (G,H) in the leaves and roots of rice. Bars represent means ± SD (n = 3). Different letters above the bars represent a significant difference at p < 0.05.
Figure 8. Effects of different nitrogen treatments on the contents of non-protein thiol compounds (A,B); cysteine (C,D); MDA (E,F); and the production of H2O2 (G,H) in the leaves and roots of rice. Bars represent means ± SD (n = 3). Different letters above the bars represent a significant difference at p < 0.05.
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Figure 9. Effects of different nitrogen treatments on the gene expression patterns of antioxidant related enzymes in the leaves (A); and roots (B) of rice. Bars represent means ± SD (n = 3). Different letters above the bars of each gene represent a significant difference at p < 0.05.
Figure 9. Effects of different nitrogen treatments on the gene expression patterns of antioxidant related enzymes in the leaves (A); and roots (B) of rice. Bars represent means ± SD (n = 3). Different letters above the bars of each gene represent a significant difference at p < 0.05.
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Chen, L.-H.; Xu, M.; Cheng, Z.; Yang, L.-T. Effects of Nitrogen Deficiency on the Photosynthesis, Chlorophyll a Fluorescence, Antioxidant System, and Sulfur Compounds in Oryza sativa. Int. J. Mol. Sci. 2024, 25, 10409. https://doi.org/10.3390/ijms251910409

AMA Style

Chen L-H, Xu M, Cheng Z, Yang L-T. Effects of Nitrogen Deficiency on the Photosynthesis, Chlorophyll a Fluorescence, Antioxidant System, and Sulfur Compounds in Oryza sativa. International Journal of Molecular Sciences. 2024; 25(19):10409. https://doi.org/10.3390/ijms251910409

Chicago/Turabian Style

Chen, Ling-Hua, Ming Xu, Zuxin Cheng, and Lin-Tong Yang. 2024. "Effects of Nitrogen Deficiency on the Photosynthesis, Chlorophyll a Fluorescence, Antioxidant System, and Sulfur Compounds in Oryza sativa" International Journal of Molecular Sciences 25, no. 19: 10409. https://doi.org/10.3390/ijms251910409

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