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Article

Metabolomics and Physiological Changes Underlying Increased Tolerance to Salt Stress Induced by Applied Nitric Oxide in Fatsia japonica Seedlings

by
Xing Hu
,
Min Zhang
,
Jiao Liu
,
Xiaomao Cheng
and
Xiaoxia Huang
*
Southwest Landscape Architecture Engineering Research Center of National Forestry and Grassland Administration, College of Landscape Architecture and Horticulture Sciences, Southwest Forestry University, Kunming 650224, China
*
Author to whom correspondence should be addressed.
Forests 2024, 15(1), 159; https://doi.org/10.3390/f15010159
Submission received: 6 December 2023 / Revised: 9 January 2024 / Accepted: 10 January 2024 / Published: 12 January 2024
(This article belongs to the Section Forest Ecophysiology and Biology)
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Abstract

:
Fatsia japonica (Thunb.) Decne. et Planch. is an important woody landscape plant, and its distribution is commonly limited by salt stress. Although the application of exogenous nitric oxide (NO) has been known to be effective in alleviating abiotic stress in plants, the underlying mechanism by which NO induces salt resistance in F. japonica remains unknown. In this study, the physiological and metabolic characteristics of F. japonica seedlings with the application of NO under salt stress conditions were investigated. We demonstrated that exogenous NO (0.1 mM sodium nitroprusside, SNP) mitigated the growth inhibition caused by 0.4% NaCl. This alleviation could be attributed to NO-induced enhancement in photosynthesis, osmotic adjustment, antioxidant enzyme activities, and a reduction in oxidative damage when exposed to salt stress. Furthermore, the metabolomic analysis revealed that salt stress significantly disrupts the growth of F. japonica by downregulating sugars, sugar alcohols, amino acids, and organic acids. However, the application of exogenous NO improves sugar metabolism, enhancing the levels of fructose, glucose, mannose, galactose, xylose, ribose, inositol, and sorbitol, as well as the metabolism of amino acids and organic acids. These findings provide new insights into the physiological and metabolic homeostasis adjustments induced by NO that promote salt stress tolerance in F. japonica, enhancing our understanding of plant resilience mechanisms.

1. Introduction

Soil salinization is widely recognized as a significant environmental challenge that seriously constrains plant growth [1]. In plants, numerous physiological and cellular processes are profoundly impacted by salt stress, including seed germination, photomorphogenesis, the growth rate of roots and leaves, and plant height [2,3,4]. Under salt stress conditions, the absorption of Na+ and Cl− is increased, which, in turn, reduces the uptake of essential minerals such as Ca2+ and K+, leading to physiological and metabolic disorders in plants [5,6]. Additionally, high concentrations of Na+ and Cl− reduce photosynthesis through both stomatal and non-stomatal factors, with Cl− further impeding photosynthetic efficiency by interfering with chlorophyll synthesis [7]. Salt stress leads to osmotic stress in plants, causing a decrease in soil water potential, hindering water uptake, and resulting in physiological drought, reduced stomatal conductance, and photosynthesis. This subsequently accelerates the accumulation of reactive oxygen species (ROS), disrupting the normal redox state and leading to oxidative stress [8,9,10]. Meanwhile, the overproduction of ROS triggered by salt stress leads to the oxidative damage of functional biomolecules, including chlorophyll, membrane lipids, proteins, and nucleic acids [11]. In response to salt stress, plants adapt through a variety of strategies, including the regulation of ion homeostasis, engagement of osmotic adjustment, modulation of plant hormone signaling, such as gibberellin (GA), ethylene, abscisic acid (ABA), and jasmonic acid (JA), and activation of antioxidant systems to scavenge reactive oxygen species [12,13,14]. However, despite these adaptations, the capacity of plants to manage stress through physiological adjustments is finite, and they are often unable to withstand severe or prolonged exposure to salt stress.
Since plants’ innate stress defense systems are not enough to provide sufficient protection in the face of severe stress conditions, the exogenous application of dynamic gaseous molecules like hydrogen sulfide (H2S), nitric oxide (NO), and carbon monoxide (CO) has been shown to play a significant role in enhancing plant resistance [15,16,17]. Among these molecules, NO is a ubiquitous gaseous signaling molecule that has broad-ranging physiological functions from seed germination to senescence. It also plays a role in enhancing plant defense capacity in the face of environmental stress conditions, including salt stress [18]. Previous findings have suggested that NO mitigates the deleterious effects of salt stress by inducing ROS metabolism and activating the antioxidant defense system [19,20]. Moreover, NO has been evidenced to increase salt resistance by regulating osmolyte levels and Na+/K+ equilibrium in plant cells [21]. Exogenous NO is utilized to improve plant photosynthesis during salt stress by alleviating the decline in chlorophyll content [22]. Furthermore, the application of exogenous NO could modulate the level of sugars, organic acids, and amino acids in barley, maintaining a higher antioxidant capacity and improving salt resistance [23]. Nonetheless, the role of NO in modulating the salt stress response in Fatsia japonica (Thunb.) Decne. et Planch. has not yet been elucidated.
F. Japonica, a subtropical evergreen shrub native to Japan and southern China, is not only widely cultivated as a horticultural ornamental plant in southern China but also possesses medicinal value [24]. Previous research has primarily focused on the cultivation, anticancer ingredients, and genes of F. japonica [25,26,27]. However, there are gaps in the physiological and metabolic mechanisms of NO-induced salt stress alleviation in F. japonica. Therefore, this research aims to investigate the mechanism of exogenous NO action under salt stress conditions in F. japonica using physiological and metabolomic approaches. It is hypothesized that NO imparts salt stress tolerance through mechanisms that impact photosynthesis, the antioxidant system, osmotic adjustment, and metabolic regulation of F. japonica.

2. Materials and Methods

2.1. Plant Materials

The experiment was conducted from September 2021 to December 2021 in the greenhouse of Southwest Forestry University (25°06′ S, 102°76′ W), Southwest China. Inside the greenhouse, the temperature ranged from 15 to 23 °C, and the humidity was 50%–70%. In September 2021, healthy half-year-old seedlings of F. japonica were selected for the experiment, with approximately the same height (8–10 cm) and in good health (no diseases and insect pests). In total, 60 seedlings were planted in a nourishment bag (0.15 m width × 0.15 m height) matched with white trays, one seedling per pot. The soil matrix was composed of red clay and humus soil (2:1), and each pot was filled with a dry weight of 550 g of the soil matrix. After one month of growth, the experiment with NaCl and SNP (sodium nitroprusside, an NO donor) application was conducted.

2.2. Treatments

The experiment utilized a fully randomized design, including four treatments (15 pots per treatment): (1) control (CK); (2) SNP treatment (NO); (3) NaCl treatment (S); and (4) NaCl + SNP treatment (S + NO). To prevent salt shock response, the NaCl solution was gradually added into the soil (25 mL for each pot every other day) nine times. The concentration of the irrigated NaCl solution was increased gradually from 50 mM to 200 mM (by 50 mM every other day) in the first 7 days, maintaining a 200 mM concentration to achieve the final salt concentrations of 0.4% on the 17th day, relative to the soil weight. Sodium nitroprusside (SNP) at a concentration of 0.1 mM was sprayed on the leaves twice a day (morning and night) from the beginning of NaCl treatment, and the non-SNP group was sprayed with an equal volume of distilled water as the control. The criterion of SNP foliar spraying was that both sides of the leaf had water droplets (approximately 15 mL for each pot). To eliminate orientation effects, the plants were rotated every 7 days during treatment. Samples were collected at 45 days (the 28th day after NaCl was completely added). At the same time, photosynthetic, the chlorophyll fluorescence parameters and growth parameters of the leaves (the third mature leaf counting from the top) were measured.

2.3. Determination Methods

2.3.1. Plant Growth Parameters

The plant was harvested on the 45th day after the first NaCl treatment. To remove the soil adhering to the plant’s surface, the plants were washed at least thrice, and the roots were kept intact. The length of the stem, root, and leaf and the width of the leaf were recorded using a metric scale (50 cm). The gravimetric method [28] was used to calculate the leaf area by tracing the outline of the fresh leaves (third leaf from the top) and calculating the area covered by it on paper (leaf area = leaf trait weight/paper weight × paper area). Then, the plants were washed twice with distilled water, and any water attached to the plants’ surface was blotted using filter paper. The plants were then weighed on an electronic balance to record the total fresh biomass. The roots and shoots were separated and transferred to an oven at 105 °C for 30 min, followed by drying at 80 °C until a constant weight was achieved. The sample was weighed again to record their respective dry biomass and total dry biomass.

2.3.2. Photosynthetic and Chlorophyll Fluorescence Parameters

Photosynthetic parameters were determined from 9:00 to 11:00 using a portable photosynthesis measurement system Li-6400 (LICOR, Lincoln, NE, USA). The data were recorded at a 400 µmol·mol−1 CO2 concentration, 800 µmol·m−2·s−1 PFD, 55% relative humidity, and at 24 °C. The parameters included the net photosynthetic rate (Pn), transpiration rate (Tr), intercellular CO2 concentration (Ci), and stomatal conductance (Gs). The chlorophyll fluorescence parameters were measured using a portable pulse-modulated chlorophyll fluorometer PAM-2100 (WALZ, Nuremberg, Germany). The measurements were performed at 0.5 µmol·m−2·s−1 PFD (measuring light) after 30 min of darkness acclimation. The initial fluorescence (F0), the maximum photochemical quantum yield of PSII (Fv/Fm), the actual photochemical quantum yield of PSII (Y(II)), the electron transport rate of PSII (ETR), the photochemical quenching coefficient (qP), and the non-photochemical quenching coefficient (NPQ) were recorded under dark conditions. Both gas exchange parameters and chlorophyll fluorescence were determined from five healthy plants in each treatment, with three leaves measured per sample and each leaf repeated five times.

2.3.3. Photosynthetic Pigments and Starch Content

The acetone-ethanol mixture method [29] was employed to calculate the content of photosynthetic pigments. Fresh leaves (0.1 g) were added to each tube, followed by a 10 mL mixture of acetone and ethanol (1:1). Then, the extraction was performed in the absence of light for at least 48 h until the leaves turned white (shaken well at 24 h intervals). The absorbance of the extraction was measured at wavelengths of 663 nm, 646 nm, and 470 nm for the chla, chlb, and carotenoid concentrations, respectively. The method for measuring the starch was consistent with that for soluble sugars, as described in Section 2.3.4.

2.3.4. Malondialdehyde (MDA) and Osmolyte Content

The thiobarbituric acid method [30] was used to estimate the MDA content. Fresh leaves (0.2 g) were crushed in 10% trichloroacetic acid (TCA) (5 mL) and then centrifuged at 8000× g for 10 min. Next, 2 mL of 6% thiobarbituric acid (TBA) was mixed with 2 mL of the supernatant. The mixture solution was heated at 100 °C for 15 min and subsequently centrifuged at 8000× g for 10 min, and the absorbance was recorded at 600, 532, and 450 nm.
The enzymolysis method [31] was used to assess the soluble sugar content of the leaves, which is based on the hydrolytic action of α-amylase and α-amyloglucosidase. A total of 1.6 mL of 0.7 M perchloric acid was used to homogenize the fresh leaves (0.2 g). Then, the soluble sugar was hydrolyzed into glucose by an enzymatic reaction. Then, 30 µL of the sample solution was mixed with GOD-POD (3 mL) to assay the glucose content, which estimated the soluble sugar content.
Coomassie Brilliant Blue staining was used to calculate the total soluble protein content [32]. In brief, 5.0 mL of extraction buffer (50 mM Na2HPO4-NaH2PO4, pH 7.0, containing 1 mM EDTA, 2% PVP) was used to homogenize the fresh leaves (0.2 g), then this was centrifuged at 12,000× g for 20 min at 4 °C. A total of 50 uL of the supernatant was mixed with Coomassie Brilliant Blue (5 mL) and pure water (950 µL). The sample reacted for 20 min, and the absorbance was recorded at 595 nm.
The proline content was estimated via the ninhydrin method [33]. A total of 5 mL of 3% sulfosalicylic acid solution was used to homogenize the fresh leaves (0.2 g). After heating for 10 min in a 100 °C water bath, each tube was centrifuged for 10 min at 8000× g. Next, 2 mL of the supernatant was added to the test tube; then, this was mixed with 2 mL of ninhydrin and 2 mL of glacial acetic acid for 1 h at 100 °C. Afterward, the reaction mixture was extracted using 5 mL of toluene (shaken vigorously) for 12 h, and the absorbance was measured at 520 nm.

2.3.5. Antioxidant Enzyme Activities

Fresh leaves (0.2 g) were homogenized with 5.0 mL of extraction buffer (50 mM Na2HPO4-NaH2PO4, pH 7.0, containing 1 mM EDTA, 2% PVP) and then centrifuged at 12,000× g for 10 min at 4 °C. Following the centrifugation, the resulting supernatant was utilized to assess the enzymatic activity. The activity of catalase (CAT, EC 1.11.1.6) was assessed according to the methods of Aebi (1984), monitoring the decrease in absorbance values at 240 nm due to H2O2 consumption [34]. The activity of ascorbate peroxidase (APX, EC 1.11.1.1) was measured as described by Knorzer et al. (1996) by monitoring the rate of ascorbate oxidation via the decline in absorbance at 290 nm [35]. The activity of peroxidase (POD, EC 1.11.1.7) was measured using the method by Lin and Wang (2002), recording the increase in absorbance at 420 nm due to the generation of tetraguaiacol [36].
The method of Giannopolitis and Ries (1977) was employed to assess the activity of superoxide dismutase (SOD, EC 1.15.1.11), which is based on the photoreduction of nitro blue tetrazolium chloride (NBT) [37]. The absorbance was detected at a wavelength of 560 nm, and the quantity of enzyme needed to inhibit 50% of the NBT photoreduction was identified to be one unit of SOD activity (U).

2.3.6. Metabolomic Profiling

The non-targeted metabolome method was used for metabolomic profiling at Wuhan Metware Biotechnology Co., Ltd. (Wuhan, China) (http://www.metware.cn/, accessed on 21 February 2022). Freeze-dried leaf samples were pulverized with a mixer mill (MM 400, Retsch, Haan, Germany) with zirconia beads at a frequency of 30 Hz for 1.5 min. A total of 100 mg of sample powder was weighted and extracted overnight at 4 °C with 1.2 mL 75% aqueous methanol, then adsorbed and filtrated before analysis using a UPLC-ESI-MS/MS system. The quantitation of the metabolites was performed using the multiple reaction monitoring (MRM) technique, following the methodology detailed by Chen et al. (2013) [38].

2.4. Statistical Analysis

The data analysis was performed using a one-way ANOVA with SPSS 19.0 to compare the differences among the treatments following Duncan’s test (p < 0.05). Data are represented as the mean ± SD of three replicates. The lowercase letters indicate statistical differences, and the graphs were plotted using Excel 2010, Origin 2023, and R Studio 2022.
To further evaluate the responses of F. japonica physiology and the key metabolites induced by NaCl stress and SNP treatment, a principal component analysis (PCA) was performed (using SPSS 19.0). This analysis helped to organize the data to reveal the treatments’ integrated impact and potential adaptation mechanisms.

3. Results

3.1. Effect of SNP (NO Donor) on the Phenotype and Growth Parameters in F. japonica under Salt Stress

After 45 days of treatment, the NaCl-treated plants showed obvious symptoms of foliar injury, evident as wilting and necrotic lesions on the leaf margins, compared to the control. The SNP-treated plants showed slight growth inhibition compared with the control plants. Notably, the NaCl + SNP-treated plants exhibited an improved phenotype with minimum stress-related damages under salt stress conditions compared with salt-treated plants. All these morphological changes verified that NO plays an effective role in increasing the tolerance of salt stress in F. japonica (Figure 1).
Salt stress reduced stem length, leaf length, leaf width, and leaf area by 39.05%, 40.21%, 42.8%, and 65.09%, respectively, with no significant decrease observed in root length. However, under salt stress, the application of exogenous SNP significantly increased leaf length, leaf width, and leaf area by 43%, 41.14%, and 78.38%, respectively, with respect to NaCl-treated plants, but no obvious effect was observed in stem length and root length. Furthermore, SNP alone decreased the stem length, leaf width, and leaf area by 27.06%, 12.84%, and 35.65%, respectively, relative to the control (Table 1).
Similarly, the biomass of F. japonica seedlings was significantly affected by NaCl treatment after 45 days. Compared to the control, the plants of root biomass, shoot biomass, total dry biomass, total fresh biomass, and root/shoot decreased by 43.35%, 29.7%, 32.84%, 29.82%, and 20%, respectively. The application of SNP notably recovered the total dry biomass by 18.2% under salt stress. Exogenous SNP also alleviated the negative effect of salt stress on root biomass, shoot biomass, total fresh biomass, and root/shoot, although not significantly (Table 2). These changes in growth indexes further support the notion that NO helps in improving the tolerance of salt stress in F. japonica.

3.2. Effect of SNP on Photosynthetic Performance in F. japonica under Salt Stress

A dramatic decline in photosynthetic characteristics indexes was observed in Pn, Gs, and Tr. Under salt stress, the value of Pn, Gs, and Tr decreased by 0.9-fold, 0.9-fold, and 1-fold, respectively, compared to the control. However, SNP-treated plants showed less negative impact from NaCl, with Pn, Gs, and Tr enhanced by 2.4-fold, 3.5-fold, and 10.3-fold, respectively (Figure 2A–C). Additionally, compared to the control, Ci was significantly higher in NaCl-stressed plants after 45 days, but SNP application significantly reduced Ci under salt stress (Figure 2D). SNP alone significantly decreased Pn after 45 days of treatment (Figure 2A).
Furthermore, chlorophyll fluorescence parameters in F. japonica leaves were negatively affected by salt stress (Figure 3). NaCl treatment greatly increased the value of F0 than the control, which was 40% higher than that of no salt stress. However, there was no significant difference in the value of F0 between the NaCl + SNP treatment and the control (Figure 3A). Fv/Fm dropped sharply under salt stress. Still, SNP application notably restored the level of Fv/Fm (Figure 3B). After 45 days of treatment, the peak values of F0 as well as the lowest values of Fv/Fm all appeared in the NaCl treatment, which corresponds with the Pn variations observed in F. japonica leaves (Figure 2A and Figure 3A,B). Moreover, YII, NPQ, ETR, and qP were remarkably decreased in NaCl-treated plants. However, the exogenous application of SNP maintained a high level of NPQ and eased the decrease in YII and ETR under salt stress but had no significant effect on qP (Figure 3C–F). Plants treated with SNP alone also showed a lower YII and ETR than non-treated plants at 45 days (Figure 3C,F). Overall, these findings imply that exogenous NO plays a role in mitigating the negative effect of salt stress on the photosynthesis of F. japonica leaves (Figure 3).

3.3. Effect of SNP on the Contents of Chlorophyll, Carotenoid, and Starch in F. japonica under Salt Stress

Salt stress resulted in a significant decline in Chla, Chlb, Car, and starch content. Compared to the control, NaCl treatment decreased Chla, Chlb, Car, and starch content by 47%, 47.9%, 47.4%, and 40.7%, respectively (Figure 4). However, plants treated with SNP maintained significantly higher Chla (38.4%), Chlb (47.6%), and starch content (120%) than non-SNP treated plants under salt stress (Figure 4A,B,D). Exogenous SNP also alleviated salt stress-induced decline in the Car content of leaves, although not significantly (Figure 4C). SNP treatment alone notably decreased Chla, Chlb, and carotenoid content (Figure 4A–C).

3.4. Effect of SNP on MDA and Osmolyte Content in F. japonica under Salt Stress

The accumulation of MDA and proline increased under salt stress, with 64.4% and 561.8% higher MDA and proline accumulation observed compared to the control plants. Nevertheless, when SNP-treated plants were exposed to salt stress, there was a significant decrease in MDA (24.9%) and proline (49.8%) accumulation compared to non-SNP-treated plants (Figure 5A,B). NaCl treatment resulted in a 38.1% decrease in soluble sugar content compared to control plants. However, this reduction was reversed by the application of SNP, as SNP-treated plants showed a significantly higher increase of 69.8% in soluble sugar content than non-SNP-treated plants under salt stress conditions (Figure 5C). Furthermore, the content of soluble protein also improved in SNP-treated plants under salt stress, although not significantly; however, treatment with SNP alone notably increased the content of soluble protein (Figure 5D).

3.5. Effect of SNP on the Activities of the Antioxidative Enzymes in F. japonica under Salt Stress

NaCl treatment significantly increased the activities of CAT, POD, and APX compared to the control, with CAT increasing by 2.2-fold, POD by 1.8-fold, and APX by 2.2-fold (Figure 6A–C). However, there was no significant difference in the SOD activity between the salt-stressed and control plants (Figure 6D). In comparison to NaCl treatment, the application of exogenous SNP remarkably increased CAT (by 7.8%) and SOD (by 15%) activity under salt stress (Figure 6A,D). Moreover, SNP treatment alone also notably increased both CAT and POD activity compared to the control (Figure 6A,B). These findings suggest a NO-mediated antioxidant system that improves the activity of antioxidant enzymes in salt-stressed F. japonica (Figure 6).

3.6. Effect of SNP on the Total Metabolomic Response of F. japonica under Salt Stress

To investigate the role of SNP in metabolite changes under salt stress, a differential metabolite analysis was conducted on F. japonica subject to various treatments. In total, 196 differential metabolites were identified, including 84 organic acids, 37 amino acids and their derivatives, 31 sugars and their derivatives, and 44 other metabolites (VIP > 1 and FC ≥ 2 or FC ≤ 0.5) (Figure 7). According to Figure 7, NaCl stress (S vs. CK) obviously downregulated the accumulation of metabolites in F. japonica, particularly organic acids, amino acids and their derivatives, and sugars and their derivatives. However, NaCl stress plus SNP treatment upregulated these differential metabolites in leaves of F. japonica compared to NaCl stress (SN vs. S) (Figure 7). As compared to control, NaCl stress did not significantly affect the total organic acids and other metabolites accumulation; it markedly decreased the accumulation of total amino acids and derivatives, sugars and derivatives in both the SNP and no SNP treated plants under salt stress (Figure 8). Notably, SNP treatment induced the total sugars, and their derivatives content improved under NaCl stress, with SNP-treated plants having a 2.7-fold higher total sugars and derivatives content than the NaCl-treated plants (Figure 8). SNP alone treatment significantly increased the accumulation of total organic acids, sugars, and sugar derivatives while decreasing the accumulation of total amino acids and derivatives (Figure 8). These changes demonstrate that NO increased the accumulation of metabolites, especially sugars and sugar derivatives, in response to salt conditions (Figure 7 and Figure 8).

3.7. Effect of SNP on Differential Metabolites Content in F. japonica under Salt Stress

To further the prior analyses, SNP-induced key differential metabolites associated with growth and stress tolerance in F. japonica were selected from S + NO vs. S (VIP > 1, FC ≥ 2 or FC ≤ 0.5, and p < 0.01) for relative content analysis (Figure 9). For changes in eight different sugars and sugar alcohols, the content of Galactose, Fructose, Glucose, inositol, Xylose, Raffinose, Ribose, and Sorbitol is reduced by NaCl. Conversely, exogenous SNP treatment notably enhanced the accumulation of Galactose (2.9-fold), Fructose (2.8-fold), Glucose (3.5-fold), inositol (3.4-fold), Xylose (1.6-fold), ribose (1-fold) and sorbitol (1.8-fold) under salt stress. Similarly, the accumulation of three amino acids and one organic acid also were significantly reduced under salt stress, but exogenous SNP treatment remarkably improved Aspartic acid (1-fold), Cystine (7.5-fold), and Abscisic acid (1.2-fold) content than the NaCl-treated plants (Figure 9). These results indicate that exogenous SNP increased the accumulation of sugars, sugar alcohols, amino acids, and organic acids in response to NaCl stress in F. japonica, consistent with total differential metabolites analysis.

3.8. Effect of SNP on Metabolic Pathways and Metabolites Response of F. japonica under Salt Stress

A total of 38 metabolites (12 organic acids, 15 amino acids, 9 sugars, and 2 sugar alcohols) were assigned to metabolic pathways mainly involved in sugar, amino acid, and organic acid metabolism (Figure 10 and Table S1). NaCl stress predominantly led to a decrease in the accumulation of 34 metabolites in the leaves of F. japonica (Figure 10 and Table S1, S vs. CK). However, compared to NaCl stress, NaCl stress plus SNP treatment caused lower metabolite downregulation in plants (Figure 10 and Table S1, SN vs. S). Exogenous SNP application upregulated the accumulation of raffinose, glucose, mannose, and fructose, leading to an increase in galactose, inositol, sorbitol, ribose, and xylose and a decrease in arabinose under NaCl stress (Figure 10 and Table S1, SN vs. S). NaCl stress inhibited the accumulation of phosphoenolpyruvate, leading to the downregulation of downstream metabolites (Figure 10 and Table S1, S vs. CK). However, SNP treatment upregulated phosphoenolpyruvate, resulting in the increased accumulation of aspartic acid, which was converted into O-acetyl serine and cystine; SNP treatment also led to citrulline, succinic semialdehyde, 4-guanidinobutyric acid, and argininosuccinic acid upregulation under salt stress (Figure 10 and Table S1, SN vs. S). Moreover, NaCl stress affected the tricarboxylic acid (TCA) cycle with decreases in malic acid and isocitric acid. Still, exogenous SNP treatment reversed the decline induced by NaCl (Figure 10 and Table S1, S vs. CK and SN vs. CK).

3.9. Principal Component Analysis

The principal component analysis (PCA) revealed a cumulative variance contribution rate of 81.7%; the first (PC1) and second principal component (PC2) explained 67.5% and 14.2% of the variance in the variables, respectively. PC1 tended to separate the effects of salt stress, and PC2 further segregated SNP treatment (Figure 11). The NaCl stress group exhibited the most pronounced divergence from the other treatments, while the NaCl + SNP treatment group was closer to the control. According to these results, SNP had an alleviating effect on NaCl stress (Figure 11).

4. Discussion

4.1. NO Alleviated the Inhibition of Salt Stress on the Growth of F. japonica

Salt stress is widely known to inhibit plant growth, primarily by disrupting photosynthesis, osmotic balance, and the cellular redox state [1,12]. Nitric oxide (NO) plays a crucial role as a signaling molecule in response to abiotic stress. Previous studies have shown that supplementation with NO could improve the growth of plants under stressful conditions [19,39,40]. In this study, the growth of F. japonica seedlings was significantly reduced by salt stress. However, this damage was mitigated by the application of exogenous NO, which resulted in improved phenotypic characteristics (Figure 1). Furthermore, NO-treated plants exhibited higher biomass compared to untreated plants under salt stress (Table 2). Alnusairi et al. (2021) have already demonstrated that the exogenous supply of NO enhances wheat growth by reinforcing photosynthetic efficiency, osmolytes, and antioxidants under salt stress [41].
Photosynthesis, the sole source of carbon required for plant growth and development, directly influences plant biomass accumulation [42]. The NO-mediated recovery in photosynthetic performance under NaCl stress involves mechanisms that include enhancing chlorophyll content, stomatal conductance, and PSII activity [18]. In this study, chlorophyll content was reduced by NaCl, which directly weakened photosynthesis, but NO application significantly alleviated the reduction (Figure 4A,B). Salt stress decreases chlorophyll content due to the excessive accumulation of Na+, which limits the absorption of Mg2+, leading to reduced chlorophyll synthesis [43]. Shams et al. (2019) reported that NO treatment could increase the uptake of nutrient elements under salt stress, implying that exogenous NO could improve plant chlorophyll synthesis by enhancing Mg uptake [44]. Like drought stress, plants close leaf stomata in response to salt stress in order to reduce transpiration and water loss, but this also decreases gas exchange and leads to a decline in photosynthesis [45]. In this study, salt stress significantly decreased Pn, Tr, and Gs, while Ci increased in F. japonica seedlings (Figure 2). Therefore, the decline of Pn induced by salt stress is attributed to both stomatal and non-stomatal limiting factors [46]. However, exogenous NO treatment increased Pn, Tr, Gs, and decreased Ci under NaCl stress (Figure 2). Higher Gs improves substrate transfer for photosynthesis, while increased Tr enhances water absorption and transport, improving plant salt tolerance [47]. Sharma et al. (2020) also reported that exogenous NO could induce stomatal opening, thus reducing salt-induced stomata limitations [48].
Furthermore, untreated F. japonica exhibited much lower Fv/Fm, YII, ETR, and NPQ than NO-treated F. japonica under salt stress conditions (Figure 3), suggesting that NO could reduce the salt-induced inhibition of electron transport in PSII while increasing the quantum yield of PSII in F. japonica seedlings [49]. Previous studies have also shown that the application of exogenous NO has an inhibitory effect on the plant photochemical activity of PSII under normal conditions [50], consistent with the variations in YII and ETR detected in this study, leading to reduced Pn (Figure 2A and Figure 3C,F). The end products of photosynthesis are primarily starch and sucrose [51], and a higher accumulation of starch further indicated that the application of exogenous NO could effectively enhance plant photosynthesis under salt stress in this study (Figure 4D). To sum up, NO mediates the improvement of photosynthesis under salt stress conditions through increasing photosynthetic pigment biosynthesis, regulating photosynthetic characteristics, and enhancing chlorophyll fluorescence.
The accumulation of osmolytes, including proline, soluble sugars, and proteins, is a strategy plants employ to withstand osmotic stress induced by salt [52]. Xue et al. (2016) reported that under stress conditions, plants increase the production of osmotic regulator substances by consuming carbon needed for growth, which raises metabolic costs [53]. This may explain the observed increase in proline accompanied by a decrease in soluble sugar in F. japonica under NaCl stress (Figure 5B,C). However, the application of SNP mainly triggered the accumulation of soluble sugar while reducing proline content in F. japonica under salt stress (Figure 5B,C). Thus, NO application may reduce the energy consumption on proline synthesis, conserving resources for plant growth under NaCl stress. Previous research has revealed that NO could increase the accumulation of soluble sugar, enhancing cell salt tolerance [52], which aligns with this research. Generally, salt stress provokes ROS accumulation, leading to membrane lipid peroxidation, as reflected by higher MDA levels [54]. To protect the cell membrane from oxidative damage, plants are equipped with a well-regulated antioxidant defense system [55]. In this study, plants significantly improved the activity of CAT, POD, and APX under salt stress, but MDA accumulation notably increased (Figure 5A and Figure 6A–C). Exogenous NO significantly increased SOD and CAT activity under NaCl stress, followed by a marked decline in MDA levels (Figure 5A and Figure 6A,D), confirming that NO induces the system of enzymatic antioxidant defense in salt-stressed plants and reduces membrane lipid peroxidation [56]. Li et al. (2008) found that exogenous NO increased the activities of SOD, APX, and CAT in barley exposed to salt stress, indicating the beneficial effect of NO on salt tolerance [20]. Therefore, based on photosynthetic pigment and starch content, photosynthetic parameters, osmolytes, and antioxidant enzyme activities, the protective mechanisms induced by exogenous SNP are highly effective against salt stress and favor plant growth under salinity conditions.

4.2. NO Regulated Metabolite Response under Salt Stress

The comparative metabolomic analysis revealed that the change in metabolites under NaCl treatment was mainly downregulated in this study. However, exogenous NO was found to upregulate multiple metabolites, including sugars, sugar alcohols, organic acids, and amino acids under NaCl stress (Figure 7). Similarly, melatonin treatment leads to significantly higher levels of amino acid, organic acid, sugar, and sugar alcohol in NaCl-treated plants to resist salt damage [57]. Therefore, the results demonstrated that exogenous NO can upregulate key metabolites in NaCl-treated F. japonica, improving salt resistance and maintaining plant growth. The metabolic pathway analysis suggested that the application of exogenous NO primarily enhanced sugar metabolism under salt stress, as evidenced by the observed variations in total sugar and sugar derivatives content in the NaCl + SNP treatment group (Table S1, Figure 8 and Figure 10). This implies the important role of sugar metabolism in NO-induced tolerance against salt stress in F. japonica.
In this study, exogenous NO upregulated the contents of glucose, galactose, fructose, xylose, ribose, mannose, inositol, and sorbitol in F. japonica under salt stress (Figure 9). Ma et al. (2021) revealed that SNP-treated barley seedlings enhanced galactose, fructose, and mannose metabolism under NaCl stress, inducing salt tolerance [23], which is consistent with our study. Previous reports have also shown that glucose, fructose, galactose, mannose, and ribose act as important osmolytes that improve salt tolerance in plants [57,58,59]. Additionally, glucose can activate antioxidant enzyme activities to improve salt stress resistance, thus decreasing MDA content in wheat [60]. Xylose and arabinose are involved in the composition of plant cell walls, with salt having a greater effect on xylose than arabinose [61,62]. In this study, only xylose content was downregulated under salt stress, while exogenous NO upregulated xylose and downregulated arabinose in the metabolic pathway under salt stress (Table S1 and Figure 10). This suggests that NO may have maintained cell wall component balance by regulating xylose and arabinose content, thereby maintaining the stability and integrity of the plant cell wall and supporting plant growth under salt stress [63]. Furthermore, as sugar derivatives, sugar alcohols may have a similar mechanism of action to sugars in plant stress responses. Nelson et al. (1999) reported that inositol and sorbitol can act as osmoregulators to enhance cellular stability under adverse stress [64]. Hence, the elevated levels of fructose, glucose, mannose, galactose, xylose, ribose, inositol, and sorbitol in F. japonica, which help to improve osmotic potential, enhance antioxidant ability and maintain the stability of plant cells during salt stress, could be indicative of their improved relative stress tolerance. This indicates that exogenous NO can enhance plant salt tolerance by upregulating sugar metabolism.
Previous studies have demonstrated that organic acids and amino acids play a role in stress tolerance by regulating energy supply, enhancing antioxidant defense, and acting as osmoprotectants in different plant species [65,66,67]. Recent studies have shown that plants require more energy produced by the TCA cycle to maintain redox homeostasis under NaCl stress, and exogenous NO aids the TCA cycle under salt conditions [23]. In this study, exogenous NO reversed the inhibition of the TCA cycle under salt stress and upregulated argininosuccinic acid and citrulline in the metabolic pathway (Table S1 and Figure 10). Interestingly, Petrack et al. (1956) reported that the conversion of argininosuccinic acid to citrulline is accompanied by ATP formation [67], suggesting that NO-induced improvement in plant salt tolerance may involve the mediation of both intermediates and non-intermediates of the TCA cycle to supply energy in F. japonica. Moreover, the accumulation of aspartic acid also increased in SNP-treated plants under salt stress in this study (Figure 9). Similar to proline and other amino acids, the accumulation of aspartic acid is associated with osmotic adjustment and membrane stabilization under salinity stress [68]. Based on the analysis presented above, the NO-induced accumulation of organic acids and amino acids in F. japonica contributes to energy supply, redox homeostasis, and osmotic adjustment, ultimately improving its salt resistance.

5. Conclusions

In summary, exogenous NO significantly alleviates the adverse effect of salt stress on the growth of F. japonica seedlings by maintaining high photosynthetic efficiency, increasing antioxidant enzyme activity, and promoting the accumulation of osmolytes. Furthermore, exogenous NO upregulates sugar metabolism and the metabolism of amino acid and organic acid under salt stress, including fructose, glucose, raffinose, mannose, galactose, xylose, ribose, inositol, sorbitol, and aspartic acid. The increased levels of these metabolites are crucial for providing osmotic protection, bolstering antioxidant capacity, supplying energy, and maintaining cell stability in F. japonica under salt stress. The results provide a theoretical basis for understanding the role of NO in conferring salt stress resistance, which could be beneficial for the protection, development, and utilization of F. japonica.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/f15010159/s1, Table S1: Different metabolites of major metabolic pathways in F. japonica leaves under NaCl treatment and exogenous SNP conditions.

Author Contributions

Conceptualization: X.H. (Xing Hu) and X.H. (Xiaoxia Huang); formal analysis: X.H. (Xing Hu) and M.Z.; investigation: X.H. (Xing Hu), M.Z. and J.L.; resources: X.H. (Xing Hu) and M.Z.; data curation: X.H. (Xing Hu), M.Z. and J.L.; writing—original draft preparation: X.H. (Xing Hu); writing—review and editing: X.C. and X.H. (Xiaoxia Huang); visualization: X.H. (Xiaoxia Huang); supervision: X.H. (Xiaoxia Huang); project administration: X.H. (Xiaoxia Huang). All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Outstanding Young Talents Support Program of Yunnan Province (YNWR-QNBJ-2020-222).

Data Availability Statement

The datasets used or analyzed during the current study are available from the corresponding author upon reasonable request.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Impact of NO treatment on the morphological changes in F. japonica seedlings under salt stress conditions. 0 DAT, plants before treatment. CK, control; NO, SNP treatment; S, NaCl treatment; S + NO, NaCl + SNP treatment (45 days after NaCl treatment).
Figure 1. Impact of NO treatment on the morphological changes in F. japonica seedlings under salt stress conditions. 0 DAT, plants before treatment. CK, control; NO, SNP treatment; S, NaCl treatment; S + NO, NaCl + SNP treatment (45 days after NaCl treatment).
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Figure 2. Impact of NO treatment on photosynthetic characteristics of F. japonica seedlings grown under salt stress conditions. (A) Pn, net photosynthetic rate. (B) Gs, stomatal conductance. (C) Tr, transpiration rate. (D) Ci, intracellular CO2 concentration. Data represent the mean ± SD (n = 3). Means followed by the same letters represent no significant differences between different treatments (p < 0.05). CK, control; NO, SNP treatment; S, NaCl treatment; S + NO, NaCl + SNP treatment; (45 days after NaCl treatment).
Figure 2. Impact of NO treatment on photosynthetic characteristics of F. japonica seedlings grown under salt stress conditions. (A) Pn, net photosynthetic rate. (B) Gs, stomatal conductance. (C) Tr, transpiration rate. (D) Ci, intracellular CO2 concentration. Data represent the mean ± SD (n = 3). Means followed by the same letters represent no significant differences between different treatments (p < 0.05). CK, control; NO, SNP treatment; S, NaCl treatment; S + NO, NaCl + SNP treatment; (45 days after NaCl treatment).
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Figure 3. Impact of NO treatment on chlorophyll fluorescence in F. japonica seedlings grown under salt stress conditions. (A) F0, the initial fluorescence. (B) Fv/Fm, the maximum photochemical quantum yield of PSII. (C) YII, the actual photochemical quantum yield of PSII. (D) NPQ, the non-photochemical quenching coefficient. (E) qP, the photochemical quenching coefficient. (F) ETR, the electron transport rate of PSII. Data represent the mean ± SD (n = 3). Means followed by the same letters represent no significant differences between different treatments (p < 0.05). CK, control; NO, SNP treatment; S, NaCl treatment; S + NO, NaCl + SNP treatment; (45 days after NaCl treatment).
Figure 3. Impact of NO treatment on chlorophyll fluorescence in F. japonica seedlings grown under salt stress conditions. (A) F0, the initial fluorescence. (B) Fv/Fm, the maximum photochemical quantum yield of PSII. (C) YII, the actual photochemical quantum yield of PSII. (D) NPQ, the non-photochemical quenching coefficient. (E) qP, the photochemical quenching coefficient. (F) ETR, the electron transport rate of PSII. Data represent the mean ± SD (n = 3). Means followed by the same letters represent no significant differences between different treatments (p < 0.05). CK, control; NO, SNP treatment; S, NaCl treatment; S + NO, NaCl + SNP treatment; (45 days after NaCl treatment).
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Figure 4. Impact of NO treatment on the contents of chlorophyll, carotenoids, and starch in F. japonica seedlings grown under salt stress conditions. (A) Chla, chlorophyll a. (B) Chb, chlorophyll b. (C) Carotenoid. (D) Starch. Data represent the mean ± SD (n = 3). Means followed by the same letters represent no significant differences between different treatments (p < 0.05). CK, control; NO, SNP treatment; S, NaCl treatment; S + NO, NaCl + SNP treatment; (45 days after NaCl treatment).
Figure 4. Impact of NO treatment on the contents of chlorophyll, carotenoids, and starch in F. japonica seedlings grown under salt stress conditions. (A) Chla, chlorophyll a. (B) Chb, chlorophyll b. (C) Carotenoid. (D) Starch. Data represent the mean ± SD (n = 3). Means followed by the same letters represent no significant differences between different treatments (p < 0.05). CK, control; NO, SNP treatment; S, NaCl treatment; S + NO, NaCl + SNP treatment; (45 days after NaCl treatment).
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Figure 5. Impact of NO treatment on MDA and osmolyte content in F. japonica seedlings grown under salt stress. (A) MDA, malondialdehyde. (B) Proline. (C) Soluble sugar. (D) Soluble protein. Data represent the mean ± SD (n = 3). Means followed by the same letters represent no significant differences between different treatments (p < 0.05). CK, control; NO, SNP treatment; S, NaCl treatment; S + NO, NaCl + SNP treatment; (45 days after NaCl treatment).
Figure 5. Impact of NO treatment on MDA and osmolyte content in F. japonica seedlings grown under salt stress. (A) MDA, malondialdehyde. (B) Proline. (C) Soluble sugar. (D) Soluble protein. Data represent the mean ± SD (n = 3). Means followed by the same letters represent no significant differences between different treatments (p < 0.05). CK, control; NO, SNP treatment; S, NaCl treatment; S + NO, NaCl + SNP treatment; (45 days after NaCl treatment).
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Figure 6. Impact of NO treatment on antioxidative enzymes activity in F. japonica seedlings grown under salt stress. (A) CAT, catalase activity. (B) POD, peroxidase activity. (C) APX, ascorbate peroxidase activity. (D) SOD, superoxide dismutase activity. Data represent the mean ± SD (n = 3). Means followed by the same letters represent no significant differences between different treatments (p < 0.05). CK, control; NO, SNP treatment; S, NaCl treatment; S + NO, NaCl + SNP treatment; (45 days after NaCl treatment).
Figure 6. Impact of NO treatment on antioxidative enzymes activity in F. japonica seedlings grown under salt stress. (A) CAT, catalase activity. (B) POD, peroxidase activity. (C) APX, ascorbate peroxidase activity. (D) SOD, superoxide dismutase activity. Data represent the mean ± SD (n = 3). Means followed by the same letters represent no significant differences between different treatments (p < 0.05). CK, control; NO, SNP treatment; S, NaCl treatment; S + NO, NaCl + SNP treatment; (45 days after NaCl treatment).
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Figure 7. Heat map of changes in differential metabolites in the leaf of F. japonica at 45 days of treatment. Log2 FC is shown in the results: green, upregulation; red, downregulation. CK, control; S, NaCl treatment; N, SNP treatment; S+NO, NaCl + SNP treatment; (45 days after NaCl treatment).
Figure 7. Heat map of changes in differential metabolites in the leaf of F. japonica at 45 days of treatment. Log2 FC is shown in the results: green, upregulation; red, downregulation. CK, control; S, NaCl treatment; N, SNP treatment; S+NO, NaCl + SNP treatment; (45 days after NaCl treatment).
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Figure 8. Changes of total relative organic acids, amino acids and their derivatives, sugars and their derivatives, and other metabolites content in the leaf of F. japonica. Data represent mean ± SD (n = 3), and different letters represent significant differences between different treatments (p < 0.05). CK, control; NO, SNP treatment; S, NaCl treatment; S + NO, NaCl + SNP treatment; (45 days after NaCl treatment).
Figure 8. Changes of total relative organic acids, amino acids and their derivatives, sugars and their derivatives, and other metabolites content in the leaf of F. japonica. Data represent mean ± SD (n = 3), and different letters represent significant differences between different treatments (p < 0.05). CK, control; NO, SNP treatment; S, NaCl treatment; S + NO, NaCl + SNP treatment; (45 days after NaCl treatment).
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Figure 9. Impact of NO treatment on key differential metabolites under salt stress. Data represent mean ± SD (n = 3). Means followed by the same letters represent no significant differences between different treatments (p < 0.05). CK, control; NO, SNP treatment; S, NaCl treatment; S + NO, NaCl + SNP treatment; (45 days after NaCl treatment).
Figure 9. Impact of NO treatment on key differential metabolites under salt stress. Data represent mean ± SD (n = 3). Means followed by the same letters represent no significant differences between different treatments (p < 0.05). CK, control; NO, SNP treatment; S, NaCl treatment; S + NO, NaCl + SNP treatment; (45 days after NaCl treatment).
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Figure 10. Assignment of 38 metabolites to metabolic pathways. No change indicates no significant change; green indicates downregulation; red indicates upregulation.CK, control; S, NaCl stress; NO, SNP treatment; SN, NaCl + SNP treatment; (45 days after NaCl treatment).
Figure 10. Assignment of 38 metabolites to metabolic pathways. No change indicates no significant change; green indicates downregulation; red indicates upregulation.CK, control; S, NaCl stress; NO, SNP treatment; SN, NaCl + SNP treatment; (45 days after NaCl treatment).
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Figure 11. Principal component analysis (PCA) plots of F. japonica physiology and key metabolites for different treatments. CK, control; S, NaCl stress; NO, SNP treatment; S+NO, NaCl + SNP treatment; (45 days after NaCl treatment).
Figure 11. Principal component analysis (PCA) plots of F. japonica physiology and key metabolites for different treatments. CK, control; S, NaCl stress; NO, SNP treatment; S+NO, NaCl + SNP treatment; (45 days after NaCl treatment).
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Table 1. Influence of NO on stem length, root length, leaf length, leaf width, and leaf area index in seedlings of F. japonica plants subjected to salt stress for 45 days.
Table 1. Influence of NO on stem length, root length, leaf length, leaf width, and leaf area index in seedlings of F. japonica plants subjected to salt stress for 45 days.
TreatmentsStem Length (cm)Root Length (cm)Leaf Length (cm)Leaf Width (cm)Leaf Area (cm2)
CK7.17 ± 0.87 a20.43 ± 3.7 a15.17 ± 2.05 a21.8 ± 2.63 a201.76 ± 21.9 a
NO5.23 ± 0.87 b21.7 ± 3.33 a13.47 ± 0.15 a19 ± 0.2 b129.83 ± 9.27 b
S4.37 ± 0.15 b15.23 ± 0.87 a9.07 ± 0.9 b12.47 ± 0.76 c70.44 ± 8.66 c
S + NO5.4 ± 0.3 b19.83 ± 5.43 a12.97 ± 0.15 a17.6 ± 0.44 b125.65 ± 1.04 b
Sig**ns********
F-value10.11.715.823.854.3
CV (%)21.621.019.921.037.8
Note: Data represent the mean ± SD (n = 3). Means followed by the same letters represent no significant differences between different treatments (p < 0.05). CK, control; NO, exogenous SNP treatment; S, NaCl treatment; S + NO, NaCl + SNP treatment; (45 days after NaCl treatment). Sig, significance (ns, no significance; ** p < 0.01; *** p < 0.001); CV, coefficient of variation.
Table 2. Influence of NO on the biomass of root, shoot, total dry, and total fresh weight of whole plant index in seedlings of F. japonica plants subjected to salt stress for 45 days.
Table 2. Influence of NO on the biomass of root, shoot, total dry, and total fresh weight of whole plant index in seedlings of F. japonica plants subjected to salt stress for 45 days.
TreatmentsShoot Biomass (g)Root Biomass (g)Total Dry Biomass (g)Total Fresh Biomass (g)Root/Shoot
CK5.73 ± 0.32 a1.73 ± 0.21 a7.46 ± 0.53 a29.58 ± 2.18 a0.3 ± 0.02 ab
NO5.25 ± 0.61 ab1.73 ± 0.1 a6.98 ± 0.56 a27.7 ± 2.93 a0.33 ± 0.05 a
S4.03 ± 0.22 c0.98 ± 0.13 b5.01 ± 0.16 c20.76 ± 0.85 b0.24 ± 0.05 b
S + NO4.69 ± 0.08 bc1.24 ± 0.09 b5.92 ± 0.17 b23.58 ± 1.35 b0.26 ± 0.01 ab
Sig**********ns
F-value12.222.522.512.03.5
CV (%)14.825.316.515.716.4
Note: Data represent the mean ± SD (n = 3). Means followed by the same letters represent no significant differences between different treatments (p < 0.05). CK, control; NO, exogenous SNP treatment; S, NaCl treatment; S + NO, NaCl + SNP treatment; (45 days after NaCl treatment). Sig, significance (ns, no significance; ** p < 0.01; *** p < 0.001); CV, coefficient of variation.
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Hu, X.; Zhang, M.; Liu, J.; Cheng, X.; Huang, X. Metabolomics and Physiological Changes Underlying Increased Tolerance to Salt Stress Induced by Applied Nitric Oxide in Fatsia japonica Seedlings. Forests 2024, 15, 159. https://doi.org/10.3390/f15010159

AMA Style

Hu X, Zhang M, Liu J, Cheng X, Huang X. Metabolomics and Physiological Changes Underlying Increased Tolerance to Salt Stress Induced by Applied Nitric Oxide in Fatsia japonica Seedlings. Forests. 2024; 15(1):159. https://doi.org/10.3390/f15010159

Chicago/Turabian Style

Hu, Xing, Min Zhang, Jiao Liu, Xiaomao Cheng, and Xiaoxia Huang. 2024. "Metabolomics and Physiological Changes Underlying Increased Tolerance to Salt Stress Induced by Applied Nitric Oxide in Fatsia japonica Seedlings" Forests 15, no. 1: 159. https://doi.org/10.3390/f15010159

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