Exogenous nitric oxide alleviates sulfur deficiency-induced oxidative damage in tomato seedlings

Exogenous nitric oxide alleviates sulfur deficiency-induced oxidative damage in tomato seedlings

Journal Pre-proof Exogenous nitric oxide alleviates sulfur deficiency-induced oxidative damage in tomato seedlings Manzer H. Siddiqui, Saud Alamri, Qa...

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Journal Pre-proof Exogenous nitric oxide alleviates sulfur deficiency-induced oxidative damage in tomato seedlings Manzer H. Siddiqui, Saud Alamri, Qasi D. Alsubaie, Hayssam M. Ali, M. Nasir Khan, Abdullah Al-Ghamdi, Abdullah A. Ibrahim, Abdullah Alsadon PII:

S1089-8603(19)30210-1

DOI:

https://doi.org/10.1016/j.niox.2019.11.002

Reference:

YNIOX 1946

To appear in:

Nitric Oxide

Received Date: 10 July 2019 Revised Date:

5 November 2019

Accepted Date: 6 November 2019

Please cite this article as: M.H. Siddiqui, S. Alamri, Q.D. Alsubaie, H.M. Ali, M.N. Khan, A. Al-Ghamdi, A.A. Ibrahim, A. Alsadon, Exogenous nitric oxide alleviates sulfur deficiency-induced oxidative damage in tomato seedlings, Nitric Oxide (2019), doi: https://doi.org/10.1016/j.niox.2019.11.002. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.

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Exogenous nitric oxide alleviates sulfur deficiency-induced oxidative damage in tomato seedlings Manzer H. Siddiquia*, Saud Alamria, Qasi D. Alsubaiea, Hayssam M. Alia, M. Nasir Khanb, Abdullah Al-Ghamdia, Abdullah A. Ibrahimc, Abdullah Alsadonc a

Department of Botany and Microbiology, College of Science, King Saud University, P.O. Box 2455,

Riyadh 11451, Saudi Arabia b

Department of Biology, Faculty of Science, College of Haql, University of Tabuk, 71491 Tabuk,

Saudi Arabia c

Department of Plant Production, College of Food and Agricultural Sciences, King Saud University,

P.O. Box 2460, Riyadh, 11451, Saudi Arabia

Corresponding: *e-mail: [email protected], [email protected]

Abstract: Despite numerous reports on the role of nitric oxide (NO) in regulating plants growth and mitigating different environmental stresses, its participation in sulfur (S) -metabolism remains largely unknown. Therefore, we studied the role of NO in S acquisition and S-assimilation in tomato seedlings under low S-stress conditions by supplying NO to the leaves of S-sufficient and S-deficient seedlings. Sstarved plants exhibited a substantial decreased in plant growth attributes, photosynthetic pigment chlorophyll (Chl) and other photosynthetic parameters, and activity of enzymes involved in Chl biosynthesis (δ-aminolevulinic acid dehydratase), and photosynthetic processes (carbonic anhydrase and RuBisco). Also, S-deficiency enhanced reactive oxygen species (ROS) (superoxide and hydrogen peroxide) and lipid peroxidation (malondialdehyde) levels in tomato seedlings. Contrarily, foliar supplementation of NO to S-deficient seedlings resulted in considerably reduced ROS formation in leaves and roots, which alleviated low S-stress-induced lipid peroxidation. However, exogenous NO enhanced proline accumulation by increasing proline metabolizing enzyme (∆1-pyrroline-5carboxylate synthetase) activity and also increased NO, hydrogen sulfide (a gasotransmitter small signaling molecule) and S uptake, and content of S-containing compounds (cysteine and reduced glutathione). Under S-limited conditions, NO improved

S utilization efficiency of plants by

upregulating the activity of S-assimilating enzymes (ATP sulfurylase, adenosine 5-phosphosulfate reductase, sulfide reductase and O-acetylserine (thiol) lyase). Under S-deprived conditions, improved S-assimilation of seedlings receiving NO resulted in improved redox homeostasis and ascorbate content through increased NO and S uptake. Application of 2-(4-carboxyphenyl)-4,4,5,5tetramethylimidazoline-1-oxy l-3-oxide (an NO scavenger) invalidated the effect of NO and again

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2 caused low S-stress-induced oxidative damage, confirming the beneficial role of NO in seedlings under S-deprived conditions. Thus, exogenous NO enhanced the tolerance of tomato seedlings to limit S-triggered oxidative stress and improved photosynthetic performance and S assimilation. Keywords: S-deficiency,

nitric oxide, hydrogen sulfide, sulfur assimilation, photosynthesis,

proline metabolism

1.

Introduction

Sulfur (S) -deficiency is one of the limiting factors that produces poor food quality and reduces yield of agricultural crops. Worldwide, an increasing S deficiency leads to a drastic deterioration of soil fertility. There are many reasons that cause S deficiency, such as (1) reduction of atmospheric sulfur dioxide caused by various industrial sources and power plants [1], (2) application of inorganic fertilizers without S, (3) low levels of organic manures use, (4) limited use of S-containing fungicides and pesticides, (5) large amount of S leaching out from soil through rain water, and (5) intensive cultivation of high yielding hybrid crops [1; 2; 3; 4]. According to the Sulfur Institute, Washington D.C., S deficiency in the soil has become a global threat to agricultural food production. S deficiency in soil and yield loss of S-deficient crops have increasingly been recorded over the last five decades [5]. However, because of the demand for profitable performance of crop, today’s challenge lies in maximizing the amount of fertilizers used. Therefore, soil fertility and exploitation of new techniques have become an essential cause of concern for sustainable agricultural yield in the new era. Also, it is very important to pay attention to optimizing S-use-efficiency in plants. As an essential nutrient, S is required at 0.1–1.0% (on a dry weight basis) for healthy growth and normal metabolic processes in plants. Reduced S plays a vital role in the function of cofactors (acetyl coenzyme A, thiamine, biotin, and lipoic acid). It is an active constituent of amino acids, proteins, and lipids, and vital for biosynthesis of photosynthetic pigments and regulation of essential enzymes, and vitamins in plants. S-starvation not only reduces agricultural food production but also threatens to human nutrition because organic S in our diets comes from plants. S deficiency, which results in a decrease in the levels of S-containing compounds, such as methionine, glutathione (GSH), phytochelatins, cysteine (Cys), hydrogen sulfide (H2S), proteins and various secondary metabolites, is evidently linked to a reduced tolerance of plants to abiotic stress [6; 7; 8; 9]. In the S assimilation pathway, S is incorporated into Cys that is metabolized to methionine or directly involved in biosynthesis of various proteins and GSH, and is apparently crucial for plant growth, development and plant stress resistance [7; 10]. With this consideration in mind, it is extremely important to comprehend the physiological and biochemical processes of plants response under increasing S-starvation conditions. Earlier studies have revealed that a well characterized signaling molecule nitric oxide (NO) not only influences plants growth and development but also acts as a scavenger of reactive oxygen species (ROS) and upregulates many genes involved in various physiological mechanisms, such as signal transduction, cell death, stress resistance, and nutrients uptake [11; 12; 13; 14]. The beneficial

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3 role of NO starts from the beginning of germination and continues to flowering, fruits ripening and senescence [11; 12; 15; 16]. In the past few years, several studies have been conducted and large number of research papers have been published revealing that NO has a beneficial role in normal growth and tolerance of plants to stresses. It is well established that NO abates the harmful effects of various environmental stresses by activating biosynthesis of thiol compounds, such as Cys, GSH and phytochelatins, indicating potential involvement of NO in S assimilation pathways. Despite the substantial development in S research in relation to plants, the association of NO in the regulation of S uptake and assimilation, particularly under S-deficiency remains largely unrevealed. Evidence from recent studies exhibited that S and NO have interactive role in plants via H2S that plays multiple roles and acts as a crucial signaling molecule involved in plant response to different environmental stress [17]. Under different abiotic stresses, exogenous sodium hydrosulfide (an H2S donor) has been observed to improve tolerance by inducing NO generation in plants [18; 19]. Proline (Pro), as a proteinogenic amino acid, is accumulated as a beneficial organic solute in plants under both stress and not-stress conditions. Under stress, the accumulation of Pro relative to other amino acids is fast and frequent to maintain osmotic balance and also regulate protein synthesis in plants [20; 21]. Pro is different from other amino acids owing to its unique structure and specific chemical properties, such as zwitterionic nature, high compatibility with internal cellular environment, neutral pH, and extreme solubility [20; 22]. It is evident from the findings of recent studies that Pro participates in growth, development, and cell differentiation throughout life span of plants by regulating many proteins in cell wall [20]. The accumulation of Pro is not affected much under S deficiency stress but increased under abiotic stress possibly because of the non-S mechanisms [23]. It reveals that plants develop an alternate mechanism of defense for biosynthesis of Pro in plant under adverse environments. Also, as we know that S-rich peptides (such as GSH) and lowmolecular-weight nitrogenous and proteinogenic amino acids or osmoprotectants (such as Pro and Cys) are synthesized to help the plants to survive under different environmental conditions [24; 25; 26]. However, both GSH and Pro are synthesized in plants from the same precursor i.e., L-glutamate [27]. Nevertheless, as no such efforts have been made to reveal the intricacies of potential metabolic relationships among Pro, GSH, and NO induction under S-deficiency conditions. However, few studies have been conducted on the influence of NO signaling on Pro and H2S in plants under low-S stress. Previously, we showed that NO abates the harmful effects of osmotic stress by enhancing Pro, glycine betaine and endogenous H2S in wheat by up-regulating enzymes related to H2S biosynthesis and antioxidant system [28]. Tomato (Solanum lycopersicum L.) is one of the species belonging to the family Solanaceae and is widely grown and consumed across the world. It is the most favored and consumed vegetable after potato owing to its chemical properties. Tomato has abundant nutrients, antioxidant (lycopene), phenolics compounds, β-carotene, and vitamin C content [29]; therefore tomato products

are

extensively used in human diet [30]. However, tomato yield is decreased owing to the imbalance in

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4 nutrient supply and unfavorable climatic conditions [31]. Despite ample research on the role of NO in abiotic stress tolerance, little or no efforts have been made to study how NO works in S-deprived plants and affects various physiological and biochemical parameters of tomato plants. The present study was therefore planned to investigate the role of NO on plant growth and regulation of enzymes related to S and Pro metabolism, H2S, and photosynthesis under limited S regimes. 2.

Material and Methods

2.1

Plant materials and culture conditions To test the given hypothesis, a sand culture pot experiment was conducted on tomato (cultivar

‘Five Star’) seedlings in a growth chamber (conditions: 250 µmol of photons m−2 s−1 light intensity, 16-h photoperiod, and 25 ± 2 °C temperature). Healthy seeds were sterilized with 0.1% HgCl2 and sown in plastic pots containing vermiculite and perlite (mixed in equal ratios).

One-week old

seedlings were transferred to plastic pots containing acid washed sand and perlite (1:1 ratio) moistened with nutrients solution, in which limited-S ( SO4; 1.5 me L-1) and optimum-S (25 me L-1) were added. Nutrients solution and concentrations of both S doses used in the present experiment were based on the earlier experiment conducted by Cerda et al. [32]. Five days after seedlings transfer, foliar application of NO [100 µM of SNP: (Na2 [Fe (CN)5NO].2H2O was used as an NO donor] with or without cPTIO [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxy l-3-oxide: an NO scavenger] was applied to the leaves of the experimental seedlings every three days up to twenty-five days. Optimum dose of S was considered as the control treatment. The dose of NO was based on our earlier findings [13]. Two tomato seedlings in each pot were maintained in each pot. After 25 days, samples were collected for physiological and biochemical analysis and also tomato seedlings were uprooted for the measurement of morphological parameters. 2.2

Growth attributes of tomato seedlings The growth characteristics of 25-days-old seedlings were measured in terms of root length

(RL), shoot length (SL), root fresh weight (RFW), shoot fresh weight (SFW), root dry weight (RDW) and shoot dry weight (SDW). 2.3

Histochemical detection of ROS in leaves We detected superoxide (O2•−) and hydrogen peroxide (H2O2) in tomato leaves according to

the method of Wang et al. [33] and Mostofa and Fujita [34], respectively. Leaves of the experimental seedlings were incubated for 12-hrs in 0.1% nitro blue tetrazolium (NBT) and 1% 3,3diaminobenzidine (DAB) solutions for the detection of O2•− and H2O2, respectively. A blue insoluble formazan for O2•− and a deep brown polymerization product for H2O2 were visualized by boiling leaves in bleaching solution [glycerol, acetic acid and ethanol (1:1:4)]. 2.4

Histochemical detection of ROS in roots We followed the method of Rodriguez-Serrano et al. [35] to visualize O2•− and H2O2 in roots

of each treated tomato seedling using fluorescence probes

dihydroethidium (DHE) and 2′,7′-

dichlorofluorescein diacetate (DCF-DA), respectively. To capture signals of DCF-DA and DHE, a

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5 fluorescence microscope (Eclipse Ni-U, Nikon, Tokyo, Japan) was used. The relative fluorescent intensity in roots of treated seedlings of the fluorescent images was analyzed using ImageJ 2.0.0-rc69/1.52p 2.5

Determination of H2O2 and O2•− and malondialdehyde (MDA) content in leaves The content of H2O2 and O2•− in leaves of each treated seedling was determined according to

the methods described by Velikova et al. [36] and Elstner and Heupel [37], respectively. The absorbance of the samples was read at 390 nm for H2O2 and 530 nm for O2•−. Fresh leaves of tomato seedlings were collected. The content of MDA in leaves was estimated colorimetrically according to the method of Dhindsa et al. [38]. The MDA concentration in the supernatant was measured by recording absorbance at 600, 532, and 450 nm, and expressed as nmole g-1 fresh weight (FW). 2.6

Determination of photosynthetic pigment and photosynthetic parameters in leave To measure chlorophyll (SPAD) in experimental seedlings, leaves were clipped with the

SPAD meter sensor (SPAD 402 PLUS chlorophyll meter, Minolta, Japan) and values were noted. Net photosynthesis rate (PN), intercellular carbon dioxide (CO2) concentration (Ci) and stomatal conductance (gS) were determined in the fully expanded topmost leaves of tomato seedlings using an infrared gas analyzer (LI-64000XT, Portable Photosynthesis System, LI-COR, NA, USA). 2.7

Determination of photosynthesis and chlorophyll (Chl) biosynthesis enzymes activity in leaves To determine the activity of carbonic anhydrase (CA) and RuBisco, fresh leaf samples were

collected from each seedling. The methods of Dwivedi and Randhawa [39], and Usuda [40] were adopted to measure the activity of CA and RuBisco, respectively. The activity of CA and RuBisco was expressed as µMol (CO2) kg−1 leaf-FW s−1 and nmol CO2 fixed mg−1 protein min−1, respectively. The activity of δ-aminolevulinic acid dehydratase (δ‐ALAD) was estimated colorimetrically by determining the amount of porphobilinogen (PBG) produced after extraction of the enzyme. One unit of δ‐ALAD activity was defined as the amount of enzyme needed to change 2 nM of δ‐ALA into 1 nM of PBG formed per hour. To estimate the PBG amount, the absorbance was recorded at 553 nm at 15 min against zero time control [41]. 2.8

Determination of proline content and its metabolizing enzyme We followed the method of Bates et al. [42] to measure Pro content in leaves of seedlings. The

content of Pro was estimated at the absorbance of 528 nm using L-Pro as a standard. In order to determine the activity of ∆1-pyrroline-5-carboxylate synthetase (P5CS) involved in Pro synthesis, the extraction was performed following the method of Sumithra et al. [43] and assayed according to the method described by Charest and Phan [44]. The activity of P5CS was measured by monitoring the rate of consumption of NADPH indicated by the decrease in absorbance at 340 nm. 2.9

Determination of ascorbate (ASC) and GSH content, and redox sate

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6 In order to estimate ASC, GSH and oxidized glutathione (GSSG) content, leaf tissue (0.25 g) was homogenized in a mixture of 2% (m/v) metaphosphoric acid and 2 mM EDTA) and thereafter centrifuged at 13,000 rpm for 15 min at 4 °C. The supernatant was collected and stored for the determination of these non-enzymatic antioxidants. The method of Takahama and Oniki [45] was followed to measure the content of ASC in leaves with slight modifications, as described by Turcsányi et al. [46]. Postassium phosphate buffer (100 mM; pH 6.1) was used to neutralize the supernatant (50 µL) and initial absorbance for ASC content was then assessed spectrophotometrically at 265 nm, followed by a second reading taken using one unit of ascorbate oxidase. The estimation of GSH and oxidized glutathione (GSSG) content was carried out according to the method of Yu et al. [47] with modifications as described by Paradiso et al. [48]. Reduced GSH was determined by an enzyme recycling process, in which it was consecutively oxidized by 5, 5´dithiobis-2-nitrobenzoic acid and reduced by NADPH in the presence of glutathione reductase. GSSG was assayed by adding 2-vinylpyridine to remove GSH, whereas H2O2 was added for the GSH assay. The details for the estimation of GSH and GSSG have been explained in Siddiqui et al. [49]. 2.10 Determination of NO, H2S, and S content We followed the methods described by Ding et al. [50] and Hu et al. [51] to estimate NO content in the leaves of tomato seedlings. Fresh leaves were collected and powdered in liquid nitrogen (LN) and 100 mg leaf powder was then homogenized in 3 mL buffer solution (50 mM acetic acid buffer pH 3.6 and 4% zinc diacetate) and centrifuged at 13,00 rpm at 4 °C for 15 min. To determine NO2− content using Griess method was used as described by Cantrel et al. [52]. The Griess reagent (1 mL) was added to 1 mL filtrate and vortexed thoroughly. After incubation for 30 min at room temperature, the absorbance of the mixture was read at 540 nm. The NO content was calculated by comparing with a standard curve of NaNO2. In order to estimate the concentration of H2S, fresh leaf tissues were powdered in LN with a mortar and pestle, and 300 mg of frozen leaf tissue powder was homogenized in potassium phosphate buffer (100 mM, pH 7.0) containing EDTA (10 mM). After centrifugation at 13,000 rpm at 4 °C for 20 min, the supernatant was collected to quantify H2S. The supernatant (100 µL) was added to the assay mixture [20 mL of 20 mM 5,5′-dithiobis(2-nitrobenzoic acid) and 1880 µL extraction buffer]. After incubation of the assay mixture at room temperature for 2 min, the absorbance was taken at 412 nm and H2S content was calculated from a calibration curve plotted using different known concentrations of NaHS as the standard. [53]. In order to estimate S content in the leaves of tomato seedlings, oven dried leaf powder (100 mg) was digested with di-acid mixture (HNO3 and HClO4 mixture; v:v = 3:1), and selenium dioxide (7.5 mg) was used as a catalyst. The resulting aliquot was collected and diluted to a constant volume and S content was determined by the turbidimetric method as described by Chesnin and Yien [54]. 2.10

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S-assimilation

7 We followed the method given by Lappartient and Touraine [55] to determine the activity of ATP sulfurylase (ATP-S) activity. The in vitro ATP-S activity was assayed in the leaves of tomato by determining molybdate-dependent formation of pyrophosphate. The details for the determination of ATP-S activity has been explained in Fatma et al. [56]. In order to determine to the activity of adenosine 5-phosphosulfate reductase (APS-R) and sulfide reductase (SiR), fresh leaves were used to extract protein using an extraction buffer [Trisacetate buffer (100 mM, pH 8.0) with sulfate (500 mM)]. We followed the detailed method of Brychkova et al. [57] and Brychkova et al. [58] to measure the activity of APS-R and SiR, respectively. The activity of O-acetylserine (thiol) lyase (OAST-L) and Cys content was determined by the methods of Gaitonde [59] as explained by Riemenschneider et al. [60] with a slight changes. The details for the determination of OAST-L activity and Cys content have been given earlier [17]. 2.11

Statistical analysis The experiment was laid down as a simple-randomized design with five replicates per

treatment. Each replicate had 2 two seedlings. The data were analyzed statistically using analysis of variance by SPSS-22 statistical software (SPSS Inc., Chicago, IL, USA), and expressed as treatment means ± standard error (n=5). Means were statistically compared by Duncan’s multiple range test at the p < 0.05% significant level. 3.

Results

3.1.

Plant growth improved by NO under low S conditions To investigate whether NO plays a significant role in the improvement of growth through the

regulation of S-assimilation in tomato seedlings under low S stress conditions, we applied SNP (an NO donor) to the tomato seedlings under low S stress and normal S (optimum S) conditions. The results presented in Table 1 exhibited that S-deficiency resulted in severe reduction in growth parameters, such as SL, RL, SFW, RFW, SDW and RDW (Fig. 1). As compared with the control, SL, RL, SFW, RFW, SDW and RDW were decreased by 47.86%, 54.07%, 53.70%, 68.04%, 50.77% and 72.22%, respectively when plants were grown under low S conditions. However, application of SNP, as an NO donor, abated the harmful effects of low S stress on growth attributes of tomato seedlings. The S-deficient-seedlings of tomato supplemented with SNP showed 86.77%, 86.59%, 104.12%, 164.51%, 68.75% and 160% higher values for SL, RL, SFW, RFW, SDW and RDW, respectively over the S-deficient-seedlings not treated with NO. On the other hand, application of cPTIO (an NO scavenger) along with NO reversed the effect on NO and confirmed its role in plant growth (Fig. 1, Table 1). Application of NO with S deficiency (SD)/or S enhanced seedlings growth characteristics. 3.2

Protection of plants from low S-stress–induced oxidative damage by NO To explore the efficacy of exogenous NO in the potential alleviation of low S stress-induced

oxidative damage, we assessed ROS formation, and MDA (an important biomarker of ROS-induced

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8 lipid peroxidation) content in leaves and root of tomato seedlings ( Figs. 2-4). To evaluate the visual effect of NO under S deprivation conditions on ROS formation in leaf and root of tomato seedlings, we used DAB and NBT to visualized in situ formation of H2O2 and O2•– in leaves, respectively, and DCF-DA and DHE fluorescence probes for the detection of H2O2 and O2•– in roots of tomato seedlings, respectively (Figs. 2 and 3). The results obtained from histochemical study reveal that in situ formation of ROS in leaves as well as roots was more in tomato seedlings grown in low S conditions than in the control (optimum S conditions). Also, biochemical quantification study clearly shows that content of H2O2, O2•– and MDA was the maximum in tomato seedlings when grown in S deficiency conditions (Fig. 4). The accumulation of H2O2, O2•–, and MDA was increased by 199.71%, 216.50% and 181.03%, respectively when tomato seedlings were grown under limited S conditions relative to that in the control. However, exogenous SNP suppressed the accumulation of H2O2, O2•– and ROS-induced lipid peroxidation (MDA) in both leaves and roots of tomato seedlings under low S stress. Conversely, addition of NO scavenger cPTIO in the growth medium confirmed the protective role of SNP

against ROS formation and ROS-induced lipid peroxidation by increasing the

accumulation of ROS and MDA as compared to SD and NO+SD. 3.3

Regulation of the activity of enzymes involved in Chl and photosynthesis by NO To recognize the beneficial role of NO in Chl biosynthesis and activity of photosynthetic

enzymes under sufficient and insufficient S conditions, we gauged Chl content, activity of δ‐ALAD, a precursor enzyme of Chl synthesis, and activity of enzymes involved in photosynthesis (CA and RuBisco) in the leaves of experimental seedlings of tomato under limited S conditions (Fig. 5 C-D). Under S-deprived conditions, the accumulation of Chl and activity of

δ‐ALAD was severely

affected and recorded lower values than the control. The content of Chl, and activity of δ‐ALAD decreased by 48.98% and 43.88%, respectively in low S-fed seedlings. Also, seedlings grown in low S media as compared to those grown in optimum S media exhibited the lowest activity of CA and RuBisco. Low-S stress suppressed the activity of CA and RuBisco by 68.94% and 50 %, respectively. However, addition of NO in growth medium containing low S increased Chl by 82.26%, and the activity of δ‐ALAD, CA, and RuBisco by 43.01%, 153.18%, and 57.97%, respectively as compared to low S-stress conditions. In the present study, these beneficial roles of NO were confirmed by adding cPTIO in the medium containing low S plus NO. We observed that cPTIO caused a decrease in Chl accumulation, and the activity of δ‐ALAD, CA and RuBisco. Thus, it indicates that NO scavenger cPTIO eliminated the ameliorating effect of NO under S deficiency conditions. 3.4

Photosynthetic parameters improved by NO

To explicate the potential role of NO in the improvement of photosynthetic parameters, we measured PN, gS and Ci under low S-stress (Fig. 6 A-C). The seedlings grown under S deficiency conditions exhibited reduced PN, gS and Ci as compared to control. As compared with control, PN, gS and Ci were decreased by 61.57%, 75.80% and 60.08%, respectively, when tomato seedlings were fed with low S. However, under the same limited S conditions, all these parameters were increased

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9 substantially when seedlings were exogenously treated with NO. The foliar spray of SNP increased PN, gS and Ci by 131.37%, 117.33%, and 89.29%, respectively under low S-stress conditions. Conversely, application of cPTIO with SNP considerably nullified the beneficial effects of SNP, resulting in the reduction of PN, gS and Ci. 3.5

NO induces Pro accumulation by regulating the activity of Pro biosynthesis enzyme In the present study, we investigated the effect of NO on the synthesis of Pro and activity of

Pro biosynthesizing enzyme (P5CS) actvity. The content of Pro and the activity of P5CS in seedlings fed with low S were lower than in the seedlings fed with optimum S. However, under limited S conditions, foliar spray of SNP enhanced Pro accumulation and P5CS activity (Fig. 7 A and B). Under S-deficiency conditions, foliar application of SNP increased Pro by 34.03% and activity of P5CS by 51.89% in comparison to S-deficient seedlings. Addition of cPTIO with SNP decreased the accumulation Pro and activity of P5CS, confirming the potential role of NO in the alleviation of low S-stress by improving Pro metabolism. 3.6

Accumulation of NO and H2S in leaves enhanced by NO To find the contribution of exogenous NO in the accumulation of NO and H2S in leaves of

tomato seedlings, we grew seedlings under S-deficient conditions and quantified the levels of NO and H2S biochemically. Under low S-stress conditions, the content of NO increased as compared to control. However, foliar application of NO increased NO and H2S content further under both Sdeprived and optimum conditions (Fig. 8 A and B). The maximum NO content was recorded under low S conditions when seedlings were subjected to NO. As compared to the control, the content of NO increased by 106.83% when seedlings were supplemented with NO. Under low S stress conditions, the tomato seedlings showed low levels of H2S as compared to control (Fig. 8 B). The content of H2S was increased when seedlings were treated with NO under limited S conditions. As compared to S deficient seedlings, leaves of seedlings treated with NO under S-deprived conditions showed content of H2S enhanced by 103.41%. The results confirmed that the inclusion of cPTIO with NO eliminated the enhancing effect of NO on the content of NO and H2S. 3.7

S uptake and biosynthesis of S-containing compound Cys enhanced by NO To elucidate whether the growth promoting efficiency of exogenous NO was associated with

S-use-efficiency of tomato seedlings under limited S conditions, we estimated S and Cys content (Fig. 8 C and D). The obtained results exhibited that seedlings grown under S deficiency conditions exhibited lower content of S and Cys as compared to control. Under low S-stress conditions, the content of S and Cys decreased by 79.05% and 47.71%, respectively as compared to normal S conditions. However, foliar application of NO was found to be markedly effective in improving the accumulation of S and the biosynthesis of Cys in leaves of S-deficient and normal S-treated seedlings of tomato (Fig. 8 C and D). The content of S and Cys in S-deprived seedlings increased by 163.48% and 113.88%, respectively as compared to optimum S-treated seedlings of tomato, when seedlings were supplemented with NO. Under low S conditions, the accumulation S and Cys biosynthesis were

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10 severely affected when seedlings were supplemented with cPTIO plus NO. Therefore, it can be postulated that NO was responsible for enhancing S uptake

and biosynthesis of S containing

compound, Cys. 3.8

Activity of S-metabolizing enzymes regulated by NO To reveal the function of NO in growth recovery of S-deficient tomato seedlings by

modulating the S-metabolizing enzymes in S assimilation, we examined the activity of ATP-R, ATPS, OAST-L, and SiR in leaves of seedlings under low S-stress conditions (Fig. 9 A-D). The obtained results reveal that the activity of ATP-R, ATP-S, OAST-L, and SiR was severely affected when seedlings were grown under limited S conditions as compared to normal conditions. The activity of APS-R, ATP-S, OAST-L and SiR markedly decreased by 60.61%, 64.58%, 45.70%, and 56.99%, respectively when seedlings were grown under low S stress conditions. However, foliar NO administration triggered the upregulation of the activity of these S-metabolizing enzymes in Sdeprived tomato seedlings. More precisely, the S-deprived tomato seedlings exhibited increased enzyme activity by 460% for APS-R, 156.37% for ATP-S, 111.32% for OAST-L and 395.38% for SiR when seedlings were supplemented with NO as compared to low S-treated seedlings. Therefore, we postulate that NO is one of the important biological molecules that could be responsible for further induction of S-assimilating enzymes; it was confirmed by the addition of NO scavenger cPTIO with NO in medium which diminished the efficiency of NO that resulted in a diminution in the activity of APS-R, ATP-S, OAST-L and SiR enzymes (Fig. 9 A-D). 3.9

GSH and ASC content and redox state (GSH/GSSG) improved by NO To understand whether exogenous NO-induced S-assimilation was associated with

the

changes in synthesis of GSH, ASC, and redox state under S-hrain we assessed the content of GSH and ASC, and GSH/GSSG in S-deprived tomato seedlings (Fig. 10 A-C). The seedlings grown in low S conditions exhibited lower values for GSH and ASC, and GSH/GSSG than the seedlings grown in optimum S levels. However, foliar application of NO increased GSH and ASC content, and GSH/GSSG under S-deficiency conditions (Fig. 10 A-C). As compared to S-deficient seedlings, the content of GSH and ASC, and redox state increased by 62.97%, 90.71% and 117.11%, respectively, when seedlings were subjected to NO under S-deprived conditions. Moreover, the inclusion of cPTIO with NO confirmed the beneficial role of NO by reversing the levels of GSH and ASC, and GSH/GSSG in low S-fed seedings. 4.

Discussion

The response of plants under adequate and inadequate nutrients supply can vary between species and individual plants. Insufficient nutrient supply seriously threatens to agricultural productivity and affects plant growth, development, and various physiological and biochemical mechanisms [6; 61]. Plant adaptation to low-S level stress is extremely complex, and soil S deficiency has become a serious global problem to the crop production owing to intensive cropping and leaching [6; 61; 62]. During the past 10 years, a large number of studies have revealed that NO boosts the plant growth and

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11 abiotic stress tolerance [12; 13; 63]. During the nutrient deficiency, NO is one of the important signaling components that is triggered by plants in response to the availability of the nutrient during the crop life cycle of the crop [63]. However, further relevant studies are to be needed to explore the function of NO in S assimilation and acquisition, especially under S starvation conditions. Interestingly, the present study clearly revealed that tomato seedlings had a significant response to exogenous NO under low level of S conditions. The obtained results exhibited that foliar application of NO resulted in stimulation of endogenous levels of NO in leaves, resulting in better growth, which could be responsible for enhanced NO-induced S uptake and assimilation, photosynthesis, Pro metabolism, and antioxidant system under limited S regimes (Table 1 and Figs. 1-10). It is well established that S, as an essential nutrient, plays a significant role in plant growth and development. Consequently, its deficiency causes several changes in plant growth and dry matter accumulation [7; 64]. Constant with the earlier reports, S-starved plants exhibited decreased plant growth attributes, such as SL, RL, SFW, RFW, SDW and RDW, whereas the foliar supply of NO to tomato seedlings showed a beneficial effect by improving growth parameters (Table 1 and Fig. 1). Under limited S conditions, a decrease in growth characteristics may be due to depression of root hydraulic conductivity and disturbance of nitrogen metabolism [7; 64]. However, exogenous NO alleviated low S-stress in tomato seedlings; it may be because of the enhanced S uptake, Chl synthesis, photosynthesis, and enzymes activity (Figs. 5 and 6). Also, NO-induced enhancement of seedlings growth attributes could be traced to the role of NO in cell growth, tissue differentiation, and accumulation of spermidine that also triggers cell division and formation of roots [13; 65; 66]. Therefore, we proposed that foliar application of NO ameliorated low S effects in tomato seedlings; it was confirmed by the application of NO scavenger cPTIO with NO, a sharp decrease in growth parameters was noted even in the presence of sufficient S supply (Table 1 and Fig. 1 ). The generation of ROS occurs owing to the imbalance in the supply of essential nutrients [7]. The deficiency of S alters the biosynthesis of S-containing compounds which leads to a metabolic oxidative burst in plant cells [7; 67]. The overproduction of ROS (H2O2 and O2•–) due to S-deficiency resulted in an increase in lipid peroxidation (MDA accumulation) (Figs. 2-4). From the foregoing talk, it is very clear that it is an important to limit the overproduction of ROS in leaves or to scavenge H2O2 and O2•– once produced. Interestingly, exogenous application of NO significantly improved tomato seedlings tolerance against low S-stress by scavenging ROS, resulting in a decrease in oxidative damage. The metabolic balance of ROS in plant cells may be due to the scavenging and multifunctional properties of NO which itself acts as an antioxidant and also regulates the various plant responses to a variety of stresses [68; 69]. Under nutrient deficiency conditions, NO is one of the common important signaling compounds that triggers the responses linked to nutrient homeostasis, which is regulated by its association with ROS, hormones and proteins [63]. Owing to the ability of NO to easily cross cellular boundaries and react quickly with ROS, thiols, and proteins, it gives a biochemical signal to regulate enzymes activity in plants, resulting in decreased oxidative

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12 damage (Fig. 4 A) [12; 70; 71]. Inclusion of cPTIO with NO confirmed the role of NO in the diminution of ROS under S deprivation conditions. In the present study, biosynthesis of Chl was substantially affected under S-deficiency stress (Fig. 5 A). This impairment in Chl accumulation might be due to downregulation of δ‐ALAD activity (a precursor enzyme for Chl synthesis) (Fig. 5 B). Under S-deficiency conditions, overproduction of ROS could be responsible for lipid peroxidation, and damage of reactions centers and enzymes activity [6; 72]. However, the foliar supply of NO resulted in a dramatic recovery in Chl accumulation in leaves of both S- sufficient and deficient tomato seedlings; it was further confirmed by the inclusion of cPTIO (Fig. 5 A). Owing to foliar application of NO, improved δ‐ALAD activity may be responsible for biosynthesis of Chl (Fig. 5 A and B). Under low levels of S, an increase in Chl accumulation may be due to the role of NO which is responsible for maintaining the development of the chloroplast by regulating iron homeostasis and also remodeling the thylakoid protein complex under S starvation conditions [73; 74; 75]. After nitrogen, phosphorus and potassium, S is a boon for plant growth and development, and triggers the photosynthetic process in plants. Consistent with earlier studies, S limitation resulted in a drastic change in activity of photosynthetic enzymes (RuBisco and CA) and other photosynthesis parameters (PN, gS, and Ci) (Figs. 5 C and D, 6 A-C); whereas, foliar supplementation of NO noticeably increased the activity of RuBisco and CA, and improved PN, gS, and Ci in tomato seedlings under S starvation conditions. Exogenous application of NO regulated gS; however, the result contradicts the finding of Mata and Lamattina [76] who reported that prolonged exogenous NO treatment induced stomatal closure. This result is consistent with the findings of earlier study of Sehar et al. [77] and Zimmer-Prados et al. [78] who reported that NO increased gS in plants. Also, regulation of gS depends on the concentrations of NO. Sakihama et al. [79] reported that application of NO at high concentrations caused stomatal opening. Also, an increase in gS might be due to the accumulation of H2S when tomato seedlings were subjected to NO [80]. Under S deficiency, a decreased in PN may be due to the decrease in the content of S and Chl in leaves (Figs. 5A and 9C). However, foliar application of NO increased RuBisco and CA activity, and both enzymes work in coordination with each other and showed a highly strong association with leaf photosynthesis. An increase in RuBisco activity may be due to the increase in Cys content when tomato seedlings were subjected to NO (Fig. 8 D) because a functional molecule of RuBisco contains Cys and methionine [81]. Owing to the application of NO, an increased CA activity might have helped various physiological mechanisms, such as ion exchange, acid-base homeostasis and diffusion of inorganic carbon within cells, and also reversible hydrogenation of CO2, thus providing its continuous supply to RuBisco. All these circumstances may explain the improved tolerance of tomato seedlings to Sstarvation stress. An increase in S content in leaves of tomato seedlings due to NO application might have increased photosynthetic CO2 uptake through stomatal conductance, resulting in increased photosynthesis (Figs. 6 A-C and 9C) [82].

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13 Pro as a proteinogenic amino acid, is not only involved in alleviation of different environmental stresses but also improves the plant growth and differentiation by triggering the cell wall proteins under both stressed and non-stressed conditions [20]. Pro accumulates in plant tissues under both conditions. Under stress, Pro accumulation contributes to buffering of cellular redox potential, subcellular structures stabilization, and scavenging of ROS [21]. As we know, S is an important constituent of many primary and secondary metabolites that help the plant for better growth and development. In this experiment, S starvation caused a significant decrease in Pro accumulation because of the downregulation of P5CS activity (Fig. 7 A and B). It may be due to the inhibition of nitrogen assimilation, and amino acid synthesis ( [7; 83]. However, application of NO markedly enhanced Pro by upregulating the enzyme P5CS activity; it may be due to the role of NO in upregulation of Pro synthesizing enzymes activity [P5CS and ornithine-δ-aminotransferase (OAT)] and suppression of Pro-degrading enzyme such as Pro dehydrogenase [16]. Both P5CS and OAT are important enzymes involved in the regulation of Pro biosynthesis through the glutamate pathway [16; 84]. Also, an increase in Pro biosynthesis due to application of NO which accelerates uptake of essential nutrients in plants (Fig. 8 C) [11]. Nitrogen and S in plants provoke a positive regulatory response on protein-amino acids; hence, it helps the plant growth and development [7]. Starvation of S produces deleterious effects on S assimilation that can’t be dissociated with general plant metabolism, and it is strongly related to the availability of other nutrients and carbohydrate metabolism [6]. Limited S in the medium caused reduced uptake of S and H2S accumulation (Fig. 8 B and C), while, the generation of NO was high in S-starved seedlings (8A), suggesting that low S-induced formation of NO was required to regulate S acquisition and assimilation which was seen in seedlings treated with NO that had improved the content of S and Cys by further increasing NO and H2S (Fig. 8 A-D). Foliar application of NO under limited S conditions increased S uptake; it may be due to increased root growth (Table 1 and Fig. 1) and root volume because NO acts like auxin and stimulates cell division and elongation, and increases adventitious root growth [71]. Also, an increase in H2S content in S-deficient seedlings treated with NO may be due to the action of NO in upregulation of OAST-L activity (Fig. 9 C) which might have increased Oacetylserine and sulfide, resulted in an increase in Cys and subsequently H2S under low S conditions (8 B and D). Under low levels of S, NO-treated tomato seedlings had higher levels of H2S, it may be due to the beneficial role of NO in the upregulation of H2S biosynthesizing enzymes, such as Lcysteine desulfhydrase and D-cysteine desulfhydrase [28]. An enhanced levels of H2S in the leaves of NO-treated tomato seedlings may be one of the reasons for improved physiological and biochemical processes. As we know that H2S is one of the S containing compounds which may have compensated S requirement of plants under low S conditions. Also, H2S is involved in the regulation of various transduction pathways, and the present study established the link between NO and H2S and revealed the mechanisms of NO and H2S -mediated signaling and tolerance to limited S stress conditions.

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14 Various primary and secondary metabolites are synthesized from sulfate, and Cys is the first organic product of S-metabolism produced through the assimilation of S to O-acetylserine in the presence of enzyme OAST-L. Being an important amino acid, it is incorporated into diverse proteins, and is a precursor of several S-containing compounds, such as antioxidants and hormones, vitamins, and enzymes cofactors [6; 17; 28; 85], which help plants perform normally. Under low S stress, seedlings exhibited low levels of Cys, whereas treated with NO seedlings exhibited increased Cys accumulation (Fig. 8 D). It may be due to NO-induced accumulation of S and upregulation of enzymes (APS-R, ATP-S, OAST-L, and SiR) activity involved in S-assimilation (Figs. 8C and 9 AD). As we mentioned above that the S metabolism can’t be separated from the general plant metabolism, as it initiates biosynthesis of S-containing macromolecules. Data of the present study exhibit that a decrease in S uptake in S-deficient seedlings resulted in a drastic downregulation of Sassimilating enzymes such as APS-R, ATP-S, OAST-L, and SiR (Fig. 9 A-D). However, foliar administration of NO under limited S conditions increased S content and NO that might have hastened the upregulation of APS-R, ATP-S, OAST-L and SiR enzymes participating in the Sassimilation pathway (Figs. 8C and 9 A-D). Moreover, the reduction in S accumulation led to a decreased activity of S-assimilating enzymes as confirmed by a substantial reduction in S compounds, such as Cys and GSH (Figs. 8 D and 10 A). It is very clear that levels of Cys and GSH were not well enough to counter the low S stress, therefore increased NO levels in NO-treated seedlings could be one of the reasons for upregulating the S-assimilating enzymes and generation of H2S (Figs. 8 A, B and 9 A-D). In the biology system, S is incorporated into S-defense compounds, such as H2S, elemental S, GSH, and phytochelatins which reduce oxidative damage by scavenging ROS under different adverse conditions. In the present study, low levels of accumulation of GSH and ASC, and redox state (GSH/GSSG) were recorded under limited S conditions, whereas foliar supplementation of NO increased the content of GSH and ASC, and redox state further (Fig. 10 A-C). Therefore, it is quite clear that NO influenced S-metabolism by regulating the NO formation in seedlings receiving NO under S-stress conditions. Innocenti et al. [86] studied on Medicago truncatula and reported that NO induces gene expression of γ-glutamylcysteine synthetase and GSH synthetase in S-metabolism and enhances the GSH production. Owing to the nature of NO which reacts with GSH and Snitrosoglutathione (GSNO) is formed which acts a reservoir of NO in plants [87]. Oxidized glutathione (GSSG) and NH3 are formed from stored GSNO, and the reaction is catalyzed by the enzyme, GSNO-reductase. The GSH-GSSG recycle could be maintained by NO through GSH pools and the ratio of GSH and GSSG. In an NADPH-dependent reaction, GSSG is reduced again to GSH in the presence of glutathione reductase enzyme. Therefore, we postulate that efficiency of exogenous NO in decreasing lipid peroxidation and ROS formation is also one of the reasons in the maintenance of higher S-assimilation and better plant growth under low S-stress conditions. Also, foliar application

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15 of NO increased ASC content in parallel with increased GSH resulting in improved S assimilation under S deprived conditions by improving the efficacy of the ASC-GSH pathway (Fig. 10). The present experiment demonstrated that NO and H2S -induced GSH and ASC levels in plants may have provided compensatory mechanism to plants by maintaining their balance during stress. Li et al. [88] reported that NO induced H2S-mediated alleviation of heavy metal toxicity in plants by modulating activity of antioxidant enzymes and their transcripts differentially.

Conclusion Until now, the regulatory function of NO in plants and plants response mediated by NO against various environmental stresses remained chiefly unidentified. Therefore, the present study reveals that foliar application of NO improved growth attributes of tomato seedlings and photosynthetic characteristics under low-S induced stress. Exogenous NO provided protection for photosynthetic pigments and organic molecules (Pro) against low S-induced oxidative membrane damage. It is postulated that NO acts as an intracellular signaling biomolecule and regulates the Smetabolic pathway that upregulates the uptake of S and S-containing compounds (Cys and GSH). Thus, the utilization of S and Cys in NO-treated seedlings under limited S conditions was found to be a vital factor in initiating the formation of gaseous-H2S generation and GSH. Also, NO alleviated the oxidative damage induced by ROS by maintaining the redox state and ASC-GSH cycle resulting in increased antioxidant system, and plant growth and development. This study offers an innovative approach to enhance the understanding of underlying mechanisms of NO-mediated regulation of Sacquisition and assimilation under low levels of S in soil. Therefore, future study should be focused on that how NO functions as an upstream signaling biomolecule with another gaseous transmitter signaling molecule, H2S, in S-assimilation pathways and production of primary and secondary metabolites by expressing different genes using detail physiological and molecular tools. The present study could be beneficial in increasing the plant growth, development, yield, and quality of crops, particularly of plants growing in S deficient soils. Acknowledgment The authors would like to thank the Deanship of Scientific Research at King Saud University for funding the Research Group No. RG-1439-041. The authors thank the Deanship of Scientific Research and RSSU at King Saud University for their technical support Conflict of Interests The authors declare that there is no conflict of interest AUTHOR CONTRIBUTIONS M.H.S conceived and designed experiment and made important contributions to the discussion and also prepared first draft of manuscript. S.A, A.A.I. M.H.S and Q.D.A assisted in the preparation of experimental pots and reagents and also in physiological and biochemical studies. H.M.A. A. A. and

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16 A. A. helped in statistical calculation and data analysis. M.N.K and S.A provided intellectual input, critical reading and editing of the manuscript References

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Table 1 Nitric oxide (NO) improves growth characteristics of tomato seedlings under low S-stress conditions. (SL – shoot length, RL – root length, SFW – shoot fresh weight, RFW – root fresh weight, SDW – shoot dry weight and RDW – root dry weight Treatments Parameters

dH2O

NO

NO+cPTO

SL (cm)

RL (cm)

SFW (g)

RFW (g)

SDW (g)

RDW (g)

S

34.06±2.61ab

20.14±0.88b

4.19±0.10b

0.97±0.03b

0.65±.03ab

0.18±.006b

SD

17.76±0.88c

09.25±0.62d

1.94±0.05c

0.31±0.02d

0.32±0.02c

0.05±0.003e

S

39.32±2.21a

27.58±0.99a

4.85±0.27a

1.26±0.03a

0.76±.03a

0.24±0.003a

SD

33.17±2.23b

17.26±0.45c

3.96±0.05b

0.82±0.02c

0.54±.04b

0.13±0.001d

S

31.35±1.66b

18.51±0.39bc

4.10±0.07b

0.77±0.04c

0.62±.04b

0.16±0.005c

SD

17.47±0.52c

08.62±0.23d

1.84±0.07c

0.27±0.08d

0.28±.04c

0.05±0.003e

Average of five determinations are presented with ± SE (n = 5). Data of each column followed by different letter are significantly different by at P < 0.05. . S-optimum sulfur, SD-sulfur deficiency, NO-nitric oxide, cPTIO-2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxy l-3oxide (an NO scavenger)

Figure 1 Foliar application of NO improves tomato seedlings growth under low sulfur (S) stress conditions. S-optimum sulfur, SD-sulfur deficiency, NO-nitric oxide, cPTIO-2(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxy l-3-oxide (an NO scavenger)

Figure 2 In situ visualization of (A) O2•− production in leaves using NBT staining and (B) H2O2 generation in leaves using DAB staining under S-defeciency conditions. Soptimum sulfur, SD-sulfur deficiency, NO-nitric oxide, cPTIO-2-(4-carboxyphenyl)4,4,5,5-tetramethylimidazoline-1-oxy l-3-oxide (an NO scavenger)

. O2 − DHE fluorescence (pixel intensity)

40

C a b

30

c

d

20

e

e f

10

0 S

SD

S

SD

dH2O

S+cPTIO SD+cPTIO SD+TMP +NO

H2O2 DCF-DA Fluorescence (pixel intensity)

Treatments

60

D

a a

50

40

b

30

c

c

20

d 10

e

0 S

SD dH2O

S

SD

S+cPTIO SD+cPTIO SD+ASC +NO

Treatments

Figure 3 Nitric oxide assuages sulfur deficiency stress-induced endogenous ROS generation in tomato seedlings. (A) superoxide (O2•−) formation (O2•−-dependent DHE fluorescence) in root, (B) hydrogen peroxide (H2O2) generation (H2O2-dependent DCF-DA fluorescence) in root, (C) DHE fluorescent pixel intensity was calculated corresponding to the images obtained from (A) and (D) DCF-DA fluorescence pixel intensity was calculated corresponding to the images obtained from (B). For negative controls, roots of Sdeficient seedlings treated with NO preincubated with tetramethyl piperidinooxy (1 mM TMP), an O2•− scavenger and ascorbate (1 mM ASC), a H2O2 scavenger. S-optimum sulfur, SD-sulfur deficiency, NO-nitric oxide, cPTIO-2-(4-carboxyphenyl)-4,4,5,5tetramethylimidazoline-1-oxy l-3-oxide (an NO scavenger)

Figure 4 Foliar application of NO attenuates limited S stress-induced accumulation of (A) malondialdehyde (MDA), (B) hydrogen peroxide (H2O2) and (C) superoxide (O2•−) in leaves of tomato seedlings. Bars followed by the same letter do not differ significantly at P < 0.05 according to Duncan Multiple Range Test. Average of five determinations are presented with bars indicating ± SE. S-optimum sulfur, SDsulfur deficiency, NO-nitric oxide, cPTIO-2-(4-carboxyphenyl)-4,4,5,5tetramethylimidazoline-1-oxy l-3-oxide (an NO scavenger)

Figure 5 Foliar application of NO enhances (A) chlorophyll (Chl), (B) δ-aminolevulinic acid dehydratase (δ-ALAD) activity, (C) RuBisco activity and (D) carbonic anhydrase (CA) activity in tomato seedlings. Bars followed by the same letter do not differ significantly at P < 0.05 according to Duncan Multiple Range Test. Average of five determinations are presented with bars indicating ± SE. S-optimum sulfur, SD-sulfur deficiency, NO-nitric oxide, cPTIO-2-(4-carboxyphenyl)-4,4,5,5tetramethylimidazoline-1-oxy l-3-oxide (an NO scavenger)

Figure 6 Foliar application of NO enhances (A) net photosynthesis (PN), (B) stomatal conductance (gS) and (C) intercellular CO2 concentration (Ci) in leaves of tomato seedlings. Bars followed by the same letter do not differ significantly at P < 0.05 according to Duncan Multiple Range Test. Average of five determinations are presented with bars indicating ± SE. S-optimum sulfur, SD-sulfur deficiency, NOnitric oxide, cPTIO-2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxy l-3oxide (an NO scavenger)

Figure 7 Foliar application of NO enhances (A) proline (Pro) content and (B) Δ1-pyrroline-5carboxylate synthetase (P5CS) in leaves of tomato seedlings. Bars followed by the same letter do not differ significantly at P < 0.05 according to Duncan Multiple Range Test. Average of five determinations are presented with bars indicating ± SE. S-optimum sulfur, SD-sulfur deficiency, NO-nitric oxide, cPTIO-2-(4-carboxyphenyl)4,4,5,5-tetramethylimidazoline-1-oxy l-3-oxide (an NO scavenger)

Figure 8 Foliar application of NO enhances the accumulation of (A) nitric oxide (NO), (B) hydrogen sulfide (H2S), (C) sulfur (S) and cysteine (Cys) in leaves of tomato seedlings. Bars followed by the same letter do not differ significantly at P < 0.05 according to Duncan Multiple Range Test. Average of five determinations are presented with bars indicating ± SE. S-optimum sulfur, SD-sulfur deficiency, NOnitric oxide, cPTIO-2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxy l-3oxide (an NO scavenger)

Figure 9 Foliar application of NO enhances the activity of (A) adenosine 5-phosphosulfate reductase (APS-R), (B) ATP sulfurylase (ATP-S), (C) O-acetylserine (thiol) lyase (OAST-L) and (D) sulfide reductase (SiR), in leaves of tomato seedlings. Bars followed by the same letter do not differ significantly at P < 0.05 according to Duncan Multiple Range Test. Average of five determinations are presented with bars indicating ± SE. S-optimum sulfur, SD-sulfur deficiency, NO-nitric oxide, cPTIO2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxy l-3-oxide (an NO scavenger)

Figure 10 Foliar application of NO enhances (A) reduced glutathione (GSH), (B) redox state (GSH/GSSG) and (C) ascorbate (ASC) content in leaves of tomato seedlings. Bars followed by the same letter do not differ significantly at P < 0.05 according to Duncan Multiple Range Test. Average of five determinations are presented with bars indicating ± SE. S-optimum sulfur, SD-sulfur deficiency, NOnitric oxide, cPTIO-2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxy l3-oxide (an NO scavenger)

Highlights • Nitric oxide, a signaling molecule, alleviated oxidative damage induced by low-sulfur stress • Nitric oxide and hydrogen sulfide crosstalk associated with the improvement of growth and photosynthetic attributes of tomato seedlings under limited-sulfur conditions. • Nitric oxide mediated modulation of enzymes activity involved in photosynthesis under low-sulfur stress conditions. • Nitric oxide efficiently improved S utilization efficiency by upregulating the activity of Sassimilating enzymes. • Nitric oxide decreased reactive oxygen species generation in sulfur-deficient tomato seedlings. • Nitric oxide and hydrogen sulfide coordinated each other and maintained redox state and ASC—GSH cycle resulting in increased antioxidant system under limited-sulfur conditions. • NO functions as an upstream signaling biomolecule with another gaseous transmitter signaling molecule, hydrogen sulfide