Exogenous nitric oxide improves salt tolerance during establishment of Jatropha curcas seedlings by ameliorating oxidative damage and toxic ion accumulation

Exogenous nitric oxide improves salt tolerance during establishment of Jatropha curcas seedlings by ameliorating oxidative damage and toxic ion accumulation

Accepted Manuscript Title: Exogenous nitric oxide improves salt tolerance during establishment of Jatropha curcas seedlings by ameliorating oxidative ...

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Accepted Manuscript Title: Exogenous nitric oxide improves salt tolerance during establishment of Jatropha curcas seedlings by ameliorating oxidative damage and toxic ion accumulation Authors: Cibelle Gomes Gadelha, Rafael de Souza Miranda, Nara L´ıdia M. Alencar, Jos´e H´elio Costa, Jos´e Tarquinio Prisco, En´eas Gomes-Filho PII: DOI: Reference:

S0176-1617(17)30050-0 http://dx.doi.org/doi:10.1016/j.jplph.2017.02.005 JPLPH 52526

To appear in: Received date: Revised date: Accepted date:

3-10-2016 10-2-2017 17-2-2017

Please cite this article as: Gadelha Cibelle Gomes, Miranda Rafael de Souza, Alencar Nara L´ıdia M, Costa Jos´e H´elio, Prisco Jos´e Tarquinio, Gomes-Filho En´eas.Exogenous nitric oxide improves salt tolerance during establishment of Jatropha curcas seedlings by ameliorating oxidative damage and toxic ion accumulation.Journal of Plant Physiology http://dx.doi.org/10.1016/j.jplph.2017.02.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.

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Exogenous nitric oxide improves salt tolerance during establishment of Jatropha curcas seedlings by ameliorating oxidative damage and toxic ion accumulation

Cibelle Gomes Gadelha1, Rafael de Souza Miranda1, Nara Lídia M. Alencar2, José Hélio Costa1, José Tarquinio Prisco1, Enéas Gomes-Filho1*

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Departamento de Bioquímica e Biologia Molecular and Instituto Nacional de Ciência e

Tecnologia em Salinidade (INCTSal) / CNPq, Universidade Federal do Ceará, 60440-970, Fortaleza, Ceará, Brasil. E-mail adresses: [email protected] (C.G. Gadelha); [email protected] (R.S. Miranda); [email protected] (J.H. Costa); [email protected] (J.T. Prisco); [email protected] (E. Gomes-Filho)

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Instituto Federal de Educação, Ciência e Tecnologia do Ceará, Crateús, Ce, Brasil. E-mail adress:

[email protected] (N.L.M. Alencar)

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Corresponding author. Tel.: +55 85 3366 9405; fax: +55 85 3366 9829.

E-mail address: [email protected] (E. Gomes-Filho).

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SUMMARY

Jatropha curcas is an oilseed species that is considered an excellent alternative energy source for fossil-based fuels for growing in arid and semiarid regions, where salinity is becoming a stringent problem to crop production. Our working hypothesis was that nitric oxide (NO) priming enhances salt tolerance of J. curcas during early seedling development. Under NaCl stress, seedlings arising from NO-treated seeds showed lower accumulation of Na+ and Cl- than those salinized seedlings only, which was consistent with a better growth for all analyzed time points. Also, although salinity promoted a significant increase in hydrogen peroxide (H2O2) content and membrane damage, the harmful effects were less aggressive in NO-primed seedlings. The lower oxidative damage in NOprimed stressed seedlings was attributed to operation of a powerful antioxidant system, including greater glutathione (GSH) and ascorbate (AsA) contents as well as catalase (CAT) and glutathione reductase (GR) enzyme activities in both endosperm and embryo axis. Priming with NO also was found to rapidly up-regulate the JcCAT1, JcCAT2, JcGR1 and JcGR2 gene expression in embryo axis, suggesting that NO-induced salt responses include functional and transcriptional regulations. Thus, NO almost completely abolished the deleterious salinity effects on reserve mobilization and seedling growth. In conclusion, NO priming improves salt tolerance of J. curcas during seedling establishment by inducing an effective antioxidant system and limiting toxic ion and reactive oxygen species (ROS) accumulation. Abreviations AsA, reduced ascorbate; AsA/(AsA + DHA), ascorbate redox state; CAT, catalase; DHA, dehydroascorbate; GR, glutathione reductase; GSH, reduced glutathione; GSSG, oxidized glutathione; GSH/(GSH + GSSG), glutathione redox state; H2O2, hydrogen peroxide; NO, nitric

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oxide; SNP, sodium nitroprusside; qPCR, quantitative PCR; ROS, reactive oxygen species; TBARS, thiobarbituric acid reactive substances; Key words: Antioxidative metabolism, gene expression, ionic homeostasis, Jatropha, salinity, seed pretreatment.

Introduction

Biofuel production from non-edible oils has emerged as an important global issue to mitigate global warming as well as a plausible alternative to fossil-based fuels. Several studies have demonstrated that Jatropha curcas biodiesel contains potential fuel properties, which may be used in replacement of diesel (Hwang et al., 2016; Kumar et al., 2016). The J. curcas is a multipurpose species of the Euphorbiaceae family, growing in environments with extreme climate and soil conditions, such as arid and semiarid areas, regions in which the large majority of plant species cannot survive and/or produce economically (Francis et al., 2005). However, cultivation of Jatropha is increasingly becoming a serious problem in northeastern Brazil due to gradual salt accumulation in the soils. High salt concentration in soil is a major factor limiting plant growth and productivity (Julkowska and Testerink, 2015). Harmful salt effects may arise primarily from decreased osmotic potential and toxic ion accumulation (Na+ and Cl-) (Ashraf, 2009). Secondarily, excessive ion accumulation can cause oxidative stress, characterized by the overproduction of reactive oxygen species (ROS), such as superoxide radical (O2•-), hydrogen peroxide (H2O2) and hydroxyl radical (OH•). In excess, ROS can result in extensive damage to proteins, DNA and lipids, thereby impairing normal cellular functions, which can result in perpetual metabolic dysfunction and plant death (Mignolet-Spruyt et al., 2016). To cope with oxidative stress, plant cells have developed elaborate defense systems, involving enzymatic and non-enzymatic components (Noctor and Foyer, 2016). The ROSscavenging enzymatic pathways include activity of catalase (CAT), superoxide dismutase (SOD),

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guaiacol peroxidase (G-POD), ascorbate peroxidase (APX) and glutathione reductase (GR), among others. The non-enzymatic compounds are generally small molecules, like ascorbate (AsA), glutathione (GSH), carotenoids and tocopherols (Zagorchev et al., 2013; Demidchik et al., 2015). It is well documented that plant species employing highly efficient antioxidant systems had improved salt stress tolerance, a response that may be signaled by some small molecules, such as H2O2 (Surówka et al., 2016), oligochitosan (Ma et al., 2012), NaCl (Pandolfi et al., 2016), proline (Rady et al., 2016), abscisic acid (Li et al., 2010) and nitric oxide (NO) (Arora et al., 2016). Thus, adopting genetic and environmental methods to induce salinity tolerance in plants is essential and has received considerable attention. Some researchers have suggested that exogenous NO is able to improve the seed germination under environmental stresses, such as Cd and Cu (Khairy et al., 2015); drought (Arc et al., 2013); salinity (Lin et al., 2013); and other conditions (Fancy et al., 2016). In the large majority of studies, the positive effects of exogenous NO are attributed to restricted regulation of ionic homeostasis, reduced oxidative damage and effective antioxidant systems, delaying salt-induced leaf senescence and regulating levels of osmolytes (Lin et al., 2012; Ahmad et al., 2016; Kong et al., 2016), as well as to indirect effects of auxin and alterations in the cell wall (Bethke et al., 2004; Hu et al., 2005; Bõhm et al., 2010). In this sense, NO priming has been found to increase the tolerance of plants against salinity by modulating multiple stress-responsive pathways (Hussain et al., 2016). Despite current knowledge about NO behaving as a signaling molecule in triggering multiple defense responses against environmental stresses in plants, the molecular mechanisms activated by NO and the manner by which this signal molecule coordinates the physiological, biochemical and molecular responses to salt stress are still elusive. Furthermore, there is no convincing evidence regarding NO priming in J. curcas seedling establishment under salt stress. Our working hypothesis was that NO priming activates cellular detoxification mechanisms and improves salt tolerance of J. curcas during early development. This hypothesis was tested

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employing a set of detailed investigations, combining biochemical analyses with molecular approaches. We report that NO priming leads to a significant mitigation of salt stress due to low toxic ion and ROS accumulation via powerful antioxidant systems, resulting in elevated growth of NO-treated seedlings under saline conditions.

Materials and Methods

Plant material and growth measurements Jatropha curcas seeds, provided by the Lagoa Seca farm, Campina Grande, Paraiba, Brazil, were disinfected with 2% Orthocid® fungicide for 10 min followed by a triple rinsing with sterile distilled water. Seeds were then distributed onto germitest paper towels moistened with distilled water (control) or pretreated with 75 µM sodium nitroprusside (SNP; an NO donor) solution (pretreatment). After 24 h, seeds were transferred to new germitest towels and moistened with distilled water or 100 mM NaCl solution (salt treatment). Thus, the experiments were divided into four groups, including a control (neither NO nor NaCl), salt stressed (no NO and NaCl treatment), NO/control (NO treatment and no NaCl) and NO/salt stressed (pretreatment with 75 µM NO and then 100 mM NaCl stress). The saline solutions were applied only once, and their volume was equal to three times the mass of the paper, as indicated by Brasil (2009). After the seed distribution on moistened germitest paper sheets, they were rolled and stored, upright, in pots covered by transparent plastic, which keep high humidity, thereby preventing water evaporation and expressive changes in NaCl concentration. The experiment was performed in a growth chamber at 30 °C (constant temperature) with a constant photoperiod of 12/12 h. Harvests were performed at 1 (only for real-time quantitative PCR [qPCR] analysis), 2, 4, 6 and 8 d after exposure to NaCl, with plant material separated into two groups. In the first group, the plant material was divided into the endosperm and embryo axis by measuring the shoot and radicle length of the embryo axis; then, after drying in a forced-air circulation oven at 60 °C for 48 h, the endosperm and embryo axis dry

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mass was determined. In the second group, the fresh endosperm and embryo axis tissues were frozen immediately in liquid nitrogen and kept at -80 °C for biochemical analysis and qPCR assays. Ion content Extracts were prepared by homogenizing 0.05 g of endosperm or embryonic axis dry mass in 5 mL of deionized water at 45 °C for 1 h. Thereafter, the homogenate was centrifuged at 3,000 x g for 15 min at room temperature, and the supernatant was used to measure Na+, K+ and Cl- concentrations. The Na+ and K+ contents were determined by flame photometry, whereas the Cl- content was estimated at 460 nm using NaCl as a standard, according to methods of Gaines et al. (1984).

Determination of H2O2, lipid peroxidation and non-enzymatic antioxidants Fresh (0.25 g) tissues were homogenized in a mortar and pestle with 3.0 mL of 5% trichloroacetic acid (TCA) at 4 °C and then centrifuged at 12,000 x g for 20 min at 4 °C. The supernatant fraction was collected and used for determination of H2O2, lipid peroxidation and non-enzymatic antioxidants. Quantitative measurement of H2O2 production was performed as described by Gay et al. (1999), with some modifications. The reaction mixture was composed of extract (100 µL), 1.0 mM FeSO4, 1.0 mM (NH4)2SO4, 100 mM H2SO4 and 1.0 mM xylenol orange. The H2O2 concentration was determined spectrophotometrically at 560 nm by comparing to a standard curve prepared with H2O2 solutions. Lipid peroxidation was determined by quantifying the thiobarbituric acid reactive substances (TBARS) according to methods of Heath and Packer (1968). The absorbance at 600 nm was subtracted from that at 532 nm, and the TBARS values were calculated using an extinction coefficient of 0.155 M-1 cm-1. Reduced AsA contents were estimated at 525 nm after reduction of Fe3+ to Fe2+, promoted by AsA in acidic solution and, subsequently, formation of a Fe2+-bipyridyl complex, according to Law et al. (1983). For measurement of total AsA [AsA + DHA (dehydroascorbate / oxidized AsA)],

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the DHA was completely reduced to AsA using dithiothreitol, followed by AsA estimation. The DHA content was determined as the difference between total AsA and AsA. The AsA redox state was established by the ratio of AsA and total AsA [AsA / (AsA + DHA)], being expressed as a percentage. The GSH content was measured using the method described by Griffith (1980). Reduced GSH was quantified at 412 nm directly in the extract of endosperm and embryo axis tissues. For the total GSH [GSH + GSSG (oxidized GSH)] assay, the GSSG was first reduced into GSH by employing the GR enzyme, followed by its measurement. Contents of GSH + GSSG and GSH were estimated using GSH as a standard. Finally, the GSSG was estimated by subtracting the GSH of total GSH. The GSH redox state was calculated as following: GSH redox state (%) = [GSH / (GSH + GSSG)].

Antioxidant enzyme assays A crude enzyme extract was prepared by homogenizing a fresh mass of endosperm (0.5 g) and embryo axis (0.1 g) with 2.0 mL of extraction buffer (100 mM potassium phosphate, pH 7.0, containing 0.1 mM EDTA) at 4 °C. In parallel, another extraction was performed for APX measurements, which involved adding 2.0 mM AsA to the extraction buffer (Nakano and Asada, 1981). The homogenate was centrifuged at 12,000 × g for 15 min at 4 °C, and the supernatant was used for the enzymatic activity assays described next. Activity of the SOD enzyme (EC 1.15.1.1) was measured according to the method of Beauchamp and Fridovich (1971), which is based on the photoreduction of nitro blue tetrazolium chloride (NBT) monitored at 560 nm. One unit of activity (UA) of SOD was defined as the amount of enzyme required to inhibit 50% of the NBT photoreduction. Activity of CAT (EC 1.11.1.6) was assayed according to methods of Havir and McHale (1987), monitoring the decrease of absorbance at 240 nm (E = 39.4 M-1 cm-1) due to H2O2 consumption. Activity of APX (EC 1.11.1.1) was measured as described by Nakano and Asada (1981) by monitoring the rate of AsA oxidation (E =

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2.8 mM-1 cm-1) through the decrease of absorbance readings at 290 nm. Activity of G-POD (EC 1.11.1.7) was assayed by the method of Kar and Mishra (1976), monitoring the increase of absorbance at 420 nm due to tetraguaiacol formation (or guaiacol oxidation). Finally, activity of GR (EC 1.6.4.2) was measured using the method proposed by Foyer and Halliwell (1976), with some modifications. The reaction medium was composed of crude extract and reaction buffer (0.1 M potassium phosphate buffer, pH 7.8, 0.1 mM EDTA, 3.0 mM GSSG). The reaction was initiated by the addition of 0.05 mM NADPH, and the decrease in absorbance (NADPH oxidation) was monitored at 340 nm. The oxidation rate was corrected for non-enzymatic oxidation of NADPH by GSSG. In order to measure the specific activity of antioxidant enzymes, soluble protein contents were determined spectrophotometrically at 595 nm in crude extracts according to methods of Bradford (1976), with bovine serum albumin as a standard.

Gene expression analysis At the harvesting time points (1, 2, 4, 6 and 8 d after salt exposure), endosperm and embryo axis organs were collected, immediately frozen in liquid nitrogen and kept at -80 °C until further processing. Total RNA was extracted by employing the Invitrogen plant RNA purification reagent as well as the Qiagen plant mini kit, according to the respective manufacturer’s protocols. The combination of the two RNA extraction procedures was essential to eliminate contaminants from RNA samples. Contaminant genomic DNA was removed using an RNase-Free DNase set (Qiagen). The concentration of total RNA was measured through a Nanodrop 2000 spectrophotometer (Thermo Scientific, Waltham, USA), and its purity was checked by analysing the A260/A280 and A260/A230 ratios. Specific primer pairs were designed for the exon–exon junctions for JcCAT1, JcCAT2, JcCAT3, JcGR1 and JcGR2 genes using PerlPrimer software and the respective annotated genes/cDNAs retrieved from public databases for J. curcas (Supplementary Table S1). The qPCR was performed on a Mastercycler ep realplex 4S (Eppendorf, Germany) employing Power SYBR-

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Green PCR Master Mix (Applied Biosystems) as an indicator, with three biological replications per sample. Relative expression level of transcripts was calculated by the cycle threshold (CT) 2-ΔΔCT method (Livak and Schmittgen 2001), and the normalized relative expression was calculated using qbase PLUS software version 1.5 (Biogazelle) (Hellemans, 2007), using as internal reference the ACT11, MPK4 and GAPDH genes, which were selected from eight tested genes (ACT, EF1α, GAPDH, MPK4, PP2A2, SAND and UBC).

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Experimental design The experimental design was completely randomized following a factorial scheme composed of two salinity levels (0 and 100 mM NaCl) and two NO treatments (0 and 75 µM SNP, NO donor), with five replications per treatment (five individual plants were considered one replication), except for qPCR (three replications were used). At each analyzed time (1, 2, 4, 6 and 8 d), the data were subjected to a two-way analysis of variance (ANOVA), and when a difference was significant (p ≤ 0.05), the mean values were compared by Tukey’s test.

Results

Effects of NO pretreatment on growth and seedling establishment Jatropha curcas seedlings were subjected to pretreatment with NO in order to evaluate its role in salt stress acclimation. No obvious differences in the growth parameters (endosperm and embryo axis dry mass; shoot and radicle length) were observed between the NO pretreated and nonpretreated seedlings under control conditions (Fig. 1). Salt stress increased the endosperm dry mass (Fig. 1a), whereas salt stress severely decreased the embryo axis dry mass and shoot and radicle lengths of J. curcas seedlings, regardless of NO treatment and analyzed time point (Fig. 1b, c, d). However, the deleterious salinity effects in seed reserve mobilization (Fig. 1a, c) and growth (Fig. 1b, d) were less severe in NO-pretreated seedlings compared to non-treated ones. In NaCl presence, the embryo axis dry mass values of NO-pretreated seedlings were found to be 65.8, 147, 43.2 and 61.6% higher than those of non-treated seedlings at 2, 4, 6 and 8 d after salt stress (DAS), respectively (Fig. 1c).

Influence of NO pretreatment on ionic homeostasis In the absence of salinity, the accumulation of K+, Na+ and Cl- was almost unaltered by NO treatment, regardless of analyzed time point (Fig. 2). In general, K+ contents were little or not

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changed by the studied NO and salt treatments during all the evaluated DAS, mainly in the embryo axis (Fig. 2a, d). Conversely, salt stress imposed an over-accumulation of Na+ and Cl- in both the endosperm and embryo axis; however, it was less intense in seedlings pretreated with NO (Fig. 2b, c, e, f).

Role of NO treatment in alleviating oxidative damage and activating the antioxidant system under salinity In order to evaluate if NO pretreatment mitigates salt-induced oxidative damage, lipid peroxidation (measured as TBARS); H2O2 content; and enzymatic and non-enzymatic antioxidants were determined. In general, non-stressed plants did not show any significant differences in TBARS and H2O2 contents as affected by NO pretreatment (Fig. 3). Salt stress promoted a significant increase in TBARS and H2O2 contents at all analyzed time points and plant tissues; nevertheless, the NO pretreatment was effective at alleviating oxidative damage and ROS accumulation in J. curcas seedlings (Fig. 3). The non-enzymatic antioxidants (GSH and AsA) were significantly altered by studied treatments (Fig. 4, 5). In endosperm, the reduced GSH contents decreased during germination (2 to 8 DAS) but were increased by salinity, mainly in the seedlings pretreated with NO (Fig. 4a). In the embryo axis, increments in the GSH content by salinity were observed at 2 and 4 DAS in NO/saltstressed seedlings and at only 2 DAS in salt-stressed ones (Fig. 4c). In the absence of NO pretreatment, the GSH redox state [GSH / (GSH + GSSG)] remained unaltered or reduced as affected by salt stress in both the endosperm and embryo axis, except at 8 DAS in endosperm (Fig. 4b, d). On the other hand, NO-pretreated stressed seedlings showed higher values of GSH redox state than the control in the endosperm and an opposite response in the embryo axis, regardless of evaluated time point. An expected increased AsA level was found in salt-stressed seedlings in both the endosperm and embryo-axis; however, this response was more intense in NO/salt-stressed seedlings, regardless

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of plant tissue (Fig. 5a, c). In endosperm, AsA redox state [AsA / (AsA + DHA)] was increased by salinity only at 6 and 8 DAS, but the NO/salt-stressed seedlings displayed values 37.5 and 31.2% higher, respectively, than those of salt-stressed seedlings (Fig. 5b). Similarly, in the embryo axis, the AsA redox state was enhanced in seedlings subjected to salinity, with more pronounced improvement under NO pretreatment (Fig. 5d). Although the SOD, CAT, APX, G-POD and GR enzymes have been cited as crucial for salt tolerance in the large majority of plant species, herein, SOD, APX and G-POD displayed no expressive role in salt acclimation of J. curcas seedlings (Supplementary Fig. S2). In general, the highest values of activity were registered for CAT (Fig. 6a, c), showing its higher relevance as an antioxidant at the beginning of embryo axis development (Fig. 6c). In salt absence (control; NO/control), CAT activity was found to be increased by the NO pretreatment at 4 and 6 DAS in the endosperm (Fig. 6a) and at 6 and 8 DAS in the embryo axis (Fig. 6c). Under salinity, CAT activity was slightly increased after the onset of stress in the endosperm (4 DAS) and later in the embryo axis (6 and 8 DAS) in salt-stressed plants (Fig. 6a, c); nevertheless, NO/salt-stressed seedlings showed higher CAT activity values in the endosperm (2, 6 and 8 DAS) and embryo axis (2 and 4 DAS) than those of non-treated seedlings. Similarly, GR activity was increased in salt-stressed seedlings, a response more intensified in NO/salt-stressed seedlings, especially later during the salt exposure (6 and 8 DAS) (Fig. 6b, d).

Effectiveness of NO pretreatment in reprogramming gene expression To investigate if the salt-responsive enzymes during germination of J. curcas are transcriptionally controlled by NO pretreatment, transcript abundance of genes encoding CAT and GR was quantified by qPCR. Three gene members of CAT (JcCAT1, JcCAT2 and JcCAT3) and two of GR (JcGR1 and JcGR2) retrieved from the J. curcas genome were analyzed. In this study, only the relative expression of genes highly expressed in the endosperm and embryo axis as well as those responsive to analyzed treatments are presented (Supplementary Table S3).

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In general, the transcript abundance of JcCAT1 and JcCAT3 gene members in the endosperm increased in response to NO pretreatment under control conditions (Fig. 7a, b). However, under salinity, NO/salt-stressed seedlings showed transcript levels higher than saltstressed seedlings at 2 DAS (for JcCAT1) and at 4 DAS (for JcCAT3). Surprisingly, no key modulation in JcGR1 and JcGR2 relative expression was observed in these reserve organs, regardless of NO or salt treatment. In the embryo axis, the JcCAT1 and JcCAT2 genes were the most responsive to treatment (Fig. 7c, d; Supplementary Table S3). Salt-stressed seedlings exhibited a slight upregulation in JcCAT2 transcript abundance after 1 and 6 d compared to the control (Fig. 7d), whereas no expressive modulation was registered for the JcCAT1 isoform (Fig. 7c). On the other hand, the JcCAT1 and JcCAT2 gene expression was strongly increased by salinity during early embryo axis development (1 DAS) of NO/salt-stressed seedlings, with values of relative expression found to be 154 and 32% higher than those of NO/control seedlings, respectively. In addition, NO/salt-stressed seedlings also displayed a 261% increase in JcCAT2 expression at 4 DAS (Fig. 7d) compared to NO/control plants. As reported for JcCAT1 and JcCAT2 genes, the JcGR1 and JcGR2 isoforms in the embryo axis were overexpressed at the beginning of germination when the seeds were pretreated with NO (1 DAS) (Fig. 8a, b), but little or no alteration was observed later, regardless of NO or salt treatment.

Discussion

Salt stress is widely known for imposing a decrease in crop productivity, mainly by drastic disturbances in germination, seedling growth, photosynthesis, ion imbalance and cell redox state alterations (Gomes-Filho et al., 2008; Gondim et al., 2013; Alencar et al., 2015). Nonetheless, exogenous use of signal molecules has been shown to improve salt tolerance to several plant species, suggesting that those species may activate biochemical and physiological mechanisms in

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order to withstand with toxic salt effects (Savvides et al., 2015). Our study shows that NO (SNP pretreatment) increases salt tolerance of J. curcas during early development and seedling establishing. The slowing of NaCl-induced early seedling development may stem from decreased water potential as well as specific toxicity of Na+ and Cl- ions (Bewley et al., 2013). Also, the impairment of embryo axis growth is a consequence of an imbalance between sink strength and degradation of seed reserves (Marques et al., 2013). We have shown previously that salinity promoted a delay in lipid and protein mobilization in salt-stressed endosperm cells (Alencar et al., 2015). Accordingly, here we observed that salinity altered the source–sink relationship (reserve transfer of endosperm to the embryo axis), suggesting a severe delay in seed reserve mobilization of J. curcas (Fig. 1a, c), which promoted a consequent restriction in the seedling growth (Fig. 1b, d). However, NO priming alleviated the harmful salt effects during reserve transfer and improved seedling growth under salinity, supporting our working hypothesis. Salt tolerance has been correlated closely with maintenance of the K+/Na+ ratio in cellular compartments, emphasized in relation to low Na+ accumulation (Miranda et al., 2016). Concordantly, a lower content of toxic Na+ ions in salt-stressed seedlings was established in the treatments of NO, in both the endosperm and embryo axis (Fig. 2b, e), without remarkable changes in K+ concentration (Fig. 2a, d). In concordance, NO pretreatment in leaves of cotton upregulated SOS1 and NHX1 gene expression, decreasing Na+ accumulation and toxicity in plant tissues (Kong et al., 2016). These findings suggest that the improved K+/Na+ of NO-treated stressed plants could be related to restrictions in Na+ influx across the root plasma membrane or to vacuolar compartmentalization, contributing to enhanced salt tolerance (Guo et al., 2009). In addition, exogenous NO also has been cited as a secondary messenger in inducing expression of H+-ATPase and H+-PPase, generating a powerful electrochemical potential gradient for secondary transporters and increasing the activity of Na+/H+ exchange (Zhao et al., 2004; Zhang et al., 2006).

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In the current study, we also analyzed the salt stress-induced oxidative damage to J. curcas and evaluated the protective effects of exogenous NO. Salinity promoted increases in TBARS and H2O2 contents (Fig. 3) in the endosperm and embryo axis, suggesting that salinity could cause injuries to the integrity of the cellular membrane and to cellular components such as lipids, proteins and nucleic acids (Demidchik, 2015). Nevertheless, we found that exogenous NO can partially prevent lipid peroxidation and ROS accumulation, thereby alleviating the oxidative damage normally caused by salinity stress. As a result, when the NO donor SNP was added to the medium of salt-stressed seedlings, better reserve mobilization (Fig. 1a, c) and seedling growth (Fig. 1b, d) were observed. Similar results also were reported for some plant species, highlighting the beneficial effects of NO in avoiding ROS accumulation and lipid peroxidation under stress, such as in tomato (Manai et al., 2014), chickpea (Ahmad et al., 2016) and maize (Ullah et al., 2016). NO-induced low H2O2 accumulation likely arises from marked upregulation of CAT activity (Fig. 3b, d, 6a, c), which seems to be transcriptionally controlled after onset of salt exposure and post-transcriptionally during later salinity. Our data suggest that exogenous NO may act as a signal molecule for activating overexpression of JcCAT1 and JcCAT2 genes during the beginning of salinity (24 h after salt exposure) (Fig. 7c, d), mainly in the embryo axis tissues. Furthermore, in the J. curcas tissues, CAT activity was around 300 times (at least) higher than that of other studied enzymes (SOD, APX, G-POD and GR), evidencing that CAT greatly acts in H2O2 scavenging (Fig. 3, 6, S2) and helps with better salt tolerance in NO-treated seedlings. Our findings are in accordance with the study by Gondim et al. (2012), who observed an essential role of CAT in the H2O2 scavenging of NaCl-stressed maize plants. Therefore, pretreatment with H2O2 induced an upregulation of CAT activity and gene expression during the early stages of salinity, decreasing ROS accumulation and improving salt tolerance of maize plants. Like CAT, GR also was positively stimulated by exogenous NO in salt-stressed seedlings (Fig. 6b, d). GR plays an important role in cell defense against ROS accumulation by converting GSSG into reduced GSH, maintaining a GSH pool in the reduced GSH state and a reducing

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environment in the cell, which is crucial for the active functioning of proteins. The GSH molecule is an abundant metabolite in plants that reacts directly with ROS; it may protect enzyme thiol groups and also is known to be involved in signal transduction (Gill et al., 2013). In this study, NOstimulated GR activity was consistent with the highest contents of GSH in salt-stressed seedlings, regardless of analyzed plant tissue (Fig. 4a, c, 6b, d), which also coincided with enhanced AsA levels (Fig. 5). As a result, NO/salt-stressed seedlings exhibited a better (and favorable) redox state than seedlings subjected to salt stress only (Fig. 4, 5). Our results are consistent with recent observations showing that NO alleviates the oxidative damage through a favorable redox balance, regulating the antioxidant defense mechanism and osmoprotection, leading to enhanced plant growth under salt stress (Kaya et al., 2015; Shi et al., 2016; Ullah et al., 2016). Our data also evidenced that exogenous NO promotes transcriptional regulation of JcGR1 and JcGR2 after the onset of salt stress, whereas it regulates post-transcriptionally the GR activity during later salinity exposure (6 and 8 DAS), as evidenced by improved GR activity in NO-treated stressed seedlings without concordant upregulation in the gene expression (Fig. 6, 8) (Medeiros et al., 2014; Zhao et al., 2014). This argument is corroborated, at least in part, by the findings of Hussain et al. (2016), who noted that NO acts in a cascade of events leading to changes in the transcriptome. The data presented here reveal that pretreatment of seeds with NO positively modulates an integrated network of responses to NaCl stress in J. curcas, eliciting transcriptional and functional regulations and influencing crop salt tolerance. Thus, the ability of NO to mitigate NaCl stress seems to rely partly on its particular signaling properties and may be used to prevent the harmful effects of salinity during growth and reserve mobilization of J. curcas.

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Conclusion

In conclusion, exogenous NO enhances the salt tolerance in establishment of J. curcas seedlings by providing a better ionic and redox homeostasis. The greater NO-induced salinity tolerance was attributed to (1) an effective enzymatic and non-enzymatic antioxidant system, especially CAT and GR enzymes, and (2) reduced Na+ and Cl- accumulation, which potentially decreased the ROS accumulation and oxidative damage. Taken together, we proposed a mechanistic explanation to show the NO-induced salt responses in J. curcas seedlings (Fig. 9). Our data reinforce that the exogenous application of NO (a sub-product of the decomposition of SNP) emerges as a good alternative to improve the seedling establishment of Jatropha crops sown in saline media. Acknowledgments

We thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Instituto Nacional de Ciência e Tecnologia em Salinidade (INCTSal) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the fellowship and financial support.

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Figure captions

Fig. 1. Dry mass of endosperm (A) and embryo axis (C) and length of shoot (B) and radicle (D) of embryo axis of Jatropha curcas beyond the establishing seedling in absence (control) and presence of 100 mM NaCl (salt stressed) and pretreated with nitric oxide (NO) at 0 and 75 µM. Values are given as the mean of five biological replications + standard error. At each time point, significant differences due to salt stress in the same NO pretreatment (control × salt stressed or NO/control × NO/salt stressed) are indicated by different capital letters, whereas significant differences due to NO pretreatment at the same salinity level (control × NO/control or salt stressed × NO/salt stressed) are denoted by different lowercase letters, using Tukey’s test (p ≤ 0.05).

Fig. 2. Contents of K+ (a, d), Na+ (b, e) and Cl- (c, f) in the endosperm and embryo axis during Jatropha curcas seedling establishment in absence (control) and presence of 100 mM NaCl (salt stressed) and pretreated with nitric oxide (NO) at 0 and 75 µM. Values represent the mean of five biological replications + standard error. Statistical details same as in Fig. 1.

Fig. 3. Contents of thiobarbituric acid reactive substances (TBARS) (a, c) and hydrogen peroxide (H2O2) (b, d) in the endosperm and embryo axis during Jatropha curcas seedling establishing in absence (control) and presence of 100 mM NaCl (salt stressed) and pretreated with nitric oxide (NO) at 0 and 75 µM. Values represent the mean of five biological replications + standard error. Statistical details same as in Fig. 1.

Fig. 4. Content of reduced glutathione (GSH) (a, c) and glutathione redox state (b, d) in the endosperm and embryo axis of Jatropha curcas during seedling establishment in absence (control) and presence of 100 mM NaCl (salt stressed) and pretreated with nitric oxide (NO) at 0 and 75 µM.

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Values represent the mean of five biological replications + standard error. Statistical details same as in Fig. 1.

Fig. 5. Content of reduced ascorbate (AsA) (a, c) and AsA redox state (b, d) in the endosperm and embryo axis of Jatropha curcas during seedling establishment in absence (control) and presence of 100 mM NaCl (salt stressed) and pretreated with nitric oxide (NO) at 0 and 75 µM. Values represent the mean of five biological replications + standard error. Statistical details same as in Fig. 1.

Fig. 6. Catalase (CAT) (a, c) and glutathione reductase (GR) (b, d) activities in the endosperm and embryo axis of Jatropha curcas during seedling establishment in absence (control) and presence of 100 mM NaCl (salt stressed) and pretreated with nitric oxide (NO) at 0 and 75 µM. Values represent the mean of five biological replications + standard error. Statistical details same as in Fig. 1.

Fig. 7. Relative expression profiles of genes JcCAT1 (a) and JcCAT3 (b) in the endosperm and JcCAT1 (c) and JcCAT2 (d) in the embryo axis of Jatropha curcas during seedling establishment in absence (control) and presence of 100 mM NaCl (salt stressed) and pretreated with nitric oxide (NO) at 0 and 75 µM. Values represent the mean + standard error of three biological replications by real-time quantitative PCR (qPCR). Statistical details same as in Fig. 1.

Fig. 8. Relative expression profiles of JcGR1 (a) and JcGR2 (b) genes in the embryo axis of Jatropha curcas during seedling establishment in absence (control) and presence of 100 mM NaCl (salt stressed) and pretreated with nitric oxide (NO) at 0 and 75 µM. Values represent the mean + standard error of three biological replications by real-time quantitative PCR (qPCR). Statistical details same as in Fig. 1.

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Fig. 9. Model proposed for responses to salt stress induced by nitric oxide (NO) pretreatment in the embryo axis of establishing Jatropha curcas seedlings. (a) Upon salt stress exposure, (1) accumulation of sodium (Na+ - yellow circles) inside the cells promotes an increase in hydrogen peroxide (H2O2 - red circles) content. In parallel, (2) a still unknown signaling pathway recognizes the H2O2 and Na+ accumulation and triggers both an upregulation of (3) JcCAT1 and (4) JcGR1 gene expression, resulting in improved (5) catalase (CAT) and (6) glutathione reductase (GR) activities. The enhanced GR activity is accomplished by greater (7) ascorbate (AsA) content. Nonetheless, these responses are not effective (8) on H2O2 scavenging, resulting in severe damage to (9) membrane lipids. (b) Yet, NO priming (1) quickly activates the signaling pathway, reprogramming the salt responses. First, (2) it recognizes Na+ accumulation and seems to activate (3) effective extrusion of Na+ back into the medium. Secondly, it induces an upregulation of (4) JcCAT1, (5) JcCAT2, (6) JcGR1 and (7) JcGR2 gene transcripts, which direct better (8) CAT and (9) GR activity. At the same time, the GR increases (10) glutathione (GSH) and (11) AsA contents. Finally, (12) the H2O2 is almost scavenged, (13) avoiding damage to membrane lipids in embryo axis cells and enhancing the salt tolerance of J. curcas seedlings. Similar responses are registered for NO-induced salt responses in endosperm of J. curcas seedlings, as described in Fig. S4.