Involvement of Ethylene in Reversal of Salt Stress by Salicylic acid in Presence of Sulfur in Mustard (Brassica juncea L.)

The involvement of ethylene in reversal of salt stress inhibited photosynthetic activity and growth by salicylic acid (SA) together with sulfur (S) was studied in mustard (Brassica juncea L.) plants. Application of SA (0.5 mM) plus SO 42- (2.0 mM) improved photosynthetic activity through markedly increased S-assimilation, antioxidant enzymes activity and optimized ethylene and glutathione (GSH) production for reduced reactive oxygen species (ROS) in plants under 50 mM NaCl stress. As SA acts as an inhibitor of ethylene, and S-assimilation is associated with ethylene synthesis, we tried to gure out the interaction of ethylene in SA and SO 42- mediated salt tolerance. The involvement of ethylene was studied by supplementing salt treated plants with 200 µL L-1 ethephon (an ethylene-releasing compound) or 100 µM norbornadiene (NBD, ethylene action inhibitor) to SA and SO 42- treatments. The ethephon application to salt treated plants suppressed stress ethylene and optimized ethylene formation and increased ethylene sensitivity to enhance photosynthesis of plants by affecting antioxidative capacity of plants. Application of NBD to plants receiving SA plus SO 4 2- in presence of salt showed inhibited photosynthetic characteristics, stomatal behavior and growth. These plants exhibited minimal capacity of S-assimilation and antioxidant enzymes activity and GSH content. This explained that ethylene was involved in the reversal of salt stress by SA plus SO 42- . Thus, the study showed that ethylene intervenes the effect of SA in the presence of SO 42- to upregulate the antioxidants that lead to increased S-assimilation, and imparted tolerance to salt in mustard plants.


Introduction
One of the most damaging abiotic stresses has been soil salinity, which has resulted in signi cant losses in cultivated land area, crop yield, and quality (Yamaguchi and Blumwald 2005;Shahbaz andAshraf 2013: Fatma et al. 2021;Jahan et al. 2021;Sehar et al. 2021;Syeed et al. 2021). It has been estimated that the rising salinization of world cultivable lands at an annual rate of 10% per year would result into salinization of more than half of all arable lands by 2050 (Jamil et al. 2011). Plants exposed to salinity have shown reduced growth and development due to oxidative stress caused by the production of reactive oxygen species (ROS) (Isayenkov and Maathuis 2019). On the other, stressed plants tend to build up their antioxidant defense system to control the cellular ROS-level and minimize ROS-accrued consequences including lipid and protein oxidation. Major antioxidant enzymes (such as ascorbate peroxidase, APX; catalase, CAT) and non-enzymatic antioxidants (such as ascorbate, AsA; reduced glutathione, GSH; and α-tocopherol, avonoids and proline), alone and/or cumulatively scavenge varied ROS (Sharma and Dietz 2009;Ashraf 2009;Mittal et al. 2012).
Markedly, major phytohormones, mineral nutrients, and/or their interaction outcomes have been widely reported to signi cantly modulate and improve the plant's defense system against stress impacts Jahan et al. 2020). Sulfur (S) is one of the most important nutrient elements for plants, and has been the subject in plant and agricultural study. Sulfur is a major constituent of methionine (Met) and cysteine (Cys), Fe-S clusters, sulfolipids, S-methylmethionine (SMM), S-adenosylmethionine (SAM), glucosinolates, vitamins (biotin and thiamine), coenzyme A, thioredoxin system and GSH, a major antioxidant metabolite and non-protein thiol (Fatma et al. 2013;Anjum et al. 2015). Applied S improved the photosynthetic capacity of the plants in salt stress by improving the cellular GSH level, modulating the major components of the ascorbate-glutathione (AsA-GSH) cycle, restricting ROS formation, and thus, minimizing oxidative stress and its consequences .
Salicylic acid (SA), a natural phenolic compound has been identi ed to control the array of growth, physiological and developmental processes. Salicylic acid also works as signaling response directly or indirectly against different stresses improving photosynthetic functions, nutrient-uptake and assimilation, proline metabolism, plant water relations, modifying antioxidant defense systems and nullifying ROS and their consequences (Iqbal et al. 2015;Tari et al. 2015;Rasheed et al. 2020). Through signaling cross-talks with S-assimilation, there is a close relationship between SA and S, where SA can govern numerous elements of plant responses in both stressful and optimal conditions ). However, it is also remarkably stated that SA acts as an inhibitor of ethylene (Ahmed et al. 2020) and S-assimilation is associated with ethylene . Ethylene, as a signaling molecule, acts as a major modulator of plant stress responses by in uencing the ROS production (Cao et al. 2013;Tao et al. 2015;Khan et al. 2016;Jahan et al. 2021;Sehar et al. 2021). It has previously been observed that ethylene supplementation increases the activity of enzymatic and non-enzymatic antioxidants, which act as a rst line of defense against abiotic stress Khan et al. 2016;Zhang et al. 2016). Recently, we have shown that ethylene protects pigment system II activity and photosynthesis in salt stress by inducing the expression of psbA and psbB and the production of GSH in wheat , and by optimization of proline metabolism and antioxidant system in mustard (Jahan et al. 2021) According to the report of Khan et al. (2015), SA-supplemented plant reduced ethylene generation in heatexposed plants by decreasing the 1-aminocyclopropane carboxylic acid (ACC) and ACC synthase (ACS) activity to an appropriate range. But the information on how ethylene is involved in SA and S-mediated salt tolerance is scanty in the literature.
As a widely cultivated species for oilseed, Indian mustard (Brassica juncea L.) stands second in world oilseed production (USDA 2018). It is cultivated mainly in the North-Western agro-climatic region of India and suffers huge losses in productivity mainly due to the salinization of the cultivable land (Yousuf et al. 2016). Therefore, in order to sustain optimum growth and produce more in saline environments, it is critical to investigate the salt tolerance mechanisms in B. juncea. Thus, the triad comprising SA, S and ethylene is hypothesized herein to in uence the response of B. juncea to salinity and helps this crop to protect growth, metabolism and photosynthetic functions, ROS-metabolism and strengthening antioxidant defense system against salinity stress-impacts. However, information on how ethylene gets involved in SA-mediated effect on salinity in the presence of S to in oilseeds such as B. juncea has remained elusive.

Plant Culture and Treatments
Experiments were performed at the Department of Botany, Aligarh Muslim University, Aligarh, India.
Healthy seeds of Brassica juncea L. Czern & Coss. cultivar Pusa Vijay were undergone surface sterilization for 15 min with 0.01% HgCl 2 followed by washings for ve times through double distilled water. Sterilized seeds were sown in a diameter of 23-cm earthen pots, lled with 5 kg of reconstituted soil including peat and compost (4:1, w/w) mixed with sand (3:1, w/w). Seeds were sown in each pot and were kept in natural day/night conditions with an average day/night temperatures of 20 ± 3ºC and 12 ± 2ºC, respectively, relative humidity of 60 ± 5%, photosynthetically active radiation (PAR) was 750 ± 25 µmol m -2 s -1 and a critical photoperiod of 10-12 h.
To assess the effect of 0.5 mM SA and 2.0 mM SO 4 2alone or in coordination in alleviation of salinity, The other experiment was performed to evaluate the involvement of ethylene in SA and S-mediated control of photosynthetic functions and growth using 200 µL L-1 ethephon (an ethylene-releasing compound) or 100 µM 2,5-norbornadiene (NBD, ethylene action inhibitor) to SA and S treatment under salt stress. The concentration of ethephon and NBD has been worked out earlier (Iqbal et al. 2017).
Ethephon was chosen because it is known to alter ethylene evolution, and NBD was selected because it inhibits ethylene activity. Ethephon or NBD was sprayed on the upper surface of the foliage at 20 DAS together with 0.5% of a teepol surfactant. Per plant 25 mL of ethephon or NBD was applied with a hand sprayer. The treatments were set up in a completely randomized block design with four replicates (n = 4) for each treatment and the parameters were studied after 30 DAS. Leaves of the same age were taken for determinations.

Photosynthetic and Growth Characteristics
On a sunny day net photosynthesis, stomatal conductance and intercellular CO 2 concentration were evaluated in completely expanded topmost second leaves of plants in every treatment around 11.00 to 12.00 at day light saturating intensity using Infra-Red Gas Analyzer (CID-340, Photosynthesis system, Bio-Science, USA). At the time of measurement, the atmospheric conditions were as follows: photosynthetically active radiation, ∼680 μmolm −2 s −1 ; air temperature, ∼22ºC and relative humidity, ∼70%.
Chlorophyll content in the leaves of plants taken from every treatment was determined using a SPAD chlorophyll meter (SPAD 502 DL PLUS, Spectrum Technologies).
Using a chlorophyll uorometer, the maximal PSII photochemical e ciency (Fv/Fm) of the fully expanded second leaf from the top of the plant was calculated (Junior-PAM, Heinz Walz, Germany). The details are given in Supplementary le S1.
Leaf area meter (LA 211; Systronics, Hyderabad, India) was used to determine the leaf area. Dry weight was estimated after drying the samples in an oven at 80°C until the water evaporated and a consistent weight was achieved.
Oxidative Stress Biomarkers Okuda et al. (1991) approach was used to determine H 2 O 2 content (1991). Lipid peroxidation was determined by estimating thiobarbituric acid reactive substances (TBARS) following Dhindsa et al. (1981). Supplementary File S1 contains the details of the procedure.

Assay of Antioxidant Enzymes
Fresh leaf tissues (0.2 g) were homogenized in a chilled mortar and pestle with an extraction buffer containing 0.05% (v/v) Triton X-100 and 1% (w/v) polyvinylpyrrolidone (PVP) in potassium-phosphate buffer (100 mM; pH 7.0). The homogenate was centrifuged at 15,000 g for 20 minutes at 4°C. After centrifugation, the supernatant was collected and activity of superoxide dismutase (SOD) and glutathione reductase (GR) was measured. For the measurement of ascorbate peroxidase (APX), the extraction buffer was supplemented with 2 mM AsA. Giannopolitis and Ries (1977) and Beyer and Fridovich (1987) methods were used for SOD (EC 1.15.1.1) by studying the suppression of photochemical reduction of nitro blue tetrazolium (NBT). Activity of APX (EC 1.11.1.11) was measured using description of Nakano and Asada (1981) by measuring the decrease in ascorbate absorbance at 290 nm owing to enzymatic breakdown. Foyer and Halliwell (1976) method was followed for GSH-dependent oxidation of NADPH at 340 nm to evaluate the activity of GR (EC 1.6.4.2). Supplementary File S1 contains the details of the procedure.

Ethylene Evolution
Ethylene evolution in leaves was assessed using gas chromatograph by the process de ned previously by Fatma et al. (2021).
Determinations of ATP-S Activity and the Content of S, Cys, GSH and Redox State The activity of ATP-S was measured using the Lappartient and Touraine method (1996). The determination of content of S, Cys and GSH was done by methods of turbimetric, Gaitonde (1967) and Gri th (1980), respectively described by Fatma et al. (2014). Supplementary File S1 contains the details of these procedures. The ratio of GSH/GSSG was used to calculate the redox status.

Assay of NR activity and N Content
Activity of nitrate reductase (EC 1.7.99.4) was evaluated by Kuo et al. (1982as described by Iqbal et al. (2012. Nitrogen content of leaves was computed by Lindner (1944) technique, and altered by Novozamsky et al. (1983). File S1 contains the details of these procedures.

Histochemical Staining Method for Visualizing the Presence of Superoxide and H 2 O 2
To visualize the presence of superoxide and H 2 O 2 in the test leaf samples, histochemical staining techniques using nitro blue tetrazolium (NBT) and 3, 3-diaminobenzidine (DAB) were utilized (Wang et al. 2011). Three leaves from each treatment were immersed in NBT (1.0 mg mL -1 ) solution made in phosphate buffer (10 mM; pH 7.8) at room temperature for six hours under light. Samples stained with NBT or DAB revealed blue or brown dots. The pigmented samples were soaked in concentrated ethanol and then run through a 70% ethanol lter. Snapshots of the leaf samples that had been cleaned were taken with a NIKON digital camera (COOLPIX110).

Scanning and Transmission Electron Microscopy
The technique of Daud et al. (2009) and Sandalio et al. (2001) with small modi cations was used to prepare leaf samples for scanning and transmission electron microscopy, respectively. File S1 contains the details of these procedures.
Statistical Analysis SPSS 17.0 for Windows was used to perform statistical analysis of variance (ANOVA), and the results were given as treatment mean SE (n = 4). The F-value was computed after an ANOVA was generated according to the experiment design. For the signi cant data, the least signi cant difference (LSD) was computed at p ≤ 0. respectively compared with the control. In plants exposed to 50 mM NaCl, application of 0.5 mM SA resulted in 15.1, 14.7, 20.7 and 12.7% increase in net photosynthesis, intercellular CO 2 concentration, stomatal conductance and chlorophyll content, respectively compared with salt treatment but lesser than the treatment of 2.0 mM SO 4 2under stress. The combined supplementation of 0.5 mM SA and 2.0 mM SO 4 2maximally increased net photosynthesis, intercellular CO 2 concentration, stomatal conductance and chlorophyll content under no stress or salt stress compared to 50 mM NaCl treatment (Table 1). PSII activity response was also similar to these treatments. Maximum increase of 18.9 % in PSII activity was noted with combined application of 0.5 mM SA plus 2.0 mM SO 4 2to the salt grown plants compared with control. Similarly, in 0.5 mM SA-supplied plants but grown without salt the increase in leaf area and plant dry mass was by 32.6, 45.1% in comparison with the control. When exposed to 50 mM NaCl, the application of 0  Effect of SA with S on the Chloroplast Ultrastructure under Salt stress Chloroplast ultrastructure was presented as TEM micrographs (Fig. 3). Micrographs showed that under no stress, chloroplasts had a normal shape with well-organized thylakoid systems (Fig. 3A), whereas disorganized thylakoids were seen in the treatment of 50 mM NaCl (Fig. 3B). The chloroplast ultrastructure of the plants receiving SA plus SO 4 2to NaCl-treated plants was signi cantly altered. As seen in Fig. 3C, chloroplast had a regular form with well-organized thylakoid systems and a signi cantly higher number of thylakoid stacks.

Effect of SA and S on Ethylene Evolution
Ethylene evolution decreased with SA, SO 4 2or their combination in salt stress. We observed maximum  Table 4).
Analysis of stomatal aperture by SEM also showed reduction in presence of NBD and the results were reversed with the application of ethephon or SA plus SO 4 2to salt treated plants as shown in the Fig. 5.
The image of the stomatal aperture was apparently visible under different treatments when SEM was done. Leaf samples under control conditions had normal stomata with the characteristic guard cells having stomatal aperture diameter of 6 µM ( Fig. 5 A-B), whereas the impact of salt stress on stomatal closure was clearly seen as the diameter of the stomatal aperture was of 1 µM (Fig. 5 C-D). The stomatal aperture improved with SA plus SO 4 2in presence of NaCl with opened stomatal aperture by 13 µM (Fig.  5 E-F). The application of ethephon counteracted the effects of NaCl for stomatal aperture (Fig. 5 G-H). However, stomatal aperture reduced in presence of NBD by closing the stomatal aperture size by 3 µM (Fig. 5 I-J), We observed in our study that individually both SA and S in the form of SO 4 2are effective in enhancing S-assimilation, photosynthesis and growth in salt stress. A common point in their alleviation strategy was ethylene because SA was inhibiting ethylene biosynthesis and S was reducing the oxidative stress by directly increasing GSH and reducing stress ethylene formation by directing the Cys to GSH and not to the ethylene pathway via Met. However, it was an interesting issue to discuss that what could be the effect of their combination and how will they in uence ethylene as this is still not discussed in any study. We observed that the combination was best in stress alleviation, and this could be explained with ethylene synthesis and signaling. In combination of SA and S, SA reduced stress ethylene formation while S increased S-assimilation and generation of ethylene by modulating its emission through the Met pathway (S-assimilation leads to Met, and SAM which is precursor for ethylene via ACC , SA caused greater reduction in ethylene but when they were applied in combination we observed a greater increase in ethylene evolution that was su cient to signal plants for increased antioxidative enzymes, S-assimilation, photosynthesis and growth in salt stress. Thus, in the second experiment we took the best dose of SA and SO 4 2combined treatment and compared it with salt and ethephon treatment. We observed signi cantly equal increase in S-assimilation, ethylene evolution, photosynthesis and growth in both the treatment suggesting ethylene to be the key molecule. The supplementation of ethephon to salt treated plants increased antioxidant activities signi cantly equal to the plants receiving salt with SA and SO 4 2- (Table 4). The results showed that the effects of SA in reversal of salt stress through S mediation also involved ethylene. The results on ethylene formation with exogenous ethephon to salt treated plants was signi cantly equal to SA plus SO 4 2in salt stress. The results were further substantiated using ethylene action inhibitor to con rm that it is ethylene signaling that affected the alleviation pathway. In the presence of NBD, salt stressed plants receiving SA plus SO 4 2exhibited decrease in the selected parameters for S-assimilation such as content of Cys, S, and GSH by 12, 13.5 and 11.9% respectively. Moreover, antioxidant enzymes activity was also minimal under NBD treatment with SA plus SO 4 2under 50 mM NaCl (Table 5). Plants receiving salt with SA and SO 4 2showed optimum ethylene evolution by 24.10% respectively compared to control which was reversed with NBD ( Fig. 6). Thus, there exists a cross-talk between SA and ethylene in reversal of salt stress in presence of S because S assimilation leads to formation of ethylene through S-adenosyl methionine and SA application induced S assimilation.

Discussion
Salicylic acid potentially regulates physiological and molecular mechanism of plants and affects S assimilation in the alleviation of the harmful effects of salt stress. So, the e ciency of SA in the alleviation of salt stress through S supplementation was determined in the present study. The roles and underlying mechanisms of SA in oxidative stress, S and N assimilation, antioxidant metabolism and photosynthetic characteristic are discussed. The individual SA or S application decreased the negative effects of salt stress, but maximum reduction resulted with combined application of SA plus S in both no stress and salt stress conditions. However, the e ciency of SA plus S in the alleviation of salt stress was due to the result of optimal ethylene formation. For this, the results were veri ed with the application of exogenous ethylene and its action inhibitor in salt stress. The present study showed that SA might be inhibiting stress ethylene, while S promoted S-assimilation to enhance optimal ethylene formation and ethylene sensitivity to affect the salt tolerance process.
In uence of SA and S in the Alleviation of Salt stress The role of SA in improving photosynthetic functions and growth in abiotic stressed plants has been widely reported (Nazar et al. 2011;Miura and Tada 2014;Gururani et al. 2015;Rasheed et al. 2020). In the present study, 50 mM NaCl-exposed plants applied with 0.5 mM SA and 2.0 mM SO 4 2− had signi cantly improved maximum chlorophyll content, PS II e ciency and gas exchange parameters. The involvement of SA in the synthesis of photosynthetic pigments, increase in PS II e ciency, rate of photosynthesis in abiotic stressed plants has been extensively studied . Enhancements in the rate of net photosynthesis and PS II e ciency were reported in SA (0.5 mM) supplied Vigna radiata under salinity exposure (Nazar et al. 2011 have shown that excess-S enhanced GSH production and improved photosynthetic e ciency and growth in salinity stressed plants in B. juncea. Yoshida and Noguchi (2009) also stated SA-mediated enhancements in S uptake and level of GSH in ozone-exposed Arabidopsis thaliana. The supplied SA-mediated protection of the photosynthetic machinery in salinity stressed B. juncea and Vigna radiata was also argued to involve increased ATP-S activity and serine acetyl transferase, and Cys and GSH content and decreased salinity-accrued oxidative stress (Nazar et al. 2015). An increased level of S-containing molecules such as Cys and GSH were previously shown associated with higher photosynthesis at varying levels, as seen here with 0.5 mM SA and 2.0 mM SO 4 2− (Wirtz and Droux 2005). Additionally, cowpea plans grown with low or optimal levels of SA showed increased net photosynthesis, up-regulated NR activity, improved chlorophyll content, carboxylation e ciency, normal thylakoid membranes, and light mediated reactions (Moharekar et al. 2003). Regarding The application of SA with SO 4 2− to NaCl-exposed plants increased the uptake of S and N through induced activity of their assimilatory enzymes ATP-S and NR in order to produce high S-containing compounds to be utilized in ROS-metabolism and thereby salinity-tolerance in the present study. Application of SA and  . Earlier, the exogenously supplied SA (0.5 mM) increased APX and GR enzymes activities, improved GSH content, and decreased leaf ROS and TBARS levels in salinity exposed plants (Nazar et al. 2011(Nazar et al. , 2015. Exogenously applied 0.5 mM SA-mediated up-regulation of the transcriptome level of antioxidant genes of key H 2 O 2 -metabolizing enzymes was suggested to protect Triticum aestivum against salinity stress in another study (Li et al. 2013). It is worthy to mention here that there occurs a close relation not only between the plant-GSH but also between GSH/GSSG redox state and with the plant SA-status. To this end, over-accumulation of SA increased GSH levels and also that of the decreasing control (GSH/GSSG ratio) (Mateo et al. 2006). Higher GR activity can restore the high ratio of GSH to GSSG that occurs under optimal growth conditions (Szalai et al. 2009). Earlier, the supply of 0.5 mM SA to 50 mM NaCl-exposed B. juncea brought signi cant enhancements in H 2 O 2 -scavenging APX activity and GSHregenerating (GR) enzymes (Nazar et al. 2015). Furthermore, increased GSH content maintained the appropriate functioning of AsA-GSH pathway enzymes, resulting in higher GR and APX activity when 0.5 mM SA was applied under salt stress (Nazar et al. 2011(Nazar et al. , 2015. Thus, it can be said that exogenously supplied SA-mediated plant health involved the control of NaCl-accrued oxidative stress via modulating O 2 •− -dismutation, H 2 O 2 -metabolizing, and GSH-regenerating enzymes; and the pool of GSH and its redox state. The effects of SA and S in the alleviation of salt stress were attributed to lowering stress ethylene to an appropriate level, and ethylene favorably controlled GSH production via control of ascorbateglutathione cycle enzyme activity and protected photosynthetic functions Sehar et al. 2021). Under salt stress, the greater reduced state (GSH/GSSG) generated as a result of ethylene-induced GSH synthesis safeguarded and enhanced photosynthetic performances and plant development ).

Involvement of Ethylene in SA and S-mediated Reversal of Salt stress
Ever since SA acts as an inhibitor of ethylene and S-assimilation is associated with ethylene synthesis , Nazar et al. 2015Fatma et al. 2016), as a result, the efforts of the present study were to determine the role of ethylene in the coordinated role of SA and SO 4 2− mediated salt tolerance.
Ethephon supplementation increases ethylene formation that helps in salinity tolerance (Jahan et al. 2021) and NBD is involved in inhibition of ethylene action. The use of both these ethylene modulators showed that ethylene synthesis and signaling are important regulators in salt tolerance effect by combined SA and SO 4 2− . Interestingly, the effects of ethephon and salt treatment were found to be signi cantly equal to the plants receiving salt with SA and SO 4 2− . However, when SA plus SO 4 2− receiving plants under salt stress were treated with NBD, inhibition in photosynthetic characteristics, stomatal behavior and growth occurred was observed. Such plants also showed reduced capacity of Sassimilation, activity of antioxidant enzymes and GSH content emphasizing that ethylene was essential to modulate the antioxidative enzymes and S-assimilation in salt stress and SA plus SO 4 2− treated plants.
Under salt stress, ethylene has been shown to maintain Na + /K + homeostasis and increased antioxidants to scavenge ROS. It also in uences nutrient uptake and enhances nitrate and sulphate assimilation (Riyazuddin et al. 2020). In Arabidopsis, both ethylene production and its signaling genes are implicated in salt tolerance (Yang andGuo 2018, Fricke 2004). Iqbal et al. (2012) reported the role of ethylene in increasing S and N-assimilation and thus photosynthesis in B. juncea cultivars differing in photosynthetic capacity. By binding to the ethylene receptors, NBD inhibited ethylene sensitivity and activity in such plants, resulting in no increase in N and S -assimilation or photosynthesis. NBD is one of the major chemical inhibitors of ethylene action (Iqbal et al. 2017).
We tried to investigate the interaction between ethylene, SA, and SO 4 2− in salt tolerance. Under stressful conditions, there is a strong relationship between SA and ACC, with SA inhibits ethylene production by restricting the conversion of ACC to ethylene and protecting plants from stress-related effects (Leslie and Romani 1986;Khan et al. 2014;Nazar et al. 2015). S-adenosyl methionine (SAM, ethylene productionprecursor) impacts ethylene biosynthesis, forming a strong relationship between S and ethylene (Masood et al. 2012;Fatma et al. 2016     Ethylene evolution in mustard (Brassica juncea L.) leaves at 30 days after sowing. Plants were treated with 0.5 mM SA and/or 2.0 mM SO42-in presence or absence of 50 mM NaCl. Data are presented as means ± SE (n = 4). Data followed by the same letter are not signi cantly different by LSD test at (p < 0.05). SA, salicylic acid.  NaCl. Data are presented as means ± SE (n = 4). Data are presented as means ± SE (n = 4). Data followed by the same letter are not signi cantly different by LSD test at (p < 0.05). NBD, norbornadiene; SA, salicylic acid.