Role of salicylic acid in regulating ethylene and physiological characteristics for alleviating salinity stress on germination, growth and yield of sweet pepper

Experiments description

During a preliminary study, effects of 0, 20, 40, and 60 mM NaCl salinity were assessed on growth and yield of sweet pepper. Owing to 60 mM NaCl of salinity, we found brutal reductions in growth and yield of sweet pepper (Preliminary study part of Ph.D Thesis). The objective of the current studies was to alleviate detrimental effects of 60 mM NaCl salinity on morphological and physiological aspects of sweet pepper by SA and determine optimal dose of SA for homeostasis of ethylene levels in bell pepper Yolo Wonder. Consequently, treatments of 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 mM SA were used for seed priming and foliar application. Each treatment was repeated thrice during lab study and four times during pot study. The cultivar of sweet pepper used was Yolo Wonder (F-1 progeny with 85% germination percentage distributed by California Production Seeds treated by producer with Thiram 50 MP dyed fungicide).

After surface sterilization with ethanol (70%), seeds were soaked in different SA solutions and deionized water then dried. The 30 primed seeds were placed over three round Whatman filter paper No. 42 (Schleicher and Schuell) in transparent petri-plates, covered by lid fitted with rubber septa and then sealed tightly with silicone gel and Para film tape (Para film “M” Laboratory Film, Pechiney Plastic Packing, Chicago, IL 60631). In each plate, 7 ml of 60 mM NaCl solution was injected through rubber septa on the lid for inducing salinity stress. All the activities were performed in laminar flow hood. After injecting water and sealing, all the plates were shifted to an incubator (Sanyo MIR 253) at 25 ± 2 °C day/night temperature with 14 h photoperiod using cool white fluorescent light.

Germinated seeds were counted at an interval of 24 h of incubation. At 312 h of incubation, the gas samples taken from each plate were analyzed for ethylene. At 312 h of incubation, seedling leaves were also sampled randomly for chlorophyll a + b and SA assay and stored at −80 °C. Seedling growth was estimated on the basis of root and shoot length and weight.

Pot trial

A pot study was also conducted with the objective to test the efficiency of SA spayed on foliage for alleviating salt stress of 60 mM NaCl on growth and yield of sweet pepper. For this purpose, thirty days old plants were transplanted in pots. The soil was sandy clay loam, alkaline (pH 8.0), deficient in total N (0.03%) and available P (5.06 mg kg⁻¹ soil). An optimal supply of N (30 mg kg⁻¹ soil), P (60 mg kg⁻¹ soil) and K (80 mg kg⁻¹ soil) was maintained using urea, diammonium phosphate, and sulfate of potash, respectively. After 15 days of transplanting, treatments of SA were sprayed on plants whereas the next spray of SA treatments was carried out 30 days after transplanting. To nullify the effect of spray, deionized water was also sprayed on plants in control. Each pot was irrigated at or near field capacity by maintaining EC at 6 dS m⁻¹. All plant protection measures were adopted throughout the entire crop period.

Growth-related morphological parameters such as number of branches, number of flowers, fresh and dry weight (g), and leaf area (cm²) were noted at 50 days after transplanting. Leaves were also sampled randomly for mineral and biochemical analysis. Fully grown fruits were picked and weighed at maturity. Leaf area was measured using a leaf area meter and then leaf area index (LAI) was also calculated. For assessing variations in chlorophyll contents, portable Chlorophyll meter (SPAD-502) was used.

Estimation of physiological processes

Infrared Gas Analyser (IRGA, model ADC, Bioscientific Ltd., England) was used to measure photosynthetic rate (P_N), Stomatal conductance (g_s) and intercellular CO₂ concentration (C_i) in fully expanded new leaves at 1100–1200 h when above the plant canopy photosynthetic active radiations (PAR; 1,060 µmol m⁻² s⁻¹) were present. The inside temperature of the leaf cuvette was set at 30 ± 2 °C. The light responses curves were carried out at ambient CO₂ concentrations (300–350 µmol mol⁻¹). Instantaneous water use efficiency (WUE) was computed as the ratio of photosynthetic rate to transpiration (Polley, 2002).

Determination of biochemical attributes

The ethylene was analyzed from gas samples collected from Petri plates using Gas Chromatography (Shimadzu, 2010) fitted with flame ionization detector (FID) and a capillary column i.e., Porapak Q 80-100 (Khalid et al., 2006).

For assay of SA in seedling, Trichloroacetic acid (TCA) based method earlier reported by Zhang et al. (2003) was used. Salicylic acid from seedling was extracted using ethyl ether extract. The residue obtained from ethyl ether exact was dissolved in a 0.02 M ammonium acetate in 50% methanol (pH 3.2). Then, 10 mL of extract + 10 mL of NH₄Fe (SO₄)₂ was mixed and shaken well and centrifuged at 2,500 rpm for 3 min. The reading of color was recorded at a wavelength of 540 nm on double beam spectrophotometer. De-ionized water instead of the sample was run as blank.

Superoxide dismutase (SOD) activity was measured using the photochemical method (Winterbourn et al., 1975). Assays were carried out under illumination. One unit SOD activity is defined as the amount of enzyme required to cause 50% inhibition of the rate of p-nitro blue tetrazolium chloride reaction at 560 nm.

Lipid peroxidation (POD) was assayed by spectrophotometry using TBA-MDA assay (Minotti & Aust, 1987). Lipid peroxides were extracted with 5 mL of 5% (w/v) metaphosphoric acid and 100 mL of 2% (w/v in ethanol) butyl hydroxytoluene. An aliquot of the supernatant was reacted with thiobarbituric acid at 95 °C and cooled to room temperature. The resulting was extracted with 1-butanol.

Total soluble sugar contents (TSS) were also assayed using the method of Dong et al. (2001). One to two drops of the supernatant, as prepared above, was placed on the prism of the digital refractometer (Model ATAGO, Japan) and TSS was reported as °Brix (noted in percentage).

Chlorophyll pigments (a, b) from leaves were extracted by the help of acetone (85%), measured at 644 and 663 nm by spectrophotometer (Metzner, Rau & Senger, 1965). Finally, chlorophyll was calculated using the following equations: ${\displaystyle \text{Chlorophyll-a}=10.3A663-0.918A644\left(\mathrm{\mu }{\text{g g}}^{-1}\right)}$ ${\displaystyle \text{Chlorophyll-b}=19.7A644-3.870A663\left(\mathrm{\mu }{\text{g g}}^{-1}\right).}$ For electrolyte leakage (EL), uniform leaf discs were added to 5 mL of deionized water in a test tube which was shaken for 4 h at 25 ± 1 °C. The EC of the solution containing leaf discs was measured before and after autoclave and finally, electrolyte leakage was calculated by using the formula: ${\displaystyle EL\left(\text{\%}\right)=\frac{\text{EC of solution before autocalve}}{\text{EC of solution after autocalve}}\times 100}$

Nutrient analysis, uptake and use efficiency

Plant samples were grounded and digested using 20 mL of H₂SO₄ (conc.) and 8 g digestion mixture (K₂SO₄: FeSO₄:CuSO₄ = 10:1:0.5) for each sample (Wolf, 1996). Nitrogen was analyzed according to Jackson (1962) while phosphorus was determined using vanadate-molybdate spectrophotometric procedure (Jones, Wolf & Mills, 1991). For potassium, the method given by Chapman & Pratt (1961) was followed. Nutrient uptake and use efficiency were calculated using the following formula: ${\displaystyle \text{Nutrient uptake}=\frac{\text{Nutrient conc.}\text{in plant}\left(\text{\%}\right)\times \text{oven dried weight}}{100}}$ ${\displaystyle \text{Agronomic NUE}\left(\mathrm{g}∕\mathrm{g}\right)=\frac{\text{Yield of treated plants}-\text{Yield of control plant}}{\text{Nutrient applied}}}$ ${\displaystyle \text{Recovery efficiency}=\frac{\text{Nutrient uptake increment per unit nutrient applied}}{\text{Nutrient applied}}\times 100}$

Statistical analysis

Data was analyzed using analysis of variance while mean values were compared using Fisher’s least significant difference (LSD) tests at 5% probability level (Steel, Torrie & Deekey, 1997).

Germination rate

Monitoring of seed germination at interval of every 24 h disclosed that signs of germination appeared at 120 h of incubation. Figure 1 shows variations in seed germination from 120 to 216 h of incubation. It was observed that salinity of 60 mM NaCl severely affected germination of seed in control treatment. The reported 85% germination of sweet pepper cv. Yolo wonder by seed supplier was reduced to 75% due to salinity of 60 mM NaCl (Table 1, Fig. 1). However, treatments of SA showed an increase in germination rate even under salinity of 60 mM NaCl (Fig. 1). Figure 1 also depicts that the seed germination improved gradually with increasing units of SA till 0.3 mM SA but onward application of SA i.e., 0.4, 0.5 and 0.6 mM SA inhibited the seed germination (Fig. 1). Treatment of 0.2 and 0.3 mM SA appeared as optimal dose as all seeds of these treatments germinated up to 312 h of incubation, compared to control treatment (Table 1).

Morph physiological characters

Salinity significantly reduced the morphological characteristics of seedlings such as length, biomass, leaf area, chlorophyll contents etc. in control treatment (Table 1). Contrary to control, the seedlings in SA treatments were more vigorous, longer in root and shoot length showing no symptoms of salinity stress (Table 1). However, changes in growth parameters of seedling varied with variations in exogenous SA rates. Application of 0.05 to 0.3 mM SA ameliorated 60 mM NaCl injury on seedling growth and vigor among SA treatments. These rates of SA increased biomass, root and shoot length of seedling by 15–20% in each, respectively compared to control treatment under salinity of 60 mM NaCl (Table 1). However, SA >0.3 mM suppressed the growth and gradually reduced biomass, root and shoot length with gradual increase in SA concentration (Table 1). It was concluded that application of SA upto 0.3 mM might be significantly reduced the detrimental effect of salinity induced by 60 mM NaCl.

A significant reduction in plant growth and yield occurred in control treatment due to salinity of 60 mM NaCl. The plants of control treatment were stunted with less number of branches, leaves, flowers, and fruits per plant, etc (Table 2). Salinity also severely affected leaf development of these plants and thus very low values of leaf area were observed in these plants. Contrary to it, the plants of SA treatments showed significant improvement in the aforementioned growth variables due to the physiological role of SA under salinity stress. Application of 0.3 mM SA appeared to be an optimal concentration of SA for sweet pepper in order to ameliorate salinity stress on its growth and yield (Table 2). Exogenous SA effectively combated salinity stress in sweet pepper and thus SA treated plants showed an increase in branches per plant by 5–15%, leaves per plant by 10–30%, leaf area by 9–25%, number of flowers per plant by 8–24% and finally fruits per plant by 3–18% (Table 2). But, reductions in these parameters were observed in plants treated by SA > 0.3 mM (Table 2).

Leaf area index and dry biomass are effective indicators of morphological characteristics. Higher values of LAI and biomass reflect improvements in morphological characteristics of plants. Results explored that salinity significantly reduced LAI and biomass but exogenous SA reversed these changes (Fig. 2). The highest LAI and biomass per plant were found in plants treated by 0.3 mM SA (Fig. 2). Figure 2 also revealed gradual increase in LAI and biomass with an increment of SA till 0.3 mM and suppression in LAI and biomass on application of SA > 0.3 mM doses (Fig. 2).

SA based ethylene regulation and electrolyte leakage

Different researchers reported dramatic increases in ethylene biosynthesis in plants under salinity stress (Morgan & Drew, 1997). However, ethylene can be regulated through endogenous SA. Salicylic acid regulates ethylene in plants by two ways; (i) Firstly by suppressing ethylene forming enzyme (EFE) and/or ACC synthase (Mattoo & Suttle, 1991; Yang & Hoffman, 1984) and (ii) Secondly by triggering biosynthesis of spermine, spermidine and putrescine and/or polyamines from SAM (Mittler et al., 2011; Freschi, 2013).

The data related to ethylene evolution from seedlings and endogenous SA is presented in Table 3. Results reveal that maximum ethylene evolution (3.19 nmole plate⁻¹) was found in control treatment, showing comparatively more endogenous ethylene biosynthesis. But contrary to control treatment, ethylene evolution from SA treated treatments ranged from 2.23 to 3.12 nmole plate⁻¹ (Table 3). Less ethylene evolution in SA treatments reveal the suppression of endogenous ethylene biosynthesis due to physiological role of SA reported under abiotic stresses (Table 3).

Electrolyte leakage is considered as one of the most brutal effects of abiotic stresses particularly of salinity stress due to physiological drought and specific ions toxicity. Significant variations in EL were observed due to varying SA treatments compared to control treatment under salinity stress. The least EL was found in treatment of 0.2 mM SA, followed by 0.3 mM SA (Table 3).

A relationship among EL, endogenous SA in seedlings and ethylene was established using Pearson’s correlation. Pearson’s correlation showed negative correlation between ethylene evolved and endogenous SA in seedlings (Fig. 3A). Ethylene biosynthesis dropped steadily as endogenous SA escalated (Fig. 3A). Likewise, Person’s correlation also indicates a negative relationship between endogenous SA in seedlings and EL (Fig. 3B), while a positive correlation between ethylene evolution and EL (Fig. 4).

Photosynthetic activity and water use efficiency

Salinity directly affected photosynthetic machinery in sweet pepper by inducing physiological drought stress. Soil salinity of 60 mM NaCl brutally affected net assimilation rate, stomatal conductance, and transpiration rate. The photosynthetic rate was observed 11.84 µmol m⁻² s⁻¹ in control which increased with application of 0.05 to 0.3 mM SA to 15.09 µmol m⁻² s⁻¹ (Table 4). It was also observed that the application of SA ≥ 0.4 mM reduced photosynthetic rate. The similar effect of SA application was observed on Stomatal conductance and transpiration rate in sweet pepper. Maximum transpiration (3.98 nmole m⁻¹ s⁻¹) was recorded in plants without SA application which gradually decreased to 2.91 nmole m⁻¹ s⁻¹ with gradual increase in SA concentration (Table 4). Likewise, salicylic acid also positively regulated the stomatal conductance, indicating physiological improvement of photosynthetic machinery under salinity stress.

Salinity reduced instantaneous WUE due to the osmotic effect of 60 mM NaCl in control treatment (Table 4). Data related to instantaneous WUE discloses that plants treated SA had more instantaneous WUE than untreated plants (Table 4). The treatment of 0.05–0.3 mM SA was found more effective than others on the basis of instantaneous WUE in sweet pepper under salinity stress.

Figure 5 collectively presents the trend of improvements in fruit yield and instantaneous WUE under salinity stress. Compared to control, plants producing higher fruit yield per plant had corresponding higher instantaneous WUE in SA treatments (Fig. 5).

Biochemical attributes regulation by SA

Salinity generally cracks down defensive system of plants either by hormonal imbalance or by inducing oxidative stress. Salicylic acid is one of those plant growth regulators that regulate antioxidant enzymes under salinity stress. It was observed that SA alleviated salt injurious effects on germination, morphological and physiological characters of sweet pepper by regulating SOD, POD, and TSS. Results of lab study revealed that salicylic acid was also protected chlorophyll a + b from injurious effects of salinity and thus led toward more accumulation of chlorophyll a + b in leaves of treated seedlings compared to control (Table 5). Among different tested rates, 0.2–0.3 mM SA were found more effective for improving chlorophyll contents (Table 5). Table 5 also depicts that SA treated seedlings showed higher SOD activity with high TSS values compared to untreated seedlings.

Results of pot study revealed that exogenous SA significantly affected the activity of SOD and POD under salinity of 60 mM NaCl (Fig. 6). The POD activity was significantly higher in untreated plants (control) while lower in SA treated plants (Fig. 6). The POD activity first gradually reduced with increment of SA concentration till 0.3 mM. Afterwards it was started to rise (Fig. 6). Similarly, SOD activity was low in plants of control treatment which was improved significantly in plants where salicylic acid was applied. The SOD activity gradually improved with the increasing rate of SA but up to 0.3 mM (Fig. 6).

Fertilizer use efficiency

Soil salinity had strong effect on K⁺ use efficiency as Na⁺ ions competes K⁺ ions for major binding sites in key metabolic processes in the cytoplasm and inhibits >50 enzymes activity, being activated by K⁺. In nutshell, K deficiency results in decrease in photosynthetic CO₂ fixation and assimilates transport and utilization. Resultantly, lower K use efficiency (agronomic and recovery) was found in plants of control treatments (Table 6). SA treatments significantly improved K use efficiency by ameliorating salinity stress. However, the decrease in K content is due to K-efflux induced by Na⁺ (Bojórquez-Quintal et al., 2014).

Salinity also had a strong effect on N and P use efficiency. But SA improved N and P use efficiency by ameliorating salinity stress (Table 6). Table 6 clearly depicts that agronomic use efficiency of N, P and K was 54, 99 and 89 g g⁻¹, respectively in control due to strong effect of salinity which increased up to 117, 212 and 190 g g⁻¹ due to SA application @ 0.3 mM, respectively.