Triacontanol regulates morphological traits and enzymatic activities of salinity affected hot pepper plants

Potential role of triacontanol applied as a foliar treatment to ameliorate the adverse effects of salinity on hot pepper plants was evaluated. In this pot experiment, hot pepper plants under 75 mM NaCl stress environment were subjected to foliar application of 25, 50, and 75 µM triacontanol treatments; whereas, untreated plants were taken as control. Salt stress had a significant impact on morphological characteristics, photosynthetic pigments, gas exchange attributes, MDA content, antioxidants activities, electrolytes leakage, vitamin C, soluble protein, and proline contents. All triacontanol treatments significantly mitigated the adversative effects of salinity on hot pepper plants; however, foliar application triacontanol at 75 µM had considerably improved the growth of hot pepper plants in terms of plant height, shoot length, leaf area, plant fresh/dry biomasses by modulating above mentioned physio-biochemical traits. While, improvement in gas exchange properties, chlorophyll, carotenoid contents, increased proline contents coupled with higher SOD and CAT activities were observed in response to 75 µM triacontanol followed by 50 µM triacontanol treatment. MDA and H2O2 contents were decreased significantly in hot pepper plants sprayed with 75 µM triacontanol followed by 50 µM triacontanol foliar treatment. Meanwhile, root and shoot lengths were maximum in 50 µM triacontanol sprayed hot pepper plants along with enhanced APX activity on exposure to salt stress. In crux, exogenous application triacontanol treatments improved hot pepper performance under salinity, however,75 µM triacontanol treatment evidently was more effective in mitigating the lethal impact of saline stress via controlling the ROS generation and increment in antioxidant enzyme activities.

. Triacontanol is a potential plant growth promoter and its foliar application has been reported to influence physiological and biochemical processes in plants under saline conditions 13,22 . Previous instances have proved that foliar supplementation of triacontanols at various growth stages resulted in improved productivity of wheat, rice, and cucumber 4,23,24 . In another study, triacontanol enhanced plant growth by modifying many metabolic processes facilitating water uptake, cell division, chlorophyll synthesis, photorespiration, photosynthesis; thereby, boosting the activities of a few key enzymes mineral nutrient status 2,23,24 . Moreover, the application of triacontanol has also been reported to enhance enzymatic and non-enzymatic antioxidants production to mitigate the negative effect of salt stress 4,17,25 . Keeping in view these facts, it is evident that triacontanol has positive impacts on plant growth even under abiotic stresses like salinity stress, and its role in comMercially and nutritionally important vegetables like hot pepper is yet to be explored. Therefore, the current study was designed to investigate the potential role of foliarly applied triacontanol in alleviating salt stress and improving the growth of hot pepper plants under salinity-induced oxidative stress.

Materials and methods
A pot experiment of hot pepper plants was conducted to evaluate the efficacy of foliar application of triacontanol under salt stress conditions. It has been confirmed that the experimental samples of plants, including the collection of plant material, complied with relevant institutional, national, and international guidelines and legislation with appropriate permissions from Institute authorities of Institute of Agricultural Sciences, University of the Punjab, Lahore Pakistan for collection of plant specimens. This study consisted of four levels (0, 25 Antioxidative enzyme activities and lipid peroxidation of hot pepper plants. Hot pepper leaves (500 mg) were homogenized in K 3 PO 4 buffer solution (pH 7.0) added with 1 mM ethylene diamine tetra-acetic acid and 1% (w/v) soluble polyvinyl pyrrolidone (PVP). Prepared solutions were centrifuged at 20,000g to separate the supernatant that was further used for the determination of superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), and ascorbate peroxidase (APX) activities. Superoxide dismutase activity (SOD) was assessed by calculating its capability to hinder the photo-chemical decline of nitro-blue tetrazolium chloride (NBT) 29 ; while Catalase (CAT) was measured by the procedure of Dhindsa et al. 30 where one unit CAT was specified as a change in absorbance of 0.01 units per min. The POD activity was calculated by the described procedure of Plewa et al. 31 and the activity of ascorbate peroxidase (APX) was analyzed by observing a reduction in ascorbic acid (ASA) with spectrophotometry 32 . Lipid peroxidation was determined by calculating malondialdehyde (MDA) from hot pepper leaves that were produced due to reaction with 2-thiobarbituric acid as scrutinized by Heath and Packer 33 . In short, supernatant that was prepared after a number of steps was taken and added to 20% TCA having 0.5% 2-thiobarbituric acid in 4 ml solution which was boiled for 30 min at 90 °C followed by centrifugation at 10,000×g and absorbance was detected at 532 and 600 nm.  34 . The ground tissue of hot pepper plants was added with 10% TCA and centrifuged to get supernatant for determination of ascorbic acid contents 35 . Soluble protein contents were measured using bovine serum albumin as a protein standard 36 . Proline contents of hot pepper plants samples were calculated by homogenized fresh leaf tissue (0.5 g) in 3% sulfosalicylic. Sample solutions prepared as by standard protocol were run at 520 nm and proline contents were determined from a standard curve 37 .

Statistical analysis.
Data for different parameters was analyzed in a factorial arrangement under complete randomized design (CRD) and results were interpreted using analysis of variance technique followed by LSD tests at a (0.05) significance level by using statistix 8.1.

Results
Growth attributes of hot pepper plants. It is evident from results that salt stress influenced plant growth and physio-biochemical attributes of hot pepper; while, maximum shoot length, root length, fresh/ dry biomass, and length were recorded in unstressed control plants. Plants subjected to 75 mM NaCl stress illustrated a decline in plant growth in terms of reduced shoot and root length, plant fresh/dry biomasses, and leaf area. Thus, exogenous application of triacontanol (25, 50, and 75 µM) significantly improved the salt tolerance of hot pepper plants and maintained better growth and biomass compared with plants in which foliar triacontanol was not applied. Among the triacontanol treatments, 75 µM triacontanol proved to be most effective followed by 50 µM and 25 to µM triacontanol in enhancing the shoot length and other growth parameters including plant fresh/dry biomasses and leaf area; whereas, root length was relatively longer in case of 50 µM triacontanol treatment (Table 1).
Gaseous exchange attributes of hot pepper plants. Unstressed  www.nature.com/scientificreports/ exposure to salt stress; whereas, the highest SPAD value 28.52 and carotenoid contents (5.28 mg g −1 FW) were recorded in unstressed hot pepper plants followed by triacontanol treatments as a foliar application of triacontanol treatments had mitigated adverse effects of salinity. Likewise, chlorophyll a and chlorophyll b pigments were also high in unstressed hot pepper plants followed triacontanol sprayed plants, Results revealed that foliar application of triacontanol (75 µM) showed maximum leaf chlorophyll content and significantly retained the highest SPAD value (23.40 mg g −1 FW) as well as produced maximum chlorophyll a (15.14 mg g −1 FW), chlorophyll b (5.07 mg g −1 FW) and carotenoid contents (4.84 mg g −1 FW) ( Table 3).
Antioxidative enzyme activities and lipid peroxidation of hot pepper plants. Moreover   www.nature.com/scientificreports/ cantly when hot pepper plants were exposed to 75 mM NaCl stress; although, hot pepper plants ameliorated the harmful effect of saline stress and maintained higher values of proline contents than control (Table 5).

Dicussion
Hot pepper is regarded as a sensitive to moderately sensitive crop to salt stress 15,38 . Growing hot pepper under saline conditions severely affects the growth and productivity of plants 14,17 . A decline in the growth of hot pepper plants grown in pots under saline stress was confirmed by our findings in Table 1. A decrease in growth and production of hot pepper plants might be due to restricted water absorption, decreased metabolic activities as a result of sodium or chloride toxicity, and specific nutrient deficiency produced via ionic intrusion 1,2 . However, foliar feeding of plant growth regulators can reduce such lethal impacts of saline stress on plants 39,40 . Our results that foliar application of triacontanol significantly improved growth attributes of stressed plants have concurred with the findings of Singh et al., 24 , where triacontanol treatment encouraged the growth of ginger plants under saline stress. It might be attributed to the synergetic role of triacontanol with gibberellic acid and cytokinins to regulate growth, metabolic processes, and yield of crops 41 . Moreover, triacontanol encouraged the development of second messenger 9-b-L(+) adenosine, which is similar to the cytokinins structure 42 that could have facilitated an increase in leaf area and photosynthesis of hot pepper correlated with a shoot and root length as well as their fresh and dry biomasses 43 . Gaseous exchange properties of hot pepper plants exposed to salt stress as presented in Table 2 were comparable with previous findings reported in different crops i.e. wheat and cucumber 2,23,45 . Impaired photosynthesis rate under salinity might be attributed to oxidative damage to imperative photosynthetic cells 2,46 or decline in stomatal conductance that ultimately restricts the availability of carbon dioxide to leaf tissues, resulted from an antagonistic imbalance of Na + ion on K + which is required for stomatal activity 45 . Exogenously applied triacontanol had significantly induced salt tolerance in hot pepper by positively modulating gas exchange properties as stated in rice crop under saline grown under saline conditions 47,48 . This improvement in gas exchange attributes by triacontanol, proved its well-established role in stomata regulation by up-regulating photosynthetic genes 49 , increasing CO 2 exchange rate 23 ; and enhanced rubisco activity which ultimately boosts photosynthesis 50 . Triacontanol treatments resulted in a rapid increase in activities of a specific secondary messenger like 9-b-L(−) adenosine, which could lead towards quick physiological responses 51 . Progressive impacts of triacontanol on photosynthesis rate may be due to improvement in the efficiency of photosystem II under saline environment and revealed that triacontanol improves stress tolerance in hot pepper by stabilizing photosynthetic pigments 52 .
Chlorophyll contents of hot pepper plants subjected to salt stress were significantly degraded as presented in Table 3; and a similar decline in chlorophyll content has previously been reported in hot pepper crops 1,53 . Salinity stress-induced accumulation of toxic ions and physiological water deficit in leaves delayed the chlorophyll biosynthesis and also accelerated the degradation of original chlorophyll 54 . However, exogenous application of triacontanol had a positive impact on chlorophyll pigments integrity, as in our results 75 µM triacontanol proved most effective in retaining the highest SPAD value as well as chlorophyll a, b, and carotenoid contents ( Table 3). The improved chlorophyll content due to foliar exposure to triacontanol is presumed to be associated with stability membrane strength, which remains intact in response to triacontanol under saline conditions. Enzymatic activities of SOD, CAT, POX, and APX were amplified hot pepper plants in response to salinity as well as foliar spray of triacontanol over the controls (Table 4); as, antioxidants are believed to have a key role in improving salt tolerance in plants 55 . The rise in antioxidant activities plays a vital role in the detoxification of ROS which leads toward the establishment of a balance between production and scavenging of ROS and prevents hot pepper plants from adverse effects of salinity. An increment in antioxidant enzyme activity under saline stress was also reported in tomato 12 and maize crop 1,2,23,56 . In this study, triacontanol-induced improvement in growth might be attributed to its influence on the actions of antioxidant enzymes, i.e., SOD, CAT, POX, and APX under salinity stress 23,57 . Our results verified that salinity-induced oxidative stress produced H 2 O 2 content and modulated lipid peroxidation in terms of enhanced malondialdehyde (MDA) content in hot pepper under saline condition (Table 4); as increment in MDA content was also reported by Ozdemir et al. 53 in hot pepper. Application of triacontanol reduced lipid peroxidation and H 2 O 2 production significantly under saline stress compared to non-sprayed plants and similar observations were reported by Verma et al. 58 where triacontanol decreased MDA in peanut crops. Triacontanol hampered MDA and H 2 O 2 production could be related to increased antioxidant activity or enhanced antioxidant production as observed in opium poppy by Khan et al. 17 . www.nature.com/scientificreports/ Imposition of salt stress to hot pepper destabilized membrane integrity and resulted in increased electrolyte leakage content as shown in hot pepper plants (Table 5), higher electrolyte leakage in salinity-induced hot pepper plants could be due to the production of reactive oxygen species that in turn might have caused oxidation of phospholipids molecules in the cell membrane. Triacontanol foliar application reduced electrolyte leakage due to enhanced water uptake, augmented cell division and membrane stability by reducing oxidative stress 2,59 . Triacontanol plays an effective role in upregulating multiple physiological and biochemical pathways in plants 49 . An increment in proline contents was observed in the current study; as it is well known that endogenous level of free proline increases under saline conditions 54 ; whereas, the concentration of soluble proteins and ascorbic acid contents of hot pepper plants were reduced upon induction of salt stress 60 . Proline has been reported to induce salt tolerance due to its role in osmotic adjustment and stabilizing the structure of organelles and macromolecules 61 . Our results illustrated that hot pepper plants treated with exogenous triacontanol showed improved leaf proline contents as well as soluble protein contents; these findings are in agreement with the observations recorded in green gram and sweet basil crops grown under saline environment 1,2,62,63 .

Conclusions
Salt stress exhibited significantly reduced plant growth and development in unsprayed hot pepper plants. All concentrations of foliar triacontanol supplement were proved beneficial for stress alleviation in hot pepper plant; however, triacontanol at 75 µM was more beneficial as it significantly improved hot pepper quality attributes like plant fresh and dry biomasses, gaseous exchange properties, activities of antioxidant enzymes, cell membrane integrity, proline, ascorbic acid, and soluble protein. Hence, it was concluded that 75 µM was the most beneficial triacontanol treatment to alleviate 75 mM NaCl stress in pot-grown hot pepper plants.