Exogenous salicylic acid and ferulic acid improve growth, phenolic and carotenoid content in tomato

Salicylic acid (SA) and ferulic acid (FA) are considered phenolic com­ pounds that act as elicitors due to their regulatory functions on plant growth, development, metabolic and physiological responses in plants. The aim of this research was to evaluate the effect of SA and FA on growth, fruit quality and synthesis of secondary metabolites in tomato (Solanum lycopersicum cultivar Santa Clara). The experiment was conducted in pots in a greenhouse. The appli­ cation of SA and FA was performed at concentration of 1.0 mmol L­1 alone and in combination, with water treated plants as control. Exogenous application of SA and FA either alone or in combination (SA + FA) resulted in increases in bio­ mass accumulation and chlorophyll contents in tomato plant; and soluble sugar, total polyphenol, flavonoids, lycopene and β­carotene contents in fruits. It was concluded that application of SA and FA resulted in higher production and concentration of secondary compounds in tomato.


Introduction
Tomato (Solanum lycopersicum L.) belongs to the Solanaceae family and it is considered as lowcalorie food. It is a good source of vitamins A, C and E, and mineral salts (Adalid et al., 2010;Kazemi, 2014). In addition to vitamins and minerals, tomato has a high lycopene and βcarotene content, which has antioxidant and anticancer properties. Carotenoids is naturally present in many fruits and vegetables and plays an important role as a functional food when consumed as part of the diet, producing specific health benefits such as reducing the risk of various diseases (MartínezHernández et al., 2016;Mehta et al., 2018) and polyphenols (flavonoids, flavanones and flavones) are also present in significant amount in tomato acting as antioxidant, antimutagenic, antiproliferati ve, antiinflammatory and antiatherogenic activities (Martí et al., 2016;Chaudhary et al., 2018).
Salicylic acid (SA), a natural plant hormone, act as an important sig nalling molecule triggering tolerance against abiotic and biotic stresses (HernándezRuiz and Arnao, 2018;Gorni et al., 2020;Gorni et al., 2021). SA plays a significant role in many physiological and biochemical processes of the plant, being able to act as growth regulators and also as an abiotic elicitor capable of increasing the synthesis of secondary compounds beneficial to human health (Javanmardi and Akbari, 2016).
Ferulic acid (FA) is a phenolic compound syn thetized from the metabolism of phenylalanine and tyrosine in plants. It is an important biological and structural component of the plant cell wall and it accumulates in soil and influences plant growth (Li et al., 2013;Paiva et al., 2013). FA is considered as non enzymatic antioxidant and acts under stress to elimi nate free radicals produced in plants (Andreasen et al., 2001;Engwa, 2018). Studies on the exogenous application of FA in plants have demonstrated that this compound acts as an important regulator of sev eral physiological processes related to plant growth and mitigating stress, such as, stomatal closure, cell division, membrane permeability, photosynthesis, respiration and many other metabolic processes (Santos et al., 2008;Li et al., 2013;Singh and Deen, 2014;Hussain et al., 2017;Cheng et al., 2018).
However, exogenous SA (Kazemi, 2014) and FA (Singh and Deen, 2014) applications were effective in inducing the growth and formation of secondary metabolites in tomato plants. Therefore, it is suggest ed that the application of SA and FA may result in combined effects of growth promotion and induction of biosynthetic pathways of secondary metabolism. Previous studies have revealed the hormonal and eliciting action of SA and FA (1.0 mmol L 1 ) in tomato plants (Hussain et al., 2017;Kumar et al., 2017). In this context, the application of SA and FA can improve the productive performance and the biosyn thesis of secondary compounds in tomato, making possible an increase in the commercial value of this crop, reaching greater market competitiveness. In this study we evaluated the effect of leaf spray of SA and FA either alone and in combination on the growth, yield and secondary compounds in tomato cultivar Santa Clara.
Trademark tomato cultivar Santa Clara seeds were placed in a germination tray and after the training period (30 days) they were planted in 18 L pots con taining soil. Soil samples were collected and submit ted to chemical analysis according to Van Raij et al. (2001) (Table 1) and was corrected by applying lime stone to increase the saturation of bases to 80% (40.0 g pots 1 ), potassium chloride (3.0 g pots 1 ) and simple super phosphate (20.0 g pots 1 ). For fertiliza tion with micronutrients it was added Yoorin Master 1 Si ® (granulated) (Si: 10%, B: 0.1%, Mn: 0.3%, Cu: 0.05% and Zn: 0.55%) (1.5 g pots 1 ), according to the recommendations of Bulletin 100 (IAC) for tomato species. The pots were irrigated by sprinklers per day at 8 a.m. and 5 p.m. in order to keep the soil mois ture and ensure the availability of water throughout the experimental period.
The application of salicylic acid (SA: 138.121 g mol 1 ) and ferulic acid (FA: 194.18 g mol 1 ) was made for 3 consecutive days after having reached 20 days from transplant. Foliar applications of SA and FA at a dose of 1.0 mmol L 1 alone and in combination, and water treated plants were used as control. SA and FA treat ments were carried out by spraying the shoots of the plants with waterbased solutions supplemented with Agral® (50 μL L 1 of solution) until the drop point (10 mL per plant), as follows: T1 plants sprayed only with water (Control); T2 applications of 1.0 mmol L 1 SA (SA); T3 applications of 1.0 mmol L 1 FA (FA); and T4 applications of 1.0 mmol L 1 SA + 1.0 mmol L 1 FA (SA + FA) by spraying the plants for three consecutive days. Plants were harvested 90 days after transplanting the seedlings by collecting leaves, roots and fruits.

Plant growth
The effect of SA and FA on the plants were evalu ated based on the fallowing: leaf area (cm 2 ), number of leaves per plant, number of fruits per plant, plant height (cm), dry weight of shoot and roots (g plant 1 ) and mean fruit weight (g plant 1 ). The leaf area was assessed using ImageJ ® Software (Powerful Image Analysis) with 5 plants per treatment. The number of leaves and fruit per plant was determined by manual counting, considering fully expanded leaves and fruits at harvest point. The plant height was deter mined by measuring tape. The shoot and root dry weight were determined after drying in an oven with air circulation at 70°C until constant weight.

Chlorophyll content
Chlorophyll content (μg mL 1 ) was determined spectrophotometrically following extraction in aceto ne, according to the method of Lichtenthaler (1987). Fresh leaf tissue (0.6 g) was added in 7 mL of 80% acetone, the tubes were shaken and left to stand for 30 minutes. The readings were performed in a spec trophotometer at λ 663 and λ 645 nm.

Total soluble sugar content
Total soluble sugar content in fruit was determi ned according to the method described by Dubois et al. (1956) using glucose as the standard. Fresh tissue (0.1 g) was added in 10 mL of 80% ethanol, and mac erated in a mortar and left to rest for 30 min. The analyzes followed with the addition of phenol (5%) and sulfuric acid (98 N). The readings were per formed in a spectrophotometer at λ 490 nm.

Preparation of fruit tomato extract
Determinations of total polyphenolic compounds and total flavonoid were obtained from the tomato juice. The fruits were ground in a low speed food pro cessor (3000 rpm) for two minutes and passed in 2 mm sieves.

Total polyphenols contents
Total polyphenols contents were determined spectrophotometrically (λ 765 nm) by FolinCiocalteu method with modification described by Stagos et al. (2012). The total polyphenols contents were report ed based on micrograms of gallic acid equivalents per milliliters (μg GAE mL 1 ). Gallic acid solutions were prepared with concentrations of 25 to 500 μg mL 1 in absolute ethyl alcohol. The assays were performed in triplicate.

Total flavonoid content
Total flavonoid content was determined spec trophotometrically (λ 510 nm) by the method of Yao et al. (2013). The total flavonoids contents were reported based on micrograms of rutin equivalents per milliliters (μg RE mL 1 ). Rutin solutions were pre pared with concentrations of 25 to 500 μg mL 1 in absolute ethyl alcohol. The assays were performed in triplicate.

Carotenoid content
Carotenoid content were extracted from fresh tomato samples after homogenisation of whole fruits. The homogenised sample was mixtured with chloroform/acetone/ethanol (2:1:1, v/v/v) (Sadler et al., 1990). For the determination of lycopene and β carotene, the absorbance was read at λ 470 and λ 450 nm. Carotenoid content were determined according to the equation described by Craft and Soares (1992), using the molar extinction coefficient of 3450 for lycopene, and 2592 for βcarotene and expressed in micrograms per grams (µg g 1 ). The assays were performed in triplicate.

Statistical analysis
The experiment was arranged in a completely ran domized design with four treatments and five repli cations per treatment. The ShapiroWilk test was used to ensure the normality assumption and the homogeneity of variances. The data were submitted to analysis of variance (p≤0.05) and Tukey test (p≤0.01) using the SISVAR software. The results were presented by means of the treatments and standard error.

Results
The results indicated that SA and FA both caused a significant increase of growth in tomato ( Table 2). The exogenous application of 1.0 mmol L 1 SA resul ted in increasing in the accumulation of root dry weight by 61% and total dry weight by 41.3% compa red to control plants. The application of 1.0 mmol L 1 FA resulted in increasing in shoot dry mass by 52.3% and total dry mass by 52% compared to control plants. However, the interaction between SA + FA resulted in increasing in shoot dry mass by 51.5%, root dry mass by 146.37% and total dry mass by 66.6%, when compared to control plants. Fruit weight increased by 16.5% in plant treated with 1.0 mmol L 1 FA.
The exogenous application of SA + FA resulted in increasing the leaf area by 47% and number of leaves by 204.3% compared to control plants (Table 3). Application of SA and FA showed increasing of 101.8 and 167.5% for the number of leaves, 70.2 and 68.3% for the number of fruits, when compared with con trol plants (Table 3). The plant height treated with SA and FA was not changed in relation to control plants.
Chlorophyll a content (Table 4) observed in toma to plants treated with SA, FA and the interaction with SA + FA were significantly higher by 26, 12 and 27.6%, respectively compared to control. Chlorophyll b and total chlorophyll decreased with the applica tion the elicitors (Table 4). The total soluble sugar content in fruits, increased up to 36% in 1.0 mmol L 1 SA and 163% in 1.0 mmol L 1 FA treated plants when compared to the control.
The exogenous application of SA, FA and the inter action with SA + FA resulted in increasing in the accu mulation of total polyphenols of 20.4, 110.3 and 12%, in relation to control plants (Table 5). Total flavonoid content in tomato were 81, 107 and 37% higher in SA, FA and the combined with SA + FA treatments when compared to control plants, respectively. In relation to carotenoid content plants treated with SA also presented increases of 157.3% (lycopene), and 157.7% (βcarotene). The combination of SA+FA showed increases of 65.25% (lycopene), and 65.3% (βcarotene), respectively, in comparison to the con trol (Table 5). Table 3 Effect of salicylic acid and ferulic acid on leaf area, number of leaves per plant, number of fruits per plant and plant height in tomato Table 4 Effect of salicylic acid and ferulic acid on the chlorophyll and fruit total soluble sugar contents in tomato

Discussion and Conclusions
Tomato plants treated with SA and FA presented higher total biomass ( Table 2). The effect of elicitors plays a key role in growth regulation, plant develop ment and these findings can be associated with the changes in hormone functions or the improvement of photosynthesis and carbohydrate accumulation in plants (Hussain et al., 2017;Gorni et al., 2020). The foliar application of SA and FA trigger a greater cellu lar activity that result in a larger number of leaves and leaf area, thus presenting a larger photosynthetic surface, improving the physiological processes (Gorni and Pahceco, 2016;Hussain et al., 2017). Regarding the increase of leaf area, number of leaves, number of fruits and fruit weight, our results showed that the application of SA and FA can alter the growing pat tern, in tomato plants (Table 3). The application of SA and FA increased the growth of leaves and roots, wit hout changing the plant height (Khan et al., 2003;de Carvalho et al., 2020). Biomass gains as an effect of SA application were also observed in Achillea millefolium (Gorni and Pacheco, 2016), Foeniculum vulgare (Gorni et al., 2017) and Mentha spicata (Kundu et al., 2018). Increase in biomass, were also observed after FA application in plants of Arabidopsis thaliana (Reigosa and PazosMalvido, 2007), Pisum sativum (Orcaray et al., 2011) and Cucumis sativus (Li et al., 2013).
Pigments are the essential components for growth and development and they can determine the health status of plants (Hussain et al., 2017;Gorni et al., 2020). Based on our results, the applica tion the elicitors SA and FA alone or in combination suggest an increase in chlorophyll a (Table 4). On the other hand, the application of SA and FA did not affect the chlorophyll b and total chlorophyll content, which directly reflected in the plant height values ( Table 3). Anway, other studies conducted on diffe rent species reported increases in chlorophyll a, chlo rophyll b and total chlorophyll contents consequen ces of SA treatment (Kazemi, 2014;Chakraborty et al., 2016) and in plants sprayed with FA (Zhu and Wakisaka, 2018). Positive effects of SA and FA on the physiological processes of tomato suggest that these elicitors may be associated with the regulation of several essential primary metabolic processes, inclu ding synthesis of chlorophylls (Zhu and Wakisaka, 2018;Gorni et al., 2020). The positive effects of the application of SA and FA on the physiological processes of tomato plants were also reflected in higher soluble sugar content (Table 4). Sugar is one of the ingredients in tomato (Hafeznia et al., 2014), and it is an important organic solute with low molecular weight in higher plants. The sugars which are accumulated under stress con ditions or as consequence of treatments with SA or FA, keeping osmotic regulation and turgor inside the plant (Orcaray et al., 2011;Gorni et al., 2020). Results show that application of SA also resulted in increa sing the total soluble sugar content in tomato plants (Kazemi, 2014), and chamomile (Zarinkamar et al., 2013); similarly to application of FA in pea (Orcaray et al., 2011) andsoybean (Ferrarese et al., 2001).
Polyphenols and flavonoids provide many physio logical functions for plant survival and are of funda mental importance for plants adaptation (Verma and Shukla, 2015;Mehta et al., 2018). Our results suggest that the application of SA and FA may modulate and alter the concentration of polyphenolic compounds (Table 5) in tomato plants. Studies report that the application of SA and FA stimulates the phenyl propanoid pathway, increasing the accumulation of polyphenols and flavonoids in plants (Salvador et al., 2013;Gorni et al., 2021).
Tomato plants have a good free radical scaven ging capacity due to the high concentration of lyco pene and βcarotene. This pigment, it is the most effective antioxidant which gives color to tomato fruit as maturity progresses (Kumar et al., 2017). Polyphenolic compounds and carotenoid act as anti oxidants because they remove singlet oxygen and other free radicals in cells (Shahidi and Ambigaipalan, 2015) due to their ability to donate hydrogen from hydroxyl groups positioned along the aromatic ring to prevent oxidation by radicals free from lipids and other biomolecules (Sousa et al., 2007).
Researchers showed an increase in carotenoid content during ripening due to the gradual degrada tion of chlorophyll (Adalid et al., 2010). Our results showed that the application of SA and FA may modu late and alter the concentration of lycopene and β carotene (Table 5) and chlorophyll a contents (Table  4) in tomato plants. Thus, our study evidenced that foliar application of SA and FA in tomato crop could enhance its antioxidant activity due to the higher concentration of these bioactive compounds. Our results corroborate with those found by Kumar et al. (2017), who also reported that foliar application of SA significantly increased lycopene content of toma to fruits. Singh et al. (2010) reported that application of Lphenylalanine and FA increased total polyphe nols in pea plants.
Therefore, it can be concluded that application of bioregulators results in higher yield and higher con centration of secondary compounds of Solanum lycopersicum.