Introduction

Tomato (Solanum lycopersicum L.) is the second chief cultivated fruit–vegetable crop in the world. However, due to fluctuating environmental conditions (global warming, population explosion, pollution), the growth and production of tomatoes are drastically affected. Therefore, one of the interesting issues to agriculture globally is to provide 70% more food by the year 2050 (Food Summit FAO 2009). Various strategies have been proposed to improve the productivity, performance and tolerance of plants under challenging environments, however, one of the approach using plant growth regulators (PGRs) proved very potential. Out of several PGRs, exogenous application of salicylic acid (SA) through seed soaking improves growth, seed germination, photosynthetic efficiency, antioxidant machinery and various other biochemical attributes (Farooq et al. 2019; Boukari et al. 2019; Mir et al. 2020a,b; Gul et al. 2020; Jelali et al. 2021; Ahmad et al. 2020). SA, chemically known as ortho-hydroxybenzoic acid, an omnipresent phenolic hormone in the plant kingdom, plays vital role in the regulation of various morphological, cytological and physio-biochemical attributes in normal and unfavourable environmental conditions (Chen et al. 2009; Wani et al. 2017; Fariduddin et al. 2018; Khan et al. 2020; Saleem et al. 2021a).

SA is regarded as the best seed primer to improve the tolerance in various crops like wheat, maize and rice by stimulating the photosynthesis, metabolism of carbon and protein, accumulation compatible solutes like proline and the antioxidant machinery in many abiotic stresses (Farooq et al. 2019; Saruhan et al. 2012; Sharma et al. 2017). Exogenous application of SA in lower doses showed beneficial for improving morpho-physiological and biochemical attributes of plant (Hayat et al. 2010). Beside this, supplementation of SA increased growth, water use efficiency (WUE), decreases reactive oxygen species (ROS) levels and yield in peanuts and maize plants (Kong et al. 2014). SA modulates several metabolic and physiological processes thereby improving the growth and development in plants (Khodary 2004; Arfan et al. 2007; Manaa et al. 2014; Saleem et al. 2021b). Furthermore, SA upregulate the activities of antioxidant enzyme and increase the tolerance of plants to various abiotic stresses (Yusuf et al. 2008; Parashar et al. 2014; Fariduddin et al. 2018; Tayyab et al. 2020).

Therefore, bearing in mind, current work was aimed to investigate the role of SA on morpho-physiological, biochemical, and the antioxidant defence system in S. lycopersicum plant through seed soaking. This study explores the SA-induced microscopic (stomatal), histochemical (ROS levels), photosynthetic, biochemical and antioxidant enzymatic changes in two varieties (S-22 and PKM-1) of tomato treated with different concentrations of SA through seed soaking. Moreover, how SA impacts the growth, photosynthetic potential and ROS-homeostasis is vital to give mechanistic-insights of SA-induced changes in plants.

Materials and methods

Plant materials

Tomato (S. lycopersicum L. varieties, PKM-1 and S-22) seeds were taken from Indian Agricultural Research Institute (IARI), New Delhi. Uniform and healthy seeds of both varieties were selected followed by sterilization with sodium hypochlorite (1% NaOCl) for about 10 min, and then repeated washing with double distilled water (DDW) before soaking. These disinfected seeds were soaked in 0, 10−4, 10−5 or 10−6 M of SA for 3, 6 or 9 h and then sown in earthen pots to create nursery. At 20 DAT, seedlings were transplanted from nursery beds to earthen pots. Two different varieties of tomato (PKM-1 and S-22) were chosen for the study based on our preliminary study depicted in Supplementary File 1. Various growth, physiological and biochemical attributes were assessed at 40 days after transplantation (DAT).

Preparation of SA

SA was bought from Sigma–Aldrich Chemicals Pvt. Ltd. India. 1 M stock of SA solution was made in 200 mL flask by solvating 27.6242 g of SA in 10 mL of ethyl alcohol, and the absolute measurements was adjusted by adding DDW. The concentrations required of SA (10−4, 10−5 or 10−6 M) were set by diluting the solution of SA stock with DDW. Tween-20 (0.5 mL) a surfactant, was also added before seed soaking. Concentrations were opted based on early findings of Fariduddin et al. (2003) and Hayat et al. (2012).

Growth conditions and experimental setup

The surface-sterilized and respective soaked seeds were propagated in nursery beds to produce the nursery. Twenty DAS, the seedlings were transplanted into pots (23 cm diameter), filled with loamy sand soil and farmyard compost in a ratio of 6:1, v/v. each pot was added with the basal dose of NP and K. An equivalent amount of tap water was provided to maintain the moisture content in the soil. The experiment was carried out in simple sequential block layout in the net house of the Department of Botany, Aligarh Muslim University (27° 34′ N and 78° 31′ E), India. Seeds were treated with three diverse concentrations 10−4, 10−5 or 10−6 M of SA for 3, 6 or 9 h. Consequently, three sets were obtained and set up for different durations, and each set was added with three different concentrations of SA. Five replicates were applied in each treatment. To reveal the effect of different concentrations and durations of seed soaking with SA, plants were assessed at 40 days after transplantation (DAT).

Analysis of growth parameters

Changes in the growth biomarkers were determined by uprooting plants cautiously from pots and these uprooted plants were gently washed to make sure that no soil particle remains with the plant. Root and shoot length, root and shoot fresh weight and leaf area were measured with the help of meter scale, electronic balance and leaf area meter (LA 211, Systronics, New Delhi, India), respectively. For root and shoot dry weight, the samples of root and shoot were retained in a hot air oven, at 80 ℃ for 72 h, then dry samples were again weighted by electronic balance.

Photosynthesis and related attributes

Determination of SPAD value of chlorophyll and maximum quantum yield of PSII (Fv/Fm)

SPAD chlorophyll meter (SPAD-502; Konica, Minolta sensing, Inc., Japan) was used to determine the chlorophyll value on extended leaves of a plant. Before determining Fv/Fm, leaf samples were adjusted in dark for 30 min. After dark adaptation, Fv/Fm was measured using a weak measuring light photosynthetic photon flux density (PPFD) of less than 1 μmol m−2 s−1 for Fo followed by a saturating pulse (PPFD = 4200 μmol m−2 s−1) for Fm.

Leaf gas exchange attributes

The gas exchange attributes were analyzed with the aid of a portable photosynthetic system Infrared gas analyser (IRGA) (LI-COR 6400, LI-COR, and Lincoln, NE, USA) during the day time (11:00 to 12:30 h) on the third fully extended leaves of a plant. Net photosynthetic rate (PN), and its related attributes like transpiration rate (E), internal CO2 concentration (Ci) and stomatal conductance (gs) were measured at ⁓25 ℃ of air temperature, 80% of relative humidity, 600 μmol mol−1 of CO2 concentration and 800 μmol mol−2 s−2 of PPFD.

Determination of electrolyte leakage (EL) and leaf water potential (LWP)

According to method of Sullivan and Ross (1979), EL was determined. Twenty leaf segments were placed in a boiling tube holding 10 mL of DDW. The samples were heated at 45 ℃ (ECa) and 55 ℃ (ECb) for 30 min each in a water container and a conductivity meter (Eutech Instruments; Part of Thermo Fisher Scientific-Singapore) was used to measure the consequent EC. Finally, the samples were boiled in a test tube at 100 ℃ for 10 min and the EC (ECc) was estimated, and EL was determined by the following formula;

$$\mathrm{Electrolyte leakage }\left(\mathrm{\%}\right)=\frac{\left({\text{ECb}}-{\text{ECa}}\right)}{{\text{ECc}}}\times 100,$$

where the ECa stands for original conductance, ECb for higher temperature conductance, and ECc for concluding conductance.

With the help of the Psypro water potential system (Wescor, Inc. USA), LWP was determined in third fully expanded leaves of plant during day time at 13:00 h which is based on the principle that loss of water via evaporation from exterior cool down the surface.

Biochemical attributes

Activity of NR and CA

The activity of nitrate reductase (NR) was estimated by the protocol proposed by Jaworski (1971), in which freshly cut leaves were placed in test tubes filled with 2.5 mL of 5% isopropanol, 2.5 mL of 0.1 M phosphate buffer at pH of 7.5 and 0.5 mL of 0.2 M potassium nitrate (KNO3) and this reaction mixture was incubated for 2 h at 30 ℃. Besides this, another set of test tubes were retained with 0.02% of N-1-napthyl-ethylendiaminhydrochloride (NED-HCl) and 0.3 mL of 1% sulfanilamide solution. About 1 mL of the incubated reaction mixture was added from each treatment to this set of test tubes. The pink colour generated was observed at absorbance of 540 nm by a spectrophotometer (Spectronic-20D, Milton Roy, USA) and expressed as fresh weight (FW) basis.

The carbonic anhydrase (CA) activity was estimated by method proposed by Dwivedi and Randhawa (1974). Leaf samples were equally weighted and cut into small pieces and placed in test tubes filled with 0.2 M of cysteine-HCl solution and incubated at 4 ℃ for 20 min, then the filtrate of this extract was filled in other test tubes having a mixture of 0.2 M sodium bicarbonate (NaHCO3) solution (2 mL), Na-phosphate buffer (2 mL) and 0.002% of bromothymol blue (0.1 mL). Finally, the test tubes were left for about 20 min and the liberation of CO2 by the action of CA was evaluated by titration of the reaction mixture versus hydrochloric acid (0.05 N HCl). The methyl red (C15H15N3O2) was used as an indicator and activity of CA activity was stated as per gram fresh weight basis by adding the following values in the equation:

$$\mathrm{Carbonic anhydrase activity}=\frac{V\times 22\times N }{W},$$

where V = Volume difference (mL of HCl used, in control and test sample titrations), 22 = Equivalent weight of CO2, N = Normality of HCl, and W = Fresh weight of leaf used.

Estimation of ROS

H2O2 and O2 •− content

Method described by Patterson et al. (1984) was used to determine H2O2 content. Fresh leaves of about 0.5 g were taken in order and ground in ice-cold mortar and pestle mixed with acetone. Homogenate formed was then centrifuged for about 15 min at 5000 g. Later on 20% of titanium chloride (TiCl4) (prepared in concentrated HCl) and 17 M of ammonia solution was added in the test tubes containing supernatant (1 mL). Once these solutions were added, the precipitate was taken and dissolved in 2 N H2SO4 (10 mL) and again centrifuged to take out undissolved substances. Finally, absorbance of the supernatant was detected at 410 nm and H2O2 content was calculated with the help of a standard curve plotted with a familiar H2O2 concentration and stated in terms of μmol g−1 FM.

Method of Wu et al. (2010) was used to determine the superoxide anion content. Fresh samples of leaves were ground in sodium phosphate buffer of about 65 mM and 1% of Poly vinyl pyrrolidone (PVP). The homogenate was set to centrifugation for 15 min at 5000 g. Later on, the supernatant was mixed with 10 mM hydroxylamine hydrochloride and 65 mM phosphate buffer in test tubes and incubated at 25 ℃ for about 30 min. Later on, 58 m of metanilic acid and 7 mM of 1-naphthyl amine was added and then again retained for incubation (20 min) at 25 ℃. At 530 nm, absorbance was recorded and content of superoxide anion was assessed with respect to the calibration curve and expressed in terms of the μmol g−1 FM.

Histochemical detection of H2O2 and O2 •− content

The method of Kaur et al. (2016) was used to determine the histochemical staining of leaves to detect the ROS (H2O2 and O2•− content) as explained by Saleem and Fariduddin (2022). The H2O2 and O2•− content was detected by dipping leaf samples in 3, 3-diaminobenzidine (DAB) (maintained at pH ⁓ 3.8) and 6 mM of nitro blue tetrazolium (NBT) dyes, respectively, for about 6 h at normal room temperature. These leaves were further immersed in 100% ethanol and boiled at 100 ℃ to remove pigments and then samples were cooled, sequentially cleaned with glycerol solution (20%). Finally, images were taken with the help of the camera (NIKON-D53).

Assessment of antioxidant enzymes and protein content

Activities of the antioxidant enzymes and the content of protein was determined following the homogenization of fresh leaves with the extraction buffer having 1 mM of EDTA (ethylenediaminetetraacetic acid), 0.5% of Triton X-100, 1 mM of PMSF (phenyl methane sulfonyl flouride) and 2% of PVP (polyvinyl pyrollidone). After that, centrifugation of homogenate was done for 20 min at 12,000 g at 4 ℃ and supernatant generated was used further to measure the total protein and activities of antioxidant enzyme.

Protein content

Protocol of Bradford (1976), was followed to determine protein content. In this method supernatant (200 mL of enzyme extract) and Bradford reagent (4 mL) were mixed intensely in test tubes. After mixing, samples were incubated at 25 ℃ for 10 min and at absorbance of 595 nm, protein content was measured with the aid of a spectrophotometer and the content of protein was stated as mg g−1 FW. Finally, the linear standard equation was attained by laying a graph of well-known concentration of BSA (bovine serum albumin) v/s absorbance.

Antioxidant enzyme activity

The activity of superoxide dismutase (SOD) was measured by following protocol of Beauchamp and Fridovich (1971) which is based on the principle of photochemical reduction inhibition of NBT (nitro blue tetrazolium). The reaction mixture having 50 mM of phosphate buffer prepared at pH of 7.8, EDTA (2 mM), L-methionine (9.9 mM), 0.02% of Triton X-100, NBT (55 mΜ) and lastly 1 mM riboflavin were mixed with extract of the enzyme. Blank and control were also measured in a similar manner and SOD activity were observed at regular intervals for 2 min, at 560 nm absorbance. The activity of SOD (one-unit) was calculated as per the quantity of requisite enzyme to cause the 50% decrease of the NBT at 25 ℃.

The catalase (CAT) activity was measured according to protocol of Aebi (1984) based on loss of H2O2. In this method, 100 μL enzyme extract was added to the reaction mixture having 50 mM phosphate buffer prepared at pH 7.0 and 15 mM of H2O2. Finally, the optical density was observed for 2 min at 25 ℃ with the short-term of 30 s at absorbance of 240 nm.

The activity of peroxidase (POX) was measured by following protocol proposed by Sanchez et al. (1995). In this method, 100 μL enzyme extract was mixed to reaction mixture having phosphate buffer (50 mM) prepared at the pH of 7.0, 20 mM guaiacol and 1.5 mM of H2O2. Finally, at 436 nm of absorbance, the activity of POX was measured with the help of spectrophotometer (Spectronic-20D, Milton Roy, USA).

Proline content

According to the protocol of Bates et al. (1973), content of proline was determined (with slight variation). Fresh leaves were ground with 3% sulphosalicylic acid (extract reagent) and then centrifugation at 10,000 g was carried for 10 min. A mixture of 2 mL each of glacial acetic acid (CH3COOH), sulphosalicylic acid (C7H6O6S) and freshly prepared acid-ninhydrin solution filled in the test tube was added with 2 mL of supernatant. Then, test tubes were kept in a hot water bath for 1 h at 100 ℃ and then transferred in an ice bath to terminate the reaction. Finally, the absorbance of the reaction mixture was observed at 528 nm and the quantity of proline was calculated on the basis of fresh weight.

Stomatal physiology

Compound microscopy

Fresh leaves were taken in which epidermal peels were removed with the aid of forceps from the abaxial surface of the leaf. These peels were then mounted on a glass slide. Then, these mounted peels were observed in a camera fixed compound microscope (OLYMPUS BX 51) at magnification of 40 ×. The aperture of stomata was revealed by micrometer scale.

Scanning electron microscopy (SEM)

The outer organization of stomata was observed via visualizing the freeze and dehydrated sections of leaf with the aid of a scanning electron microscope (JEOL JSM-JSM 6510 SEM) as explained by Khan et al. (2022). Leaf samples were cut (2 × 2 mm segments) and fixed for 2 h in smallest polyoxymethylene paraformaldehyde (2%), glutaraldehyde (2.5%) and buffer 0.1 M of sodium cacodylate (pH of 7.3). After fixation, the segments of leaf samples were compressed by aid of 1% osmium oxide. Finally, the samples were dried out sequentially by graded series of 50%, 70%, 80%, 90%, and 100% of ethanol. With the help of gold–palladium, the samples were glazed in a coater splatter instrument (JEOL JFC-1600). At a magnification about 4000 × and 15 kV voltage images of stomata were taken.

Statistical analysis

With the aid of the SPSS version 17 for windows (IBM Corporation, New York, USA) statistical analysis was done. Analysis of variance (ANOVA) and standard error was calculated on the data taking five replicates (n = 5). For comparison among means for significant differences Duncan Multiple Range test (DMRT) was performed at P ≤ 0.05. Means have been separated by the Duncan’s multiple range test (DMRT). Beside this the Pearson’s correlation between the various attributes and non-metric multidimensional scaling (NMDS) was done with the aid of software Past.

Results

Growth parameters

Plants upraised from seed soaking with SA under three different durations (3, 6 or 9 h) and concentrations (10−4, 10−5 or 10−6 M) improved growth traits like shoot and root length, fresh and dry weight of shoot and root and leaf area with respect to control plants. Soaking of seeds with SA for 6 h generated significant impact on growth as compared to other sets of seed soaking and the response was more prominent in S-22 variety than PKM-1. The maximum length of shoot and root (53 and 48%), the fresh weight of shoot and root (51 and 36%), dry weight of shoot and root (52 and 45%) and leaf area (50%) of S-22 variety was observed by soaking of seeds (10−5 M of SA for 6 h) as compared to control (Figs. 1, 2A). Soaking of seeds with 10−5 M of SA for 6 h considerably improved all parameters of growth.

Fig. 1
figure 1

Effect of salicylic acid (SA) through seed soaking on A shoot and B root length; C shoot and D root fresh weight; E shoot and F root dry weight; and G phenotype of two varieties of tomato seedlings at 40 DAT. All the data are the means of five replicates (n = 5); vertical bars show standard errors (± SE). Means with different letters above the bars are significantly different at P ≤ 0.05

Full size image
Fig. 2
figure 2

Effect of SA through seed soaking on A leaf area, B SPAD Chlorophyll content, C maximum quantum yield of PSII (Fv/Fm), D net photosynthetic rate (PN), E stomatal conductance (gs) and F internal CO2 concentration (Ci) in two different varieties of Solanum lycopersicum L. plants at 40 DAT. All the data are the means of five replicates (n = 5); vertical bars show standard errors (± SE). Means with different letters above the bars are significantly different at P ≤ 0.05

Full size image

Photosynthesis and stomatal traits

Seed soaking with SA increased the chlorophyll content (SPAD value) and Fv/Fm as compared to their respective control. However, the plants raised from seed soaking with 10−5 M of SA for 6 h had significantly increased SPAD level and Fv/Fm than control, and the impact was more pronounced in S-22 than the PKM-1 variety. The values of SPAD and Fv/Fm were increased by (53 and 52%) and (45 and 43%) in S-22 and PKM-1 variety by seed soaking with 10−5 M of SA for 6 h (Fig. 2B, C).

Furthermore, treatment of SA by soaking of seeds triggered a substantial increase in gas exchange parameters (net photosynthetic rate (PN), internal CO2 concentration (Ci), stomatal conductance (gs) and the transpiration rate (E)). But soaking of seeds with 10−5 M of SA for 6 h triggered a significant increase in comparison to their respective control, and the outcome was seen more pronounced in S-22 than the PKM-1 variety. Pre-sowing treatment of seed with 10−5 M of SA for 6 h increased about PN by 52 and 39%, gs by 43 and 28%, Ci by 51 and 38% and E by 45 and 36% in S-22 and PKM-1 variety, respectively (Figs. 2D–F, 3A).

Fig. 3
figure 3

Effect of SA through seed soaking on A transpiration rate, B leaf water potential, C electrolyte leakage, D carbonic anhydrase, E nitrate reductase and F proline content in two different varieties of S. lycopersicum L. plants at 40 DAT. All the data are the means of five replicates (n = 5); vertical bars show standard errors (± SE). Means with different letters above the bars are significantly different at P ≤ 0.05

Full size image

Soaking of seeds for different durations and concentrations of SA significantly decreased the EL and increased the LWP with respect to control. But, the plants raised from treatment of seeds for 6 h with 10−5 M of SA decreased the EL by 21 and 16% as compared to control in S-22 and PKM-1 variety, respectively, (Fig. 3C). Besides this, the pre-sowing seed treatment with 10−5 M of SA for 6 h significantly increased the LWP by 50 and 32% in S-22 and PKM-1 variety, respectively, with respect to control plant (Fig. 3B).

Stomatal physiology

Treatment of seeds with SA and the developed plant had improved width of stomata with respect to control. However, the outstanding improvement in the stomatal width was observed in the plants raised from soaking for 6 h in SA (10−5 M) of as evident in the (Fig. 4A). The compound microscopy examinations were corroborated from SEM observations (Fig. 4B).

Fig. 4
figure 4

Stomatal response of two different varieties of S. lycopersicum L. plants at 40 DAT under control (a), 10−5 M of SA soaking for 3 h (b), 10−5 M of SA soaking for 6 h (c) 10−5 M of SA soaking for 9 h (d), at 40 × using compound microscope (A) and at 3000 × using scanning electron microscope (B)

Full size image

Biochemical traits

Activity of carbonic anhydrase (CA) and nitrate reductase (NR)

Plants raised with the soaking of seeds with SA had substantial increase in the activities of CA and NR as compared to their respective control. However, out of various concentrations and duration of soaking treatment, 10−5 M of SA for 6 h maximally improved the CA and NR activity by 45 and 49% in the S-22 variety, as compared to their respective control. Although similar impact was observed in PKM-1 variety, but S-22 was more responsive than PKM-1 where activity of CA and NR increased by 34 and 33%, respectively, as compared to their respective control (Fig. 3D, E).

ROS (Reactive oxygen species) indicators

H2O2 and O2 •− content

Plants raised from the soaking of seeds with SA showed lesser content of both H2O2 and superoxide anion as compared to the control. But soaking with 10−5 M of SA for 6 h decreased the H2O2 and superoxide anion by 26 and 20% in S-22 variety; 21 and 16% in PKM-1 variety, respectively, as compared to the respective control (Figs. 5A, 6A).

Fig. 5
figure 5

Effect of SA through seed soaking on A hydrogen peroxide content and B histochemical detection of hydrogen peroxide in leaves by DAB staining at 40 DAT in different varieties of S. lycopersicum L. plants under control (a), 10−5 M of SA soaking for 3 h (b), 10−5 M of SA soaking for 6 h (c) 10−5 M of SA soaking for 9 h (d). All the data are the means of five replicates (n = 5); vertical bars show standard errors (± SE). Means with different letters above the bars are significantly different at P ≤ 0.05

Full size image
Fig. 6
figure 6

Effect of SA through seed soaking on A superoxide anion content and B histochemical detection of superoxide anion content in leaves by NBT staining at 40 DAT in different varieties of S. lycopersicum L. plants under control (a), 10−5 M of SA soaking for 3 h (b), 10−5 M of SA soaking for 6 h (c) 10−5 M of SA soaking for 9 h (d). All the data are the means of five replicates (n = 5); vertical bars show standard errors (± SE). Means with different letters above the bars are significantly different at P ≤ 0.05

Full size image

Histochemical staining for H2O2 and O2 •−

Histochemical staining of plants depicted the level of H2O2 and superoxide anion, where H2O2 was observed by brownish spots, whereas superoxide anion by blue spots on leaf discs. It was observed that plants raised from seed soaking with SA had lesser stained spots than the control. However, less number of spots (accretion of H2O2 and O2•−) were observed in the leaf discs produced by treatment of SA (10−5 M) for 6 h as compared to the respective controls (Figs. 5A–D, 6A–D) and S-22 was more responsive than PKM-1 variety.

Total protein content

Pre-sowing seed treatment with SA enhanced the protein content with respect to control. However, soaking of seeds with 10−5 M of SA for 6 h triggered maximum increase in the protein content by 25 and 18% in S-22 and PKM-1 variety, respectively, as compared to the control (Fig. 7A).

Fig. 7
figure 7

Effect of SA through seed soaking on A protein content, B catalase, C peroxidase and D superoxide dismutase in two different varieties of S. lycopersicum L. plants at 40 DAT. All the data are the means of five replicates (n = 5); vertical bars show standard errors (± SE). Means with different letters above the bars are significantly different at P ≤ 0.05

Full size image

Activities of antioxidant enzymes

Pre-sowing seed treatment of SA via seed soaking increased the antioxidant enzyme activities like CAT, POX and SOD as compared to control. However, the maximum activities of POX (63%), CAT (57%) and SOD (61%) were noticed in the S-22 variety subjected to soaking with 10−5 M of SA for 6 h, whereas, 45%, 54% and 49% rise in the activity of CAT, POX and SOD was observed in PKM-1 variety by soaking for 6 h with the 10−5 M of SA as compared to the control (Fig. 7B–D).

Proline content

The content of proline amino acid observed was improved by all the treatments of seed soaking with SA with respect to control. But, plants raised from soaking by 10−5 M of SA for 6 h showed maximum accretion in proline by 48 and 36% in S-22 and PKM-1 varieties, respectively, then the respective control (Fig. 3F).

A strong correlation among the several growth biomarkers, photosynthetic and biochemical attributes was observed in our experiment on tomato plants treated with SA via seed soaking. The correlation-matrix of both varieties (S-22 and PKM-1) are depicted in Fig. 8, respectively, which portrays a significant positive correlation (red colour) and negative correlation (blue colour). We observed a significant positive relationship between the various growth, photosynthetic and biochemical attributes except EL, H2O2 and superoxide anion content which exhibited a negative correlation with all these attributes. Moreover, we found that photosynthetic gas exchange attributes were positively correlated with the growth attributes, SPAD chlorophyll value and antioxidant enzymes but, negatively correlated with EL, H2O2 and O2•− content in both S-22 and PKM-1 (Fig. 8) variety. The impact of SA on two different varieties of tomato has been statistically seen by NMDS analysis (Fig. 9). It shows the effect of SA within and between two different varieties of tomato was significant in all growth, physiological and biochemical attributes. SA enhanced the growth and photosynthesis in both varieties but the response was different in both varieties, as depicted in NMDS diagram that mean value of various parameters of both varieties clustered separately as shown in Fig. 9 cluster a (S-22 variety; black colour) and cluster b (PKM-1 variety; blue colour). Variety S-22 was more responsive to exogenous treatment of SA than PKM-1 variety.

Fig. 8
figure 8

Pearson’s correlation matrix of two different varieties of tomato at 40 DAT S-22 and PKM-1 are depicted. It portrays a significant positive correlation (red colour) and negative correlation (blue colour). All parameters except electrolyte leakage, hydrogen peroxide content and superoxide anion content shows positive correlation. Various abbreviations used are SL (shoot length), RL (root length), SFW (shoot fresh weight), RFW (root fresh weight), SDW (shoot dry weight), RDW (root dry weight), LA (leaf area), SPAD (chlorophyll content), PN (net photosynthetic rate), Ci (internal CO2 concentration), gs (stomatal conductance), E (transpiration rate), FV/FM (represents the maximum potential quantum efficiency of Photosystem II), LWP (leaf water potential), EL (electrolyte leakage), CA (carbonic anhydrase), NR (nitrate reductase), POX (peroxidase), SOD (superoxide dismutase), CAT (catalase), PRO (proline content), HPC (hydrogen peroxide content), SOC (superoxide anion content) and PC (protein content)

Full size image
Fig. 9
figure 9

Non-metric multidimensional scaling ordination diagram (NMDS with Bray–Curtis similarity measure) of two different varieties of tomato which assembled separately in two clusters (a) S-22 and (b) PKM-1 and it signifies the differential response of both varieties of tomato to SA at 40 DAT

Full size image

Discussion

In the current study, the potentiality of PGR, SA through the exogenous application by seed soaking was assessed, and it was hypothesized to enhance the morphological and physio-biochemical traits of tomato plants in normal condition. Growth of tomato plants, i.e., the length of shoot and root, fresh and dry weight of shoot and root and leaf area were increased by SA treatment as depicted in Figs. 1 and 2A. Tayyab et al. (2020) and Mohammed (2020) revealed that SA applied exogenously by seed soaking improved the growth biomarkers like dry weight of root and shoot, plant height and leaf area in maize and oat plants. Beside this, SA also promoted the morphological attributes in various plants like mustard, tomato, soybean, wheat, alfalfa and violet plants in a concentration-dependent manner (Gutiérrez-Coronado et al. 1998; Fariduddin et al. 2003, 2018; Shakirova et al. 2003; Hayat et al. 2005; Yusuf et al. 2012; Parashar et al. 2014; Boukari et al. 2019; El-Mergawi and Abd El-Wahed 2020). Our results were analogous with the findings of Rady and Mohamed (2015) and Kaydan et al. (2007) in which it was exposed that soaking of seeds with SA enhanced growth traits like shoot length, number and leaf area per plant, and plant dry mass in common bean and wheat. The possible reason for this increase in the morphological characters could be a due increase in cell division, photosynthetic attributes and nutrient uptake efficiency by exogenous application of SA (Zhu 2001; El-Tayeb 2005; Pacheco et al. 2013; Rady and Mohamed 2015; Pradhan et al. 2016; Jelali et al. 2021).

The photosynthetic attributes like SPAD value, Fv/Fm (quantum yield of PSII), gas exchange parameters, stomatal behaviour were increased along with the decrease in the EL and increase in LWP by SA supplementation through seed soaking as depicted in Figs. 2, 3A–C and 4. Our results are in agreement with the findings of Kohli et al. (2018) in Brassica juncea, Boukari et al. (2019) in alfalfa and Jelali et al. (2021) in Sulla carnosa plants. It is revealed that SA increased the rate of photosynthesis by accretion of α-ALA (α-aminolevulinic acid) a close precursor molecule of biosynthetic pathway of chlorophyll (Kumar et al. 2010). Potentiality of SA in enhancing the content of chlorophyll pigments might be due to its positive impact on leaf and chloroplast organization, stomatal behaviour, activation of ATPase pump that facilitates the iron uptake, and ability to inhibit the chlorophyll degrading enzyme chlorophyllase by decreasing the expression of CHLASE gene (Uzunova and Popova 2000; Kong et al. 2014; Kohli et al. 2018). The increased morpho-physiological traits including gas exchange attributes and ionic uptake were enhanced by SA seed priming in Medicago sativa which are in accordance with our findings (Boukari et al. 2019). SA possibly maintains the stability and functionality of PSII reaction centers by increasing the expression of D1, D2 and LHC protein, and hence oxygen evolving complex (oec) (Luo et al. 2009; Wang et al. 2010). SA is recognized as a vital regulator of photosynthesis as it increases photosynthesis, water relations and other related attributes (Uzunova and Popova 2000; Ashraf et al. 2010; Parashar et al. 2014; Janda et al. 2014; Fariduddin et al. 2018; Singh et al. 2021). An increase in the photosynthesis by SA could be probably due to its ability to control the Rubisco enzyme and PSII activity (Pacheco et al. 2013; Alam et al. 2022). Study of Krantev et al. (2008), Shao et al. (2018) and Sharma et al. (2018) revealed that SA pre-treatment increases the efficiency of photosynthesis by enhancing the activities of regulatory enzymes of CO2 assimilation like Rubisco, Rubisco activase, CA, phosphoenol pyruvate carboxylase (PEPC) and various transcription factors (Rbc L, ZmRCAβ mRNA and ZmRCAα) involved in photosynthesis.

NR and CA activity was increased by supplementation of SA exogenously as depicted in Fig. 3D–E. Our results were analogous with the findings of Khalil et al. (2021) in Phaselous vulgaris, Aftab et al. (2011) in Artemesia; Zanganeh et al. (2019) in Zea mays. The main reason for elevation in NR activity by SA could be due to enhancement in the activity of H+-ATPase pump associated with plasma membrane, membrane stability index thereby facilitating the nutrient uptake particularly NO3̅̅̅̅̅, which act as the stimulator for NR activity (Campbell 1999; Agarwal et al.2005; Dong et al. 2016; Khalil et al. 2021). Our present outcomes are concurrent with the study of Hayat et al. (2005), Yusuf et al. (2008), Idrees et al. (2012) and Fariduddin et al. (2018). In addition soaking of seeds with SA improved the growth of Z. mays L. and P. vulgaris plants by increasing CO2 fixation, activity of Rubisco, PEPC, photosynthetic pigments, content of nitrogen and carbohydrate, along with increased activities of CA and NR (Gunes et al. 2005; Krantev et al. 2008; Khalil et al. 2021).

In the current study, SA has been revealed to enhance the activities of antioxidant enzymes like CAT, SOD and POX and proline content while decreasing the H2O2 and superoxide anion content in the plants (Fig. 7). SA enhanced the activities of SOD, glutathione reductase (GR), CAT and ascorbate peroxidase (APX) as reported in soybean plants by Chen et al. (2016) and Sharma et al (2018). In present study, SA treatment decreased the hydrogen peroxide and superoxide anion content (Figs. 5, 6) which improved the growth and photosynthetic potential of plant by regulating balance between ROS and antioxidant enzymes (Mutlu et al. 2016; Tayyab et al. 2020). Enhancement in the activities of antioxidant enzymes by pre-treatment of seeds with SA might be due to regulation of amino acid content and hence the proteins of ascorbate and glutathione cycle (Zanganeh et al. 2019). Besides this, elevation in antioxidative enzymes could be due to accrual of osmolytes (proline, sugar alcohol, etc.) and enhanced activity of aldose reductase. SA is known to reduce the oxidative burst by increasing the antioxidant enzymatic activities and accumulation of osmolytes (Kazemi et al. 2010; Agami 2013; Hussain et al. 2016; Faghih et al. 2017). Application of SA by exogenous mode decreases the accrual of ROS and maintains the homeostasis of plant, and analogous results were observed in several plants like Artemisia anua L., mustard and wheat (Singh and Usha 2003; Fariduddin et al. 2003; Aftab et al. 2010; Yadu et al. 2017; Dutra et al. 2017). Moreover, SA increase the tolerance against stress by enhancing the antioxidant machinery (Mutlu et al. 2009; Li et al. 2014; Anaya et al. 2017; El-Esawi et al. 2017; Fariduddin et al. 2018; Saleem et al. 2021a). In addition, it was reported by Briache et al. (2020) in faba bean that out of three different modes (foliar spray; FS, seed soaking; SS and combined; FS + SS) of SA application, seed soaking in SA induced the maximum resistance against Orobanche crenata a parasitic weed as compared to other modes of SA application or untreated control plants.

Apart from inducing antioxidant machinery, we also observed that the content of proline and protein was increased by exogenous treatment of SA. The interaction of SA with other hormones could contribute to the enhancement of protein content and other osmolytes (Song et al. 2011; Anosheh et al. 2014). Proline, stress amino acid maintains the osmoticum of the plant cell and function as molecular chaperons which protect the protein integrity and activate various vital enzymes essential for sustenance of plant life (Hong et al. 2000; Anosheh et al. 2014; Naliwajski and Skłodowska 2014). As proline acts as an osmolyte which acts together with other proteins and enzymes of the cell and helps in upregulation, activation and stabilization of other proteins (Efimova et al. 2014; Fariduddin et al. 2018). Accumulation of proline was observed in various plants by SA application, and this increased level of proline could be due to the regulation of amino acid content or inhibition of hydrolyzing enzymes thereby causing accrual of osmolytes (Agami 2013; Anosheh et al. 2014; Fariduddin et al. 2018). The soaking of common bean seeds in SA increased the overall growth by enhancing membrane stability index, relative water content (RWC), osmolytes concentrations, and activity of antioxidant enzymes, along with decreased EL and peroxidation of lipids (Sadeghipour and Aghaei 2012; Semida and Rady 2014).

Conclusion

Finally, our results signify that the exogenous application of SA increased the growth, photosynthesis, stomatal physiology, water balance and chlorophyll content. Further the performance of plant is enhanced by SA via increasing activity of antioxidant enzymes, CA, NR, proline content, total protein content and decreasing the ROS level. Therefore, the pre-treatment of SA via seed soaking improves the morphological and physio-biochemical attributes with respect to control in two varieties of tomato plants. However, out of two different varieties of tomato and different concentrations and durations of seed soaking the supplementation of S-22 variety with 10−5 M of SA for 6 h generated promising impact as compared to all the concentrations tested. Response was more prominent in S-22 than PKM-1 variety. Therefore, in conclusion, SA improved the growth of tomato plants by controlling various morphological and physio-biochemical attributes. SA application through seed soaking could be a promising technique for crop improvement and these outcomes would be vital for understanding the molecular and physiological mechanisms associated with performance of plants in changing environment.

Author contribution statement

Study conception, design and data collection, analysis and interpretation of manuscript, draft manuscript preparation by MS; overall supervision by QF. Both authors reviewed and approved the final version of the manuscript.