EB stress has been declared to inhibit cell and plant growth factors. ANOVA analysis of FW and DW data of tomato calli showed that EB stress resulted in a significant decrease in the mass gain of the studied tomato calli (Fig. 1A-B, Tables S1-6). However, the three PAs applied to EB-stressed calli alleviated this growth deficiency. EB in the nutrient medium caused a significant decrease in callus biomass in terms of FW and DW compared to B-unstressed calli, and the reduction was 46.97% and 42.76%, respectively. PA treatments increased callus biomass compared to EB-stressed calli and recorded high increases in moderate concentrations of benzoate, gallate, and salicylate, respectively. Compared to EB-stressed calli, the use of moderate concentrations of benzoate, gallate, and salicylate increased the FW and DW of EB-stressed calli by approximately 158.75%, 66.21%, 48.70% for FW, 83.53%, 32.53%, 43.86% for DW, respectively. Under MS-B conditions, our results indicated that the use of BA (0.676*, 0.392, 0.809**), GA (0.897**, 0.811**, 0.559), and SA (0.461, -0.788**, 0.586*) resulted, in most cases, a strong relation between free, semi-bound, and bound boron content and DW of callus, respectively. As expected, under excess boron stress, BA (-0.805**, -0.658*, -0.722**), GA (-0.544, -0.729**, -0.586*), and SA (-0.614*, -0.883**, -0.872**) showed strong negative correlations between free, semi-bound and bound boron content and DW of callus, respectively.
The H2O2 contents, a product of redox metabolites, were evaluated within tomato calli that underwent various treatments to assess the degree of oxidative regulation resulting from EB stress and the influences of benzoate, gallate, and salicylate in reducing this negative damage (Fig. 2A, Tables S1-6). As revealed in Fig. 2A, EB stress increased hydrogen peroxide content in tomato calli by 60.22% compared to EB-unstressed calli. PA treatments of boron-stressed calli reduced the effect of stimulating EB stress on H2O2 content. Compared to EB-stressed calli, calli treated with moderate concentrations of benzoate, gallate, and salicylate showed reductions in H2O2 content of 23.00%, 47.94%, and 25.77%, respectively. The higher concentration of BA and SA caused an insignificant decrease in H2O2 content; whereas, GA significantly reduced its content.
Under MS-B-conditions, the low and moderate levels of the BA and GA treatments did not alter the H2O2 content; whereas, its content increased with the higher levels in the MS medium. Moreover, the H2O2 content gradually increased as the SA level increased in the MS medium. Interestingly, the application of BA (0.848**, 0.607*, 0.770**), GA (0.900**, 0.931**, 0.967**), and SA (0.552, 0.916**, 0.893**) to EB-stressed calli showed strong positive correlations between H2O2 and free, semi-bound and bound B content.
To test whether the positive effects of PA treatments on EB-stressed calli were related to their ability to reduce membrane damage, LOX activity was assessed in calli exposed to EB stress with or without PAs application (Fig. 2B, Tables S1-6). LOX activity in calli was increased with an increase of B in the MS nutrient medium and the increase was 65.96% compared to the control calli. Treating EB-stressed calli with the three tested PAs attenuated the negative impact of EB stress on LOX activity. LOX activity decreased significantly, in most cases, and recorded the highest drops in moderate levels of benzoate, gallate, and salicylate; whereas, compared to EB-stressed calli the decrease was 36.43%, 25.64%, and 15.65%, respectively.
Under MS-boron conditions, significant increments in LOX activity were noticed with the application of PAs, and only BA recorded non-significant changes. Moreover, the application of benzoate, gallate, and salicylate induced strong positive relations between LOX activity and H2O2 content in calli-treated with or without EB.
Superoxide dismutase activity
SOD activity was estimated as a remarkable scavenger for ROS (Fig. 3A, Tables S1-6). SOD activity in callus cells significantly increased when the callus was exposed to EB, compared to the control calli, and the recorded increase was 23.63%. The use of gallate and salicylate did not considerably alter SOD activity in EB-stressed calli; whereas benzoate statistically reduced its activity.
Under MS-boron conditions, benzoate and gallate treatments did not alter SOD activity, however, its activity gradually increased with increasing salicylate level in the nutrient medium. Furthermore, the relations between SOD activity and H2O2 content were minimal in calli-treated with PAs and stressed or unstressed with EB, only the correlations in calli-treated with SA (0.895**) without EB and BA (0.579*) with EB were significant.
CAT activity was estimated in tomato calli subjected to different levels of PAs with or without EB because it aids in the decomposition of H2O2 into H2O and O2 (Fig. 3B, Tables S1-6). As appeared in Fig. 3B, it can be observed that the EB had a considerable catalytic effect on CAT activity in the callus (about 2-fold above the control calli). However, applications of the three concentrations of benzoate, gallate, or salicylate to EB-stressed calli reduced the stimulating effect of EB on CAT activity.
Treatment of EB-stressed calli with a moderate concentration of GA showed a higher decrease in CAT activity (60.51%) compared to moderate concentrations of benzoate and SA, which recorded a decrease of 39.46% and 34.44%, respectively, of B-stressed calli.
Under MS-B cases, the three phenolic acids applied, in the most cases, significantly increased CAT activity in callus cells compared to MS-B-unstressed calli. Interestingly, PA applications caused strong positive relations between CAT activity and H2O2 content in calli stressed or unstressed with EB, only GA without EB stress caused a non-significant relationship.
POD activity was determined because it induces oxidation by H2O2 for a wide range of organic materials (Fig. 3A, Tables S1-6). POD activity was stimulated with increased B in the MS-nutrient medium which showed a 35.11% increase in POD activity compared to MS-B-unstressed calli. Supplementation of benzoate and gallate at different concentrations with excess boron increased POD activity, while salicylate treatments decreased its activity significantly. Compared to EB-stressed calli, moderate levels of BA and GA showed increases in POD activity of 26.16% and 83.47%, respectively, while SA at moderate concentration reduced its activity by 43.63%.
Under MS-B conditions, benzoate and gallate treatments progressively improved POD activity in the corresponding absolute controls. In contrast, POD activity decreased significantly with the application of SA levels in the MS medium. Furthermore, in the callus treated with excess boron and benzoate, gallate, and salicylate the correlation between POD activity and H2O2 content was significant (-0.603*, -0.789**, and +0.865**, respectively), while it was insignificant in the PAs-treated-callus only.
Ascorbate peroxidase activity
APX activity was determined because it induces the hydrogen peroxide dependent oxidation of AsA in plants (Fig. 3B, Tables S1-6). Treating tomato callus with 2 mM B positively affected APX activity. Under EB states, the increase in APX activity, compared to MS controls, was 34.99%. Treatment with different levels of benzoate and gallate plus excess boron enhanced APX activity (10.46%, 22.34%, 16.23% for benzoate and 10.25%, 9.16%, 20.32 for gallate, respectively, over B-stressed calli), while salicylate treatments did not significantly alter its activity.
Under MS-B states, BA treatments reduced APX activity in calli, only the higher level did not alter its activity. GA treatments catalyzed APX activity, in most cases, under MS conditions. However, salicylate applications did not alter APX activity, only the higher level enhanced its activity, under MS conditions. Additionally, our results showed that APX activity in calli treated with only SA (0.655*) and BA with EB (-0.744**) showed a strong association with H2O2 content; whereas, in other PA treatments with or without excess boron, the correlations were insignificant.
Phenylalanine ammonia-lyase activity
PAL activity was examined to mark whether co-applications of PAs with or without EB treatments affected phenolic biosynthesis in tomato calli (Fig. 4A, Tables S1-6). The data showed that EB significantly failed to enhance PAL activity in tomato calli. Nonetheless, the moderate and high concentrations of the tested PAs significantly enhanced PAL activity compared to EB-stressed calli. Combined application of BA, GA, SA, and EB at the highest level resulted in increased PAL activity, which showed a higher increase, in most cases, of 16.83%, 42.03%, and 18.95%, respectively, than that found in EB-stressed calli. Also, our results manifested that correlations between PAL activity and H2O2 content were not significant in the existence of PAs with EB, only there was a significant relationship in the BA case with EB (-0.684*).
Correspondingly, under MS-B conditions, PAL activity decreased by 21.74% and 29.82%, respectively, at moderate and high concentrations of BA compared to MS- calli. However, gallate and salicylate treatments did not affect PAL activity in calli exposed to MS-B conditions; only the highest level of SA boosted its activity (46.39%). Moreover, under BA without EB treatments, PAL activity showed a negatively strong association with H2O2 content (-0.673*); whereas, the same relationship was positively strong in cases of GA (0.714**) and SA (0.820**) without EB.
Polyphenol oxidase activity
PPO activity in calli was examined to look at the degree of phenolic oxidation induced by PAs with or without EB treatments (Fig. 4B, Tables S1-6). EB negatively affected PPO activity in calli, with PPO activity reduced by 31.86%, compared to EB-unstressed calli. The PA treatments had, in most cases, significant catalytic effects on PPO activity in calli under EB conditions. GA in high concentration was the most active PA in stimulating PPO activity in callus tissues; the stimulation rate was 179.19%, compared to EB-stressed calli. Moreover, the results manifested that the PAs (BA, GA, and SA) together with the EB treatments caused significant relationships between PPO activity and H2O2 content in calli (-0.777**, -0.918**, and -0.763**, respectively).
PA treatments without EB led to altered effects on PPO activity in callus tissues. The applications of different levels of benzoate significantly reduced PPO activity; however, gallate greatly enhanced its activity. In contrast, SA did not considerably alter PPO activity under MS-B states. Additionally, the results revealed that the PAs without EB treatments induced non-significant relationships between PPO activity and H2O2 content in calli.
Under MS-B cases, B was found mainly in free form in the tomato callus, with free B present at 68.4%, semi-bound B at 23.4%, and bound B at 8.2% of total B. However, in cases of EB, free and semi-bound B decreased by 6.90% and 4.38% to increase bound B by 11.28%. It can also be seen that EB caused significant increases in free, semi-bound, and bound B contents in B-stressed calli higher than in non-stressed calli (Fig. 5A-C, Tables S1-6). EB stress increased the free, semi-bound, and bound B content in calli by 74.55%, 57.74%, and 362.39%, respectively compared with the control callus. Benzoate, gallate, and salicylate applications reduced free, semi-bound, and bound B accumulation in EB-stressed calli as indicated by 40.35%, 28.67%, 14.93% for free B, 14.43%, 25.02%, 42.28% for semi-bound B, 41.46%, 35.06%, and 30.83% for bound B at moderate concentrations compared to EB-stressed calli, respectively. Under MS-B cases, benzoate and gallate treatments resulted in significant or non-significant increases in different concentrations of B form in calli compared to the corresponding absolute control. However, the different SA treatments did not alter the different forms of B in calli.
Transmission electron microscopy
TEM enables an estimate of the cell's microstructure, particularly in the internal features of cells and organelles. Hence, this technique was harnessed to look at the differences in ultrastructural resulting from excessive B stress and whether applications of phenolic acid enhanced callus cell resistance (Fig. 6 and 7). Imaging obtained from TEM showed that EB-untreated (control) cells have a normal shape, large nuclei, large vacuoles, and thin cell walls (ranging from 333 to 559 nm). The EB caused the cell walls to be thicker between 1167 and 1347 nm, which means 2.4 to 3.5 times more than the control cell wall. However, the moderate concentration of benzoate, gallate, and salicylate treatments reduced cell wall thickness in EB-stressed calli. Compared to B-stressed calli, the highest decrease in cell wall thickness was found at 78.17-85.00%, 42.54-64.52%, 83.59-83.72%, respectively after exposure to benzoate, gallate, and salicylate. Moreover, the addition of benzoate, gallate, and salicylate alone without increasing B to the nutrient medium reduced the thickness of the cell walls.