Roles of Calcium Signaling in Gene Expression and Photosynthetic Acclimatization of Solanum lycopersicum Micro-Tom (MT) after Mechanical Damage

A momentary increase in cytoplasmic Ca2+ generates an oscillation responsible for the activation of proteins, such as calmodulin and kinases, which interact with reactive oxygen species (ROS) for the transmission of a stress signal. This study investigated the influence of variations in calcium concentrations on plant defense signaling and photosynthetic acclimatization after mechanical damage. Solanum lycopersicum Micro-Tom was grown with 0, 2 and 4 mM Ca2+, with and without mechanical damage. The expression of stress genes was evaluated, along with levels of antioxidant enzymes, hydrogen peroxide, lipid peroxidation, histochemistry, photosynthesis and dry mass of organs. The ROS production generated by mechanical damage was further enhanced by calcium-free conditions due to the inactivation of the oxygen evolution complex, contributing to an increase in reactive species. The results indicated that ROS affected mechanical damage signaling because calcium-free plants exhibited high levels of H2O2 and enhanced expression of kinase and RBOH1 genes, necessary conditions for an efficient response to stress. We conclude that the plants without calcium supply recognized mechanical damage but did not survive. The highest expression of the RBOH1 gene and the accumulation of H2O2 in these plants signaled cell death. Plants grown in the presence of calcium showed higher expression of SlCaM2 and control of H2O2 concentration, thus overcoming the stress caused by mechanical damage, with photosynthetic acclimatization and without damage to dry mass production.


Introduction
Mechanical damage in plants activates a cascade of defense reactions closely related to plant defenses against herbivory [1]. Mechanically damaged plant tissues produce O 2 and H 2 O 2 , which are ROS involved in a wide range of biological processes such as growth, development and responses to biotic and abiotic stimuli [2][3][4]. Increased ROS production promotes Ca 2+ -channel opening, resulting in increased cytoplasmic Ca 2+ [5,6]. This cytoplasmic calcium activates the respiratory burst oxidase homolog-D (RBOHD), the enzyme responsible for ROS wave formation, thus aiding ROS propagation from stimulated to unstimulated tissues during stress [6,7].
A momentary increase in cytoplasmic Ca 2+ generates an oscillation responsible for the activation of proteins such as calmodulin (CaMs), CaM-likes, calcinerin B-like and calciumdependent protein kinases (CDPKs) [8]. High levels of Ca 2+ and ROS activate MAPKs (mitogen-activated protein kinases) and calmodulin to regulate ROS accumulation [5]. SlCaM2 damage x calcium p < 0.005, (B). RBOH1 calcium × damage p < 0.016, (C). MPK1 damage × calcium p < 0.023 and (D). MPK2 time × damage × calcium p < 0.027 in Solanum lycopersicum "cv. Micro-Tom" grown with variation in calcium concentration (mM) and with (w/MD) or without (wo/MD) mechanical damage. Different lowercase letters indicate a significant difference in calcium levels within wo/MD or w/MD plants. Capital letters test wo/MD and w/MD plants within the same calcium level. ns = not significant at 5% Tukey test. Bars correspond to averages, whiskers to ± SE (n = 3). SlCaM2 damage x calcium p < 0.005, (B). RBOH1 calcium × damage p < 0.016, (C). MPK1 damage × calcium p < 0.023 and (D). MPK2 time × damage × calcium p < 0.027 in Solanum lycopersicum "cv. Micro-Tom" grown with variation in calcium concentration (mM) and with (w/MD) or without (wo/MD) mechanical damage. Different lowercase letters indicate a significant difference in calcium levels within wo/MD or w/MD plants. Capital letters test wo/MD and w/MD plants within the same calcium level. ns = not significant at 5% Tukey test. Bars correspond to averages, whiskers to ± SE (n = 3).
Calcium and mechanical damage contributed to increased SlCaM2 expression ( Figure 1A), whereas the absence of Ca 2+ with mechanical damage increased RBOH1 expression ( Figure 1B). MPK1 showed higher gene expression in plants with mechanical damage, independent of Ca 2+ level ( Figure 1C). However, MPK2 showed higher gene expression in plants with Ca 2+ at 0.5 h and with mechanical damage, indicating that the presence of calcium increased MPK2 expression ( Figure 1D).

Ca 2+ Absence with Mechanical Damage Activated Antioxidant Enzymes and Increased Hydrogen Peroxide and Lipid Peroxidation
The time since mechanical damage was not significant (p < 0.05), except for SOD, CAT and hydrogen peroxide, and so only calcium and mechanical damage showed an interaction, and are thus represented in Figure 2.
In general, Ca 2+ -free plants had higher activities of the enzymes SOD, POD and APX when subjected to mechanical damage. In general, there was no variation in the activities of these enzymes with 2 and 4 mM Ca 2+ , independent of mechanical damage (Figure 2A,B,D). Specifically, the highest activities of the enzyme CAT were for Ca 2+ -free and 4 mM Ca 2+ -cultivated plants, with and without damage ( Figure 2C). concentration of hydrogen peroxide immediately after damage, and its concentration varied little in subsequent evaluations ( Figure 2E).
Lipid peroxidation was higher for Ca 2+ -free plants with mechanical damage. Lipid peroxidation did not differ among plants grown in the presence of Ca 2+ ( Figure 2F).
Plants grown in the absence of Ca 2+ and with mechanical damage resisted the deficiency of the element longer. . Ascorbate peroxidase (APX) damage × calcium p < 0.006, (E). Hydrogen peroxide (H2O2) concentration time × damage × calcium p < 0.001 and (F). Lipid peroxidation (expressed by the formation of malonaldehyde, MDA) damage × calcium p < 0.017, in Solanum lycopersicum "cv. Micro-Tom" grown with variation in calcium concentration (mM) and with (w/MD) or without (wo/MD) mechanical damage. Different lowercase letters indicate a significant difference in calcium levels within wo/MD or w/MD plants. Capital letters test wo/MD and w/MD Figure 2. Activity of antioxidants (A). Superoxide dismutase (SOD) time × damage × calcium p < 0.001, (B). Peroxidase (POX) damage × calcium p < 0.045 and (C). Catalase (CAT) time × damage × calcium p < 0.033 and (D). Ascorbate peroxidase (APX) damage × calcium p < 0.006, (E). Hydrogen peroxide (H 2 O 2 ) concentration time × damage × calcium p < 0.001 and (F). Lipid peroxidation (expressed by the formation of malonaldehyde, MDA) damage × calcium p < 0.017, in Solanum lycopersicum "cv. Micro-Tom" grown with variation in calcium concentration (mM) and with (w/MD) or without (wo/MD) mechanical damage. Different lowercase letters indicate a significant difference in calcium levels within wo/MD or w/MD plants. Capital letters test wo/MD and w/MD plants within the same calcium level. ns = not significant at 5% Tukey test. Bars correspond to averages, whiskers to ± SE (n = 3).
Plants grown with 2 and 4 mM Ca 2+ showed no variation in hydrogen peroxide concentration, regardless of mechanical damage, and had the lowest concentrations. Plants grown in the absence of Ca 2+ and with mechanical damage presented a higher concentration of hydrogen peroxide immediately after damage, and its concentration varied little in subsequent evaluations ( Figure 2E).
Lipid peroxidation was higher for Ca 2+ -free plants with mechanical damage. Lipid peroxidation did not differ among plants grown in the presence of Ca 2+ ( Figure 2F).
Plants grown in the absence of Ca 2+ and with mechanical damage resisted the deficiency of the element longer.

Hydrogen Peroxide Was Evidenced in Leaves of Plants Grown in the Presence of Ca 2+ and with Mechanical Damage
Thirty minutes after mechanical damage, Ca 2+ -free plants without mechanical damage showed discrete staining from reactive 3,3 -diaminobenzidine (DAB) restricted to the walls of parenchymal cells of the endoderm on the abaxial surface of the midrib, as well as the phloem and vessel elements of the midrib and smaller veins ( Figure 3A-D   Plants grown in the presence of 2 mM Ca 2+ and without mechanical damage had staining within some cells of the palisade parenchyma and spongy parenchyma ( Figure 3E). There was intense DAB staining on the walls of the vessel elements of the midrib ( Figure 3F) and of the smaller veins. Otherwise, plants grown in the presence of 2 mM Ca 2+ and with mechanical damage revealed more intense hydrogen peroxide staining by DAB inside some palisade and spongy parenchyma cells ( Figure 3G) and in the head cells of glandular trichomes ( Figure 3H). Staining on the walls of vessel elements of the midrib ( Figure 3H) and in the smaller veins was also observed.
At 21 days, Ca 2+ -free plants without mechanical damage showed subtle staining for hydrogen peroxide within some cells of the palisade parenchyma ( Figure 4a) and on the walls of vessel elements of the midrib (Figure 4b) and of smaller veins, while Ca 2+ -free plants with mechanical damage showed staining on the walls of the vessel elements of the smallest veins ( Figure 4c) and the midrib (Figure 4d). Staining was also detected inside the head cells of glandular trichomes (Figure 4d).     as revealed at the end of the evaluations, these plants had similar total dry mass and and stem dry mass (Figure 8), although leaf and root dry mass were higher for grown in the presence of 4 mM Ca 2+ .
Plants grown in the presence of 2 and 4 mM of Ca 2+ and with mechanical da showed lower carbon assimilation rates, RuBisCO carboxylation and wate efficiencies, as well as low stem, fruit and total dry mass ( Figures 7 and 8), compa the same without mechanical damage.
Thus, the results for chlorophyll a fluorescence, gas exchange and plant bi reveal that the photosynthetic performance of Ca 2+ -free plants with mechanical da differs from that of those grown in the presence of Ca 2+ and with mechanical damag Ex of Ca 2+ -free plants with mechanical damage was low, while that of plants grown presence of Ca 2+ with mechanical damage was elevated ( Figure 6). The A/Ci of Ca plants with mechanical damage was higher than that of Ca 2+ -free plants w mechanical damage seven days after damage ( Figure 7). The A/Ci of plants grown presence of 2 and 4 mM Ca 2+ and with mechanical damage was lower than that of grown in the presence of 2 and 4 mM Ca 2+ and without mechanical damage at 14, 2 28 days after mechanical damage.
Micro-Tom" grown with variation in calcium concentration (mM) and with (w/MD) or without (wo/MD) mechanical damage. Different lowercase letters indicate a significant difference in calcium levels within wo/MD or w/MD plants. Capital letters test wo/MD and w/MD plants within the same calcium level. ns = not significant at 5% Tukey test. Bars correspond to averages, whiskers to ± SE (n = 3).
(wo/MD) mechanical damage. Different lowercase letters indicate a significant d levels within wo/MD or w/MD plants. Capital letters test wo/MD and w/MD pla calcium level. ns = not significant at 5% Tukey test. Bars correspond to averages, = 3). . Photosystem II efficiency (Fv'/Fm') damage × calcium < 0.001; (B rate (ETR) time × calcium p < 0.008; (C). effective quantum yield (ΦPSII) time (D). photochemical quenching (qL) time × damage × calcium p < 0.002; (E). fra excitation energy in the antenna that cannot be used for photochemical phase (Ex p < 0.005; (F). light fraction absorbed by the PSII antenna that is dissipated as 0.001, in Solanum lycopersicum "cv. Micro-Tom" grown with variation in calcium and with (w/MD) or without (wo/MD) mechanical damage. Different lowerca significant difference in calcium levels within wo/MD or w/MD plants. Capital and w/MD plants within the same calcium level. ns = not significant at 5% correspond to averages, whiskers to ± SE (n = 3). . electron transport rate (ETR) time × calcium p < 0.008; (C). effective quantum yield (ΦPSII) time × calcium p < 0.008; (D). photochemical quenching (qL) time × damage × calcium p < 0.002; (E). fraction of dissipated excitation energy in the antenna that cannot be used for photochemical phase (Ex) damage × calcium p < 0.005; (F). light fraction absorbed by the PSII antenna that is dissipated as heat (D) calcium p < 0.001, in Solanum lycopersicum "cv. Micro-Tom" grown with variation in calcium concentration (mM) and with (w/MD) or without (wo/MD) mechanical damage. Different lowercase letters indicate a significant difference in calcium levels within wo/MD or w/MD plants. Capital letters test wo/MD and w/MD plants within the same calcium level. ns = not significant at 5% Tukey test. Bars correspond to averages, whiskers to ± SE (n = 3).
Plants of Solanum lycopersicum cv. Micro-Tom grown in the absence of Ca 2+ and with or without mechanical damage, in the dark, showed decreased effective quantum efficiency (ΦPSII), electron transport rate (ETR), potential quantum efficiency (Fv/Fm) and PSII efficiency (Fv'/Fm') over time, with a high fraction of light absorbed by the PSII antenna and dissipated as heat (D) (Figures 5 and 6). In the presence of light, these same plants showed low PSII efficiency (Fv'/Fm'), photochemical quenching (qL), electron transport rate (ETR), and effective quantum efficiency (ΦPSII), and the fraction of excitation energy did not dissipate in the antenna. It did not use photochemistry (Ex), and with the fraction of light absorbed by the PSII antenna, it dissipated most as heat (D) (Figures 5 and 6). Plants grown in the presence of 2 and 4 mM of Ca 2+ and with or without mechanical damage presented high non-photochemical extinction coefficients (NPQ) throughout the evaluations (Figures 5 and 6).
Plants grown in the absence of Ca 2+ and with or without mechanical damage showed low carbon assimilation (A), stomatal conductance (Gs), transpiration (E), instantaneous water use efficiency (iWUE), the efficiency of RuBisCO carboxylation (A/Ci) and high concentrations of intracellular carbon (Ci) (Figure 7). These conditions probably contributed to smaller leaf area and lower total dry mass and lower root, stem, leaf, flower and fruit dry mass ( Figure 8). It is noteworthy that Ca 2+ -free plants without mechanical damage died before Ca 2+ -free plants with mechanical damage. rate (ETR) time × calcium p < 0.008; (C). effective quantum yield (ΦPSII) time × (D). photochemical quenching (qL) time × damage × calcium p < 0.002; (E). frac excitation energy in the antenna that cannot be used for photochemical phase (Ex) p < 0.005; (F). light fraction absorbed by the PSII antenna that is dissipated as h 0.001, in Solanum lycopersicum "cv. Micro-Tom" grown with variation in calcium co and with (w/MD) or without (wo/MD) mechanical damage. Different lowercas significant difference in calcium levels within wo/MD or w/MD plants. Capital l and w/MD plants within the same calcium level. ns = not significant at 5% correspond to averages, whiskers to ± SE (n = 3). transpiration rate (E) calcium p < 0.001; (E). Instant carboxylation efficiency of ribulose enzyme 1,5diphosphate carboxylase (RuBisCO) (A/Ci) time × damage × calcium p < 0.016; (F). water use efficiency (iWUE) time × calcium p < 0.001 in Solanum lycopersicum "cv. Micro-Tom" grown with variation in calcium concentration (mM) and with (w/MD) or without (wo/MD) mechanical damage. Different lowercase letters indicate a significant difference in calcium levels within wo/MD or w/MD plants. Capital letters test wo/MD and w/MD plants within the same calcium level. ns = not significant at 5% Tukey test. Bars correspond to averages, whiskers to ± SE (n = 3).
(D). transpiration rate (E) calcium p < 0.001; (E). Instant carboxylation efficiency of ribulose enzyme 1,5-diphosphate carboxylase (RuBisCO) (A/Ci) time × damage × calcium p < 0.016; (F). water use efficiency (iWUE) time × calcium p < 0.001 in Solanum lycopersicum "cv. Micro-Tom" grown with variation in calcium concentration (mM) and with (w/MD) or without (wo/MD) mechanical damage. Different lowercase letters indicate a significant difference in calcium levels within wo/MD or w/MD plants. Capital letters test wo/MD and w/MD plants within the same calcium level. ns = not significant at 5% Tukey test. Bars correspond to averages, whiskers to ± SE (n = 3).
Plants grown in the presence of 2 and 4 mM of Ca 2+ and without damage had similar carbon assimilation rates, RuBisCO carboxylation and water use efficiencies. Moreover, as revealed at the end of the evaluations, these plants had similar total dry mass and fruit and stem dry mass (Figure 8), although leaf and root dry mass were higher for plants grown in the presence of 4 mM Ca 2+ .
Plants grown in the presence of 2 and 4 mM of Ca 2+ and with mechanical damage showed lower carbon assimilation rates, RuBisCO carboxylation and water use efficiencies, as well as low stem, fruit and total dry mass (Figures 7 and 8), compared to the same without mechanical damage.
Thus, the results for chlorophyll a fluorescence, gas exchange and plant biomass reveal that the photosynthetic performance of Ca 2+ -free plants with mechanical damage differs from that of those grown in the presence of Ca 2+ and with mechanical damage. The Ex of Ca 2+ -free plants with mechanical damage was low, while that of plants grown in the presence of Ca 2+ with mechanical damage was elevated ( Figure 6). The A/Ci of Ca 2+ -free plants with mechanical damage was higher than that of Ca 2+ -free plants without mechanical damage seven days after damage (Figure 7). The A/Ci of plants grown in the presence of 2 and 4 mM Ca 2+ and with mechanical damage was lower than that of plants grown in the presence of 2 and 4 mM Ca 2+ and without mechanical damage at 14, 21 and 28 days after mechanical damage.

Two Clusters Were Found, One for Plants Grown in the Absence of Ca 2+ and One for Those Grown in the Presence of Ca 2+ , and a Positive Correlation between H2O2, Gene Expression and Enzyme Activity Is Highlighted
Hierarchical cluster analysis (HCA) revealed the formation of two clusters representing treatments with the absence of Ca 2+ and with the presence of Ca 2+ . The presence of Ca 2+ with mechanical damage exhibited a strong positive correlation with SlCaM2 and MPK1 expression and CAT enzyme activity. On the other hand, the absence of Ca 2+ with mechanical damage exhibited a strong positive correlation with H2O2 and RBOH1 and MPK1 expression, POX and APX activity, lipid peroxidation and Ci, and a strong negative correlation with iWUE and A/Ci ( Figure 9A).
The paired correlation heat map revealed that H2O2 is positively correlated with RBOH1 and MPK1 expression, SOD and POX activity, lipid peroxidation (MDA) and Ci, and negatively correlated with SlCaM2 and MPK2 expression and E, Gs, A, A/Ci and iWUE ( Figure 9B).

Discussion
Plants of Solanum lycopersicum "cv. Micro-Tom" grown in the absence of Ca 2+ showed signs of element deficiency, according to those recorded by Kalaji et al. [24] and Tang and The paired correlation heat map revealed that H 2 O 2 is positively correlated with RBOH1 and MPK1 expression, SOD and POX activity, lipid peroxidation (MDA) and Ci, and negatively correlated with SlCaM2 and MPK2 expression and E, Gs, A, A/Ci and iWUE ( Figure 9B).

Discussion
Plants of Solanum lycopersicum "cv. Micro-Tom" grown in the absence of Ca 2+ showed signs of element deficiency, according to those recorded by Kalaji et al. [24] and Tang and Luan [28]. Several studies have revealed that plants grown in the absence of Ca 2+ and with mechanical damage show greater expression of the RBOH1 gene after mechanical damage. This gene is activated by calcium, and the protein it expresses is responsible for the production and control of hydrogen peroxide, which is involved in signaling and signal propagation of plant defenses [29]. These studies show that RBOH1 is involved with injury recognition and the rapid and systemic cell-to-cell signaling induced by injury [20][21][22][23].
Accompanied by, and dependent on, the production and accumulation of H 2 O 2 in extracellular spaces [4], the signaling described above can be converted into a radial signal, propagated among xylem cells by the release of Ca 2+ from transporter glutamate receptorlike channels (GRLs), which is a mechanism that can interconnect signals generated by Ca 2+ and reactive oxygen species (ROS) [30]. Accordingly, H 2 O 2 markings by DAB suggest that this signal is propagated via xylem in plants grown in the absence of Ca 2+ and with mechanical damage, a condition that suggests that RBOH1 signal propagation may depend on, and be linked to, the presence of hydrogen peroxide in the absence of calcium. It is important to note that chloroplasts are a source of Ca 2+ [31], and it was necessary for plants to use the element for the activation of RBOH1.
The heat map showed a positive influence on the expression of RBOH1, MPK1 and MPK2 in plants grown in the presence of 2 mM Ca 2+ and with mechanical damage, compared to plants grown in the presence of 2 mM Ca 2+ and without mechanical damage, which revealed a low concentration of H 2 O 2 , efficiently controlled by CAT. Plants grown in the absence of Ca 2+ -and with mechanical damage showed high expression of RBOH1 and accumulation of H 2 O 2 . However, the increase in activity of the chloroplast enzyme APX should be noted, as it indicates an alteration possibly due to the activation of RBOH1 with the use of Ca 2+ . The change in nutrient utilization priority negatively influenced photosynthesis with energy accumulation in the photosystem, which may have caused an increase in APX activity.
Plants grown in the absence of Ca 2+ and without damage showed a negative influence on gas exchange, despite the low Ci. It is important to highlight the low correlation between Gs and Ci, which suggests stress in the photosynthetic system, probably due to the need for Ca 2+ in the chloroplast for signaling and increasing APX and POX at the expense of SOD and CAT. Plants grown in the absence of Ca 2+ and with mechanical damage showed higher Ci, which suggests the need for Ca 2+ in the electron transport chain for CO 2 incorporation.
Plants grown in the presence of 2 mM Ca 2+ and with mechanical damage showed higher CAT activity, which controlled H 2 O 2 .
In plants grown in the absence of Ca 2+ and with mechanical damage, a higher concentration of H 2 O 2 may have contributed to the signaling of defense mechanisms, with the expression of MPK1, which, by acting in cascade, activates MPK2. This is supported by the positive correlations between H 2 O 2 and MPK1 and between MPK1 and MPK2, which are genes involved in the recognition and overcoming of stress [15,16]. These genes transcribe MPK1 and MPK2 kinases involved with the activation of antioxidant enzymes, which may explain the greater activity of antioxidant enzymes in plants grown in the absence of calcium and with mechanical damage. In addition, it is known that silencing of MPK1 and MPK2 can result in decreased levels of Cu/Zn-SOD, APX, GR1 and CAT1 transcription [18].
In addition, Ca 2+ -free plants with mechanical damage had no activation of calmodulin, as the heat map reveals lower expression of the SICaM2 gene, probably influenced by the damage. Calmodulin is necessary for the activation of MPK2 and catalase, the first enzyme to neutralize H 2 O 2 , which revealed low activity in these plants right after mechanical damage. Even though catalase increased during the evaluations, it did not control the H 2 O 2 concentration in these plants, as confirmed by the presence of hydrogen peroxide in plant tissue as indicated by DAB, which may have been responsible for the accumulation of malondialdehyde because of lipid peroxidation. Studies have demonstrated the need for calmodulin activation to coordinate the response to mechanical damage by activating MAPK kinases and antioxidant enzymes involved in regulating H 2 O 2 levels [5,10,32,33].
Explained by the greater activity of peroxidases in plants with mechanical damage, the marking of hydrogen peroxide by DAB in Ca 2+ -free plants with mechanical damage was acuter than in Ca 2+ -free plants without mechanical damage. The DAB markings still suggest the need for the Ca 2+ ion in the activation of peroxidases that neutralize H 2 O 2 in the cell walls of the main veins. Studies have reported that calcium application stimulates cell wall peroxidase activity [34][35][36].
Plants grown in the absence of Ca 2+ and without mechanical damage did not present stress response mechanisms, and the expression of target genes was not detected. The H 2 O 2 concentration in these plants increased with time but was lower than in Ca 2+ -free plants with damage and was insufficient to signal stress and activate the genes (RBOH1, SlCaM2, MPK1 and MPK2) necessary for CAT activation for H 2 O 2 neutralization, resulting in structural damage such as malondialdehyde accumulation and tissue necrosis.
The absence of Ca 2+ also interfered with the photosynthetic process and the accumulation of dry mass. Solanum lycopersicum plants grown in the absence of Ca 2+ , regardless of mechanical damage, showed impairment in the functioning of photosystems with decreases in potential quantum yield (Fv/Fm), photosystem II efficiency in light (Fv'/Fm') and photochemical quenching in light (qL). These results indicate difficulty in capturing light and suggest that calcium deficiency interferes with the stability of photosystems [37,38].
In addition, the low effective quantum yield (ΦPSII) and electron transport rate (ETR) in Ca 2+ -free plants suggest that calcium deficiency in electron transport is influencing quantum productivity responsible for reducing carbon due to its channel energy. It has been suggested that calcium is important for the stability of the oxygen evolution complex when inactivated, as it interrupts the flow of electrons in the photosystems [24,31]. This, in turn, explains the greater energy dissipation in the form of heat in the light (D) and the lower energy not dissipated or used in the photochemical phase in the light (Ex), found in the present study.
Undissipated energy (Ex) in Ca 2+ -free plants with mechanical damage may have been responsible for the production of H 2 O 2 in the chloroplast, as it has been reported by Choudhury et al. [39] and may explain the DAB staining of cells of the palisade parenchyma of these plants. For them, the lower energy produced in the photochemical phase, with less production of reducing agents, may explain the low assimilation rate (A), the low rubisco carboxylation efficiency (A/Ci) and the greater internal carbon accumulation. Previous studies embracing stress revealed the same response when plants were subjected to water deficiency [40,41]. Reduced stomatal conductance (Gs), transpiration (E) and water use efficiency (iWUE) may be explained by the fact that stomatal opening requires signaling with rapid calcium oscillation, as already addressed by previous studies [42,43], and this movement does not happen in Ca 2+ -free plants, resulting in the stomatal closure.
Thus, it should be noted that growth in the absence of Ca 2+ promoted an increase in RBOHD expression and H 2 O 2 concentration, being higher when the plants were subjected to mechanical damage, which may have caused oxidative stress contributing to stomatal closure and reduced conductance stomatal (Gs). Moreover, the functions of calcium in cell division, cell wall formation, pollen tube to flower fertilization and fruit development [44] explain why plants grown in the absence of Ca 2+ , regardless of mechanical damage, had a low leaf, flower, fruit, stem, and root mass.
Plants grown in the presence of Ca 2+ and with mechanical damage showed greater expression of SlCaM2 (Calmodulin gene) six hours after mechanical damage, which suggests that the activation of this gene depends on a lower-level interaction between Ca 2+ and reactive oxygen species, as revealed in these plants. Thus, calmodulin may be involved with coordinating the action of kinases and antioxidant enzymes in order to make the plant's response to damage more efficient and rapid, as it has been observed in other studies, which demonstrates that CaM2 acts in the coordination of kinases and antioxidant enzymes [10,45]. Thus, this may indicate that calcium is essential to control the activity of antioxidant enzymes. The expression of RBOH1, MPK1 and MPK2 in plants grown with Ca 2+ and with mechanical damage suggests that the presence of this ion enabled molecular adjustments so that the plants had increased CAT enzyme activity, low levels of hydrogen peroxide and low lipid peroxidation, which can lead to overcoming stress from mechanical damage. Calcium-dependent protein kinases are involved with the RBOH1 regulation pathway due to its phosphorylation [20][21][22].
Plants grown in the presence of Ca 2+ showed a reaction to DAB, suggesting that the presence of calcium may have improved peroxidase activity, as indicated by this reaction in tissues [18,46]. In plants without mechanical damage, the main veins and glandular trichomes were marked by DAB, while in those with mechanical damage, the main vein, xylem, palisade parenchyma and glandular trichomes were marked. Staining of trichomes and palisade parenchyma can be explained by high metabolic activity, as presented by Balcke et al. [47], in which case, an increase in peroxidase activity may occur.
As for the photosynthetic process, plants grown in the presence of Ca 2+ and without mechanical damage showed low minimum fluorescence adapted to the dark (Fo), high capacity to absorb light (Fv'/Fm'), low light fraction absorbed by the PSII antenna dissipated as heat (D) and low fraction of excitation energy not dissipated in the antenna and not used for photochemistry in the light (Ex), all of which are variables that indicate that the presence of Ca 2+ allowed, in the light, normal electron flow (ETR) and high quantum productivity (ΦPSII), resulting in the production of reducing agents for carbon reduction [48]. These plants also showed low energy dissipation (Ex and D), as observed in previous stress studies, due to different causes, among them cadmium [31,37,49,50]. According to the literature, it is suggested that Ca 2+ triggers the signaling of defense pathways to prevent damage to the photosystem [13]. These conditions contribute to high rates of carbon assimilation, rubisco activity and better control of stomatal opening, with high water use efficiency, as found in other studies [42,43], which is a condition not verified in plants grown in the absence of calcium.

Study Species and Cultivation
Solanum lycopersicum "cv. Micro-Tom" seeds were supplied by Lázaro E. P. Peres and germinated in trays with expanded clay of medium texture, according to [51]. Fourteen days after sowing, young plants were grown in standard nutrient solution containing 4 mM Ca 2+ (control treatment), and Hoagland and Arnon [52]

Gene Expression
Gene expression of RBOH1, MPK1, MPK2 and SlCaM2 (Table 1) in plants grown in the presence of 0 (absence of Ca 2+ or Ca 2+ -free) and 2 mM Ca 2+ were evaluated at 0.5 and 3, 6 and 24 h after mechanical damage to plants, representing the time of greatest gene expression [5,9]. For this, 50 mg of leaves in the region where the mechanical damage was performed were collected at 9:00 a.m., packed in plastic bags, wrapped in aluminum foil and frozen in liquid nitrogen to immediately stop all metabolic reactions. The study of gene expression was performed by Quantitative Polymerase Chain Reaction (* RT-qPCR). The extracted RNA was used to make cDNA by reverse transcription. The presence or absence of a transcript (mRNA) was determined by * RT-qPCR reaction. The extraction of its transcriptome followed the protocol of the manufacturer of the TriZOL Reagent (Thermo Scientific, Waltham, MA, USA). The obtained total RNA samples were quantified and then properly treated with DNase RNase free, according to recommendations (Promega, Madison, WI, USA). The treated RNA samples were used in cDNA synthesis using the High-Capacity kit, according to Thermo Fischer protocol. Subsequently, the cDNA samples were treated with RNase and then used for analysis of RBOH1, MPK1, MPK2 and SlCaM2 by qPCR using the enzymatic system GoTaq ® qPCR and RT-qPCR ( Table 1). The 2 −∆∆Ct method was used to calculate the level of gene expression (mRNA) of the referred genes. Genes already described in the literature and that had constitutive expression within each treatment were used as normalizers (Table 1). Three biological repetitions for each treatment and three technical repetitions for each biological repetition were performed.

Histochemical Analysis of Hydrogen Peroxide
Sections of the median portion of fully expanded leaf blades with mechanical damage at 0.5 h and 21 days after mechanical damage were submitted to histochemical testing with 3,3 -diaminobenzidine (DAB) to locate hydrogen peroxide (H 2 O 2 ), following Thordal-Christensen [46], with three biological repetitions for each treatment.

Chlorophyll a Fluorescence, Gas Exchange and Plant Biomass
Chlorophyll a fluorescence was measured between 9:00 a.m. and 11:00 a.m. at 0.5 h and 7, 14, 21 and 28 days after mechanical damage in fully expanded leaves located in the stem region, below mechanical damage, and stored in the dark for 30 min. Measurements were made with a pulse amplitude portable fluorometer (Jr PAM, Walz) under 1150 PPFD saturating irradiance. Minimum dark-adapted fluorescence (Fo), maximum quantum efficiency of photosystem II (Fv/Fm), non-photochemical quenching and estimates of the constant rate of heat loss from PSII [NPQ = (Fm − Fm')/Fm'] were determined, with four biological repetitions for each treatment.
Chlorophyll a fluorescence and gas exchange were evaluated at the same time and in leaves located in the stem region, below mechanical damage, before destructive sampling, using open-system photosynthesis equipment with CO 2 and water-vapor infrared analyzer and a coupled fluorometer (Infra-Red Gas Analyzer-IRGA, model GFS 3000 FL with LED-Array/PAM-Fluorometer 3055-FL, Walz) at 0.5 h and 7, 14, 21 and 28 days after mechanical damage with saturant light of 1200 µmol m −2 s −1 , 401.75 ± 23.95 PPM and VPD 1.834 ± 0.183 kPa at the moment of analysis. The fluorescence variables measured were effective quantum yield (ΦPSII), electron transport rate (ETR), photosystem II efficiency (Fv'/Fm'), photochemical quenching (qL), light fraction absorbed by the PSII antenna that is dissipated as heat (D) and fraction of dissipated excitation energy in the antenna that cannot be used for photochemical phase (Ex) [62,63]. The gas exchange variables measured were net CO 2 assimilation rate (A, µmol CO 2 m −2 s −1 ), transpiration rate (E, mmol water vapor m −2 s −1 ), stomatal conductance (Gs, mmol m −2 s −1 ) and internal leaf CO 2 concentration (Ci, µmol CO 2 mol −1 ar). Instantaneous water use efficiency (iWUE, µmol CO 2 (mmol H 2 O −1 ) was determined as the ratio between CO 2 assimilation rate and transpiration rate (A/E), while instant carboxylation efficiency of ribulose enzyme 1,5-diphosphate carboxylase (RuBisCO) was calculated as the ratio between net CO 2 assimilation rate and internal leaf CO 2 concentration (A/Ci) [64], with four biological repetitions for each treatment.
The leaves, stems, roots, flowers, and fruits were subjected to drying at 38 • C until constant dry mass and total dry mass were calculated as the sum of all organs. Leaf area was determined by a leaf area integrator LI-3100C area meter LI-COR at 7, 14, 21 and 28 days after damage, with four biological repetitions for each treatment.

Statistical Analysis
Homogeneity of variances was checked using Levene's test. The variables were submitted to analysis of variance (three-way ANOVA) and means compared by Tukey's test at 5% probability (SigmaPlot 12.0) [65]. Heat map generation, hierarchical cluster analysis (HCA) and pairwise correlation heat maps were performed using MetaboAnalyst 4.0 software [66].

Conclusions
Based on the results presented here and on other studies reported in the literature, damage may be important in activating stress response mechanisms in Ca 2+ -free plants. The signaling of mechanical damage must have been performed by reactive oxygen species, as high levels of H 2 O 2 and expression of genes involved in stress control and signaling were observed in these plants, which revealed low photosynthetic performance.
Regarding gas exchange, plants grown in the presence of Ca 2+ and with mechanical damage showed a decrease in the efficiency of instantaneous carboxylation of the activity of the enzyme ribulose 1,5-diphosphate carboxylase (RuBisCO). This result suggests that mechanical damage increased H 2 O 2 , thus generating a signal to activate defensive pathways to reverse stress. Although they did not show the best results in terms of chlorophyll a fluorescence and gas exchange, plants cultivated in the presence of Ca 2+ and subjected to mechanical damage recovered since mechanical damage did not lead to a decrease in total dry mass, which is in agreement with other studies.
We conclude that the plants grown in the absence of a calcium supply recognized the mechanical damage but did not survive. The highest expression of the RBOH1 gene and the accumulation of H 2 O 2 in these plants signaled cell death. Plants grown in the presence of calcium showed higher expression of SlCaM2 and control of H 2 O 2 concentration, overcoming the stress caused by mechanical damage, with photosynthetic acclimatization and without damage to dry mass production.