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Article

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

by
Felipe Girotto Campos
1,
Diana Pacheco Seixas
1,
Gustavo Ribeiro Barzotto
2,
Letícia Galhardo Jorge
1,
Karina Renostro Ducatti
2,
Gisela Ferreira
1,
Tatiane Maria Rodrigues
1,
Edvaldo Aparecido Amaral da Silva
2 and
Carmen Sílvia Fernandes Boaro
1,*
1
Biostatistics, Plant Biology, Parasitology and Zoology Department, Institute of Biosciences, São Paulo State University (UNESP), Campus Botucatu, Street Prof. Dr. Antônio Celso Wagner Zanin 250, Botucatu 18618-970, SP, Brazil
2
Plant Production Department, School of Agriculture, São Paulo State University (UNESP), Campus Botucatu, Av. Universitária n° 3780, Altos do Paraíso, Botucatu 18610-307, SP, Brazil
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2022, 23(21), 13571; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms232113571
Submission received: 19 August 2022 / Revised: 28 October 2022 / Accepted: 2 November 2022 / Published: 5 November 2022
(This article belongs to the Special Issue State-of-the-Art Molecular Plant Sciences in Brazil)
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Versions Notes

Abstract

:
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.

1. Introduction

Mechanical damage in plants activates a cascade of defense reactions closely related to plant defenses against herbivory [1]. Mechanically damaged plant tissues produce O2 and H2O2, 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 Ca2+-channel opening, resulting in increased cytoplasmic Ca2+ [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 Ca2+ generates an oscillation responsible for the activation of proteins such as calmodulin (CaMs), CaM-likes, calcinerin B-like and calcium-dependent protein kinases (CDPKs) [8]. High levels of Ca2+ and ROS activate MAPKs (mitogen-activated protein kinases) and calmodulin to regulate ROS accumulation [5]. Furthermore, expression of the calmodulin gene (SlCaM2) present in tomato leaves is dependent on, among other factors, mechanical damage, an important intermediate in signaling abiotic and biotic stresses, along with kinases [9].
Calcium-dependent calmodulins play an important role in signaling and activating kinases in response to stress [10] since MAPKs translate extracellular stimuli to intracellular responses [1]. Calcium application activates Calmodulin, provides better coordination and stimulates antioxidant enzymes, which reduce lipid peroxidation, allowing tolerance to stress induced by, for example, the element nickel [10,11,12]. The signals of calcium trigger transduction for gene expression and activation of biochemical responses, which enable faster and more efficient neutralization of ROS by antioxidant enzymes, thereby protecting photosystems and maintaining a high photosynthetic rate [13].
Among kinases activated by calmodulins, MAPKs are signaling modules conserved in eukaryotes that link the perception of stimuli to cellular responses. This signaling combines at least three sequentially phosphorylation-activated protein kinases, MAP3K and MAP2K [14]. In plants, MAPK cascades are mainly associated with drought, salinity, cold, injury, ozone, ROS and hormonal stimuli, as well as developmental processes such as cell division and differentiation or abscission [15,16]. In Arabidopsis, MPK1 and MPK2 are activated by stresses induced by wounding and H2O2 [17].
Specifically, it is known that stress conditions caused by cold, drought and Paraquat®® application induce increased MPK1 and MPK2 transcription in leaves of tomato plants inoculated with tobacco mosaic virus. In addition, silencing of the RBOH1 gene reduced the expression of MPK1 and MPK2 genes by 70%, suggesting that H2O2 from RBOH1 in the apoplast is essential for the activation of MPK1 and MPK2 [18].
RBOHD regulation by C-terminal ubiquinone phosphorylation is mediated by calcium-dependent protein kinases (CPKs), also preserved in plants and animals due to H2O2 synthesis regulation, and is related to cell death [19,20,21,22]. Arabidopsis mutants kept in the dark and with RBOHD absence or overexpression revealed that RBOHD is responsible for damage recognition and H2O2 control [23].
The mentioned genes are directly related to ROS, calcium signaling pathways and stress responses. In this way, the evaluation of gene expression can provide evidence of mechanical damage interfering with the use of endogenous calcium to overcome stress at the expense of photosynthesis, which also requires calcium [24].
Stress by calcium deficiency has been found to interfere with the photosynthetic process, such as with electron flow, since it destabilizes the oxygen evolution complex and causes greater energy dissipation in the form of heat by the antennas (D), as observed in corn and tomatoes plant [24].
On the other hand, exogenous calcium in Pennisetum americanum (L.) K. Schum was found to contribute to the regulation of low H2O2 levels, allowing the restoration of chlorophyllous tissues, maintaining high expression of the D1 protein, and adjusting glutamine-S-transferase, which prevented photoinhibition of PSII [25].
There are reports of the involvement of calcium–calmodulin in the synthesis of violaxanthine deepoxidase, which contributes to the xanthophyll cycle, an important pathway for the dissipation of non-photochemical energy (NPQ). Thus, plants with calcium deficiency and under conditions of abiotic stress can direct this element for signaling and stress response pathways, which can lead to energy accumulation in photosystem II, thereby limiting the photosynthetic process [26].
Solanum lycopersicum Micro-Tom (MT) is a model plant for studies of gene expression and physiological processes [27] since its genome is sequenced and its signaling pathways are well known. This condition allows the investigation of the roles of Ca2+ and H2O2 in signaling and gene expression involved with antioxidant enzyme activity, thereby contributing to the maintenance of the photosynthetic process in plants subjected to mechanical damage.
Therefore, this study aimed to investigate the influence of variation in calcium concentration on signaling of defense against mechanical damage, as well as restoration and photosynthetic acclimation, using Solanum lycopersicum Micro-Tom (MT) as a model plant.

2. Results

2.1. Differentiated Expression of RBOH1, MPK1, MPK2 and SlCaM2 under Ca2+ Presence/Absence (Ca2+-Free) and with/without Mechanical Damage

The time since mechanical damage was not significant (p < 0.05), except for MPK2, and so only calcium and mechanical damage showed an interaction, and are thus represented in Figure 1.
Calcium and mechanical damage contributed to increased SlCaM2 expression (Figure 1A), whereas the absence of Ca2+ with mechanical damage increased RBOH1 expression (Figure 1B). MPK1 showed higher gene expression in plants with mechanical damage, independent of Ca2+ level (Figure 1C). However, MPK2 showed higher gene expression in plants with Ca2+ at 0.5 h and with mechanical damage, indicating that the presence of calcium increased MPK2 expression (Figure 1D).

2.2. Ca2+ 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, Ca2+-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 Ca2+, independent of mechanical damage (Figure 2A,B,D). Specifically, the highest activities of the enzyme CAT were for Ca2+-free and 4 mM Ca2+-cultivated plants, with and without damage (Figure 2C).
Plants grown with 2 and 4 mM Ca2+ showed no variation in hydrogen peroxide concentration, regardless of mechanical damage, and had the lowest concentrations. Plants grown in the absence of Ca2+ 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 Ca2+-free plants with mechanical damage. Lipid peroxidation did not differ among plants grown in the presence of Ca2+ (Figure 2F).
Plants grown in the absence of Ca2+ and with mechanical damage resisted the deficiency of the element longer.

2.3. Hydrogen Peroxide Was Evidenced in Leaves of Plants Grown in the Presence of Ca2+ and with Mechanical Damage

Thirty minutes after mechanical damage, Ca2+-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). No staining was observed in the mesophyll cells under these conditions.
Plants grown in the presence of 2 mM Ca2+ 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 Ca2+ 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, Ca2+-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 Ca2+-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).
Plants grown in the presence of 2 mM Ca2+ and without mechanical damage showed intense staining for hydrogen peroxide on the walls of the vessel elements of the midrib (Figure 4f) and smaller veins. No staining of parenchymal cells was observed in the mesophyll (Figure 4e). Plants grown in the presence of 2 mM Ca2+ and with mechanical damage had more intense and generalized staining for hydrogen peroxide. The staining was evident in the head cells of glandular trichomes (Figure 4g), inside palisade and spongy parenchyma cells (Figure 4g) and on the walls of the vessel elements of the midrib (Figure 4h) and smaller veins.
Cross sections of the mesophyll region and midrib of a leaf blade of Solanum lycopersicum “cv. Micro-Tom” without DAB treatment are shown in Supplementary Figure S1.

2.4. Performance of Chlorophyll a Fluorescence Does Not Differ between Plants Grown in the Absence of Ca2+ with and without Damage, but There Is an Influence Resulting in Low Gas Exchange and Plant Biomass

Plants grown in the absence of Ca2+ with and without mechanical damage did not differ in chlorophyll a fluorescence performance (Figure 5 and Figure 6). Likewise, plants grown in the presence of 2 or 4 mM Ca2+ and with and without damage did not differ in chlorophyll a fluorescence performance (Figure 5 and Figure 6).
Plants of Solanum lycopersicum cv. Micro-Tom grown in the absence of Ca2+ 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) (Figure 5 and Figure 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) (Figure 5 and Figure 6). Plants grown in the presence of 2 and 4 mM of Ca2+ and with or without mechanical damage presented high non-photochemical extinction coefficients (NPQ) throughout the evaluations (Figure 5 and Figure 6).
Plants grown in the absence of Ca2+ 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 Ca2+-free plants without mechanical damage died before Ca2+-free plants with mechanical damage.
Plants grown in the presence of 2 and 4 mM of Ca2+ 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 Ca2+.
Plants grown in the presence of 2 and 4 mM of Ca2+ 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 (Figure 7 and Figure 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 Ca2+-free plants with mechanical damage differs from that of those grown in the presence of Ca2+ and with mechanical damage. The Ex of Ca2+-free plants with mechanical damage was low, while that of plants grown in the presence of Ca2+ with mechanical damage was elevated (Figure 6). The A/Ci of Ca2+-free plants with mechanical damage was higher than that of Ca2+-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 Ca2+ and with mechanical damage was lower than that of plants grown in the presence of 2 and 4 mM Ca2+ and without mechanical damage at 14, 21 and 28 days after mechanical damage.

2.5. Two Clusters Were Found, One for Plants Grown in the Absence of Ca2+ and One for Those Grown in the Presence of Ca2+, 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 Ca2+ and with the presence of Ca2+. The presence of Ca2+ with mechanical damage exhibited a strong positive correlation with SlCaM2 and MPK1 expression and CAT enzyme activity. On the other hand, the absence of Ca2+ 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).

3. Discussion

Plants of Solanum lycopersicum “cv. Micro-Tom” grown in the absence of Ca2+ 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 Ca2+ 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 H2O2 in extracellular spaces [4], the signaling described above can be converted into a radial signal, propagated among xylem cells by the release of Ca2+ from transporter glutamate receptor-like channels (GRLs), which is a mechanism that can interconnect signals generated by Ca2+ and reactive oxygen species (ROS) [30]. Accordingly, H2O2 markings by DAB suggest that this signal is propagated via xylem in plants grown in the absence of Ca2+ 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 Ca2+ [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 Ca2+ and with mechanical damage, compared to plants grown in the presence of 2 mM Ca2+ and without mechanical damage, which revealed a low concentration of H2O2, efficiently controlled by CAT. Plants grown in the absence of Ca2+- and with mechanical damage showed high expression of RBOH1 and accumulation of H2O2. 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 Ca2+. 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 Ca2+ 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 Ca2+ in the chloroplast for signaling and increasing APX and POX at the expense of SOD and CAT. Plants grown in the absence of Ca2+ and with mechanical damage showed higher Ci, which suggests the need for Ca2+ in the electron transport chain for CO2 incorporation.
Plants grown in the presence of 2 mM Ca2+ and with mechanical damage showed higher CAT activity, which controlled H2O2.
In plants grown in the absence of Ca2+ and with mechanical damage, a higher concentration of H2O2 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 H2O2 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, Ca2+-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 H2O2, which revealed low activity in these plants right after mechanical damage. Even though catalase increased during the evaluations, it did not control the H2O2 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 H2O2 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 Ca2+-free plants with mechanical damage was acuter than in Ca2+-free plants without mechanical damage. The DAB markings still suggest the need for the Ca2+ ion in the activation of peroxidases that neutralize H2O2 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 Ca2+ and without mechanical damage did not present stress response mechanisms, and the expression of target genes was not detected. The H2O2 concentration in these plants increased with time but was lower than in Ca2+-free plants with damage and was insufficient to signal stress and activate the genes (RBOH1, SlCaM2, MPK1 and MPK2) necessary for CAT activation for H2O2 neutralization, resulting in structural damage such as malondialdehyde accumulation and tissue necrosis.
The absence of Ca2+ also interfered with the photosynthetic process and the accumulation of dry mass. Solanum lycopersicum plants grown in the absence of Ca2+, 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 Ca2+-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 Ca2+-free plants with mechanical damage may have been responsible for the production of H2O2 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 Ca2+-free plants, resulting in the stomatal closure.
Thus, it should be noted that growth in the absence of Ca2+ promoted an increase in RBOHD expression and H2O2 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 Ca2+, regardless of mechanical damage, had a low leaf, flower, fruit, stem, and root mass.
Plants grown in the presence of Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ 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.

4. Materials and Methods

4.1. 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 Ca2+ (control treatment), and Hoagland and Arnon [52] nutrient solution N° 2 modified to provide concentrations of 2 and 0 mM Ca2+ (absence of Ca2+ or Ca2+-free). Seven days later, plants that received mechanical damage had their leaf limb margins cut with the aid of scissors, without removal of plant tissue (Supplementary Figure S2) and kept in the same solutions until later analysis.
The experiment was conducted at the Biostatistics, Plant Biology, Parasitology and Zoology Department of the Biosciences Institute, UNESP, Campus Botucatu, São Paulo, Brazil (22°49′10″ S, 48°24′35″ W, 800 m above sea level) in a paddy fan-greenhouse, with sunrise at 6:20 a.m., temperature maintained at 25.62 ± 2.62 °C and air humidity at 50.35 ± 4.94%. At the moment of all analyses, the photosynthetic photon flux density (PPFD) was at 495.00 ± 75.00 µmol m−2 s−1 and environmental CO2 was at 401.75 ± 23.95 PPM. The experiment used a randomized complete block design with four replications of 16 plants in a 3 × 2 × 5 factorial scheme: 0 (absence of Ca2+ or Ca2+-free), 2 and 4 mM Ca2+, with/without mechanical damage and evaluation times.

4.2. Gene Expression

Gene expression of RBOH1, MPK1, MPK2 and SlCaM2 (Table 1) in plants grown in the presence of 0 (absence of Ca2+ or Ca2+-free) and 2 mM Ca2+ 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−ΔΔCt 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.

4.3. Activity of Antioxidant Enzymes and Quantification of Hydrogen Peroxide and Lipid Peroxidation

Leaves were collected between 9:00 and 11:00 a.m. at 0.5 h and 7, 14 and 21 days after mechanical damage, and samples were frozen in liquid nitrogen and stored until evaluation. Extract was obtained from 100 mg of fresh leaf mass and separated into microtubes and stored at −20 °C for later determination. The activities of the enzymes superoxide dismutase (SOD, EC 1.15.1.1) [55], total peroxidase (POX, EC 1.11.1.7) [56], catalase (CAT, EC: 1.11.1.6) [57] and ascorbate peroxidase (APX, EC: 1.11.1.1) [58] were determined. Total proteins were quantified according to [59], hydrogen peroxide according to Alexieva et al. [60] and lipid peroxidation according to Devi and Prasad [61], with four biological repetitions for each treatment.

4.4. 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 (H2O2), following Thordal-Christensen [46], with three biological repetitions for each treatment.

4.5. 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 CO2 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 CO2 assimilation rate (A, μmol CO2 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 CO2 concentration (Ci, µmol CO2 mol−1 ar). Instantaneous water use efficiency (iWUE, μmol CO2 (mmol H2O−1) was determined as the ratio between CO2 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 CO2 assimilation rate and internal leaf CO2 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.

4.6. 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].

5. 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 Ca2+-free plants. The signaling of mechanical damage must have been performed by reactive oxygen species, as high levels of H2O2 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 Ca2+ 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 H2O2, 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 Ca2+ 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 H2O2 in these plants signaled cell death. Plants grown in the presence of calcium showed higher expression of SlCaM2 and control of H2O2 concentration, overcoming the stress caused by mechanical damage, with photosynthetic acclimatization and without damage to dry mass production.

Supplementary Materials

The following supporting information can be downloaded at: https://0-www-mdpi-com.brum.beds.ac.uk/article/10.3390/ijms232113571/s1.

Author Contributions

Conceptualization, F.G.C. and C.S.F.B.; methodology, F.G.C., D.P.S., G.R.B., L.G.J. and K.R.D.; validation, F.G.C., G.F., T.M.R., E.A.A.d.S. and C.S.F.B.; formal analysis, F.G.C., D.P.S., G.R.B. and L.G.J.; investigation, F.G.C., D.P.S., G.R.B. and L.G.J.; data curation, F.G.C., G.F., C.S.F.B., T.M.R. and E.A.A.d.S.; writing—original draft preparation, F.G.C., D.P.S., T.M.R., E.A.A.d.S. and C.S.F.B.; writing—review and editing, F.G.C., D.P.S., L.G.J., G.R.B. and C.S.F.B.; supervision, G.F., T.M.R., E.A.A.d.S. and C.S.F.B. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

The authors thank the National Council for Scientific and Technological Development (CNPq) for financial support (Process number 140103/2016-3) and for the research productivity fellowship (Process number 312900/2021-0) to T.M. Rodrigues. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil—CAPES (Finance Code 001), Pro-Rectory of Research (PROPe-Unesp) for support to scientific publication.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Seo, S.; Katou, S.; Seto, H.; Gomi, K.; Ohashi, Y. The Mitogen-Activated Protein Kinases WIPK and SIPK Regulate the Levels of Jasmonic and Salicylic Acids in Wounded Tobacco Plants. Plant J. 2007, 49, 899–909. [Google Scholar] [CrossRef] [PubMed]
  2. Baxter, A.; Mittler, R.; Suzuki, N. ROS as Key Players in Plant Stress Signalling. J. Exp. Bot. 2013, 65, 1229–1240. [Google Scholar] [CrossRef] [PubMed]
  3. Pandey, S.; Fartyal, D.; Agarwal, A.; Shukla, T.; James, D.; Kaul, T.; Negi, Y.K.; Arora, S.; Reddy, M.K. Abiotic Stress Tolerance in Plants: Myriad Roles of Ascorbate Peroxidase. Front. Plant Sci. 2017, 8, 581. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Suzuki, N.; Mittler, R. Reactive Oxygen Species-Dependent Wound Responses in Animals and Plants. Free Radic. Biol. Med. 2012, 53, 2269–2276. [Google Scholar] [CrossRef]
  5. Takahashi, F.; Mizoguchi, T.; Yoshida, R.; Ichimura, K.; Shinozaki, K. Calmodulin-Dependent Activation of MAP Kinase for ROS Homeostasis in Arabidopsis. Mol. Cell 2011, 41, 649–660. [Google Scholar] [CrossRef]
  6. Gilroy, S.; Białasek, M.; Suzuki, N.; Górecka, M.; Devireddy, A.R.; Karpiński, S.; Mittler, R. ROS, Calcium, and Electric Signals: Key Mediators of Rapid Systemic Signaling in Plants. Plant Physiol. 2016, 171, 1606–1615. [Google Scholar] [CrossRef]
  7. Stael, S.; Kmiecik, P.; Willems, P.; van der Kelen, K.; Coll, N.S.; Teige, M.; van Breusegem, F. Plant Innate Immunity—Sunny Side Up? Trends Plant Sci. 2015, 20, 3–11. [Google Scholar] [CrossRef] [Green Version]
  8. Bouché, N.; Scharlat, A.; Snedden, W.; Bouchez, D.; Fromm, H. A Novel Family of Calmodulin-Binding Transcription Activators in Multicellular Organisms. J. Biol. Chem. 2002, 277, 21851–21861. [Google Scholar] [CrossRef] [Green Version]
  9. Peng, H.; Yang, T.; Jurick, W.M., II. Calmodulin Gene Expression in Response to Mechanical Wounding and Botrytis cinerea Infection in Tomato Fruit. Plants 2014, 3, 427–441. [Google Scholar] [CrossRef] [Green Version]
  10. Yang, T.; Poovaiah, B.W. Hydrogen Peroxide Homeostasis: Activation of Plant Catalase by Calcium/Calmodulin. Proc. Natl. Acad. Sci. USA 2002, 99, 4097–4102. [Google Scholar] [CrossRef]
  11. Siddiqui, M.H.; Al-Whaibi, M.H.; Sakran, A.M.; Basalah, M.O.; Ali, H.M. Effect of Calcium and Potassium on Antioxidant System of Vicia Faba L. under Cadmium Stress. Int. J. Mol. Sci. 2012, 13, 6604–6619. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  12. Xu, C.; Li, X.; Zhang, L. The Effect of Calcium Chloride on Growth, Photosynthesis, and Antioxidant Responses of Zoysia japonica under Drought Conditions. PLoS ONE 2013, 8, e68214. [Google Scholar] [CrossRef]
  13. Yang, S.; Wang, F.; Guo, F.; Meng, J.J.; Li, X.G.; Wan, S.B. Calcium Contributes to Photoprotection and Repair of Photosystem II in Peanut Leaves during Heat and High Irradiance. J. Integr. Plant Biol. 2014, 57, 486–495. [Google Scholar] [CrossRef] [PubMed]
  14. Colcombet, J.; Hirt, H. Arabidopsis MAPKs: A Complex Signalling Network Involved in Multiple Biological Processes. Biochem. J. 2008, 413, 217–226. [Google Scholar] [CrossRef] [PubMed]
  15. Danquah, A.; de Zelicourt, A.; Colcombet, J.; Hirt, H. The Role of ABA and MAPK Signaling Pathways in Plant Abiotic Stress Responses. Biotechnol. Adv. 2014, 32, 40–52. [Google Scholar] [CrossRef]
  16. Zelicourt, A.; Colcombet, J.; Hirt, H. The Role of MAPK Modules and ABA during Abiotic Stress Signaling. Trends Plant Sci. 2016, 21, 677–685. [Google Scholar] [CrossRef]
  17. Colcombet, J.; Sözen, C.; Hirt, H. Convergence of Multiple MAP3Ks on MKK3 Identifies a Set of Novel Stress MAPK Modules. Front. Plant Sci. 2016, 7, 1941. [Google Scholar] [CrossRef] [Green Version]
  18. Zhou, J.; Xia, X.-J.; Zhou, Y.-H.; Shi, K.; Chen, Z.; Yu, J.-Q. RBOH1-Dependent H2O2 Production and Subsequent Activation of MPK1/2 Play an Important Role in Acclimation-Induced Cross-Tolerance in Tomato. J. Exp. Bot. 2014, 65, 595–607. [Google Scholar] [CrossRef] [Green Version]
  19. Maeda, K.; Kimura, S.; Demura, T.; Takeda, J.; Ozeki, Y. DcMYB1 Acts as a Transcriptional Activator of the Carrot Phenylalanine Ammonia-Lyase Gene (DcPAL1) in Response to Elicitor Treatment, UV-B Irradiation and the Dilution Effect. Plant Mol. Biol. 2005, 59, 739–752. [Google Scholar] [CrossRef]
  20. Lee, D.H.; Lal, N.K.; Lin, Z.J.D.; Ma, S.; Liu, J.; Castro, B.; Toruño, T.; Dinesh-Kumar, S.P.; Coaker, G. Regulation of Reactive Oxygen Species during Plant Immunity through Phosphorylation and Ubiquitination of RBOHD. Nat. Commun. 2020, 11, 1838. [Google Scholar] [CrossRef]
  21. Pan, G.; Zhang, H.; Chen, B.; Gao, S.; Yang, B.; Jiang, Y.-Q. Rapeseed Calcium-Dependent Protein Kinase CPK6L Modulates Reactive Oxygen Species and Cell Death through Interacting and Phosphorylating RBOHD. Biochem. Biophys. Res. Commun. 2019, 518, 719–725. [Google Scholar] [CrossRef] [PubMed]
  22. Kimura, S.; Hunter, K.; Vaahtera, L.; Tran, H.C.; Citterico, M.; Vaattovaara, A.; Rokka, A.; Stolze, S.C.; Harzen, A.; Meißner, L.; et al. CRK2 and C-Terminal Phosphorylation of NADPH Oxidase RBOHD Regulate Reactive Oxygen Species Production in Arabidopsis. Plant Cell 2020, 32, 1063–1080. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  23. Yu, Y.; Zhang, Q.; Sun, S.; Yang, J. Upregulated Expression of RESPIRATORY BURST OXIDASE HOMOLOG D Underlies Lesion-Mimic Phenotype in Dark-Treated Arabidopsis Pheide a Oxygenase Mutant Leaves. Planta 2022, 255, 110. [Google Scholar] [CrossRef] [PubMed]
  24. Kalaji, H.; Oukarroum, A.; Alexandrov, V.; Kouzmanova, M.; Brestic, M.; Zivcak, M.; Samborska, I.A.; Cetner, M.D.; Allakhverdiev, S.I.; Goltsev, V. Identification of Nutrient Deficiency in Maize and Tomato Plants by in Vivo Chlorophyll a Fluorescence Measurements. Plant Physiol. Biochem. 2014, 81, 16–25. [Google Scholar] [CrossRef] [PubMed]
  25. Erinle, K.O.; Jiang, Z.; Ma, B.; Li, J.; Chen, Y.; Ur-Rehman, K.; Shahla, A.; Zhang, Y. Exogenous Calcium Induces Tolerance to Atrazine Stress in Pennisetum Seedlings and Promotes Photosynthetic Activity, Antioxidant Enzymes and PsbA Gene Transcripts. Ecotoxicol. Environ. Saf. 2016, 132, 403–412. [Google Scholar] [CrossRef]
  26. Wang, Q.; Yang, S.; Wan, S.; Li, X. The Significance of Calcium in Photosynthesis. Int. J. Mol. Sci. 2019, 20, 1353. [Google Scholar] [CrossRef] [Green Version]
  27. Aoki, K.; Yano, K.; Suzuki, A.; Kawamura, S.; Sakurai, N.; Suda, K.; Kurabayashi, A.; Suzuki, T.; Tsugane, T.; Watanabe, M.; et al. Large-Scale Analysis of Full-Length CDNAs from the Tomato (Solanum lycopersicum) Cultivar Micro-Tom, a Reference System for the Solanaceae Genomics. BMC Genom. 2010, 11, 210. [Google Scholar] [CrossRef] [Green Version]
  28. Tang, R.J.; Luan, S. Regulation of Calcium and Magnesium Homeostasis in Plants: From Transporters to Signaling Network. Curr. Opin. Plant Biol. 2017, 39, 97–105. [Google Scholar] [CrossRef]
  29. Li, X.; Zhang, H.; Tian, L.; Huang, L.; Liu, S.; Li, D.; Song, F. Tomato SlRbohB, a Member of the NADPH Oxidase Family, Is Required for Disease Resistance against Botrytis cinerea and Tolerance to Drought Stress. Front. Plant Sci. 2015, 6, 463. [Google Scholar] [CrossRef] [Green Version]
  30. Steinhorst, L.; Kudla, J. Signaling in Cells and Organisms—Calcium Holds the Line. Curr. Opin. Plant Biol. 2014, 22, 14–21. [Google Scholar] [CrossRef]
  31. Hochmal, A.K.; Schulze, S.; Trompelt, K.; Hippler, M. Calcium-Dependent Regulation of Photosynthesis. Biochim. Biophys. Acta BBA Bioenerg. 2015, 1847, 993–1003. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  32. Hashimoto, K.; Kudla, J. Calcium Decoding Mechanisms in Plants. Biochimie 2011, 93, 2054–2059. [Google Scholar] [CrossRef]
  33. Nie, W.-F.; Wang, M.-M.; Xia, X.-J.; Zhou, Y.-H.; Shi, K.; Chen, Z.; Yu, J.Q. Silencing of Tomato RBOH1 and MPK2 Abolishes Brassinosteroid-Induced H2O2 Generation and Stress Tolerance. Plant Cell Environ. 2013, 36, 789–803. [Google Scholar] [CrossRef]
  34. Mika, A.; Buck, F.; Lüthje, S. Membrane-Bound Class III Peroxidases: Identification, Biochemical Properties and Sequence Analysis of Isoenzymes Purified from Maize (Zea Mays L.) Roots. J. Proteom. 2008, 71, 412–424. [Google Scholar] [CrossRef] [PubMed]
  35. Minibayeva, F.; Beckett, R.P.; Kranner, I. Roles of Apoplastic Peroxidases in Plant Response to Wounding. Phytochemistry 2015, 112, 122–129. [Google Scholar] [CrossRef] [PubMed]
  36. Plieth, C.; Vollbehr, S. Calcium Promotes Activity and Confers Heat Stability on Plant Peroxidases. Plant Signal. Behav. 2012, 7, 650–660. [Google Scholar] [CrossRef] [Green Version]
  37. Amao, Y.; Takai, K.; Ohashi, A. Effect of Manganese and Calcium Ions on the Photoinduced Water Oxidation with Photosynthesis Organ Grana from Green Plant. Appl. Catal. B 2010, 97, 36–40. [Google Scholar] [CrossRef]
  38. Ramalho, J.C.; Rebelo, M.C.; Santos, M.E.; Antunes, M.L.; Nunes, M.A. Effects of Calcium Deficiency on Coffea Arabica. Nutrient Changes and Correlation of Calcium Levels with Some Photosynthetic Parameters. Plant Soil 1995, 172, 87–96. [Google Scholar] [CrossRef]
  39. Choudhury, S.; Panda, P.; Sahoo, L.; Panda, S.K. Reactive Oxygen Species Signaling in Plants under Abiotic Stress. Plant Signal Behav. 2013, 8, e23681. [Google Scholar] [CrossRef] [Green Version]
  40. Da Silva, L.; Marchiori, P.E.R.; Maciel, C.P.; Machado, E.C.; Ribeiro, R.V. Fotossíntese, Relações Hídricas e Crescimento de Cafeeiros Jovens Em Relação à Disponibilidade de Fósforo. Pesqui. Agropecu. Bras. 2010, 45, 965–972. [Google Scholar] [CrossRef]
  41. Ribeiro, R.V.; Machado, E.C.; Santos, M.G.; Oliveira, R.F. Seasonal and Diurnal Changes in Photosynthetic Limitation of Young Sweet Orange Trees. Environ. Exp. Bot. 2009, 66, 203–211. [Google Scholar] [CrossRef]
  42. Buchanan, B.B.; Gruissem, W.; Jones, R.L. (Eds.) Biochemistry & Molecular Biology of Plants; John Wiley & Sons, Ltd.: Chichester, UK, 2015; ISBN 9780470714218. [Google Scholar]
  43. Evans, N.H.; McAinsh, M.R.; Hetherington, A.M. Calcium Oscillations in Higher Plants. Curr. Opin. Plant Biol. 2001, 4, 415–420. [Google Scholar] [CrossRef]
  44. Himschoot, E.; Beeckman, T.; Friml, J.; Vanneste, S. Calcium Is an Organizer of Cell Polarity in Plants. Biochim. Biophys. Acta BBA Mol. Cell Res. 2015, 1853, 2168–2172. [Google Scholar] [CrossRef] [PubMed]
  45. Yang, S.-L.; Lan, S.-S.; Deng, F.-F.; Gong, M. Effects of Calcium and Calmodulin Antagonists on Chilling Stress-Induced Proline Accumulation in Jatropha curcas L. J. Plant Growth Regul. 2016, 35, 815–826. [Google Scholar] [CrossRef]
  46. Thordal-Christensen, H.; Zhang, Z.; Wei, Y.; Collinge, D.B. Subcellular Localization of H2O2 in Plants. H2O2 Accumulation in Papillae and Hypersensitive Response during the Barley-Powdery Mildew Interaction. Plant J. 1997, 11, 1187–1194. [Google Scholar] [CrossRef]
  47. Balcke, G.U.; Bennewitz, S.; Bergau, N.; Athmer, B.; Henning, A.; Majovsky, P.; Jiménez-Gómez, J.M.; Hoehenwarter, W.; Tissier, A. Multi-Omics of Tomato Glandular Trichomes Reveals Distinct Features of Central Carbon Metabolism Supporting High Productivity of Specialized Metabolites OPEN. Plant Cell 2017, 29, 960–983. [Google Scholar] [CrossRef] [Green Version]
  48. Neill, S.; Barros, R.; Bright, J.; Desikan, R.; Hancock, J.; Harrison, J.; Morris, P.; Ribeiro, D.; Wilson, I. Nitric Oxide, Stomatal Closure, and Abiotic Stress. J. Exp. Bot. 2008, 59, 165–176. [Google Scholar] [CrossRef] [Green Version]
  49. Drazkiewicz, M.; Baszyński, T. Calcium Protection of PS2 Complex of Phaseolus Coccineus from Cadmium Toxicity: In Vitro Study. Environ. Exp. Bot. 2008, 64, 8–14. [Google Scholar] [CrossRef]
  50. Pospíšil, P.; Prasad, A. Formation of Singlet Oxygen and Protection against Its Oxidative Damage in Photosystem II under Abiotic Stress. J. Photochem. Photobiol. B 2014, 30, 168–179. [Google Scholar] [CrossRef]
  51. Pino, L.E.; Peres, L.E.P. Manual do Modelo Vegetal Micro-Tom Capítulo 1: Especificações da Casa de Vegetação. 2010. Available online: https://docplayer.com.br/68592185-Manual-do-modelo-vegetal-micro-tom.html (accessed on 27 January 2021).
  52. Hoagland, D.R.; Arnon, D.I. The Water-Culture Method for Growing Plants without Soil. Calif. Agric. Exp. Stn. 1950, 397, 32. [Google Scholar]
  53. González, R.M.; Ricardi, M.M.; Iusem, N.D. Atypical Epigenetic Mark in an Atypical Location: Cytosine Methylation at Asymmetric (CNN) Sites within the Body of a Non-Repetitive Tomato Gene. BMC Plant Biol. 2011, 11, 94. [Google Scholar] [CrossRef]
  54. Alfenas-Zerbini, P.; Maia, I.G.; Fávaro, R.D.; Cascardo, J.C.M.; Brommonschenkel, S.H.; Zerbini, F.M. Genome-Wide Analysis of Differentially Expressed Genes During the Early Stages of Tomato Infection by a Potyvirus. Mol. Plant Microbe Interact. 2009, 22, 352–361. [Google Scholar] [CrossRef] [Green Version]
  55. Misra, N.; Gupta, A.K. Effect of Salinity and Different Nitrogen Sources on the Activity of Antioxidant Enzymes and Indole Alkaloid Content in Catharanthus Roseus Seedlings. J. Plant Physiol. 2006, 163, 11–18. [Google Scholar] [CrossRef]
  56. Teisseire, H.; Guy, V. Copper-Induced Changes in Antioxidant Enzymes Activities in Fronds of Duckweed (Lemna Minor). Plant Sci. 2000, 153, 65–72. [Google Scholar] [CrossRef]
  57. Peixoto, H.P.P.; Cambraia, J.; Sant, A.R.; Mosquim, R.; Moreira, M.A. Aluminum Effects on Lipid Peroxidation and on the Activities of Enzymes of Oxidative Metabolism in Sorghum 1. Braz. J. Plant Physiol. 1999, 11, 137–143. [Google Scholar]
  58. Nakano, Y.; Asada, K. Hydrogen Peroxide is Scavenged by Ascorbate-Specific Peroxidase in Spinach Chloroplasts. Plant Cell Physiol. 1981, 22, 867–880. [Google Scholar] [CrossRef]
  59. Bradford, M.M. A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Anal. Biochem. 1976, 72, 248–254. [Google Scholar] [CrossRef]
  60. Alexieva, V.; Sergiev, I.; Mapelli, S.; Karanov, E. The Effect of Drought and Ultraviolet Radiation on Growth and Stress Markers in Pea and Wheat. Plant Cell Environ. 2001, 24, 1337–1344. [Google Scholar] [CrossRef]
  61. Devi, S.R.; Prasad, M.N.V. Copper Toxicity in Ceratophyllum Demersum L. (Coontail), a Free Floating Macrophyte: Response of Antioxidant Enzymes and Antioxidants. Plant Sci. 1998, 138, 157–165. [Google Scholar] [CrossRef]
  62. Demmig-Adams, B.; Adams, W.W., III; Barker, D.H.; Logan, B.A.; Bowling, D.R.; Verhoeven, A.S. Using Chlorophyll Fluorescence to Assess the Fraction of Absorbed Light Allocated to Thermal Dissipation of Excess Excitation. Physiol. Plant 1996, 98, 253–264. [Google Scholar] [CrossRef]
  63. Murchie, E.H.; Lawson, T. Chlorophyll Fluorescence Analysis: A Guide to Good Practice and Understanding Some New Applications. J. Exp. Bot. 2013, 64, 3983–3998. [Google Scholar] [CrossRef] [Green Version]
  64. Zhang, S.; Li, Q.; Ma, K.; Chen, L. Temperature-Dependent Gas Exchange and Stomatal/Non-Stomatal Limitation to CO2 Assimilation of Quercus Liaotungensis under Midday High Irradiance. Photosynth. Res. 2001, 39, 383–388. [Google Scholar] [CrossRef]
  65. Zar, J.H. Biostatistical Analysis, 5th ed.; Pearson Prentice Hall: Upper Saddle River, NJ, USA, 2010. [Google Scholar]
  66. Chong, J.; Wishart, D.S.; Xia, J. Using MetaboAnalyst 4.0 for Comprehensive and Integrative Metabolomics Data Analysis. Curr. Protoc. Bioinform. 2019, 68, e86. [Google Scholar] [CrossRef]
Ijms 23 13571 g001 550
Figure 1. Gene expression, (A). 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).
Figure 1. Gene expression, (A). 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).
Ijms 23 13571 g001
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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 (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 plants within the same calcium level. ns = not significant at 5% Tukey test. Bars correspond to averages, whiskers to ± SE (n = 3).
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 (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 plants within the same calcium level. ns = not significant at 5% Tukey test. Bars correspond to averages, whiskers to ± SE (n = 3).
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Figure 3. Photomicrographs of the median cross sections of the leaf blade of Solanum lycopersicum “cv. Micro-Tom” stained with 3,3′-diaminobenzidine (DAB) at 0.5 h after mechanical damage. (A,B). absence of Ca2+ and without mechanical damage. (C,D). absence of Ca2+ and with mechanical damage. (E,F). presence of 2 mM Ca2+ and without mechanical damage. (G,H). presence of 2 mM Ca2+ and with mechanical damage. (A,C,E,G). Mesophyll region. (B,D,F,H). Midrib. PP—Palisade parenchyma; SP—Spongy parenchyma; Xy—Xylem; Ph—Phloem; ↑ (arrow)—Glandular trichome. Bars: ((A–C,E,G) = 100 μm); ((D,F,H) = 150 μm).
Figure 3. Photomicrographs of the median cross sections of the leaf blade of Solanum lycopersicum “cv. Micro-Tom” stained with 3,3′-diaminobenzidine (DAB) at 0.5 h after mechanical damage. (A,B). absence of Ca2+ and without mechanical damage. (C,D). absence of Ca2+ and with mechanical damage. (E,F). presence of 2 mM Ca2+ and without mechanical damage. (G,H). presence of 2 mM Ca2+ and with mechanical damage. (A,C,E,G). Mesophyll region. (B,D,F,H). Midrib. PP—Palisade parenchyma; SP—Spongy parenchyma; Xy—Xylem; Ph—Phloem; ↑ (arrow)—Glandular trichome. Bars: ((A–C,E,G) = 100 μm); ((D,F,H) = 150 μm).
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Figure 4. Photomicrographs of the median cross sections of the leaf blade of Solanum lycopersicum “cv Micro-Tom” stained with 3,3′-diaminobenzidine (DAB) at 21 days after mechanical damage. (a,b). absence of Ca2+ and without mechanical damage. (c,d). absence of Ca2+ and with mechanical damage. (e,f). presence of 2 mM Ca2+ and without mechanical damage. (g,h). presence of 2 mM Ca2+ with mechanical damage. (a,c,e,g). Mesophyll region. (b,d,f,h). Midrib. PP—Palisade parenchyma; SP—Spongy parenchyma; Xy—Xylem; Ph—Phloem; SV—Smaller veins; ↑ (arrow)—Glandular trichome. Bars: ((c,e,g) = 100 μm); ((a,b,d,f,h) = 150 μm).
Figure 4. Photomicrographs of the median cross sections of the leaf blade of Solanum lycopersicum “cv Micro-Tom” stained with 3,3′-diaminobenzidine (DAB) at 21 days after mechanical damage. (a,b). absence of Ca2+ and without mechanical damage. (c,d). absence of Ca2+ and with mechanical damage. (e,f). presence of 2 mM Ca2+ and without mechanical damage. (g,h). presence of 2 mM Ca2+ with mechanical damage. (a,c,e,g). Mesophyll region. (b,d,f,h). Midrib. PP—Palisade parenchyma; SP—Spongy parenchyma; Xy—Xylem; Ph—Phloem; SV—Smaller veins; ↑ (arrow)—Glandular trichome. Bars: ((c,e,g) = 100 μm); ((a,b,d,f,h) = 150 μm).
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Figure 5. (A). Maximum quantum efficiency of photosystem II (Fv/Fm) time × damage × calcium p < 0.007; (B). minimum dark-adapted fluorescence (Fo) damage × calcium p < 0.028; (C). non-photochemical quenching (NPQ) time × damage × calcium p < 0.033 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).
Figure 5. (A). Maximum quantum efficiency of photosystem II (Fv/Fm) time × damage × calcium p < 0.007; (B). minimum dark-adapted fluorescence (Fo) damage × calcium p < 0.028; (C). non-photochemical quenching (NPQ) time × damage × calcium p < 0.033 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).
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Figure 6. (A). Photosystem II efficiency (Fv’/Fm’) damage × calcium <0.001; (B). 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).
Figure 6. (A). Photosystem II efficiency (Fv’/Fm’) damage × calcium <0.001; (B). 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).
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Figure 7. (A). CO2 assimilation rate (A) time × damage × calcium p < 0.005; (B). stomatal conductance (Gs) calcium p < 0.001; (C). internal leaf CO2 concentration (Ci) time × damage × calcium p< 0.001; (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).
Figure 7. (A). CO2 assimilation rate (A) time × damage × calcium p < 0.005; (B). stomatal conductance (Gs) calcium p < 0.001; (C). internal leaf CO2 concentration (Ci) time × damage × calcium p< 0.001; (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).
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Figure 8. (A). Leaf area (dm2) damage × calcium p < 0.011; (B). leaf dry mass (g) time × damage × calcium p < 0.004; (C). flower dry mass (g) damage × calcium p < 0.022; (D). fruit dry mass (g) time × calcium p < 0.037; (E). stem dry mass (g) damage × calcium p < 0.008; (F). root dry mass (g) damage × calcium p < 0.010; (G). total dry mass (g) time × damage × 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).
Figure 8. (A). Leaf area (dm2) damage × calcium p < 0.011; (B). leaf dry mass (g) time × damage × calcium p < 0.004; (C). flower dry mass (g) damage × calcium p < 0.022; (D). fruit dry mass (g) time × calcium p < 0.037; (E). stem dry mass (g) damage × calcium p < 0.008; (F). root dry mass (g) damage × calcium p < 0.010; (G). total dry mass (g) time × damage × 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).
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Figure 9. (A). Heat map and (B). Correlation analysis with expression of RBOH1, MPK1, MPK2, SlCaM2 genes; enzymatic activity of SOD, POD, CAT and APX; hydrogen peroxide concentration; malonaldehyde; carbon assimilation; stomatal conductance; transpiration; internal carbon concentration; water use efficiency; and carboxylation efficiency of RuBisCO, in Solanum lycopersicum “cv. Micro-Tom” grown with variation in calcium concentration (mM) and with (w/MD) or without (wo/MD) mechanical damage at 0.5 h after damage. Values correspond to average ± SE (n = 3).
Figure 9. (A). Heat map and (B). Correlation analysis with expression of RBOH1, MPK1, MPK2, SlCaM2 genes; enzymatic activity of SOD, POD, CAT and APX; hydrogen peroxide concentration; malonaldehyde; carbon assimilation; stomatal conductance; transpiration; internal carbon concentration; water use efficiency; and carboxylation efficiency of RuBisCO, in Solanum lycopersicum “cv. Micro-Tom” grown with variation in calcium concentration (mM) and with (w/MD) or without (wo/MD) mechanical damage at 0.5 h after damage. Values correspond to average ± SE (n = 3).
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Table
Table 1. Primers Used in Real-Time Quantification (qRT-PCR).
Table 1. Primers Used in Real-Time Quantification (qRT-PCR).
GeneOligonucleotidesGene IDReference
RBOH1 F5′-GGAGCTCCAGCACAAGATTA-3′Sl08g081690 [18]
RBOH1 R5′-CTTGTTGCAGCACTCATGTC-3′
MPK1 F5′-GCTGACAGATTGTTGCAGGT-3′Sl12g019460
MPK1 R5′-TCCACCCCATAAAGATACATCA-3′
MPK2 F5′-TACTCGCTCGTTTGCTGTTG-3′Sl08g014420
MPK2 R5′-TTGGAGTACAGGAAAACAATGG-3′
SlCaM2-F5′-TCTGAGGAGGAGTTGAAAGAGG-3′Solyc10g081170 [9]
SlCaM2-R5′-TCAACATCAGCTTCCCTAATCA-3′
Actin F5′-GGGATGATATGGAGAAGATATGG-3′Actina-185 pb [53]
Actin R5′-AAGCACAGCCTGGATAGC-3′
β-6-tubulin F5′-TTGGTTTTGCACCACTGACTTC-3′Tub6-65 pb [54]
β-6-tubulin5′-AAGCTCTGGCACTGTCAAAGC-3
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Campos, F.G.; Seixas, D.P.; Barzotto, G.R.; Jorge, L.G.; Ducatti, K.R.; Ferreira, G.; Rodrigues, T.M.; Silva, E.A.A.d.; Boaro, C.S.F. Roles of Calcium Signaling in Gene Expression and Photosynthetic Acclimatization of Solanum lycopersicum Micro-Tom (MT) after Mechanical Damage. Int. J. Mol. Sci. 2022, 23, 13571. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms232113571

AMA Style

Campos FG, Seixas DP, Barzotto GR, Jorge LG, Ducatti KR, Ferreira G, Rodrigues TM, Silva EAAd, Boaro CSF. Roles of Calcium Signaling in Gene Expression and Photosynthetic Acclimatization of Solanum lycopersicum Micro-Tom (MT) after Mechanical Damage. International Journal of Molecular Sciences. 2022; 23(21):13571. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms232113571

Chicago/Turabian Style

Campos, Felipe Girotto, Diana Pacheco Seixas, Gustavo Ribeiro Barzotto, Letícia Galhardo Jorge, Karina Renostro Ducatti, Gisela Ferreira, Tatiane Maria Rodrigues, Edvaldo Aparecido Amaral da Silva, and Carmen Sílvia Fernandes Boaro. 2022. "Roles of Calcium Signaling in Gene Expression and Photosynthetic Acclimatization of Solanum lycopersicum Micro-Tom (MT) after Mechanical Damage" International Journal of Molecular Sciences 23, no. 21: 13571. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms232113571

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