Calcium in Photosynthetic Restoration and Growth of Annona emarginata after Mechanical Damage

Calcium, an essential element with structural function in the cell wall and plasma membrane, in addition to being a secondary messenger, is responsible for the regulation of physiological processes in plant development and responses to biotic and abiotic stresses. This study investigated the effects of calcium variation on photosynthetic performance, growth, and enzymatic antioxidant defense system in A. emarginata subjected to mechanical damage. The experimental design was in 6 × 5 factorial randomized blocks. A. emarginata plants were submitted to the six treatments: plants grown in solution with 0 mM Ca2+ without mechanical damage, 0 mM Ca2+ with mechanical damage, 2 mM Ca2+ without mechanical damage, 2 mM Ca2+ with mechanical damage, 4 mM Ca2+ without mechanical damage, and 4 mM Ca2+ with mechanical damage, as well as five evaluation periods at 0, 15, 30, 60, and 90 days after mechanical damage. The fluorescence of chlorophyll a, gas exchange, total dry mass, quantitative growth, and lipid peroxidation was studied. It is concluded that the A. emarginata plants showed better performance in restoration after mechanical damage in the presence of Ca2+ and was more sensitive in the absence of the mineral. Cultivation of the species with 2 mM Ca2+ in complete nutrient solution was sufficient to guarantee the efficiency of the enzymatic antioxidant defense system, and photosynthetic restoration of plants subjected to


Introduction
Calcium (Ca 2+ ), an essential element with structural function in the cell wall and plasma membrane, is also a secondary messenger and is responsible for physiological process regulation in vegetal development and responses to biotic and abiotic stress [1]. Ca 2+ deficiency can compromise plant growth and recovery [2,3], especially after damage and stress.
One of the pathways to stress signaling and perception is the change in calcium and reactive oxygen species (ROS) concentration in the cytoplasm, which initiates a cascade responsible for activating antioxidant enzymes that regulate ROS accumulation [4] and membrane damage, contributing to the maintenance of the photosynthetic process. This cascade coordinates intracell responses in plants and long-distance signaling [5] and can influence plant growth and development.
Calcium acts on photosystem integrity because its fast oscillation in the cytoplasm regulates the energy ratio between the quantum efficiency of photosystem II (ΦPSII) and 2.1.1. Chlorophyll A Fluorescence, Gas Exchange, Biomass and Growth Index Chlorophyll a fluorescence was measured in fully expanded leaves dark-adapted for 30 min, from the region below to mechanical damage, using a pulse-amplitude modulated fluorometer (Jr PAM, Walz) with saturating irradiance of 1150 PPFD, between 9:00 and 11:00 a.m. Maximum quantum yield of PSII photochemistry (Fv/Fm), minimum Chl a fluorescence in the dark-adapted state (Fo), and non-photochemical quenching (NPQ = (Fm-Fm')/Fm'), were measured.
The gas exchange and chlorophyll a fluorescence in the light were measured between 9:00 and 11:00 a.m. in leaves with the same conditions using an infrared gas analyzer GFS 3000 FL, Walz. The potential quantum efficiency of open PSII center (Fv'/Fm'), effective quantum efficiency of PSII photochemistry (ΦPSII), electron transport rate (ETR = PPFD × ∆F/Fm' × 0.5 × 0.84), light fraction absorbed by PSII antenna that is dissipated as heat (D), and energy fraction not dissipated in the antenna that cannot be used for photochemistry stage (Ex) were measured [11]. For the gas exchange evaluation, CO 2 assimilation rate (A, µmol CO 2 m −2 s −1 ), transpiration rate (E, mmol H 2 O m −2 s −1 ), and stomatal conductance (Gs, mmol H 2 O m −2 s −1 ) were measured. The ribulose 1,5-difosfato carboxylase oxygenase (RuBisCO) efficiency was calculated between the ratio of A and internal CO 2 concentration in leaves (A/Ci) [12].
Leaf area was measured using a leaf area integrator Area Meter 3100C LICOR. Growth indices were calculated through the relationship between dry mass and leaf area, and the net assimilation rate (NAR), which reflects liquid photosynthesis, and the relative growth rate (RGR), which reflects growth in relation to pre-existing plant material [13], were determined.

Calcium Concentration in Plant Tissue
The foliar and stem Ca 2+ concentrations were determined in growth plants at 0 and 2 mM Ca 2+ , with or without mechanical damage, 90 days after damage. Ca 2+ determination was realized through flame atomic absorption instruments, model Varian 55B-Agilent [21].

Statistical Analysis
Levene's test was used to test homogeneity of variances between treatments using the software Sigmaplot 12.5. The results of variables were subjected to analysis of variance. The averages of treatments 0 mM Ca 2+ intact, 0 mM Ca 2+ w/MD, 2 mM Ca 2+ intact, 2 mM Ca 2+ w/MD, 4 mM Ca 2+ intact and 4 mM Ca 2+ w/MD, in 0 (30 min), 15, 30, 60, and 90 days after mechanical damage were compared using the Tukey test at the level of 5% probability [22].
Software used to create the heat map was MetaboAnalyst (v4.0, https://www.meta boanalyst.ca/ (accessed on 23 May 2022). Variables were standardized and the Euclidean distance between treatments was considered.

Fluorescence Analysis of Chlorophyll A, Gas Exchange and Biomass
Plants 0 mM Ca 2+ intact showed lower Fv/Fm ( Figure 1A) and higher Fo ( Figure 1B

Fluorescence Analysis of Chlorophyll A, Gas Exchange and Biomass
Plants 0 mM Ca 2+ intact showed lower Fv/Fm ( Figure 1A) and higher Fo ( Figure 1B  Lower NPQ was observed in plants 2 mM Ca 2+ intact compared to plants 0 mM Ca 2+ intact at time 0 ( Figure 1C). At         Plants grown with 0 mM Ca 2+ , regardless of mechanical damage, showed constant total dry mass throughout the evaluation period ( Figure 5). However, at 90 days, plants with 0 mM Ca 2+ showed signs of deficiency in this element, such as small and necrotic leaves and apical stem and root death ( Figure 6B). The difference between the total dry mass was observed at 60 days and plants with 4 mM Ca 2+ intact had higher total dry mass than the others, with the exception of those cultivated with 2 mM Ca 2+ intact. Plants grown with 0 mM Ca 2+ , regardless of mechanical damage, showed constant total dry mass throughout the evaluation period ( Figure 5). However, at 90 days, plants with 0 mM Ca 2+ showed signs of deficiency in this element, such as small and necrotic leaves and apical stem and root death ( Figure 6B). The difference between the total dry mass was observed at 60 days and plants with 4 mM Ca 2+ intact had higher total dry mass than the others, with the exception of those cultivated with 2 mM Ca 2+ intact.

Hydrogen Peroxide Concentration (H2O2)
Plants grown with 0 and 2 mM Ca 2+ and mechanical damage showed high concentrations of H2O2 compared to the intact. Plants grown with 4 mM Ca 2+ showed the same concentration of H2O2, regardless of mechanical damage (Figure 8).

Antioxidant Enzyme Activity
Plants grown with 0 mM Ca 2+ intact and 4 mM Ca 2+ w/MD showed lower SOD activity compared to 2 mM Ca 2+ plants regardless of damage at time 0 ( Figure 9A). At 90 days, plants with 0 mM Ca 2+ intact showed the highest SOD and POD activity of the evaluated period ( Figure 9A,B).

Hydrogen Peroxide Concentration (H2O2)
Plants grown with 0 and 2 mM Ca 2+ and mechanical damage showed high concentrations of H2O2 compared to the intact. Plants grown with 4 mM Ca 2+ showed the same concentration of H2O2, regardless of mechanical damage (Figure 8).

Antioxidant Enzyme Activity
Plants grown with 0 mM Ca 2+ intact and 4 mM Ca 2+ w/MD showed lower SOD activity compared to 2 mM Ca 2+ plants regardless of damage at time 0 ( Figure 9A). At 90 days, plants with 0 mM Ca 2+ intact showed the highest SOD and POD activity of the evaluated period ( Figure 9A,B).

Antioxidant Enzyme Activity
Plants grown with 0 mM Ca 2+ intact and 4 mM Ca 2+ w/MD showed lower SOD activity compared to 2 mM Ca 2+ plants regardless of damage at time 0 ( Figure 9A). At 90 days, plants with 0 mM Ca 2+ intact showed the highest SOD and POD activity of the evaluated period ( Figure 9A,B).
The CAT activity was constant in plants grown with 0 mM Ca 2+ , regardless of damage ( Figure 9C). In damaged 2 mM Ca 2+ plants, CAT activity was higher at 90 days than at 15 days. Plants with 4 mM Ca 2+ intact showed higher CAT activity at 60 days compared to 15, 30, and 90 days. Plants with 4 mM Ca 2+ w/MD showed higher CAT activity at time 0 compared to 15 and 90 days.
At 90 days, plants grown with 2 mM Ca 2+ w/MD showed higher CAT activity compared to plants with 0 and 2 mM Ca 2+ intact and plants 4 mM Ca 2+ w/MD.

Lipid Peroxidation Quantification
Regardless of the calcium supplied, plants without mechanical damage showed no change in MDA concentration over the period ( Figure 9D). Plants 0 mM Ca 2+ w/MD showed higher MDA at 30 and 90 days compared to time 0. Plants 2 mM Ca 2+ w/MD showed higher MDA at 60 days. Plants 4 mM Ca 2+ w/MD showed lower MDA at 15 days, compared to 30, 60 and 90 days.
At 60 days, among plants that received mechanical damage, plants 2 mM Ca 2+ showed higher MDA. At 90 days, MDA did not differ in plants.

Calcium Concentration in Plant Tissue
A. emarginata plants had an equal leaf calcium concentration when grown in the presence of 2 mM Ca 2+ or absence of the ion. Lower leaf calcium concentrations were found in plants subjected to mechanical damage compared to intact plants. A higher Ca 2+ concentration in the stems was observed in plants grown in the presence of 2 mM Ca 2+ , and with no variation verified through mechanical damage (Table 1). The CAT activity was constant in plants grown with 0 mM Ca 2+ , regardless of damage ( Figure 9C). In damaged 2 mM Ca 2+ plants, CAT activity was higher at 90 days than at 15 days. Plants with 4 mM Ca 2+ intact showed higher CAT activity at 60 days compared to 15, 30, and 90 days. Plants with 4 mM Ca 2+ w/MD showed higher CAT activity at time 0 compared to 15 and 90 days.
At 90 days, plants grown with 2 mM Ca 2+ w/MD showed higher CAT activity compared to plants with 0 and 2 mM Ca 2+ intact and plants 4 mM Ca 2+ w/MD.

Lipid Peroxidation Quantification
Regardless of the calcium supplied, plants without mechanical damage showed no change in MDA concentration over the period ( Figure 9D)

Calcium Concentration in Plant Tissue
A. emarginata plants had an equal leaf calcium concentration when grown in the presence of 2 mM Ca 2+ or absence of the ion. Lower leaf calcium concentrations were found in plants subjected to mechanical damage compared to intact plants. A higher Ca 2+ concentration in the stems was observed in plants grown in the presence of 2 mM Ca 2+ , and with no variation verified through mechanical damage (Table 1).

Heat Map
The heat map of the variables shows two large groups at time 0 ( Figure 10A). One group with plants 0 mM Ca 2+ intact and 4 mM Ca 2+ w/MD, and another group, divided into two sub-groups, which showed greater similarity in responses to each other. The first group (0 mM Ca 2+ intact and 4 mM Ca 2+ w/MD) showed similarity in chlorophyll a fluorescence and gas exchange responses, with a negative influence on these variables.

Discussion
Manifestation of deficiency signs in A. emarginata was late, a result consistent with the low nutritional demand of the species [8]. Over time, plants grown in the absence of Ca 2+ showed death of apical buds, a condition aggravated in the last evaluation. NPQ and Fo were high, while ΦPSII did not vary. However, plants 0 mM Ca 2+ intact revealed A and A/Ci similar to plants 2 mM Ca 2+ , which may be an indication that these plants have the capacity to store enough calcium to maintain baseline functions [23].
In plants 0 mM Ca 2+ intact, the concentration of H2O2 was low, contributing to the efficiency of the enzymes SOD, CAT, and POD in controlling membrane damage. The development of these plants was compromised, since the relative growth rate showed constant behavior and a net assimilatory rate increasing, indicating that the photoassimilates were mainly directed to leaf production. Although plants grown without Ca 2+ were kept in this condition for 120 days, the ion amount stored in the leaf did not differ from that of plants 2 mM Ca 2+ . In the absence of calcium, there was no limitation in the accumulation of total dry mass at the end of the evaluation period, and the growth was constant, as observed by the growth analysis. Same calcium concentration in the stems was observed in intact plants and in those with mechanical damage, perhaps due to its low mobility. These verified results agree with those found in the literature that describe that a low Ca 2+ concentration can interfere with development [24]. The low concentration of H2O2 contributed to the efficiency of antioxidant enzymes and was not enough to signal

Discussion
Manifestation of deficiency signs in A. emarginata was late, a result consistent with the low nutritional demand of the species [8]. Over time, plants grown in the absence of Ca 2+ showed death of apical buds, a condition aggravated in the last evaluation. NPQ and Fo were high, while ΦPSII did not vary. However, plants 0 mM Ca 2+ intact revealed A and A/Ci similar to plants 2 mM Ca 2+ , which may be an indication that these plants have the capacity to store enough calcium to maintain baseline functions [23].
In plants 0 mM Ca 2+ intact, the concentration of H 2 O 2 was low, contributing to the efficiency of the enzymes SOD, CAT, and POD in controlling membrane damage. The development of these plants was compromised, since the relative growth rate showed constant behavior and a net assimilatory rate increasing, indicating that the photoassimilates were mainly directed to leaf production. Although plants grown without Ca 2+ were kept in this condition for 120 days, the ion amount stored in the leaf did not differ from that of plants 2 mM Ca 2+ . In the absence of calcium, there was no limitation in the accumulation of total dry mass at the end of the evaluation period, and the growth was constant, as observed by the growth analysis. Same calcium concentration in the stems was observed in intact plants and in those with mechanical damage, perhaps due to its low mobility. These verified results agree with those found in the literature that describe that a low Ca 2+ concentration can interfere with development [24]. The low concentration of H 2 O 2 contributed to the efficiency of antioxidant enzymes and was not enough to signal and stimulate growth, considering that these ROS are reported in the literature as signaling agents in plants [25].
As observed in the heat map, at 90 days, plants 0 mM Ca 2+ w/MD showed high Fv/Fm and Fv'/Fm' and low NPQ and Fo, conditions that, according to the literature, can lead to membrane damage [26,27]. These ROS, generated by low energy dissipation in the form of heat and fluorescence, with a lower ETR and ΦPSII, can cause damage to photosystems, according to studies in which ROS were generated by interruption of electron flow in photosystems [28,29]. This condition, in the present study, may justify low A, Gs, and E in plants 0 mM Ca 2+ with mechanical damage, since, with the reduction in the ETR, there is a decrease in the synthesis of NADPH + H + , which is required for CO 2 reduction [30].
While some studies demonstrate that the presence of ROS induces a cascade of signals, with the involvement of ABA and kinases, which contributes to stomatal closure [31] and limits H 2 O vapor output and CO 2 input, other studies reveal that ROS act in the signaling of physiological processes, which can accelerate plant growth and development [32]. In this study, the accumulation of ROS may have acted both in signaling stomatal closure and in growth [33,34], since, even with less stomatal conductance, plants cultivated without calcium and with mechanical damage revealed a marked decrease in the curves of the net assimilation rate and relative growth rate, indicative conditions of self-shading and, therefore, rapid decline. This can explain the lower Ca 2+ concentration in the leaves of plants grown without calcium and subjected to mechanical damage, indicating that this element may have been used to overcome stress from mechanical damage and not to increase the total dry mass.
Plants 2 mM Ca 2+ w/MD showed ΦPSII and A similar to those presented by plants 4 mM Ca 2+ intact, and with high Fv/Fm. This indicates high efficiency in energy use and less need for energy dissipation as a protection mechanism [28,35], which indicates that plants 2 mM Ca 2+ , even with mechanical damage, showed highly efficient photochemistry.
In these plants, 2 mM Ca 2+ w/MD, a low variation of the total dry mass was observed, in addition to constant NAR and RGR, indicating that their photoassimilates were not reversed for growth and development. The high concentration of H 2 O 2 may have signaled the directing of photosynthetic resources to overcome stress from mechanical damage, as previously observed [33]. In addition, antioxidant enzymes were effective in controlling the concentration of H 2 O 2 , preventing membrane damage, observed at 90 days.
Plants 2 mM Ca 2+ intact showed, in general, high ΦPSII, which estimates the amount of energy available for carbon reduction. This condition may have favored high A, indicating that 2 mM Ca 2+ was sufficient for plants without damage to maintain their quantum yield without photoinhibition [36], since these plants presented adequate use of the luminous resource to produce reducing agents and carbon assimilation, without limiting stomatal conductance. In these plants, the amount of H 2 O 2 was low, and the activity of the enzymes SOD, CAT and POD efficiently neutralized ROS, avoiding membrane damage [37]. The rapid decrease in curves of NAR and RGR also indicate good performance of these plants, which agrees with other studies carried out with the same species [9]. Thus, 2 mM Ca 2+ is sufficient to guarantee intact plant growth. A. emarginata recovers from mechanical damage in the presence of 2 mM Ca 2+ but stoppage growth.
Plants 4 mM Ca 2+ intact showed a high ΦPSII. This allows the production of reducing agents, which together with the high Gs and A, contributed to high A/Ci, indicative of appropriate functioning of the photosynthetic apparatus as observed in other studies [36,38]. In these plants, the high concentration of H 2 O 2 , which may be due to photochemical activity, did not cause an increase in membrane damage. The activity of antioxidant enzymes controls reactive oxygen species, especially catalase, which is the main enzyme involved in the detoxification of H 2 O 2 [34,37], as observed at 60 days.
These plants with 4 mM Ca 2+ intact showed a high accumulation of total dry mass, although the slight decreases in the NAR and RGR revealed late self-shading and slow growth.
Plants 4 mM Ca 2+ w/MD showed high NPQ, attributed to a photoprotection mechanism, to avoid damage to photosystems, and is in accordance with the literature [28,35]. Such protection may have contributed to the A that increased over time and with antioxidant enzymes, efficient in combatting the high concentration of H 2 O 2 , avoiding damage to membranes [37].
In addition, these reactive species may have participated in mechanical damage stress signaling [39], directing photoassimilates to overcome and restore the plant. This can be confirmed by the practically constant relative growth rate and the increasing net assimilation rate, indicative of leaf investment.
According to the literature, mechanical damage and photosynthesis can generate ROS [27], that were efficiently neutralized by the antioxidant system which depend on calcium signaling. This system's action can be confirmed by the evaluation of lipid peroxidation. In the present study, the absence of significant variation in the concentration of malondialdehyde, at 90 days, suggests that A. emarginata, without supplying Ca 2+ and with damage, may have stored the ion in the form of calcium oxalate crystals when necessary [23] and as already verified for other Annona species [40], or it may have been stored in chloroplast, since this element is essential for the functioning of the photosynthetic apparatus [28]. This is in line with what was observed in the present study, in which the plants, regardless of the Ca 2+ concentration, did not alter the concentration of the leaf element and in the condition of mechanical damage presented less accumulation of ions in the leaf, a condition that may have contributed to signaling and defense against the stress generated by mechanical damage, although other functions exerted by calcium, such as growth, may be impaired. It is known that the species A. emarginata presents strategies for overcoming abiotic stress, such as high energy dissipation in the form of heat, observed by the NPQ and maintaining effective quantum efficiency, conditions already observed in a study with water deficiency [8]. The high NPQ can also be indicative of calcium storage in the tissue, and this ion is involved in the energy balance between heat dissipation and the production of reducing agents [7].
The greater resistance of A. emarginata plants to mechanical damage may be the result of their slower growth, demonstrated by growth rate curves, with growth arrest when calcium was not supplied. In plants subjected to mechanical damage and with calcium, photoassimilates were used for restoration. Intact plants without calcium stopped growth, and their slower metabolism contributed to the restoration of the photosynthetic rate. In these plants, the stored calcium may have been sufficient to maintain the antioxidant system. Mechanical damage in the absence of calcium may have stimulated the use of the element previously stored in plant tissue.
We suggest that mechanical damage and calcium are related to activation of the defense system in A. emarginata. In absence of the element, the acceleration of growth did not influence the dry mass production, which was low due to the investment of resources to overcome this damage. In that case, calcium may have come from storage as calcium oxalate crystals, or from calcium stored in the chloroplast. The presence of calcium in plants with mechanical damage contributed to a signaling cascade, allowing photosynthetic restoration with growth arrest.

Conclusions
It was concluded that Annona emarginata showed better performance in restoration after mechanical damage in the presence of Ca 2+ and was more sensitive in the absence of the mineral.
The cultivation of the species with 2 mM of Ca 2+ in the complete nutrient solution was sufficient to guarantee the efficiency of the enzymatic antioxidant defense system, and photosynthetic reestablishment of plants subjected to mechanical damage.