Chlorophyll a fluorescence as an indicator of water stress in Calophyllum brasiliense

The objective of this study was to evaluate chlorophyll a fluorescence as a stress indicator in Calophyllum brasiliense Cambess seedlings grown with different concentrations of abscisic acid (ABA) under intermittent water deficit condition: daily irrigation without ABA (I); daily irrigation + 10 μM ABA (I 10); daily irrigation + 100 μM ABA (I 100); suspension of daily irrigation without ABA (SI); suspension of daily irrigation + 10 μM ABA (SI 10) and suspension of daily irrigation + 100 μM ABA (SI 100). The intermittent water deficit reduces water status and impairs the photochemical apparatus functioning and seedling quality. The fluorescence measurements helped identify the stress condition of water deficit in the cultivation of C. brasiliense and the beneficial effect of the application of 10 μM of ABA in minimizing stress and facilitating the recovery of seedlings after re-irrigation, while maintaining the integrity and function of the photosynthetic apparatus.


Introduction
Calophyllum brasiliense Cambess. (Clusiaceae), commonly known as 'guanandi', is a tree species native to Central and South America. In Brazil, it is found in several states, especially in the Amazon region and in the 211 Cerrado, occurring spontaneously in alluvial soils with poor drainage, periodically flooded humid places, sandy to loamy soils, and acidic soils (pH 4.5-6.0). It has ornamental, apicultural, and medicinal applications, and also has potential application in recovery of degraded areas, thus, facilitating the establishment of other species. Its wood is used in the construction of furniture, frames, and ships among other applications (Carvalho, 2003;Kalil Filho et al., 2007).
Although there are some reports on the water and light requirements of this species, no information is available on its potential to tolerate water deficit when induced by abscisic acid (ABA).This information is important to extend its use economically and ecologically, especially in the recovery of degraded areas subject to seasonal periods of water deficit.
The water deficit can change the organelle, pigment concentration and metabolites, as well as stomatal regulation (Mohammadi et al., 2015). The closing of stomatal is considered a primary mechanism to regulate the water content when the plant is under dry conditions (Kowitcharoen et al., 2015;Zhao et al., 2015).
Studies suggest that stomatal closure under water deficit can occur under the influence of the water content of the soil and/or due to the hormonal levels of the plant (Damour et al., 2010;Brodribb and McAdam, 2013). Thus, the exogenous application of some phytohormones like the ABA, you can enable the plant to adapt to hydric deficit, to mediate adaptive responses, stimulating the biosynthesis of proline and the translocation of photo assimilation (Sarafraz et al., 2014).
The chlorophyll a fluorescence (ChlF-a) parameters can be used to understand the processes of tolerance and/or physiological plasticity to different water conditions (Kalaji et al., 2018), since, as the only photochemical functions reflect a reduction in the efficiency of plants to capture, transfer, and convert energy (Nesterenko et al., 2019) and the preservation of integrity or photo inhibitory damage in the reaction centers of PS II due to some stress factor.
Considering the natural habitat of this species, we hypothesized that their seedlings are sensitive to water deficit, which can be minimized by the application of ABA, and that ChlF-a can indicate stress condition, which reflects on the quality of the seedling. Therefore, the aim of the present study was to evaluate ChlF-a as a stress indicator in C. brasiliense seedlings grown with different concentrations of ABA and under intermittent water deficit condition.

Plant and cultivation material
The experiment was carried out in a protected environment where the seedlings of C. brasiliense Cambess were maintained under 30% shade and protected from rainfall using plastic cover. Seven-month old seedlings (after emergence), of mean height 14.32 cm and with 9.33 leaves, were grown in 7L pots. All the pots were irrigated at 70% water retention capacity (WRC) of the substrate until the characterization of the seedlings at time zero, with two seedlings per pot.
The treatments included suspension of daily irrigation without the addition of ABA (SI 0 ABA), suspension of daily irrigation + 10 μM ABA (SI 10 ABA), suspension of daily irrigation + ABA 100 μM (SI 100 ABA), daily irrigation without ABA (I 0 ABA), daily irrigation + 10 μM ABA (I 10 ABA),and daily irrigation + 100 μM ABA (I 100 ABA). Each treatment consisted of 22 pots, in addition to the six separate seedlings used for evaluation at time zero. For the irrigation treatments, the seedlings were divided into two groups. The first group was irrigated daily throughout the experimental period and the soil was maintained at 70% WRC; this group was considered control based on the concentration of ABA. The second group was subjected to water deficit until the photosynthetic rate approached zero, considered the first zero photosynthesis (1 st P0).
From the 1 st P0 period, all the pots were irrigated daily, and the plants were maintained at 70% WRC until recovery (REC), which was considered the stage when the seedlings under water deficit presented photosynthesis rates similar to those of irrigated seedlings (data not shown). After REC, another cycle of irrigation suspension was performed, and the seedlings were evaluated until the photosynthetic rate approached zero again, considered the second photosynthesis zero (2 nd P0).To determine the 1 st P0 and 2 nd P0, photosynthesis was monitored every two days using portable LCIPro-SD (IRGA -Ifra Red Gas Analyzer) (ADC BioScientific Ltd model), considering favorable climatic conditions. Subsequently, the pots were irrigated again until REC, according to the previously established standard and final evaluation that occurred 165 days after the start of the experiment (END).
On day 17, based on the photosynthetic rate of approximately 2 μmolm -2 s -1 , according to pre-tests, ABA was applied at the predetermined concentrations. The results were evaluated at five periods: T0, time zero (beginning of experiment); 1 st P0, first photosynthesis zero (day 23); 2 nd P0, second photosynthesis zero (day 82); REC, recovery (day 120); and END, final evaluation (day 165). The following parameters were evaluated:

Evaluations
Chlorophyll a fluorescence: Using a portable fluorometer (OS-30p;Opti-Sciences Chlorophyll Fluorometer, Hudson, NY, USA), we determined the potential quantum efficiency of photosystem II (PS II) (Fv/Fm), efficiency of the effective photosystem in the conversion of absorbed energy (Fv/F0), and basal quantum production of non-photochemical processes in PSII (F0/Fm). Fluorescence was determined between 8:00 and 11:00 AM was the second pair of fully expanded leaves. To determine their fluorescence, the leaves were subjected to a 30 min period of dark adaptation using adaptive clips, to ensure that all the reaction centers in this leaf region were open, that is, complete oxidation of the photosynthetic system of electron transport.
Relative water content (RWC): The relative water content in the leaves was determined using four leaves of each treatment, using the formula: RWC = 100 × (fresh mass-dry mass/saturated mass -dry mass). The leaves were collected between 7:00 and 10:00 AM and cut into discs of known area. After weighing the fresh mass, they were placed in Petri dishes with distilled water for 24 h for saturation. After weighing the saturated discs, they were dried for determine the dry mass.
Chlorophyll index and leaf area: The chlorophyll index was determined using achlorophyll meter (SPAD 502; MINOLTA) (8:00 and 11:00 AM), and leaf area was determined using a leaf area integrator (Li 3100, Area Meter).

Experimental design and statistical analysis
The data were evaluated in a completely randomized design with subdivided plots, where the plots represented the form of irrigation (daily irrigation -I and irrigation suspension -SI); each subplot included the three concentrations of ABA (0, 10, and 100 μM ABA); and the sub-sub-plot included the five evaluation periods (T0, 1 st P0, 2 nd P0, REC, and END). The results were subjected to the analysis of variance (ANOVA), and when significant effect was observed according to the F test, the means of the plots were subjected to the ttest of Bonferroni (p≤0.05) and the averages of subplots and sub-subplots were subjected to Tukey test (p≤0.05), using the SISVAR statistical program (Ferreira, 2014).
Results The Fv/Fm was lower in the seedlings under water deficit at the 1 st P0 period in relation to that of the irrigated seedlings ( Figure 1A). The non-irrigated seedlings treated with 10 μM ABA presented a higher Fv/Fm (0.797) than that of the seedlings subjected to other treatments, including SI 0 ABA (0.768) and SI 100 μM ABA (0.738), and was close to that of the control I 10 μM ABA treatment (0.801) ( Figure 1B). The seedlings subjected to the SI 100 μM ABA treatments presented the lowest Fv/Fm values, especially at the 1 st P0 period, with an average of < 0.75. In the END evaluation, this ratio tended to increase in the seedlings of all ABA treatments, with 0.785, 0.845, and 0.781, and reached a maximum of 0.896, 0.845 and 0.830, respectively. Furthermore, the Fv/Fm values of seedlings without ABA application varied significantly only at the END evaluation period ( Figure  1C). Stressed seedlings recovered to the values close to those of the control plants when treated with 10 μM ABA. The Fv/F0 was higher at T0 (4.47) that at the 1 st and 2 nd P0 (3.76 and 4.02, respectively) ( Figure 2A). The Fv/F0 value of seedlings subjected to the SI 0 ABA treatment was lower (3.75) than that of the seedlings subjected tothe I 0 ABA control (4.65) ( Figure 2B). Stressed seedlings recovered only after re-irrigation at the end of evaluation and when treated with ABA. The F0/Fm was higher at the 1 st P0 period, but not significantly different from that at the 2 nd P0, REC, and END periods ( Figure 2C). The seedlings subjected to the SI 0 ABA treatment presented the F0/Fm value (0.016) higher than that of the seedlings subjected to the SI10 ABA treatments (0.195), which reached values close to that of the seedlings subjected the I 0 ABA control treatment (0.192 higher) ( Figure 2D). We also observed that the F0/Fm values of the seedlings subjected to the SI 0 ABA treatment was 0.031-times higher than those of the seedlings subjected to the I 0 ABA treatment. Stressed plants recovered to values close to that of control seedling after re-irrigation when treated with ABA.
The RWC of the leaves decreased in the seedlings under water deficit with the lowest values in the seedlings subjected to the SI without ABA and SI 100 μM ABA treatments; however, the seedlings subjected to the 10μM ABA treatment presented values similar to those of the control ( Figure 3A). There was a significant reduction in RWC at both 1 st and 2 nd P0 periods in the seedlings grown under water deficit with elevation after re-irrigation, although it did not reach the values close to those of control seedlings ( Figure 3B).
The leaf area of seedlings subjected to the 10 μM ABA treatment and those subjected to irrigation treatments increased and was maintained relatively high throughout the experiment (Figures 3C, D). The highest leaf area was observed in the END evaluation, and the irrigated seedlings (I) presented 105.35 cm² more area than that of the stressed seedlings (SI). Stressed seedlings did not recover the leaf area after re-irrigation, presenting significantly lower values than that of the control plants.
The non-irrigated seedlings without ABA application presented relatively low chlorophyll index, which was low even at the 1 st P0, 2 nd P0, and REC periods, although the index increased, it did not reach the values to 215 that of the control seedlings. The application of ABA favored the maintenance of relatively high chlorophyll index when cultivated under water stress ( Figures 4A, B, C).
The lowest chlorophyll index was observed at the 1 st P0 evaluation period in all the treatments with or without ABA application, especially in the stressed seedlings, which was 44.51% lower than that in the irrigated seedlings ( Figures 4B, C).
Dickson Quality Index (DQI) increased during the experiment under both irrigation conditions. In the irrigated treatments, the values were higher than the stressed seedlings with significant differences at 2 nd P0, REC, and END evaluation periods. However, stressed seedlings even after rehydration did not attain values close to those of the control seedlings ( Figure 4D), suggesting that the period might not have been enough for the seedlings to recover.

Discussion
The intermittent water deficit reduces the water status of C. brasiliense, which impairs the functioning of the photochemical apparatus and the quality of the seedlings. After the reirrigation of the seedlings and until the end of the evaluations, most of the characteristics evaluated reached values close to that of the control seedlings, showing that the damage was not irreversible. However, for the quality of the seedlings and the leaf area, the period evaluated may not have been sufficient for the metabolic adjustment, which may have occurred after the second reirrigation, to reflect the growth and consequently the quality of the seedlings.
Studies have provided reference values related to chlorophyll a fluorescence, which has been used to predict stress condition. The reference values for the Fv/Fm range between 0.750 and 0.850 (Baker and Rosenqvst, 2004). However, these values are subject to a range of variation that depends, on the species, its physiological mechanisms and growth (Li et al., 2004;Zanandrea et al., 2006). For the Fv/F0, the values that reflect the maintenance of good state of functionality of the PS II reaction centers are between 4 and 6; values 217 below this range indicate stress. This characteristic can be used as an indicator of the maximum efficiency of the photochemical process in PSII and/or potential photosynthetic activity. Similarly, the reference for the F0/Fm is between 0.14 and 0.20, suggesting that the increase in this ratio is indicative of stress (Rohácek, 2002).
Thus, we can use these fluorescence characteristics as reliable parameters to evaluate the cultivation condition of C. brasiliense seedlings, which indicated the stress condition in the present study due to water deficit.This was reinforced by the changes in other characteristics evaluated, such as leaf area, chlorophyll index, and seedling quality.
The higher Fv/Fm reduction observed at the 1 st P0 period reinforces the stress condition of the nonirrigated C. brasiliense seedlings. However, after re-irrigation, the Fv/Fm ratio was restored, and the ABA concentration in the END evaluation period was higher than 0.75, indicating that there were no permanent damages to the photosynthetic apparatus.
The Fv/F0 ratio was also lower at the 1 st P0 evaluation period. However, at the REC and END evaluation periods, the seedlings presented values close to those of the control, indicating the efficiency of C. brasiliense seedlings in tolerating stress. The F0/Fm ratio increased significantly in the 1 st P0 period, and after re-irrigation, the tendency of reduction was observed. However, the values were maintained above the reference values and those of the control, except in the seedling treated with 10 µM ABA.
Calophyllum brasiliense, S. macrophylla and H. albus showed higher sensitivity to water deficit than that of other species. This was reflected by higher reductions in gas exchange and the photochemical efficiency of PS II (Campelo et al., 2015). Freitas et al. (2018) working on H. coubaril with different concentrations of ABA, verified water stress attenuation, thus, a reduction in the functions of PS II.
The seedlings of C. brasiliense under water deficit condition presented reduced leaf area in relation to the plants irrigated from the 1 st P0; although the leaf area increased at the end of the experiment, the seedlings under stress did not reach values close to those of the control plants (I).
Abscisic acid increased leaf area and did not cause leaf abscission in C. brasiliense seedlings. The ABA alters the growth and development of plants and regulates the adaptive responses under conditions of low water availability, such as opening and closure of the stomata, leaf abscission, and root growth (Zhu, 2002;Tardieu et al., 2010;Vieira et al., 2017). The leaf area of C. brasiliense seedlings treated with 10 μm ABA was higher than that of the seedlings not treated with ABA.
Furthermore, Tardieu et al. (2010) suggest that the positive effect of ABA on leaf expansion is attributed to the increase in the hydraulic conductivity of the root system at the same time as it leads to the stomatal closure and consequently to the maintenance of leaf turgescence.
In the present study, the plants under water deficit condition exhibited reduced leaf area at the 2 nd P0 period. Although the leaf area of seedlings under stress increased, which can be attributed to the natural growth of the plant, it did not attain the control values. The reduction in the leaf area of woody plants as a response to low soil water availability has been proven in several species. Similar results were observed for Guazuma ulmifolia (Scalon et al., 2011) and Vatairea macrocarpa (Benth.) Ducke (Vieira et al., 2017).
The chlorophyll index varied according to the treatments, however, stressed seedlings treated with 10 μM ABA maintained higher SPAD index in relation to that of the seedlings subjected to other treatments under stress, observing also hormonal action between the evaluation periods. The 1 st P0 was the time that presented the lowest chlorophyll index in all the treatments with or without ABA application. At the dose of 10 μM ABA, the values were higher than that of the treatment without ABA at the 2 nd P0 and REC periods. At the other periods of evaluation, there was an increase in the SPAD index; however, they did not attain the control values. Generally, plants under water stress present reduced photosynthetic pigment content due to oxidative damages, thus affecting photosynthesis (Asharaf and Harris, 2013) and consequently the production of dry mass and leaf area expansion, which reflected the reduction of DQI, to C. brasiliense seedlings.
Seedlings ofHymenaea coubaril treated with 10 μM ABA both in the photosynthesis close to zero and in the recovery period, presented SPAD index close to that of the control seedlings. However, chlorophyll index values showed reduced for plants treated with 100 μM ABA, independent of water availability and the DQI did not differ significantly among the different treatments throughout the experimental period (Freitas et al., 2018).
Dickson quality index of C. brasiliense seedlings under water deficit condition at T0 and 1 st P0 periods did not change significantly from that of the seedlings subjected to irrigation treatments. At the other evaluation periods, the seedlings under stress presented lower DQI, which might be due to lower biomass production for growth and target metabolism for defense mechanisms, such as increased enzyme activity (data not shown). Dickson Quality Index indicates robustness of seedlings; the higher the value, the better the quality (Moraes et al., 2012;Gordin et al., 2016).
Schinus terebinthifolius at different irrigation depths (8, 10, 12, and 14 mm) also showed lower DQI when they received the lowest amount of water (Moraes et al., 2012). Similar results were observed in Hancornia speciosa seedlings when grown at 25%, 50%, 75% and 100% substrate water retention capacity, they presented lower DQI at 25% and 50% RWC (Gordin et al., 2016). As stressed seedlings, even after rehydration, did not attain quality indexes similar to those of control seedlings, we believe that the period might not have been sufficient for them to recover.
In natural habitat, C. brasiliense is found in phytophysiognomy that present hyper seasonality, that is, dry and rainy seasons throughout the year, causing oscillations in the soil water status , causing the species to grow plants subjected to the water deficit condition at certain periods. Under reduced soil water availability (dry season -1 st P0 and 2 nd P0), C. brasiliense seedlings reduced the efficiency of activities in PSII, indicating stress conditions for maintenance of metabolic processes, as well as physiological plasticity by reversing photochemical damage, at the end of evaluation period.
The application of ABA in adequate amounts is a practice that substantially contributed to the mitigation of photochemical damage, maintaining the integrity of photosynthetic apparatus until the rainy season, characterized by re-irrigation, ensuring the development of seedlings and restoration of ecosystem services. Thus, study of plant photochemical responses to environmental variants contributes understanding of processes resulting from PSII under adverse conditions, aiming at the in situ and ex situ conservation.

Conclusions
The fluorescence measurements helped identify the stress condition of water deficit in the cultivation of C. brasiliense and the beneficial effect of the application of 10 μM ABA in minimizing stress and in facilitating the recovery of seedlings after re-irrigation, while, maintaining the integrity and function of the photosynthetic apparatus.