INFLUENCE OF EXOGENOUS APPLICATION OF ABSCISIC ACID ON GAS EXCHANGES IN Hymenaea courbaril L . ( FABACEAE ) SEEDLINGS SUBJECTED TO WATER DEFICIT

The study was performed in the greenhouse of the Federal University of Grande Dourados (UFGD), from April to May, 2015. The aim of this study was to assess the effect of abscisic acid (ABA) different concentrations on Hymenaea courbaril L. seedlings water stress response and recovery after the rehydration. The following six treatments were evaluated: 1) Irrigation 0 μM ABA; 2) Irrigation + 10 μM ABA; 3) Irrigation + 100 μM ABA; 4) Irrigation without 0 μM ABA; 5) Irrigation without + 10 μM ABA and 6) Irrigation without + 100 μM ABA. The irrigation without treatment was performed by withouting water until the photosynthetic rate was close to zero, after which the plants were rehydrated. The plants were then irrigated daily to maintain 70% of the water holding capacity of the soil for a 12-day recovery period. The ABA doses applied exogenously during the irrigation without period neither prevented the reduction of photosynthetic activity nor optimized the recovery of the gas exchanges and the carboxylation efficiency of Rubisco after water and seedling quality restoration. The 100 μM ABA concentration promoted the degradation of chlorophylls independent of irrigation, showing that high concentrations of ABA may cause a residual effect on this species. The seedlings exhibited normal metabolic recovery after about 12 days of re-irrigation of the soil, which we attribute to species characteristics and not the tested treatments.


INTRODUCTION
Hymenaea courbaril L. (Fabaceae) is an arboreal species with a wide geographical distribution and can be found in phytogeographical areas of the Amazon, Caatinga, Cerrado, Atlantic Forest, and Pantanal (LIMA;PINTO, 2016).This species has the ability to develop in different edaphoclimatic environments and is of forestry and environmental importance because of its potential as a carbon fixing and storage plant (CARVALHO, 2007).
The knowledge of the abiotic factors that affect natural regeneration or reforestation programs with native plants of the Cerrado are indispensable to the restoration of native vegetation.The growing concern about reforestation highlights the importance of studies on the tolerance or resistance of native species in both flooded and water deficit conditions, whereas the biome is characterized by seasonal rains and strong irradiance (LESSA et al., 2016).
Depending on the importance of water in the life cycle of plants, the deficit or excess of that element occupies a prominent position in studies on the environmental stresses.When exposed to water stress situations plants exhibit a wide range of molecular, cellular, physiological, and metabolic responses (FLOWERS, 2004) which can ensure their survival under such adverse conditions.
Plant responses to water deficit may include leaf area reduction, leaf abscission, stomatal closure, deep root growth, photosynthesis reduction, and increased leaf wax deposition (TAIZ et al., 2017).Many plants are able to delay dehydration by maintaining the hydration of their tissues through reduction of transpiration or increased water absorption; others tolerate dehydration due to their ability to keep their metabolism while dehydrated; other plants avoid drought by completing their cycle during the wet season, before the onset of drought (NASCIMENTO et al., 2011).
Plants under water stress typically exhibit greater accumulation of abscisic acid (ABA), which has been identified as a messenger in response pathways to perceived water stress and other environmental stresses and has several physiological effects on plant development.Some studies showed that application of ABA in intact plants could increase their tolerance to stress (YIN et al., 2004).
ABA is known as the stress hormone, triggering plant changes under water stress such as osmotic adjustment and stomatal closure upon increase in CO2 concentration or decrease in humidity (AASAMAA; SÕBER, 2011).The same authors used exogenous abscisic acid at 15-µM concentration on fast, slow, and intermediate growing plants in order to assess its effect on different stomatal sensitivities.They found that ABA affected stomatal sensitivityparticularly the leaf water potential in plants subjected to water deficitand accelerated stomatal closure.
Based on several reports in the literature where ABA shows a "low persistence" in the plant, we postulated that the application of higher doses allows for a longer lasting effect.Thus, the aim of this study was to evaluate the effect of abscisic acid doses throughout the irrigation without period as well as in the water stress response and recovery of H. courbaril seedlings after the rehydration.

MATERIAL AND METHODS
The study was performed in the greenhouse of the Federal University of Grande Dourados (UFGD).Five month old H. courbaril (Herbarium of the Universidade Federal da Grande Dourados, nº 3487) seedlings were transplanted into 5 kg capacity pots containing Carolina ® substrate (having in its composition: vermiculite, charred rice husk, Canadian peat and fertilizer; pH: 5.65; total porosity: 76%)+ soil (1:1), were acclimatized for 30 days with irrigation for a 70% water holding capacity, and were kept in the greenhouse under 40% shade being that these conditions were determined on the basis of pretesting).The plants were protected from rainfall by a plastic cover throughout the experiments.
The pots were divided into two groups, as follows: 1 -Control, where irrigation of the plants was periodically performed in order to maintain 70% of the water holding capacity of the soil and 2 -Treatment, where irrigation was withheld until photosynthetic rate was close to zero (Gomes et al. 2008), after which the plants were rehydrated and irrigated on a daily basis, maintaining 70% of the water holding capacity of the soil, for a 12-day recovery period (period in which the seedlings without irrigation presented values of photosynthesis similar the irrigated, after the rehydration).
Solutions of 0, 10 µM, and 100 µM abscisic acid were prepared and plants were pulverized to the drip point early in the morning.The plants of treatments 1 and 2 were or not sprayed with ABA.The application of ABA was held at the beginning of the experiment when the suspension of irrigation.
Every gas exchange evaluation was performed in two pairs of visually healthy and fully expanded leaves in two individuals from each treatment batch until the photosynthetic values approached zero.The gas exchange measurements were carried out on clear days without clouds, from 9 to 10 am, in order for the gas exchange to coincide with the period of maximum photosynthesis.
The quantum efficiency of the photosystem II (Fv/Fm) and the efficiency of the effective photosystem on the absorbed energy conversion (Fv/F0) were analyzed with a portable fluorometer (model OS-30p) (Opti-Sciences Chlorophyll Fluorometer, Hudson, USA).The fluorescence determination was carried out between 8 and 11 am on the same leaves used for gas exchanges evaluation.The leaves were subjected to 30 minutes of dark adaptation using adapter clips, so that all reaction centers in the leaf were "open," that is, the electron transport photosynthetic system was completely oxidized.
Chlorophyll index (SPAD) was determined using a SPAD Konica-Minolta 502 chlorophyll meter.Growth was analyzed using height, stem diameter, and shoot and root biomass (BENINCASA, 2003).The Dickson Quality Index was then calculated according to the equation DQI = [total dry weight/(height/diameter ratio + shoot dry weight ratio/root dry weight)] ( DICKSON et al., 1960).
The gas exchanges assessment was completely randomized and contained the following six treatments: 1) Irrigation 0 µM ABA; 2) Irrigation + 10 µM ABA; 3) Irrigation + 100 µM ABA; 4) irrigation without 0 µM ABA; 5) irrigation without + 10 µM ABA and 6) irrigation without + 100 µM ABA.Each treatment was performed with 5 repetitions, each repetition corresponding to a new seedling.Analysis of variance was performed and the means of each treatment were submitted to the t test at 5% probability when statistical effects were found.
A completely randomized design in a 6 treatments x 3 evaluation periods factorial was used for the other evaluations, with 5 repetitions, where each repetition corresponded to a seedling.Analysis of variance (ANOVA, p < 0.05) was performed and the means were compared by the Scott-Knott test (p < 0.05) using the statistical program SISVAR (FERREIRA, 2011).

RESULTS
The water deficit caused by irrigation without influenced some physiological parameters of the H. courbaril seedlings.The seedlings under water deficit, with or without ABA, exhibited significant variations in their photosynthetic rates.The onset of photosynthetic rate decreases occurred at the 20 th day with a subsequent intensification until the 28 th day, when the rates approached zero.After re-irrigation, seedlings recovered their photosynthetic metabolism in 12 days, demonstrating higher levels than those of irrigated seedlings without irrigation without (Figure 1A).These results suggest that the species possesses adaptation mechanisms after a 28-day water deficit period.
The transpiration rate (Figure 1B) for seedlings under water deficit was followed by reductions in stomatal conductance (Figure 1C), which is considered one of the first responses to drought because it prevents dehydration of plant tissues.
Seedlings submitted to water deficit without ABA and with 10 µM ABA showed a significant drop in internal CO2 concentration (Ci) starting at the 24 th day of irrigation without.However, seedlings without irrigation + 100 µM ABA did not differ statistically from seedlings with constant irrigation with or without ABA (Figure 1D).After restoration of irrigation, a recovery in Ci values was observed, being most significant in seedlings subjected to water deficit + 100 µM ABA treatment.This elevation in the Ci values for treatments involving water deficit related to increases in photosynthetic rate (Figure 1A).
Carboxylation efficiency fluctuated throughout the evaluation period, but a significant reduction was observed at the 28 th day in the treatments involving water deficit and ABA application.After 12 days of soil reirrigation, the treatments did not vary statistically among them, except for the treatment involving water deficit without ABA (Figure 2A).A reduction in water use efficiency (WUE) (Figure 2B) was observed in treatments with water deficit 12 days after the restoration of irrigation, although these same treatments subsequently exhibited a transpiration enhancement (Figure 1D).A significant reduction in effective efficiency of conversion of energy absorbed by the photosystem (Fv/F0) was observed in the irrigation without + 10 μM ABA treatment (Figure 3A).
The seedlings subjected to irrigation without exhibited a reduction in quantum efficiency of the photosystem II (Fv/Fm) (Figure 3B) when the photosynthetic rates reached zero.Lower chlorophyll index values (Figure 4A) were observed for plants treated with 100 μM ABA, independent of water availability and the DQI (Figure 4B) did not differ significantly among the different treatments throughout the experimental period.These results show that the water deficit did not affect the quality of the seedlings of this species during the period of study.FLORESTA,Curitiba,PR,v. 48,n. 2,abr

DISCUSSION
The reduction of intercellular CO2 concentration is related with stomatal closure, caused either by dehydration of the guard cells or by hormonal response (FILHO et al., 2008).Reduction of the photosynthetic rate in plants subjected to water deficit conditions is associated with stomatal closure, induced by changes in leaf water status (SILVA et al., 2009), as well as the reduction in stomatal conductance may represent a vantage immediate prevent dehydration of plant tissues (EGEA et al., 2011).
Similar results were observed in Calendula officinalis plants subjected to water deficit and treated with ABA doses, where the 100-µM dose did not increase the water stress tolerance of the species (PACHECO et al., 2011).
Most of the water absorbed by roots evaporates from leaf surfaces by transpiration, while a small part remains in the plant meeting growth, photosynthetic, and other metabolic demands.The WUE of certain species reflects their ability to fix carbon through transpiration Moreover, after water restoration the seedlings did not reach the same values of the irrigated treatments.Because Fv/F0 is an indicator of the electron transport chain status and its effectiveness, this decrease indicates possible damage on the electron transport chain.Although it contains information similar to the reason Fv/Fm, this right extends small variations detected (AZEVEDO et al., 2011).
The reason Fv/Fm is an estimate of the quantum efficiency of photochemical activity of PSII in maximum, when all reaction centers are open (BAKER, 2008).This relationship has been widely used to detect disturbances in photosynthetic system caused by stress, characterized as a strong fluorescence study parameter of the chlorophyll-a, since your decrease indicates a decline in the efficiency of PSII in photochemistry, suggesting damage or disturbance in the photosynthetic apparatus (SCHANSKER et al., 2011).
In this study, only the treatments with irrigation without showed Fv/Fm ratios below the ideal values when the photosynthetic rates were close to zero.However, when the soil irrigation was restored, the values increased again, and no statistical difference was observed relative to the treatments without irrigation without.This is owing to the fact that in some species, application of ABA can promote the production of ethylene (KERBAUY, 2008), a hormone involved in chlorophyll degradation (TAIZ et al., 2017).The determination of the leaf chlorophyll content is important because it permits inferences about a plant's photosynthetic capacity (CARVALHO et al., 2015).Hunt (1990) suggested that seedlings of satisfactory planting quality have a DQI of 0.2.In our study, the seedlings in all treatments exhibited DQIs above the recommended values, reinforcing the idea that despite a reduction in photosynthetic activity, stress did not damage the quality of the seedlings.
Thus, it is expected that the Cerrado plants acquire, over the generations, gene that allows heritage to respond efficiently to a number of limiting factors, whereas this environment naturally has policies that require high plants stomatal and photochemistry efficiency even in low water availability (RUGGIERO et al., 2006).

CONCLUSION
The analyses carried out lead to the conclusion that: • The ABA applied exogenous at the time of the suspension of irrigation not avoided reducing the photosynthetic activity and neither optimized recovery of gas exchange and improvement of carboxylation of Rubisco after restoring normal water and quality of seedlings.• The concentration 100 µ M promoted degradation of chlorophylls water condition independent, showing that high concentrations of ABA can cause residual effect on this species.• The seedlings showed normal your metabolism is restored with the re-irrigation of the soil, which occurred around 12 days due to characteristics of the species and not on the basis of the treatments tested.