Short-Time of Rehydration is not Effective to Re-Establish Chlorophyll Fluorescence and Gas Exchange in Two Cowpea Cultivars Submitted to Water Deficit

Low water supply frequently interferes on chlorophyll fluorescence and gas exchange. This study aimed to answer if a short-time of rehydration is efficient to re-establish chlorophyll fluorescence and gas exchange in cowpea plants. The experiment used four treatments (sensitive / water deficit, sensitive / control, tolerant / water deficit and tolerant / control). The sensitive and tolerant cultivars after water restriction had significant changes in gas exchange. On the third day, the stress caused lower for PN and gs in sensitive cultivar of 67% and 45%, respectively. After rehydration these parameters were not recovered significantly to two cultivars. In relation to chlorophyll fluorescence, water stress caused significant changes in all parameters evaluated of cultivars, being observed effects more intense on sensitive cultivar in the parameters Fv/Fm (38%) and Fo (69%). Rehydration did not promote recovery of the values of Fv/Fm and Fo to sensitive cultivar. Therefore, our study revealed that a short-time of rehydration is not effective to re-establish chlorophyll fluorescence and gas exchange in cowpea plants submitted to water deficit.


Introduction
Low water supply frequently interferes on water relations of the plants and causes reversible alterations on stomatal mechanism (gs), photosynthesis (PN), transpiration (E) and intercellular CO2 concentration (Ci), reflecting negatively on water use efficiency (WUE) and also on yield (Souza et al., 2004;Wang et al., 2003).Thus, the adequate work of the photosynthetic apparatus in plants under water deficit is extremely important aiming tolerance to water deficit (Zlatev, 2013).
Water deficit reduces the photochemical efficiency of photosystem II (PSII) and the transport rate of electrons used to photosynthesis, causing higher energy excess followed by photoinhibition of the PSII reaction center (Bai et al., 2008).In addition, this abiotic stress induces the overproduction of reactive oxygen species (ROS), with consequent increase in lipid peroxidation and electrolyte leakage of the chloroplast membranes (Catola et al., 2016).Souza et al. (2004) reported increase in F o accompanied by reductions in Fm and Fv/Fm in V. unguiculata plants.Additionally, Pereira et al. (2016) studying six populations V. unguiculata under water deficit also found changes in chlorophyll fluorescence, being described reductions in Fv/Fm, ΦPSII, qP and ETR, and increases to EXC, NPQ and ETR/PN.Based on this overview, our hypothesis is that the short-time of rehydration might recover the gas exchange and chlorophyll fluorescence modified by the water deficit.Therefore, this study aimed to answer if a short-time of rehydration is efficient to re-establish chlorophyll fluorescence and gas exchange in cowpea plants, being used two contrasting cultivars in relation to water deficit.

Location and growth conditions
The experiment was performed at the Paragominas Campus of the Universidade Federal Rural da Amazônia, Paragominas, Brazil (2°55' S, 47°34' W).The study was conducted in a fluorometer (model OS5p; Opti-Sciences).Chlorophyll fluorescence was measured using fully expanded leaves under light conditions.Preliminary tests determined the location of the leaf, the part of the leaf and the time required to obtain the greatest Fv/Fm ratio; consequently, the third acropetal leaf from the middle third of the plant adapted to the dark for 30 min was used in the evaluation.The intensity and duration of the saturation light pulse were 7,500 µmol m -2 s -1 and 0.7 s, respectively.

Evaluation of gas exchange
The net photosynthetic rate (PN), transpiration rate (E), stomatal conductance (gs), and intercellular CO2 concentration (Ci) were evaluated using an infrared gas analyser (model LCPro + ; ADC BioScientific).These parameters were measured on the adaxial surface of fully expanded leaves that were collected from the middle region of the plant.The water-use efficiency (WUE) was estimated according to Ma et al. (2004), and the instantaneous carboxylation efficiency (PN/Ci) was calculated using the formula described by Aragão et al. (2012).Gas exchange was evaluated in all plants under constant conditions of CO2 concentration, photosynthetically active radiation, air-flow rate and temperature in a chamber set at 360 μmol mol -1 CO2, 800 μmol photons m -2 s -1 , 300 µmol s -1 and 28 °C, respectively, between 10:00 and 12:00 h.

Leaf water potential
The leaf water potential (Ψw) was measured using fully expanded leaves located in the middle region of the plant and exposed to light, during the period between 11:30 to 12:00 h, which corresponded to midday potential.To determinate the Ψw, one leaf per plant and five plants per treatment were measured using an analogue plant moisture system (PMS Instrument Company, model 600).This system is based on the pressure chamber technique (Scholander et al., 1964), and the procedure outlined by Turner (1988) was followed.

Determination of superoxide concentration
To determine O2 -, 1 ml of extract was incubated with 30 mM phosphate buffer [pH 7.6] and 0.51 mM hydroxylamine hydrochloride for 20 min at 25 °C.Then, 17 mM sulphanilamide and 7 mM α-naphthylamine were added to the incubation mixture for 20 min at 25 °C.After the reaction, an identical volume of ethyl ether was added and centrifuged at 3,000 × g for 5 min.The absorbance was measured at 530 nm (Elstner and Heupel 1976).

Extraction of nonenzymatic compounds
Nonenzymatic compounds (H2O2 and MDA) were extracted as described by Wu et al. (2006).Briefly, a mixture to extract H2O2 and MDA was prepared by homogenising 500 mg of fresh leaf material in 5 mL of 5% (w/v) trichloroacetic acid.Then, the samples were centrifuged at 15,000 × g for 15 min at 3 °C to collect the supernatant.

Determination of hydrogen peroxide concentration
To measure H2O2, 200 µL of supernatant and 1800 µL of reaction mixture (2.5 mM potassium phosphate buffer [pH 7.0] and 500 mM potassium iodide) were mixed, and the absorbance was measured at 390 nm (Velikova et al., 2000).
greenhouse under controlled temperature and humidity conditions; the minimum, maximum, and median temperatures were 23, 32 and 26.5 °C, respectively.The air relative humidity during the experimental period varied between 60% and 80%.

Plants, containers and acclimation
The seeds of Vigna unguiculata (L.) Walp were obtained from cultivar 'BR3-Tracuateua' and 'Pingo de Ouro 1-2'.The cultivars were chosen according to the characteristics of tolerance to water deficit verified by Bastos et al. (2011) evaluating twenty genotypes submitted to water deficit, being 'BR3-Tracuateua' sensitive to water deficit and 'Pingo de Ouro 1-2' tolerant.The seeds were germinated and grown in 1.2-L pots (0.15 m in height and 0.10 m in diameter) filled with a mixed substrate of sand and vermiculite in a 3:1 ratio.Plants were cultivated under semihydroponic conditions, and the pots had one hole at the bottom, which was covered with mesh to maintain the substrate and aerate the roots.Solution absorption occurred by capillarity; these pots were placed into other containers (0.15 m in height and 0.15 m in diameter) containing 500 mL of distilled water for five d.Modified Hoagland and Arnon's (1950) solution was used as a source of nutrients; the ionic strength started at 50% and was modified to 100% after one day.Subsequently, the nutrient solution remained at total ionic strength.

Experimental design
The experiment was set up using a completely randomized design with four treatments (sensitive / water deficit, sensitive / control, tolerant / water deficit and tolerant / control).The experiment was assembled with five replicates for each of four treatments, a total of 20 experimental units were used in the experiment, with one plant in each unit.

Plant conduction, water deficit and rehydration
The plants received the following macro-and micronutrients from the nutritive solution: 8.75 mmol KNO3, 7.5 mmol Ca(NO3)2•4H2O, 3.25 mmol NH4H2PO4, 1.5 mM MgSO4•7 H2O, 62.50 µmol KCl, 31.25 µM H3BO3, 2.50 μmol MnSO4•H2O, 2.50 μM ZnSO4•7H2O, 0.63 μmol CuSO4•5H2O, 0.63 μmol NaMoO4•5H2O, and 250.0 μmol NaEDTAFe•3H2O.Twenty four-day-old plants were used to simulate the water deficit and rehydration, the solution was totally removed, being the root system placed in equal pots without water/solution, which was applied the water deficit by three day and one day after to establish the water/solution.During the study, the solutions were changed at 07:00h at 3-day intervals, with the pH adjusted to 5.5 using HCl or NaOH.On day 28 of the experiment, physiological parameters were measured for all plants, and leaf tissues were harvested for biochemical analyses.

Measurement of chlorophyll fluorescence
The minimal fluorescence yield of the dark-adapted state (F0), maximal fluorescence yield of the dark-adapted state (Fm), variable fluorescence (Fv), maximal quantum yield of PSII photochemistry (Fv/Fm), effective quantum yield of PSII photochemistry (ΦPSII), photochemical quenching coefficient (qP), nonphotochemical quenching (NPQ), electron transport rate (ETR), relative energy excess at the PSII level (EXC) and the ratio between electron transport rate and net photosynthetic rate (ETR/PN) were determined using an modulated chlorophyll Quantification of malondialdehyde concentration MDA was determined by mixing 500 µL of supernatant with 1,000 µL of the reaction mixture, which contained 0.5% (w/v) thiobarbituric acid in 20% trichloroacetic acid.The mixture was incubated in boiling water at 95 °C for 20 min, after which the reaction was terminated by placing the reaction container in an ice bath.The samples were centrifuged at 10,000 x g for 10 min, and the absorbance was measured at 532 nm.The nonspecific absorption at 600 nm was subtracted from the absorbance data.The MDA-TBA complex (red pigment) amount was calculated based on the method of Cakmak and Horst (1991), with minor modifications, and an extinction coefficient of 155 mM -1 cm -1 was used.

Determination of electrolyte leakage
Electrolyte leakage was measured according to the method of Gong et al. (1998), with minor modifications.Fresh leaves (200 mg) were cut into pieces 1 cm in length and placed in containers with 8 mL of distilled deionised water.The containers were incubated in a water bath at 40 °C for 30 min, and the initial electrical conductivity of the medium (EC1) was measured.Then, the samples were boiled at 95 °C for 20 min to release the electrolytes.After cooling, the final electrical conductivity (EC2) was measured (Gong et al., 1998).The percentage of electrolyte leakage was calculated using the formula EL (%) = (EC1/EC2) × 100.

Determination of photosynthetic pigments
The chlorophyll and carotenoid determinations were performed using 40 mg of leaf tissue.The samples were homogenised in the dark with 8 mL of 90% methanol (Nuclear).The homogenate was centrifuged at 6,000 × g for 10 min at 5 ºC.The supernatant was removed, and the chlorophyll a (Chl a) and b (Chl b), and carotenoid (Car) and total chlorophyll (total Chl) contents were quantified using a spectrophotometer (model UV-M51; Bel Photonics) according to the methodology of Lichtenthaler and Buschmann (2001).

Data analysis
The data were subjected to analysis of variance, and significant differences between the means were determined using the F test.Standard deviations were calculated for each treatment at all the times evaluated.The statistical analyses were performed using Assistat software.

Reductions in water potential promoted by water deficit
Reductions in water potential promoted by water deficit.The Ψw presented significant reductions of 393% and 267% after three days of water deficit in sensitive and tolerant cultivars, respectively (Fig. 1).After rehydration, significant differences of 220% and 147% wereobserved when compared to control treatment of sensitive and tolerant cultivars, respectively.

Insufficient recovery of the chlorophyll fluorescence after rehydration
The water deficit induced significant reductions in F v/ F m, F o and F m for both cultivars, in which the sensitive cultivar after three days of water deficit had decreases of 38%, 69% and 37%, respectively (Fig. 1).Rehydration did not promote recovery of the values of F v/ F m and F o to sensitive cultivar, as well as not recovered F m in both cultivars.The three days of water stress caused significant changes to ΦPSII, qP, NPQ, ETR, EXC and ETR/PN in both cultivars (Fig. 2), being observed effects more intense on sensitive cultivar.

Short-time of rehydration not recovered gas exchange
The sensitive and tolerant cultivars after water restriction had significant changes, being detected in the sensitive cultivar decreases by 67%, 31%, 45%, 52% and 80% for P N, E, g s, WUE and P N/ C i, respectively, and 63% increase for C i (Fig. 3).After rehydration these parameters were not recovered significantly to two cultivars.

Overproduction of oxidative composts and cell damages after water deficit
The O2 -, H2O2, MDA and EL concentrations of the two cultivars suffered more intense changes on the 3 rd day under water deficit, in which the sensitive cultivar showed increases of 227%, 142%, 140% and 35%, respectively (Fig. 4).Rehydration not recovered H2O2 and MDA significantly in both cultivars, while tolerant cultivar had recovery only to O2 -and EL.
Water deficit reduced the photosynthetic pigments Plants under water deficit presented significant reductions in Chl a, Chl b, total Chl and Car in both cultivars (Fig. 5), being more intense the effects on the sensitive cultivar.Rehydration did not allow Significant recovery to Chl b and the total Chl in sensitive cultivar.

Discussion
The reduction in Ψw is related to limited absorption and transport of water by the roots and water loss through E (Grzesiak et al., 2006).This behavior promotes the loss of cellular turgor, higher stomatal resistance and decrease in osmotic potential.Decrease in Ψw was described by Miyashita et al. (2005) in Phaseolus vulgaris exposed to water deficit of 8 days.

241
The reductions in the Fv/Fm and ΦPSII values are related to decreases in the photochemical efficiency of the PSII, in which the water deficit reduces the capacity of the plastoquinone (PQ) to transport electrons (Drozdova et al., 2004).Increases in F o accompanied by reductions in Fm can be explained by the damages caused to the reaction center of PSII (Zlatev, 2009).The NPQ was increased in response to higher EXC values, indicating that V. unguiculata plants dissipated in the form of heat the excess energy.In addition, ETR/PN increased due to the increase previously described to NPQ, suggesting that occurred lower electron transfer to CO2 fixation, with consequent use of these electrons to photorespiration and other metabolic processes (Silva et al., 2012).Similar behavior was reported to EXC in Saccharum spp plants (Sales et al., 2015) and Jatropha curcas (Silva et al., 2015).Rivas et al. (2016) studying two cultivars of V. unguiculata, 'Pingo de Ouro 1-2' and Santo Inácio, found reductions for NPQ in 80% and 200%, respectively, after 10 days of irrigation suspension.Similar results for ETR/PN were found by Santos et al. (2009) evaluating five genotypes of Phaseolus vulgaris after 10 days of water deficit.The reduction in qP reveals lower photochemical efficiency in the energy conversion due to increase of NPQ, previously detected in this study.In addition, reductions in ETR indicate that the water deficit caused disorders in the electron transport chain present into membranes of thylakoids (Chagas et al., 2008).Catola et al. (2016) evaluating Punica granatum plants under water deficit found reduction of approximately 50% in qP.Souza et al. (2004) studying V. unguiculata plants reported reductions of 82% and 72% for qP and ETR, respectively, after two days of water restriction, corroborating our results.
The water deficit induced lower P N and E, and had as consequence decrease in the absorption and diffusion of CO2 inside the plant, besides to promote CO2 accumulation in the mesophyll region (Dutra et al., 2015), corroborating with increases detected to C i in the sensitive cultivar.Decreases in P N (83%) and E (85%) were reported by Endres et al. (2010) in Vigna unguiculata plants under water deficit conditions.For C i, Silva et al. (2010) evaluating Vigna unguiculata behavior in three irrigation regimes (25%, 50% and 100%) detected higher values reported in treatment with lower water regime.
The decrease described to P N/ C i was influenced by the reduction in the CO2 absorption (PN) and reductions in the ETR observed in this study.The decrease in ETR interferes on the supplies of ATP and NADPH, that will be used in fixation reactions of CO2 (Zlatev, 2013), resulting in the increase of the intercellular CO2 concentration (Ci) due this molecule not be fixed into Calvin cycle.The decrease obtained to WUE is explained by the reduction induced by water deficit mainly in P N and also E, because this variable is obtained from the ratio between P N and E.
The increases in O2 -and H2O2 reveal the water limitation into plant cells, and consequent formation of reactive oxygen species (ROS), being that these composts promote the lipid peroxidation (Siddiqui et al., 2015), and induce the deterioration of the membranes, described by the increases of MDA and EL.Increases in ROS levels under water deficit conditions are associated with low efficiency in the excitation energy dissipation, through the photosynthetic apparatus, which it is considered an important source of overproduction of ROS under abiotic stress (Hura et al., 2015).
These results indicate that water deficiency resulted in an inhibition of the synthesis and/or degradation of the chlorophylls, as well as proteins involved in chloroplast synthesis, possibly to prevent the excessive absorption of UV radiation, during progressive stress (Jin et al., 2015).The recovery of pigments in the tolerant cultivar after rehydration is an indication that was recovered the physiological competence, after significant losses of water fraction in tissues.Trujillo et al. (2013) also observed negative impacts on the pigments of two cultivars of Phaseolus vulgaris after eight days of water deficit, and it observed a slight recovery of these parameters after one day of rehydration, corroborating the results obtained in this study.

Conclusions
Our results described that both cultivars after water restriction suffered significant changes in gas exchange and chlorophyll fluorescence, being more intense in sensitive cultivar.Additionally, this study revealed that a short-time of rehydration is not effective to reestablish chlorophyll fluorescence and gas exchange in cowpea plants submitted to water deficit.

Fig. 1 .
Fig. 1.Leaf water potential, maximal fluorescence yield of the dark-adapted state, minimal fluorescence yield of the darkadapted state and maximal quantum yield of PSII photochemistry in two cowpea cultivars submitted to water deficit and rehydration.Symbols described the mean values, bars represent the standard deviations from five repetitions and the arrow indicates the rehydration point

Fig. 2 .
Fig. 2. Effective quantum yield of PSII photochemistry, photochemical quenching coefficient, nonphotochemical quenching, electron transport rate, relative energy excess at the PSII level and the ratio between electron transport rate and net photosynthetic rate in two cowpea cultivars submitted to water deficit and rehydration.Symbols described the mean values, bars represent the standard deviations from five repetitions and the arrow indicates the rehydration point Fig. 3. Net photosynthetic rate, transpiration rate, stomatal conductance, intercellular CO 2 concentration, water-use efficiency, and carboxylation instantaneous efficiency in two cowpea cultivars submitted to water deficit and rehydration.Symbols described the mean values, bars represent the standard deviations from five repetitions and the arrow indicates the rehydration point