Drought tolerance in cowpea species is driven by less

South African Journal of Botany 103 (2016) 101–107

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Drought tolerance in cowpea species is driven by less sensitivity of leaf gas exchange to water deficit and rapid recovery of photosynthesis after rehydration R. Rivas a, H.M. Falcão a, R.V. Ribeiro b, E.C. Machado c, C. Pimentel d, M.G. Santos a,⁎ a

Departamento de Botânica, Universidade Federal de Pernambuco, Recife, PE, Brazil Department of Plant Biology, Institute of Biology, University of Campinas (UNICAMP), Campinas, SP, Brazil c Centro de Pesquisa e Desenvolvimento em Ecofisiologia e Biofísica, Instituto Agronômico, Campinas, SP, Brazil d Departamento de Fitotecnia, Instituto de Agronomia, Universidade Federal Rural do Rio de Janeiro, Seropédica, RJ, Brazil b

a r t i c l e

i n f o

Article history: Received 18 December 2014 Received in revised form 28 July 2015 Accepted 25 August 2015 Available online xxxx Edited by BS Ripley Keywords: A/Ci curves Chlorophyll fluorescence Gas exchange Vigna unguiculata Water deficit

a b s t r a c t Cowpea grains are the main protein source for many people in semi-arid regions. The goal of this study was to study the in vivo photosynthetic behavior of two cowpea cultivars under well-watered, drought stress, and recovery conditions. The cultivars differ in their sensitivity to drought stress, being classified as tolerant and sensitive based on grain production. After 10 days of water deficit, the leaf water potential of the tolerant cultivar was higher than in the sensitive cultivar, suggesting a mechanism of drought tolerance related to the maintenance of shoot water status. During drought stress, the leaf gas exchange and chlorophyll fluorescence parameters decreased faster in the sensitive cultivar as compared to the tolerant one. After 48 h of rehydration, the stressed plants of both cultivars did not recover the maximum rates of carboxylation, the maximum rate of electron transport driving the RuBP regeneration and photosynthetic capacity. However, tolerant cultivar recovered all photosynthetic parameters faster than the sensitive cultivar after 60 h of rehydration. Our results suggest that the tolerant cultivar was able to maintain higher photochemical activity and leaf gas exchange during water deficit for a longer period than the sensitive cultivar does, which could alleviate the stress effects to the photosynthetic machinery and improve its recovery ability. The consequences of this behavior are discussed. © 2015 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction In some tropical areas of developing countries, such as the Africa Sahelian zones and the Brazilian Northeast region, grain legumes are grown only during the rainy season and drought stress represents the most important threat to biomass production for many small farmers (Donohue et al., 2013). For the people of these regions, cowpea grains are an important protein source. Although studies with cowpea are numerous and several important agronomic traits have been revealed (Bastos et al., 2011; Nascimento et al., 2011; Oliveira et al., 2012), the knowledge about the photosynthetic metabolism of cowpea cultivars

Abbreviations: AES, alternative electron sink; Ca, air CO2 concentration; Rd, day respiration; ETR, electron transport rate; Ci, intercellular CO2 concentration; RWC, leaf relative water content; Ψl, leaf water potential; Vc,max, maximum rate of RuBisCO carboxylation; Jmax, maximum rate of electron transport driving RuBP regeneration; A, net CO2 assimilation; NPQ, non-photochemical quenching; NSL, non-stomatal limitation; qP, photochemical quenching; PPFD, photosynthetic photon flux density; Fv′/Fm′, PSII effective efficiency (Fv′/Fm′); Amax, photosynthetic capacity; ϕPSII, quantum yield of PSII; SMC, soil water content; SS, soluble sugars; Sta, starch; gs, stomatal conductance; SL, stomatal limitation; TFA, total free amino acids; TP, total protein. ⁎ Corresponding author. Tel.: +55 81 2126 8844; fax: +55 81 2126 7803. E-mail address: [email protected] (M.G. Santos).

http://dx.doi.org/10.1016/j.sajb.2015.08.008 0254-6299/© 2015 SAAB. Published by Elsevier B.V. All rights reserved.

under drought stress and recovery is limited (Singh and Reddy, 2011; Souza et al., 2004). The predicted changes in climate may lead to extreme events, such as increased precipitation and long drought periods (IPCC, 2007). In the Brazilian Northeast region, cowpea is the main cultivated legume due to its morphological and physiological plasticity under drought stress (Anyia and Herzog, 2004; Bastos et al., 2011; Hamidou et al., 2007; Souza et al., 2004). Cowpea plants can produce more than 1,000 kg grain ha−1, but the drought stress reduces such potential to approximately 360 kg ha−1 (Bastos et al., 2011; Nascimento et al., 2011), especially when stress occurs during pre-flowering. To avoid lethal water potential and other water deficit effects, stomatal control is the major physiological trait to prevent excessive water loss in C3 plants, such as cowpea (Anyia and Herzog, 2004; Boyer, 1978; Hamidou et al., 2007; Singh and Reddy, 2011; Souza et al., 2004). Under low soil water availability, stomatal closure is induced by root signals, leading to decreases in leaf gas exchange, which in turn causes loss of productivity (Saradadevi et al., 2014; Tang et al., 2002; Tardieu and Simonneau, 1998). Besides low mesophyll CO2 concentration, which reduces net CO2 assimilation rate of stressed cowpea plants (Singh and Reddy, 2011; Souza et al., 2004), leaf photochemical and biochemical responses are also changed in different

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ways among cowpea cultivars (Singh et al., 2010). Some aspects of leaf photosynthetic metabolism in cowpea plants under constraining conditions are still unclear. For instance, we do not know about the photosynthetic recovery capacity of drought-stressed cowpea cultivars after soil rehydration. Besides the stomatal limitation, severe or even mild water deficit can lead to biochemical limitation of photosynthesis in cowpea plants in a cultivar-dependent manner, including reductions in phosphorylation, ribulose-1,5-bisphosphate (RuBP) regeneration, and Rubisco activity (Singh and Reddy, 2011). Whether differences in photosynthetic metabolism between tolerant and sensitive P. vulgaris cultivars to drought are clear (Santos et al., 2009), we do not know the recovery capacity of photosynthesis in V. unguiculata cultivars with contrasting performance under drought. In fact, such recovery capacity is an important physiological trait in drought-tolerant genotypes, enabling rapid CO2 supply during regrowth after rehydration. Thus, this study aims to explore physiological mechanisms that contribute to drought tolerance in two cowpea cultivars with differential sensitivity to water deficit. 2. Materials and methods 2.1. Plant material and experimental conditions The experiment was conducted under greenhouse conditions, where two cowpea [Vigna unguiculata (L.) Walp.] cultivars were grown: Pingo de Ouro 1-2 (PO) and Santo Inácio (SI), which are drought tolerant and sensitive, respectively (Bastos et al., 2011). Seven plants of each cultivar were grown in pots (10 L) with the substrate being composed by organic horizon soil (blacksoil), clayhorizon soil (redearth), and washed sand (3:1:2). The pots were fully randomized and kept watered through daily irrigation. After 17 and 25 days of plant emergence, nitrogen fertilizer was applied using soluble urea equivalent to 20 kg ha−1on each date (Santos et al., 2006). From 35 to 45 days after emergence, the water deficit treatment was imposed through water withholding. Then cowpea cultivars were subjected to two water regimes: control (well watered) and water deficit. The maximum water stress was reached after 10 days of treatment, when plants presented predawn leaf water potential (Ψl) around − 1.5 MPa. As 2.5 MPa is considered the lethal Ψl value for cowpea (Boyer, 1978), plants were subjected to mild drought stress. After the maximum water deficit, plants were rehydrated for 5 days through daily irrigation. During the experiment, the mean air temperature and relative humidity inside the greenhouse were 28 ± 1 °C and 66 ± 3%, respectively. Maximum photosynthetic photon flux density was around 2,000 μmol m−2 s−1.

(NPQ), photochemical quenching (qP), electron transport rate (ETR), and quantum yield of PSII (ϕPSII) were measured using an IRGA (Li6400, Li-Cor, Lincoln NE, USA) attached to a modulated fluorometer (6400-40), and a gas flow of 500 μmol s−1. The activity of alternative electron sinks (AES) was estimated through the relationship between PSII effective quantum efficiency (ΔF/Fm') and the quantum efficiency of CO2 assimilation [ΦCO2 = A/(PPFD × 0.84)], as done by Ribeiro et al. (2004). Chl fluorescence parameters were calculated according to Schreiber et al. (1994) and Maxwell and Johnson (2000), and measurements were taken simultaneously to the gas exchange under PPFD of 800 μmol m−2 s− 1 and natural variation of air temperature and relative humidity. The response of A to increasing intercellular CO2 concentration (Ci) was measured in the same leaves that were evaluated for daily gas exchange and Chl fluorescence. The A/Ci curves were made under saturating PPFD of 800 μmolm− 2 s− 1as determined previously (data not shown). The air temperature and vapor pressure deficit were maintained at 25 ± 1 °C and 1.0 ± 0.1 kPa. The Ci values were obtained by changing the air CO2 concentration (Ca) from 390 to 125 μmol mol–1 and then increasing from 390 to 1500 μmol mol–1in a 16 steps procedure. The maximum rates of RuBisCO carboxylation (Vc.max), the maximum rates of electron transport driving RuBP regeneration (Jmax), the maximum CO2 assimilation (Amax), and the day respiration (Rd) were calculated following Farquhar et al. (1980) and Bernacchi et al. (2001). The stomatal limitation of A (SL) was estimated by considering the relationship between the actual photosynthetic rate A′ at Ca of 390 μmol mol−1 and the hypothetical photosynthetic rate A” obtained when Ci = Ca: SL = (A”–A′)/A”. Furthermore, the non-stomatal limitation (NSL = 1–SL) was also estimated (Long and Bernacchi, 2003). The A/Ci curves were performed on control plants and in drought-stressed plants after 60 h of rehydration. 2.4. Leaf biochemical analyses One fully expanded and mature leaf from each of the seven plants per treatment was collected. Immediately after sampling, leaves were packed in aluminum foil, frozen in liquid nitrogen, and stored at − 20 °C for biochemical analyses. Samples were collected at 15:00 h on the day of maximum water stress (10th day) and after plant rehydration (15th day). The leaf content of soluble sugar (SS), starch (Sta), total free amino acid (TFA), and total soluble proteins (TSP) were quantified following the classical methods described by Dubois et al. (1956), Farrar (1995), Moore and Stein (1948), and Bradford (1976), respectively. All analyses used a spectrophotometer (model 10S UVVIS Genesys, Thermo Scientific, Waltham, MA, USA), with a dual beam adjusted to the specific wavelength for the organic compound considered.

2.2. Soil and plant water status

2.5. Statistical analysis

The soil moisture content (SMC) was measured using a Falker HFM2030 meter (HidroFarm, Porto Alegre RS, Brazil) between 08:00 h and 09:00 h. The leaf relative water content (RWC) was measured every 2 days from the beginning of the experiment until 48 h after rehydration (12th day of the experiment), following the procedure described by Barrs and Weatherley (1962). The Ψl was measured at predawn (05:00 h) using a pressure chamber (Scholander et al., 1965), with three replications at days 0 (beginning), 10 (maximum water deficit), and 15 (recovery) of the experiment.

The pots were arranged in a completely randomized design, and the data were submitted to the analysis of variance (ANOVA), with the interaction of two factors being considered: cowpea cultivar and water regime. Significant differences were compared by the Student–Newman– Keuls test at a 5% probability. The replicates were taken from different plants, with one plant in each pot. Three replicates were used for the measurements of water status and photosynthesis, whereas five replicates were used for the biochemical data. 3. Results

2.3. Gas exchange and chlorophyll fluorescence 3.1. Leaf and soil water status Leaf gas exchange and chlorophyll (chl) fluorescence were measured from 08:00 h to 10:00 h in the middle leaflet of the fifth trifoliate leaf from the plant base, which was a mature but not senescent leaf. Stomatal conductance (gs), net CO2 assimilation rate (A), PSII potential quantum efficiency (Fv′/Fm′), non-photochemical quenching

Water withholding reduced significantly the soil moisture content, reaching values lower than 5% after 10 days of treatment (Fig. 1A). Under such limiting condition, reduction in RWC of PO plants was only apparent at the maximum water stress, while reductions of RWC


(A)

(B)

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(A)

(B)

(C)

Fig. 2. Stomatal conductance (gs, in A) and net CO2 assimilation (A, in B) in two Vigna unguiculata cultivars Pingo de Ouro (PO, circles) and Santo Inácio (SI, squares) subjected to well-watered conditions (closed symbols) and water deficit (open symbols). Hatched area indicates the recovery time, when plants were re-irrigated. All measurements were taken from 08:00 h to 10:00 h. Each symbol represents the mean value of three replicates (±SE).

Fig. 1. Soil moisture content (SMC, in A), leaf relative water content (RWC, in B), and leaf water potential (Ψl, in C) in two Vigna unguiculata cultivars Pingo de Ouro (PO, circles) and Santo Inácio (SI, squares) subjected to well-watered conditions (closed symbols) and water deficit (open symbols). Hatched area indicates the recovery time, when plants were re-irrigated. Each symbol represents the mean value of three replicates (±SE).

in SI plants were apparent from 3 days of treatment (Fig. 1B). At the maximum water stress, the lowest Ψl values were found in SI plants (Fig.1C). During the rehydration period, Ψl increased along with the RWC and SMC for both cultivars, with control and water-stressed plants showing similar values of RWC after 2 days of recovery and similar values of Ψl after 5 days of rehydration (Fig. 1). 3.2. Changes in leaf stomatal conductance, CO2 assimilation rate, and photochemistry under water deficit and rehydration From the third day of water withholding, gs and A of SI plants decreased significantly (P b 0.05) as compared to plants under wellwatered conditions (Fig. 2). On the other hand, the water deficit decreased (P b 0.05) gs and A of PO cultivar after 5 days of treatment (Fig. 2). At this time, A in PO cultivar was eight-fold higher than in SI cultivar under water deficit (Fig. 2B). In fact, PO cultivar presented higher A than SI cultivar until the tenth day of treatment (maximum water stress), when similar values were found in both cultivars. After

5 days of rehydration, gs and A recovered to the control values in PO plants (Fig. 2), while SI plants did not recover the control A values (Fig. 2B). The photochemical parameters Fv′/Fm′, qP, ϕPSII, and ETR decreased (P b 0.05) during water deficit, with SI cultivar showing higher sensitivity as compared to PO cultivar (Fig. 3A–C, E). An opposite pattern was observed for NPQ, with drought-induced increases from the third day until the tenth day in both cultivars (Fig. 3D). Such increase in NPQ occurred earlier in SI cultivar than in PO cultivar. A rapid increase in AES was found in SI plants under water shortage, with AES in SI plants being around 4.5-fold higher than in PO plants after 5 days of treatment (Fig. 3F). Afterward, AES was significantly reduced in SI plants, while PO plants maintained AES higher than 30 until the maximum water deficit (Fig. 3F).

3.3. Stomatal and non-stomatal limitations of photosynthesis after plant rehydration After 60 h of rehydration, Rd and Vc,max were similar between cultivars and presented full recovery (Fig. 4A, B). Only PO cultivar showed recovery of Jmax and Amax after the water deficit (Fig. 4C, D). The Jmax/ Vc,max ratio was similar in both cultivars and did not change after the water stress, varying between 2.0 and 2.7. The proportion between the stomatal and non-stomatal limitation of photosynthesis varied between cultivars during recovery period after water deficit (Table 1). While the stomatal limitation increased in SI cultivar, PO cultivar presented increases in non-stomatal limitation of photosynthesis during the recovery period.

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(A)

(B)

(C)

(D)

(E)

(F)

Fig. 3. PSII maximum efficiency (Fv′/Fm′, in A), quantum yield of PSII (ФPSII, in B), non-photochemical quenching (NPQ, in C), photochemical quenching (qP, in D), electron transport rate (ETR, in E) and alternative electron sink (AES, in F) in two Vigna unguiculata cultivars Pingo de Ouro (PO, circles) and Santo Inácio (SI, squares) subjected to well-watered conditions (closed symbols) and water deficit (open symbols). Hatched area indicates the recovery time, when plants were re-irrigated. All measurements were taken from 08:00 h to 10:00 h. Each symbol represents the mean value of three replicates (±SE).

3.4. Leaf biochemical analyses At the maximum water stress, PO and SI cultivars showed increases in leaf content of free amino acids and total proteins compared to control plants (Table 2). Regarding the leaf carbohydrates, only stressed SI plants showed increases in soluble sugar concentration as compared to the control plants. Leaf starch concentration was increased due to water deficit only in PO cultivar (Table 2). After plant rehydration, significant differences were found only in soluble sugar concentration of SI cultivar, being the lowest values found in previously stressed plants. Non-significant changes in starch concentration were found between cultivars or water regimes after rehydration.

4. Discussion This study was carried out to evaluate the behavior of the photosynthetic machinery in two cowpea cultivars with differential drought tolerance, and also to reveal differences between them during the water deficit and rehydration periods. Our results confirmed that PO plants had higher photosynthetic performance than SI plants under water deficit, also showing faster recovery of photosynthesis as compared to SI cultivar. Such drought sensitivity of SI cultivar was in accordance with its leaf water status, with this cultivar showing lower Ψl and relative water

content than PO cultivar under moderate drought (Fig. 1). As SI cultivar had larger leaf area than PO (data not shown), the total amount of water transpired by SI cultivar was likely higher than in PO cultivar, causing lower Ψl and relative water content (Fig. 1). As leaf area modulates water demand by plant canopy and then affects soil water availability and defines the onset of water deficit, selection for drought tolerance in cowpea should also consider the water use efficiency besides photosynthetic performance under varying water conditions. Regarding the ways to maintain shoot hydration, many cowpea cultivars previously assessed (Singh and Reddy, 2011; Souza et al., 2004) showed no osmotic adjustment, suggesting that differences at the maximum water deficit were not originated by the accumulation of osmotically active solutes. Therefore, the stomatal behavior in cowpea plants is important to preserve shoot water status under dehydration conditions. In addition, there must be other mechanisms in the tolerant PO cultivar to save water, because the water potential at the end of 10 days of water withholding was higher than in the sensitive SI cultivar (Fig. 1C). In fact, common bean cultivars showed different performance under abiotic stress due to different physiological, biochemical, and anatomical characteristics (Santos et al., 2009; Wentworth et al., 2006). The photosynthetic machinery of common bean plants (Santos et al., 2006, 2009) or cowpea is sensitive to water deficit (Souza et al., 2004), as reduction in A due to stomatal closure is found shortly after the imposition of drought. In general, cowpea cultivars under water deficit exhibit a rapid reduction in gs followed by low values of A and transpiration


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Fig. 4. Day respiration (Rd, in A), maximum rate of RuBisCO carboxylation (Vc.max, in B), maximum rate of electron transport driving RuBP regeneration (Jmax, in C), and photosynthetic capacity (Amax, in D) in two Vigna unguiculata cultivars Pingo de Ouro (PO) and Santo Inácio (SI) subjected to well-watered conditions and after 60 h of rehydration. Each symbol represents the mean value of three replicates (±SE).

(Bastos et al., 2011). However, our results showed that reduction of gs does not maintain high Ψl in the sensitive SI cultivar, which was likely caused by high canopy transpiration, as discussed previously. This is in agreement with field measurements, where SI plants always show lower Ψl and gs than PO plants under same environmental conditions. This finding also suggests a greater water loss through the epidermis and thus leaf area with its morpho-anatomical aspects should be investigated in further studies. In fact, White and Montes-R (2005) have found significant differences in leaf thickness among common bean cultivars. As SI plants were more sensitive to water deficit as compared to PO plants, they showed slower recovery of photosynthetic activity, considering both carboxylation and photochemical activity (Figs. 3 and 4). From the existing knowledge, this early reduction in A due low gs under drought and a slow recovery after stress may be among the major causes of reduction in grain yield of SI cultivar in field experiments (Bastos et al., 2011). The time course of photochemical parameters revealed that stressed PO plants had decreases in photochemistry at least 4 days later than

Table 1 Stomatal (SL) and non-stomatal (NSL) limitation (%) of photosynthesis in two Vigna unguiculata cultivars Pingo de Ouro (PO) and Santo Inácio (SI) subjected to well-watered conditions (Control) and after 60 h of rehydration (Recovery). Water regime

Cultivar

SL

NSL

Control

PO SI PO SI

26.77 26.17 14.19 39.56

73.94 78.40 84.88 77.62

Recovery

stressed SI plants did (Fig. 3A, B, C, E). At the same time that gs decreased in SI plants, NPQ and AES increased two and four times, respectively, due to water deficit (Fig. 3D, F). The ability to quickly increase AES could represent a protective strategy to avoid excess of energy at PSII and then oxidative damage (Ribeiro et al., 2004; Santos et al., 2009). As stressed SI plants exhibited decreases in AES from fifth day after water withholding, we may argue that such protective capacity was overcome by stressful conditions, which probably affected the alternative electron sinks such as nitrogen metabolism, photorespiration, and Mehler reaction. On the other hand, the tolerant PO cultivar maintained AES and photosynthesis until the maximum water deficit, thus avoiding excessive energy pressure at PSII and reducing the potential damage caused by oxidative stress at chloroplasts (Ribeiro et al., 2004; Santos et al., 2009). Measurements of chl fluorescence and CO2 assimilation in Phaseolus vulgaris plants under water deficit showed that reduction of photosynthesis was due to internal CO2 depletion caused by stomatal closure (Cornic and Briantais, 1991; Santos et al., 2009). In addition, low photosynthesis is related to substantial decrease in ATP synthesis under water deficit, which is a consequence of reduced ATP synthase activity and also to the inability of chloroplasts to regenerate RuBP (Tezara et al., 1999). Such analysis of photosynthesis in cowpea plants under water deficit is scarce and there is no detailed data about the photosynthesis recovery after stressful events. Photosynthetic analyses after plant rehydration helped us to clarify the differential behavior of studied cowpea cultivars under water deficit. Our data indicated that carboxylation was not a limiting factor for both cultivars (Fig. 4). In fact, the regeneration of RuBP dependent on electron transport (Jmax) was the main difference between cultivars, with the sensitive plants not showing recovery even after 60 h of

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Table 2 Soluble sugar (SS), total free amino acid (FFA), total soluble protein (TSP), and starch concentrations in leaves two Vigna unguiculata cultivars Pingo de Ouro (PO) and Santo Inácio (SI) subjected to well-watered conditions (control) and water deficit (stress). Measurements were taken at the maximum water deficit (10 days of water withholding) and after 60 h of rehydration. Each value represents the mean of five replicates (±SE). Means followed by the same letter (lowercase on maximum stress and capital on recovery) did not differed by the Student–Newman–Keuls test (5%). Time

Cultivar

Water regime

SS (mmol kg−1 DW)

FAA (mmol kg −1 DW)

TSP (g kg−1 DW)

Starch (mmol kg−1 DW)

Maximum water deficit

PO

Control Stress Control Stress Control Stress Control Stress

96.1 ± 7.0 b 119.4 ± 7.1 b 83.0 ± 6.6 b 150.4 ± 15.1 a 102.2 ± 9.0 A 94.2 ± 8.5 A 88.0 ± 3.0 A 74.0 ± 7.9 B

8.8 ± 1.1 c 24.5 ± 1.4 b 9.3 ± 4.6 c 44.0 ± 6.2 a 15.4 ± 2.1 A 18.9 ± 1.1 A 19.6 ± 2.0 A 20.9 ± 0.8 A

9.0 ± 1.9 c 13.8 ± 1.7 ab 6.7 ± 0.7 c 12.7 ± 0.6 b 8.4 ± 0.8 A 11.5 ± 1.3 A 8.8 ± 1.1 A 9.3 ± 0.9 A

63.6 ± 6.0 b 80.7 ± 6.0 a 61.3 ± 7.1 b 74.7 ± 2.8 ab 60.2 ± 5.1 A 61.5 ± 5.2 A 51.6 ± 6.3 A 60.8 ± 7.0 A

SI Recovery

PO SI

rehydration. Such metabolic dysfunction found in SI cultivar previously stressed was responsible for reduced photosynthetic capacity (Amax) as compared to well-watered plants. The present study demonstrates that the in vivo photosynthetic metabolism components were affected differentially in cowpea cultivars with contrasting sensitivity to drought. The PO cultivar quickly recovered from water stress, reducing stomatal limitation of photosynthesis as compared to the SI cultivar (Table 1). Such response suggests a higher dependence of photosynthesis on mesophyll processes and indicates that the ability of PO cultivar in maintaining biochemical reactions of photosynthesis under water deficit is crucial for plant performance, which could have been achieved due to the higher leaf water potential maintained. On the other hand, SI cultivar was less dependent on nonstomatal factors after recovering of water deficit. This pattern points out that the importance of CO2 availability through stomatal aperture, suggesting that CO2 supplying should be maintained to avoid extensive photosynthetic damage under water stress (Pimentel et al., 1999; Souza et al., 2004). The sensitive cultivar assimilated more CO2 than the tolerant cultivar in the absence of water deficit, a similar behavior found in P. vulgaris cultivars (Pimentel et al., 1999). However, the higher photosynthetic performance of SI plants was down-regulated under moderate water stress and did not recover even after plant rehydration, which suggests a trade-off between photosynthetic capacity and drought tolerance. Photochemical and carboxylation patterns led to different leaf primary metabolites when comparing cultivars, with stressed SI plants showing the highest leaf SS and FA contents at the maximum water deficit (Table 2). An increase in leaf SS concentration of SI cultivar under water deficit without significant changes in starch concentration suggests that starch synthesis was impaired by drought. In addition, high leaf SS concentration may down-regulate photosynthesis through the repression of photosynthetic-related genes (Farrar et al., 2000; Stitt and Zeeman, 2012), being an additional factor causing higher sensitivity of photosynthesis in SI cultivar as compared to PO cultivar. Under limiting conditions, when plants have reduced growth, photo assimilates are stored as starch and this pattern was found only in PO cultivar. Our data also reveal that such starch in PO plants was metabolized during plant rehydration, providing carbon and energy to resume the plant growth. Concluding, our results indicate that the maintenance of leaf water status and photosynthesis under drought stress conditions are important traits found in cowpea species, enabling plants to overcome the stressful conditions and to recover more rapidly after rehydration.

Acknowledgments The authors acknowledge the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil) for the scholarship and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil) for fellowships granted.

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