Traditional Varieties of Caupi Submitted to Water Deficit : Physiological and Biochemical Aspects

The cowpea (Vigna unguiculata (L.) Walp) it is a leguminous widely cultivated in Northeast of Brazil. In the state of Ceara, its cultivation is performed mainly by family farms who make use of traditional varieties of good adaptation to the growing region. Thus, characterizing traditional varieties with characteristics of adaptation to regions with water shortage is essential for the production of food in the world, especially in semi-arid regions. In this sense, the objective was to evaluate the physiological and biochemical responses in three genotypes of cowpea, being two traditional varieties grown in Ceara (Sempre-Verde and Cabeça-de-Gato) and a genotype characterized as a standard of drought tolerance (Pingo-de-Ouro-1,2) under three water regimes: irrigated, moderate deficit and severe water deficit. The parameters evaluated were: gas exchange, chlorophyll a fluorescence, photosynthetic pigments, organic solutes (proline, total carbohydrates, reducing and non-reducing carbohydrates), starch and enzyme activity (APX, G-POD, CAT and SOD). The genotype Pingo-de-Ouro-1,2 confirmed its tolerance pattern in a water deficit condition, presenting greater water potential, higher photosynthetic rate, high levels of total carbohydrates and high accumulation of proline. Among the traditional varieties, the Cabeça-de-Gato presented superior photosynthesis to Sempre-Verde higher Electron Transport Rate (ETR), reflecting in a greater photochemical quenching (qP) and a greater accumulation of proline, indicating that this variety presents more pronounced adaptive characteristics for water restriction conditions, which is a common condition to the Brazilian semiarid.


Introduction
The cowpea (Vigna unguiculata (L.) Walp.) is a legume originating in West Africa, having great nutritional and economic importance where it is cultivated, such as the semi-arid tropics, Asia, Africa, south-east Europe, and Central and South America.Its cultivation is justified by its development and productive capacity in areas where other crops do not produce satisfactorily, due to high temperatures and irregular rains (Akibode & Maredia, & Bailey, 2012), modulation of gas exchange and alterations to biochemical level simultaneously (Goufo et al., 2017;Rivas et al., 2016), in addition to morphological changes such as the development of deeper roots (Araus et al., 2002), decrease of the growth rate and reduction of leaf area (Cardona-Ayala et al., 2013).At the biochemical level, plants that present a standard tolerance to water deficit seek the maintenance of tissue turgidity through osmotic adjustment, through the accumulation of inorganic or organic solutes, being that the synthesis and/or accumulation of these solutes will depend on the water status of the plant and the genotype (Blum, 2017;Rivas et al., 2016).
The role of osmoprotection in cowpea is not well established and presents divergences between the different genotypes.In some cultivars under water stress, rapid and significant changes in proline levels are observed, favoring osmotic adjustment (Hamidou, Zombre, & Braconnier, 2007;Costa et al., 2011).In other cultivars, proline does not accumulate or only increases after several days of the imposition of the water deficit (Singh & Reddy, 2011;Shui et al., 2013).This delayed response may be linked to the protection of the photosynthetic apparatus (Goufo et al., 2017), once this solute acts on the reduction of NADPH from glutamate (proline precursor), thus avoiding the generation of singlet oxygen (Cecchini et al., 2011).In addition to proline, other organic solutes may be directly involved in osmotic adjustment and may contribute of differential form in tolerance to water stress in cowpea.
Due to these variations between rapid and late responses, the physiological and biochemical changes in cowpea in a water deficiency condition are not yet fully understood.However, these late responses can be more specific and can be directly related to the mechanisms induced by the diffusive and biochemical limitations of photosynthesis in order to protect the photosynthetic apparatus against excess reactive oxygen species.In general, atmospheric CO 2 diffuses through the stomata into the intercellular spaces and then through the mesophyll to the carboxylation sites.The limitations to the assimilation of CO 2 imposed by the stomatal closure in the leaves during the water stress can lead to an imbalance between the generation of electrons in photosystem II (PSII) and the electron requirement for photosynthesis.In turn, this could lead to hyperexcitation and subsequent photoinhibitory damage of the PSII reaction centers from the mesophyll and the biochemical limitations of photosynthesis.
All this divergence between the answers, resulting from the great genetic diversity of the cowpea, is the object of study by many researchers who seek to elucidate the interaction between the physiological and biochemical processes to deal with drought and to identify promising genotypes (Singh & Reedy, 2011).The objective of this work was to study the effects of water stress on physiological and biochemical responses in three genotypes of cowpea with differences and responses that are important for the Brazilian semi-arid region.

Plant Material, Growing Conditions and Experimental Design
The experiment was conducted in a greenhouse belonging to the Federal University of Ceara (UFC), in Fortaleza, from June to August 2016, where the flux density of photosynthesizing photons at noon was approximately 1.300 mol m -2 s -1 and average temperature of 32.0±2 °C.Three genotypes were used, two traditional varieties being collected in the state of Ceará/Brazil: Sempre-Verde (from Tururu-CE/Brazil) and Cabeça-de-Gato (originally from Juazeiro do Norte-CE/Brazil); and the standard genotype for drought tolerance Pingo-de-Ouro-1,2 (CE-1019).The seeds were pre-germinated on pre-weighed "germitest" type filter paper and moistened with distilled water and maintained in a chamber under controlled conditions (temperature at 25 ºC and photoperiod of 12 hours) until the emergence of the radicles.Subsequently the seeds with the emerged radicles (germinated) were transferred to 3 dm 3 filled with sand, humus and vermiculite (6:3:1), previously irrigated, to field capacity (CC).The plants were maintained in the CC with daily irrigation with distilled water and, weekly, fertigated with Hoagland nutrient solution until the imposition of the water deficit that occurred at 32 days after seeding (DAS).
The treatments were applied when the plants reached the V4 stage (pre-flowering) and consisted of three water regimes: Irrigated (absence of water stress); moderate water deficit (5 days of stress, having an irrigation with 100 mL on the third day); and severe water deficit (5 consecutive days of water stress), following the completely randomized design (DIC), in a 3 × 3 factorial arrangement (3 varieties × 3 water regimes) with 5 repetitions.The evaluations were performed after 5 days of the beginning of the irrigation suspension using the third and fourth trefoil fully expanded for the physiological and biochemical evaluations.For the biochemical analyzes, the leaves were collected and frozen in liquid N 2 , lyophilized and macerated for later use.

Potential Leaf Water and Biometric Parameters
The leaf water potential was measured in the morning (05:00 a.m.-06:00 a.m.) using the fourth trefoil with the aid of a Scholander type pressure pump.
The following biometric parameters were measured: plant height using a ruler graduated in cm; number of leaves by direct counting; leaf area with the aid of an area integrator (LI-3100, Li-COR, Inc., Lincoln, NE, USA); and the dry mass of leaves using a forced air circulation greenhouse at 60 °C for 72 hours and analytical balance.

Gas Exchange, Chlorophyll a Fluorescence
The gas exchange measurements were performed between 08:00 and 11:00 am on the central leaflet of the third sheet completely expanded in all plants using an infrared gas analyzer (IRGA, model LI-6400XT, LI-COR, Lincon, Nebraska, USA).Liquid photosynthesis (A), stomatal conductance (g s ), transpiration rate (E), ratio between internal concentration and CO 2 environment (Ci/Ca) were evaluated.For these parameters, the photosynthetically active radiation (PAR) constant of 1200 μmol photons m -2 s -1 , constant concentration of CO 2 (400 ppm), temperature and ambient humidity.
The chlorophyll a fluorescence was performed using the fluorometer coupled to IRGA (6400-40, LI-COR, USA) on the same sheet in which the gas exchanges were evaluated.The plants were acclimatized in the dark for 30 minutes, obtaining the minimum fluorescence parameters (Fo) and after a pulse of saturating light, the maximum fluorescence (Fm) was obtained.Then, the potential photochemical efficiency of PSII, expressed by the Fv/Fm ratio, was calculated.Then, the potential photochemical efficiency of PSII, expressed by the Fv/Fm ratio, was calculated.With the fluorescence parameters collected in the clear (at the same moment of determination of the gas exchanges) were determined the effective quantum yield of FSII (ɸFSII), electron transport rate (ETR), photochemical quenching (qP), non-photochemical quenching (qN) and the non-photochemical extinction coefficient (NPQ).

Photosynthetic Pigments
For the determination of photosynthetic pigments (chlorophyll a, b, total and carotenoids), leaf discs were immersed in dimethylsulfoxide solution (DMSO) saturated with CaCO 3 being kept in the dark at room temperature until quantification.The absorbances of the extracts were measured in a UV/visible spectrophotometer at wavelengths 480, 649 and 665nm, and the concentrations were calculated using equations based on the specific absorption coefficients, according to Wellburn (1994).

Soluble Carbohydrates and Starch
The extracts for determination of soluble carbohydrates were prepared from 30 mg of lyophilized leaves that were added to 5 mL of ethanol (80%) and placed in a water bath at 75 °C for 1 h and then centrifuged at 3000 × g at 4 °C, being the supernatant collected and the extraction steps repeated 2×.The total carbohydrate levels and reducing carbohydrate were quantified according to the methods proposed by Dubois (1956) and Nelson (1945), respectively.The non-reducing carbohydrates were obtained from the subtraction of the aforementioned parameters.The results were expressed as μmol of dry matter carbohydrate g -1 .
The extracts for determination of carbohydrates were prepared with the precipitate remaining of ethanolic extract of soluble carbohydrates with respect to the precipitate 4 mL of perchloric acid (30%) with subsequent stirring and centrifugation.The determination followed the method proposed by Hodge and Hofreiter (1962) and the concentration was expressed in μmol glucose g -1 dry matter.

Proline Content
The extracts for proline quantification were prepared using 20 mg of lyophilized sheets added to 2.0 mL deionized water where they remained for 1h with shaking every 10 m.After centrifugation at 3,000 × g for 15 min, the supernatant was collected for quantification.The quantification was determined according to Bates et al. (1973) and the result expressed in μmol proline g -1 dry matter.

Extraction and Antioxidant Enzyme Activity Assays
The enzymatic extracts were prepared from 1 g of fresh leaf, macerated in 4 mL of the potassium phosphate buffer (50 mM and pH 7).From this extract, the enzymatic activities of ascorbate peroxidase (APX), guaiacol peroxidase (GPOD), catalase (CAT) and superoxide dismutase (SOD) were measured, according to the methods of Nakano and Asada (1981), Kar and Mirsha (1976), Havir and McHale (1987) and Beauchamp and Fridovich (1971), respectively.The protein contents were quantified in the same extract of the enzymatic activities, from the Coomassie Blue reagent by the method proposed by Bradford (1976), the enzymatic activities being expressed in mmol of H 2 O 2 min -1 g -1 protein. jas.ccsenet.

Statisti
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