A patatin-like protein with galactolipase activity is induced by drought stress in Vigna unguiculata leaves

This paper reports the cloning of a cDNA (Vupat1) expressed in Vigna unguiculata leaves coding for a protein with 48% sequence homology to patatin, the major protein from potato tuber which has lipolytic acylhydrolase activity. Two cultivars differing in drought tolerance were examined in Northern-blot analyses. Expression of Vupat1 is stimulated by drought stress, especially in the drought-sensitive cultivar. Vupat1 was expressed in the baculovirus system as a fusion protein secreted in the culture medium. The recombinant protein displays lipolytic activity towards monogalactosyldiacylglycerol, digalactosyldiacylglycerol and sulphoquinovosyldiacylglycerols.     

Proteomic analysis of salt stress and recovery in leaves of Vigna unguiculata cultivars differing in salt tolerance

Key message

Cowpea cultivars differing in salt tolerance reveal differences in protein profiles and adopt different strategies to overcome salt stress. Salt-tolerant cultivar shows induction of proteins related to photosynthesis and energy metabolism.

Abstract

Salinity is a major abiotic stress affecting plant cultivation and productivity. The objective of this study was to examine differential proteomic responses to salt stress in leaves of the cowpea cultivars Pitiúba (salt tolerant) and TVu 2331 (salt sensitive). Plants of both cultivars were subjected to salt stress (75 mM NaCl) followed by a recovery period of 5 days. Proteins extracted from leaves of both cultivars were analyzed by two-dimensional electrophoresis (2-DE) under salt stress and after recovery. In total, 22 proteins differentially regulated by both salt and recovery were identified by LC–ESI–MS/MS. Our current proteome data revealed that cowpea cultivars adopted different strategies to overcome salt stress. For the salt-tolerant cultivar (Pitiúba), increase in abundance of proteins involved in photosynthesis and energy metabolism, such as rubisco activase, ribulose-5-phosphate kinase (Ru5PK) (EC 2.7.1.19), glycine decarboxylase (EC 1.4.4.2) and oxygen-evolving enhancer (OEE) protein 2, was observed. However, these vital metabolic processes were more profoundly affected in salt-sensitive cultivar (TVu), as indicated by the down-regulation of OEE protein 1, Mn-stabilizing protein-II, carbonic anhydrase (EC 4.2.1.1) and Rubisco (EC 4.1.1.39), leading to energy reduction and a decline in plant growth. Other proteins differentially regulated in both cultivars corresponded to different physiological responses. Overall, our results provide information that could lead to a better understanding of the molecular basis of salt tolerance and sensitivity in cowpea plants.     

Effect of drought stress on proteolytic activities in Phaseolus and Vigna leaves from sensitive and resistant plants

Although much information is available concerning the effect of senescence on cell proteolytic activities, few reports are devoted to the impact of drought stress. Our aim was to study the influence of water deficit on the cell proteolytic potential, and to determine whether or not it could be used as a physiological parameter for screening varieties for tolerance to drought. We have used Phaseolus and Vigna species differing in their senstivity to water deficit: V. unguiculata L. Walp. cv. EPACE (resistant), V. unguiculata L. Walp. cv. IT83D (moderately senstive) and P. vulgaris L. cv. Carioquinha (sensitive). The plants were subjected to controlled water conditions. Proteolytic activities were assayed using azocasein in the case of leaf extracts and [¹⁴C]-methylated casein in the case of cell compartments: soluble fraction, membrane fraction and isolated, purified chloroplasts. The results indicate that the leaf extracts contained 3 groups of proteinases with optimum pH at 4.5, 5-6 and 8.5 for the Vigna cultivars and 5.0, 6.5 and 9.0 for the Phaseolus cultivar. The sensitive P. vulgaris Carioquinha showed higher caseinolytic activities than the other tow cultivars in response to water deficit. As regards cell fractions, proteolytic activities were determined for pH values of 4.5, 6.0 and 9.0. In soluble fractions of stressed plants, proteolytic activities increased at all the pH values tested; this clearly correlated with the drought sensitivity level of the plants, especially at pH 4.5. The same phenomenon was observed in the case of membranes and purified chloroplasts of the sensitive cultivar. Under drought stress, the proteolytic potential of the cell increased especially in the vaculoar sap (soluble fraction). The higher activities observed for all the cell compartments in the sensitive cultivar could be responsible, at least partly, for the rapid degradation of leaf and chloroplast proteins under drought. The use of [¹⁴C]-methylated casein and soluble cell fractions seem to allow a clear differentiation between cultivars with respect to the drought tolerance at the cellular level.     

Morphological features and inheritance of Foliaceous Stipules of primary leaves in cowpea (Vigna unguiculata)

BACKGROUND AND AIMS: The presence of connate foliaceous stipules of primary leaves and their inheritance in cowpea (Vigna unguiculata) genotype EC394736 is reported for the first time. METHODS: The development of foliaceous stipules (FS) and their persistence were examined throughout the growth and developmental stages of the plants of the genotype EC394736. The shape, size, colour, texture and other parameters were examined in the field during the period 15-50 d after sowing. The area of FS was measured using image analysis software. The inheritance of FS was studied by making a cross between the genotype EC394763 with rudimentary stipules (RS) and the genotype EC394736, which has connate foliaceous stipules of primary leaves. The presence or absence of FS in plants of the F1, F2 and F3 generations was recorded. KEY RESULTS: The stipules developed along with the primary leaves in the genotype EC394736. One stipule of each primary leaf fused with the adjacent stipule of the other primary leaf forming a foliaceous structure. These stipules persisted on the plants for >50 d, even after the primary leaves had withered off. The F1 plants showed an absence of FS indicating the rudimentary stipules to be dominant over foliaceous stipules. The F2 segregation into 15 (RS) : 1 (FS) indicated that duplicate recessive genes controlled the presence of the FS. This was confirmed from the segregation pattern in the F3 generation. CONCLUSIONS: The presence of FS is a unique feature in cowpea genotype EC394736 and duplicate recessive genes govern it. The FS can be used as a morphological marker for identification of cowpea varieties.     

Barriers to interspecific hybridization between Vigna unguiculata and Vigna vexillata

Summary Interspecific hybridization between Vigna unguiculata and V. vexillata always failed: no seed was obtained in both crossing directions. Two different barriers to crossability were found: a pre-zygotic barrier and a post-zygotic one. Many abnormalities were observed in pollen-tube development, which reduced the percentage of fertilization to 18–30%. Differences in the percentage of fertilization were detected between the two accessions of V. vexillata involved in the interspecific crosses. The development of the interspecific embryo was analyzed and the embryo and endosperm nuclei always degenerated 5–8 days after pollination. The growth of the embryo stopped at a globular stage, which is too early for excision and in vitro culturing.     

Responses of cowpeas (Vigna unguiculata (L.) Walp.) to progressive soil drought

Summary Greenhouse-grown cowpeas, Vigna unguiculata (L.) Walp., were subjected either to well-watered or to progressive soil drought conditions between 10–40 days after emergence. Stomatal closure was found to correlate with the progressive drying of soil while leaf water potentials were not very different from the well-watered plants. Reduction in leaf turgor resulted in a reduced rate of leaf extension but increased that of root. Stomatal conductance and transpiration rates of soil-drought plants were similar to well-watered plants in the morning, but were greatly reduced in the afternoon till evening. It is suggested that the maintenance of transpiration rates per unit leaf area of soil-drought cowpeas in the morning is due to the reduction in the leaf area per plant and possibly the hydration of the plants in the night through enhanced root growth.     

Enzymatic conversions of polar lipids. Principles, problems and solutions

This text provides a brief overview of the principles of enzymatic lipid conversion and some recent advances in the enzymatic conversion of glycerophospholipids and galactolipids. Lipases and phospholipases are used to exchange fatty acids or the polar group in the lipids. The reactions can be carried out either as hydrolysis–esterification sequences or as one-step transferase reactions. The scope and limitations of the different methods are discussed.     

Biosynthesis and accumulation of D-ononitol in Vigna umbellata in response to drought stress

Metabolic responses to water deficit that lead to an accumulation of cyclitols, have been examined in rice bean ( Vigna umbellata [Thunb.] Ohwi et Ohashi). Imposition of drought stress by withholding water from the soil for 9 days led to an accumulation of D-ononitol (lD-4- O -methyl- myo -inositol) which was most pronounced in leaves (from 33 to 88 umol g⁻¹ dry mass). However, the activity of the enzyme myo-inositol 6-O -methyltransferase (m6OMT, EC 2.1.1.X), which catalyzes the synthesis of ononitol from myo -inositol and S -adenosyl-L-methionine (AdoMet), increased in stems but not in leaves during the drought stress experiment. Detailed analysis of different plant parts revealed that the accumulation of ononitol in leaves was linearly related to stem m6OMT activity during drought stress, indicating that m6OMT may control the in vivo biosynthetic rate of this cyclitol. The availability of myo -inositol, required for enhanced rates of ononitol synthesis by m6OMT, increased during the stress experiment, while the capacity to synthezise AdoMet by S -adenosyl-L-methionine synthetase (SMS, EC 2.5.1.6) decreased. However, the high capacity for degradation of S -adenosyl-L-homocysteine (AdoHcy; a potent competitive inhibitor of m6OMT) by the enzyme S -adenosyl-L-homocysteine hydrolase (SHH, EC 3.3.1.1) provided favourable conditions for ononitol biosynthesis during the whole stress treatment.     

Light dependence of catalase synthesis and degradation in leaves and the influence of interfering stress conditions

The enzyme catalase (EC 1.11.1.6) is light sensitive and subject to a rapid turnover in light, similar to the D1 reaction center protein of photosystem II. After 3 h of preadaptation to darkness or to different light intensities (90 and 520 μmol m⁻² s⁻¹ photosynthetic photon flux density), sections of rye leaves (Secale cereale L.) were labeled for 4 h with l-[³⁵S]methionine. From leaf extracts, catalase was immunoprecipitated with an antiserum prepared against the purified enzyme from rye leaves. Both incorporation into catalase and degradation of the enzyme polypeptide during a subsequent 16-h chase period increased with light intensity. At a photon flux density of 520 μmol m⁻² s⁻¹, the apparent half-time of catalase in rye leaves was 3 to 4 h, whereas that of the D1 protein was much shorter, about 1.5 h. Exposure to stress conditions, such as 0.6 m NaCl or a heat-shock temperature of 40°C, greatly suppressed both total protein synthesis and incorporation of the label into catalase and into the D1 protein. Immunoblotting assays indicated that in light, but not in darkness, steady-state levels of catalase and of the D1 protein strongly declined during treatments with salt, heat shock, or translation inhibitors that block repair synthesis. Because of the common property of rapid photodegradation and the resulting dependence on continuous repair, declines in catalase as well as of the D1 protein represent specific and sensitive indicators for stress conditions that suppress the translational activities of leaves.     

Mapping QTL for drought stress-induced premature senescence and maturity in cowpea [Vigna unguiculata (L.) Walp.]

Cowpea is an important crop for subsistence farmers in arid regions of Africa, Asia, and South America. Efforts to develop cultivars with improved productivity under drought conditions are constrained by lack of molecular markers associated with drought tolerance. Here, we report the mapping of 12 quantitative trait loci (QTL) associated with seedling drought tolerance and maturity in a cowpea recombinant inbred (RIL) population. One hundred and twenty-seven F₈ RILs developed from a cross between IT93K503-1 and CB46 were screened with 62 EcoR1 and Mse1 primer combinations to generate 306 amplified fragment length polymorphisms for use in genetic linkage mapping. The same population was phenotyped for maintenance of stem greenness (stg) and recovery dry weight (rdw) after drought stress in six greenhouse experiments. In field experiments conducted over 3 years, visual ratings and dry weights were used to phenotype drought stress-induced premature senescence in the RIL population. Kruskall–Wallis and multiple-QTL model mapping analysis were used to identify QTL associated with drought response phenotypes. Observed QTL were highly reproducible between stg and rdw under greenhouse conditions. Field studies confirmed all ten drought-response QTL observed under greenhouse conditions. Regions harboring drought-related QTL were observed on linkage groups 1, 2, 3, 5, 6, 7, 9, and 10 accounting for between 4.7 and 24.2% of the phenotypic variance (R ²). Further, two QTL for maturity (R ² = 14.4–28.9% and R ² = 11.7–25.2%) mapped on linkage groups 7 and 8 separately from drought-related QTL. These results provide a platform for identification of genetic determinants of seedling drought tolerance in cowpea. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Variation in polar-group content in lipids of cowpea (Vigna unguiculata) Cell cultures as a mechanism of haloadaptation

Callus and cell suspension cultures of cowpea (Vigna unguiculata) were induced with 2,4-dichlorophenoxyacetic acid and grown at different NaCl concentrations. The cell biomass yield and its total lipid content decreased with increasing salinity. However, while the hexose content in lipids was higher, the amount of lipid phosphorus was significantly lower in both agar and cell suspension cultures. Ion-transport rates with artificial membranes prepared with different lipid fractions showed that lipids from cells grown in a saline medium were less permeable to Na⁺ and to Cl^- than those grown in a non-saline medium. Also the permeability of membranes prepared with glycolipids was lower than those prepared with phospholipids and whole lipids. Apparently, the increase of hexose/phosphorus ratio in membrane lipids is induced in response to the halo-adaptation process.     

Nitric oxide increases tolerance responses to moderate water deficit in leaves of Phaseolus vulgaris and Vigna unguiculata bean species

Drought stress is one of the most intensively studied and widespread constraints, and nitric oxide (NO) is a key signaling molecule involved in the mediation of abiotic stresses in plants. We demonstrated that a sprayed solution of NO from donor sodium nitroprusside increased drought stress tolerance responses in both sensitive (Phaseolus vulgaris) and tolerant (Vigna unguiculata) beans. In intact plants subjected to halting irrigation, NO increased the leaf relative water content and stomatal conductance in both species. After cutting leaf discs and washing them, NO induced increased electrolyte leakage, which was more evident in the tolerant species. These leaf discs were then subjected to different water deficits, simulating moderate and severe drought stress conditions through polyethylene glycol solutions. NO supplied at moderate drought stress revealed a reduced membrane injury index in sensitive species. In hydrated discs and at this level of water deficit, NO increased the electron transport rate in both species, and a reduction of these rates was observed at severe stress levels. Taken together, it can be shown that NO has an effective role in ameliorating drought stress effects, activating tolerance responses at moderate water deficit levels and in both bean species which present differential drought tolerance.     

Isolation and metabolism of Vigna unguiculata root nodule protoplasts

Axenic cultures of bacteroid-containing protoplasts were isolated from root nodules of Vigna unguiculata L. Walp. Dimensions of the protoplasts were 35 to 135 m long x 35 to 95 m wide. Yields were about 30 to 50 mg dry weight per gram fresh weight of nodules. About 5x10⁸ protoplasts packed into 1 ml of basal medium under the influence of gravity. When incubated in hypertonic, nitrogen-free media, freshly isolated protoplasts began to reduce acetylene to ethylene after a lag period of 24 to 48 h. Various additions to the basal medium showed that the system possessed functional glycolytic and tricarboxylic acid pathways. Endogenous application of various intermediary metabolites stimulated both acetylene reduction and respiration, though not often equally. As acetylene reduction, but not respiration, was inhibitable by both asparagine and glutamine, the system appears suitable for the study of mechanisms controlling symbiotic nitrogen fixation.Abbreviations BSA bovine serum albumine - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - PEP phospho(enol)pyruvate - UMKL 76 University of Malaga, Kuala Lumpur, Rhizobium, No. 76 - TCAC tricarboxylic acid cycle     

Development and polymorphism of Vigna unguiculata ssp. unguiculata microsatellite markers used for phylogenetic analysis in asparagus bean (Vigna unguiculata ssp. sesquipedialis (L.) Verdc.)

Asparagus bean (V. unguiculata ssp. sesquipedialis), a specific form of cowpea (V. unguiculata L. Walp.), is cultivated as a vegetable crop throughout eastern and southern Asia for its tender long pods. Little is known about the genetic relationship between asparagus bean and the broader species, particularly the dominant ssp. unguiculata. We report here the development and transferability of simple sequence repeat (SSR) markers, over 40% of which are EST-derived, from ssp. unguiculata to asparagus bean and the use of a subset of the polymorphic markers to assess the genetic diversity of asparagus bean cultivars from diverse geographic origins across China. A total of 410 EST derived SSR (eSSR) markers and 600 SSR markers derived from cowpea genespace sequences (GSS) were developed, with a cross-subspecies transferability of 100% and 98.5%, respectively. In a recombinant inbred line population of asparagus bean, a 1:1 segregation was observed for most loci. Principal coordinate analysis (PCA) and phylogenetic clustering based on 62 alleles detected by 14 polymorphic SSR markers distinguished ssp. unguiculata and sesquipedialis into separate groups. Improved asparagus bean cultivars in China generally have a narrow genetic basis compared with landraces varieties. This suggests that asparagus bean breeding programs need to consider utilizing landrace germplasm to enhance genetic variability and ensure long-term gains from selection and reduce genetic vulnerability to pathogen/pest epidemics. Because of their transferability across subspecies, the SSR markers described in this study could be effectively employed in cross-subspecies trait introgression breeding from ssp. unguiculata to sesquipedialis.     

Germination stimulant from root exudates of Vigna unguiculata

Root exudate of Vigna unguiculata was extracted from a soil system consisting of charcoal and vermiculite. Germination stimulating activity for Striga gesnerioides was found in extracts of the soil system, and an active compound was isolated. The chemical structure of the active ingredient was determined to be (+)-4-O-acetylorobanchol, based on analysis of the spectral data of 1-D and 2-D NMR together with nuclear Overhauser effect (NOE) experiments. Application of the active compound to the seeds of S. gesnerioides at a concentration of 0.35 × 10⁻⁹ mol/disk led to 69% germination. The germination observed with application of GR-24, a positive control, at 0.57 × 10⁻¹⁰ mol/disk was 80%.     

Spatio-temporal heterogeneity in Arabidopsis thaliana leaves under drought stress

Using chlorophyll (chl) fluorescence imaging, we studied the effect of mild (MiDS), moderate (MoDS) and severe (SDS) drought stress on photosystem II (PSII) photochemistry of 4-week-old Arabidopsis thaliana. Spatio-temporal heterogeneity in all chl fluorescence parameters was maintained throughout water stress. After exposure to drought stress, maximum quantum yield of PSII photochemistry (F(v)/F(m)) and quantum efficiency of PSII photochemistry (Φ(PSΙΙ)) decreased less in the proximal (base) than in the distal (tip) leaf. The chl fluorescence parameter F(v) /F(m) decreased less after MoDS than MiDS. Under MoDS, the antioxidant mechanism of A. thaliana leaves seemed to be sufficient in scavenging reactive oxygen species, as evident by the decreased lipid peroxidation, the more excitation energy dissipated by non-photochemical quenching (NPQ) and decreased excitation pressure (1-q(p)). Arabidopsis leaves appear to function normally under MoDS, but do not seem to have particular metabolic tolerance mechanisms under MiDS and SDS, as revealed by the level of lipid peroxidation and decreased quantum yield for dissipation after down-regulation in PSII (Φ(NPQ)), indicating that energy dissipation by down-regulation did not function and electron transport (ETR) was depressed. The simultaneous increased quantum yield of non-regulated energy dissipation (Φ(NO)) indicated that both the photochemical energy conversion and protective regulatory mechanism were insufficient. The non-uniform photosynthetic pattern under drought stress may reflect different zones of leaf anatomy and mesophyll development. The data demonstrate that the effect of different degrees of drought stress on A. thaliana leaves show spatio-temporal heterogeneity, implying that common single time point or single point leaf analyses are inadequate.