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This work was conducted to assess whether or not a natural occurring polysaccharide from Adenanthera pavonina seeds (PLSAp) and its chemically sulfated derivative galactomannan (SPLSAp), which did not act as fungicides, induce defense proteins and protect a susceptible cowpea (Vigna unguiculata) genotype (BR3-Tracuateua) against the phytopathogenic fungus Colletotrichum gloeosporioides. Twelve-day-old plants were sprayed to run-off with PLSAp or SPLSAp, both at 100 and 200 mg L-1, dissolved in 0.01% Triton X-100, which served as control. The primary leaves were collected at 0, 6, 12, 24, and 48 h after the carbohydrate treatments and the activities of guaiacol peroxidase (POX), β-1,3-glucanase (βGLU), and chitinase (CHI) were determined to verify the ability of the studied galactomannans to induce alterations in their kinetics. In addition, the primary leaves of a plant group previously sprayed with SPLSAp and 6 h later inoculated with C. gloeosporioides were collected at 0, 6, 12, 24, and 48 h after the polysaccharide treatment, the enzyme activities measured, and the severity of the fungal disease evaluated. SPLSAp induced more prominently POX, βGLU, and CHI activity than PLSAp. Importantly, the combined treatment of SPLSAp and C. gloeosporioides was even more effective as enzyme inductor than SPLSAp alone, and the necrotic lesions of the anthracnose disease caused by the fungus were much less severe compared to control plants. The results indicate that SPLSAp induces plant defense proteins and increases the cowpea tolerance to C. gloeosporioides. These findings indicate that SPLSAp merits further investigation as a promising alternative or a supplemental environmentally friendly protector agent against anthracnose. [ABSTRACT FROM AUTHOR]

Abstract Correlations between the transcriptional responses of genes that encode superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and peroxiredoxin (Prx) enzymes and Colletotrichum gloeosporioides development in cowpea leaves were assessed. Each of these genes is involved in the redox metabolism and hydrogen peroxide balance. Although electron microscopy revealed that conidia adhered to and germinated on the leaf cuticle, the inoculated cowpea leaves did not show any characteristic anthracnose symptoms. The adhered and germinated conidia showed irregular surfaces and did not develop further. This was apparently due to increased leaf H 2 O 2 levels in response to inoculation with C. gloeosporioides. During the early stages post inoculation, cowpea leaves elevated the H 2 O 2 content and modulated the defense gene expression, as well as associated pathways. During the later stages, the increased expression of the CuZnSODI and CuZnSODII genes suggested an active superoxide dismutation to further elevate H 2 O 2 levels, which indicated that higher H 2 O 2 content may function as a toxic agent that kills the fungus. The second increase in H 2 O 2 production above the threshold level was correlated with the expression of the APXI , CATI , CATII , PrxIIBCD , and PrxIIE genes, which resulted in a coordinated pattern to establish an appropriate balance between H 2 O 2 generation and scavenging. Therefore, appropriate H 2 O 2 content in cowpea leaves inhibited C. gloeosporioides development and maintained intracellular redox homeostasis to avoid uncontrolled programmed cell death and necrosis in cowpea leaves. [ABSTRACT FROM AUTHOR]

The physiological and biochemical responses of a drought tolerant, virus-susceptible cowpea genotype exposed to drought stress (D), infected by Cowpea severe mosaic virus (CPSMV) (V), and to these two combined stresses (DV), at 2 and 6 days post viral inoculation (DPI), were evaluated. Gas exchange parameters (net photosynthesis, transpiration rate, stomatal conductance, and internal CO 2 partial pressure) were reduced in D and DV at 2 and 6 DPI compared to control plants (C). Photosynthesis was reduced by stomatal and biochemical limitations. Water use efficiency increased at 2 DPI in D, DV, and V, but at 6 DPI only in D and DV compared to C. Photochemical parameters (effective quantum efficiency of photosystem II and electron transport rate) decreased in D and DV compared to C, especially at 6 DPI. The potential quantum efficiency of photosystem II did not change, indicating reversible photoinhibition of photosystem II. In DV, catalase decreased at 2 and 6 DPI, ascorbate peroxidase increased at 2 DPI, but decreased at 6 DPI. Hydrogen peroxide increased at 2 and 6 DPI. Peroxidase increased at 6 DPI and chitinase at 2 and 6 DPI. β-1,3-glucanase decreased in DV at 6 DPI compared to V. Drought increased cowpea susceptibility to CPSMV at 2 DPI, as verified by RT-PCR. However, at 6 DPI, the cowpea plants overcome this effect. Likewise, CPSMV increased the negative effects of drought at 2 DPI, but not at 6 DPI. It was concluded that the responses to combined stresses are not additive and cannot be extrapolated from the study of individual stresses. [ABSTRACT FROM AUTHOR]

Plant-fungus interactions usually generate H 2 O 2 in the infected plant tissue. H 2 O 2 has a direct antimicrobial effect and is involved in the cross-linking of cell walls, signaling, induction of gene expression, hypersensitive cell death and induced systemic acquired resistance. This has raised the hypothesis that H 2 O 2 manipulation by pharmacological compounds could alter the lifestyle of Colletotrichum gloeosporioides during interaction with the BR-3-Tracuateua cowpea genotype. The primary leaves of cowpea were excised, infiltrated with salicylic acid (SA), glucose oxidase + glucose (GO/G), catalase (CAT) or diphenyliodonium chloride (DPI), followed by spore inoculation on the adaxial leaf surface. SA or GO/G-treated plantlets showed increased H 2 O 2 accumulation and lipid peroxidation. The fungus used a subcuticular, intramural necrotrophic strategy, and developed secondary hyphae associated with the quick spread and rapid killing of host cells. However, CAT or DPI-treated leaves exhibited decreased H 2 O 2 concentration and lipid peroxidation and the fungus developed intracellular hemibiotrophic infection with vesicles, in addition to primary and secondary hyphal formation. These results suggest that H 2 O 2 plays an important role in the cowpea ( C. gloeosporioides ) pathosystem given that it affected fungal lifestyle during interaction. [ABSTRACT FROM AUTHOR]

Highlights: [•] Cowpea leaf proteome was studied following challenge with the fungus Colletotrichum gloeosporioides. [•] Up- and down-regulated proteins were identified by 2D-PAGE in association with ESI-TOF/TOF MS/MS and database-searching. [•] Up- and down-regulated proteins were related to metabolism, photosynthesis, response to stress, oxidative burst, defense signaling, and pathogenesis-related proteins. [ABSTRACT FROM AUTHOR]

Plants encode specific class III peroxidases (POX) as large multigene families that are involved in lignification, cell elongation, stress defense, and seed germination. However, many functions associated with these plant enzymes are not fully understood. Here, a class III peroxidase (Vu-RPOX) was purified from cowpea (Vigna unguiculata) roots with 0–90% ammonium sulfate precipitation, chitin column and Resource Q anion-exchange chromatography. Vu-RPOX is a glycoprotein composed by a 58 kDa polypeptide chain and pI of 3.79. Two peptide sequences of Vu-RPOX (RGLDVVNDIKT and RCSTFINRL) generated by Nano ESI-Q-TOF MS/MS analysis showed variable identity with seed coat peroxidase of Glycine max , peroxidase E5 of Vigna radiata , hypothetical peroxidase of Phaseolus vulgaris , and peroxidase E5-like of Vigna unguiculata. Vu-RPOX follows the Michaelis-Menten kinetics, optimal activity at pH 6.0, and was high thermo-stable when incubated up to 75 °C for 90 min. The catalytic activity of Vu-RPOX decreased in the presence of the classic peroxidase inhibitors, but was not inhibited by MgCl 2 , NH 4 Cl, MnCl 2 , CaCl 2 , and NaCl. Vu-RPOX delay the vegetative growth of the phytopathogenic fungi Colletrotrichum gloeosporioides and Fusarium oxysporum. Based on its properties Vu-RPOX is a potentially useful molecule for application in biomedical, biotechnological, industrial, and agricultural areas particularly because it showed high stability when exposed to a wide range of temperatures and performance at pH 4–7 and impairment of mycelial growth of C. gloeosporioides and F. oxysporum. • A peroxidase (Vu-RPOX) was purified using chitin and Ion exchange column. • The enzyme is a glycoprotein with 58 kDa and pI 3.75. • Vu-RPOX has Km 0.120–33.44 mM and Vmax 0.15–0.93 AU/min for different substrates. • The peroxidase showed high thermo and chemical stability under several conditions. • Vu-RPOX has inhibitory activity on phytopathogenic fungi. [ABSTRACT FROM AUTHOR]

Abstract: Background: Peroxiredoxins have diverse functions in cellular defense-signaling pathways. 2-Cys-peroxiredoxins (2-Cys-Prx) reduce H2O2 and alkyl-hydroperoxide. This study describes the purification and characterization of a genuine 2-Cys-Prx from Vigna unguiculata (Vu-2-Cys-Prx). Methods: Vu-2-Cys-Prx was purified from leaves by ammonium sulfate fractionation, chitin affinity and ion exchange chromatography. Results: Vu-2-Cys-Prx reduces H2O2 using NADPH and DTT. Vu-2-Cys-Prx is a 44kDa (SDS-PAGE)/46kDa (exclusion chromatography) protein that appears as a 22kDa molecule under reducing conditions, indicating that it is a homodimer linked intermolecularly by disulfide bonds and has a pI range of 4.56–4.72; its NH2-terminal sequence was similar to 2-Cys-Prx from Phaseolus vulgaris (96%) and Populus tricocarpa (96%). Analysis by ESI-Q-TOF MS/MS showed a molecular mass/pI of 28.622kDa/5.18. Vu-2-Cys-Prx has 8% α-helix, 39% β-sheet, 22% of turns and 31% of unordered forms. Vu-2-Cys-Prx was heat stable, has optimal activity at pH 7.0, and prevented plasmid DNA degradation. Atomic force microscopy shows that Vu-2-Cys-Prx oligomerized in decamers which might be associated with its molecular chaperone activity that prevented denaturation of insulin and citrate synthase. Its cDNA analysis showed that the redox-active Cys52 residue and the amino acids Pro45, Thr49 and Arg128 are conserved as in other 2-Cys-Prx. General significance: The biochemical and molecular features of Vu-2-Cys-Prx are similar to other members of 2-Cys-Prx family. To date, only one publication reported on the purification of native 2-Cys-Prx from leaves and the subsequent analysis by N-terminal Edman sequencing, which is crucial for construction of stromal recombinant 2-Cys-Prx proteins. [Copyright &y& Elsevier]

Cowpea severe mosaic virus (CPSMV) causes significant losses in cowpea ( Vigna unguiculata ) production. In this present study biochemical, physiological, and proteomic analysis were done to identify pathways and defense proteins that are altered during the incompatible interaction between the cowpea genotype BRS-Marataoã and CPSMV. The leaf protein extracts from mock- (MI) and CPSMV-inoculated plantlets (V) were evaluated at 2 and 6 days post-inoculation (DPI). Data support the assumptions that increases in biochemical (high hydrogen peroxide, antioxidant enzymes, and secondary compounds) and physiological responses (high photosynthesis index and chlorophyll content), confirmed by label-free comparative proteomic approach, in which quantitative changes in proteasome proteins, proteins related to photosynthesis, redox homeostasis, regulation factors/RNA processing proteins were observed may be implicated in the resistance of BRS-Marataoã to CPSMV. This pioneering study provides information for the selection of specific pathways and proteins, altered in this incompatible relationship, which could be chosen as targets for detailed studies to advance our understanding of the molecular, physiological, and biochemistry basis of the resistance mechanism of cowpea and design approachs to engineer plants that are more productive. Biological significance This is a pioneering study in which an incompatible relationship between a resistant cowpea and Cowpea severe mosaic virus (CPSMV) was conducted to comparatively evaluate proteomic profiles by Gel-free/label-free methodology and some physiological and biochemical parameters to shed light on how a resistant cowpea cultivar deals with the virus attack. Specific proteins and associated pathways were altered in the cowpea plants challenged with CPSMV and will contribute to our knowledge on the biological process tailored by cowpea in response to CPSMV. [ABSTRACT FROM AUTHOR]