Maria da Guia Silva Lima

Salt modulation of vacuolar H+-ATPase and H+-Pyrophosphatase activities in Vigna unguiculata

Summary Salt modulation of the tonoplast H + -pumping V-ATPase and H + -PPase was evaluated in hypocotyls of Vigna unguiculata seedlings after 3 and 7 days of treatment. In 3-day-old seedlings, treatment with 100 mmol/L NaCl decreased the proton transport and hydrolytic activities of both the V-ATPase and the H + -PPase. After 7 days, the proton transport and hydrolysis activities of the V-ATPase were higher, while the H + -PPase activities were lower in seedlings. Western blot analysis of A- and B-subunits of V-ATPase revealed that the protein content of the two subunits varied in parallel with their activities, i.e. to a higher activity corresponded a higher protein content of the subunits and vice versa. Contrarily, Western blot analysis of H + -PPase levels failed to show any correlation with PPase activity, suggesting a partial enzyme inactivation. The results indicate that salt stress induces V-ATPase expression in V. unguiculata with concomitant enhancement of its activity as a homeostatic mechanism to cope with salt stress. Under the same conditions PPase is inhibited.

Stress-induced co-expression of two alternative oxidase (VuAox1 and 2b) genes in Vigna unguiculata

Cowpea (Vigna unguiculata) alternative oxidase is encoded by a small multigene family (Aox1, 2a and 2b) that is orthologous to the soybean Aox family. Like most of the identified Aox genes in plants, VuAox1 and VuAox2 consist of 4 exons interrupted by 3 introns. Alignment of the orthologous Aox genes revealed high identity of exons and intron variability, which is more prevalent in Aox1. In order to determine Aox gene expression in V. unguiculata, a steady-state analysis of transcripts involved in seed development (flowers, pods and dry seeds) and germination (soaked seeds) was performed and systemic co-expression of VuAox1 and VuAox2b was observed during germination. The analysis of Aox transcripts in leaves from seedlings under different stress conditions (cold, PEG, salicylate and H2O2 revealed stress-induced co-expression of both VuAox genes. Transcripts of VuAox2a and 2b were detected in all control seedlings, which was not the case for VuAox1 mRNA. Estimation of the primary transcript lengths of V. unguiculata and soybean Aox genes showed an intron length reduction for VuAox1 and 2b, suggesting that the two genes have converged in transcribed sequence length. Indeed, a bioinformatics analysis of VuAox1 and 2b promoters revealed a conserved region related to a cis-element that is responsive to oxidative stress. Taken together, the data provide evidence for co-expression of Aox1 and Aox2b in response to stress and also during the early phase of seed germination. The dual nature of VuAox2b expression (constitutive and induced) suggests that the constitutive Aox2b gene of V. unguiculata has acquired inducible regulatory elements.

Allergenicity and tolerance to proteins from brazil nut (Bertholletia excelsa H. B. K.)

Allergenicity to Brazil nut protein fractions and to a purified methionine‐rich 2S protein, considered to be similar to the allergens of other oilseeds, has been followed through IgG1 and IgE synthesis after mouse subcutaneous immunization. The allergenic capacities of all protein fractions and of the 2S protein are not as prominent. Oral intake of Brazil nuts in several doses was also tested in mice and rats as a possible route of immunization, but instead of immunization, a systemic tolerance was induced. This state of immunological tolerance was more efficiently induced by a scheme of multiple feeding than by a single feeding.

Differential responses of ribulose-1,5-bisphosphate carboxylase/oxygenase activities of two Vigna unguiculata cultivars to salt stress

Vita 3 and Vita 5 are two Vigna unguiculata cultivars that differ in their capacities for survival in saline environments; Vita 3 is more tolerant and Vita 5 more sensitive. Both cultivars were submitted to salt stress with 0.1 M NaCl. After 8 days, root and shoot growth from both cultivars was reduced but reduction was more pronounced in Vita 5. Furthermore, leaf area was also reduced in this cultivar. Chlorophyll content and chlorophyll fluorescence parameters were not affected by salt stress, but the specific activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) decreased in Vita 3 and increased in Vita 5. The use of immunological techniques also revealed that the Rubisco content from Vita 3 decreased while that of Vita 5 increased. The discussion of these results is aimed at reaching a better understanding of the differences between these cultivars in relation to salt stress.

Effect of pH on CN-resistant respiratory activity and regulation on Vigna uniguiculata mitochondria

Abstract Mitochondria from Vigna unguiculata cv. Vita 5 have a cyanide insensitive alternative oxidase cross-reacting with a monoclonal antibody raised against the alternative oxidase of Sauromatum guttatum. In the presence of NADH as substrate and dithiothreitol, the CN-resistant respiration of V. unguiculata intact mitochondria was found to be significantly influenced by assay pH with an optimum value of 6.25. This effect was still observed when nigericin, known to abolish ΔpH between the matrix and the intermembrane space, was present in the reaction medium. This pH effect was shown to be reversible. Alternative oxidase activation by pyruvate, also appeared to be pH dependent with an optimum pH value of 7.25. This modulation of the alternative oxidase activity by pH has been tentatively attributed to a new protonated-deprotonated status of this protein.

Effects of Mg2+, Na+ and K+ on the F1ATPase of mitochondria of Vigna sinensis (L.) cv. seridó

Abstract 1. 1. Mitochondria from the serido bean Vigna sinensis have an ATPase which is inhibited by oligomycin. 2. 2. Mg 2+ does not activate the ATPase, but is essential for forming the substrate MgATP – . 3. 3. Na + and K + function as activators of the ATPase in intact and lyzed mitochondria. Different to Na + , K + has a more pronounced active effect on the ATPase in lyzed mitochondria than in intact mitochondria. 4. 4. MgATP 2- is able to penetrate the membranes of the mitochondria.