Atividade ribonucleásica de Vigna unguiculata (L.) Walp durante a germinação e estabelecimento da plântula: distribuição nos órgãos, propriedades cinéticas e efeitos da salinidade na ativação

Abstract

In this work, Pitiúba cowpea [Vigna unguiculata (L.) Walp] seeds were sown in distilled water (control treatment) or in 0.1M (salt treatment). The results showed that salinity inhibited growth and cotyledon reserve mobilization to others seedling parts. The study on salinity effect on total and specific RNase activities in cotyledon extract showed that although activities increased during the first days after sowing, saline stress reduced the specific RNase activity and caused a delay in the total activity. The protein extract content showed that reserve mobilization was strongly decreased by salinity, mainly on the 3rd and 5th days after sowing although in the last experimental day these values were the same for control and salt-stressed seedlings. To carry out the immunological studies, RNase was purified from the crude extract from cotyledons of seedlings (5 days after sowing). The enzyme was purified by extract precipitation using ammonium sulfate (fraction F45-75), following purification through CM-Celulose (fraction FcM), DEAESephadex A-25 (fraction FDEAE) and Sephadex G-50 (fraction FG50) columns. The latter fraction was submitted to a SDS-polyacrylamide gel electrophoresis and showed a unique band with molecular mass of 16.3 kDa. For specific RNase antibodies production, "swiss" mice were immunized with FGso fraction extracted from polyacrylamide gel after SDSelectrophoresis. The ELISA test showed that RNase is also present in cotyledon of quiescent seeds as an inactive form (or low activity) and it is activated during germination and seedling establishment. So, it indicates that salinity reduces RNase activity due to enzyme activation and not through de novo synthesis. The ELISA tests also showed that roots, stem and leaves RNases are similar to the cotyledons from an immunological point of view and these results were confirmed by the passive cutaneous anaphylaxis (PCA). The study concerning linear DNA hydrolytic capacity of F45-75 fractions from several seedling parts (roots, stem, leaves and cotyledons) was evaluated through measurements of the hydrolysis product absorbance at 260 nm and results showed a lower DNase activity (as compared to RNase activity) and only traces of this activity in the FcM. Nevertheless, using the electrophoresis agarose gel to detect DNA or hydrolysis product, DNase activity was clearly detected in FcM fractions from the several parts of the seedling. In the general, the DNase activity of these fractions was inhibited by EDTA and stimulated by MgC12, mainly those in cotyledons, suggesting the existence of nucleases or a mixture of RNase and nucleases and/or DNases in these samples. The most purified fraction of cotyledonary enzyme (FGso) also showed DNase activity, being in reality, a nuclease and not an RNase. This enzyme was inhibited by EDTA and by Cue+, Hg2+ and Zn2+ ions, but stimulated by MgCl2. The DNase activity from the FGso fraction was inhibited by increasing concentrations of RNA in the reaction mixture, suggesting the existence of a competition between RNA and DNA by the enzyme active center. Indeed, the fact of FGso fraction showed circular DNA (plasmid) hydrolytic capacity, it demonstrates that enzyme is specifically an endonuclease. Therefore, a purified cotyledon RNase from Vigna unguiculata is in reality a nuclease that shows a higher affinity by RNA than DNA.