Polifenoloxidase como fator de resistencia da soja a nematoides e na oxidação do palmito

AUTOR(ES)
DATA DE PUBLICAÇÃO

2004

RESUMO

Most of the studies on polyphenol oxidase (PPO) are concerned with its participation in the response of plants to insect attack and its importance in food quality. The first chapter of this thesis presents the study of PPO from roots of two soybean (Glycine max) varieties differing in their resistance to nematodes. The aim was to study the role of PPO at the time of inoculation and consequently analyses were carried out at 6, 12, 24, 48 and 72 h after inoculation. Roots of Cristalina (susceptible) and Hartwig (resistant) were subjected to mechanical damage, treated with methyl jasmonate and inoculated with the nematodes Meloidogyne javanica and Heterodera glycines. Initial tests showed that the presence of triton X-100 in the extraction buffer increased the efficiency of PPO extraction. Sodium dodecylsufate (SDS) did not affect the enzyme activity and chlorogenic acid was the best substrate. Plants subjected to mechanical damage and treated with methyl jasmonate showed an increase of PPO activity, mainly in Hartwig. This variety also showed the highest activity following nematode inoculation. Analyses of phenolic compounds did not show quantitative or qualitative differences among the treatments. Using degenerate primers and PCR we isolated five clones of PPO from soybean root, but only two (JH1 and JH2) were used for Southern blot analysis. Probes made from these clones indicated 4 genes coding for PPO in soybean. Only JH1 showed an increase of PPO transcription with semi-quantitative RT-PCR based expression analysis. The results suggest that different stimuli might induce transcription of different PPO genes in soybean. Cristalina treated with methyljasmonate showed an increase of resistance to both nematodes, while no changes were observed with Hartwig. In the second chapter of this thesis PPO was studied in palm plants which differ in the oxidation of the extracted heart of palm (palmito). Analyses of PPO in young and mature leaves, roots and heart of palm showed that activity was higher in juçara (Euterpe edulis) and açaizeiro (Euterpe oleraceae). In general, the activity was higher in young tissues. Enzyme assay showed chlorogenic acid (CGA) as the best substrate and the best temperature reaction was 25ºC for açaízeiro and pupunha (Bactris gassipae) and 30ºC for juçara. The best activities of PPO were between pH 5.5 and 6.0. PPO was inhibited by specific inhibitors, such as salicylhydroxamic acid, cetyltrimethylammonium bromide and tropolone. Ki s were determined for salicylhydroxamic acid (1,96 µM for açaí and 2.82 µM for juçara) and tropolone (1.25 µM for açaí and 6.55 µM for juçara) with the PPOs from juçara and açaizeiro, respectively. SDS in the reaction medium led to the loss of activity of the enzyme. PPO of juçara and açaizeiro were relatively resistant to the temperature. The Km for chlorogenic acid was lower for pupunheira (0.53 mM), followed by juçara (1.19 mM) and açaizeiro (1.917 mM). Analyses of total soluble phenols and chlorogenic acid showed that pupunha has a considerable amount of phenols but a siginificantly lower amount of chlorogenic acid when compared with the other two palms. "Tissue printings" of cut stems of pupunha showed an absence of oxidation indicating that low activity of PPO and not low phenol was responsible two PPO clones were isolated, one from heart of palm of juçara (PPOEd17) and other from açaizeiro (PPOAc45). We were not able to isolate any clone from pupunha. These clones had low similarity (52%) and they were used as probes in Southern blot analysis. The results indicated that only one PPO gene was present in the three palms. Using semi-quantitative RTPCR based expression analysis it was possible to detect significant PPO transcription only in the heart of palm of juçara and açaizeiro, confirming that that low the oxidation observed in pupunha is due to low expression of the gene coding for PPO. However, the low chlorogenic acid content cannot be excluded as another factor contributing to the lower oxidation in this palm, since other phenols present would not be as good substrates as CGA.

ASSUNTO(S)

soja oxidação nematoides fenois palmito

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