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Synthesis of dehydrogenation polymers of ferulic acid with high specificity by a purified cell-wall peroxidase from French bean (Phaseolus vulgaris L.).

ABSTRACT: A cationic (pI 8.3) wall-bound peroxidase has been purified to homogeneity from suspension-cultured cells of French bean (Phaseolus vulgaris L.). The enzyme was a glycoprotein and its M(r) was 46,000 as determined by SDS/Page and h.p.l.c. gel filtration. It was localized biochemically to microsomes and the cell wall, and the latter subcellular distribution was confirmed by immunogold techniques. The native enzyme showed absorption maxima at 403, 500 and 640 nm, with an RZ (A405/A280) of 3.3. The peroxidase oxidized guaïacol and natural phenolic acids. By desorption-chemical-ionization mass spectrometry the enzyme was found to oxidize the model compound, ferulic acid, into dehydrodiferulic acid. Kinetics studies indicated an apparent Km of 113.3 +/- 22.9 microM and a Vmax of 144 mumol.min-1.nmol-1 of protein at an H2O2 concentration of 100 microM. In comparison with a second French-bean peroxidase (FBP) and horseradish peroxidase, as a model, it acted with a 6-10-fold higher specificity in this capacity. It is a member of the peroxidase superfamily of bacterial, fungal and plant haem proteins by virtue of its highly conserved amino acid sequence within the proximal and distal haem-binding sites. This is good evidence that this particular FBP may function in constructing covalent cross-linkages in the wall during development and response to pathogens.

SUBMITTER: Zimmerlin A

PROVIDER: S-EPMC1138084 | biostudies-other | 1994 May

REPOSITORIES: biostudies-other

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Project description:Common bean (Phaseolus vulgaris L.) is a staple food in Brazil with both nutritional and socioeconomic importance. As an orphan crop, it has not received as much research attention as the commodity crops. Crop losses are strongly related to virus diseases transmitted by the whitefly Bemisia tabaci, one of the most important agricultural pests in the world. The main method of managing whitefly-transmitted viruses has been the application of insecticides to reduce vector populations. Compared to chemical vector control, a more sustainable strategy for managing insect-borne viruses is the development of resistant/tolerant cultivars. RNA interference has been applied to develop plant lines resistant to the whitefly in other species, such as tomato, lettuce and tobacco. Still, no whitefly-resistant plant has been made commercially available to date. Common bean is a recalcitrant species to in vitro regeneration; therefore, stable genetic transformation of this plant has been achieved only at low frequencies (<1%) using particle bombardment. In the present work, two transgenic common bean lines were obtained with an intron-hairpin construct to induce post-transcriptional gene silencing against the B. tabaci vATPase (Bt-vATPase) gene, with stable expression of siRNA. Northern blot analysis revealed the presence of bands of expected size for siRNA in leaf samples of the line Bt-22.5, while in the other line (11.5), the amount of siRNA produced was significantly smaller. Bioassays were conducted with both lines, but only the line Bt-22.5 was associated with significant mortality of adult insects (97% when insects were fed on detached leaves and 59% on the whole plant). The expression of the Bt-vATPase gene was 50% lower (p < 0.05) in insects that fed on the transgenic line Bt-22.5, when compared to non-transgenic controls. The transgenic line did not affect the virus transmission ability of the insects. Moreover, no effect was observed on the reproduction of non-target organisms, such as the black aphid Aphis craccivora, the leafminer Liriomyza sp. and the whitefly parasitoid Encarsia formosa. The results presented here serve as a basis for the development of whitefly-tolerant transgenic elite common bean cultivars, with potential to contribute to the management of the whitefly and virus diseases.

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Synthesis of dehydrogenation polymers of ferulic acid with high specificity by a purified cell-wall peroxidase from French bean (Phaseolus vulgaris L.).

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