Hydrogen peroxide generated by copper amine oxidase involved in adventitious root formation in mung bean hypocotyl cuttings

The role of hydrogen peroxide (H2O2) generated by copper-containing diamine oxidases (CuAO) in adventitious rooting in mung bean hypocotyl was studied. The physiological data show that aminoguanidine (AG), an irreversible inhibitor of CuAO, inhibited adventitious rooting significantly. Among major catalysed and metabolised products or substrates of CuAO, only H2O2 obviously induced adventitious rooting and notably reversed the inhibitory effect of AG on adventitious rooting. The spatio-temporal change of CuAO activity in the rooting region of hypocotyls was histochemically measured using a peroxidase-coupled assay with the artificial substrate 3,3-diaminobenzidine tetrahydrochloride (DAB) as chromogen. DAB staining was detected in the parenchyma tissue between the vascular bundles, where roots originated before the emergence of root primordium, and then in the forming root primordium. With the origination of adventitious roots, the density and area of DAB staining increased rapidly. Most cells in root primodium had an evidently DAB staining, and strong staining mostly distributed in root meristem. H2O2, which was detected with the H2O2-sensitive probe 2,7-dichlorofluorescein diacetate, had a similar change to CuAO activity in this process. AG largely suppressed DAB staining and H2O2 fluorescence, and prevented root primordium formation. Taken together, these results clearly prove that, CuAO is responsible for H2O2 production during adventitious rooting, and H2O2 generated by CuAO is required for adventitious rooting in mung bean hypocotyls.

Bean dwarf mosaic virus BV1 Protein Is a Determinant of the Hypersensitive Response and Avirulence in Phaseolus vulgaris

The capacities of the begomoviruses Bean dwarf mosaic virus (BDMV) and Bean golden yellow mosaic virus (BGYMV) to differentially infect certain common bean (Phaseolus vulgaris) cultivars were used to identify viral determinants of the hypersensitive response (HR) and avirulence (avr) in BDMV. A series of hybrid DNA-B components, containing BDMV and BGYMV sequences, was constructed and coinoculated with BDMV DNA-A (BDMV-A) or BDMVA-green florescent protein into seedlings of cv. Topcrop (susceptible to BDMV and BGYMV) and the BDMV-resistant cvs. Othello and Black Turtle Soup T-39 (BTS). The BDMV avr determinant, in bean hypocotyl tissue, was mapped to the BDMV BV1 open reading frame and, most likely, to the BV1 protein. The BV1 also was identified as the determinant of the HR in Othello. However, the HR was not required for resistance in Othello nor was it associated with BDMV resistance in BTS. BDMV BV1, a nuclear shuttle protein that mediates viral DNA export from the nucleus, represents a new class of viral avr determinant. These results are discussed in terms of the relationship between the HR and resistance.

Purification and Characterization of a Polygalacturonase-inhibiting Protein from Grapefruit Flavedo

Polygalacturonase-inhibiting proteins (PGIPs) are believed to be one component of plants inherent defense mechanisms against fungal pathogens. We have purified a PGIP from mature grapefruit (Citrus paradisi cv. Marsh) flavedo using ammonium sulfate precipitation, preparative isoelectric focusing and ion exchange chromatography. Two peaks of PGIP activity were separated by isoelectric focusing, one at pH 6–7 and one at pH 9–10. The basic protein was more abundant than the neutral protein and was selected for further purification. The basic protein binds to S Sepharose at pH 6.1 and has an apparent Mr of ≈43,000 based on SDS-PAGE analysis. The protein is glycosylated as revealed by binding to ConA sepharose and is serologically similar to PGIPs from bean hypocotyl and pear fruit. Two dimensional PAGE analysis revealed the presence of two bands of similar Mr but with slightly different pIs (≈9.0–9.5). The N-terminal amino acid sequence of grapefruit PGIP shows high homology with PGIPs from fruit of other species and with a cDNA clone of PGIP that was isolated from a Citrus sinensis cv. Hamlin expression library. Grapefruit PGIP inhibits polygalacturonases from Aspergillus niger, and the citrus pathogen Penicillium italicum. We are interested in the role of PGIP in resistance of citrus fruit to postharvest decay fungi.