Cell suspension cultures of parsley (Petroselinum crispum) have previously been used as a suitable system for studies of the nonhost resistance response to Phytophthora sojae. In this study, we replaced the penetrating fungus by local mechanical stimulation by using a needle of the same diameter as a fungal hypha, by local application of a structurally defined fungus-derived elicitor, or by a combination of the two stimuli. Similar to the fungal infection hypha, the local mechanical stimulus alone induced the translocation of cytoplasm and nucleus to the site of stimulation, the generation of intracellular reactive oxygen intermediates (ROI), and the expression of some, but not all, elicitor-responsive genes. When the elicitor was applied locally to the cell surface without mechanical stimulation, intracellular ROI also accumulated rapidly, but morphological changes were not detected. A combination of the mechanical stimulus with simultaneous application of low doses of elicitor closely simulated early reactions to fungal infection, including cytoplasmic aggregation, nuclear migration, and ROI accumulation. By contrast, cytoplasmic rearrangements were impaired at high elicitor concentrations. Neither papilla formation nor hypersensitive cell death occurred under the conditions tested. These results suggest that mechanical stimulation by the invading fungus is responsible for the observed intracellular rearrangements and may trigger some of the previously demonstrated changes in the activity of elicitor-responsive genes, whereas chemical stimulation is required for additional biochemical processes. As yet unidentified signals may be involved in papilla formation and hypersensitive cell death.
A protein consisting of 60 kDa subunits (As-P60) was isolated from etiolated oat seedlings (Avena sativa L.) and characterized as avenacosidase, a beta-glucosidase that belongs to a preformed defence system of oat against fungal infection. The enzyme is highly aggregated; it consists of 300-350 kDa aggregates and multimers thereof. Dissociation by freezing/thawing leads to complete loss of enzyme activity. The specificity of the enzyme was investigated with para-nitrophenyl derivatives which serve as substrates, in decreasing order beta-fucoside, beta-glucoside, beta-galactoside, beta-xyloside. The corresponding orthonitrophenyl glycosides are less well accepted. No hydrolysis was found with alpha-glycosides and beta-thioglucoside. An anti-As-P60 antiserum was prepared and used for isolation of a cDNA clone coding for As-P60. A presequence of 55 amino acid residues was deduced from comparison of the cDNA sequence with the N-terminal sequence determined by Edman degradation of the mature protein. The presequence has the characteristics of a stroma-directing signal peptide; localization of As-P60 in plastids of oat seedlings was confirmed by western blotting. The amino acid sequence revealed significant homology (> 39% sequence identity) to beta-glucosidases that are constituents of a defence mechanism in dicotyledonous plants. 34% sequence identity was even found with mammalian and bacterial beta-glucosidases of the BGA family. Avenacosidase extends the occurrence of this family of beta-glucosidases to monocotyledonous plants.
The esterification kinetics of chlorophyllide, obtained by a single flash of light, were investigated in etiolated barley (Hordeum vulgare L.) and oat (Avena sativa L.) leaves. A rapid phase, leading to esterification of 15% of total chlorophyllide within 15-30 s, was followed by a lag-phase of nearly 2 min and a subsequent main phase, leading to esterification of 85% of total chlorophyllide within 30-60 min. The presence of additional protochlorophyllide, produced in the leaves by incubation with 5-aminolevulinate, did not change the esterification kinetics. The rapid phase was identical after partial (11-15%) and full (>80%) photoconversion of protochlorophyllide; the ability for a second rapid esterification phase was restored in a dark period of at least 10 min. Cooling the leaves to 0°C abolished the esterification of the main phase while the rapid phase remained unchanged. The prolamellar bodies were already in part transformed into prothylakoid-like structures within 2-5 min after a full flash but not after a weak flash (11% photoconversion); in the latter case, the corresponding transformation required a dark period of about 45 min. The existence of subcomplexes of prolamellar bodies containing NADPH:protochlorophyllide oxidoreductase and chlorophyll synthase in the ratio 7:1 is discussed.
Summary.We have found 5 profilin cDNAs in cultured parsley cells, representing a small gene family of about 5 members in parsley. Specific antibodies were produced using heterologously expressed parsley profilin as antigen. Western blot analysis revealed the occurrence of similar amounts of profilin in roots and green parts of parsley plants. Immunocytochemical staining of parsley cells infected with the oomycetous plant pathogen Phytophthora infestans clearly revealed that profilin accumulates at the site on the plasma membrane subtending the oomycetous appressorium, where the actin cables focus. We also observed the accumulation of Rop GTPases around this site, which might point to a potential function in signaling to the cytoskeleton.
A 60 kDa protein (P60) co-purified with phytochrome was identified as avenacosidase, a fl-glucosidase which is part of the defense system of Arena sativa. An antiserum raised against P60 was used to isolate a eDNA done coding for the complete amino acid sequence of P60. The eDNA-derived amino acid sequence contained the partial sequences described before for a protein kinase [(1989) Planta 178, 199-206] and for a TCPl-related molecular chaperone [(1993) Nature 363, 644-647] co-purified with phytochrome. We conclude that these activities were related to minor contaminants and that only sequences of avenacosidase had been obtained.
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