Membrane‐bound hydroxylases in elicitor‐treated bean cells

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Abstract
1. Treatment of cell‐suspension cultures of bean (Phaseolus vulgaris cv. Canadian Wonder) with an elicitor preparation heat‐released from the cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum resulted in rapid changes in the activities of two microsomal oxygenases, cinnamic acid 4‐hydroxylase, involved in accumulation of wall‐bound phenolics and phytoalexins, and proline 2‐oxoglutarate dioxygenase (prolylhydroxylase) involved in the post‐translational modification of hydroxyproline‐rich glycoproteins.2. An anti‐(cytochrome P‐450) monoclonal antibody, originally raised against rat cytochrome P‐450 isoform c, has been shown to bind to bean microsomes and recognise in Western blots an Mr‐48 000 polypeptide, which comigrates with a haeme‐containing protein on SDS/polyacrylamide gel analysis and which has been tentatively identified as a cytochrome P‐450 capable of the hydroxylation of cinnamic acid.3. A preparation of proline 2‐oxoglutarate dioxygenase purified to homogeneity was used to immunise rabbits for the production of antiserum. An elicitor‐induced polypeptide of Mr 65000 was identified as prolyl hydroxylase while an antigenically related polypeptide of Mr 60000 was also immunoprecipitated but not induced by elicitor treatment.4. Use of the two antibodies has demonstrated rapid transient increases in the synthesis of the Mr 48000 and Mr 65000 oxygenases in vivo and for mRNAs as measured in in vitro translations, particularly for the putative cytochrome P‐450. These increases slightly precede corresponding changes in the synthesis of the soluble enzyme phenylalanine ammonia‐lyase, in common with which these oxygenases probably share a mechanism of gene activation underlying the increased activities seen in response to elicitor treatment.