Involvement of diamine oxidase and peroxidase in insolubilization of the extracellular matrix: Implications for pea nodule initiation by Rhizobium leguminosarum

Rhizobium leguminosarum colonizes host cells and tissues through infection threads, which are tubular in-growths of the plant cell wall. Monoclonal an tibody MAC265 recognizes a plant matrix glycoprotein (MGP) associated with the lumen of these infection threads. This glycoprotein is also released in soluble form from the root tips of pea seedlings. In the presence of hydro gen peroxide, release of glycoprotein from root tips was not observed. Extr actability from root tips was therefore used as the basis for investigating the peroxide-driven insolubilization of MGP and the possible involvement o f two extracellular enzymes, peroxidase (POD) and diamine oxidase (DAO), wa s investigated. Release of MGP from root tips was enhanced by application o f POD and DAO inhibitors (salicylhy-droxamic acid and o-phenanthroline, res pectively). Furthermore, release of MGP was inhibited by pretreatment of ro ots with putrescine (the substrate of DAO) and also by application of a par tially purified extract of DAO from pea shoots. Following inoculation of pe a roots with R. leguminosarum, elevated levels of DAO transcript were obser ved by reverse transcriptase-polymerase chain reaction (RT-PCR), but these then dropped to a low level from 4 to 10 days post inoculation, rising agai n in more mature nodules, In situ hybridization studies indicated that the bulk of the transcription was associated with the infected tissue in the ce nter of the nodule, On the basis of these observations, we postulate that D AO may be involved in the peroxide-driven hardening of MGP in the lumen of infection threads and in the intercellular matrix.