1.9.38
RELEASE OF GENISTEIN BY AN ISOFLAVONE-SPECIFIC GLUCOSIDASE ACTIVATES A PEROXIDASE-LIKE NADH OXIDASE WHICH TRIGGERS THE DEFENSE COMPETENCY OF SOYBEAN CELLS

TL GRAHAM, MY GRAHAM, AR ROSE and RS POLING

Department of Plant Pathology, Ohio State University, Columbus, OH, 4-J210, USA

Background and objectives
Soybean defense responses to the glucan elicitor from Phytophthora sojae include both proximal and distal cell reactions. Proximal cell reactions occur in cells immediately adjacent to wounded or hypersensitively dying (HR) cells and include the release of the isoflavones daidzein and genistein from their preformed 6"-malonyl-7-0-glucosides, phenolic polymer deposition and phytoalexin (glyceollin) accumulation. Distal cell reactions occur in cells as far as 30 cells beyond the proximal cells and include a massive buildup of newly synthesized isoflavone conjugates. Proximal cell responses to the glucan require preconditioning of cells by factors from dying cells which are termed competency factors [1]. Two competency factors, CF- 1 and CF-2 can be separated and differentially enable the phenolic polymer and glyceollin responses to elicitor, respectively. Although CF- 1 is currently being purified and characterized, CF-2 is unstable to fractionation. However, we have learned that events which mimic HR cell death, such as shifts in transmembrane ion flux or redox status can trigger CF-2 competency. In particular, tetrazolium redox dyes of relatively low potential can clamp cells into the CF-2 state. The target for these dyes appears to be a plasma membrane-associated peroxidase functioning as an NADH oxidase (Nox 11). The oxidation of NADH by Nox 11 generates superoxide which inactivates the enzyme. Specific phenolic molecules, such as ferulic acid, catalyze the dismutation of superoxide and regeneration of the active peroxidase, allowing the continuation of the cycle and the concomitant generation of large quantities of hydrogen peroxide, which may subsequently be involved in phenolic polymer formation. Here we compare the effects of various soybean flavonoids on soybean Nox II activity and report the highly specific activation of the enzyme by the isoflavone genistein. We also report the distribution of Nox 11 in various soybean tissues and compare this to the distribution of the isoflavone-specific glucosidase [2] which releases genistein from its pre-formed conjugates.

Results and discussion
The major phenolic metabolises present in soybean tissues are the isoflavones genistein and daidzein and the flavonols kaempferol, quercetin and isorhamnetin. Genistein was of particular interest to us because the net hydrolysis of genistein conjugates in elicited tissues shows a very strong correlation to glyceollin accumulation, despite the fact that genistein is not a glyceollin precursor. We thus compared the effects of the various soybean flavonoids on Nox II with that of the standard activator of the enzyme, ferulic acid, over a concentration range of luM - 2 mM. Half maximal reaction rates of soybean Nox 11 were seen at 1 mM NADH with 1 mM ferulic acid, consistent with established values in the literature. In contrast, comparable reaction rates of Nox 11 were seen at less than 100 uM NADH with 100 uM genistein and maximal reaction rates over 10 fold those seen with ferulate could be achieved. Other soybean flavonoids were inactive as effectors of Nox 11, as was biochanin A, a very close structural analog of genistein. Thus, genistein is a highly specific and unusually potent endogenous activator of soybean Nox 11. Genistein is released during infection or elicitation of glyceollin from apoplastic preformed conjugates through the action of a highly specific extracellular 7-0-isoflavone glucosidase (ISG,[2]). Thus, we compared the distributions of Nox 11 and ISG in soybean seedlings. The enzymes showed similar distributions, being localized predominantly in the root tip and hypocotyl hook of etiolated seedlings, the same tissues which show race-specific resistance to P. sojae. These findings suggest that release of genistein from preformed conjugates may play a key regulatory role in the activation of Nox 11 and establishment of competency for soybean defense responses.

References
1. Graham MY, Graham, TL 1994. Plant Physiology 105, 571-578.
2. Hsieh MC, 1997. Ph.D. Thesis, Ohio State University.