Submitted to: Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/6/1999
Publication Date: N/A
Citation: Interpretive Summary: Over 245,000 bales of cotton were lost to wilt diseases by US producers in 1998. To reduce losses to these pathogens, we are attempting to increase the plant's natural resistance. One method to increase resistance is to increase the potency of the compounds that the plant already makes to fight off these pathogens. We found that the potency of one of the defense compounds is reduced by a process called methylation. Our long-range goal is to "shut down" the methylation gene in cotton. To this end, we now report the characterization and purification of the enzyme that controls the methylation process. We will use this information to identify the gene that codes for this enzyme.
Technical Abstract: A SAM dependent O-methyltranferase, S-adenosyl-L-methionine: desoxyhemigossypol-6-O-methyltransferase (dHG-6-OMT), which specifically methylated the dHG to form dMHG, was purified to homogeneity from the cotton stele tissue inoculated with Verticillium dahliae by an anion exchange, a gel filtration, and three affinity columns. An overall 851 fold purification has been achieved. The purified enzyme showed a single band at 41.2 kDa. on SDS-PAGE. It had a native molecular weight of 81.4 kDa. and consisted of two identical subunits. The enzyme exhibited high substrate specificity. For instance, hemigossypol, 2,7-dihydroxycadalene, caffeic acid, 4-methylcatechol, and 2,3-dihydroxynaphthalene were not active as substrates for the enzyme. Mg2+ ion is not required for the enzyme activity. Thiol group blocking reagent, p-chloromercuribenzoate, inhibited the enzyme activity by 30% at 1 mM and 98% at 10 mM concentrations, indicating possible involvement of thiol groups in the enzyme¿s active center. Substrate-saturation kinetic data were obtained with the dHG OMT preparations purified to homogeneity, and were typical Michaelis-Menten type. A km of 4.6 micro M and a km/kcat of 5.08 x 104 s-1 (mol/L)-1 were determined for dHG and a km of 81.4 micro M and a km/kcat of 1.83 x 103 s-1 (mol/L)¿1 were determined for SAM. The enzyme showed strong affinity toward dHG.