Glycosylation of DIMBOA by tissue-culture-isolated enzyme

In an associated note it is reported that DIMBOA is produced in maize callus. For the majority of callus the breakdown product of DIMBOA, MBOA, was found at higher concentrations. This abnormal occurrence is unexplained. One possible explanation is that once DIMBOA is synthesized it is not properly handled by the cell. DIMBOA normally exists in the cell as a glucoside which is relatively stable. The question then arose whether the enzyme responsible for glycosylating DIMBOA might be inactive in tissue culture resulting in the observed breakdown of the aglycone to MBOA. The present study concerns the identification of glucosyltransferase activity capable of adding glucose to the carbon 2 position of DIMBOA using callus tissue.

The conditions for callus culture are reported in the associated report. Enzyme was extracted from the C131A and Oh43 callus using 2ml of buffer (50mM HEPES, 2mM DTE, pH 7.5) from which the particulates were removed by centrifugation for 20' at 26,000 X G. The assay conditions included 20µg protein, 372µM DIMBOA, 1mM UDPG and 7mM CaCl2 in 200µl extraction buffer (pH 8.2). The assays were incubated at 37C for 5' and reactions were terminated by addition of 0.8ml of a 2:1 chloroform: methanol solution (1% HCl). DIMBOA glucoside produced in each assay was identified by HPLC methods and quantitated using standard curves based on peak height. The specific activity (nm/mg protein/min) was then determined for each assay.

The enzyme isolated from callus did include a significant amount of glucosyltransferase capable of adding glucose to DIMBOA. Specific activities of 84.5 and 123 were observed for C131A and Oh43 callus respectively. The enzyme activity obtained from callus is similar to levels found in seedling tissue (manuscript in preparation). The success found in identifying DIMBOA glucosyltransferase activity suggests that other enzymes in the pathway for DIMBOA production may be studied using callus. These results also suggest that the breakdown of DIMBOA to MBOA in callus is not due to the failure of glucosyltransferase to protect the aglycone by glycosylation.

B.A. Bailey, J. Bussard and R.L. Larson

Please Note: Notes submitted to the Maize Genetics Cooperation Newsletter may be cited only with consent of the authors.

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