--Anita S. Klein and Carol M. O'Brien
Cassab and Varner developed an elegant cytochemical method called tissue-printing to examine localization of the glycoprotein extensin (J. Cell Biol. 105:2581, 1987). We attempted to adapt this technique to localize the Bronze protein and by chance discovered endogenous alkaline phosphatase activity in the embryo and aleurone of maize kernels.
In Cassab and Varner's tissue-printing method, free-hand tissue sections are blotted onto nitrocellulose. The 'print' is dried and extensin is detected as in a typical Western blot procedure: the nitrocellulose is blocked with gelatin or bovine serum albumin, incubated with rabbit anti-extensin antibodies and subsequently alkaline phosphatase [AP] conjugated goat-anti-rabbit IgG. Chromogenic substrates for AP are used to visualize the antibody binding sites on the blot. Cassab and Varner report that the soybean tissues in their study lacked endogenous AP activity. Controls in which the blots were not treated with primary antisera or those treated with preimmune sera did not stain positive for AP activity. Given the availability of antibodies to other plant proteins, tissue-printing should have general applicability to immunolocalization of those proteins.
We have prepared an anti-peptide antiserum against the N-terminal 12 amino acids of UDPglucose flavonol glucosyl transferase (UFGT), the Bronze gene product (Klein, Miller, Labonte and Laudano, unpublished data). The peptide was conjugated to bovine serum albumin [BSA] to facilitate recognition of the antigen; therefore the antiserum also reacts strongly with BSA.
Mature kernels (sh Bz-McC, R-r) were hydrated for 10-30 minutes in distilled deionized water and sectioned in both horizontal and longitudinal directions. Tissue prints were prepared according to the method of Cassab and Varner and processed by the Bio-Rad immuno-blot method. AP-conjugated goat anti-rabbit IgG was purchased from Bio-Rad Laboratories. Binding of the second antibody was detected using Fast Red/Naphthol Phosphate (FR/NP) color development as specified by the manufacturer. Dot blots of a dilution series of BSA (25ug to 25pg) were treated in a similar fashion to monitor the sensitivity of the assay. The antisera reacted strongly with the BSA, for which the most dilute dot (25pg) stained a pink-red color.
In preliminary experiments with antisera treated blots, both the embryo and aleurone layer stained positive for AP. However control blots treated with preimmune sera also stained positively for alkaline phosphatase. New prints were prepared, blocked with gelatin and incubated directly with the FR/NP reagents. Again the embryo and the aleurone stained positive for AP activity. In other experiments prints were prepared from nonshrunken kernels, Sh, blocked and treated directly with the FR/NP reagents. In these prints the Sh endosperm also stained positive, albeit faintly, for AP activity.
An attempt was made to inactivate the endogenous AP activity so that the goat-anti-rabbit alkaline phosphatase IgG could be used with the tissue-printing method. Control prints were autoclaved to denature protein and treated directly with the FR/NP reagent. Autoclaving reduced but did not eliminate endogenous AP activity on the control prints. Autoclaving did, however, substantially reduce the sensitivity of detection of BSA in the dot blot assay.
We have observed that both the embryo and aleurone of maize kernels
contain alkaline phosphatase activity and this AP is stable to the processes
of seed desiccation and maturation and is heat-resistant. For kernels,
another type of enzyme-linked second antibody for immuno-blot development
may be more suitable for immunolocalization via the tissue-printing method.
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