Immunocytochemical localization of phenolic compounds in pollen walls using antibodies againstp-coumaric acid coupled to bovine serum albumin

Summary Two different antibodies against bovine serum albumin (BSA)-p-coumaric acid-conjugates were produced and used to localize phenolic compounds in exines of pollen from different species,p-Coumaric acid (pC) was coupled to BSA either via the carboxy group (BSA-pC) or directly to the aromatic ring system (BSA-azopC). The polyclonal antibodies raised in rabbits were characterized by ELISA with homologous and heterologous antigens using turkey ovalbumin as carrier protein. The results showed that the two immune sera directed against BSA-pC and BSA-azo-pC, respectively, were specific forp-coumaric acid and structurally similar compounds. Only a very poor binding by acetic acid-ovalbumin-conjugates and no binding by turkey ovalbumin was detectable. The antibodies reacted with partially purified pollen walls and with highly purified exines. The intensity of the immune reaction was proved to be dependent upon the pollen source and the preparation of the pollen walls. Using light and electron microscopy, it was shown for the first time that, in the exines ofCucurbita maxima, antibody binding was predominantly observed in the region of the germ pore apertures, the outer foot layers, and in the micro- and macrospines. We conclude from this and other earlier published data that phenols are important structural compounds of sporopollenin.Abbrevations AA acetic acid - BA benzoic acid - BSA bovine serum albumin - BSA-azo-pC p-coumaric acid coupled in meta position to BSA by a diazo reaction - BSA-azo-pC I immune serum against BSA-azo-pC - BSA-pC p-coumaric acid coupled to BSA via the COOH-group - BSA-pC I immune serum against BSA-pC - FA ferulic acid - OVA ovalbunin from turkey - pC p-coumaric acid - pHY p-hydroxybenzoic acid - SA sinapic acid - SYA syringic acid - TAT TBS-azide-Tween-buffer - TFA trifluoroacetic acid - VA vanillic acid