Cytokinin oxidase activity and cytokinin content in roots of sunflower under water stress.

Contents of trans-zeatin riboside (ZR), dihydrozeatin riboside (DZR) and N6-(delta2-isopentenyl) adenosine (iPA) was quantified by an indirect ELISA using polyclonal antibodies, in the roots, xylem sap and leaves of pot grown sunflower plants subjected to water stress (RWC of leaves approximately 65 per cent). The delivery rates of all three cytokinins decreased significantly under stress. Cytokinin levels also decreased in roots and in leaves of stressed plants. Three-fold increase in cytokinin oxidase activity was observed in stressed roots after polymin P-ammonium sulphate fractionation. Further purification using Con A agarose resulted in elution of protein with cytokinin oxidase activity and was found to be 30 kDa protein on SDS-PAGE.

Unusual discrimination against carrier protein antibodies during partial purification of hapten-protein polyclonal antibodies to plant stress hormones.

Polyclonal antibodies (PcAb) were raised against t-zeatin riboside (t-ZR) and abscisic acid (ABA). t-ZR-BSA and ABA-BSA antibodies had high titre and specificity to haptens but also contained BSA specific antibodies as observed in double immuno diffusion and quantitative precipitation tests. Partial purification of antiserum by precipitation, desalting, and ion-exchange chromatography almost completely eliminated interference from BSA specific antibodies. Consequently, very little or no reaction was observed in dot-immunoblot assays even when high concentrations of BSA were probed with partially purified t-ZR-BSA IgG. Further studies with ABA antiserum showed that discrimination against BSA occurred during chromatography and not during salt fractionation. Because antibodies to both hapten and carrier protein were predominantly of IgG class, this unusual discrimination against carrier protein Ab was possibly influenced by two approaches followed for DEAE chromatography, viz. (i) adsorption of IgG at pH 8 followed by elution; or (ii) adsorption of contaminating proteins at neutral pH while specific IgG comes off as unbound fraction.