Novel cytokinins: The predominant forms in mature buds of Pinus radiata

To elicit the roles of cytokinins in the regulation of maturation of Pinus radiata D, Don, the spectrum of endogenous cytokinins and their concentrati on in the mature buds were analysed using double-solvent extraction, column complex purification and separation, a novel immunoaffinity purification m ethod, normal and reverse phase high-pressure liquid chromatography, enzyma tic treatment, radioimmunoassay and electrospray MS/MS spectrometry. We hav e isolated two novel cytokinin glycosides whose proposed structures are iso pentenyladenine-9-(glucopyranosyl riboside), dihydrozeatin-9-(glucopyranosy l riboside) and confirmed the presence of zeatin-9-(glucopyranosyl riboside ), We have also found the presence of novel phosphorylated forms of these 3 cytokinin ribosyl-linked glycosides, Quantitative analyses revealed that t he cytokinin ribosyl-linked glycosides predominate in P, radiata mature bud s, Although cytokinin free base, riboside and nucleotide forms are also pre sent, we could find no evidence of the traditional cytokinin O- or N-glucos ides in the conifer buds, Thus, cytokinin metabolism in mature buds of P, r adiata is very different from other species previously examined.