Cytokinins and phase change in Pinus radiata : morphological, physiological and molecular studies : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Biology at Massey University, Palmerston North, New Zealand
Phase change in higher plants is a developmental process during which changes occur at morphological, physiological and molecular levels. In Pinus radiata, buds of juvenile trees produce photosynthetically functional primary needles while buds from mature trees do not produce such primary needles. Cytokinin, however, causes production of primary needles from mature buds in vitro (Horgan, 1987). Pursuing this observation, morphological and anatomical examinations of the buds were carried out using light microscopy. The results showed that the cytokinin-induced transition from mature to juvenile bud morphology may be through resetting the fate of fascicle meristems and/or foliar primordia. To determine if a correlation existed between the endogenous cytokinin content and the maturation status of the buds, buds from the juvenile and mature P. radiata were analysed using a range of modern techniques, including column complex purification, immunoaffinity purification, normal and reverse HPLC, radioimmunoassay and electrospray tandem mass spectrometry. A wide spectrum of endogenous cytokinins were detected in the bud tissues, including five novel forms discovered in this work. Quantitative analyses revealed a general trend with seedling buds > juvenile (J4) buds > mature (M4) buds > mature (M8) buds for the combined concentration of free base and riboside cytokinins. High concentrations of phosphorylated cytokinins were found in the mature buds but not the juvenile buds. Novel cytokinin glucosides were the most abundant forms in the buds, with zeatin-9-(glucopyranosyl-1,3-ribosyl) and dihydrozeatin-9-(glucopyranosyl-1,3-ribosyl) being higher in the mature buds and isopentenyladenine-9-(glucopyranosyl-l,3-ribosyl) being higher in the juvenile buds. Overall, particular patterns of cytokinins in the field buds reflected the maturation status of the buds. Extensive metabolism of 6-benzylaminopurine occurred, including the production of the novel forms, 6-benzylaminopurine-9-(glucopyranosyl-1,3-ribosyl) and phosphorylated 6-benzylaminopurine-9-(glucopyranosyl-1,3-ribosyl), during the in vitro 'rejuvenation' of mature buds to the juvenile phenotype. Among the metabolites, the abundance of 6-benzylaminopurine, 6-benzylaminopurine riboside and 6-benzylaminopurine-9-(glucopyranosyl-1,3-ribosyl) was high while phosphorylated forms were very low over the duration of the experiment. The patterns of metabolites reflected the patterns of endogenous cytokinins observed in juvenile buds. The results also indicated that 6-benzylaminopurine did not regulate phase-specific traits by increasing endogenous cytokinins. Molecular tools were used to clone cytokinin-responsive genes which may also be involved in the regulation of phase change. A cDNA sequence (Prcr5) was cloned using a modified mRNA differential display technique. Northern analyses showed that cytokinin promoted and maintained the expression of Prcr5 at a high level during rejuvenation of the mature buds in vitro. The deduced PrCR5 protein sequence displays homology to Ginseng RNases and PR-10. A possible function of the Prcr5 gene in the regulation of phase change is discussed. A cDNA sequence (Prcab) coding for a chlorophyll a/b binding protein was also cloned. Although expression of the cab gene has been reported to be associated with phase change in other species, no such change was observed in P. radiata.