The mode of action of Cytokinin in the moss Physcomitrella Patens

Different aspects of the mode of action of cytokinin in the moss Physcomitrella patens were studied. In the search for a cytokinin receptor, a particulate fraction was isolated which had cytokinin-binding activity . Cytokinin-binding a c t ivi ty is described as the amount of binding of a radioactive cytokinin that is prevented from binding by excess unlabel led cytokinin. The cytokinin-binding activity was only detected with a tritiated cytokinin of high specific activity. Two assays were used, an equil ib r ium d ia ly s i s and a c ent r i fugat ion assay. These assays were used to show that the cytokinin-binding a c t iv i ty was heatlab i le and can be solubilised by the detergent Triton X100, but not by acetone. Cytokinin-binding a c t iv i ty was higher in phosphate-starved t is sue which consists mainly of caulonemata, the target cells for cytokinin action. A 13 ,0 0 0-8 0 ,000g pel let appeared to contain a component which has some of the characteristic s expected for a cytokinin receptor that was a membrane bound protein. Mutagenised spores were screened for cytokinin nonresponding mutants. No such mutants were obtained from 25,000 plants. A temperature-sensitive mutant, ove 409, was isolated which produced normal leafy shoots at I7°C, but many abnormal buds at 2 4 ° C . T h i s mutant was found to over-produce isopentenyladenine at the higher temperature. The level of cytokinin production in wild type and ove 78 was also found to be temperature-dependent. The change in cytokinin production by ove 409 was over twice that with wild type and ov.e 78, and dropped to wi ld type levels at 17°C. By the use of protoplast fusion, ove 409 was found to be recessive to wild type and in the same complementation group as ove A78. The role of light was studied in the induction of the gametophore. In the presence of supplied cytokinin, bud induction was caused by red light around 613 to 687nm. When the cytokinin-induced bud induction occurs in the dark after exposure to red light , bud induction is reduced by a short exposure to far-red light or a dark period before the addit ion of the cytokinin- This indicates that phytochrome is involved in bud induction. However, another factor appears to be involved in bud induction as this was greater when the cytokinin was applied to the cultures 2 hours after the exposure to red light. The possible role of calcium ions in bud induction was investigated. Calcium ions have been reported to induce the first stages of buds in the moss Funaria hygrometrica. Using the calcium ionophore A23187, calcium ions were shown to induce chloronemal branching in Physcomitre 11a patens. Therefore calcium ions appear to have a role in branching, but not in the differentiation of a bud.