The transduction of light signals controlling floral induction in Pharbitis nil

The control of floral induction in the short day plant, Pharbitis nil, is based on a circadian rhythm. At certain times of the circadian (24 h) cycle, a brief light pulse, acting through the photoreceptor, phytochrome, can cause inhibition of induction, whilst at other time points, a brief light pulse promotes a change in phase of the rhythm. The aim of the research presented in this thesis was to investigate the signal transduction pathway linking perception of light by phytochrome to its physiological action on floral induction in Pharbitis nil, and how the circadian oscillator interacts with and influences this pathway. Experimental evidence has implicated calcium in both the regulation of circadian rhythms and in phytochrome action, and more specifically, in the control of floral induction. It was therefore decided to investigate whether the phosphatidylinositol cycle, which, via the key signalling component, l(l,4,5)P3, releases calcium from intracellular stores in animal cells, is involved in the transduction of light signals physiologically active in floral induction. In physiological studies, the calcium channel blocker Verapamil and the calcium chelator EGT A both enhanced the inhibition of floral induction by a sub-maximal light pulse at the ninth hour of an otherwise inductive period. EGT A applied before an inductive dark period significantly reduced the level of flowering, but did not affect flowering if applied at the end of induction. these findings imply that calcium is necessary for induction to take place. In studies investigating the metabolism of I( 1,4,S)P 3 by a soluble extract from Pharbitis nil cotyledons, elevated calcium levels were found to slow the metabolism of l(l,4,5)P3• Lithium, which blocks inositol-1-phosphatase activity in animal cells, causing inositol phosphates to accumulate and blocking the PI cycle, reduced the sensitivity of the response of inhibition to a night break, and caused a decrease in flowering when given before or after an inductive period. These results were interpreted as being a consequence of the characteristic period lengthening effect of lithium. In studies on the metabolism of I( 1,4,5)P 3 by a soluble extract from Pharbitis nil cotyledons, no effect of LiCl was observed, either on the profile of inositol phosphates obtained, or on the rate of metabolism. Therefore it is likely that the effects of lithium on floral induction are not brought about via perturbation of the PI signal transduction cascade. Components of the PI cycle were identified from Pharbitis nil cotyledons (the inductive organs) by radiolabelling with [32P]-orthophosphate and subsequent separation of the aqueous components by HPLC and the lipid components by TLC, and comparison with authentic standards. However, radiolabelling did not permit identification of any light stimulated changes in levels of I( 1,4,5)P 3• This was because of the large variation found in label uptake by the·seedlings. Use of a radio-ligand binding assay specific for I(l ,4,5)P 3, however, permitted identification of an increase in I( 1,4,5)P 3 in response to a 30 s light pulse at the ninth hour of an inductive dark period, which causes inhibition of flowering without affecting the phase of the rhythm. Light did not affect levels of I( 1,4,5)P 3 when given at the third hour of an inductive period, when it has no physiological effect, or at the sixth hour which causes phase shifting of the circadian rhythm. The implications of these findings, that light acting through phytochrome only instigates an increase in I( 1,4,5)P 3 at certain discrete time points of the circadian cycle, are discussed in relationship to the phytochrome multi-gene family, and the multiple modes of phytochrome action, with the implication that there must be other additional signalling systems for transducing light signals perceived by the photoreceptor phytochrome, and that more than one phytochrome species must be active in the control of floral induction