Characterisation of PHYA mutants of Arabidopsis thaliana.

Phytochrome, a R/FR reversible photoreceptor that regulates plant photomorphogenesis, is encoded by a gene family of which there are at least five members. The best characterised member is phytochrome A which accumulates to high levels in etiolated tissue but is degraded in light, unlike other members of the family which are light-stable. A two-stage screen was designed for isolation of phyA mutant seedlings. The mutant phenotype was illustrated by deficiency in FR-HIR-mediated inhibition of hypocotyl elongation, which is exclusively mediated by phytochrome A while retaining R-HIR-mediated inhibition of hypocotyl elongation, which is mediated by one or more of the light-stable phytochrome species. Mutants isolated using this screen were divided into three complementation classes, fhy1, fhy2 and fhy3. Immunoblot and spectrophotometric analyses of these three mutant lines demonstrated that one of them, fhy2, was deficient in phytochrome A protein. Southern analysis of several alleles of the fhy2 mutation indicated that deficiency in phytochrome A resulted from structural alterations in the PHYA gene. Subsequently, the fhy2 mutants were designated as phyA mutants. The other two classes of long hypocotyl mutants isolated in this screen, fhy1 and fhy3, showed no structural alteration in the PHYA gene and had normal immunochemically and spectrophotometrically detectable levels of phytochrome A. Therefore, fhy1 and fhy3 are putative transduction chain mutants. Comparison of the photophysiology of phytochrome A mutants with that of wild-type seedlings allowed conclusions to be drawn about the roles of phytochrome A in selected, assayable photomorphogenetic responses. phyA mutant seeds exhibit wild-type germination responses to R but do not germinate in FR, suggesting that phytochrome A mediates a promotory response to FR. The importance of phytochrome A as the principal photoreceptor in FR is extended to de-etiolated plants where phytochrome A retains an inhibitory role in hypocotyl elongation, under supplementary FR or low R:FR ratio conditions. Phytochrome A also plays a role in flowering which becomes apparent under low fluence-rate incandescent day extensions, suggesting that in fluence-rate limiting conditions, phytochrome A is an important component of daylength perception. fhy1 and fhy3 mutants, selected for long hypocotyls in FR alongside phyA mutants, were also characterised and shown to have wild-type levels of phytochrome A protein with no apparent rearrangement of the PHYA gene. The mutation at FHY1 affects hypocotyl elongation but not germination, de-etiolation or flowering, suggesting that hypocotyl elongation is on a different branch of the transduction chain to these other responses and that FHY1 is a component of that branch. Hypocotyl elongation of fhy3 seedlings is affected in R, as well as in FR which may suggest that the transduction chain component encoded by FHY3 is shared by the phytochrome, or phytochromes, that control hypocotyl elongation in R. Characterisation of phyA and fhy mutants of Arabidopsis has led to elucidation of the roles of phytochrome A in different photomorphogenetic responses and some preliminary investigation of components of the transduction chain. This reseach has also further clarified the roles of other phytochromes and the extent of overlap of the spheres of action of distinct molecular species of phytochrome.