Blue-light photoreceptors and development of the garden pea

Developmental responses of plants to their light environment are of obvious importance to their fitness and survival, and plant photomorphogenesis has been the focus of much study over the past decade. Great advances have been made in our understanding of the photoreceptors involved in photomorphogenic responses, their roles, their biochemical and photochemical nature, and signal transduction processes initiated by the activated photoreceptors. Much of this study has been focussed on the red/far-red phytochrome photoreceptors, for which mutants have now been isolated in a number of model species. The cryptochrome photoreceptors are comparatively less well characterised, with mutants so far only identified in the Arabidopsis CRYJ and CRY2 genes, and the tomato CRY1a gene. The aim of the current research was to extend the characterisation of the structure and function of the cryptochrome gene family to include another major member of the eudicots, the Fabaceae, represented by the garden pea (Pisum sativum L.). The cryptochrome gene family in the garden pea was found to be represented by 3 members: one CRYJ homologue, and two CRY2 homologues. The latter were found to encode full-length predicted cryptochrome products, containing all major functional domains previously identified to be important in cryptochrome function. They appear to have arisen from a gene duplication event that occurred around the time of divergence of the Hologalegina from the Millettioids/Phaseoloids. One member (PsCRY2B) appears to be rapidly diverging from the ancestral sequence, though whether this is a result of random drift or active selection is not known. Sequence alignments with other available angiosperm cryptochrome sequences in the databases (both monocot and dicot) provide support for many of the previously identified conserved motifs potentially responsible for cryptochrome function, and in addition identified a number of motifs in both the N- and C-terminal domains which are conserved in one of the CRYJ or CRY2 subfamilies, but not the other. Many of these motifs contain potential phosphorylation sites introduced or removed from one of the subfamilies. Expression of the three pea cryptochromes was differentially regulated by light. PsCRY1 was up-regulated by blue light (both high- and low fluence-rate), but not white or red light, suggesting some autoregulatory role of the cryptochromes. This was supported by studies of a Pscry1-deficient mutant (see below). PsCRY2A showed a down-regulation of expression under low- but not high fluence-rate blue light, and under white light. The PsCRY2B gene showed a similar pattern of up-regulation to PsCRY1 under blue light, however expression was down regulated under white light as opposed to showing no appreciable change. To further characterise the role of the pea cryptochromes, the M2 of EMS-treated phyA-1 seed was screened for mutants deficient in their blue-light responses. One mutant line was isolated with selective defects in responses to all fluence-rates of blue light. This line was subsequently found to have a G - A substitution at position 749 of the PsCRY1 mRNA coding sequence. This is predicted to mutate a highly conserved glycine residue (Gly250) to glutamic acid. This mutation is in an equivalent residue to that mutated in the Arabidopsis fha-2 mutant, which contains a G - R substitution. The Pscryl-1 mutant displayed similar defects in de-etiolation and flowering responses to previously identified Arabidopsis and tomato cry1 mutants (longer epicotyledonous internodes, smaller leaflets), however they did not show the general cell expansion in all organs seen in the Arabidopsis mutants, suggesting that CRY1 does not mediate a general inhibition of cell expansion in the wild type. The Pscry1-1 single mutant also displayed a much weaker phenotype than its Arabidopsis counterpart, possibly suggesting the cryptochromes have a lesser role in photomorphogenesis of peas. However, a phyAphyBcry1 triple mutant was found to be lethal, failing to show any appreciable de-etiolation even under high-irradiance white light. This suggests that these photoreceptors are collectively necessary for photomorphogenesis in pea, and therefore that PsCRY1 may simply show a higher degree of redundancy with the phytochromes than is seen in Arabidopsis. Screening of the M2 population also yielded a novel late-flowering mutant of pea which shows selective de-etiolation defects to blue, but not red, light. This mutant was named lfp1-1 for its late-flowering photomorphogenic phenotype. This mutant was found to be late flowering under both long- and short days, and to cause a dramatic increase in the extent of aerial branching, along with an increase in internode length, smaller leaflets and darker green foliage. These effects were only seen on a phyA-deficient background in the Torsdag cultivar of pea. On a heterozygous Terese background, the effect of lfp1-1 was also seen in heterozygous PHYA phyA-1 plants. The lfp1 phenotype showed no linkage with the PsCRY2A or PsCRY2B genes, and possibly represents a novel class of signalling intermediate specific to cryptochrome. A single-gene recessive mutant has also been identified from a screen of EMS treated cv. Torsdag seed which shows a hyper-phototropic and hyper-gravitropic phenotype. This mutant displays an increase in the curvature attained under continuous phototropic or gravitropic stimuli due to an extension of the phase of curvature. Mutant plants also display longer epicotyls under all light qualities tested, which was accompanied by a pronounced spiral curving of the internodes. Despite their hyper-phototropic phenotype, mutant plants were found to have an increased down-regulation of PsPK5 expression (a PHOTJ orthologue) on transfer to light. These plants display many similarities to the recently characterised mdr mutants of Arabidopsis, which contain defects in basipetal auxin transport. In summary, examination of the cryptochrome gene family in the garden pea has revealed a novel gene duplication in the CRY2 gene lineage. Alignments of the pea cryptochromes with published sequences has revealed a number of sites potentially involved in cry1 and cry2 regulation, however further work is required to test this. Characterisation of a mutant deficient in the PsCRY1 gene has revealed that cry1 plays a similar role in pea to other species, but does not mediate the general inhibition of cell expansion seen in the Arabidopsis cry1 mutant. In addition, phyA, phyB and cry1 are almost solely responsible for the de-etiolation response of peas. Screening has also identified a number of other mutants with affected photomorphogenic responses that may aid in the investigation of transduction of the light signals.