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Blue-light photoreceptors and development of the garden pea

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Platten, John Damien 2003 , 'Blue-light photoreceptors and development of the garden pea', PhD thesis, University of Tasmania.

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Abstract

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.

Item Type: Thesis - PhD
Authors/Creators:Platten, John Damien
Keywords: Plants, Peas, Pisum, Plant photoreceptors
Copyright Holders: The Author
Copyright Information:

Copyright 2003 the author - The University is continuing to endeavour to trace the copyright
owner(s) and in the meantime this item has been reproduced here in good faith. We
would be pleased to hear from the copyright owner(s).

Additional Information:

Includes notes in back pocket. Thesis (Ph.D.)--University of Tasmania, 2004. Includes bibliographical references

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