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Molecular genetics and regulation of gibberellin biosynthesis in pea

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Davidson, Sandra Elizabeth (2003) Molecular genetics and regulation of gibberellin biosynthesis in pea. PhD thesis, University of Tasmania.

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Abstract

The molecular identity of the final two classical gibberellin (GA)
biosynthesis mutants of Pisum sativum, na and lh, have been determined. NA and
LH encode adjacent cytochrome P450 monooxygenases in the GA biosynthesis
pathway and together oxidise the six steps from ent-kaurene to GA l2.
The GA deficient dwarf na mutant in pea has severely reduced internode
elongation, reduced root growth, and decreased leaflet size. However, the seeds
develop normally. Two genes, PsKA01 and PsKA02, encoding cytochrome P450
monooxygenases of the subfamily CYP88A were isolated. Both PsKA01 and
PsKA02 had ent-kaurenoic acid oxidase (KAO) activity; catalysing the 3 steps of
the GA biosynthetic pathway from ent-kaurenoic acid to GA l2 when expressed in
yeast.
The NA gene encodes PsKA01, since in two independent mutant alleles, na-
1 and na-2, PsKA01 had altered sequences and the 5 base deletion in PsKA01
associated with the na-1 allele co-segregated with the dwarf na phenotype. PsKA01
was expressed in the stem, apical bud, leaf, pod and root, organs in which GA levels
have previously been shown to be reduced in na plants. PsKA02 was expressed only
in seeds and this may explain the normal seed development and normal GA
biosynthesis in seeds of na plants.
The pea gene associated with the GA deficient lh dwarf mutant was isolated
and found to encode a cytochrome P450 monooxygenase, of the subfamily
CYP701A. This gene had high similarity to genes encoding ent-kaurene oxidases in
Arabidopsis and pumpkin, AtK01 and CmK01 respectively. The PsK01 gene
sequence was altered in all three of the dwarf pea mutant alleles, lh-1, lh-2 and lh-3.This confirmed previous work that showed that the lh-2 mutant blocked the 3 step
metabolism of ent-kaurene to ent-kaurenoic acid. The phenotypes of the lh-1 and lh-
3 alleles differ from 1h-2 in a tissue specific manner. In the seed, the lh-1 and lh-3
alleles had little effect while the lh-2 allele phenotype had smaller seeds and
decreased seed survival. However, in the vegetative parts of the plants all three
alleles are of similar dwarf stature. However, the lh-2 mutant is up to 30 times more
sensitive to the KO specific inhibitor paclobutrazol than is the lh-1 or lh-3 alleles.
The different phenotypes appear to reflect the different natures of the aberrant
proteins produced by the different alleles.
With the isolation of genes encoding the two major cytochrome P450
monooxygenases from the GA biosynthesis pathway in pea, control of the early
sections of the pathway was investigated. Northern blot analysis was used to
measure the mRNA transcript levels of genes encoding enzymes at three steps
including the first committed step of GA biosynthesis, cyclization of GGDP to
copalyl diphosphate (CPS), and the two monooxygenases which oxidise entkaurene,
(KO), and ent-kaurenoic acid, (KA0s).
ent-kaurenoic acid oxidase activity is encoded by a 2 membered gene family
with tissue specific expression patterns. PsKA02 is only expressed in developing
seed and therefore potentially allows differential regulation of GA biosynthesis in
seed. In contrast, the single copy ent-kaurene oxidase, PsK01, is expressed at high
levels in all tissue and developmental stages tested. Although copalyl diphosphate
synthase, PsCPS, was expressed at low levels in all tissues tested, it had higher
expression in seed and stem tissue.
There is evidence of developmental regulation of the genes in seeds at the
transcript level. All four genes examined, PsCPS, PsK01, PsKA01 and PsKA02,
have a transcript level peak around the time of contact point coinciding with a peak
of GA20accumulation in maturing seeds. PsK01 expression levels were also high in
young seeds (11 days after anthesis) coinciding with the tail end of the peak of GA 1
content in young seeds. However, genes for these steps appear unaffected at the transcript level by maturation of the pea shoot. PsCPS, PsK01 and PsKA01 were
expressed in mature stem and leaf tissue, even though the mature tissue contained
less bioactive GA1and had ceased expanding.
Unlike some genes later in the GA biosynthesis pathway, those early in the
pathway, PsCPS, PsK01, PsKA01 and PsKA02, are not under feed-back regulation
by GA1activity. Expression levels were unaltered in samples from plants with
reduced stature and decreased bioactive GA levels as in GA deficient mutants or
plants treated with the GA biosynthesis inhibitor, paclobutrazol.

Item Type: Thesis (PhD)
Keywords: Plants, Peas
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, 2003. Includes bibliographical references

Date Deposited: 09 Dec 2014 00:08
Last Modified: 09 May 2016 06:27
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