Molecular genetics and regulation of gibberellin biosynthesis in pea
Creators: name: Davidson, Sandra Elizabeth
Publisher: name: University Of Tasmania
Year: 2023

description: 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.