Gibberellin Metabolism: New Insights Revealed by the Genes

In a review article entitled, “Gibberellin metabolism: new insights revealed by the genes”,  published in the December 2000 issue of Trends in Plant Science, Dr. Peter Hedden [peter.hedden@bbsrc.ac.uk] and Andrew L. Phillips at the Long Ashton Research Station, UK, enumerate the current status of our knowledge in GA metabolism. After a brief historical introduction, the authors cover the topics of gibberellin biosynthetic pathways, the genes and gene families involved, the regulation of gibberellin metabolism by endogenous and environmental factors and the potential for genetic modification of GA metabolism.

The authors begin with a short historical overview of gibberellins metabolism.  Gibberellins (GAs) were originally shown to be endogenous growth regulators by restoring the height of dwarf mutants of pea and maize by application of GA3, obtained from the fungus, Gibberella fujikuroi. Technological advancements, made in the field of gas chromatography and mass spectrometry have made it possible to identify as many as 126 GAs from plants, fungi and bacteria. Despite the large number of forms, only a few have since been shown to be biologically active. Work with GA-deficient mutants has shown that active GAs are involved not just in stem elongation, but play a role in most of the developmental stages of plant growth. It has been shown that selected members of this group of hormones also regulate stem elongation and flowering in response to light quality and photoperiod.

As a result of careful metabolic studies over many years, the biosynthetic pathways to GAs have been elucidated in plants and fungi. The early pathway from trans-geranylgeranyl disphosphate (GGPP) up to the formation of the intermediate GA12-aldehyde is the same in all systems that have so far been investigated. The first steps by which GGPP is converted to the tetracyclic hydrocarbon ent-kaurene via ent-copalyl diphosphate are catalysed by two enzymes, CPP (copalyl diphosphate) synthase (CPS) and ent-kaurene synthase (KS) in higher plants whereas only one enzyme is involved in fungi.

The recent discovery of the genes and gene families has made it possible to gain insights into the molecular mechanisms that control GA metabolism. These insights include the realization that many of the enzymes that participate in this pathway are multifunctional and therefore fewer enzymes than might have been anticipated are required to synthesize the various GA structures.

The formation of ent-kaurene from GGPP has been shown in pea and wheat to take place in proplastids within the shoot meristem. The pathway appears to be under tight developmental control. The identification and characterization of the genes, involved in the early and final steps of GA biosynthesis, have been greatly facilitated by the study of a number of dwarf – (ga1, ga2, ga3) and semi dwarf (ga4, ga5) loci in GA-deficient mutants in Arabidopsis. Similar work has used mutants of maize and pea. Further confirmation of the involvement of these genes in early and final stages of biosynthesis was obtained by their heterologous expression* in a different organism, E. coli. In Arabidopsis, single genes encoding enzymes for the early reactions in the pathway have been identified. Mutations at these sites produce severe dwarf phenotypes. In contrast, small multigene families encode the dioxygenases. Interestingly, of the three enzymes, GA20ox, GA3ox and GA2ox, which participate in the later stages of the biosynthesis, the first two are encoded by a minimum of four and the last by at least five genes in Arabidopsis. The GA biosynthesis genes are distributed throughout the Arabidopsis genome and are not clustered except for two genes AtGA3ox2 and AtGA3ox4, located side by side on chromosome 1.

The authors then review recent research on the regulation of gibberellin metabolism by development, hormones and light.

Several genes appear under tight developmental control. Members of the dioxygenase gene families show tissue-specific patterns of expression. For example, in Arabidopsis, expression of the GA20ox1 and GA3ox1 genes occurs in growing vegetative tissues and in some flower organs. GA3ox1 is also expressed in developing siliques (pods) but different GA20ox genes are expressed in this tissue.

The authors elaborate also on the hormonal regulation of GA biosynthesis through the use of examples. Treatment of plants with bioactive GAs reduces the levels of transcripts for GA20ox and GA3ox genes. Some, but not all genes in the pathway are involved in negative feedback regulation. This is illustrated by the gene GA3ox2 which is involved in the germination of Arabidopsis seeds and is not feedback regulated. For germination, high levels of GA are produced for a short period and as such no feedback regulation is necessary.

Light also plays a significant role in GA biosynthesis. For instance, the GA levels in potato grown under short days are reduced leading to tuberization. Exposure of potato plants to long days increases the levels of GA inhibiting tuber formation.

GA is extensively used in agriculture and horticulture. Thus, the potential for modification of its metabolism for human benefit is of considerable interest. There are many possibilities for this. For example, seedless grapes with large berry size on an elongated peduncle, are commercially produced by application of GA3 to the developing inflorescences. In other situations in which the growth of crops and ornamentals is intended to be restricted, the material is treated with growth retardants. The authors suggest that instead of using chemicals, a better approach will be to regulate GA biosynthesis in specific tissues, by targeting the particular gene(s) that are expressed in those tissues. Such a strategy would work if it involves enzymes such as GA20ox, GA3ox and GA2ox which function in the later stages of biosynthesis. Manipulation of expression of the genes for the early stages of biosynthesis might have a less significant effect on the overall rate of biosynthesis.

The authors then mention that a number of antisense RNAs of GA biosynthetic genes have been used to produce transgenics. Studies of the latter have helped investigators identify the roles of individual members of the gene family. For instance, in a transgenic line of potato in which a GA20ox gene was suppressed by the relevant antisense RNA, tuberization increased concomitant with a reduction in stem length. Overexpression of a bean (Phaseolus coccineus) GA2ox gene led to reduced stem elongation in Arabidopsis, ornamentals and wheat.

Although almost all the genes for the GA-biosynthetic enzymes have been identified in Arabidopsis, the authors point out that a lot of ground is yet to be covered in gibberellin metabolism research. They hope that more basic information will soon be available with the advancement of recombinant enzyme and other advanced technologies. They anticipate that such information will not only lead to the design of enzymes with altered substrate specificities but also provide a clearer picture of the molecular mechanisms involved in the regulation of GA biosynthesis by endogenous and environmental factors.

Epilogue

Last Remaining Steps of the Gibberellin Biosynthesis Pathway Revealed

Home page of Dr. Jim Peacock:
http://www.pi.csiro.au/Research/Office_Chief/office_chief.htm

Home page of Dr. Andrew Phillips:
http://www.lars.bbsrc.ac.uk/plantsci/molbiol/andy.html

CSIRO:
http://www.pi.csiro.au//url]

In their review article, “Gibberrellin Metabolism: New Insights Revealed by the Genes” published in the December issue of Trends in Plant Science Peter Hedden and [url=http://www.lars.bbsrc.ac.uk/plantsci/molbiol/andy.html]  Andrew Phillips mention that there remains a gap in our knowledge about the genes involved in the four steps between ent-kaurenoic acid and GA 53 in gibberellin biosynthesis. Since publication of their review, Dr. Hedden has informed us there has been a more recent publication by Chris Helliwell and colleagues at Dr. W. J. Peacock‘s laboratory at CSIRO, Australia in the February 13 issue of PNAS about three genes that encode ent-kaurenoic acid oxidase. The authors have shown that this enzyme is a member of the CYP88A subfamily of cytochrome P450 enzymes and it catalyzes the three steps of the gibberellin biosynthetic pathway from ent-kaurenoic acid to GA(12). With the discovery of this gene, the working mechanism of the entire gebberellin pathway has been unraveled. In a gibberellin-responsive grd5 barley mutant the above enzyme is accumulated.

Three independent grd5 mutants contained mutations in a gene encoding a member of the CYP88A subfamily of cytochrome P450 enzymes, defined by the maize Dwarf3 protein. Mutation of the Dwarf3 gene gives rise to a gibberellin-responsive dwarf phenotype, but the lesion in the gibberellin biosynthesis pathway had not been identified. Arabidopsis has two CYP88A genes, both of which are expressed, explaining the lack of an Arabidopsis mutant comparable to the maize Dwarf3 and barley grd5 mutants.
The above authors demonstrated that yeast strains expressing cDNAs encoding each of the two Arabidopsis and the barley CYP88A enzymes catalyze the three steps of the GA biosynthesis pathway from ent-kaurenoic acid to GA(12)

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Heterologous expression = Expression of a gene in a different species.

 

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