Gibberellin 2-oxidase

Details Gibberellin 2-oxidase Gibberellin 2-oxidase

2001-05-25

2003-12-30

Fox, David T. (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

The polynucleotide alters plant part growth

C800S278000, C800S287000, C536S023600, C435S468000

Reexamination Certificate

active

06670527

ABSTRACT:

CROSS REFERENCE TO RELATED APPLICATIONS
The present application is the U.S. National Phase of International Application No. PCT/GB9901857, International Application filing date Jun. 11, 1999, which was published in English on Dec. 23, 1999 as WO99/66029, and claims priority under 35 U.S.C. § 119 to United Kingdom applications GB 9812821.8, filed Jun. 12, 1998, and GB 9815404.0, filed Jul. 15, 1998.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a novel enzyme involved in the control of plant growth, DNA sequences coding for the enzyme and uses of the nucleotide sequence coding for the enzyme in the production of transgenic plants with improved or altered growth characteristics.
2. Related Art
The gibberellins (GAs) are a large group of diterpenoid carboxylic acids that are present in all higher plants and some fungi. Certain members of the group function as plant hormones and are involved in many developmental processes, including seed germination, stem extension, leaf expansion, flower initiation and development, and growth of the seeds and fruit. The biologically active GAs are usually C
19
compounds containing a 19-10 lactone, a C-7 carboxylic acid and a 3&bgr;-hydroxyl group. The later stages of their biosynthesis involve the oxidative removal of C-20 and hydroxylation at C-3. Hydroxylation at the 2&bgr; position results in the production of biologically inactive products. This reaction is the most important route for GA metabolism in plants and ensures that the active hormones do not accumulate in plant tissues. The GA biosynthetic enzymes 7-oxidase, 20-oxidase, 3&bgr;-hydroxylase and 2&bgr;-hydroxylase are all 2-oxoglutarate-dependent dioxygenases. These are a large group of enzymes for which 2-oxoglutarate is a co-substrate that is decarboxylated to succinate as part of the reaction (see review by Hedden, P. and Kamiya, Y., in
Annu. Rev. Plant Physiol. Plant Mol. Biol
. 48 431-460 (1997)).
Chemical regulators of plant growth have been used in horticulture and agriculture for many years. Many of these compounds function by changing the GA concentration in plant tissues. For example, growth retardants inhibit the activity of enzymes involved in GA biosynthesis and thereby reduce the GA content. Such chemicals are used commonly, for example, to prevent lodging in cereals and to control the growth of ornamental and horticultural plants. Conversely, GAs may be applied to plants, such as in the application of GA
3
to seedless grapes to improve the size and shape of the berry, and to barley grain to improve malt production. Mixtures of GA
4
and GA
7
are applied to apples to improve fruit quality and to certain conifers to stimulate cone production. There are several problems associated with the use of growth regulators. Some of the growth retardants are highly persistent in the soil making it difficult to grow other crops following a treated crop. Others require repeated applications to maintain the required effect. It is difficult to restrict application to the target plant organs without it spreading to other organs or plants and having undesirable effects. Precise targeting of the growth-regulator application can be very labour intensive. A non-chemical option for controlling plant morphology is, thus, highly desirable.
Developing seeds often contain high concentrations of GAs and relatively large amounts of GA-biosynthetic enzymes. Mature seeds of runner bean (
Phaseolus coccineus
) contain extremely high concentrations of the 2&bgr;-hydroxy GA, GA
8
, as its glucoside, indicating that high levels of 2&bgr;-hydroxylase activity must be present. This has been confirmed for the related species
Phaseolus vulgaris
in which there is a rapid increase in GA 2&bgr;-hydroxylase activity shortly before seeds reach full maturity (Albone et al.,
Planta
177 108-115 (1989)). 2&bgr;-Hydroxylases have been partially purified from the cotyledons of
Pisum sativum
(Smith, V. A. and MacMillan, J.,
Planta
167 9-18 (1983)) and
Phaseolus vulgaris
(Griggs et al
Phytochemistry
30 2507-2512 (1991) and Smith, V. A. and MacMillan, J.,
J. Plant Growth Regul
. 2 251-264 (1984)). These studies showed that there was evidence that, for both sources, at least two enzymes with different substrate specificities are present. Two activities from cotyledons of imbibed
P. vulgaris
were separable by cation-exchange chromatography and gel-filtration. The major activity, corresponding to an enzyme of M
r
26,000 by size exclusion HPLC, hydroxylated GA
1
and GA
4
in preference to GA
9
and GA
20
, while GA
9
was the preferred substrate for the second enzyme (M
r
42,000). However, attempts to purify the enzyme activity to obtain N-terminal information for amino acid sequencing have proved impossible because of the low abundance of the enzyme in the plant tissues relative to other proteins and the co-purification of a contaminating lectin with the enzyme activity rendering N-terminal amino acid sequencing impossible.
The regulation of gibberellin deactivation has been examined in
Pisum sativum
(garden pea) using the sln (slender) mutation as reported in Ross et al (
The Plant Journal
7 (3) 513-523 (1995)). The sln mutation blocks the deactivation of GA
20
which is the precursor of the bioactive GA
1
. The results of these studies indicated that the sln gene may be a regulatory gene controlling the expression of two separate structural genes involved in GA deactivation, namely the oxidation of GA
20
to GA
29
by 2&bgr;-hydroxylation at C-2 followed by the further oxidation of the hydroxyl group to a ketone (GA
29
to GA
29
-catabolite). The conversion of GA
29
to GA
29
-catabolite in pea seeds was inhibited by prohexadione-calcium, an inhibitor of 2-oxoglutarate-dependent dioxygenases (Nakayama et al
Plant Cell Physiol
. 31 1183-1190 (1990)), indicating that the reaction was catalysed by an enzyme of this type. Although the slender (sln) mutation in peas was found to block both the conversion of GA
20
to GA
29
and of GA
29
to GA
29
-catabolite in seeds, the inability of unlabeled GA
20
to inhibit oxidation of radiolabelled GA
29
, and vice versa, indicated that the steps were catalysed by separate enzymes. Furthermore, in shoot tissues, the slender mutation inhibits the 2&bgr;-hydroxylation of GA
20
, but not the formation of GA
29
-catabolite. These observations lead to the theory that there were two separate enzymes involved in this metabolic pathway controlling the deactivation of GA in plants (Hedden, P. and Kamiya, Y., in
Annu. Rev. Plant Physiol. Plant Mol. Biol
. 48 431460 (1997)).
BRIEF SUMMARY OF THE INVENTION
However, it has now surprisingly been found that a single enzyme can, in fact, catabolise these different reactions. The present invention represents the first reported cloning of a cDNA encoding a GA 2&bgr;-hydroxylase that acts on C
19
-GAs and for which 2&bgr;-hydroxylation is its only hydroxylase activity. A cDNA clone from pumpkin seed encodes an enzyme that has both 2&bgr;- and 3&bgr;-hydroxylase activities (Lange et al.
Plant Cell
9 1459-1467 (1997)), but its major activity is 3&bgr;-hydroxylation and it acts as a 2&bgr;-hydroxylase only with tricarboxylic acid (C
20
) substrates; it does not 2&bgr;-hydroxylate C
19
-GAs. Since the new enzyme of the present invention catalyses both the &bgr;-hydroxylation and further oxidation of the substituted hydroxyl group to a ketone group at C-2, the enzyme has been termed a “GA 2-oxidase”.
DETAILED DESCRIPTION OF THE INVENTION
According to a first aspect of the present invention there is provided an isolated, purified or recombinant nucleic acid sequence encoding a gibberellin 2-oxidase enzyme comprising a nucleic acid sequence as shown in
FIG. 1
or a functional derivative thereof, or its complementary strand or a homologous sequence thereto.
A system of nomenclature for the GA-biosynthesis genes has now been introduced (Coles et al
The Plant Journal
17(5) 547-556 (1999). References in the present application to the gibberellin 2-oxidase gene of
Phaseolus coccineus
should b

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