Induction of dwarfism in transgenic Solanum dulcamara by over-expression of a gibberellin 20-oxidase cDNA from pumpkin.

The gibberellin (GA) 20-oxidase (CmGA20ox1) from immature pumpkin seed produces predominantly inactive tricarboxylic acid GAs. We expressed CmGA20ox1 under the control of the CaMV 35S promoter in Solanum dulcamara to assess the usefulness of this gene for reducing GA content in transgenic plants. All transgenic plants obtained were semi-dwarfs with smaller, deep-green leaves and highly pigmented stems compared to the wild-type. Such transformants flowered earlier than the wild-type plants and produced more fruit and more seeds per fruit. The transgene was efficiently expressed, producing high levels of CmGA20ox1 transcript and protein. Furthermore, the concentration of GA(1) was reduced in leaves of the transformants to approximately 20% or less of that in the wild-type and to about 40% or less in stems. The concentrations of other 13-hydroxylated GAs were also reduced, except for the tricarboxylic acid, GA(17), which accumulated in the transformants due to 13-hydroxylation of GA(25). By contrast, the concentrations of non-13-hydroxylated GAs, GA(4) and GA(34), were not consistently reduced, indicating that the effect of expressing the pumpkin gene may not be predictable. Transcript abundance for a native GA 20-oxidase gene was higher in the leaves and stems of S. dulcamara transformed with the pumpkin gene than in wild-type, reflecting the feedback control of 20-oxidase gene expression that serves as a homeostatic mechanism for GAs.

Modification of gibberellin production and plant development in Arabidopsis by sense and antisense expression of gibberellin 20-oxidase genes.

Gibberellin (GA) 20-oxidase catalyses consecutive steps late in GA biosynthesis in plants. In Arabidopsis, the enzyme is encoded by a gene family of at least three members (AtGA20ox1, AtGA20ox2 and AtGA20ox3) with differential patterns of expression. The genes are regulated by feedback from bioactive GAs, suggesting that the enzymes may be involved in regulating GA biosynthesis. To investigate this, we produced transgenic Arabidopsis expressing sense or antisense copies of each of the GA 20-oxidase cDNAs. Over-expression of any of the cDNAs gave rise to seedlings with elongated hypocotyls; the plants flowered earlier than controls in both long and short days and were 25% taller at maturity. GA analysis of the vegetative rosettes showed a two- to threefold increase in the level of GA4, indicating that GA 20-oxidase normally limits bioactive GA levels. Plants expressing antisense copies of AtGA20ox1 had short hypocotyls and reduced rates of stem elongation. This was reflected in reduced levels of GA4 in both rosettes and shoot tips. In short days, flowering was delayed and the reduction in the rate of stem elongation was greater. Antisense expression of AtGA20ox2 had no apparent effects in long days, but stem growth in one transgenic line grown in short days was reduced by 20%. Expression of antisense copies of AtGA20ox3 had no visible effect, except for one transgenic line that had short hypocotyls. These results demonstrate that GA levels and, hence, plant growth and development can be modified by manipulation of GA 20-oxidase expression in transgenic plants.

Identification of three C20-gibberellins: GA97 (2 beta-hydroxy-GA53), GA98 (2 beta-hydroxy-GA44) and GA99 (2 beta-hydroxy-GA19).

Three new C20-gibberellins, GA97 (2 beta-hydroxy-GA53), GA98 (2 beta-hydroxy-GA44) and GA99 (2 beta-hydroxy-GA19), have all been isolated from spinach, GA97 also from tomato root cultures and pea pods, and GA98 from maize pollen. The structures of these compounds were established by GC-mass spectrometric comparisons of the trimethylsilylated methyl esters with authentic samples prepared from gibberellic acid (GA3).

Stomatal Closure in Flooded Tomato Plants Involves Abscisic Acid and a Chemically Unidentified Anti-Transpirant in Xylem Sap.

We address the question of how soil flooding closes stomata of tomato (Lycopersicon esculentum Mill. cv Ailsa Craig) plants within a few hours in the absence of leaf water deficits. Three hypotheses to explain this were tested, namely that (a) flooding increases abscisic acid (ABA) export in xylem sap from roots, (b) flooding increases ABA synthesis and export from older to younger leaves, and (c) flooding promotes accumulation of ABA within foliage because of reduced export. Hypothesis a was rejected because delivery of ABA from flooded roots in xylem sap decreased. Hypothesis b was rejected because older leaves neither supplied younger leaves with ABA nor influenced their stomata. Limited support was obtained for hypothesis c. Heat girdling of petioles inhibited phloem export and mimicked flooding by decreasing export of [14C]sucrose, increasing bulk ABA, and closing stomata without leaf water deficits. However, in flooded plants bulk leaf ABA did not increase until after stomata began to close. Later, ABA declined, even though stomata remained closed. Commelina communis L. epidermal strip bioassays showed that xylem sap from roots of flooded tomato plants contained an unknown factor that promoted stomatal closure, but it was not ABA. This may be a root-sourced positive message that closes stomata in flooded tomato plants.

Gibberellin concentration and transport in genetic lines of pea : effects of grafting.

Effects of the Na and Le loci on gibberellin (GA) content and transport in pea (Pisum sativum L.) shoots were studied. GA(1), GA(8), GA(17), GA(19), GA(20), GA(29), GA(44), GA(8) catabolite, and GA(29) catabolite were identified by full-scan gas chromatography-mass spectrometry in extracts of expanding and fully expanded tissues of line C79-338 (Na Le). Quantification of GAs by gas chromatography-single-ion monitoring using deuterated internal standards in lines differing at the Na and Le alleles showed that na reduced the contents of GA(19), GA(20), and GA(29) on average to <3% and of GA(1) and GA(8) to <30% of those in corresponding Na lines. In expanding tissues from Na le lines, GA(1) and GA(8) concentrations were reduced to approximately 10 and 2%, respectively, and GA(29) content increased 2- to 3-fold compared with those in Na Le plants. There was a close correlation between stem length and the concentrations of GA(1) or GA(8) in shoot apices in all six genotypes investigated. In na/Na grafts, internode length and GA(1) concentration of nana scions were normalized, the GA(20) content increased slightly, but GA(19) levels were unaffected. Movement of labeled GAs applied to leaves on Na rootstocks indicated that GA(19) was transported poorly to apices of na scions compared with GA(20) and GA(1). Our evidence suggests that GA(20) is the major transported GA in peas.

Identification, quantitation and distribution of gibberellins in fruits of Pisum sativum L. cv. Alaska during pod development.

In addition to the previously-reported gibberellins: GA1; GA8, GA20 and GA29 (García-Martínez et al., 1987, Planta 170, 130-137), GA3 and GA19 were identified by combined gas chromatography-mass spectrometry in pods and ovules of 4-d-old pollinated pea (Pisum sativum cv. Alaska) ovaries. Pods contained additionally GA17, GA81 (2α-hydroxy GA20) and GA29-catabolite. The concentrations of GA1, GA3, GA8, GA19, GA20 and GA29 were higher in the ovules than in the pod, although, with the exception of GA3, the total content of these GAs in the pod exceeded that in the seeds. About 80% of the GA3 content of the ovary was present in the seeds. The concentrations of GA19 and GA20 in pollinated ovaries remained fairly constant for the first 12 ds after an thesis, after which they increased sharply. In contrast, GA1 and GA3 concentrations were maximal at 7 d and 4-6 d, respectively, after anthesis, at about the time of maximum pod growth rate, and declined thereafter. Emasculated ovaries at anthesis contained GA8, GA19 and GA20 at concentrations comparable with pollinated fruit, but they decreased rapidly. Gibberellins a1 and A3 were present in only trace amounts in emasculated ovaries at any stage. Parthenocarpic fruit, produced by decapitating plants immediately above an emasculated flower, or by treating such flowers with 2,4-dichlorophenoxyacetic acid or GA7, contained GA19 and GA20 at similar concentrations to seeded fruit, but very low amounts of GA1 and GA3 Thus, it appears that the presence of fertilised ovules is necessary for the synthesis of these last two GAs. Mature leaves and leaf diffusates contained GA1, GA8, GA19 and GA20 as determined by combined gas chromatography-mass spectrometry using selected ion monitoring. This provides further evidence that vegetative tissues are a possible alternative source of GAs for fruit-set, particularly in decapitated plants.

Comparison of gibberellins in normal and slender barley seedlings.

Gibberellins A(1), A(3), A(8), A(19), A(20), and A(29) were identified by full scan gas chromatography-mass spectrometry in leaf sheath segments of 7-day-old barley (Hordeum vulgare L. cv Golden Promise) seedlings grown at 20 degrees C under long days. In a segregating population of barley, cv Herta (Cb 3014), containing the recessive slender allele, (sln 1) the concentration of GA(1) and GA(3) was reduced by 10-fold and 6-fold, respectively, in rapidly growing homozygous slender, compared with normal, leaf sheath segments. However, the concentration of the C(20) precursor, GA(19), was nearly 2-fold greater in slender than in normal seedlings. There was little difference in the ABA content of sheath segments between the two genotypes. The gibberellin biosynthesis inhibitor, paclobutrazol, reduced the final sheath length of normal segregants (50% inhibition at 15 micromolar) but had no effect on the growth of slender seedlings at concentrations below 100 micromolar. There was a 15-fold and 4-fold reduction in GA(1) and GA(3), respectively, in sheath segments of 8-day-old normal seedlings following application of 10 micromolar paclobutrazol. The same treatment also reduced the already low concentrations of these gibberellins in slender segregants. The results show that the pool sizes of gibberellins A(1) and A(3) are small in slender barley and that leaf sheath extension in this genotype appears to be gibberellin-independent. The relationship between gibberellin status and tissue growth-rate in slender barley is contrasted with other gibberellin nonresponsive, but dwarf, mutants of wheat (Triticum aestivum) and maize (Zea mays).