Early GA Research Articles

Regulation of the early GA biosynthesis pathway in pea.

The early steps in the gibberellin (GA) biosynthetic pathway are controlled by single copy genes or small gene families. In pea (Pisum sativum L.) there are two ent-kaurenoic acid oxidases, one expressed only in the seeds, while ent-copalyl synthesis and ent-kaurene oxidation appear to be controlled by single copy genes. None of these genes appear to show feedback regulation and the only major developmental regulation appears to be during seed development. During shoot maturation, transcript levels do not change markedly with the result that all the three genes examined are expressed in mature tissue, supporting recent findings that these tissues can synthesise GAs. It therefore appears that the regulation of bioactive GA levels are determined by the enzymes encoded by the 2-oxoglutarate-dependent dioxygenase gene families controlling the later steps in GA biosynthesis. However the early steps are nonetheless important as a clear log/linear relationship exists between elongation and the level of GA1 in a range of single and double mutants in genes controlling these steps.

Distinct cell-specific expression patterns of early and late gibberellin biosynthetic genes during Arabidopsis seed germination.

Gibberellins (GAs) are biosynthesized through a complex pathway that involves several classes of enzymes. To predict sites of individual GA biosynthetic steps, we studied cell type-specific expression of genes encoding early and late GA biosynthetic enzymes in germinating Arabidopsis seeds. We showed that expression of two genes, AtGA3ox1 and AtGA3ox2, encoding GA 3-oxidase, which catalyzes the terminal biosynthetic step, was mainly localized in the cortex and endodermis of embryo axes in germinating seeds. Because another GA biosynthetic gene, AtKO1, coding for ent-kaurene oxidase, exhibited a similar cell-specific expression pattern, we predicted that the synthesis of bioactive GAs from ent-kaurene oxidation occurs in the same cell types during seed germination. We also showed that the cortical cells expand during germination, suggesting a spatial correlation between GA production and response. However, promoter activity of the AtCPS1 gene, responsible for the first committed step in GA biosynthesis, was detected exclusively in the embryo provasculature in germinating seeds. When the AtCPS1 cDNA was expressed only in the cortex and endodermis of non-germinating ga1-3 seeds (deficient in AtCPS1) using the AtGA3ox2 promoter, germination was not as resistant to a GA biosynthesis inhibitor as expression in the provasculature. These results suggest that the biosynthesis of GAs during seed germination takes place in two separate locations with the early step occurring in the provasculature and the later steps in the cortex and endodermis. This implies that intercellular transport of an intermediate of the GA biosynthetic pathway is required to produce bioactive GAs.

Cone enhancement in Abies amabilis using GA 4/7, fertilizer, girdling and tenting

The demand for Abies amabilis (Dougl. Ex Loud) Dougl. Ex Forbes, Pacific silver fir seed has increased in British Columbia in recent years but the supply of wild seed is limited due to periodic cone crops and difficulty and high cost of cone harvest. Several clonal seed orchards have been established but cone production is very low. A study was undertaken to enhance cone production in a clonal seed orchard grafted in 1984. Five treatments were applied with 20 trees per treatment. Treatments began on 20 May 1998. Each tree was given two applications of GA 4/7 at 1 week interval, 20 and 27 May (early application) or 27 May and 3 June (late application). Ramets in treatment 2 received early GA 4/7 application+ fertilizer+ girdling . Ramets in treatment 3 received a similar treatment but given late. Ramets in treatment 4 received early GA 4/7 application+ fertilizer+ girdling+ tenting . Ramets in treatment 5 received a similar treatment but given late. Ramets in treatment 1 received one injection of 80% ethanol but no GA 4/7 on 30 May. Each treatment had 10 trees in which vegetative bud burst was early and 10 trees in which bud burst was late. Pollen and seed cone abundance was measured in April 1999. Only 10% of control ramets produced seed cones (0.75 cones/ramet). In treatment 2, 45% of ramets produced seed cones (8.4 cones/ramet). In treatment 3, 35% of ramets produced seed cones (2.3 cones/ramet). In treatment 4, 70% of ramets produced seed cones (23.1 cones/ramet). In treatment 5, 75% of ramets produced seed cones (21.2 cones/ramet). Pollen cone enhancement was statistically significant for treatments 2–5 over controls but production was not as dramatic as for seed cones because most control ramets produced some pollen cones.

Gibberellins in internodes and ears of wheat containing different dwarfing alleles

Gibberellins (GAs) belonging to the main early 13-hydroxylation GA pathway (GA 1, GA 3, GA 8, GA 17, GA 19, GA 20, GA 29, GA 44 and GA 53) were identified by full-scan GC-mass spectrometry in rapidly expanding stem internodes and young ears of wheat. In addition, GA 97 (2β-hydroxy GA 53) and GA 98 (2β-hydroxy GA 44) were detected in reproductive, but not vegetative, tissues. Conversely, some members of the non-13-hydroxylation GA pathway (GA 4, GA 9 and GA 34) were present in stem internodes but not in young ears. Later members of the main GA pathway (GA 19 → GA 20 → GA 1 → GA 8) were quantified by GC-selected ion monitoring in stem internodes and ears. In the rht (tall) line, GA 19 ⋙ GA 20 ≈ GA 1 ⪡ GA 8 during the main phase of expansion growth of both stem internodes and ears, suggesting that the conversion of GA 19 to GA 20 is a rate-limiting step on the GA pathway. In vegetative stem internodes, GA 1 concentration increased in proportion to the severity of the Rht1 (semi-dwarf) and Rht3 (dwarf) phenotypes but, at certain stages of Rht3 internode development, the expected decrease in GA 19 content, relative to the GA-responsive, rht (tall) line, did not occur and both GA 19 and GA 1 accumulated. In very young ears of both tall and dwarf lines, the concentrations of GA 20 and GA 8 were high, suggesting low 3β-(GA 20 to GA 1) and high 2β-(GA 1 to GA 8) hydroxylase activities prior to the period of rachis elongation. The Rht alleles had no effect on either GA content or rates of ear rachis elongation during the main phase of ear growth, providing indirect evidence that Rht genes are not expressed in expanding wheat ears.

Flowering on long and short shoots of Larix laricina in response to differential timing of GA(4/7) applications.

In Larix, reproductive buds most often occur terminally on short shoots, but they can also differentiate in lateral positions on long shoots. The phenology of long and short shoots differs considerably, with short shoots breaking bud and expanding about 5 weeks before the start of long shoot extension. Foliar sprays of GA(4/7) were applied to 160 branches on 10 greenhouse-grown Larix laricina (Du Roi) K. Koch grafts either before (early) of after (late) the start of long shoot extension, or during both periods, to test whether the timing of GA(4/7) application affects flowering on short and long shoots. All three treatments induced flowering on both long and short shoots. The early GA(4/7) treatment led to slightly, but not significantly, more flowering on short shoots than on long shoots, whereas the late GA(4/7) treatment resulted in increased flowering on both types of shoots, but primarily on long shoots. Application of GA(4/7) during both periods did not result in increased flowering over the early or late treatment alone. Based on the fact that gibberellins are metabolized rapidly in conifers and our finding that GA(4/7) applied before shoot elongation, when the bud primordia were at a very early stage of development (detectable bud differentiation only occurred several weeks later), induced flowering on long shoots, we conclude that the early GA(4/7) treatment did not affect differentiation as it was occurring, but somehow predisposed the bud primordia to differentiate reproductively.

Gibberellins and ?-amylase gene expression in germinating wheat grains

Gibberellins (GAs), GA8, GA17, GA19, GA20, GA29, and GA79 were identified by full-scan gas chromatography-mass spectrometry in a purified acidic fraction and GA8, GA20, GA79, and GA90 in a hydrolysed conjugate fraction from mature wheat grains. Gibberellin A20-13-O-glucoside was also quantified directly as the permethyl derivative in dry seed. The scutellum was identified as the major site of de novo GA biosynthesis by measuring ent-kaurene accumulation in vivo in grains treated with 2S,3S-paclobutrazol. Several GAs of the early 13-hydroxylation GA pathway began to accumulate in the axis and scutellum between 24 and 48 hours in untreated grains germinated at 15°C. Bioactive GA1 and GA3 also increased in the endosperm during this period, whereas abscisic acid contents of embryo and endosperm declined rapidly over 48 hours following imbibition. Treating grains with 2S,3S-paclobutrazol reduced GA1 plus GA3 content of scutella by 70–80% over 4 days without affecting significantly the steady-state pool of α-amylase mRNA transcripts. In contrast, a 50–80% reduction in the content of bioactive GAs in the endosperm was associated with a 70–78% decrease in transcripts for both α-amylase gene families in aleurones of paclobutrazol-treated grains. It was concluded that the initiation of α-amylase gene expression in wheat scutella was independent of de novo GA biosynthesis, whereas that in the aleurone was largely dependent on embryo-produced GAs.

Identification of gibberellins in leaf tissues of strawberry (Fragaria � ananassa Duch.) grown under different photoperiods

In studies of the effect of long or short-day photoperiod treatments on the qualitative gibberellin (GA) content of mature leaves of a facultative short-day (SD) strawberry cultivar (Fragaria × ananassa Duch. cv. Elsanta), GA1, GA8, GA17, GA19, GA20, GA29 and GA44 were identified by full-scan gas chromatography - mass spectrometry (GC-MS) in extracts from plants grown under long-day (LD) conditions, and GA1, GA5, GA8, GA19, GA20 and GA29 in similar extracts from plants subjected to eight SD cycles after growth under LD conditions. The early 13-hydroxylation GA biosynthetic pathway thus appeared to predominate, with the apparent absence of GA5 in LD and of GA17 and GA44 in SD extracts providing evidence of modulation of this pathway by photoperiod. A search, including GC-MS with selected ion monitoring, failed to detect GA3, or the polyhydroxylated GA85, GA86, GA87 or GA32 for which some extracts were specifically purified.

Endogenous gibberellins in flushing buds of three deciduous trees: Alder, aspen, and birch

Endogenous gibberellins (GAs) were extracted from flushing (expanding) vegetative buds of river alder (Alnus tenuifolia), European white birch (Betula pendula), and aspen (Populus tremuloides) and identified by gas chromatography-mass spectrometry with full scans and/or selected ion monitoring. Five 13-hydroxylated GAs were detected from the three trees: GA1, 8, and 20 from alder, GA1, 8, 19 and 20 from aspen and GA1, 8, 19, 20, and 29 from birch. Thirteen other GAs previously detected in Salix or common in other plants were specifically investigated but not detected. The presence of GA1, its probable precursors GA19 and GA20, and its probable metabolite, GA8, suggests that the early 13-hydroxylated GA biosynthetic pathway is dominant in vegetative buds of these trees. Abundant endogenous GAs of these trees are similar to the principal GAs of willows (various Salix spp.) and poplars (various Populus spp.). This suggests similarities in the GA physiology and is consistent with a common role of GA1 as a regulator of shoot growth in woody angiosperms.

Effects of applications of gibberellic acid on late growth, maturation and pigmentation of the Clementine mandarin

Gibberellic acid (GA 3) applications were performed periodically from late September to late November on seedless Clementine mandarin ( Citrus clementina Hort. ex Tanaka) trees, and their influence on the fruit growth and pigmentation determined. Irrespective of the time of application, GA 3 had no effect on the growth of the fruit and its anatomical parts nor on juice content and composition, but early GA 3 applications reduced peel thickness at maturation. The pigment changes in the peel were retarded by GA 3, and this response was maximal when GA 3 was applied during the 10 day interval between the onset of chlorophyll degradation and the onset of carotenoid accumulation.

Implication of Gibberellins in Head Smut (Sporisorium reilianum) of Sorghum bicolor

The head smut fungus, Sporisorium reilianum ([Kuhn] Landon and Fullerton), was shown to reduce plant height in infected Sorghum bicolor ([L.] Moench) plants. The major reductions occurred in the internodes nearest the panicle and were more severe in naturally infected than in inoculated plants. Less affected plants developed reproductively sterile panicles, and eventually smutted panicles developed phyllodied growths which progressed into leafy shoots. Extracts of smutted, sterile, and healthy (control) panicles of field-grown plants exhibited gibberellin (GA)-like activity in the dwarf rice bioassay. When extracts were purified and assayed with deuterium-labeled GA standards by gas chromatography-mass spectrometry-selected ion monitoring (GC-MS-SIM), GA(1), GA(3), GA(19), GA(20), and GA(53) were detected based on coelution with the standards, identical Kovats retention index values, and matching ion masses and relative abundances for three major ions. In addition, based on published Kovats retention index values, ion masses, and relative abundance values, GA(4), GA(7), GA(8), GA(14), GA(29), and GA(44) were tentatively identified. Quantitative analysis revealed that panicles of healthy control plants contained from 60 to 100% higher total concentrations of GAs than panicles of smutted plants. These comparisons were most striking for the early 13-hydroxylation pathway precursors GA(53), GA(44), and GA(19) but not for GA(20). Extracts of S. reilianum sporidia and culture medium exhibited GA-like bioactivity, and GA(1) and GA(3) were detected based on GC-MS-SIM assay with (2)H-labeled internal standards. Quantitative analysis of these GAs showed increasing concentrations from 4 to 7 to 10 days of culture and a decline at 20 days. This is the first GC-MS-SIM detection of GAs in a non-Ascomycete fungus, and the disease symptoms and quantitative data suggested that fungal infection may interfere with biosynthesis of GAs by the host plant.

Off-season Bloom in ‘Temple’ Orange Repressed by Gibberellin1

Abstract One early treatment (late May) with 25 ppm gibberellic acid (GA3) strongly depressed summer and fall bloom and resulting off-season fruits of ‘Temple’ orange (Citrus reticulata Blanco × C. sinensis (L.) osbeck). A late June treatment failed to repress and even tended to increase the number of off-season fruits. A repeated treatment at both dates, had a smaller effect than a single early treatment. A possible effect of a late GA application on the persistence of off-season fruitlets is suggested. Girdling of a part of scaffold branches gave a response similar to that of the early GA treatment.

Ionic currents in the uterine smooth muscle.

1. Short segments of isolated longitudinal myometrium from the pregnant rate uterus have been studied in a double sucrose-gap voltage-clamp arrangement. The clamped segment averaged 65 mum times 240 mum times 100 mum, has an average total capacitance of 0-14 muF, and may contain 50-200 individual myometrial cells. 2. A significant resistance exists in series with the membrane, and limits theprecision of the quantitative information. However, it is argued that some qualitative and some comparative information is useful. 3. In Krebs-bicarbonate solution, depolarizing steps produced initial transient inward currents followed by delayed outward currents. 4. When [Na+]o was reduced by 50%, the equilibrium potential Ea shifted by an average of -17-6 mV, the maximum inward current was reduced to 0-5, the time to peak of the early current was delayed by 1-1 msec, and the maximum chord conductances for the early(Ga) and late (GK) currents remained unchanged as compared with those in normal [Na+]o. 5. When [Ca2+] was reduced to 25% of normal, Ea shifted by an average of -20-3 mV, the maximum inward current was reduced to 0-5, the time to peak was delayed 3-1 msec, and Ga was significantly reduced, while GK was unaffected. 6. The early current, and its tail when repolarization was imposed, reversed direction from inward to outward when [Na+]o was reduced from 143 mM to zero, with [Ca2+]o remaining constant at 1-9 mM. 7. From the observations in 4, 5 and 6, it was concluded that Na+ is the main charge carrier for the early current, and that Ca2+ is important in regulating Ga. 8. The late current is outwards when [K+]o equals 5-9 mM, but inwards in some voltage range when [K+]o was elevated to 120 or 148 mM. K+ is the main charge carrier for the late current. 9. The equilibrium potential for the late current, EK, is about 15 mV more negative than the natural resting potential. 10. Prolonged holding of the preparations at voltages that differ significantly from the natural resting potential tends to shift EK in a way consistent with passive changes in [K+]i by the holding current. 11. The steady-state inactivation of the early current, h, is unusual. Inward current is macimum around the resting potential, and declines with both hyperpolarizing and depolarizing changes. Half-inactivation occurred with about 9 mV depolarization and 15 mV hyperpolarization. 12. The instantaneous current-voltage relations of both early and late currents are linear. The chord conductances Ga and GKare similar in form to those in other tissues.