Stem elongation and gibberellins in alpine and prairie ecotypes of Stellaria longipes

The potential for gibberellins (GAs) to control stem elongation and itsplasticity (range of phenotypic expression) was investigated inStellaria longipes grown in long warm days. Gibberellinmetabolism and sensitivity was compared between a slow-growing alpine dwarfwithlow stem elongation plasticity and a rapidly elongating, highly plastic prairieecotype. Both ecotypes elongated in response to exogenous GA₁,GA₄ or GA₉, but surprisingly, the alpine dwarf wasrelatively unresponsive to GA₃. Endogenous GA₁,GA₃, GA₄, GA₅, GA₈, GA₉and GA₂₀ were identified and quantified in stem tissue harvested atcommencement, middle and end of the period of most rapid elongation. Theconcentration of GAs which might be expected to promote shoot elongation washigher during rapid elongation than toward its end for both ecotypes. Whilethere was a trend for certain GAs (GA₃, GA₄,GA₉, GA₂₀) to be higher in stems of the alpine ecotypeduring rapid elongation, that result does not explain the slower growth of thealpine ecotype and the faster growth of the prairie ecotype under a range ofconditions. To determine if the two ecotypes metabolized GA₂₀differently, plants were fed [²H]- or[³H]-GA₂₀. The metabolic products identified included[²H₂]-GA₁, -GA₈, -GA₂₉,-GA₆₀, -3-epi-GA₁, GA₁₁₈(-1-epi-GA₆₀) and -GA₇₇. The concentration of[²H₂]-GA₁ also did not differ between the twoecotypes and metabolism of [²H₂]- or[³H]-GA₂₀ was also similar. In the same experiments thepresence of epi-GA₁, GA₂₉, GA₆₀,GA₁₁₈ and GA₇₇ was indicated, suggesting that these GAsmay also occur naturally in S. longipes, in addition tothose described above. Collectively, these results suggest that while stemelongation within ecotypes is likely regulated by GAs, differences in GAcontent, sensitivity to GAs (GA₃ excepted), or GA metabolism areunlikely to be the controlling factor in determining the differences seen ingrowth rate between the two ecotypes under the controlled environmentconditionsof this study. Nevertheless, further study is warranted especially underconditions where environmental factors may favour a GA:ethylene interaction.     

The apple gene responsible for columnar tree shape reduces the abundance of biologically active gibberellin

Ectopic expression of the apple 2-oxoglutarate-dependent dioxygenase (DOX, 2ODD) gene, designated MdDOX-Co, is thought to cause the columnar shape of apple trees. However, the mechanism underlying the formation of such a unique tree shape remains unclear. To solve this problem, we demonstrated that Arabidopsis thaliana overexpressing MdDOX-Co contained reduced levels of biologically active gibberellin (GA) compared with wild type. In summary: (i) with biochemical approaches, the gene product MdDOX-Co was shown to metabolize active GA A₄ (GA₄) to GA₅₈ (12-OH-GA₄) in vitro. MdDOX-Co also metabolized its precursors GA₁₂ and GA₉ to GA₁₁₁ (12-OH-GA₁₂) and GA₇₀ (12-OH-GA₉), respectively; (ii) Of the three 12-OH-GAs, GA₅₈ was still active physiologically, but not GA₇₀ or GA₁₁₁; (iii) Arabidopsis MdDOX-Co OE transformants converted exogenously applied deuterium-labeled (d₂)-GA₁₂ to d₂-GA₁₁₁ but not to d₂-GA₅₈, whereas transformants converted applied d₂-GA₉ to d₂-GA₅₈; (iv) GA₁₁₁ is converted poorly to GA₇₀ by GA 20-oxidases in vitro when GA₁₂ is efficiently metabolized to GA₉; (v) no GA₅₈ was detected endogenously in MdDOX-Co OE transformants. Overall, we conclude that 12-hydroxylation of GA₁₂ by MdDOX-Co prevents the biosynthesis of biologically active GAs in planta, resulting in columnar phenotypes.     

Interconversion of gibberellin A4 to gibberellins A1 and A34 by dwarf rice,cultivar Tan-ginbozu

Summary Interconversion of GA₄ to GA₁ and GA₃₄ occurred within 24 h of application of 1,2-[³H]-GA₄ to seedlings of dwarf rice, cv. Tan-ginbozu. Identification was made by direct comparison of the trimethylsilyl ether derivatives of the methyl esters of Silica-gel partition-column fractions on gas-liquid radiochromatography with derivatized GA₁ and GA₃₄ standards on three columns: 2% QF-1, 2% SE-30, and 1% XE-60. GA₂, an artifact of the purification and chromatography system, may also be formed by the plant. The conversions from GA₄ to GA₁ and GA₃₄ are single hydroxylations. At least two unidentified radioactive products were also formed by the plant. Interconversions were in the order of 0.3 to 0.8% of applied [³H]-GA₄.Abbreviations GA gibberellin - GLC gas-liquid chromatography - GLRC gasliquid radiochromatography - TMSMe trimethylsilyl ether of methyl ester - TLC thin-layer chromatography     

Lack of influence of photoperiod on the metabolism of gibberellin A20 in Salix pentandra

The influence of photoperiod on the metabolism of GA₂₀ in Salix pentandra was studied by feeding [³H]-GA₂₀ to seedlings which had been grown previously under long day (LD) or short day (SD) conditions. After 48 h in LD or SD, metabolites were separated on sequential, silica gel partition columns and reversed-phase C₁₈ HPLC. The principal metabolite co-chromatographed with [³H]-GA₁ and this conversion was confirmed by feeding [²H]-GA₂₀, which was converted to [²H]-GA₁ as identified by gas chromatography-selected ion monitoring. Chromatographic evidence also indicated the minor conversion of [³H]-GA₂₀ to [³H]-GA₈ (via [³H]-GA₁) and trace conversion to [³H]-GA₂₉ (GAs A_1.8,20.29 are native in Salix). Ethyl acetate-insoluble [³H] metabolites were formed and could be cleaved by cellulase to release putative [³H]-GA₂₀ and [³H]-GA₁ suggesting the conversion to glucosyl conjugates of these GAs. Metabolism of [³H]-GA₂₀ was slightly more rapid in plants previously grown under LD than SD, an effect which reflected the generally increased shoot growth under LD. However, altering the photoperiod after [³H]-GA₂₀ addition had only a slight effect on the metabolism of [³H]-GA₂₀ in Salix seedlings. This indicates that the conversion of GA₂₀ to GA₁ is not a controlling step in the photoperiodic regulation of growth cessation in Salix.     

Endophytes Aspergillus caespitosus LK12 and Phoma sp. LK13 of Moringa peregrina produce gibberellins and improve rice plant growth

Two new strains of endophytic fungi were isolated from the bark of Moringa peregrina and identified as Aspergillus caespitosus LK12 and Phoma sp. LK13. These endophytes were identified through amplifying polymerase chain reaction (PCR) and sequencing the 18S internal transcribed spacer of DNA extracted from both endophytes. Pure cultures of endophytic fungi were subjected to extract and isolate gibberellins (GAs). Deuterated standards of [17,17-²H₂]-GA₁, [17,17-²H₂]-GA₃, [17, 17-²H₂]-GA₄ and [17, 17-²H₂]-GA₇ were used to quantify the endophytic fungal GAs. The analysis revealed that both the endophytes are producing bioactive GAs in various quantities (ng mL^?1). A. caespitosus LK12 was producing GA₁ (54.51 ± 1.23), GA₄ (26.5 ± 0.65), and GA₇ (2.87 ± 1.23) while Phoma sp. LK13 was secreting GA₁ (4.8 ± 0.12), GA₃ (8.65 ± 0.21), GA₄ (23.7 ± 0.98), and GA₇ (22.7 ± 0.73). The culture filtrate (CF) of A. caespitosus and Phoma sp. significantly increased the shoot length of GAs-deficient mutant waito-c and normal Dongjin-beyo rice seedlings as compared to control. Application of such growth-promoting and GAs-producing endophytes can ameliorate poorly growing crop plants.     

Bacterial endophyte Sphingomonas sp. LK11 produces gibberellins and IAA and promotes tomato plant growth

Plant growth promoting endophytic bacteria have been identified as potential growth regulators of crops. Endophytic bacterium, Sphingomonas sp. LK11, was isolated from the leaves of Tephrosia apollinea. The pure culture of Sphingomonas sp. LK11 was subjected to advance chromatographic and spectroscopic techniques to extract and isolate gibberellins (GAs). Deuterated standards of [17, 17-²H₂]-GA₄, [17, 17-²H₂]-GA₉ and [17, 17-²H₂]-GA₂₀ were used to quantify the bacterial GAs. The analysis of the culture broth of Sphingomonas sp. LK11 revealed the existence of physiologically active gibberellins (GA₄: 2.97 ± 0.11 ng/ml) and inactive GA₉ (0.98 ± 0.15 ng/ml) and GA₂₀ (2.41 ± 0.23). The endophyte also produced indole acetic acid (11.23 ± 0.93 μM/ml). Tomato plants inoculated with endophytic Sphingomonas sp. LK11 showed significantly increased growth attributes (shoot length, chlorophyll contents, shoot, and root dry weights) compared to the control. This indicated that such phyto-hormones-producing strains could help in increasing crop growth.     

Light effects on endogenous levels of gibberellins in photoblastic lettuce seeds

Gibberellin A₁ (GA₁), 3-epi-GA₁ GA₁₇, GA₁₉, GA₂₀, and GA₇₇ were identified by Kovats retention indices and full-scan mass spectra from gas chromatography-mass spectrometry analysis of a purified extract of mature seeds of photoblastic lettuce (Lactuca sativa L. cv. Grand Rapids). Non-13-hydroxylated GAs such as GA₄ and GA₉ were not detected even by highly sensitive radioimmunoassay. These results show that the major biosynthetic pathway of GAs in lettuce seeds is the early-13-hydroxylation pathway leading to GA₁, which is suggested to be physiologically active in lettuce seed germination. Quantification of endogenous GAs in the lettuce seeds by gas chromatography-selected ion monitoring using deuterated GAs as internal standards indicated that the endogenous level of GA₁ increased to a level about three times that of dark control 6 h after a brief red light irradiation, and that far-red light given after red light suppressed the effect of red light. The contents of GA₂₀ and GA₁₉ were not affected by the red light irradiation. Evidence is also presented that 3-epi-GA₁ is a native GA in the lettuce seeds.     

Conversion of gibberellin A1 into gibberellin A3 by the mutant R-9 of Gibberella fujikuroi

A mutant R-9 of Gibberella fujikuroi has been isolated and shown to be blocked for GA₁ and GA₃ biosynthesis, but not for GA₄, G_A7 and other gibberellins. Cultures of this mutant convert low concentrations of [1,2-³H2]-GA₁ into GA₃ in a radiochemical yield of 2·7 %.     

Effect of Photoperiod on Metabolism of [H] Gibberellins A(1), 3-epi-A(1), and A(20) in Agrostemma githago L

To determine whether daylength influences the rate of metabolism of gibberellins (GAs) in the long-day (LD) rosette plant Agrostemma githago L., [³H]GA₂₀ and [³H]GA₁ were applied under short day (SD) and LD. Both were metabolized faster under LD than under SD. [³H]GA₂₀ was metabolized to a compound chromatographically identical to 3-epi-GA₁. [³H]GA₁ was metabolized to two acidic compounds, the major metabolite having chromatographic properties similar to, but not identical with GA₈. [³H]3-epi-GA₁ applied to plants under LD was metabolized much more slowly than was [³H]GA₁, and formed a very polar metabolite which did not partition into ethyl acetate at pH 2.5. Very polar metabolites were also formed after the feeds of [³H]GA₂₀ and [³H]GA₁. It was not possible to characterize these very polar compounds further because of their apparent instability. The results obtained suggest that in Agrostemma GA₂₀ is the precursor of 3-epi-GA₁, but there is at present no evidence indicating the precursor of GA₁.     

The Synthesis of (3H)-Gibberellin A9 with High Specific Activity

The synthesis of 2,3-(³H)-gibberellin A₉ (GA₉) with a specific activity of 47 Ci mmole^?1 is described. △^2,3GA₉ methyl ester epoxide was converted to (³H)-GA₉ methyl ester epoxide using carrier-free tritium gas. This product was de-epoxidized then hydrolysed to yield (³H)-GA₉.     

Stability and bioactivity of gibberellic acid in different solvents

Summary HPLC chromatograms indicated that transformation of gibberellic acid (GA₃) to the corresponding dicarboxylic acid via iso-GA₃ occurred in weak alkaline solution. The bioactivity of this dicarboxylic acid was about 20% that of GA₃ and above 0.3 M NaOH this compound appeared to decompose. Aqueous solutions of NaOH cannot, therefore, be used as solvent in bioassays of GA₃ activity.     

Decomposition of aqueous solutions of gibberellic acid on autoclaving

A qualitative and quantitative analysis of the decomposition of unbuffered and buffered (pH 3–8) aqueous solutions of gibberellic acid (GA₃) on autoclaving is recorded. The identified products, which vary in composition with pH, are iso-GA₃ (II), iso-GA₃ hydroxy acid (III), gibberellenic acid (IV), allogibberic acid (V), epiallogibbe detected after autoclaving in all cases. The identified products, in all cases, account for not less than 95% of the decomposition product, Dehydroallogibberic acid has not previously been recorded as an aqueous decomposition product of GA₃ and its biological activity in the lettuce hypocotyl test is recorded.     

The effect of photoperiod on the levels of seven endogenous gibberellins in the long-day plant Agrostemma githago L.

The following seven gibberellins (GAs) have been identified by gas chromatography-mass spectrometry in shoots and leaves of the long-day plant Agrostemma githago: GA₅₃, GA₄₄, GA₁₉, GA₁₇, GA₂₀, GA₁, and 3-epi-GA₁. The levels of these compounds were measured, using selected ion monitoring, during photoperiodic induction. The levels of GA₄₄, GA₁₉, GA₁₇, and GA₂₀ all increased to a peak at eight long days (LD), followed by a decline, while the levels of GA₁ and 3-epi-GA₁ did not reach a peak until 12 LD. The level of GA₅₃ remained steady over the first 10–12 LD. Later in the LD treatment the levels of GA₅₃, GA₄₄, GA₁₉, and GA₁₇ increased again. The rate of metabolism of all GAs except GA₅₃ was higher after 12–16 LD than under short days. These data thus provide indirect evidence for an effect of photoperiodic induction on GA turnover in A. githago.Abbreviations AMO-1618 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine-carboxylate methyl chloride - GA(s) gibberellin(s) - GC-MS gas chromatography-mass spectrometry - HPLC high performance liquid chromatography - LD long day(s) - MeTMS trimethylsilylether of the methyl ester - SD short day(s) - SIM selected ion monitoring     

Gibberellin biosynthetic pathway and the physiologically active gibberellin in the shoot ofCucumis sativus L.

[²H₂]Gibberellin A₂₄ (GA₂₄) and [²H₄]-GA₉ were applied to the apices of normal-type cucumber (Cucumis sativus L. cv. Yomaki) seedlings treated with uniconazole, an inhibitor of GA biosynthesis. The metabolites from these feeds were identified by full-scan gas chromatography-mass spectrometry (GC-MS) to confirm the conversions of [²H₂]GA₂₄ to [²H₂]GA₉ and of [²H₄]GA₉ to [²H₄]GA₄. The results show that GA₄ is biosynthesized from GA₂₄ via GA₉. In a cucumber hypocotyl elongation bioassay using cv. Yomaki, prohexadione (DOCHC), an inhibitor of 2-oxoglutaratedependent dioxygenase, inhibited the hypocotyl elongation caused by application of GA₉, while DOCHC enhanced the elongation caused by application of GA₄. These results indicate that GA₄ is a physiologically active GA and that the activity of GA₉ is due to its conversion to GA₄ in cucumber shoots.     

The transport and metabolism of gibberellins A1 and A5 in excised segments from internodes of Phaseolus coccineus

Jacobs, W. P., Beall, F. D. and Pharis, R. P. 1988. The transport and metabolism of gibberellins A₁ and A₅ in excised segments from internodes of Phaseolus coccineus. -Physiol. Plant. 72: 529–534. The transport and metabolism of gibberellins (GAs) ([³H]-GA, and [³H]-GA₅) of high specific radioactivity were investigated in excised segments from young internodes of Phaseolus coccineus L. Both GA₁ and GA₅ are native to this species and present in shoot tissue. The segments, 5.1 mm long, were incubated for 6 h in the horizontal position with agar donor blocks containing the [³H]-GA on the morphological apical or basal ends and with plain agar receiver blocks on the opposite end. At the end of incubation, the individual agar blocks were analyzed immediately for total radioactivity, or both blocks and intervening tissue were frozen and freeze-dried for later chromatographic analysis. The movement of both [³H]-GA, and [³H]-GA₅ was found to be consistently without polarity. However, approximately 5-fold more [³H]-GA, than [³H]-GA₅ was transported through the Phaseolus segments into receivers when equal amounts were in the donors. The extractable radioactivity from receiver blocks was primarily that of the donor GA. No putative GA conjugates were found in any class of receivers, but more GA metabolites were found in the free acid fraction from acropetal than basipetal receivers. Chromatographic analysis by reversed phase C₁₈ high performance liquid chromatography of the tissue segments showed that [³H]-GA, was metabolized more than [³H]-GA₅. Tissue adjacent to receiver blocks contained not only the precursor GA from the donor, but also polar ‘free GA metabolites’ and putative GA glucosyl conjugates. These results provide evidence that GA., which is the known ‘effector’ GA for elongation in shoot tissue of several species, is more effectively transported than GA₅ (a known precursor of GA₁) or than GA₁s more polar metabolites.     

Qualitative and Quantitative Analysis of Endogenous Gibberellins in Onion Plants and Their Effects on Bulb Development

Evidence has been reported that bulb development in onion plants (Allium cepa L.) is controlled by endogenous bulbing and anti-bulbing hormones, and that gibberellin (GA) is a candidate for anti-bulbing hormone (ABH). In this study, we identified a series of C-13-H GAs (GA₁₂, GA₁₅, GA₂₄, GA₉, GA₄, GA₃₄, and 3-epi-GA₄) and a series of C-13-OH GAs (GA₄₄, GA₂₀, GA₁ and GA₈) from the leaf sheaths including the lower part of leaf blades of onion plants (cv. Senshu-Chuko). These results suggested that two independent GA biosynthetic pathways, the early-non-hydroxylation pathway to GA₄ (active GA) and early-13-hydroxylation pathway to GA₁ (active GA), exist in onion plants. It was also suggested that GA₄ and GA₁ have almost the same ability to inhibit bulb development in onion plants induced by treatment with an inhibitor of GA biosynthesis, uniconazole-P. The endogenous levels of GA₁ and GA₄, and their direct precursors, GA₂₀ and GA₉, in leaf blades, leaf sheaths, and roots of 4-week-old bulbing and non-bulbing onion plants were measured by gas chromatography/selected ion monitoring with the corresponding [²H]labeled GAs as internal standards. In most cases, the GA levels in long-day (LD)-grown bulbing onion plants were higher than those of short-day (SD)-grown non-bulbing onion plants, but the GA₁ level in leaf blades of SD-grown onion plants was rather higher than that of LD-grown onion plants. Relationship between the endogenous GAs and bulb development in onion plants is discussed.     

Interconversion of gibberellin A5 to gibberellin 3 in seedlings of dwarf Pisum sativum

[³H]-Gibberellin A₅ ([³H]-GA₅) applied to seedlings of dark-grown dwarf pea (Pisum sativum L. cv. Meteor), was converted to two acidic compounds, GA₃ and a chromatographically similar unknown. Identification of GA₃ was made by gas-liquid radiochromatography using three stationary phases.     

Gibberellin biosynthetic pathway and the physiologically active gibberellin in the shoot ofCucumis sativus L.

[²H₂]Gibberellin A₂₄ (GA₂₄) and [²H₄]-GA₉ were applied to the apices of normal-type cucumber (Cucumis sativus L. cv. Yomaki) seedlings treated with uniconazole, an inhibitor of GA biosynthesis. The metabolites from these feeds were identified by full-scan gas chromatography-mass spectrometry (GC-MS) to confirm the conversions of [²H₂]GA₂₄ to [²H₂]GA₉ and of [²H₄]GA₉ to [²H₄]GA₄. The results show that GA₄ is biosynthesized from GA₂₄ via GA₉. In a cucumber hypocotyl elongation bioassay using cv. Yomaki, prohexadione (DOCHC), an inhibitor of 2-oxoglutaratedependent dioxygenase, inhibited the hypocotyl elongation caused by application of GA₉, while DOCHC enhanced the elongation caused by application of GA₄. These results indicate that GA₄ is a physiologically active GA and that the activity of GA₉ is due to its conversion to GA₄ in cucumber shoots.     

The relative significance for stem elongation and flowering in Lolium temulentum of 3β-hydroxylation of gibberellins

In previous experiments with many gibberellins (GAs) and GA derivatives applied to Lolium temulentum L., quite different structural requirements were evident for stem elongation on the one hand and for the promotion of flowering on the other. Whereas hydroxylation at carbons 12, 13 and 15 enhanced flowering relative to stem growth, the reverse was the case at carbon 3 (L.T. Evans et al. 1990, Planta 182, 97–106). The significance of hydroxylation at carbon 3 is examined in this paper. The application of inhibitors of 3β-hydroxylation, including C/D-ring-rearranged GAs, reduced stem growth but, in the case of the two acylcyclohexanediones, increased the flowering response when applied on the inductive long day. Later applications of the acylcyclohexanediones, made after floral initiation had occurred, were inhibitory to flowering, suggesting that subsequent inflorescence development requires 3β-hydroxylated GAs. Applications of the 3α-hydroxy epimers of GA₁, GA₃ and GA₄ gave slightly less promotion of flowering in comparison with the 3β-hydroxy GAs, but far less promotion of stem elongation, except in the case of 3-epi-GA₄, which was comparable to GA₄. The 3α-hydroxy epimer of 2,2-dimethyl GA₄ gave less promotion of flowering than its 3β-hydroxy epimer but almost no promotion of stem elongation. The 3α-hydroxy epimers of GA₃ and 2,2-dimethyl GA₄ did not act as competitive inhibitors of the stem elongation elicited by GA₃ and 2,2-dimethyl GA₄, respectively. These results extend the differences in GA structure which favour flowering as opposed to stem elongation, and indicate that 3-hydroxylation and its epimeric configuration are of much greater importance to stem elongation than to flower initiation in Lolium.     

Activity of synthetic gibberellin A15 and (±)-gibberellin A15isolactone

The activities of (±)-gibberellin A₁₅ ((±)-GA₁₅) and (±)-gibberellin A₁₅-isolactone ((±)-iso-GA₁₅) which were obtained by stereocontrolled total synthesis and gibberellin A₁₅ (E-GA₁₅) synthesized by interconversion of enmein were assayed by the rice seedling test. As expected, (±)-GA₁₅ showed half the activity of natural gibberellin A₁₅ (GA₁₅). E-GA₁₅ which has a natural configuration showed the same activity as natural gibberellin A₁₅ while (±)-iso-GA₁₅ was almost inactive. These samples were also submitted to the cucumber hypocotyl assay. Contrary to what has already been reported, they were almost inactive.