Gibberellins in immature seeds and dark-grown shoots of Pisum sativum

Gibberellins (GAs) A₁₇, A₁₉, A₂₀, A₂₉, A₄₄, 2OH-GA₄₄ (tentative) and GA₂₉-catabolite were identified in 21-day-old seeds of Pisum sativum cv. Alaska (tall). These GAs are qualitatively similar to those in the dwarf cultivar Progress No. 9 with the exception of GA₁₉ which does not accumulate in Progress seeds. There was no evidence for the presence of 3-hydroxylated GAs in 21 day-old Alaska seeds. Dark-grown shoots of the cultivar Alaska contein GA₁, GA₈, GA₂₀, GA₂₉, GA₈-catabolite and GA₂₉-catabolite. Dark-grown shoots of the cultivar Progress No.9 contain GA₈, GA₂₀, GA₂₉ and GA₂₉-catabolite, and the presence of GA₁ was strongly indicated. Quantitation using GAs labelled with stable isotope showed the level of GA₁ in dark-grown shoots of the two cultivars to be almost identical, whilst the levels of GA₂₀, GA₂₉ and GA₂₉-catabolite were significantly lower in Alaska than in Progress No. 9. The levels of these GAs in dark-grown shoots were 10²- to 10³-fold less than the levels in developing seeds. The 2-epimer of GA₂₉ is present in dark-grown-shoot extracts of both cultivars and is not thought to be an artefact.Abbreviations cv cultivar - GA_n gibberellin A_n - GC gas chromatography - GC-MS combined gas chromatographymass spectrometry - HPLC high-pressure liquid chromatography - KRI Kovats retention index - MeTMSi methyl ester trimethylsilyl ether     

Immunoaffinity techniques applied to the purification of gibberellins from plant extracts

The use of immunoaffinity columns containing anti-gibberellin (GA) antibodies for the selective purification of GAs in plant extracts is described. GA₁, GA₃, GA₄, GA₅, GA₇, and GA₉ conjugates to bovine serum albumin were synthesized and used to elicit anti-GA polyclonal antibodies (Abs) in rabbits. Protein A purified rabbit serum, containing a mixture of anti-GA Abs, was immobilized on matrices of Affi-gel 10 or Fast-Flow Sepharose 4B. Columns of these immunosorbents retained a wide range of C-19 GA methyl esters, but no C-20 GA methyl esters. Quantitative recovery of C-19 GA methyl esters was achieved from the columns, which, after reequilibration in buffer, could be reused up to 500 times. The immunosorbents were tested by examination of extracts from immature soybean and pea seeds. GAs were initially purified by passing the extracts through DEAE-cellulose and concentrating them on octadecylsilica. The extracts were methylated and further purified on the mixed anti-GA immunoaffinity columns. GAs were detected and quantified as methyl esters or methyl ester trimethylsilyl ethers by gas chromatography-mass spectrometry-selected ion monitoring. GA₇ was found in soybean seeds, 17 days after anthesis, at low levels (8.8 nanograms per gram fresh weight). C-19 GAs were examined in cotyledons, embryonic axes, and testae of G2 pea seeds harvested 20 days after anthesis. High levels of GA₂₀ and GA₂₉ were found in cotyledons (3580 and 310 nanograms per gram fresh weight, respectively) and embryonic axes (5375 and 1430 nanograms per gram) fresh weight, respectively). Lower levels of GA₉ were found in cotyledons and embryonic axes (147 and 161 nanograms per gram fresh weight, respectively). GA₉ was the major GA of testae at levels of 195 nanograms per gram fresh weight. Trace quantities of GA₂₀ and GA₅₁ were also observed in testae.     

Quantification of GA1, GA3, GA4, GA7, GA8, GA9, GA19 and GA20; and GA20 metabolism in dormant and non-dormant beechnuts

The endogenous levels of GA₁, GA₃, GA₄, GA₇, GA₈, GA₉, GA₁₉ and GA₂₀ were determined in beech seeds (Fagus sylvatica L.) treated with different dormancy breaking treatments. Gibberellins were analysed separately in cotyledons and embryo axes. After purification of the extracts, GAs were quantified by GC-MS-selected ion monitoring (GC-MS-SIM) with deuterated GAs as internal standards. The results showed that GAs corresponding to the 13-OH pathway seemed to be involved in dormancy breaking. Strong differences in GA₁, GA₃, GA₈, GA₁₉ and GA₂₀ levels between embryo axes and cotyledons of dormant and non-dormant beechnuts were detected with less pronounced differences for GA₄, GA₇ and GA₉ levels. Both the quantitative differences between dormant and non-dormant seeds in the analysed GAs corresponding to the 13-OH pathway, and the capacity of non-dormant seeds to carry out metabolic conversions when labelled GA₂₀ was injected into the seeds, reveal a dynamic role of GAs in dormancy release.     

Metabolism of gibberellins in a cell-free system from immature seeds of Phaseolus vulgaris L.

The biosynthetic steps from gibberellin A₁₂-aldehyde (GA₁₂-aldehyde) to C₁₉-GAs were studied by means of a cell-free system from the embryos of immature Phaseolus vulgaris seeds. Stable-isotope-labeled GAs were used as substrates and the products were identified by gas chromatography-mass spectrometry. Gibberellin A₁₂-aldehyde was converted to GA₄ via non-hydroxylated intermediates and to GA₁ via 13-hydroxylated intermediates. 13-Hydroxylation took place at the beginning of the pathway by the conversion of GA₁₂-aldehyde to GA₅₃-aldehyde. The conversion of GA₂₀ to GA₅ and GA₆ was also shown but no 2-hydroxylating activity was found. Endogenous GAs from embryos and testas of 17-dold seeds were re-examined by gas chromatography-selected ion monitoring using stable-isotopelabeled GAs as internal standards. Gibberellins A₉, A₁₂, A₁₅, A₁₉, A₂₃, A₂₄, and A₅₃ were identified for the first time in P. vulgaris, in addition to GA₁, GA₄, GA₅, GA₆, GA₈, GA₁₇, GA₂₀, GA₂₉, GA₃₇, GA₃₈ and GA₄₄, which were previously known to occur in this species. The levels of all GAs, except the 2-hydroxylated ones, were greater in the embryos than in the testas. Conversely, the contents of GA₈ and GA₂₉, both 2-hydroxylated, were much higher in the testas than in the embryos.Abbreviations GA_n gibberellin A_n - GC-MS gas chromatography-mass spectrometry - GC-SIM gas chromatography-selected ion monitoring - HPLC high-performance liquid chromatography - TLC thin-layer chromatography - m/z ion of mass     

Endophytic Cephalotheca sulfurea AGH07 reprograms soybean to higher growth

Abstract

Gibberellins (GAs) are well known for plant growth promotion. GAs production by fungi has received little attention, although substantial work has been carried out on other aspects of plant growth-promoting fungi (PGPF). We investigated GAs production and plant growth-promoting capacity of an endophytic fungus isolated from the roots of soil grown soybean plants. The endophytic fungus is reported as GAs producer and as PGPF for the first time in this study. Nine endophytic isolates were collected from the roots of soybean, and culture filtrates (CFs) obtained from their pure cultures were screened on Waito-C, a dwarf rice cultivar, for the presence of GAs. Of these, seven fungal isolates promoted shoot length as compared to control (distilled water), while one inhibited it. Three fungal isolates were selected on the basis of higher shoot elongation as compared to wild type Gibberella fujikuroi, which was used as positive control. The growth-prompting capacity of selected fungal isolates SB5-1, SB3-2, and SB3-3 was bio-assayed on soybean cv. Hwangkeumkong. Fungal isolate SB5-1 provided maximum plant height (31.6 cm), shoot length (21.1 cm), whole plant fresh biomass (2.41 g), shoot fresh biomass (1.99 g), and leaf area (24.37 cm²). The CF of isolate SB5-1 was analyzed for the presence of GAs, and it was found that all physiologically active GAs were present (GA₁, 0.15 ng/ml, GA₃, 1.2 ng/ml, GA₄, 7.37 ng/ml, and GA₇, 3.18 ng/ml) in conjunction with physiologically inactive GA₅, GA₉, GA₁₅, GA₁₉, GA₂₀, and GA₂₄. The fungal isolate SB5-1 was identified as a new strain of Cephalotheca sulfurea through molecular and phylogenetic approaches.     

Gibberellins in Leaves of Alstroemeria hybrida: Identification and Quantification in Relation to Leaf Age

In alstroemeria (Alstroemeria hybrida), leaf senescence is retarded effectively by the application of gibberellins (GAs). To study the role of endogenous GAs in leaf senescence, the GA content was analyzed by combined gas chromatography and mass spectrometry. Five 13-hydroxy GAs (GA₁₉, GA₂₀, GA₁, GA₈, and GA₂₉) and three non-13-hydroxy GAs (GA₉ and GA₄) were identified in leaf extracts by comparing Kováts retention indices (KRIs) and full scan mass sprectra with those of reference GAs. In addition, GA₁₅, GA₄₄, GA₂₄, and GA₃₄ were tentatively identified by comparing selected ion monitoring results and KRIs with those of reference GAs. A number of GAs were detected in conjugated form as well. Concentrations of GAs in alstroemeria changed with the development of leaves. The proportion of biologically active GA₁ and GA₄ decreased with progressive senescence and the fraction of conjugated GAs increased. Received May 26, 1997; accepted August 12, 1997     

Overexpression of 20-Oxidase Confers a Gibberellin-Overproduction Phenotype in Arabidopsis

In the gibberellin (GA) biosynthesis pathway, 20-oxidase catalyzes the oxidation and elimination of carbon-20 to give rise to C₁₉-GAs. All bioactive GAs are C₁₉-GAs. We have overexpressed a cDNA encoding 20-oxidase isolated from Arabidopsis seedlings in transgenic Arabidopsis plants. These transgenic plants display a phenotype that may be attributed to the overproduction of GA. The phenotype includes a longer hypocotyl, lighter-green leaves, increased stem elongation, earlier flowering, and decreased seed dormancy. However, the fertility of the transgenic plants is not affected. Increased levels of endogenous GA₁, GA₉, and GA₂₀ were detected in seedlings of the transgenic line examined. GA₄, which is thought to be the predominantly active GA in Arabidopsis, was not present at increased levels in this line. These results suggest that the overexpression of this 20-oxidase increases the levels of some endogenous GAs in transgenic seedlings, which causes the GA-overproduction phenotype.     

Gibberellin content of immature apple seeds from paclobutrazol-treated trees over three seasons

Seeds from heavily fruiting (on-year), mature untreated, and paclobutrazol-treated apple trees (Malus domestica Borkh. cv. Spartan) were sampled in mid-June 1987, mid-July 1987, and mid-July 1990. After seeds were freeze-dried, gibberellins (GAs) were extracted, purified, and fractionated via C₁₈ reversed-phase high-performance liquid chromatography (HPLC). Nine GAs (GA₁, GA₃, GA₄, GA₇, GA₈, GA₉, GA₁₉, GA₂₀, and GA₅₃) were quantified by the use of deuterated GA internal standards. Paclobutrazol trunk drench treatments reduced vegetative shoot elongation in the seasons that seeds were sampled by 55% or more. Between June 17, 1987 and July 15, 1987, the dry weight of seeds from both untreated and treated trees increased about 2.5 times and there were reductions, on a per seed basis, of GA₄ in seeds from both untreated and treated trees, of GA₇ in seeds from treated trees, and of GA₉ in seeds from untreated trees. However, GA₉ increased in seeds from treated trees. Changes in levels of some of the early-13-hydroxylation pathway GAs (GA₁₅ GA₃, GA₈, GA₁₉, GA₂₀, and GA₅₃) also occurred during the month. For mid-July harvested seeds, the pattern, with some exceptions, was that 2 years after paclobutrazol treatment (1987), levels of early-13-hydroxylation pathway GAs in seeds from treated trees were lower compared to controls but after 5 years (1990) their levels tended to increase. For the non-13-hydroxylated GAs (GA₄, GA₇, and GA₉), 2 years after paclobutrazol treatment, GA₄ levels were equal in seeds from untreated and treated trees, GA₇ decreased in seeds from treated trees compared with controls, but GA₉ levels increased. Levels of these three GAs were higher in seeds from treated trees 5 years after treatment (1990) but levels of GA₄, GA₇, and GA₉ dramatically increased in seeds from treated trees 4 years after treatment (1989), as we previously reported.     

Endogenous gibberellins in the shoots of normal- and bush-typeCucumis sativus L.

Endogenous gibberellins (GAs) in the shoots of normal- (cv. Yomaki, YO) and bush-type (cv. Spacemaster, SP) cultivars of cucumber (Cucumis sativus L.) grown under natural conditions were analyzed. From both YO and SP grown for 40 days, after sowing, a series of C-13-H GAs including GA₄, GA₉, GA₁₅, GA₂₄, GA₂₅, GA₃₄, and GA₅₁ were identified by gas chromatography-mass spectrometry (GC-MS; full scan). In addition to the above GAs, GA₁₂ and GA₇₀ were similarly identified from both YO and SP grown for 61 days after sowing. The endogenous levels of GA₄ and GA₉, which are highly active in promoting cucumber hypocotyl elongation, were quantified by GC-selected ion monitoring (GC-SIM) using [²H₂]GA₄ and [²H₄]GA₉ as internal standards. No remarkable difference in terms of endogenous levels of GA_4/9 was observed between YO and SP in both growth stages (40 and 61 days after sowing).     

<Emphasis Type="Italic">Chrysosporium pseudomerdarium</Emphasis> produces gibberellins and promotes plant growth

We isolated 10 endophytic fungi from the roots of drought stressed soybean cultivar Hwangkeumkong and bioassyed on waito-c rice and soybean seedlings, in order to identify plant growth-promoting fungi. The fungal isolate D-2-1 provided the best result for plant height and biomass promotion as compared to wild type Gibberella fujikuroi. The D-2-1 culture filtrate (CF) was analyzed for the presence of gibberellins (GAs) and it was observed that all physiologically active GAs, especially gibberellic acid, were present in higher amounts (GA₁, 0.24 ng/ml; GA₃, 8.99 ng/ml; GA₄, 2.58 ng/ml and GA₇, 1.39 ng/ml) in conjunction with physiologically inactive GA₅, GA₉, GA₁₅, GA₁₉, and GA₂₄. The fungal isolate D-2-1 was identified as a new strain of Chrysosporium pseudomerdarium through phylogenetic analysis of 18S rDNA sequence. Plant growth promotion and GAs production capacity of genus Chrysosporium have been reported for the first time in this study.     

Accumulation of C19-gibberellins in the gibberellin-insensitive dwarf mutantgai ofArabidopsis thaliana (L.) Heynh

The endogenous gibberellins (GAs) from shoots of the GA-insensitive mutant,gai, ofArabidopsis thaliana were analyzed and compared with the GAs from the Landsberg erecta (Ler) line. Twenty GAs were identified in Ler plants by full-scan gas chromatography-mass spectrometry (GC-MS) and Kovats retention indices (KRI's). These GAs are members of the early-13-hydroxylation pathway (GA₅₃, GA₄₄, GA₁₉, GA₁₇, GA₂₀, GA₁, GA₂₉, and GA₈), the non-3,13-hydroxylation pathway (GA₁₂, GA₁₅, GA₂₄, GA₂₅, GA₉, and GA₅₁), and the early-3-hydroxylation pathway (GA₃₇, GA₂₇, GA₃₆, GA₁₃, GA₄, and GA₃₄). The same GAs, except GA₅₃, GA₄₄, GA₃₇, and GA₂₉ were detected in thegai mutant by the same methods. In addition, extracts fromgai plants contained GA₄₁ and GA₇₁. Both lines also contained several unknown GAs. In Ler plants these were mainly hydroxy-GA₁₂ derivatives, whereas in thegai mutant hydroxy-GA₂₄, hydroxy-GA₂₅, and hydroxy-GA₉ compounds were detected. Quantification of seven GAs by GC-selected ion monitoring (SIM), using internal standards, and comparisons of the ion intensities in the SIM chromatograms of the other thirteen GAs, demonstrated that thegai mutant had reduced levels of all C₂₀-dicarboxylic acids (GA₅₃, GA₄₄, GA₁₉, GA₁₂, GA₁₅, GA₂₄, GA₃₇, GA₂₇, and GA₃₆). In contrast,gai plants had increased levels of C₂₀-tricarboxylic acid GAs (GA₁₇, GA₂₅, and GA₄₁) and of all C₁₉-GAs (GA₂₀, GA₁, GA₈, GA₉, GA₅₁, GA₄, GA₃₄, and GA₇₁) except GA₂₉. The 3β-hydroxylated GAs, GA₁ and GA₄, and their respective 2β-hydroxylated derivatives, GA₈ and GA₃₄, were the most abundant GAs found in shoots of thegai mutant. Thus, thegai mutation inArabidopsis results in a phenotype that resembles GA-deficient mutants, is insensitive to both applied and endogenous GAs, and contains low levels of C₂₀-dicarboxylic acid GAs and high levels of C₁₉-GAs. This indicates that theGAI gene controls a step beyond the synthesis of an active GA. Thegai mutant is presumably a GA-receptor mutant or a mutant with a block in the transduction pathway between the receptor and stem elongation. We thank Dr. L.N. Mander, Australian National University, Canberra, for providing [²H]gibberellins, Dr. B.O. Phinney, University of California, Los Angeles, USA for [¹³C]GA₈, and Dr. D.A. Gage, MSU-NIH Mass Spectrometry Facility (grant No. DRR00480), for advice with mass spectrometry. This work was supported by a fellowship from the Spanish Ministry of Agriculture (I.N.I.A.) to M.T., by the U.S. Department of Energy under Contract DE-ACO2-76ERO-1338, and by U.S. Department of Agriculture grant No. 88-37261-3434 to J.A.D.Z.     

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.     

Gibberellins and antheridiogen on sex in Blechnum spicant L.

The role of gibberellins (GAs) in determining sex in the gametophyte of the fern Blechnum spicant L. was studied through (a) the effect of exogenous GA₄₊₇ and GA₃ (b) quantitation of the endogenous levels of GA_1, GA₃, GA₄, GA₇, GA₉, and GA₂₀ in male and female gametophytes, and (c) the effect of flurprimidol, a GAs biosynthesis inhibitor of the steps of oxidation of ent-kaureno to ent-kaurenoic acid. Our results show that GA₄₊₇ had a slight effect of inducing either male or female sexual organs, antheridia and archegonia, respectively. The endogenous GAs content was not significantly different between sexes, but the GA₄, GA₇, and GA₂₀ levels were raised above those of the other GAs in both sexes. Neither antheridiogen biosynthesis nor antheridia formation was inhibited by flurprimidol. Gametophytes regenerated from homogenized mature gametophytes (HG) show a different physiological behavior than spore-derived gametophytes. In the first case, gametophytes are males and synthesize antheridiogen before they attain maturity, in contrast to what occurs in spore-derived gametophytes which are females and synthesize antheridiogen when mature.     

Identification of endogenous gibberellins, and metabolism of tritiated and deuterated GA4, GA9 and GA20, in Scots pine (Pinus sylvestris) shoots

The application of gibberellin A_4/7 (GA_4/7) to the stem of previous-year (1-year-old) terminal shoots of Scots pine (Pinus sylvestris) seedlings has been observed to stimulate cambial growth locally, as well as at a distance in the distal current-year terminal shoot, but the distribution and metabolic fate of the applied GA_4/7, as well as the pathway of endogenous GA biosynthesis in this species, has not been investigated. As a first step, we analysed for endogenous GAs and monitored the transport and metabolism of labelled GAs 4, 9 and 20. Endogenous GAs from the elongating current-year terminal shoot of 2-year-old seedlings were purified by column chromatography and high-performance liquid chromatography and analysed by combined gas chromatography-mass spectrometry (GC-MS). GAs 1, 3, 4, 9, 12 and 20 were identified in the stem, and GAs 1, 3 and 4 in the needles, by full-scan mass spectrometry (GAs 1, 3, 4, 9 and 12) or selected-ion monitoring (GA₂₀) and Kovats retention index. Tritiated and deuterated GA₄, GA₉ or GA₂₀ were applied around the circumference at the midpoint of the previous-year terminal shoot, and metabolites were extracted from the elongating current-year terminal shoot, the application point, and the 1-year-old needles and the cambial region above and below the application point. After purification, detection by liquid scintillation spectrometry and analysis by GC-MS, it was evident that, for each applied GA, unmetabolised [²H₂]GA and [³H]radioactivity were present in every seedling part analysed. Most of the radioactivity was retained at the application point when [³H]GA₉ and [³H]GA₂₀ were applied, whereas the largest percentage of radioactivity derived from [³H]GA₄ was recovered in the current-year terminal shoot. It was also found that [²H₂]GA₉ was converted to [²H₂]GA₂₀ and to both [²H₂]GA₄ and [²H₂]GA₁, [²H₂]GA₄ was metabolised to [²H₂]GA₁, and [²H₂]GA₂₀ was converted to [²H₂]GA₂₉. The data indicate that for Pinus sylvestris shoots (1) GAs applied laterally to the outside of the vascular system of previous-year shoots not only are absorbed and translocated extensively throughout the previous-year and current-year shoots, but also are readily metabolised, (2) the GA metabolic pathways found are closely related to the endogenous GAs identified, and (3) GA₉ metabolism follows two distinctly different routes: in one, GA₉ is converted to GA₁ through GA₄, and in the other it is converted to GA₂₀, which is then metabolised to GA₂₉. The results suggest that the late 13-hydroxylation pathway is an important route for GA biosynthesis in shoots of Pinus sylvestris, and that the stimulation of cambial growth in Scots pine by exogenous GA_4/7 may be due to its conversion to GA₁, rather than to it being active per se.     

Thermo- and Photoperiodicity and Involvement of Gibberellins during Day and Night Cycle on Elongation Growth of Begonia x hiemalis Fotsch

The effects of thermo- and photoperiodicity on elongation growth and on endogenous level of gibberellins (GAs) in Begonia x hiemalis during various phases of the day-night cycle have been studied. Plant tissue was harvested during the day and night cycle after temperature and photoperiodic treatments and analyzed for endogenous GAs using combined gas chromatography and mass spectrometry. Elongation growth increased when the difference between day and night temperature (DIF = DT − NT) increased from a negative value (−9.0 and −4.5°C) to zero and with increasing photoperiod from 8 to 16 h. When applied to the youngest apical leaf, gibberellins A₁, A₄, and A₉ increased the elongation of internodes and petioles. GA₄ had a stronger effect on elongation growth than GA₁ and GA₉. In relative values, the effect of these GAs decreased when DIF increased from −9 to 0°C. The time of applying the GAs during a day and night cycle had no effect on the growth responses. In general, endogenous levels of GA₁₉ and GA₂₀ were higher under negative DIF compared with zero DIF. The level of endogenous GA₁ in short day (SD)-grown plants was higher under zero DIF than under negative DIF, but this relationship did not appear in long day (LD)-grown plants. The main effects of photoperiod seem to be a higher level of GA₁₉ and GA₁ at SD compared with LD, whereas GA₂₀ and GA₉ show the opposite response to photoperiod. No significant differences in endogenous level of GA₁, GA₉, GA₁₉, and GA₂₀ were found for various time points during the diurnal day and night cycle. Endogenous GA₂₀ was higher in petiole and leaf compared with stem, whereas there were no differences of GA₁, GA₉, and GA₁₉ between plant parts. No clear relationship was found between elongation of internodes and petioles and levels of endogenous GAs. Received December 26 1996; accepted July 1, 1997     

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.     

Endogenous Gibberellins in Aralia cordata

Eight gibberellins (GAs) were identified in extracts of buds of Aralia cordata by full scan GC/MS and by Kovats retention indices. These GAs comprised five GAs on the early-13-hydroxylation pathway [GA₁, GA₁₉, GA₂₀, GA₄₄, and GA₅₃] and three other GAs [GA₄, GA₁₅, and GA₃₇]. The major GAs were GA₁₉ and GA₄₄.     

Identification of Ten Gibberellins from Sporophytes of the Tree fern, Cyathea australis

Ten gibberellins (GAs) have been identified by Kovats retention indices and full mass spectra from GC-MS analysis of purified extracts of sporophytes of the tree-fern, Cyathea australis. These include the known GA₁, GA₄, GA₉, GA₁₅, GA₂₄, GA₃₅, and GA₅₈ and three new GAs, 12β-hydroxyGA₉ (GA₆₉), 12α-hydroxyGA₉ (GA₇₀) and 12β-hydroxyGA₄ (GA₇₁). The structure of GA₇₁ was established by the preparation and characterization of its methyl ester (as a metabolite of GA₄ methyl ester in a culture of prothallia of Lygodium japonicum).     

Changes in levels of gibberellins,their putative conjugates,and ABA in zygotic embryos during late maturation and dry seed stages of <Emphasis Type="Italic">Brassica napus</Emphasis> cv Topaz

Two late stages [days 35 and 40 after pollination (DAP)] in zygotic embryo (ZE) development of Brassica napus were utilized to quantify, by the stable isotope-labeled dilution method, levels of “free” and “aglycone” gibberellins (GAs), as well as abscisic acid (ABA), during the programmed dehydration of the seed. GAs from both the early 13 hydroxylation and early non-hydroxylation pathways were present in these ZEs of B napus. Between 35 and 40 DAP endogenous ABA dropped precipitously (almost 30-fold) and this drop in ABA was accompanied by a significant reduction in levels of GA₁ and even in levels of the inactive GA catabolites, GA₈ and GA₂₉. Levels of GA₄ and putative GA₈₅ also dropped appreciably, though not significantly. In contrast, the levels of GA₂₀ and GA₉ (the immediate precursors of GA₁ and GA₄, respectively) did not change in the ZEs during this transition. A fungal-derived cellulase was used to hydrolyze the highly water-soluble fraction, which will contain GA conjugates. Relatively high levels of several GAs (GA₉, GA₂₀) were thus quantified after hydrolysis as the aglycones, e.g., 56 and 25 ng/g DW of GA₂₀ and 23 and 5 ng/g DW, of GA₉, respectively at DAP 35 and DAP 40. Other GAs found after hydrolysis of the highly water-soluble fraction remained relatively constant between 35 and 40 DAP. An exception was the putative GA₈₅ aglycone, which increased sixfold (free GA₈₅ decreased by ca. half). The transition to the dry seed stage for ZEs of B. napus is thus accompanied not only by the expected reduction in ABA, but also by reduced levels of many “free” GAs, especially the bioactive, 3β-hydroxylated GAs. In contrast, levels of 3-deoxy GAs remain relatively high, implying a partial block in the 3β-hydroxylation “activation” step of GA biosynthesis.     

Gibberellins and the photoperiodic control of leaf growth in Poa pratensis

The role of gibberellins (GAs) in photoperiodic control of leaf elongation in Poa pratensis was studied by both application of exogenous GAs and analysis of endogenous GAs. Leaf elongation was strongly increased under long day (LD, 24 h) conditions at both 9 and 21°C, leaf length at 9°C LD being similar to that in plants grown in short days (SD, 8 h) at 21°C. However, even at 21°C leaf elongation was enhanced by LD. Exogenous GA₁ could completely compensate for LD at both 9 and 21°C. Gibberellins A₂₀, A₁₉ and A₄₄ could also partly replace LD, but they were significantly less active than GA₁, GA₅₃ was inactive when applied to plants grown at 9°C in SD and exhibited only marginal activity at 9°C LD and 21°C SD. The total level of GAs of the early 13-hydroxylation pathway (A₅₃, A₄₄, A₁₉, A₂₀ and A₁) increased rapidly when plants were transferred from SD to LD at 9°C. After transfer from 9 to 21°C, there was an increase in GA levels at both LD and SD, followed by a decrease under LD conditions. In all cases, GA₁₉ was the predominant GA, accounting for 60 to 80% of the analysed GAs. Levels of the bioactive GA₁ were low and increased transiently by LD four days after transfer from SD to LD. At both temperatures, the ratio GA₁₉ to GA₂₀ and GA₂₀ to GA₁ at 9°C was enhanced by LD compared with SD. Taken together, these results support the hypothesis that photoperiodic regulation of leaf elongation in Poa pratensis is GA-mediated, and they indicate a photoperiodic control of oxidation of GA₅₃ to GA₄₄ and GA₁₉ to GA₂₀, and perhaps also of 3β-hydroxylation of GA₂₀ to GA₁.