Early 13-hydroxylation Research Articles

One-pot sample preparation approach for profiling spatial distribution of gibberellins in a single shoot of germinating cereal seeds.

Sample preparation remains a bottleneck in the rapid and reliable quantification of gibberellins (GAs) for obtaining an insight into the physiological processes mediated by GAs. The challenges arise from not only the extremely low content of GAs in complex plant matrices, but the poor detectability of GAs by mass spectrometry (MS) in negative ion mode. In an effort to solve these urgent difficulties, we present a spatial-resolved analysis method to investigate the distribution of GAs in tiny plant tissues based on a simplified one-pot sample preparation approach coupled with ultrahigh-performance liquid chromatography-tandem MS. By integrating extraction and derivatization into one step, target GAs were effectively extracted from plant materials and simultaneously reacted with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide, the sample preparation time was largely shortened, the probability of sample loss was minimized and the detection sensitivity of MS was also greatly improved compared with underivatized GAs. Under optimal conditions, the method was validated from the quantification linearity, limits of detection and limits of quantification in the presence of plant matrices, recoveries, and precision. With the proposed method, 15 endogenous GAs were detected and, among these, 11GAs could be quantified in 0.50mg fresh weight (FW) wheat shoot samples, and fiveGAs were quantified in only 0.15mg FW developing seed samples of Arabidopsis thaliana. The distribution patterns of GAs along both the non-13-hydroxylation pathway and the early 13-hydroxylation pathway in a single shoot of germinating wheat, rice and maize seeds were finally profiled with a spatial resolution down to approximately 1mm2 .

Quantification of near-attomole gibberellins in floral organs dissected from a single Arabidopsis thaliana flower.

There remains a methodological bottleneck in the quantification of ultra-trace plant hormones in very tiny plant organs at fresh weights below a milligram. The challenge becomes even more serious in the determination of endogenous gibberellins (GAs), which are a class of compounds that are difficult to separate and detect. Herein, a quantification method using ultra-high-performance liquid chromatography-tandem mass spectrometry was developed, combined with a derivatization technique in which GAs react with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide in ethanol. The method was validated as capable of determining GAs in floral organs (about 80-250μg) - pistil, stamens, petals, sepals and receptacle - which were dissected from only one flower of Arabidopsis thaliana. Substantially different abundance patterns of GAs were measured from the floral organs at floral stages 13, 14 and 15 along the non-13-hydroxylation pathway and the early 13-hydroxylation pathway in plants. This allows sub-flower-level insights into how GAs affect floral development. The method exhibited excellent limit of detection and limit of quantification down to 5.41 and 18.0 attomole, respectively, and offered a fairly wide linear range from 0.01 to 25 femtomole with linear coefficients above 0.9961. The precision of the method was evaluated with relative standard deviations below 10.6% for intra-day and 11.4% for inter-day assays, and recoveries ranged from 64.0% to 107%.

Regulation of endogenous gibberellins and abscisic acid levels during different seed collection periods in Panax ginseng

Ginseng (Panax ginseng Meyer) is an important oriental plant that has been used for medicinal purposes for centuries. Propagation of ginseng is frequently problematic due to its prolonged seed germination time. In this study tested the seasonal regulation of endogenous hormones (GAs and ABA) and hormone treatments in order to reduce the duration of the seed germination period. The embryo growth ratio increased from 10.2 to 33.7% between July 30, 2009 to October 27, 2009 and abruptly increased to 71.6% of the that. The seed germination rate assessed for different seed sizes revealed that approximately 60.2%–63.0% of the seeds had germinated by April 20, 2010; however, no significant differences in germination rate were observed among the different seed sizes. Analysis of GA showed that endogenous GAs were synthesized in the ginseng seeds through a non-13-hydroxylation pathway and the early 13-hydroxylation pathway. The GA contents were significantly increased from December 2009 to February 2010, while endogenous ABA contents were significantly reduced. However, the GA contents from August 2009 to December 2009 were lower than or comparable to the ABA contents, which were significantly increased. Thus, the total concentration of GAs and ABA during different seed growth stages suggests antagonism between these hormones. Similarly, the embryo growth ratio during the seed collection period revealed a similar trend in which the total GA and ABA contents were inversely related.

Hormonal Profile in Vegetative and Floral Buds of Azalea: Levels of Polyamines, Gibberellins, and Cytokinins

The floral transition includes a complex system of factors that interact and involve various biochemical signals, including plant growth regulators. The physiological signals involved in the control of the floral transition have been sparsely studied and mainly in plant species whose genetics are poorly known. In this work, the role of polyamines, gibberellins, and cytokinins was investigated by analyzing their endogenous content in vegetative and floral buds of azalea. The results showed that there is a clear distinction between floral and vegetative buds with respect to the levels of these plant hormones, with floral buds containing higher amounts of conjugated polyamines, gibberellins (GAs) from the non-13-hydroxylation pathway (GA9, GA7, and GA4), and cytokinins (particularly isopentenyl-type species), and vegetative buds containing higher amounts of free polyamines and gibberellins from the early 13-hydroxylation pathway and fewer cytokinins. In conclusion, there is a specific pattern of endogenous hormone profiles in both vegetative and floral bud development in azalea, which may be relevant for future research on the control of flowering by exogenous hormone applications.

Effect of light on endogenous levels of gibberellin and abscisic acid in seed germination of photoblastic weedy rice (Oryza sativa L.)

The effect of red (R) and R/far-red (FR) lights on endogenous gibberellin (GA) and abscisic acid (ABA) content was first investigated during the germination of photoblastic black-hulled weedy rice (PBWR) seeds. The R light-treated PBWR seeds germinated after 36–48 h and germination was increased to 63% at 72 h. However, the FR light-treated seeds after R light treatment, suppressed the R light effect showing only 11% germination even at 72 h after the light treatment. The PBWR seed treated with R light rapidly increased the endogenous level of GA1 to about 200 times at 12 h before seed germination as compared with R/FR (control) which suppressed the effect of R light. The contents of other GAs like GA12, GA53, GA19, GA20, and GA8 were not affected by the R light irradiation. These results showed that the major biosynthetic pathway of GAs in PBWR seeds is the early 13-hydroxylation pathway leading to GA1, which was suggested to be physiologically active in the PBWR seed germination. The decrease in the level of ABA in the R light-treated seeds was greater than the R/FR light-treated seeds, indicating that the balance of endogenous GA1 and ABA is responsible for the induction of germination in the PBWR seed.

Involvement of gibberellins in the stem elongation of sun and shade ecotypes of Stellaria longipes that is induced by low light irradiance

Plants from two ecotypes of Stellaria longipes, alpine (an open, sunny habitat) and prairie (where adjacent plants provide a shaded habitat), were grown under normal and reduced levels of photosynthetically active radiation (PAR). Growth under low PAR is significantly promoted in both ecotypes. When quantified by the stable isotope dilution method, endogenous gibberellins (GAs) (GA1, GA8, GA20, GA19) were significantly elevated under low PAR in both 'sun' and 'shade' ecotypes, as was GA53 in the shade ecotype. Changes in endogenous GA1 levels were significantly correlated with stem growth during a 28 d growth cycle and with relative growth rate (RGR) for height under low PAR for both ecotypes. Interestingly, under low irradiance PAR, changes (both increases and decreases) in GA8, the 2beta-hydroxylated 'inactive' catabolite of GA1, closely parallel bidaily stem growth changes for both ecotypes. Because the significantly greater stem elongation of both ecotypes in response to low irradiance PAR is associated with significant increases in the endogenous levels of five GAs (GA53, GA19, GA1, GA8) in the early 13-hydroxylation GA biosynthesis pathway (measured at days 7,14 and 21), we conclude that the low irradiance PAR has very likely induced an overall increase in GA biosynthesis.

Developing Inflorescences of Male and Female Rumex acetosa L. Show Differences in Gibberellin Content

Rumex acetosa L. (common sorrel) is a dioecious perennial in the family Polygonaceae. Gibberellins (GAs) of the early 13-hydroxylation pathway and the putative early 3β, 13-hydroxylation pathway were previously identified in young R. acetosa inflorescences by GC-MS. In this investigation to examine the GA content of individual inflorescences ELISAs were used for quantitative analysis. Significant differences were revealed between the sexes in the GA content of young inflorescences, and GC-SRM was used to validate the observed trends. Males had higher levels of the 3β, 13-hydroxylated C20-GA GA18 and the 2β, 13-hydroxylated C19-GA GA29, whereas females had higher levels of the 13-hydroxylated C20-GAs GA53 and GA19. It is suggested that the conversion from C20-GAs to C19-GAs is under tighter control in the inflorescences of females compared to male plants and therefore there is accumulation of the C20-GAs in the females. Results from flowering bioassays using authentic GAs indicate that differences in GA content between the sexes are unlikely to be a consequence of sex determination.

3β,13-Dihydroxylated C 20 gibberellins from inflorescences of Rumex acetosa L.

Using full scan GC–MS a wide range of gibberellins (GAs) was identified in the young inflorescences of the dioecious species Rumex acetosa L., consistent with the ubiquitous early 13-hydroxylation pathway in both male and female plants. In addition, R. acetosa is the first species in which all three 3β,13-dihydroxylated C 20-GAs—GA 18, GA 38 and GA 23—have been identified in the same organism, suggesting an early 3β,13-dihydroxylation biosynthesis pathway in this species. Authentic GA 18, GA 38 and GA 23 were synthesized and their effects and that of GA 1, a GA common to both pathways, on the time to inflorescence emergence was investigated. GA 1 accelerated the emergence of inflorescences in both male and female plants. In addition some evidence for biological activity per se of the C 20-GA 38 was obtained.

Abscisic acid, phaseic acid and gibberellin contents associated with dormancy and germination in barley.

Analyses of abscisic acid (ABA), ent-kaurenoids and gibberellins (GAs) showed that there were major changes in the contents of these compounds associated with germination of after-ripened barley (Hordeum vulgare cv. Schooner and cv. Proctor) grain but not in hydrated dormant grain. Embryos from dormant and after-ripened dry grain contained similar amounts of ABA, of ent-kaurenoids and of GAs, determined by gas chromatography-mass spectrometry-selected ion monitoring. In embryos of after-ripened grain, ABA content decreased rapidly after hydration and ABA appeared to be metabolized (inactivated) to phaseic acid (PA) rather than diffusing into the endosperm or the surrounding medium as previously thought. Similar changes in ABA occurred in hydrated dormant grain during germination in darkness. Accumulation of ent-kaurenoids and GAs, including GA1, the first biologically active GA in the early 13-hydroxylation biosynthetic pathway, occurred to a much greater extent in after-ripened than in dormant grain and these changes occurred mainly after 18 h of hydration when ABA had already decreased and germination was occurring. The block in ent-kaurenoid and GA synthesis in dormant grain appeared to occur prior to ent-kaurene in the biosynthetic pathway. These results are consistent with the view that ABA is the primary effector of dormancy and that after-ripening involves the development of the ability to reduce the amount of ABA quickly following hydration. Accumulation of GAs does not appear to be causally related to loss of dormancy but it does appear to be related to germination.

Cytokinins and gibberellins in sap exudate of the oil palm

Exudates were collected from stumps of pre-anthesis inflorescences of oil palm and analysed for cytokinin and gibberellin content using combined HPLC-ELISA techniques. Three antisera, for zeatin-type, dihydrozeatin-type and isopentenyladenine-type cytokinins, were used in ELISAs to identify members of these three groups of cytokinins. Ribotides, 9-glucosides, free bases and ribosides were detected for each of the groups with zeatin riboside the most abundant cytokinin identified in the exudate. Isopentenyladenine-type and dihydrozeatin-type cytokinins were also identified but at lower levels. In addition, two monoclonal antibodies were used in the development of novel ELISAs for members of the 13-hydroxylated and non-13-hydroxylated families of gibberellins. The new ELISAs allow the determination of gibberellins in smaller amounts of tissue than are required for GC–MS. The most abundant gibberellins identified in exudates were GA 19 and GA 44, as well as other members of the early 13-hydroxylation pathway. Gibberellins were confirmed by GC–MS. The presence of these types of growth regulators in exudate supplying immature inflorescences suggest they have a role in growth and development of these structures.

Cloning of gibberellin 3 beta-hydroxylase cDNA and analysis of endogenous gibberellins in the developing seeds in watermelon.

We have isolated Cv3h, a cDNA clone from the developing seeds of watermelon, and have demonstrated significant amino acid homology with gibberellin (GA) 3 beta-hydroxylases. This cDNA clone was expressed in Escherichia coli as a fusion protein that oxidized GA(9) and GA(12) to GA(4) and GA(14), respectively. The Cv3h protein had the highest similarity with pumpkin GA 2 beta,3 beta-hydroxylase, but did not possess 2 beta-hydroxylation function. RNA blot analysis showed that the gene was expressed primarily in the inner parts of developing seeds, up to 10 d after pollination (DAP). In the parthenocarpic fruits induced by treatment with 1-(2-chloro-4-pyridyl)-3-phenylurea (CPPU), the embryo and endosperm of the seeds were undeveloped, whereas the integumental tissues, of maternal origin, showed nearly normal development. Cv3h mRNA was undetectable in the seeds of CPPU-treated fruits, indicating that the GA 3 beta-hydroxylase gene was expressed in zygotic cells. In our analysis of endogenous GAs from developing seeds, GA(9) and GA(4) were detected at high levels but those of GA(20) and GA(1) were very low. This demonstrates that GA biosynthesis in seeds prefers a non-13-hydroxylation pathway over an early 13-hydroxylation pathway. We also analyzed endogenous GAs from seeds of the parthenocarpic fruits. The level of bioactive GA(4 )was much lower there than in normal seeds, indicating that bioactive GAs, unconnected with Cv3h, exist in integumental tissues during early seed development.

Gibberellins in relation to flowering in Polianthes tuberosa.

Endogenous gibberellins (GAs) in corms of Polianthes tuberosa L. (cv. Double) were isolated and identified by high performance liquid chromatography, bioassay and combined capillary gas chromatography-mass spectrometry (GC-MS). Gibberellins A1, A19, A20 and A53 were quantified at the vegetative, early floral initiation and flower development stages. The identification of 13-hydroxylated GAs indicates the presence of the early 13-hydroxylation pathway in P. tuberosa corms. An increase in GA1 and GA20, and a decrease in GA19 levels, coincided with the transition from the vegetative phase to the stages of early floral initiation and flower development. GA53 stayed at constant levels at the 3 different growth stages. The absence of GA1 in vegetative corms and its presence in corms at early floral initiation and flower development stages suggest that GA1 is a causal factor in inducing floral initiation in P. tuberosa. When GA1, GA3, GA4, GA20 and GA32 were applied to corms at the vegetative stage (plants about 5 cm in height), floral initiation was promoted by all of the GAs used, GA32 being the most active. In contrast with the other GAs, GA32 had no effect on stem elongation. Therefore, it is suggested that hydroxylated C-19 GAs play an important role in flower induction in P. tuberosa.

Role of gibberellins in parthenocarpic fruit development induced by the genetic system pat-3/pat-4 in tomato.

The role of gibberellins (GAs) in the induction of parthenocarpic fruit-set and growth by the pat-3/pat-4 genetic system in tomato (Lycopersicon esculentum Mill.) was investigated using wild type (WT; Cuarenteno) and a near-isogenic line derived from the German line RP75/59 (the source of pat-3/pat-4 parthenocarpy). Unpollinated WT ovaries degenerated but GA3 application induced parthenocarpic fruit growth. On the contrary, parthenocarpic growth of pat-3/pat-4 fruits, which occurs in the absence of pollination and hormone treatment, was not affected by applied GA3. Unpollinated pat-3/pat-4 fruit growth was negated by paclobutrazol, an inhibitor of ent-kaurene oxidase, and this inhibitory effect was negated by GA3. The quantification of the main GAs of the early 13-hydroxylation pathway (GA1, GA8, GA19, GA20, GA29 and GA44) in unpollinated ovaries at 3 developmental stages (flower bud, FB; pre-anthesis, PR; and anthesis, AN), by gas chromatography-selected ion monitoring, showed that the concentration of most of them was higher in pat-3/pat-4 than in WT ovaries at PR and AN stages. The concentration of GA1, suggested previously to be the active GA in tomate, was 2-4 times higher. Unpollinated pat-3/pat-4 ovaries at FB, PR and AN stages also contained relatively high amounts (5-12 ng g-1) of GA3, a GA found at less than 0.5 ng g-1 in WT ovaries. It is concluded that the mutations pat-3/pat-4 may induce natural facultative parthenocarpy capacity in tomato by increasing the concentration of GA1 and GA3 in the ovaries before pollination.

Correlation of Endogenous Gibberellic Acid with Initiation of Mango Shoot Growth

Stems of mango (Mangifera indica L.) rest in a non-growing, dormant state for much of the year. Ephemeral flushes of vegetative or reproductive shoot growth are periodically evoked in apical or lateral buds of these resting stems. The initiation of shoot growth is postulated to be primarily regulated by a critical ratio of root-produced cytokinins, which accumulate in buds and by leaf-produced auxin, which decreases in synthesis and transport over time. Exogenously applied gibberellic acid (GA 3 ) delays initiation of bud break but does not determine whether the resulting flush of growth is vegetative or reproductive. We tested the hypothesis that endogenous GA 3 , which influences release of these resting buds, may decrease in stem tips or leaves with increasing age of mango stems. GA 3 and several other GAs in stem tip buds and leaves were identified and quantified in stems of different ages. The major endogenous GAs found in apical buds and leaves of vegetative mango stems were early 13-hydroxylation pathway gibberellins: GA 1 , epi-GA 1 , GA 3 , GA 19 , GA 20 , and GA 29 , as identified by gas chromatography-mass spectrometry (GC-MS). A novel but unidentified GA-like compound was also present. The most abundant GAs in apical stem buds were GA 3 and GA 19 . Contrary to the hypothesis, the concentration of GA 3 increased within buds with increasing age of the stems. The concentrations of other GAs in buds were variable. The concentration of GA 3 did not change significantly with age in leaves, whereas that of most of the other GAs declined. GA 1 levels were greatest in leaves of elongating shoots. These results are consistent with the concept that rapid shoot growth is associated with synthesis of GAs leading to GA 1 . The role of GA 3 in delaying bud break in mango is not known, but it is proposed that it may enhance or maintain the synthesis or activity of endogenous auxin. It, thereby, maintains a high auxin/cytokinin ratio similar to responses to GA 3 that maintain apical dominance in other plant species.

Gibberellin biosynthesis: metabolic evidence for three steps in the early 13-hydroxylation pathway of rice

[ 14C 4]GA 53, [ 14C 4]GA 44, and [ 2H 2 / 14C 4]GA 19 were injected separately into seedlings of rice ( Oryza sativa) using a dwarf mutant ( d 35) that has low levels of endogenous gibberellins (GAs). After 8 h incubation, the shoots were extracted and the labeled metabolites were identified by full-scan gas chromatography–mass spectrometry (GC–MS) and Kovats retention indices (KRIs). Our results document the metabolic sequence, GA 53→GA 44→GA 19→GA 20 and the presence of endogenous GA 53, GA 44, GA 19, GA 20 and GA 1. Previous metabolic studies have shown the presence of the step, GA 20→GA 1 in rice. Taken together, the data establish in vegetative shoots of rice the presence of the early 13-hydroxylation pathway, a pathway that originates from GA 12 and leads to bio-active GA 1.

Gibberellin Biosynthesis in Maize. Metabolic Studies with GA(15), GA(24), GA(25), GA(7), and 2,3-Dehydro-GA(9).

[17-(14)C]-Labeled GA(15), GA(24), GA(25), GA(7), and 2,3-dehydro-GA(9) were separately injected into normal, dwarf-1 (d1), and dwarf-5 (d5) seedlings of maize (Zea mays L.). Purified radioactive metabolites from the plant tissues were identified by full-scan gas chromatography-mass spectrometry and Kovats retention index data. The metabolites from GA(15) were GA(44), GA(19), GA(20), GA(113), and GA(15)-15,16-ene (artifact?). GA(24) was metabolized to GA(19), GA(20), and GA(17). The metabolites from GA(25) were GA(17), GA(25) 16alpha,17-H(2)-17-OH, and HO-GA(25) (hydroxyl position not determined). GA(7) was metabolized to GA(30), GA(3), isoGA(3) (artifact?), and trace amounts of GA(7)-diene-diacid (artifact?). 2,3-Dehydro-GA(9) was metabolized to GA(5), GA(7) (trace amounts), 2,3-dehydro-GA(10) (artifact?), GA(31), and GA(62). Our results provide additional in vivo evidence of a metabolic grid in maize (i.e. pathway convergence). The grid connects members of a putative, non-early 3,13-hydroxylation branch pathway to the corresponding members of the previously documented early 13-hydroxylation branch pathway. The inability to detect the sequence GA(12) --> GA(15) --> GA(24) --> GA(9) indicates that the non-early 3,13-hydroxylation pathway probably plays a minor role in the origin of bioactive gibberellins in maize.

Dwarf (di)haploid pito mutants obtained from a tetraploid potato cultivar ( Solanum tuberosum subsp. tuberosum) via anther culture are defective in gibberellin biosynthesis

Nine dwarf (di)haploid lines (2 n=2×=24) were obtained from the tetraploid (2 n=4×=48), long day-adapted potato cultivar ‘Pito’ ( Solanum tuberosum subsp. tuberosum) through anther culture. They grew slowly, had very short internodes, compact and ball-shaped appearance, and dark green leaves. Dwarfism was due to a recessive gene, designated pito. Endogenous gibberellin contents were measured in the leaves of dwarf and wild-type lines by gas chromatography linked to mass spectrometry (GC-MS). High amounts of GA 19, GA 20, GA 29, GA 1, and GA 8 were detected in the wild-type plants, which indicated that the early 13-hydroxylation pathway was predominantly used for GA biosynthesis in S. t. subsp. tuberosum. Also GA 53, GA 15 and GA 9 were detected but not quantified. Very low endogenous amounts of all analysed GAs were detected in the pito mutants, indicating a block at an early part of the GA biosynthesis pathway. The dwarf lines strongly and quickly responded to the exogenous application of low amounts (79 nM) of bioactive GA (GA 3), which restored normal growth and confirmed that the pito dwarfs were synthesis mutants and not GA response mutants.

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.

The metabolism of GA 9 in maize ( Zea mays)

[17- 13C, 3H 2]GA 9 was administered to 4-week-old seedlings of normal, dwarf 1 and dwarf 5 seedlings at the 3–4 leaf-stage. After 12 or 24 hr incubation, shoots were harvested, extracted with methanol and the extracts purified by solvent partitioning and HPLC. The resulting radiolabelled fractions were analysed by full-scan GC mass spectrometry with Kovats Retention Indices (R l ). The major metabolite was [ 13C]GA 20, with [ 13C]GA 51, [ 13C]GA 69, [ 13C]GA 70, [ 13C]3-epi- GA 4 and [ 13C]3-epi- GA 1 being identified as minor metabolites. The metabolism of GA 9 to GA 20 demonstrates the possibility of late convergence of the two biosynthetic branch pathways, the presumptive non-early 3,13-hydroxylation pathway and the early 13-hydroxylation pathway, both of which originate from GA 12. There was no evidence for the metabolism of GA 9 to GA 1, GA 4, GA 5, GA 3, GA 7, or 2,3-dehydro-GA 9.

Role of Endogenous Gibberellins in Cold-Induced Stem Elongation and Flowering of Japanese Radish (Raphanus sativus L.).

Eighteen endogenous gibberellins (GAs) which belong to the early 13-hydroxylation pathway and the early non-hydroxylation pathway of GA-biosynthesis were identified by combined gas chromatography/mass spectrometry (GC/MS) in shoots of a cold-requiring plant, Raphanus sativus L. Contents of GA1 and GA4, the probable endogenous biologically-active GAs, increased in the stem during a long-day condition (LD) both with or without a prior cold treatment (CT); however, the increase was greater with CT than without it. In contrast to the contents of 3β-hydroxylated GAs (i.e. GA1 and GA4), that of GA34, a 2β-hydroxylated GA, was decreased significantly by CT and LD, indicating that CT and LD activated the 3β-hydroxylation and inactivated the 2β-hydroxylation during the course of GA-biosynthesis. GA1 and GA4 contents in the leaf decreased during LD subsequent to CT, whereas they increased with LD alone. The increase in GA1 and GA4 contents in the stem probably promoted the cold-induced stem elongation and flowering of R. sativus.