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.     

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.     

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.     

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 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     

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.     

Effect of Gibberellin Biosynthesis Inhibitors on Native Gibberellin Content, Growth and Floral Initiation in Sorghum bicolor

CCC, uniconazol, ancymidol, prohexadione-calcium (BX-112), and CGA 163′935, which represent three groups of gibberellin (GA) biosynthesis inhibitors, were applied as a soil drench to Sorghum bicolor cultivars 58M (phyB-1, phytochrome B-deficient mutant) and 90M (phyB-2, equivalent phenotypically to wild type, PHYB, except for small differences in flowering dates). The inhibitors that block steps before GA₁₂ (CCC, uniconazol, and ancymidol) lowered the concentrations of all endogenous early-C13α-hydroxylation pathway GAs found in sorghum: GA₁₂, GA₅₃, GA₄₄, GA₁₉, GA₂₀, GA₁, and GA₈. In contrast, the inhibitors that block the conversion of GA₂₀→ GA₁, (CGA 163′935 and BX-112) drastically reduced GA₁ and GA₈ levels, but they either did not change or caused accumulation of intermediates from GA₁₂ to GA₂₀. Combinations of pre-GA₁₂ inhibitors and GA₃ plus GA₁ strongly reduced GAs other than GA₁ and GA₃. Each of these compounds inhibited shoot growth in both cultivars and delayed floral initiation in 90M. Floral initiation of 58M was also delayed by CCC, uniconazol, and ancymidol but not by CGA 163`935 and BX-112. This separation of shoot elongation from floral initiation in sorghum is novel. Both inhibition of shoot growth and delayed floral initiation were almost completely relieved by a mixture of GA₃ and GA₁ in both 58M and 90M. This observation, plus the much lower levels of endogenous GA₃ than of GA₁ observed in these experiments, implies that GA₁ is the major endogenous GA active in shoot elongation. CGA 163′935 and BX-112 also failed to promote tillering in 58M, whereas inhibitors active before GA₁₂ did so. The possibility that the GA₂₀→ GA₁ inhibitors fail to block flowering and promote tillering in 58M because biosynthetic intermediates between GA₁₂ and GA₂₀ accumulate and/or because 58M is altered in GA metabolism in this same region of the biosynthetic pathway is discussed. Received April 7, 1998; accepted July 31, 1998     

Gibberellins and parthenocarpic ability in developing ovaries of seedless mandarins

Satsuma (Citrus unshiu [Mak] Marc.) and Clementine (Citrus reticulata [Hort.] Ex. Tanaka, cv Oroval) are two species of seedless mandarins differing in their tendency to develop parthenocarpic fruits. Satsuma is a male-sterile cultivar that shows a high degree of natural parthenocarpy and a high fruit set. Seedless Clementine varieties are self-incompatible, and in the absence of cross-pollination show a very low ability to set fruit. The gibberellins (GAs) GA53, putative 17-OH-GA₅₃, GA₄₄, GA₁₇, GA₁₉, GA₂₀, GA₂₉, GA₁, 3-epi-GA₁, GA₈, GA₂₄, GA₉, and GA₄ have been identified from developing fruits of both species by full-scan combined gas chromatography-mass spectrometry. Using selected ion monitoring with [²H₂]- and [¹³C]-labeled internal standards, the levels of GA₅₃, GA₄₄, GA₁₉, GA₂₀, GA₁, GA₈, GA₄, and GA₉ were determined in developing ovaries at anthesis and 7 days before and after anthesis, from both species. Except for GA8, levels of the 13-hydroxy-GAs were higher in Satsuma than in Clementine, and these differences were more prominent for developing young fruits. At petal fall, Satsuma had, on a nanograms per gram dry weight basis, higher levels of GA₅₃ (10.4x), GA₄₄ (13.9x), GA₁₉ (3.0x), GA₂₀ (11.2x), and GA₁ (2.0x). By contrast, levels of GA₈ were always higher in Clementine, whereas levels of GA₄ did not differ greatly. Levels of GA₉ were very low in both species. At petal fall, fruitlets of Satsuma and Clementine contained 65 and 13 picograms of GA₁, respectively. At this time, the application of 25 micrograms of paclobutrazol to fruits increased fruit abscission in both varieties. This effect was reversed by the simultaneous applications of 1 microgram of GA₃. GA₃ alone improved the set in Clementine (13x), but had little influence on Satsuma. Thus, seedless fruits of the self-incompatible Clementine mandarin may not have adequate GA levels for fruit set. Collectively, these results suggest that endogenous GA content in developing ovaries is the limiting factor controlling the parthenocarpic development of the fruits.     

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.     

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.     

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.     

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.     

Function and Expression Analysis of Gibberellin Oxidases in Apple

Three cDNAs, encoding gibberellin (GA) 20-oxidase (MdGA20ox1, identical to AB037114), 3-oxidase (MdGA3ox1), and 2-oxidase (MdGA2ox1), were isolated from apple cv. Fuji (Malus x domestica). Southern blot analysis indicated that each of these genes belongs to a gene family. Standard enzyme assays show that the MdGA20ox1-MBP fusion protein can sequentially oxidize three times at C-20 position of GA₁₂ and GA₅₃ and generate GA₉ and GA₂₀; the MdGA3ox1-MBP fusion protein converts GA₂₀ and GA₉ to GA₄ and GA₁, and the MdGA2ox1-MBP fusion protein converts GA₄ and GA₁ to GA₃₄ and GA₈, respectively. In addition, we confirmed that MdGA20ox1 is strongly expressed in immature seeds and scarcely detected in other tissues, whereas MdGA3ox1 and MdGA2ox1 are mainly expressed in flowers. Therefore, all the three cDNAs are localized in reproductive tissues. Functional and expression analysis of the three GA oxidases would provide fundamental molecular information to analyze GA metabolic regulation in apple.     

Over-expression of a tobacco homeobox gene, NTH15 , decreases the expression of a gibberellin biosynthetic gene encoding GA 20-oxidase

Ectopic expression of the homeobox gene, NTH15 ( Nicotiana tabacum homeobox 15) in transgenic tobacco leads to abnormal leaf and flower morphology, accompanied by a decrease in the content of the active gibberellin, GA₁. Quantitative analysis of intermediates in the GA biosynthetic pathway revealed that the step from GA₁₉ to GA₂₀ was blocked in transgenic tobacco plants overexpressing NTH15 . To investigate the relationship between the expression of NTH15 and genes involved in GA biosynthesis, we isolated three cDNA clones from tobacco encoding two types of GA 20-oxidase and a 3β-hydroxylase. RNA gel blot analysis revealed that the expression of one gene ( Ntc12 , encoding GA 20-oxidase), which in wild-type tobacco plants was abundantly expressed in leaves, was strongly suppressed in the transformants. The expression level of Ntc12 decreased with increasing severity of phenotype of transgenic tobacco leaves. The abnormal leaf morphology was largely overcome by treatment with GA₂₀ or GA₁ but not by GA₁₉. These data strongly suggest that overexpression of NTH15 inhibits the expression of Ntc12 , resulting in reduced levels of active GA and abnormal leaf morphology in transgenic tobacco plants. In situ hybridization in wild-type tobacco revealed that expression of Ntc12 occurred mainly in the rib meristem, cells surrounding the procambium and in leaf primordia. Expression was not seen in the tunica, corpus and procambium, tissues in which NTH15 was predominantly expressed. The contrasting expression patterns of these genes may reflect their antagonistic functions in the formation of lateral organs from the shoot apical meristem.     

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.     

Identification of Gibberellins in Spinach and Effects of Light and Darkness on their Levels

The endogenous gibberellin (GA) content of spinach (Spinacia oleracea) was reinvestigated by combined gas chromatography-mass spectrometry analysis. The 13-hydroxy GAs: GA₅₃, GA₄₄, GA₁₉, GA₁₇, GA₂₀, GA₅, GA₁, GA₂₉, and GA₈; the non-3, 13-hydroxy GAs: GA₁₂, GA₁₅, GA₉, and GA₅₁; and the 3β-hydroxy GAs: GA₄, GA₇, and GA₃₄, were identified in spinach extracts by comparing full-scan mass spectra and Kovats retention indices with those of reference GAs. In addition, spinach plants contained GA₇-isolactone, 16,17-dihydro-17-hydroxy-GA₅₃, GA₂₉-catabolite, 3-epi-GA₁, and 10 uncharacterized GAs with mass spectra indicative of mono- and dihydroxy-GA₁₂, monohydroxy-GA₂₅, dihydroxy-GA₂₄, and dihydroxy-GA_g. The effect of light-dark conditions on the GA levels of the 13-hydroxylation pathway was studied by using labeled internal standards in selected ion monitoring mode. In short day, the GA levels were higher at the end of the light period than at the end of the dark period. Levels of GAs at the end of each short day were relatively constant. During the first supplementary light period of long day treatment, GA₅₃ and GA₁₉ declined dramatically, GA₄₄ and GA₁ decreased slightly, and GA₂₀ increased. During the subsequent high-intensity light period, the GA₂₀ level decreased and the levels of GA₅₃, GA₄₄, GA₁₉, and GA₁ increased slightly. Within 7 days after the beginning of long day treatment, similar patterns for GA₅₃ and GA₁₉ occurred. Furthermore, when these plants were transferred to darkness, an increase in the levels of GA₅₃ and GA₁₉ was observed. These results are compatible with the idea that in spinach, the flow through the GA biosynthetic pathway is much enhanced during the high-intensity light period, although GA turnover occurs also during the supplementary period of long day, both effects being responsible for the increase of GA₂₀ and GA₁ in long day.     

Metabolism of gibberellin a(12)-7-aldehyde by soybean cotyledons and its use in identifying gibberellin a(7) as an endogenous gibberellin

The level of gibberellin(GA)-like material in cotyledons of soybean (Glycine max L.) was highest at mid-pod fill—about 10 nanograms GA₃ equivalents per gram fresh weight of tissue, assayed in the immersion dwarf rice bioassay. This amount is about 1000-fold less than levels in Pisum and Phaseolus seed, other legume species whose spectrum of endogenous gibberellins (GAs) is well known. The metabolism of [¹⁴C]-GA₁₂-7-aldehyde (GA₁₂ald)—the universal GA precursor—by intact, mid-pod-fill, soybean cotyledons and their cell-free extracts was investigated. In 4 hours, extracts converted GA₁₂ald to two products—[¹⁴C]GA₁₂ (42% yield) and [¹⁴C]GA₁₅ (7%). Within 5 minutes, intact embryos converted GA₁₂ald to [¹⁴C]GA₁₂ and [¹⁴C]GA₁₅ in 15% yield; 4 hour incubations afforded at least 22 products (96% total yield). The putative [¹⁴C]GA₁₂ was identified as a product of [¹⁴C]GA₁₂ald metabolism on the basis of co-chromatography with authentic GA₁₂ on a series of reversed and normal phase high pressure liquid chromatography (HPLC) and thin-layer chromatography (TLC) systems, and by a dual feed of the putative [¹⁴C]GA₁₂ and authentic [¹⁴C]GA₁₂ to cotyledons of both peas and soybeans. The [¹⁴C]GA₁₅ was identified as a metabolite of [¹⁴C]GA₁₂ald by capillary gas chromatography (GC)-mass-spectrometry-selected ion monitoring, GC-radiocounting, HPLC, and TLC. By adding the [¹⁴C] metabolites of [¹⁴C]GA₁₂ald to a different and larger extract (about 0.2 kg fresh weight of soybean reproductive tissue) and purifying endogenous substances co-chromatographing with these metabolites, at least two GA-like substances were obtained and one identified as GA₇ by GC-mass spectrometry. Since [¹⁴C]GA₉ was not found as a [¹⁴C]metabolite of [¹⁴C]GA₁₂ald, soybean embryos might have a pathway for biosynthesis of active, C-19 gibberellins like that of the cucurbits; GA₁₂ald → GA₁₂ → GA₁₅ → GA₂₄ → GA₃₆ → GA₄ → GA₇.     

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.     

GA4 Does Not Require Conversion into GA1 to Delay Senescence of Alstroemeria hybrida Leaves

The biological activity and metabolism of applied GA₁ and GA₄ were studied in leaves of alstroemeria (Alstroemeria hybrida). It appeared that GA₄ was 2 orders of magnitude more active in delaying leaf senescence than GA₁. GA₃-13-OMe, a GA analog that cannot be hydroxylated on the 13-C position, also retarded chlorophyll loss, although less efficiently. Tritiated and deuterated GA₁, GA₄, and GA₉ were applied to leaves, and their metabolites were analyzed. According to high performance liquid chromatography and gas chromatography-mass spectrometry analyses, GA₉ was converted into GA₄ and GA₃₄, and GA₄ was converted into GA₃₄ and more polar components. No evidence was found for the conversion of both GA₉ and GA₄ into GA₁, even at the relatively high concentrations that were taken up by the leaf. The results strongly suggest that GA₄ is recognized directly by a receptor involved in regulation of leaf senescence in alstroemeria. Received November 24, 1997; accepted February 17, 1998