Quantitation of gibberellins A1, A3, A4, A9 and an A9-conjugate in good- and poor-flowering clones of Sitka spruce (Picea sitchensis) during the period of flower-bud differentiation

The levels of endogenous gibberellin A₁ (GA₁), GA₃, GA₄, GA₉ and a cellulase-hydrolysable GA₉-conjugate in needles and shoot stems of Sitka spruce [Picea sitchensis (Bong.) Carr.] grafts with different coning or flowering histories were estimated by combined gas chromatography-mass spectrometry selected ion monitoring using deuterated GA₃, GA₄ and GA₉ as internal standards. The samples were taken at the approximate time of the start of flower-bud differentiation, i.e. when the shoots had elongated approx. 95% of the final length. The needles of the good-flowering clones contained 11–12 ng per g fresh weight (FW) and 15–28 ng· (g FW) ^–1 of GA₉-conjugate and GA₉, respectively. The shoot stems of the same material contained no detectable amounts of GA₉-conjugate and 11–15 ng-(g FW)^–1 of GA₉. The amounts of GA₉-conjugate and GA₉ were apparently lower in the poor-flowering clones, the needles containing 4–9 ng-(g FW)^–1 and 7–17 ng·(g FW)^–1, respectively. Also in this material the shoot stems contained no detectable amounts of GA₉-conjugate. The amounts of GA₄ were very small in both materials, ranging from 1–1.6 ng-(g FW)^–1. The good-flowering clones contained no detectable amounts of the more polar gibberellins, GA₁ and GA₃. The poor-flowering clones, on the other hand, contained high levels of GA₁₅ 17–19ng·(gFW)^–1 in the needles and 10–13 ng·(g FW) ^–1 in the shoot stems, and also smaller amounts of GA₃, 2–3 ng·(g FW)^–1 in the needles and approx. 1 ng·(g FW)^–1 in the shoot stems. The results demonstrate differences in GA-metabolism between the poor- and the good-flowering clones. The higher amounts of GA₉-conjugate and GA₉ might indicate a higher capacity for synthesizing GA₄ in the good-flowering material. This synthesis does not, however, result in a build-up of the GA₄-pool, maybe because of a high rate of turnover. Gibberellin A₄ was apparently neither hydroxylated to GA₁ nor converted to GA₃ in the goodflowering material, as was the case in the poor-flowering material. This might indicate that gibberellin metabolism in the poor-flowering material is directed towards GA₁ and GA₃, GAs preferentially used in vegetative growth.Abbreviations FW fresh weight - GA_n gibberellin A_n - HPLC high-performance liquid chromatography     

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.     

Identification of Gibberellins A(1), A(5), A(29), and A(32) from Immature Seeds of Apricot (Prunus armeniaca L.)

Gibberellins (GAs) A₁, A₅, and A₂₉ were identified, and also GA₃₂ was confirmed, as endogenous GAs of immature seeds (3-4 weeks after anthesis, 0.25-0.5 gram fresh weight) of apricot (Prunus armeniaca L.) based on capillary gas chromatography (GC), retention time (Rt), and selected ion monitoring (SIM), in comparison with authentic standards. Fractions subjected to GC-SIM were purified and separated using sequential solvent partitioning → paper chromatography → reverse phase C₁₈ high performance liquid chromatography (HPLC) → bioassay on dwarf rice cv Tan-ginbozu. Two other peaks of free GA-like bioactivity (microdrop and immersion dwarf rice assays) were eluted from C₁₈ HPLC at Rts where GA_4/7 and GA₈ (or other GAs with similar structures) would elute. Also, three unidentified GA glucoside-like compounds (based on bioactivity on the immersion assay, and no bioactivity on the microdrop assay) were noted. There were very high amounts of GA₃₂ (112 ng of GA₃ equivalents per gram fresh weight), and minor amounts (0.5 ng of GA₃ equivalents) for each of GA₁ and GA₅, respectively, based on the microdrop assay.     

Metabolism of Gibberellin A(12) and A(12)-Aldehyde in Developing Seeds of Pisum sativum L

Metabolism of [¹⁴C]gibberellin (GA) A₁₂ (GA₁₂) and [¹⁴C]gibberellin A₁₂-aldehyde (GA₁₂-aldehyde) was examined in cotyledons and seed coats from developing seeds of pea (Pisum sativum L.). Both were metabolized to only 13-hydroxylated GAs in cotyledons but to 13-hydroxylated and non-13-hydroxylated GAs in seed coats. The metabolism of [¹⁴C]GA₁₂ was slower in seed coats than in cotyledons. [¹⁴C]GA₁₂-aldehyde was also metabolized to conjugates in seed coats. Seed coat [¹⁴C]-metabolites produced from [¹⁴C]GA₁₂-aldehyde were isolated by high-performance liquid chromatography (HPLC). Conjugates were base hydrolyzed and the free GAs reisolated by HPLC and identified by gas chromatography-mass spectrometry. [¹⁴C]GA₅₃-aldehyde, [¹⁴C]GA₁₂-aldehyde conjugate, and [¹⁴C]GA₅₃-aldehyde conjugate were major metabolites produced from [¹⁴C]GA₁₂-aldehyde by seed coats aged 20-22 days or older. The dilution of ¹⁴C in these compounds by ¹²C, as compared to the supplied [¹⁴C]GA₁₂-aldehyde, indicated that they are endogenous. Feeding [¹⁴C]GA₅₃-aldehyde led to the production of [¹⁴C]GA₅₃-aldehyde conjugate in seed coats and shoots and also to 13-hydroxylated GAs in shoots. Labeled GAs, recovered from plant tissue incubated with either [¹⁴C]GA₁₂, [¹⁴C]GA₁₂-aldehyde, or [³H]GA₉, were used as appropriate markers for the recovery of endogenous GAs from seed coats or cotyledons. These GAs were purified by HPLC and identified and quantified by gas chromatography-mass spectrometry. GA₁₅, GA₂₄, GA₉, GA₅₁, GA₅₁-catabolite, GA₂₀, GA₂₉, and GA₂₉-catabolite were detected in seed coats, whereas GA₉, GA₅₃, GA₄₄, GA₁₉, GA₂₀, and GA₂₉ were found in cotyledons. The highest GA levels were for GA₂₀ and GA₂₉ in cotyledons (783 and 912 nanograms per gram fresh weight, respectively) and for GA₂₉ and GA₂₉-catabolite in seed coats (1940 and > 1940 nanograms per gram fresh weight, respectively).     

Gibberellin physiology of safflower: endogenous gibberellins and response to gibberellic acid

Endogenous gibberellins (GAs) were extracted from safflower (Carthamus tinctorius L.) stems and detected by capillary gas chromatography-mass spectrometry from which GA₁, GA₃, GA₁₉,, GA₂₀, GA₂₉, and probably, GA₄₄ were detected. The detection of these GAs suggests that the early 13-OH biosynthetic pathway is prevalent in safflower shoots. Deuterated GAs were used as internal standards and GA concentrations were determined in stems harvested at weekly intervals. GA₁ and GA₁₉ levels per stem increased but concentrations per gram dry weight decreased over time. GA₂₀ was only detected in young stem tissue.Gibberellic acid (GA₃) was also applied in field trials and both GA₃ and the GA biosynthetic inhibitor, paclobutrazol, were applied in growth chamber tests. GA₃ increased epidermal cell size, internode length, and increased internode cell number causing stem elongation. Conversely, paclobutrazol reduced stem height, internode and cell size, cell number and overall shoot weight. In field tests, GA₃ increased total stem weight, but decreased leaf weight, flower bud number and seed yield. Thus, GA₃ promoted vegetative growth at the expense of reproductive commitment. These studies collectively indicate a promotory role of GAs in the control of shoot growth in safflower, and are generally consistent with gibberellin studies of related crop plants. Author for correspondence     

Measurement of Indole-3-Acetic Acid in Peach Fruits (Prunus persica L. Batsch cv Redhaven) during Development

The amount of indole-3-acetic acid (IAA) was measured in peach fruits by gas chromatography-mass spectrometry-selective ion monitoring using an isotope dilution assay with [¹³C₆]IAA as an internal standard throughout the growing season. Ethylene evolution of the fruit was also measured. IAA levels were 25 nanograms per gram fresh weight, 18 days after anthesis. Both IAA levels and rates of ethylene evolution declined to their lowest levels (7 nanograms IAA per gram fresh weight and 0.01 nanoliter ethylene per gram per hour) in the second stage of fruit growth. Endogenous levels of free-IAA and ethylene evolution increased in the last stage of peach fruit development to 32 nanograms per gram fresh weight and 0.27 nanoliter per gram per hour, respectively. IAA amounts peaked in the ovules 67 days after anthesis.     

Endogenous Gibberellins in Elongating Shoots of Clones of Salix dasyclados and Salix viminalis

Elongating shoots of rapidly growing clones of Salix viminalis L. (clone 683-4) and Salix dasyclados Wimm. (clone 908) harvested in early August were analyzed for endogenous gibberellins (GA). Distribution of GA-like activity, determined by Tan-ginbozu dwarf rice microdrop bioassay after reverse phase C₁₈ high performance chromatography, was similar for both species. For S. dasyclados, combined gas chromatography-selected ion monotoring (GC-SIM) yielded identifications of GA₁, GA₈, GA₁₉, GA₂₀, and GA₂₉. Identifications of GA₄ and GA₉ were also made using co-injections of known amounts of [17, 17-²H₂]GAs. By bioassay, the main activity was GA₁₉-like in both species. Gibberellin A₁, GA₁₉, and GA₂₀ concentrations were approximated by GC-SIM using co-injections of known amounts of [17,17-²H₂]GAs. Both bioassay and GC-SIM results indicated very high concentrations of GA₁₉ and GA₂₀ (about 6000 nanograms per kilogram fresh weight shoot tissue using GC-SIM: 800 ng using bioassay), compared to the concentration of GA₁ (about 130 nanograms per kilogram fresh weight using either GC-SIM or bioassay).     

Levels of Indole-3-Acetic Acid in Lemna gibba G-3 and in a Large Lemna Mutant Regenerated from Tissue Culture

Large changes in indole-3-acetic acid (IAA) levels occur during growth of Lemna gibba G-3 in sterile culture. The levels of IAA were measured in plants during a 45 day growth cycle using HPLC and isotope dilution analysis followed by selected ion current monitoring GC-MS analysis with ¹³C₆-IAA as the internal standard. Even though the rate of plant growth remained constant over the entire growth period, IAA levels ranged from a high of 222 to a low of 6 nanograms per gram fresh weight. A Lemna mutant (jsR₁) which has a giant phenotype was obtained by regeneration from primary callus cultures. Microspectrofluorometry of diamidino-2-phenylindole stained cells showed that jsR₁ has the same amount of DNA per nucleus as the parent line (PL). All jsR₁ cell types measured are about 1.5 times larger than in PL. The endogenous levels of IAA per gram fresh weight were higher in jsR₁ at several stages of the plant culture cycle as compared to PL. This difference ranged from 1.2 to over 100 times as much. While PL showed only one high peak at day 9, jsR₁ had IAA levels of 480 and 680 nanograms per gram fresh weight at days 9 and 45, respectively. Throughout the midculture stage of the growth cycle (20-28 days) both jsR₁ and PL had IAA levels in the range of 9 to 14 nanograms per gram fresh weight. In contrast to PL, at day 45, jsR₁ had no detectable ester or amide conjugates of IAA. These changes in IAA levels were determined in sterile plant cultures and thus cannot be attributed to bacterial or fungal activity.     

A Gibberellin-Deficient Brassica Mutant-rosette

A single-gene mutant (rosette [ros/ros]) in which shoot growth and development are inhibited was identified from a rapid cycling line of Brassica rapa (syn campestris). Relative to normal plants, the mutant germinated slowly, had delayed or incomplete floral development, and reduced leaf, petiole, and internode growth. The exogenous application of GA₃ by foliar spray or directly to the shoot tip of rosette resulted in rapid flowering, bolting (shoot elongation), and viable seed production. Shoots of rosette contained endogenous levels of total gibberellin (GA)-like substances (`Tan-ginbozu' dwarf rice assay) of about one-tenth of that of the normal rapid-cycling line of B. rapa which consisted almost entirely of a very nonpolar, GA-like substance which yielded GA₁ and GA₃ upon mild acid hydrolysis. In a normal rapid-cycling B. rapa line, the nonpolar putative GA₁ and GA₃ conjugates were present, but additionally, free GA₁ and GA₃ were abundant and identified by gas chromatography-mass spectrometry-selected ion monitoring. The quantities of free GA₁ and GA₃ in the normal line and in rosette were quantified by GC-MS-SIM using [²H₂]GA₁ as an internal standard. Fourteen-day-old rosette and normal seedlings contained 5.3 and 23.2 ng GA₁ per plant, respectively. At day 21 the rosette plants contained 7.7 and 26.1 nanograms per plant of GA₁ and GA₃, while normal plants contained 31.1 and 251.5 nanograms per plant, respectively. Thus, normal plants contained from four to ten times higher levels of total GA-like substances, GA₁, or GA₃, than rosette. The ros allele results in reduced GA level, yielding the rosette phenotype whose delayed germination and flowering, and reduced shoot growth responses indicate a probable role for endogenous GA₁ and GA₃ in the regulation of these processes in Brassica.     

Relationship between Indole-3-Acetic Acid Levels in Apple (Malus pumila Mill) Rootstocks Cultured in Vitro and Adventitious Root Formation in the Presence of Indole-3-Butyric Acid

In vitro rooting response and indole-3-acetic acid (IAA) levels were examined in two genetically related dwarfing apple (Malus pumila Mill) rootstocks. M.26 and M.9 were cultured in vitro using Linsmaier-Skoog medium supplemented with benzyladenine (BA), indole-3-butyric acid (IBA), and 1,3,5-trihydroxybenzoic acid (PG). Rooting response was tested in Lepoivre medium supplemented with IBA and PG. IBA concentrations of 12.0 and 4.0 micromolar induced the maximum rooting percentages for M.9 and M.26, respectively. At these concentrations rooting response was 100% for M.26 and 80% for M.9. Free and conjugated IAA levels were determined in M.26 and M.9 shoots prior to root inducing treatment by high performance liquid chromatography with fluorescence detection and validated by gas chromatography-mass spectrometry using ¹³[C₆]IAA as internal standard. Basal sections of M.26 shoots contained 2.8 times more free IAA than similar tissue in M.9 (477.1 ± 6.5 versus 166.6 ± 6.7 nanograms per gram fresh weight), while free IAA levels in apical sections of M.26 and M.9 shoots were comparable (298.0 ± 4.4 versus 263.7 ± 9.3 nanograms per gram fresh weight). Conjugated IAA levels were significantly higher in M.9 than in M.26 indicating that a greater proportion of total IAA was present as a conjugate in M.9. These data suggest that differences between M.26 and M.9 rooting responses may be related to differences in free IAA levels in the shoot base.     

A Developmental Pattern of Flowering in Colored <Emphasis Type="Italic">Zantedeschia</Emphasis> spp.: Effects of Bud Position and Gibberellin

The restricted flowering of colored cultivars ofZantedeschia is a consequence of developmental constraints imposed by apical dominance of the primary bud on secondary buds in the tuber, and by the sympodial growth of individual shoots. GA₃ enhances flowering inZantedeschia by increasing the number of flowering shoots per tuber and inflorescences per shoot. The effects of gibberellin on the pattern of flowering and on the developmental fate of differentiated inflorescences along the tuber axis and individual shoot axes were studied in GA₃ and Uniconazole-treated tubers. Inflorescence primordia and fully developed (emerged) floral stems produced during tuber storage and the plant growth period were recorded. Days to flowering, percent of flowering shoots and floral stem length decreased basipetally along the shoot and tuber axes. GA₃ prolonged the flowering period and increased both the number of flowering shoots per tuber and the differentiated inflorescences per shoot. Activated buds were GA₃ responsive regardless of meristem size or age. Uniconazole did not inhibit inflorescence differentiation but inhibited floral stem elongation. The results suggest that GA₃ has a dual action in the flowering process: induction of inflorescence differentiation and promotion of floral stem elongation. The flowering pattern could be a result of a gradient in the distribution of endogenous factors involved in inflorescence differentialtion (possibly GAs) and in floral stem growth. This gradient along the tuber and shoot axes is probably controlled by apical dominance of the primary bud. Online publication: 7 April 2005     

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.     

Metabolism of Tritiated Gibberellins A(4) and A(9) in Norway Spruce, Picea abies (L.) Karst. : Effects of a Cultural Treatment Known to Enhance Flowering

Shoots of mature grafted propagules of Picea abies (L.) Karst. metabolized [³H]gibberellin A₄ (GA₄) to at least 14 acidic substances, two of which were tentatively identified by gas-liquid radiochromatography as GA₂ (possibly an artifact) and GA₃₄. [³H]GA₉ was converted into a number of metabolites, one of which was chromatographically similar to, but not identical with, GA₄. Metabolism was maximally 61 and 57% over 48 hours for GA₄ and GA₉, respectively, and was correlated with the rate of change (i.e. increase followed by decrease) in endogenous GA-like substances as shoot elongation progressed. Propagules covered with a clear plastic film, a treatment which promotes flowering, metabolized [³H]GA₄ more slowly than did control plants in the open. Inasmuch as a GA_4/7 mixture can also promote flowering in P. abies, the retarded metabolism of [³H]GA₄ may reflect the manner in which trees under plastic metabolize endogenous GA-like substances. If so, then the stimulating effect of this cultural treatment on flowering may come about through an increased level of endogenous, less polar GA-like substances.     

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.     

Light intensity, gibberellin content and the resolution of shoot growth in Brassica

The role of gibberellins (GAs) in the regulation of shoot elongation is well established but the phytohormonal control of dry-matter production is poorly understood. In the present study, shoot elongation and dry-matter production were resolved by growing Brassica napus L. seedlings under five light intensities (photon flux densities) ranging from 25 to 500 μmol m⁻² s⁻¹. Under low light, plants were tall but produced little dry weight; as light intensity was increased, plants were progressively shorter but had increasing dry weights. Endogenous GAs in stems of 16- and 17-d-old plants were analyzed by gas chromatography-selected ion monitoring with [²H₂] internal standards. The contents of GAs increased dramatically with decreasing light intensity: GA₁, GA₃, GA₈ and GA₂₀ were 62, 15, 16 and 32 times higher, respectively, under the lowest versus highest light intensities. Gibberellin A₁₉ was not measured at 25 μmol m⁻² s⁻¹ but was 9␣times greater in the 75 compared to 500 μmol m⁻² s⁻¹ treatment. Shoot and hypocotyl lengths were closely positively correlated with (log) GA concentration (for example: r ² = 0.93 for GA₁ and hypocotyl length) but shoot dry matter was negatively correlated with GA concentration. The application of gibberellic acid (GA₃) produced elongation of plants grown under high light, indication that their low level of endogenous GA was limiting shoot elongation. Although endogenous GA₂₀ showed the greatest influence of light treatment, metabolism of [³H]GA₂₀ and of [³H]GA₁ was only slightly influenced by light intensity, suggesting that neither 2β- nor 3β-hydroxylation were points of metabolic regulation. The results of this study indicate that GAs control shoot elongation but are not directly involved in the regulation of shoot dry weight in Brassica. The study also suggests a role of GAs in photomorphogenesis, serving as an intermediate between light condition and shoot elongation response. Received: 18 June 1998 / Accepted: 29 July 1998     

Endogenous Gibberellins and Shoot Growth and Development in Brassica napus

Greenhouse-grown oilseed rape (Brassica napus, annual Canola variety `Westar') plants were harvested at six dates from the vegetative phase until the early pod (silique)-fill/late flowering stage. Endogenous gibberellin (GA)-like substances were extracted from stems, purified, and chromatographed on silica gel partition columns prior to bioassay in serial dilution using the `Tan-ginbozu' dwarf rice microdrop assay. The concentrations of total endogenous GA-like substances were low during vegetative stages (1 nanogram GA₃ equivalents/gram dry weight), and rose 300-fold by the time of floral initiation. After floral initiation the concentration of GA-like substances fell, then rose again during bolting to maximal levels during the early pod-fill stage (940 nanograms per gram dry weight). The qualitative profiles of GA-like substances varied across harvests, with higher proportions of a GA₁-like substance at the early pod-fill stage. In a second study stems were similarly harvested at eight dates and the concentrations of endogenous GA₁, the principal bioactive native GA of oilseed rape, were determined by gas chromatography-selected ion monitoring using [17,17-²H]GA₁ as a quantitative internal standard. The concentration of GA₁ increased at about the time of floral initiation and then subsequently fell, thus confirming the pattern noted above for total GA-like substances. The exogenous application of paclobutrazol (PP333), a persistent triazole plant growth regulator (PGR) which blocks GA biosynthesis, or another triazole, triapenthenol (RSW0411), prevented flowering as well as bolting; plants remained at the vegetative rosette stage. These results imply a causal role for endogenous GA, in the control of bolting, which normally precedes anthesis. Further, the rise in the concentration of total endogenous GA-like substances, including GA₁, which was associated with floral initiation, and the prevention of visable floral development by the triazole PGRs, also indicates a role for endogenous GAs in the regulation of flowering in B. napus.     

Gibberellins and Heterosis in Maize : II. Response to Gibberellic Acid and Metabolism of [H]Gibberellin A(20)

Two maize inbreds, CM7 and CM49, and CM7 × CM49, their F₁ hybrid (which displayed significant heterosis), were examined with regard to response to exogenous gibberellin A₃ (GA₃), and in their ability to metabolize GA₂₀, a native GA of maize. The leaf sheath elongation response to GA₃ was far greater for the imbreds than for their hybrid. The inbreds also displayed significant elongation of the leaf blades in response to GA₃, whereas the hybrid was unaffected. Promotion of cell division in the leaf sheath of CM7 and the hybrid was effected by GA₃, but no promotion of cell elongation was observed in CM49, even though significant leaf sheath elongation occurred. Shoot dry weight of both inbreds was significantly increased by GA₃, but response by the hybrid in this parameter was slight and variable. Root dry weight of CM7 was significantly increased by GA₃, but was unchanged in CM49 and the hybrid. Thus, inbred shoot dry weight increases effected by GA₃ were not at the expense of the root system. Rapid metabolism of [2,3-³H]GA₂₀ occurred in all genotypes, although genotypic differences were observed. The hybrid had the highest rates of metabolism to GA glucosyl conjugate-like substances. Oxidative metabolism was also fastest in the hybrid, followed by CM7, and slowest in CM49, the slowest-growing inbred. Thus, rate of GA₂₀ metabolism is under genetic control in normal (i.e. not dwarfed) maize genotypes. These results, taken together with previous reports that the hybrid has significantly enhanced levels of endogenous GA-like substances, suggest that GA play a role in the expression of heterosis in maize.     

Genetic Regulation of Development in Sorghum bicolor: V. The ma(3) Allele Results in Gibberellin Enrichment

Sorghum bicolor genotypes, near isogenic with different alleles at the third maturity locus, were compared for development, for responsiveness to GA₃ and a GA synthesis inhibitor, and occurrence and concentrations of endogenous GAs, IAA, and ABA. At 14 days the genotype 58M (ma₃^Rma₃^R) exhibited 2.5-fold greater culm height, 1.75-fold greater total height, and 1.38-fold greater dry weight than 90M (ma₃ma₃) or 100M (Ma₃Ma₃). All three genotypes exhibited similar shoot elongation in response to GA₃, and 58M showed GA₃-mediated hastening of floral initiation when harvested at day 18 or 21. Both 90M and 100M had exhibited hastening of floral initiation by GA₃ previously, at later application dates. Tetcyclacis reduced height, promoted tillering, and delayed flowering of 58M resulting in plants which were near phenocopies of 90M and 100M. Based on bioassay activity, HPLC retention times, cochromatography with ²H₂-labeled standards on capillary column GC and matching mass spectrometer fragmentation patterns (ions [m/z] and relative abundances), GA₁, GA₁₉, GA₂₀, GA₅₃, and GA₃ were identified in extracts of all three genotypes. In addition, based on published Kovats retention index values and correspondence in ion masses and relative abundances, GA₄₄ and GA₁₇ were detected. Quantitation was based on recovery of coinjected, ²H₂-labeled standards. In 14 day-old-plants, total GA-like bioactivity and GA₁ concentrations (nanograms GA/gram dry weight) were two- to six-fold higher in 58M than 90M and 100M in leaf blades, apex samples, and whole plants while concentrations in culms were similar. Similar trends occurred if data were expressed on a per plant basis. GA₁ concentrations for whole plants were about two-fold higher in 58M than 90M and 100M from day 7 to day 14. Concentrations of ABA and IAA did not vary between the genotypes. The results indicate the mutant allele ma₃^R causes a two- to six-fold increase in GA₁ concentrations, does not result in a GA-receptor or transduction mutation and is associated with phenotypic characteristics that can be enhanced by GA₃ and reduced by GA synthesis inhibitor. These observations support the hypothesis that the allele ma₃^R causes an overproduction of GAs which results in altered leaf morphology, reduced tillering, earlier flowering, and other phenotypic differences between 58M and 90M or 100M.     

Gibberellins and Gravitropism in Maize Shoots : Endogenous Gibberellin-Like Substances and Movement and Metabolism of [3H]Gibberellin A20

[³H]Gibberellin A₂₀ (GA₂₀) of high specific radioactivity (49.9 gigabecquerel per millimole) was applied equilaterally in a ring of microdrops to the internodal pulvinus of shoots of 3-week-old gravistimulated and vertical normal maize (Zea mays L.), and to a pleiogravitropic (prostrate) maize mutant, lazy (la). All plants converted the [³H]GA₂₀ to [³H]GA₁⁻ and [³H]GA₂₉-like metabolites as well as to several metabolites with the partitioning and chromatographic behavior of glucosyl conjugates of [³H]GA₁, [³H]GA₂₉, and [³H]GA₈. The tentative identification of these putative [³H]GA glucosyl conjugates was further supported by the release of the free [³H]GA moiety after cleavage with cellulase. Within 12 hours of the [³H]GA₂₀ feed, there was a significantly higher proportion of total radioactivity in lower than in upper halves of internode and leaf sheath pulvini in gravistimulated normal maize. Further, there was a significantly higher proportion of putative free GA metabolites of [³H]GA₂₀, especially [³H]GA₁, in the lower halves of normal maize relative to upper halves. The differential localization of the metabolites between upper and lower halves was not apparent in the pleiogravitropic mutant, la. Endogenous GA-like substances were also examined in gravistimulated maize shoots. Forty-eight hours after gravistimulation of 3-week-old maize seedlings, endogenous free GA-like substances in upper and lower leaf sheath and internode pulvini halves were extracted, chromatographed, and bioassayed using the `Tanginbozu' dwarf rice microdrop assay. Lower halves contained consistently higher total levels of GA-like activity. The qualitative elution profile of GA-like substances differed consistently, upper halves containing principally a GA₂₀-like substance and lower halves containing mainly GA₁-like and GA₁₉-like substances. Gibberellins A₁ (10 nanograms per gram) and A₂₀ (5 nanograms per gram) were identified from these lower leaf sheath pulvini by capillary gas chromatography-selected ion monitoring. Results from all of these experiments are consistent with a role for GAs in the differential shoot growth that follows gravitropism, although the results do not eliminate the possibility that the redistribution of GAs results from the gravitropic response.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of northern red oak (<Emphasis Type="Italic">Quercus rubra</Emphasis> L.)

In vitro propagation of northern red oak (Quercus rubra) shoots was successful from cotyledonary node explants excised from 8-wk-old in vitro grown seedlings. Initially, four shoots per explant were obtained on Murashige and Skoog (MS) medium supplemented with 4.4 μM 6-benzylaminopurine (BA), 0.45 μM thidiazuron (TDZ), and 500 mg l⁻¹ casein hydrolysate (CH) with a regeneration frequency of 64.7% after 3 wk. Subculturing explants (after harvesting shoots) to fresh treatment medium significantly increased shoot bud regeneration (16.6 buds per explant), but the buds failed to develop into shoots. A higher percentage (73.3%) of the explants regenerated four shoots per explant on woody plant medium (WPM) supplemented with 4.4 μM BA, 0.29 μM gibberellic acid (GA₃), and 500 mg l⁻¹ CH after 3 wk. Explants subcultured to fresh treatment medium after harvesting shoots significantly increased shoot regeneration (16 shoots per explant). Shoot elongation was achieved (4 cm) when shoots were excised and cultured on WPM supplemented with 0.44 μM BA and 0.29 μM GA₃. In vitro regenerated shoots were rooted on WPM supplemented with 4.9 μM indole-3-butyric acid. A higher percentage regeneration response and shoot numbers per explant were recorded on WPM supplemented with BA and GA₃, than on MS medium containing BA and TDZ. Lower concentrations of BA and GA₃ were required for shoot elongation and prevention of shoot tip necrosis. Each cotyledonary node yielded approximately 20 shoots within 12 wk. Rooted plantlets were successfully acclimatized.