OsEXPA4 and OsRWC3 are involved in asymmetric growth during gravitropic bending of rice leaf sheath bases

Expression of an expansin gene ( OsEXPA4 ) and a plasma membrane aquaporin ( OsRWC3 ) were differentially induced in the abaxial (lower) side of rice leaf sheath bases during gravitropism. Expression of both of these genes was induced by GA₃. β-glucuronidase (GUS) activity in transgenic rice expressing a GUS reporter gene under the control of the gibberellin (GA)-responsive OsRWC3 promoter was investigated during gravitropic curvature and found to be enhanced in the abaxial side of graviresponding leaf sheath bases. Ancymidol, a GA biosynthesis inhibitor, reduced the expression gradient of OsEXPA4 and OsRWC3 . The osmotic potential was transiently lower on the abaxial side of gravistimulated leaf sheath bases, and the gravitropic curvature was inhibited by HgCl₂ and phloretin, two known inhibitors of aquaporins. These data suggest that asymmetric GA redistribution following gravistimulation results in the localized expression of OsEXPA4 and OsRWC3 , that in turn might bring about differential cell wall loosening, cell expansion and water influx via aquaporins, thus leading to gravitropic bending.     

Effects of exogenous gibberellins and paclobutrazol on floral stalk growth of tulip sprouts isolated from cooled and non-cooled tulip bulbs

The involvement of gibberellins (GAs) in the regulation of floral stalk elongation and flower development has been studied in tulip. The biological activity of GA₄ and GA₉, both endogenous in tulip bulb sprouts, and GA₁, was tested in vitro on sprouts of cooled and non-cooled tulip bulbs ( Tulipa gesneriana L. cv. Apeldoorn), in the presence or absence of the GA biosynthesis inhibitor paclobutrazol. At early starting dates of incubation, floral stalks from both cooled and non-cooled bulbs hardly showed any elongation in the absence of exogenous GA. Paclobutrazol had no effect on floral stalk elongation, and the response to GAs of sprouts from cooled bulbs was greater than that of sprouts from non-cooled bulbs. At later starts of incubation, considerable floral stalk elongation occurred without GA application. Paclobutrazol inhibited this floral stalk elongation, and the growth of sprouts from both cooled and non-cooled bulbs was stimulated by GA application. The effect of paclobutrazol was reversed by simultaneous application of GA₄ or GA₉. Application of GA with and without paclobutrazol resulted in the same elongation of the floral stalk, indicating the absence of substantial side effects of the inhibitor. The isolated sprouts did not develop a full-grown flower without the addition of GA. GA₄ was more effective than GA₉ in stimulating this flower development. The results demonstrate that both sprouts from cooled and non-cooled bulbs are responsive to exogenous GAs in vitro, and may be a site of GA biosynthesis.     

Interaction of gibberellins and phytochrome in the control of cowpea epicotyl elongation

The physiological response of cowpea ( Vigna sinensis L.) epicotyl explants to far‐red light (FR) and its interaction with gibberellins (GAs) have been investigated. The effect of FR and GA₁ varied with the age of the seedlings from which the explants were made: for FR, it decreased progressively with age (though the sensitivity of the epicotyls to FR did not change significantly until at least day 11), whereas it remained essentially constant for applied GA₁ between days 5 and 9 after sowing. This indicates that the loss of response to FR may be due to a decrease in endogenous GA levels in the epicotyl. For a range of GA₁ and GA₂₀ (0.01–1 µg explant⁻¹), both hormones were more active in FR than in R irradiated epicotyls, suggesting that phytochrome may affect GA sensitivity besides GA metabolism. The location of the epicotyl region most sensitive to FR (between 5 and 20 mm below the apex) was different from that to GAs (the upper 10 mm). Nevertheless, FR extended the region responsive to applied GAs, even in paclobutrazol‐treated epicotyls where elongation was due entirely to exogenous GAs. This means that modulation of epicotyl elongation by phytochrome, that occurs in a zone different from though overlapping with the GA‐sensitive subapical zone, is also mediated by GAs. Growth in the most FR‐sensitive region of the epicotyl stimulated by FR or GA₁ was due to cell elongation, and in the most GA‐sensitive region to both cell division and elongation. The effect of FR and GA₁ was negated by colchicine, indicating that microtubules may be involved in the response to both factors.     

Internode length in Pisum. The lrs mutation reduces gibberellin response and levels

We describe a new mutation, lrs , which reduces internode length in Pisum sativum L. The mutation appears to act by reducing both GA synthesis and the response to GA₁. The levels of the 13‐hydroxylated GAs, GA₅₃, GA₄₄, GA₁₉, GA₂₀, GA₁, and GA₈ in the lrs mutant were greatly reduced compared with the wild‐type. The extent of the reduction in GA₁ content in the apical tissues would, at least in part, account for the dwarf phenotype of the mutant. The reduced GA responsiveness of the new mutant was indicated by the inability of applied GA₁ to remove the difference in elongation between lrs and LRS plants. The lrs mutant appears to be unique amongst internode length genotypes, possessing characteristics of both GA synthesis and GA response mutants.     

Biological activity, identification and quantification of gibberellins in seedlings of Norway spruce (Picea abies) grown under different photoperiods

Short photoperiod induces growth cessation in seedlings of Norway spruce ( Picea abies (L.] Karst.). Application of different gibberellins (GAS) to seedlings growing under a short photoperiod show that GA₉ and GA₂₀ can not induce growth. In contrast application of GA, and GA₄ induced shoot elongation. The results indicate that 3β-hydroxylation of GA₉ to GA₄ and of GA₂₀ to GA₁ is under photoperiodic control. To confirm that conclusion, both qualitative and quantitative analyses of endogenous GAs were performed. GA₁, GA₃, GA₄, GA₇, GA₉, GA₁₂, GA₁₅, GA₁₅, GA₂₀, GA₂₉, GA₃₄ and GA₅₁ were identified by combined gas chromatography-mass spectrometry in shoots of Norway spruce seedlings. The effect of photoperiod on GA levels was determined by using deuterated and ¹⁴C-labelled GAs as intermal standards. In short days, the amounts of GA₉, GA₄ and GA₁ are less than in plants grown in continuous light. There is no significant difference in the amounts of GA₃, GA₁₂, and GA₂₀ between the different photoperiods. The lack of accumulation of GA₉ and GA₂₀ under short days is discussed.     

Gibberellin substitution for long day secondary induction of flowering in Poa pratensis

Plants of Poa pratensis cv. Holt initiate inflorescence primordia when exposed to short days (SD) and low temperature, but require a secondary induction by at least 4 long days (LD) for further inflorescence development and stem elongation. Single or double applications of 10 µg per plant of gibberellins A₁, A₃, A₅ and 16,17‐dihydro A₅ (DHGA₅) induced inflorescence development in a high proportion of plants in SD, but only if the plants were detillered to a single stem. Exposure to 2 LD cycles did not cause heading and flowering alone but enhanced the effect of exogenous gibberellins (GAs), bringing flowering to 100%. GA₅ and DHGA₅ were less effective than GA₁ and GA₃ in SD, especially with double applications, but were more effective than GA₁ and GA₃ when given together with 2 LD. The GAs had differential effects on vegetative growth and flowering, GA₅ and DHGA₅ causing much less leaf and stem growth than the other two GAs. Marginal induction, whether by LD or GA application, resulted in a high proportion of spikelets with viviparous proliferation. Thus, whereas GAs are inhibitory to the primary induction by SD, they can replace secondary induction by LD when vegetative growth is limited.     

Gibberellic acid-stimulated and auxin-stimulated cell growth in relation to RNA synthesis, protein synthesis and development in the wheat coleoptile

Young excised coleoptiles from dark grown wheat have their cell growth promoted by gibberellic acid (GA₃), while sections from older coleoptiles have their cell growth promoted by auxin. The GA₃ response has a much longer lag period than that of auxin. Neither GA₃ nor auxin has any effect on ¹⁴C-leucine and ¹⁴C-uridine incorporation and uptake after 1 h, indicating that the lag in growth stimulation following GA₃ application is not associated with changes in protein or RNA synthesis. Following a 6 h incubation there are small increases in ¹⁴C-leucine and ¹⁴C-uridine incorporation in response to both GA₃ and auxin, and in the case of auxin this is associated with increased uptake. Studies on protein and RNA turnover using pulse-chase experiments have shown that both GA₃ and auxin have no effect on protein and RNA stability. There are, however, developmental changes in RNA and protein synthesis that should be considered in any explanation of the mechanism of action of these hormones on cell growth. Young GA₃-sensitive tissue has high rates of RNA synthesis and low protein and RNA turnover, while auxin-sensitive tissue has low rates of RNA synthesis, slightly higher rates of RNA turnover and much higher rates of protein turnover. The evidence overall favours more effective utilisation by GA₃ and auxin of a basal control level of RNA and protein synthesis and turnover in coleoptile tissue.     

The end-of-day far-red irradiation increases gibberellin A1 content in cowpea (Vigna sinensis) epicotyls by reducing its inactivation

It has been shown previously that gibberellins (GAs) mediate the phytochrome (Phy) control of cowpea ( Vigna sinensis L.) epicotyl elongation induced by end-of-day (EOD)-far-red light (FR). In the present work, the EOD-FR effect on GA metabolism and GA levels in cowpea has been investigated. GA₁, GA₈, GA₁₉ and GA₂₀ were identified in epicotyls, and GA₁, GA₁₉, GA₂₀ and GA₂₉-catabolite in leaves of 6-day-old cowpea seedlings. The content of GA₁ in the epicotyl paralleled the decrease of its growth rate, supporting the hypothesis that this is the GA bioactive in controlling cowpea epicotyl elongation. FR enhanced both the amount of [3H]GA₁ in the epicotyl produced from applied [3H]GA₂₀, and that of applied [3H]GA₁ that remained unmetabolized in epicotyl explants, suggesting that Phy may regulate the inactivation of GA₁. In agreement with this effect of light on GA₁ metabolism, the contents of GA₁ in the epicotyl remained higher in FR-treated than in R-treated explants. Moreover, in intact seedlings EOD-FR treatment increased both epicotyl elongation and GA₁ content in the responsive epicotyl, whereas it was not altered in the leaves. These results show, for the first time, that photostable Phys modulate the stem elongation in light-grown plants by locally controlling the GA₁ levels through regulation of its inactivation.     

Gibberellin levels and cold-induced floral stalk elongation in tulip

To investigate the role of gibberellins (GAs) in the cold requirement of tulip ( Tulipa gesneriana L. cv. Apeldoorn), bulbs were dry-stored at 5°C or at 17°C for 12 weeks prior to planting at 20°C. Only precooled bulbs showed rapid sprout growth and developed a full-grown flower. Endogenous GA levels were measured in sprouts and basal plates at the time of planting and in the second week after planting, by combined gas chromatography-mass spectrometry using deuterated internal standards. GA₄ was the major gibberellin. while GA₁, GA₉ and GA₃₄ were present in lower amounts. At the time of planting, sprouts from non-cooled bulbs contained significantly more GA₄ and GA₁, per sprout than those from precooled bulbs. Hence, there is no direct correlation between rapid sprout growth after planting and high GA levels at planting. In the second week after planting, floral stalks of precooled bulbs contained 2 to 3 times more GA₄ and its metabolite GA₃₄ per floral stalk and per g fresh weight than those of non-cooled bulbs. The results are discussed with regard to the role of gibberellins in the cold-induced floral stalk elongation of tulip.     

Identification and quantification of endogenous gibberellins in apical buds and the cambial region of Eucalyptus

Endogenous gibberellins (GAs) were extracted and purified from apical buds of Eucalyptus nitens (Deane and Maid.) Maid. and the cambial region of E. globulus (Labill.). then analysed by capillary gas chromatography-mass spectrometry. GA₁ GA₁₉ GA₂₀ and GA₂₉ were identified by full scan mass spectra. Kovats retention indices and high resolution selected ion monitoring. Using deuterated internal standards. GA₁. GA₁₉. GA₂₀ and putative GA₂₉ and GA₅₃ were quantified in the apical buds, while GA₄. GA₈. GA₉ and GA₄₄ were shown to be either absent or present at very low levels. From the cambial region. GA₁ and GA₂₀ were quantified at levels of 0.30 ng (g fresh weight)-¹ and 8.8 ng (g fresh weight)-¹ respectively. These data suggest that the early 13-hydroxylation pathway is the dominant pathway for GA biosynthesis in Eucalyptus .     

Detection and identification of gibberellins in Sitka spruce (Picea sitchensis) of different ages and coning ability by bioassay, radioimmunoassay and gas chromatography – mass spectrometry

Gibberellins Al (GA₁), GA₃, GA₄, GA₉, and after enzymatic hydrolysis of GA-conjugate-like fractions, GA₉ and GA₁₅, were identified in shoots of Sitka spruce [ Picea sitchensis (Bong.) Carr.] of different ages by combined gas chromatography-mass spectrometry (GC-MS). The purification and separation of the GAs involved the use of reverse phase and normal phase high performance liquid chromatography (HPLC). The Tan-ginbozu dwarf rice bioassay and binding to antibodies raised against GA₁, GA₄ and GA₉ were used for detection of GA-like substances. The qualitative differences between the three ages of plant material were the presence of GA₃ and GA₁ in the 48-year-old material and the absence of detectable amounts of GA₄ in the same material. This indicates a difference in GA metabolism which may reflect the difference in ability to form reproductive buds.     

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 .     

An acylcyclohexadione retardant inhibits gibberellin A1 metabolism, thereby nullifying phytochrome-modulation of cowpea epicotyl explants

The regulation by phytochrome of stem elongation in light-grown plants depends on gibberellins (GAs). To investigate whether this is mediated by a change in GA metabolism, the effect of the GA biosynthesis inhibitor LAB 198 999 (an acylcyclohexadione derivative) on the end-of-day far-red (FR) response in cowpea ( Vigna sinensis L.) epicotyl explants has been investigated. Growth of epicotyl explants of light-grown seedlings was enhanced when treated with far-red light before incubation in the dark (end-of-day FR effect). Low doses of LAB 198 999 (0.05 and 0.5 μg explant⁻¹) reduced the effect of FR, whereas 5 to 50 μg explant⁻¹ stimulated elongation of both red light (R)- and FR-treated epicotyl explants while nullifying the differences between R and FR treatments. In paclobutrazol-treated epicotyl explants, FR enhanced the response to applied GA₁ and GA₂₀, whereas LAB 198 999 increased the activity of GA₁ and decreased that of GA₂₀, [³H]Gibberellin A₁, injected into the basal part of the epicotyl, was transported and metabolized mainly to [³H]GA₈ in the apical 20 mm of the epicotyl. The conversion of [³H]GA₁ to [³H]GA₈ was dramatically reduced by both end-of-day FR treatments and LAB 198 999 applications. In addition, both treatments enhanced epicotyl elongation. It is proposed that the regulation of cowpea epicotyl growth by phytocrome is mediated, at least partially, by modifying GA₁ degradation.     

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.     

The plant-growth-promoting rhizobacteria Bacillus pumilus and Bacillus licheniformis produce high amounts of physiologically active gibberellins

The plant-growth-promoting rhizobacteria (PGPR), Bacillus pumilus and Bacillus licheniformis, isolated from the rhizosphere of alder ( Alnus glutinosa [L.] Gaertn.) have a strong growth-promoting activity. Bioassay data showed that the dwarf phenotype induced in alder seedlings by paclobutrazol (an inhibitor of gibberellin [GA] biosynthesis) was effectively reversed by applications of extracts from media incubated with both bacteria and also by exogenous GA₃. Full-scan gas chromatography-mass spectrometry analyses on extracts of these media showed the presence of GA₁, GA₃, GA₄and GA₂₀, in addition to the isomers 3- epi -GA₁ and iso -GA₃. Isotope dilution analysis indicated that epi -GA₁ was an artefact. Likewise, iso -GA₃ is also probably an artifact spontaneously formed during extraction and/or analysis. In both culture media, GA₁ was present in higher concentrations (130–150 ng ml⁻¹) than GA₃ (50–60 ng ml⁻¹), GA₄ (8–12 ng ml⁻¹) and GA₂₀ (2–3 ng ml⁻¹). The data indicated that culture of both bacteria accumulate bioactive C19-gibberellins in relative high amounts and that these GAs appear to be physiologically active in the host plant. The evidence suggests that the promotion of stem elongation induced by the PGPR could be mediated by bacterial GAs.     

Gibberellin-regulated XET is differentially induced by auxin in rice leaf sheath bases during gravitropic bending

The asymmetric distribution of auxin plays a fundamental role in plant gravitropism, yet little is understood about how its lateral distribution stimulates growth. In the present work, the asymmetric distribution not only of auxin, but also that of gibberellins (GAs), was observed in rice leaf sheath bases following gravistimulation. Gravistimulation induced the transient accumulation of greater amounts of both IAA and GA in the lower halves of the leaf sheath bases of rice seedlings. OsGA3ox1, a gene of active GA synthesis, was differentially induced by gravistimulation. Furthermore, 2,3,5-tri-iodobenzoic acid (TIBA), an inhibitor of auxin transport, substantially decreased the asymmetric distribution of IAA and the gradient of OsGA3ox1 expression. Externally applied GA(3) restored the gravitropic curvature of rice leaf sheaths inhibited by either TIBA or by ancymidol, a GA synthesis inhibitor. The expression of XET (encoding xyloglucan endotransglycosylase) was differentially induced in the lower halves of gravistimulated leaf sheath bases and was also up-regulated by exogenous IAA and GA(3). Both ancymidol and TIBA decreased the gradient of XET expression. These data suggest that the asymmetric distribution of auxin effected by gravistimulation induced a gradient of GAs via asymmetric expression of OsGA3ox1 in rice leaf sheath bases, and hence caused the asymmetric expression of XET. Cell wall loosening in the curvature site of the leaf sheath triggered by the expression of XET would contribute to gravitropic growth.     

Effects of the triazole plant growth retardant BAS 111¨W on gibberellin levels in oilseed rape, Brassica napus

Three-week-old shoots of the spring oilseed rape cv. Petranova ( Brassica napus L. ssp. napus ) were found by combined gas chromatography-mass spectrometry to contain GA₁, GA₈, GA₁₅, GA₁₇, GA₁₉, GA₂₀, GA₂₄, GA₂₉, 3-epi-GA₁ and a previously uncharacterised C₁₉ dicarboxylic acid that is probably structurally related to GA₂₄. Shoots of the winter cultivar Belinda, harvested at the early flowering stage, contained the same GAs with the exception of the C₁₉ dicarboxylic acid and, in addition, GA₃₄ and GA₅₁ were identified. All material contained higher levels of GA₂₀ than of GA₁; the ratio of GA₁ to GA₂₀ was highest in shoots containing the largest proportion of young immature tissues. Soil treatment of cv. Petranova seedlings with the growth retardant BAS 111¨W [1-phenoxy-5,5-dimethyl-3-(1,2,4-triazol-1-yl)-hexan-4-ol] caused 80% reduction in height 18 days after treatment and the levels of all GAs were 20% or less that of control plants. Foliar treatment at the same dosage reduced height by 50% and caused an 85% or greater reduction in the concentrations of the GA₁ precursors GA₂₀, GA₁₉ and GA₄₄. However, the levels of GA₁, GA₈ and GA₂₉ were affected to a much smaller extent. Foliar application of BAS 111¨W to cv. Belinda 1 month after sowing resulted in only a 20% height reduction at flowering, but no uniform decrease in the concentrations of endogenous GAs at this stage.     

A localised decrease of GA1 in shoot tips of Salix pentandra seedings precedes cessation of shoot elongation under short photoperiod

By application of a recently developed method allowing analysis of gibberellins (GAs) in mg amounts of tissue, the effect of photoperiod on levels of GAs in shoot tips of individual seedlings of the woody species Salix pentandra was studied. In elongating long day-grown seedlings, maximum levels of GA₁ were found 5–20 mm below the apex, approximately twice the levels in other segments. After exposure of plants to 5 or 15 short days, the levels of GA₁ were about 50% lower within this specific region of the stem, as compared with seedlings grown under long days. Short day-induced cessation of shoot elongation also correlated with overall declines in the levels of GA₅₃, GA₁₉, GA₂₀ and GA₈, Within each photoperiodic treatment the levels of these GAs were generally relatively similar throughout the upper 35 mm of stems. No differences in internode lengths or in lengths of pith or epidermal cells were found in plants grown under long days compared with those exposed to 5 short days. In both cases, cells in mitosis were observed in the subapical stem tissues of shoot tips. After 15 short days, stem elongation was completed, and dividing cells were generally not found in the subapical part of the stem. However, short day exposure did not prevent elongation of internodes and cells differentiated before the treatment was started. Thus, the localised decrease in level of GA₁ in shoot tips under short days precedes the morphological and anatomical changes connected with the short day-induced cessation of elongation growth. This supports the hypothesised role for GA₁ in photoperiodic control of shoot elongation in S. pentandra .