Ethylene-Mediated Regulation of Gibberellin Content and Growth in Helianthus annuus L

Elongation of hypocotyls of sunflower can be promoted by gibberellins (GAs) and inhibited by ethylene. The role of these hormones in regulating elongation was investigated by measuring changes in both endogenous GAs and in the metabolism of exogenous [³H]- and [²H₂]GA₂₀ in the hypocotyis of sunflower (Helianthus annuus L. cv Delgren 131) seedlings exposed to ethylene. The major biologically active GAs identified by gas chromatography-mass spectrometry were GA₁, GA₁₉, GA₂₀, and GA₄₄. In hypocotyls of seedlings exposed to ethylene, the concentration of GA₁, known to be directly active in regulating shoot elongation in a number of species, was reduced. Ethylene treatment reduced the metabolism of [³H]GA₂₀ and less [²H₂]GA₁ was found in the hypocotyls of those seedlings exposed to the higher ethylene concentrations. However, it is not known if the effect of ethylene on GA₂₀ metabolism was direct or indirect. In seedlings treated with exogenous GA₁ or GA₃, the hypocotyls elongated faster than those of controls, but the GA treatment only partially overcame the inhibitory effect of ethylene on elongation. We conclude that GA content is a factor which may limit elongation in hypocotyls of sunflower, and that while exposure to ethylene results in reduced concentration of GA₁ this is not sufficient per se to account for the inhibition of elongation caused by ethylene.     

Comparison of the role of gibberellins and ethylene in response to submergence of two lowland rice cultivars, Senia and Bomba

We examined the gibberellin (GA) and ethylene regulation of submergence-induced elongation in seedlings of the submergence-tolerant lowland rice (Oryza sativa L.) cvs Senia and Bomba. Elongation was enhanced after germination to facilitate water escape and reach air. We found that submergence-induced elongation depends on GA because it was counteracted by paclobutrazol (an inhibitor of GA biosynthesis), an effect that was negated by GA₃. Moreover, in the cv Senia, submergence increased the content of active GA₁ and its immediate precursors (GA₅₃, GA₁₉ and GA₂₀) by enhancing expression of several GA biosynthesis genes (OsGA20ox1 and -2, and OsGA3ox2), but not by decreasing expression of several OsGA2ox (GA inactivating genes). Senia seedlings, in contrast to Bomba seedlings, did not elongate in response to ethylene or 1-aminocyclopropane-1-carboxylic-acid (ACC; an ethylene precursor) application, and submergence-induced elongation was not reduced in the presence of 1-methylcyclopropene (1-MCP; an ethylene perception inhibitor). Ethylene emanation was similar in Senia seedlings grown in air and in submerged-grown seedlings following de-submergence, while it increased in Bomba. The expression of ethylene biosynthesis genes (OsACS1, -2 and -3, and OsACO1) was not affected in Senia, but expression of OsACS5 was rapidly enhanced in Bomba upon submergence. Our results support the conclusion that submergence elongation enhancement of lowland rice is due to alteration of GA metabolism leading to an increase in active GA (GA₁) content. Interestingly, in the cv Senia, in contrast to cv Bomba, this was triggered through an ethylene-independent mechanism.     

Interaction between Biological Activities on Rice Seedlings of Dehydroabietic Acid Derivatives and Gibberellin A3

Effects of hydrofluorene and hydrophenanthrene compounds derived from dehydroabietic acid on the second leaf sheath growth of rice seedlings were examined in the presence and absence of gibberellin A₃(GA₃). In the absence of GA₃, nineteen compounds at 100 ppm inhibited more than 20% the growth of normal rice seedlings. In the presence of GA₃ (1.5 ppm) with dwarf rice seedlings, nine compounds at 500 ppm suppressed the elongation caused by the hormone, and a compound was slightly promotive. Then, three compounds were selected and subjected to the bioassay under various conditions.     

Effects of colchicine and lumicolchicine on hypocotyl elongation,respiration rates and microtubules in gibberellic-acid-treated lettuce seedlings

Gibberellic-acid (GA₃)-induced hypocotyl elongation of intact lettuce (Lactuca sativa L.) seedlings was inhibited by colchicine (4×10^-4 M) but not by lumicolchicine (4×10^-4 M). In excised lettuce hypocotyls, GA₃ (10^-5 M) increased respiration over water controls, while both colchicine and lumicolchicine alone, or in combination with GA₃, reduced respiration. Microtubules were present in the hypocotyls of lumicolchicine-treated seedlings but absent in those treated with colchicine. It is suggested that lumicolchicine is a useful drug to discriminate between the metabolic and microtubule-mediated processes in cell morphogenesis.     

Phytochrome Regulation of the Response to Exogenous Gibberellins by Epicotyls of Vigna sinensis

The elongation rate of cowpea epicotyls from whole cowpea (Vigna sinensis) seedlings and derooted and debladed plants (explants) increased after the main light period (8-hour duration) was extended with either continuous low intensity tungsten light or brief (5 minutes) far-red (FR) irradiation. This end-of-day FR effect was reversed by red (R) irradiation suggesting the involvement of phytochrome. These results confirm and extend those obtained previously with other species. Localization studies indicate the epicotyl to be the site of the photoreceptor. Treatment of cowpea seedlings with paclobutrazol, a gibberellin (GA) biosynthetic inhibitor, abolished the FR promoted epicotyl elongation, indicating a role for GAs in this process. There was no significant difference in epicotyl elongation rates of R plus FR irradiated explants treated with GA₁ or GA₂₀ and R irradiated explants treated with GA₁. However, R irradiation inhibited subsequent epicotyl elongation of GA₂₀ treated explants. Moreover, the observation, using GC-MS, that GA₁ and GA₂₀ are native GAs in cowpea lends support to the concept that phytochrome may control the conversion of endogenous GA₂₀ to GA₁ in cowpea.     

Gibberellic Acid stimulation of cucumber hypocotyl elongation : effects on growth, turgor, osmotic pressure, and cell wall properties

Recently developed techniques have been used to reinvestigate the mechanism by which gibberellic acid (GA₃) stimulates elongation of light-grown cucumber (Cucumis sativus L.) seedlings. Osmotic pressure and turgor pressure were slightly reduced in GA₃-treated seedlings, which elongated 3.5 times faster than control seedlings. This indicated that GA₃ enhancement of growth was not controlled by changes in the osmotic properties of the tissues. Stress/strain (Instron) analysis revealed that plastic extension of the cell walls of GA₃-treated seedlings increased by up to 35% above the control values. Stress-relaxation measurements on frozen-thawed tissue showed that T₀ the minimum relaxation time, was reduced following application of GA₃. In vivo wall relaxation (measured by the pressure block technique) showed that the wall yield coefficient was increased, and the yield threshold was slightly reduced. Thus GA₃ affected both the mechanical (viscoelastic) and biochemical (chemorheological) properties of the cell walls of light-grown cucumber. The previous hypothesis, that GA₃ stimulates cucumber hypocotyl growth by increasing osmotic pressure and cell turgor, is contradicted by our results.     

Growth-active gibberellins overcome the very slow shoot growth of Hancornia speciosa, an important fruit tree from the Brazilian “Cerrado”

Shoot elongation of Hancornia speciosa, an endangered tree from the Brazilian savannah “Cerrado”, is very slow, thus limiting nursery production of plants. Gibberellins (GAs) A₁, A₃, and A₅, and two inhibitors of GA biosynthesis, trinexapac-ethyl and ancymidol were applied to shoots of Hancornia seedlings. GA₁ and GA₃ significantly stimulated shoot elongation, while GA₅ had no significant effect. Trinexapac-ethyl and ancymidol, both at 100 μg per seedling, inhibited shoot elongation up to 45 days after treatment, though the effect was statistically significant only for ancymidol. Somewhat surprisingly, exogenous GA₃ more effectively stimulated shoot elongation in SD-grown plants, than in LD-grown plants. The results from exogenous application of GAs and inhibitors of GA biosynthesis imply that Hancornia shoot growth is controlled by GAs, and that level of endogenous growth-active GAs is likely to be the limiting factor for shoot elongation in Hancornia. Application of GAs thus offer a practical method for nursery production of Hancornia seedlings for outplanting into the field.     

Effect of exo-16,17-dihydro-gibberellin A5 on gibberellin A20 metabolism in seedlings of dwarf rice (Oryza sativa L. cv. Tan-ginbozu)

Several of the 16,17-dihydro gibberellins (GAs) inhibit elongation in a variety of species. In a study of their mechanism of action we have investigated the effect of exo-16,17-dihydro-Ga₅ (diHGA₅) on the metabolism of GA₂₀ in dwarf rice (Oryza sativa cv. Tan-ginbozu). A mixture of [³H]- and [³H]-GA₂₀ (100 ng per plant) was applied in microdrops to 4 d old seedlings which were harvested 72 h later. Concurrent treatment with diHGA₅ at 100 ng or 333 ng per plant reduced GA₂₀-induced elongation of the second leaf sheath by 41–66%. There was a concomitant reduction in the amount of [²H₂]GA₁ present at harvest, measured by gas chromatography-mass spectrometry-selected ion monitoring. The [²H₂]GA₂₉ content was also reduced. There was no clear effect of diHGA₅ on the total radioactivity recovered, or on conversion of the [³H]GA₂₀ to putative [³H]GA conjugates, or on the amount of [²H₂]GA₂₀ found. No free [²H₂]GA₈ was detected. In other experiments there was little effect of diHGA₅ on elongation induced by treatment with GA₁. We conclude that diHGA₅ inhibited GA₂₀-induced elongation in dwarf rice shoots at least partly by reducing the 3-hydroxylation of GA₂₀ to GA₁.Abbreviations diHGA₅ = exo- 16, 17-dihydro-gibberellin A₅ - GA = gibberellin - GC-MS-SIM = gas chromatography-mass spectrometry-selected ion monitoring     

Detection of endogenous gibberellins and their relationship to hypocotyl elongation in soybean seedlings

Four gibberellins, GA₅₃, GA₁₉, GA₂₀, and GA₁, were detected by bioassay, chromatography in two HPLC systems, and combined gas chromatography-mass spectroscopy-selected ion monitoring (GC-MS-SIM) in etiolated soybean (Glycine max [L.] Merr.) hypocotyls. GC-MS-SIM employed [²H₂]-labeled standards for each endogenous gibberellin detected, and quantities estimated from bioassays and GC-MS-SIM were similar. This result plus the tentative detection of GA₄₄ and GA₈ (standards not available) indicates that the early-C-13-hydroxylation pathway for gibberellin biosynthesis predominates in soybean hypocotyls. Other gibberellins were not detected. Growth rates decreased after transfer to low water potential (ψ_w) vermiculite and were completely arrested 24 hours after transfer. The GA₁ content in the elongating region of hypocotyls had declined to 38% of the 0 time value at 24 hours after transfer to low ψ_w vermiculite, a level which was only 13% of the GA₁ content in control seedlings at the same time (24 hours posttransfer). Rewatering seedlings following 24 hours growth in low ψ_w vermiculite resulted in a complete recovery in elongation rate, an increase in GA₁ (20% at 2 hours, two-fold at 8 hours, eightfold at 24 hours), and a decrease in ABA levels (tenfold at 2 hours). Treatment of well-watered seedlings with the GA-synthesis inhibitor tetcyclacis (TCY) resulted in lowered GA₁ levels and increased ABA levels. When seedlings grown 24 hours in low ψ_w vermiculite were rewatered with TCY, recovery of the elongation rate was delayed and reduced, and the decline in ABA levels was slowed. Addition of GA₃ restored the elongation rate inhibited by TCY. Seedlings were growth responsive to exogenous GA₃, and this GA₃-promoted growth was inhibited by exogenous ABA. The data are consistent with the hypothesis that changes in GA₁ and ABA levels play a role in adjusting hypocotyl elongation rates. However, the changes observed are not of sufficient magnitude nor do they occur rapidly enough to suggest they are the primary regulators of elongation rate responses to rapidly changing plant water status.     

Relationship of IAA-oxidase Activity to Gibberellinand IAA-induced Elongation of Light-grown Cucumber Seedlings

The growth and IAA-oxidase activity of light-grown cucumber seedlings (cv. Aonagajibae) were investigated in response to GA₃ and IAA. Both GA₃ and IAA induced significant elongation of the hypocotyl. The fresh and dry weights of the hypocotyl increased due to GA₃ or IAA treatment, whereas no significant change was observed in the cotyledons of GA₃-treated seedlings as compared with the controls. The fresh and dry weights of IAA-treated cotyledons were both lower than those of controls. Treatment with GA₃ or IAA resulted in retardation of IAA-oxidase activity in the hypocotyl and cotyledons. The degree of retardation was less in the cotyledons than in the hypocotyl. An inverse relationship was recognized between GA₃- or IAA-induced elongation and IAA-oxidase activity in the hypocotyl. The auxin-mediated mechanism for gibberellin action was discussed.     

Control by Light of Hypocotyl Elongation and Levels of Endogenous Gibberellins in Seedlings of Lactuca sativa L.

Four 13-hydroxygibberellins, gibberellin A₁ (GA₁), 3-epi-GA₁,GA₁₉ and GA₂₀ were identified by full-scan GC/MS in extractsof lettuce seedlings (Lactuca sativa L. cv. Grand Rapids). Theresults suggest that the early-13-hydroxylation biosyntheticpathway to GA₁ functions in the lettuce seedlings. It was alsofound that GA₁ is active per se in the control of hypocotylelongation in lettuce seedlings. To investigate the relationshipbetween control by light of hypocotyl elongation and levelsof endogenous GAs in lettuce, endogenous levels of GAs werequantified by radioimmunoassay in seedlings that had been grownfor 5 days in the dark (5D) and in those that had been grownfor 4 days in the dark and then under white light for 1 day(4D1L). The endogenous level of GA₁ in the upper and lower partsof hypocotyls in 5D seedlings was about four times higher thanthat in 4D1L seedlings. The response of explants (hypocotylsegments with cotyledons) from dark-grown seedlings to GA₁ isknown to be similar in the dark and under white light when theexplants are treated with inhibitors of the biosynthesis ofGA. Therefore, the above information suggests that the highlevel of GA₁ in hypocotyls of dark-grown seedlings is responsiblefor the rapid elongation of hypocotyl, while irradiation bywhite light decreases the endogenous level of GA₁ in the hypocotylswith a resultant decrease in the rate of hypocotyl elongation. (Received March 13, 1992; Accepted May 21, 1992)     

Interaction of growth retardants,daylength, and gibberellins A19, A20, and A1 on shoot elongation in birch and alder

Gibberellins A₁₉, A₂₀, and A₁ were applied to seedlings of birch (Betula pubescens Ehrh.) and alder (Alnus glutinosa (L.) Gaertn.) in order to test their ability to counteract growth inhibition induced by growth retardants (ancymidol and BX-112) or short day (SD, 12 h) photoperiod. Ancymidol inhibits early and BX-112 inhibits late steps in gibberellin biosynthesis. BX-112 inhibited stem elongation in both species while ancymidol, applied as a soil drench, was effective in alder only. Growth retardants affected stem elongation mainly by inhibiting elongation of internodes. All three gibberellins were equally active when applied to seedlings treated with ancymidol; however, only GA₁ was able to counteract the growth inhibition induced by BX-112. SD-induced cessation of elongation growth in birch was counteracted by GA₁, and to some degree, by GA₂₀, while GA₁₉ was inactive. SD treatment did not induce cessation of apical growth in alder. These results are consistent with the hypothesis that of gibberellins belonging to the early C-13 hydroxylation pathway, GA₁ is the only active gibberellin for stem elongation.     

Genotypic variation in the responsiveness to GA3 within tall (rht1) and semi-dwarf (Rht1) spring wheat

The effect of GA₃ on coleoptile-and first leaf elongation of tall (rht1) and semi-dwarf (Rht1) nearly-isogenic genotypes, within each of 25 random F₉ wheat families, was determined on seedlings grown in a growth room at 18 °C. Conspicuous and very significant inter-family variation in the response of the first leaf to GA₃ application was found in both the rht1 and Rht1 genotypes. The magnitudes of the response of the different families within genotypes to GA₃ were not related to the leaf length of their untreated seedlings. It is suggested that, under given environmental conditions, background genotypic effects, inducing inter-family variation in responsiveness to GA₃, regulate the elongation growth up to the limits set by the Rht alleles.     

The roles and interactions of ethylene with gibberellins in the far-red enriched light-mediated growth of <Emphasis Type="Italic">Solanum lycopersicum</Emphasis> seedlings

Wild type (WT) tomato seedlings responded to a low red to far-red (R/FR) ratio with increased stem elongation, similar leaflet area expansion and lower shoot ethylene levels. The levels of endogenous growth-active GA₁ and its immediate precursor GA₂₀ were decreased by low R/FR ratio, whereas the levels of GA₁ catabolite, GA₈, increased. To examine the interaction of ethylene with GAs in regulating tomato shoot growth under low R/FR ratio, transgenic (T) seedlings bearing Le-ACS2 and Le-ACS4 antisense mRNA were utilized. Low R/FR ratio increased stem elongation and decreased ethylene levels in T tomato shoots, as it did in WT shoots. However, T stems were significantly taller than the WT stems under low R/FR ratio. Leaflet areas were significantly larger for T, than WT seedlings under both R/FR ratios. Low R/FR ratio did not decrease endogenous levels of GA₁ and GA₂₀ in T shoots, but did increase GA₈ levels, which were higher than in WT shoots. These results, and hormone/inhibitor application studies, showed that in tomato shoots subjected to low R/FR ratio, GAs play a growth-promotive role in stem elongation, whereas ethylene is growth-inhibitory. Further, these results may imply that decreasing ethylene production under low R/FR ratio causes increases in stem elongation and GA levels.     

Gibberellic-acid-induced cell elongation in pea epicotyls: Effect on polyploidy and DNA content

In gibberellic-acid(GA₃)-treated epicotyls of dwarf peas (Pisum sativum L.) grown in the light, DNA (per cell and per epicotyl) is followed. Histofluorometric DNA determinations show that GA₃-promoted cell elongation is not accompanied by increased endomitosis, but chemical estimations show an increased DNA content per epicotyl. This difference must therefore be the result of increased mitotic activity in the GA₃-treated tissue. Epicotyls of seedlings grown with or without cotyledons under continuous light with GA₃ are tetraploid, as are those of ecotylized embryos grown in darkness. These epicotyls reach no more than half the length of octaploid epicotyls of seedlings grown in darkness. This result provides evidence for a relationship between polyploidy and final possible cell length.     

Effect of Gibberellin A3 on the Elongation, α-Amylase Activity, Reducing Sugar Content and Oxygen-uptake of the Leaf Sheath of Dwarf Maize Seedlings

GA₃-treatment of dwarf maize seedlings resulted in the elongation of the leaf sheath and also an increase in α-amylase activity. Excised leaf sheaths did not respond to GA₃ in leaf shealh length and α-amylase activity. Increase in the enzyme activity is always accompanied by an increase in the length of the leaf sheath. α-Amylase activity gradually increased as the growth of the first leaf proceeded, and a parallelism was found between the length of the leaf sheath and the enzyme activity, suggesting that the degree, of the enzyme activity depends on the length of the leaf sheath. On the other hand, IAA did not affect α-amylase activity while it promoted leaf sheath elongation. This suggests that elongation per se is not associated with the increase in α-amylase activity and that the enzyme-promoting effect is specific to gibberellin. Higher α-amylase activity and lower content of reducing sugars were detected in the older tissue of the leaf sheath, that is, in the upper half. This was the same for GAlrealed seedlings. The amount of reducing sugars was less in GA₃-trealcd seedlings. Oxygen-uptake of the leaf sheath was higher in the upper half in both controls and GA₃-treated seedlings. It was slightly higher in the latter than in the former. From these results it was discussed 1o conclude that the processes of the GA₃-induced elongation and increase in α-amylase activity of the leaf sheath are independent of one another.     

Growth Responses and Allocation of Assimilates of Rice Seedlings by Paclobutrazol and Gibberellin Treatment

Paclobutrazol [(2RS,3RS)-1-(4-chlorophenyl)methyl-4,4-dimethyl-2-(1h-1,2,4-trizol-1-yl)penten-3-ol] effectively decreased vegetative growth of rice (Oryza sativa L.) seedlings and increased the chlorophyll content. The number of veins in a leaf, the calculated number of stomata per leaf, and the length of guard cells were not altered by the paclobutrazol treatment, suggesting an effect on cell elongation. The allocation pattern of carbohydrates was changed by either gibberellin (GA) or paclobutrazol treatment. GA₃ induced more shoot growth and less accumulation of starch than the control and paclobutrazol-treated seedlings. Photosynthetic ability was not affected by either paclobutrazol or GA₃ treatment. Paclobutrazol-treated plants allocated a smaller amount of photosynthates for vegetative shoot growth and stored more as starch in the crowns than the control and GA₃-treated plants. The same starch degrading activity in the crown tissue of paclobutrazol-treated seedlings as in control plants suggests that the accumulated starch is utilized in a normal activity for growth including leaf emergence, tiller formation, and root production, resulting in improved seedling quality. Received May 30, 1996; accepted December 10, 1996     

Effects of concentration and treatment duration upon dwarf pea response to gibberellic acid root treatments in solution culture

Gibberellic acid (GA₃) root treatments stimulated internode elongation of hydroponically grown dwarf pea seedlings (Pisum sativum L.,cv. Little Marvel) When the GA₃ concentration in the solution was at least 2.9 M.Both GA₃ concentration and the duration of the root-treatment period significantly affected internode elongation. This is attributed to a limited availability or saturation of active sites for gibberellin-induced cell elongation. The amount of GA₃ taken up through the roots in 1 day from a 29 M GA₃ solution apparently equaled or exceeded the amount which could be metabolized during the first four days after treatment, although higher concenrations and longer treatment periods produced a more prolonged response, conceivably due to 1) initial saturation of gibberellin active sites, 2) storage of surplus gibberellin in the plant, and 3) subsequent utilization of the stored gibberellin. GA₃-induced stem elongation in hydroponically grown Little Marvel peas seemed to be limited initially by apparent saturation of active sites when the GA₃ concentration exceeded 29 M.     

Regulation of cell division in the subapical shoot meristem of dwarf watermelon seedlings by gibberellic acid and polyethylene glycol 4000

The stimulatory effects of gibberellic acid (GA₃) and the inhibitory effects of polyethylene glycol 4000 (PEG) on hypocotyl elongation and cell cycle kinetics in subapical pith cells of dwarf watermelon seedlings (Citrullus lanatus [Thunb.] Matsu and Nakai) were investigated. Mitotic indices (MI) were determined from direct counts of pith cells stained by a modified Feulgen technique. Labeling indices (LI) were determined from direct counts of labeled pith cells sampled 1.5 h after apical applications of³H-thymidine. Root application of 0.32 mM GA₃ at 96, 120, or 144 h after sowing resulted in significant increases in both mitotic and labeling indices within 4.5 to 7.5 h following treatment. A single mitotic peak at 13.5 h occurred in all three treatment periods. Labeling peaks were often less defined than mitotic peaks; however, a relatively high proportion of labeled nuclei were usually observed between 7.5 and 9 h after GA₃ treatment and at 16.5 h, the latter period coinciding with progression of cells into S phase from the peak period of mitosis. The results suggest that GA₃ increases the proportion of rapidly dividing cells in the subapical meristem by increasing the probability that slowly cycling or nonproliferative cells in both 2C and 4C DNA states will enter the proliferative pool. The addition of PEG (200 g/l, = 1.5 mPA) to the rooting medium of dwarf watermelon seedlings inhibited hypocotyl elongation and reduced both mitotic and labeling indices simultaneously within 4.5 h after treatment. Within 24–28 h after PEG treatment, mitotic and labeling indices approached 0. Seedlings transferred from PEG to either water or GA₃ exhibited rapid recovery of cell division and hypocotyl elongation. Mitotic and labeling indices increased within 4.5–7.5 h into the recovery period in either water or GA₃ and reached control values within 10.5 h. GA₃ hastened the recovery from PEG-induced stress. It is concluded that water stress imposed by PEG 4000 causes arrest of cell division in meristematic cells of watermelon seedlings in both G₁ and G₂ periods. PEG and GA treatments resulted in only a partial and transitory synchronization of the cell cycle.