Effects of Gibberellin and Growth-retarding Chemicals on Respiration and Catalase Activity in Various Organs of Cucumber Seedlings

Cucumber seedlings were grown in darkness on filter paper saturatedwith water, gibberellin (GA), growth-retarding chemicals (mainlyAmo-1618) or both GA and Amo-1618. The effects on oxygen consumptionand catalase activity were studied on cotyledons, radicles,and hypocotyl tips. GA increased respiration and catalase activity of hypocotyland cotyledons, decreased radicle respiration, and had no significanteffect on its catalase activity. Amo-1618 decreased respirationof hypocotyl and cotyledons and increased that of the radicles.It stimulated catalase activity in all three organs. The inverse effects of GA and Amo-1618 on oxygen uptake andcatalase activity were mutually antagonized when both compoundswere applied at concentrations counteracting their oppositeeffects on hypocotyl elongation. No stimulating effect on catalase activity was found with mostother growth-retarding chemicals. Some of them had no significanteffect on the enzyme, and one even decreased its activity.     

The Effect of CCC and Amo-1618 on Growth, Catalase, Peroxidase and Indoleacetic Acid Oxidase Activity of Young Barley Seedlings

Barley seedlings were grown in darkness on filter paper saturated with phosphate buffer or CCC and Amo-1618 buffered solutions. The effects of CCC and Amo-1618 on growth, catalase, peroxidase and lAA-oxidase were studied on coleoptile and primary leaves. Both growth-retarding chemicals cause an inhibition of growth, Amo-1618 being more effective than CCC. They have no effect on catalase activity. Increasing concentrations of CCC and Amo-1618 progressively stimulate peroxidase and IAA-oxidase activities (Amo-1618 more than CCC). The enzymatic activity in short-treated plants is higher than in the corresponding control plants of the same height. It is proposed that CCC and Amo-1618 exert their effect on the growth of barley by acting on auxin catabolism.     

Kinetics of growth retardant and hormone interactions in affecting cucumber hypocotyl elongation

The capacities of indole-3-acetic acid (IAA) and gibberellin A₃ (GA₃) to counteract the inhibitory effects of (2-chloroethyl) trimethylammonium chloride (CCC), 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidinecarboxylate methyl chloride (Amo-1618), and N,N-dimethylaminosuccinamic acid (B-995) on hypocotyl elongation in light-grown cucumber (Cucumis sativus L.) seedlings were investigated. One μg of GA₃ applied to the shoot tip was sufficient to completely nullify the effect of 10 μg of Amo-1618 or 25 μg of B-995 applied simultaneously to the shoot tip, and 10 μg of GA₃ completely counteracted the effect of 10⁻³ m CCC added to the root medium. One μg of IAA counteracted the effect of 10⁻³ m CCC in the root medium, but IAA did not nullify the action of either Amo-1618 or B-995. Experiments were conducted using 2 growth retardants simultaneously, which indicated that Amo-1618 and CCC inhibit a common process, namely GA biosynthesis, essential to hypocotyl elongation. However, since the effect of CCC was overcome by applications of both GA and IAA, growth retardation resulting from treatment with CCC apparently is not due solely to inhibition of GA biosynthesis. B-995 did not interact additively with either Amo-1618 or CCC, which suggests that B-995 affects a process different from those affected by the other 2 retardants. Thus, while inhibition evoked by B-995 is reversible by applied GA, the action of B-995 does not appear to be inhibition of GA biosynthesis.     

EFFECTS OF GIBBERELLIN AND AMO-1618 ON GROWTH,DRY-MATTER ACCUMULATION,CHLOROPHYLL CONTENT AND PEROXIDASE ACTIVITY OF CITRUS SEEDLINGS

Monselise , S. P., and A. H. Halevy . (Hebrew U., Rehovot, Israel.) Effects of gibberellin and AMO–1618 on growth, dry-matter accumulation, chlorophyll content and peroxidase activity of citrus seedlings. Amer. Jour. Bot. 49(4): 405–412. Illus. 1962.—Sweet-lime seedlings, 6 months old, were sprayed with gibberellic acid (GA) and a growth retardant, AMO–1618, alone and in combination, at concentrations ranging between SO and 1600 ppm. Increasing concentrations of GA progressively increased shoot and internode length, did not influence number of leaves, and decreased leaf area. Dry weight of shoots was progressively increased up to 400 ppm, while dry weights of leaves and roots were decreased over all GA concentrations. Total dry weight of plants was increased by GA when related to leaf area or weight and to total chlorophyll content, which indicates a higher synthetic efficiency of leaves. This could not be detected by manometric determinations using leaf discs. It is suggested that a short determination period and/or work with detached leaves are responsible for failures to detect increased photosynthetic activity of GA-treated leaves. Chlorophyll content of leaves was decreased by increasing GA concentrations; it is shown that this is not due to “dilution” over a larger area of leaves. Peroxidase activity of leaves was only slightly reduced by GA, while it was increased by AMC–1618, acting as antagonist to GA. This is remarkable, since AMO–1618 did not clearly affect other procperties of citrus seedlings which are only slightly responsive to this chemical.     

Comparative effects of some Amo-1618 analogs on gibberellin production in Fusarium moniliforme and on growth in higher plants

Summary The plant growth retardant 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidinecarboxylate methyl chloride (Amo-1618) and three analogs (Carvadan, XII, XIII) were tested for ability to inhibit gibberellin production in Fusarium moniliforme and to suppress stem elongation in three species of higher plants.Amo-1618 and compound XII were highly effective in suppressing GA production in Fusarium whereas Carvadan and XIII were inactive. These inactive analogs were not degraded to any appreciable extent by Fusarium cultures.All four compounds suppressed stem growth although the relative effectiveness varied with the species tested. Carvadan was most active in dwarfing Phaseolus vulgaris and Pharbitis nil; compound XII was most active in Helianthus annuus.The lack of correlation between the ability to inhibit gibberellin production in Fusarium and to cause dwarfed growth in higher plants is interpreted to indicate that enzymes involved in gibberellin biosynthesis in different organisms exhibit certain variations which make them more or less sensitive to inhibitors such as the four growth retardants tested.     

Effects of Growth Retardants on Norway Spruce (Picea abies)

Young seedlings of Picea abies Karst, grown in nutrient solution were treated with the growth retardants Amo-1618, B-995, and CCC. These were added to the nutrient medium. B-995 and CCC retarded root and shoot growth in the concentrations 100, 10, and 1 mg/l. Growth was almost entirely inhibited by 300 mg/l, obviously due to toxicity. The effects of Amo-1618 were similar but more varying. GA counteracted the effects of all the retardants on shoot growth, but not on root growth.     

EVIDENCE ON THE SITE OF ACTION OF GROWTH RETARDANTS

1. The ability of five growth retardants to inhibit the GA-inducedand endogenous growth of Avena leaf sections has been investigated.The retardants vary in effectiveness. The order, from most effectiveto least, is Phosfon D, Amo-1618, C011, CCC and B995. 2. The inhibition of growth caused by Phosfon D and Amo-1618is not reversed by GA. It is apparent that the retardants donot compete with GA at the site of GA-action. 3. Addition of IAA will partially reverse the inhibition inducedby Phosfon D or Amo-1618. It is concluded that the retardantsact in part in Avena leaf sections by interfering with the auxinmetabolism of the tisssue. ¹ Supported in part by grants G-14578 and GB-1950 from the NationalScience Foundation ² Present address: Department of Botany, University of Washington,Seattle, Washington     

Inhibition of Gibberellin Production in the Fungi Gibberella fujikuroi and Sphaceloma manihoticola by Plant Growth Retardants

The effect of different types of plant growth retardants on fungal gibberellin (GA) formation has been studied in cultures of Gibberella fujikuroi and Sphaceloma manihoticola. Quaternary ammonium compounds (chlormequat chloride, mepiquat chloride, Amo-1618), triazoles (uniconazole and several experimental compounds), and the norbornanodiazetine tetcyclacis inhibited GA biosynthesis in both fungal species. Concentrations between 2 × 10⁻⁴ and 10⁻⁹m were required for a 50% inhibition of the production of gibberellin A₃ in Gibberella fujikuroi and of giberellin A₄ in Sphaceloma manihoticola. The formation of other prominent GAs was affected at a similar degree of intensity. Tetcyclacis was the most active compound in both fungi. Compared to the growth retardants mentioned above, the biological activity of chlorphonium chloride was low. The acylcyclohexanediones prohexadione and LAB 198 999 had virtually no activity. Most likely, this lack of activity is due to a rapid metabolism of the compounds in the cultures. For the triazole-type compounds and tetcyclacis, a relatively distinct correlation exists in their ability to inhibit GA formation in fungal cultures, to block ent-kaurene oxygenase in a cell-free system, and to reduce shoot growth of rice seedlings. Due to differences in their metabolic fate and species specificities, such conclusions cannot be made for the other compounds.     

Effect of inhibitors of gibberellin biosynthesis on the induction of α-amylase in embryoless caryopses of Hordeum vulgare cv. Himalaya

The induction of -amylase by exogenously supplied gibberellin A₁ (GA₁) and GA₄ in embryoless caryopses of Hordeum vulgare (cv. Himalaya) was determined indirectly by measuring reducing sugars released from the endosperm. The presence of the inhibitors of GA biosynthesis, 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidinecarboxylate methyl chloride (Amo 1618), Ancymidol, 2-chloroethyl trimethyl ammonium chloride (CCC) or (R,S)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,3-triazolyl)pentan-3-ol (PP333) did not inhibit -amylase production by either GA₁ or GA₄.Abbreviations Amo-1618 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidinecarboxylate methyl chloride - CCC 2-chloroethyl trimethyl ammonium chloride - cv. cultivar - GA gibberellin - GC gas chromatography - GC-MS combined gas chromatography-mass spectrometry - PP333 (R,S)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,3-triazolyl) pentan-3-01     

INHIBITORY EFFECT OF GROWTH RETARDANTS ON THE INDUCTION OF FLOWERING IN WINTER WHEAT

1. Growth retardants, CCC, Amo-1618, Phosfon-D and B-995, appliedduring seed vernalization inhibited the ear development of winterwheat. CCC applied during green plant vernalization inhibitedflowering,but it had no appreciable effect on the final stemlength. Theinhibition by CCC was reversed by foliar applicationof gibberellin.On the other hand, CCC applied after the vernalizationperiodaffected the final stem length but not flowering.
2. Theamount of endogenous gibberellin-like substance(s) was greaterin the vernalized plant than in the non-vernalized plant. Nogibberellin-like substance was detected in the CCC-treated plant.
3. Endogenous gibberellin-like substance(s), whose biosynthesisis inhibited by some growth retardants, may play a part in thevernalization process in winter wheat.

¹Present address: National Institute of Agricultural Sciences,Nishigahara, Kitaku, Tokyo     

Role of growth regulators in the bean hypocotyl hook opening response

Summary The opening of the hypocotyl hook in bean seedlings is due to a rapid elongation of cells on the inner side of the hook elbow. Red light promotes hook opening by inducing this cell elongation.Opening is inhibited by low concentrations of indoleacetic acid (IAA) and 2,4-dichlorophenoxyacetic acid (2,4-D), and higher concentrations of these auxins cause a closure of the hook. In darkness, opening is induced slightly by p-chlorophenoxyisobutyric acid (PCIB), whereas in red light this auxin antagonist promotes opening only when IAA is added simultaneously to inhibit opening.The amount of diffusible auxin released by the hook tissue is not affected by red illumination that is sufficient to induce maximal hook opening.Gibberellic acid (GA) promotes the hook opening. The magnitude of its effect is, however, rather small, especially in darkness. (2-Chloroethyl)-trimethylammonium chloride (CCC) and 2-isopropyl-4-(trimethylammonium-chloride)-5-methylphenyl piperidine-1-carboxylate (Amo-1618) inhibit hook opening in red light, and this inhibition is completely overcome by addition of GA.Cytokinins and abscisic acid at rather high concentrations inhibit hook opening in light but produce no significant effect in darkness.Hook opening is promoted by Ca⁺⁺ and K⁺, and notably by Co⁺⁺ and Ni⁺⁺.It is concluded that 1. endogenous gibberellin assists in hook opening, but light does not act by changing the gibberellin level; 2. light does not act by decreasing the endogenous auxin level; and 3. cytokinins or abscisic acid do not seem to have a special role in the response.     

Comparison of the enzymatic properties of ent-copalyl diphosphate synthases in the biosynthesis of phytoalexins and gibberellins in rice

The rice genome contains two ent-copalyl diphosphate synthase genes: OsCPS1 acts in gibberellin (phytohormone) biosynthesis, and OsCPS2/OsCyc2 acts in the synthesis of oryzalexins A-F and phytocassanes A-E (phytoalexins). We characterized the enzymatic properties of recombinant OsCPS2/OsCyc2 fused with a tag-protein at the N-terminus, and compared them to those of OsCPS1. Several enzymatic properties of OsCPS2/OsCyc2, including the optimal pH, optimal temperature, divalent cation requirement, and kinetic values for the geranylgeranyl diphosphate (GGDP) substrate, were almost the same as those of OsCPS1. However, OsCPS2/OsCyc2 activity was not inhibited by 50-60 muM GGDP substrate, by which the OsCPS1 activity was inhibited. Furthermore, the OsCPS1 activity exhibited approximately 70% inhibition by 100 muM Amo-1618 (a gibberellin biosynthetic inhibitor), whereas the OsCPS2/OsCyc2 activity exhibited approximately 10% inhibition. These results indicate that the properties of OsCPS2/OsCyc2 were partially different from those of OsCPS1, although OsCPS2/OsCyc2 catalyzes the same reaction step as OsCPS1.     

Effects of AMO-1618 on Growth, Morphology, and Gibberellin Content of Phaseolus Cocineus Seedlings

A study was made of the effects of AMO-1618 on the endogenousgibberellin (GA) levels and stem, leaf, and root growth of Phaseoluscoccineus seedlings. The data establishes that some of the effectsof AMO-1618 on the growth of Phaseolus seedlings are mediatedby factors other than an inhibition of GA biosynthesis.     

Distribution of Gibberellins in Lathyrus odoratus L. and Their Role in Leaf Growth

In sweet pea (Lathyrus odoratus L.) the mutant allele l reduced the level of gibberellin A1 (GA1) in expanding leaflets and resulted in smaller, more oval leaflets compared with the wild type. The apical portions of 6-d-old wild-type (L) seedlings also contained less GA1 and produced smaller, more oval leaflets than did comparable 20-d-old L seedlings. Application of GA1 markedly altered leaflet shape and, at certain dosages, restored the wild-type shape and size to leaflets of the l (dwarf) mutant. Taken together, these observations indicate that GA1 performs a regulatory role in the control of leaf growth in this species. The levels of GA1 precursors in the wild type were also determined. Rapidly expanding internodes contained much more gibberellin A19 (GA19) than gibberellin A20 (GA20), whereas the opposite was true for expanding leaflets. Although in entire apical portions of established seedlings the level of GA20 exceeded that of GA19, apical portions of very young seedlings contained more GA19 than GA20. Basal stem tissue of established seedlings also contained substantially more GA19 than GA20 or GA1. Both stems and leaflets from the basal portion of the plant contained much less GA20 and GA1 than did the rapidly expanding apical tissue. The implications of these results for the regulation of GA1 biosynthesis are discussed.     

The effect of growth retardants on phytochrome-induced lettuce seed germination

Experiments were carried out to explore the involvement of the plant hormone gibberellin (GA) in the light-induced germination of lettuce seeds. Three growth retardants known to be inhibitors of GA biosynthesis were tested for their effect on red-light-induced germination. Chlormequat chloride (CCC) and AMO-1618 had no effect, but ancymidol was strongly inhibitory. Moreover, the inhibition caused by ancymidol was completely overcome by GA₃. CCC and AMO-1618 inhibit the formation ofent-kaurene, while ancymidol blocks the oxidation ofent-kaurene toent-kaurenoic acid. Ancymidol also was found to inhibit GA-induced dark germination of lettuce seeds, and this inhibition was partially reversed by higher levels of GA. Therefore, the results suggest two possibilities for the relationship between phytochrome and GA in this system: first, the rate-limiting step in the germination of light-sensitive lettuce seeds, that which is regulated by phytochrome, is the oxidation ofent-kaurene toent-kaurenoic acid. Alternatively, red-light treatment may result in the release of active GAlike substances which, in turn, induce germination. In either case the results presented here support the view that phytochrome exerts its effect on lettuce seed germination by means of GA rather than via an independent pathway.     

CONTROL OF FLOWER FORMATION BY GROWTH RETARDANTS AND GIBBERELLIN IN SAMOLUS PARVIFLORUS,A LONG-DAY PLANT

The growth retardants AMO–1618 and CCC inhibited flower formation and stem elongation in Samolus parviflorus, a long-day rosette plant, under inductive conditions. The vegetative growth of the plants, as measured by leaf formation, was affected only slightly, or not affected at all. Application of gibberellic acid (GA₃) reversed completely the inhibition both of flower formation and of stem elongation caused by AMO, but relatively larger amounts of GA were required to reverse the CCC inhibition of stem elongation than that of flower formation. When applied under short-day conditions, AMO had no effect on the level of applied GA required for flower induction. When applied following long-day treatment the retardant caused some reduction of flower formation after marginal numbers of long days, but had no effect when enough long days to cause 100% flower formation were given. Other evidence indicates that the growth retardants act by inhibiting the synthesis of endogenous gibberellin. In LD plants, at least part of the action of inductive environmental conditions consists in causing an increase of gibberellin synthesis, supporting the hypothesis that relatively high GA levels are necessary for the production of the floral stimulus in this group of plants, as in long-short-day plants. The experiments with CCC indicate that stem elongation and flower formation in Samolus can be separated, and that the effect of GA on flower formation is not necessarily dependent on its effect on stem elongation.     

毛竹茎秆伸长过程中赤霉素生物合成、降解和信号转导关键基因的鉴定及表达分析

赤霉素(Gibberellin)是一类非常重要的植物激素,在高等植物生命活动的整个周期都起着重要的调控作用。从毛竹Phyllostachys edulis基因组中共鉴定出23个赤霉素途径基因,包括赤霉素生物合成相关的8个GA20ox和1个GA3ox基因、降解相关的8个GA2ox基因、参与赤霉素感知的2个GID1基因以及信号转导的2个GID2基因和2个DELLA基因。拟南芥、水稻和毛竹的系统进化树和保守基序分析显示赤霉素的合成代谢与信号转导在这些物种中是高度保守的。利用外源赤霉素处理毛竹种子和幼苗,发现赤霉素能显著提高种子的萌发率和幼苗的茎秆伸长,并且有着最佳的作用浓度。在GA3处理后,毛竹体内赤霉素生物合成基因GA20ox和GA3ox表达量均下调而降解活性赤霉素的GA2ox基因表达量上调;赤霉素受体GID1和正调控基因GID2的转录水平显著提高而负调控基因DELLA的表达受到抑制。这些基因在竹笋茎秆的不同形态学位置表达差异明显,大部分赤霉素生物合成与降解的相关基因GA20ox、GA3ox和GA2ox以及赤霉素受体GID1和正调控基因GID2都在竹笋的形态学上端大量表达,而赤霉素信号转导的阻遏基因DELLA在笋体形态学底端大量积累而顶端基本不表达。     

Biosynthesis of (-)-Kaurene in Cell-free Extracts of Immature Pea Seeds

Mevalonate-¹⁴C was incorporated into (—)-kaurene-¹⁴C in cell-free extracts of immature pea (Pisum sativum L.) seeds. The identification of ¹⁴C-product as (—)-kaurene was based on: A) comparison with authentic (—)-kaurene on thin-layer and gas-liquid chromatography; and B) oxidation of ¹⁴C-product and (—)-kaurene with osmium tetroxide to form the common derivative kaurane-16,17-diol. The enzyme system is heat labile and is dependent upon ATP and Mg²⁺ or Mn^2-, with Mn²⁺ being a more effective activator than Mg²⁺. The reaction rate was proportional to enzyme concentration in reaction mixtures containing 0.45 to 1.8 mg protein n/ml, and was linear with time through 120 minutes in standard reaction mixtures. Enzyme preparations from immature seeds of tall and dwarf peas appeared to synthesize (—)-kaurene at the same rate. Synthesis of (—)-kaurene was readily inhibited by Amo-1618. (2-Chloroethyl)-trimethylammonium chloride (CCC) also inhibited (—)-kaurene synthesis; however, approximately 1000-fold higher concentrations of CCC were required to evoke the same percentages of inhibition as Amo-1618.     

Gibberellins and the photoperiodic control of stem elongation in the long-day plant Agrostemma githago L.

Agrostemma githago is a long-day rosette plant in which transfer from short days (SD) to long days (LD) results in rapid stem elongation, following a lag phase of 7–8 d. Application of gibberellin A₂₀ (GA₂₀) stimulated stem elongation in plants under SD, while 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine-carboxylate methyl chloride (AMO-1618, an inhibitor of GA biosynthesis) inhibited stem elongation in plants exposed to LD. This inhibition of stem elongation by AMO-1618 was overcome by simultaneous application of GA₂₀, indicating that GAs play a role in the photoperiodic control of stem elongation in this species. Endogenous GA-like substances were analyzed using reverse-phase high-performance liquid chromatography and the d-5 corn (Zea mays L.) assay. Three zones with GA-like activity were detected and designated, in order of decreasing polarity, as A, B, and C. A transient, 10-fold increase in the activity of zone B occurred after 8–10 LD, coincident with the transition from lag phase to the phase of rapid stem elongation. After 16 LD the activity in this zone had returned to a level similar to that under SD, even though the plants were elongating rapidly by this time. However, when AMO-1618 was applied to plants after 11 LD, there was a rapid reduction in the rate of stem elongation, indicating that continued GA biosynthesis was necessary following the transient increase in activity of zone B, if stem elongation was to continue under LD. It was concluded that control of stem elongation in A. githago involves more than a simple qualitative or quantitative change in the levels of endogenous GAs, and that photoperiodic induction alters both the sensitivity to GAs and the rate of turnover of endogenous GAs.Abbreviations AMO-1618 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine-carboxylate methyl chloride - GA(s) gibberellin(s) - LD long day(s) - LDP long-day plant(s) - SD short day(s)