The Effect of Gibberellin A3 and (2-Chloroethyl)-trimethylammoniuin chloride on Assimilate Distribution in Gladiolus in Relation to Corm Growth1

The influence of GA and CCC on growth and assimilate translocationto the various plant organs in gladiolus was studied by labellinga single leaf with ¹⁴CO₂ and following the distribution of theassimilates. GA promotes inflorescence growth by directing assimilatemovement towards the inflorescence at the expense of the corm.CCC has a similar but smaller effect. GA and CCC both promoteassimilate translocation from the labelled leaf during the periodof inflorescence growth. When this period is over and the cormbecomes the main sink, translocation from the labelled leafis promoted by CCC but inhibited by GA. The effect of CCC maybe only partly explained on the basis of an increase in GA turnoverin gladiolus.     

Effect of CCC on the gibberellin content of excised sunflower organs

Summary CCC was shown to be effective in retarding stem growth of sunflower; this effect was overcome by gibberellic-acid application. Using an agar-diffusion technique, the gibberellin (GA) content of sunflower apices treated with CCC was found to be significantly reduced as compared to controls. Similarly, the GA content of agar diffusates obtained from 2-day-old sunflower root tips treated with CCC was also significantly reduced as compared to controls.Root exudate or bleeding sap obtained from mature CCC treated sunflower plants contained no measurable GA-like substance, although it could not be argued that this was due to suppression of GA synthesis in the root systems.     

CCC-Induced increase of gibberellin levels in pea seedlings

Summary Pea seedlings (cv. Alaska), were treated with two concentrations of (2-chloroethyl)trimethylammonium chloride (CCC) and choline chloride. Treatment with 1 mg/l CCC resulted in as much as a 150fold increase in endogenous gibberellin (GA) levels without there being any parallel stimulation of growth. Plants grown in 1,000 mg/l CCC were severely dwarfed but contained GA levels not significantly different from control plants grown in distilled water. CCC also retarded GA₃-induced growth of pea seedlings. These effects appear to be CCC specific as the CCC analogue choline chloride affected neither the GA content of pea seedlings nor their response to GA₃. The lack of correlation between endogenous GA levels and stem height suggests that in peas the predominant factor in CCC-induced inhibition of stem growth is not related to an effect of CCC on GA biosynthesis.Supported by National Research Council (Canada) grant A-5727.Supported by a Postdoctoral Fellowship from NRC Grant A-2585 to R.P. Pharis.     

The influence of light,gibberellic acid and CCC on sprout growth and mobilization of tuber reserves in the potato (Solanum tuberosum L.)

Summary Warm white fluorescent light inhibited the elongation of potato sprouts and reduced the rate at which reserve materials in the mother tuber were mobilized. The evidence indicated that the influence of light on mobilization was independent of light effects on the magnitude of the sink for translocates in the sprout. However, dark-grown sprouts exerted a greater directive influence than illuminated sprouts on the translocation of the mobilized materials.The rate of elongation and increase in dry weight of sprouts was promoted by exogenous GA independently of the effects of light on these processes, and the absence of any effects of CCC on sprout length and dry weight in these experiments was interpreted to indicate that light did not influence these parameters by controlling the level of endogenous gibberellins. Neither GA nor CCC modified the effects of light on the dry matter content of the sprouts, suggesting that growth in darkness and growth induced by GA were qualitatively different. The results are compared with the effects of light and GA on growth and stem elongation in other species.     

Enhancement of lateral bud growth in Coleus blumei BENTH. by (2-chloroethyl) trimethylammonium chloride (CCC)

The relationship of GA to apical dominance in Coleus was examinedby substituting 1 % IAA, in lanolin, for the shoot apex of CCC-treated,control and GA-treated plants containing, theoretically, hyponormal,normal and hypernormal GA levels, respectively. The greatestinhibition of lateral bud growth was obtained in the treatmentcombining 1 % IAA and 100 ppm GA, suggesting that GA may beimportant in the apical dominance of Coleus. CCC inhibited main axis growth, reduced the level of endogenousGA and caused a marked release of lateral buds from apical dominance. The significant stimulation of lateral bud growth by CCC couldnot be ascribed to reduced endogenous GA since it was not reversedby exogenous GA, or by GA plus IAA, whereas 100 ppm GA overcamethe inhibition of main axis growth by CCC. It was also shownthat the CCC stimulation was not a result of compensatory growth,that is, enhanced lateral bud growth resulting from reducedapical bud growth. The CCC effect on lateral buds was interpretedas involving a system independent of auxin and GA or else apossible immobilization of auxin in addition to inhibition ofGA biosynthesis. (Received December 5, 1967; )     

Gibberellin and auxin contents and ethylene production in the hypocotyls of green and etiolated bean plants treated with chlorocholine chloride

The effect of CCC treatment on the GA and IAA contents of the hypocotyls of green and etiolated bean plants and on their distribution was studied. In the treated plants the GA content of the hypocotyls was higher than that of the control. On the application of CCC the distribution of the hormone content within the organ altered: in the basal part of the hypocotyls of the treated plants the hormone content increased. In the hypocotyls of the treated plants the IAA content was less than in the controls and it was accumulated in the basal part. The ethylene production of the aplcal and basal parts of the hypocotyls proved to be correlated with their IAA content. Presented at the International Symposium “Plant Growth Regulators” held on June 18–22, 1984 at Liblice, Czechoslovakia.     

Modification of growth habit of Majestic potato by growth regulators applied at different times

The effect of gibberellic acid, CCC (2-chloroethyltrimethylammonium chloride) and B 9 (N-dimethylaminosuccinamic acid) was studied on growth of potato plants in pots. Growth was analysed on four occasions and changes in habit defined in terms of internode lengths, leaf areas and growth of lateral branches. Soaking seed pieces for 1 hr. in GA solution caused six internodes to elongate greatly, an effect not prevented by CCC applied when the shoot emerged from the soil. The effects on internode extension were determined by the length of the interval between GA treatment and CCC treatment. Treatment at emergence with CCC shortened all internodes and more CCC applied 4 weeks later had no effect. Late application of CCC or B9 shortened the top two lateral branches, usually very long in untreated plants. The regulators affected leaf growth differently from internode growth: usually growth regulators had less effect on leaf growth. Effects on growth depended on when the regulators were applied. Treatment with GA alone inhibited bud development at higher nodes than in untreated plants; when followed by late treatment with CCC, lateral growth at higher nodes was also less. CCC retarded development of lateral branches especially when applied early. B9 had a similar effect to CCC applied late. When regulators retarded growth of lower laterals, upper laterals often grew more than in untreated plants. Treatments did not affect the number of leaves on the main stem at first but later GA hastened senescence. GA increased the number of leaves on lateral stems, and the effect was enhanced by CCC. CCC alone increased the number of first- and second-order lateral leaves. GA lengthened and CCC shortened stolons. The effect of CCC persisted throughout the life of the plant. CCC or B 9 shortened stolons whenever applied. CCC hastened tuber initiation but slowed tuber growth. CCC at first retarded formation of lateral tubers but had no effect on the ultimate numbers of lateral and terminal tubers. The value of E (net assimilation rate) did not alter with time. CCC applied at emergence increased E, probably because it hastened tuber initiation and temporarily increased sink capacity. Although tubers formed earlier with CCC, their growth was slower and their demand for carbohydrate was also less. The increase in second-order laterals in CCC-treated plants indicates that they utilize carbohydrate that would normally go to tubers. This experiment also demonstrates that crowding leaves by shortening stems did not diminish E, possibly because another over-riding process (bigger sinks) offsets the effect of shading.     

The plant growth retardant CCC as inhibitor of gibberellin biosynthesis inFusarium moniliforme

Summary Gibberellin (GA) production inFusarium moniliforme (Gibberella fujikuroi) is suppresed by adding the plant growth retardant CCC [(2-chloroethyl)trimethylammonium chloride] to the culture medium. A concentration of 0.1 mg/l of CCC causes 50% inhibition whereas 10 mg/l and higher concentrations fully suppress GA production. Dry weight of the mycelium is not, or only slightly reduced in the presence of CCC.Thin-layer chromatography of acidic fractions of CCC-free cultures reveals fluorescent spots at 4 differentR _f values. No fluorescent spots can be detected on chromatograms of acidic fractions obtained from CCC cultures, thus demonstrating that production of all GA's is inhibited by CCC.If CCC is added to the medium 2 or 3 days after inoculation, further GA production is blocked, but the level of GA present at the time of CCC application is maintained. CCC does not enhance inactivation of GA₃ in sterile culture medium, nor in the presence of the fungus. It is therefore concluded that CCC inhibits the biosynthesis of GA in the fungus.Transfer of thoroughly washed mycelium from medium with CCC to fresh medium does not result in GA production because sufficient CCC is carried over in the mycelium to block GA biosynthesis completely.     

Effects of CCC and GA on internodal development of barley

Summary The effect of applying (2-chloroethyl)trimethylammonium chloride (CCC) or gibberellic acid (GA) as foliar sprays on internodal development of barley was studied. CCC applied to the whole plant at main tiller leaf stages 1, 2 or 3 decreased shoot elongation, and prevented elongation of internode 6 (internode 5 subtended the head). CCC at all stages delayed senescence of the lower leaves. CCC sprayed at all 6 leaf stages and GA sprayed at main tiller leaf stages 1, 2, 3 or 4 reduced plant height at maturity. GA treatment at leaf stages 2, 3 or 4 initially stimulated internodal elongation; elongation of later developed internodes was inhibited resulting in shorter plants at maturity. Only the treatment with GA at leaf stages 5 and 6 resulted in increased plant height.     

Effect of chlorocholine chloride and gibberellic acid on the anthocyanin synthesis in radish seedlings

Anthocyanin synthesis in radish ( Raphanus sativus L. cv. Scarlet Globe) seedlings after treatment with chlorocholine chloride (CCC) and gibberellic acid (GA) has been investigated. CCC promotes and GA₃ inhibits the synthesis. When both substances are given together, CCC reverses the inhibition caused by GA₃. Simultaneous external feeding of anthocyanin precursors (sucrose and phenylalanine) reverses the GA₃ inhibition. A higher amount of total free amino acids, in particular phenylalanine, was present in CCC-treated seedlings compared to controls grown on distilled water. The amount of phenylalanine was lower in seedlings treated with both CCC and GA₃ as compared to seedlings treated with CCC alone, and total free sugars (reducing plus non-reducing) was lower in CCC treated seedlings than in controls grown on distilled water. We conclude that CCC and GA3 control the anthocyanin synthesis at the level of precursors.     

Gibberellins are not essential for photoperiodic flower induction of Pharbitis nil

The influence on photoperiodic flowering of (2-chloroethyl)trimethylmmonium chloride (CCC), an inhibitor of gibberellin (GA) biosynthesis, was studied in the short-day plant Pharbitis nil cv. Violet. The cotyledons contained high levels of endogenous bioactive gibberellins, whereas in the plumules and first leaves the levels were low or undetectable. The first leaf responded to a single'dark treatment by inducing flowering when it was 10 mm or wider. Similar seedlings, but without cotyledons, were used as the assay plants to study the effect of CCC on photoperiodic flowering. Treatment with CCC had no effect on flowering of seedlings without cotyledons, although stem elongation was inhibited. By contrast. CCC inhibited flowering of the intact seedlings with cotyledons. Gibberellic acid applied to the shoot apex or to the first leaf promoted flowering in the CCC-treated seedlings without cotyledons. The results indicate thai gibberellins are not essential for the flower induction process in leaves, but that they promote flower initiation and/or later processes in the shoot apices.     

Suppression of floral induction inBryophyllum daigremontianum by a growth retardant

Summary Flower formation in the long-short-day plantBryophyllum daigremontianum induced by the shift from long to short day is fully suppressed by applying the growth retardant CCC [(2-chloroethyl)-trimethylammonium chloride]via the roots during short day treatment at a level of at least 1 g per plant. At the same time CCC applications strongly reduce the internode length, but the rate of leaf formation does not seem to be affected. CCC also causes the production of more anthocyanin in the leaves.Gibberellin A₃ appliedvia the apices and youngest leaves at a dose of 1.5 g per plant, completely overcomes the inhibition of flower formation caused by CCC. Ten times higher quantities are needed for a complete reversal of the reduction in stem elongation.The growth retardant CCC seems to keep the level of physiologically active GA below that which is normally required for floral initiation and stem elongation. Thus, the results are in agreement with the hypothesis that a high GA level is necessary for the production of floral stimulus inBryophyllum daigremontianum.With 2 Figures in the TextThis work was supported by the National Science Foundation, grants G-16408 and G-17483.     

Studies in seed dormancy

Summary When dormant hazel seeds were subjected to six weeks chilling at 5° C their subsequent transfer to 20° C resulted in the accumulation of gibberellin (GA) followed by germination. In the presence of either phosphon D or -chlorethyltrimethylammonium chloride (CCC) at 20° C there was inhibition of both GA accumulation and germination, a finding consistent with the hypothesis that GA biosynthesis is a necessary prerequisite for the germination of chilled hazel seeds. As abscisic acid showed a strong inhibition of germination but had little effect on GA accumulation it is presumed not to have affected GA biosynthesis but to have inhibited GA action. These conclusions were supported by experiments in which the interaction of exogenous GA₃ with growth retardants and ABA was tested on the germination of chilled hazel seeds. Experiments in which the embryonic axes and cotyledons of chilled seeds were incubated separately at 20° C established that GA biosynthesis de novo occurred in the embryonic axis and indicated that in the intact seed some of the GA would have been translocated to the cotyledons. The isolated cytoledons showed no GA biosynthesis de novo but gave some release of GA from one or more bound forms.Abbreviations ABA abscisie acid - AMO 1618 2-isopropyl-4-(trimethylammonium chloride)-5-methylphenyl piperidine carboxylate - CCC -chlorethyltrimethylammonium chloride - GA gibberellin, phosphon - D tributyl-2,4-dichlorobenzylphosphonium chloride - TLC thin-layer chromatography     

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.     

Reduction of the Gibberellin Content of Pharbitis Seeds by CCC and After-Effects in the Progeny

Plants of Pharbitis nil were treated with the growth retardant (2-chloroethyl) trimethylammonium chloride (CCC) shortly before and after anthesis. Fresh and dry weight of immature seeds were not affected by the CCC treatment.

The level of gibberellin-like activity in Pharbitis seeds as compared to control seeds was strongly reduced by CCC application. The progenies of the treated plants also had a much reduced GA content in the seedling stage. These results are interpreted to indicate that CCC blocks gibberellin biosynthesis in higher plants, as it does in the fungus Fusarium.

CCC applied via the roots accumulated in the immature seeds and was carried over to the following generation. Consequently, growth of CCC progenies was dwarfed and flower formation inhibited. Both phenomena were overcome by application of gibberellin A₃.

Three gibberellin-like substances (called fractions I, II, and III) were present in Pharbitis seeds and could be separated by thin-layer chromatography. All 3 fractions were also present in seeds treated with CCC. Fractions II and III were present in much higher quantities than fraction I. Both fractions II and III promoted growth of d5 corn but only fraction II was active in dwarf peas grown under red light.

     

Effect of Gibberellin on Growth, Protein Secretion, and Starch Accumulation in Maize Endosperm Suspension Cells

The physiologic effect of gibberellins (GA) in seed development is poorly understood. We examined the effect of gibberellic acid (GA₃) on growth, protein secretion, and starch accumulation in cultured maize (Zea mays L.) endosperm suspension cells. GA₃ (5 and 30 μm) increased the fresh weight, dry weight, and protein content of the cultured cells, but the effect of GA₃ at 50 μm was not significantly different. However, the protein content in the culture medium was increased by these three concentrations of GA₃. The effect of GA₃ on the amount of cellular structural polysaccharides was not significant, but GA₃ had a dramatic effect on the starch content. At 5 μm, GA₃ caused an increase in the starch content, but at 50 μm the starch accumulation was reduced. Chlorocholine chloride (CCC), an inhibitor of GA biosynthesis, significantly increased the starch content and decreased the structural polysaccharide content of the cultured cells. The effects of CCC at 500 μm on the starch and polysaccharide content were partially reversed by 5 μm GA₃ applied exogenously. Based on these results we suggest that GA does not favor starch accumulation in the cell cultures and that the addition of lower concentrations of GA₃ in the medium may provide an improved balance among the endogenous GA in the cultured cells. Received October 31, 1995; accepted March 25, 1997     

Role of Endogenous Plant Growth Regulators in Seed Dormancy of Avena fatua: II. Gibberellins

Gibberellin A₁ (GA₁) was identified by combined gas chromatographymass spectrometry as the major biologically active gibberellin (GA) in seeds of wild oat (Avena fatua L.) regardless of the depth of dormany or stage of imbibition. Both unimbibed dormant and nondromant seeds contained similar amounts of GA₁ as estimated by the d5-maize bioassay. During imbibition, the level of GA₁ declined in both dormant and non-dormant seeds, although the decline was more rapid in dormant seeds. Only in imbibing nondormant seeds did the GA biosynthesis inhibitor, 2-chloroethyltrimethyl ammonium chloride (CCC), cause a reduction in the level of GA₁ from that observed in control seeds. These results are interpreted as an indication that while afterripening does not cause a direct change in the levels of GAs during dry storage, it does induce a greater capacity for GA biosynthesis during imbibition.

Nondormant seeds imbibed in the presence of 50 millimolar CCC germinated equally as well as untreated seeds. When wild oat plants were fed CCC throughout the entire life cycle, viable seeds were produced that lacked detectable GA-like substances. These seeds afterripened at a slightly slower rate than the controls. Moreover, completely afterripened (nondormant) seeds from plants fed CCC continuously contained no detectable GA-like substances, and when these seeds germinated, dwarf seedlings were produced, indicating GA biosynthesis was inhibited during and after germination. In total, these results suggest that the increased capacity for GA biosynthesis observed in imbibing nondormant seeds is not a necessary prerequisite for germination. It is therefore possible that GA biosynthesis in imbibing nondormant seeds is one of many coordinated biochemical events that occur during germination rather than an initiator of the processes leading to germination.

     

Effects of Growth Retardants on the Growth of Excised Roots of Dolichos lablab L. in Culture

The effects of various growth-retarding chemicals on the growthof excised roots of Dolichos lablab in sterile culture are investigated.Application of a range of concentrations of CCC, phosfon, andB-995 inhibited growth in length of the roots by reducing thefrequency of cell division, with little or no effect on cellelongation. Treated roots had generally lower DNA, RNA, andprotein contents than controls, although CCC-treatment significantlyenhanced the soluble nitrogen content of the roots. The inhibitoryeffects of growth retardants were not reversed by IAA or GA,but substances like choline chloride and pyridoxine hydrochloridewere partially effective in reinstating normal growth in rootsgrowing in concentrations of CCC producing about 50 percentinhibition of growth in length.     

The role of auxin in growth of the plumular hook section of etiolated pea seedling

Externally applied GA greatly promoted elongation of the plumularhook section of the etiolated Alaska pea seedling, but IAA hadno such effect when given either alone or with GA. PCIB inhibitedelongation of the plumular hook section both in the presenceand absence of applied GA. The PCIB effect in the absence ofGA was partially overcome by IAA, but not completely. On theother hand, the PCIB effect in the presence of GA was completelyovercome by IAA. No antagonic response was, however, obtainedbetween GA and PCIB. CCC also retarded elongation of the sectionand this inhibition was completely overcome by GA, but not byIAA. There was little difference in the amount of endogenous auxindetectable in GA treated and untreated sections. These resultssuggest that auxin is necessary for the growth of both GA treatedand untreated plumular hook sections and that auxin and gibberellinact differently on the growth of the section. (Received April 24, 1968; )