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Hormone interactions and regulation of Unifoliata, PsPK2, PsPIN1 and LE gene expression in pea (Pisum sativum) shoot tips.

The Unifoliata (Uni) gene plays a major role in development of the compound leaf in pea, but its regulation is unknown. In this study, we examined the effects of plant hormones on the expression of Uni, PsPK2 (the gene for a pea homolog of Arabidopsis PID, a regulator of PIN1 targeting), PsPIN1 (the major gene for a putative auxin efflux carrier) and LE (a gibberellin biosynthesis gene, GA3ox), and also examined mutual hormonal regulation of these genes, in pea shoot tips, including a number of mutants. The Uni promoter possessed putative auxin and gibberellin response elements. The PsPIN1 mRNA levels were increased in afila, which replaces leaflets with branched tendrils; and reduced in tendrilless, which replaces tendrils with leaflets, compared with the wild type (WT). In contrast, mRNA levels of LE were increased in uni and tendrilless and decreased in afila compared with the WT. Uni, PsPK2 and PsPIN1 are positively regulated by gibberellin and auxin, and were induced to higher levels by simultaneous application of auxin and gibberellin. Auxin induction of Uni, PsPK2 and PsPIN1 did not require de novo protein synthesis. LE was positively regulated by auxin and cytokinin. In conclusion, these results support the hypothesis that auxin and gibberellin positively regulate Uni, which controls pea compound leaf development. Also, Uni, PsPIN1, PsPK2 and LE are expressed differentially in the leaf mutants, suggesting that mutual regulation by auxin and gibberellin promotes compound leaf development.

Cytokinins↗

Hormonal regulation of cell elongation in the hypocotyl of rootless soybean: an evaluation of the role of DNA synthesis.

A method was developed where soybean seedlings were grown without roots to study the influence of hormones of root origin on shoot growth. Excision of the root resulted in inhibition of apical section growth and DNA synthesis and inhibited elongating section growth. A synthetic cytokinin restored DNA synthesis in the apical section, but did not influence growth in either the apical or elongating sections. Low concentrations of gibberellin with the cytokinin restored growth in the apical section. Gibberellin alone was sufficient to restore growth in the elongating section. An inhibitor of DNA synthesis, 5-fluorodeoxyuridine, inhibited the increase in apical section DNA without inhibiting control or gibberellin-induced growth in the elongating section. Experiments with (14)C-thymidine resulted in no DNA labeling differences in the elongating section under conditions where gibberellin-induced elongation varied from 50% to 73% above controls. It was concluded that gibberellin-induced elongation in soybean hypocotyl occurred in the absence of DNA synthesis. Gibberellin does stimulate DNA synthesis in the apical tissue apart from its effect on cell elongation. Excised soybean hypocotyl elongated maximally at 10(-6)m auxin. At higher auxin concentrations, fresh weight and ethylene production increased, but elongation was reduced. Addition of GA to the higher auxin concentrations resulted in a 50% inhibition in auxin-induced ethylene production and resumption in maximal elongation. Added ethylene inhibited elongation 30% at 2 mul/l. Addition of up to 100 mul/l ethylene did not inhibit elongation with GA present in the incubation medium. Thus GA may counteract ehtylene inhibition of cell elongation in addition to inhibiting ethylene production in auxin-treated tissues.

DNA↗

ent-Kaurene and squalene synthesis in Fusarium fujikuroi cell-free extracts.

Sterols and gibberellins are the main terpenoids in the Ascomycete Fusarium fujikuroi. Their respective precursors squalene and ent-kaur-16-ene (henceforth called kaurene) were the main terpenoids synthesised from radioactive mevalonate by extracts of F. fujikuroi in vitro. Kaurene predominated when the extracts were obtained from mycelia engaged in gibberellin production. Squalene predominated in all other cases, and particularly when the extracts were obtained from mutants with various defects in gibberellin synthesis or nitrogen-fed wild-type cultures. New protein synthesis was required to maintain the production of gibberellins in vivo and of kaurene in vitro, but not to maintain the capacity to produce squalene in vitro. Addition of a nitrogen source to cultures engaged in gibberellin production caused a large, transient increase in the mycelial concentration of L-glutamine and abolished the accumulation of gibberellins immediately and the capacity to produce kaurene later.

Cell-Free System↗

Domains of expansin gene expression define growth regions in the shoot apex of tomato.

Expansins are members of a multigene family of extracellular proteins, which increase cell wall extensibility in vitro and thus are thought to be involved in cell expansion. The major significance of the presence of this large gene family may be that distinctly expressed genes can independently regulate cell expansion in place and time. Here we report on LeExp9, a new expansin gene from tomato, and compare its expression in the shoot tip with that of LeExp2 and LeExp18. LeExp18 gene is expressed in very young tissues of the tomato shoot apex and the transcript levels are upregulated in the incipient primordium. LeExp2 mRNA accumulated in more mature tissues and transcript levels correlated with cell elongation in the elongation zone. In situ hybridization experiments showed a uniform distribution of LeExp9 mRNA in submeristematic tissues. When gibberellin-deficient mutant tomatoes that lacked elongation of the internodes were treated with gibberellin, the phenotypic rescue was correlated with an increase in LeExp9 and LeExp2, but not LeExp18 levels. We propose that the three expansins define three distinct growing zones in the shoot tip. In the meristem proper, gibberellin-independent LeExp18 mediates the cell expansion that accompanies cell division. In the submeristematic zone, LeExp9 mediates cell expansion at a time that cell division comes to a halt. LeExp9 expression requires gibberellin but the hormone is not normally limiting. Finally, LeExp2 mediates cell elongation in young stem tissue. LeExp2 expression is limited by the available gibberellin. These data suggest that regulation of cell wall extensibility is controlled, at least in part, by differential regulation of expansin genes.

Binding Sites↗

Differential expression of acid invertase genes during seed germination in Arabidopsis thaliana.

In Arabidopsis thaliana (L.) Heynh ecotype Landsberg, levels of soluble acid invertase activity are closely related to the progress of seed germination. To study the mechanism(s) of the development of these enzymes, two cDNA clones that encode putative vacuolar acid invertases were isolated from germinating seeds and very young seedlings using reverse-transcription polymerase chain reactions with degenerate primers. These fragments corresponded to the genes At beta fruct3 and At beta fruct4 from the Columbia ecotype. An apoplasmic invertase gene corresponding to At beta fruct1/ATCWINV1 was also isolated from these samples. Northern blot analyses showed that At beta fruct3 and At beta fruc4 are expressed concomitantly with germination and the subsequent seedling growth. In contrast, the At beta fruct1/AtcwINV1 mRNA is translated before germination. These expression patterns are regulated by phytochrome, which perceives red light and in turn triggers de novo synthesis of gibberellin, initiating Arabidopsis seed germination. To test the effects of gibberellin on the expression of these genes, seed were treated with a gibberellin biosynthesis inhibitor, uniconazole or prohexadione. These chemicals inhibited both seed germination and expression of the above genes, but subsequently applied GA(4), an active gibberellin, reversed the inhibition. These results suggest that the transcription of genes encoding the vacuolar invertases, At beta fruct3 and At beta fruct4 and a gene encoding the apoplasmic invertase, At beta fruct1/AtcwINV1, are induced by gibberellin synthesized de novo following irradiation with red light.

Arabidopsis↗

Energy Metabolism of Rumex Leaf Tissue in the Presence of Senescence-regulating Hormones and Sucrose.

Hormones which inhibit senescence of Rumex leaf tissue in the dark include gibberellin A(3), and the cytokinins 6-benzylamino purine and zeatin. These hormones inhibit respiratory metabolism in this tissue, but do not change the pattern or total amount of oxygen consumption during senescence. Abscisic acid, a senescence accelerator, correspondingly stimulates oxygen consumption. This correlation of senescence rate and respiration rate holds with regard to the hormone concentrations effective and the continued activity of the hormones when added after the lag phase of chlorophyll breakdown. Transfer experiments show that the respiratory inhibition due to gibberellin A(3) and the promotion due to abscisic acid become established within 3 hours of hormone addition. When gibberellin A(3) and zeatin were rapidly added to narrow strips of tissue, no inhibitions of oxygen uptake were observed in the first 12 minutes. Senescence-inhibiting concentrations of sucrose strongly stimulate respiratory meabolism, raise the respiratory quotient, and cause inhibition of chlorophyll and protein breakdown which is distinct from the effect of gibberellins or cytokinins.It is proposed that the gibberellins and cytokinins may act by inhibiting a rate-limiting process in the metabolism of starving leaf tissue. This rate-limitation hypothesis envisions a possibly subtle action of the hormone which retards but does not alter the pattern of macromolecular changes during cell autolysis.

Journal Article↗

Glucose repression of alpha-amylase in barley embryos is independent of GAMYB transcription.

The induction of alpha-amylase triggered by gibberellic acid in barley embryos is repressed by sugars. We investigated the effects of glucose on the gibberellin signal transduction pathway to localize the site of interaction of the sugar/hormone signalling pathways. Our results indicate that glucose represses gibberellin signalling late along this hormone transduction pathway, downstream of transcription of the gibberellin-modulated transcriptional activator (GAMYB) needed for alpha-amylase induction. This result suggests either that glucose repression is transduced by a pathway independent of gibberellin signalling or that repression occurs at the level of GAMYB translation.

Gene Expression Regulation, Enzymologic↗

Low temperature sensing in tulip (Tulipa gesneriana L.) is mediated through an increased response to auxin.

Tulip (Tulipa gesneriana L.) is a bulbous plant species that requires a period of low temperature for proper growth and flowering. The mechanism of sensing the low temperature period is unknown. The study presented in this paper shows that the essential developmental change in tulip bulbs during cold treatment is an increase in sensitivity to the phytohormone auxin. This is demonstrated using a model system consisting of isolated internodes grown on tissue culture medium containing different combinations of the phytohormones auxin and gibberellin. Using mathematical modelling, equations taken from the field of enzyme kinetics were fitted through the data. By doing so it became apparent that longer periods of low temperature resulted in an increased maximum response at a lower auxin concentration. Besides the cold treatment, gibberellin also enhances the response to auxin in the internodes in this in vitro system. A working model describing the relationship between the cold requirement, gibberellin action and auxin sensitivity is put forward. Possible analogies with other cold-requiring processes such as vernalization and stratification, and the interaction of auxin and gibberellin in the stalk elongation process in other plant species are discussed.

Cold Temperature↗

Photo and hormonal control of meristem identity in the Arabidopsis flower mutants apetala2 and apetala1.

We have analyzed the contributions of phytochrome and gibberellin signal transduction to the control of flower meristem identity in the Arabidopsis mutants apetala1 (ap1) and apetala2 (ap2). ap1 flowers are partially defective for the establishment of flower meristem identity and are characterized by the production of ectopic secondary or axillary flowers and by branching. Axillary flower production is also induced in ap2-1 flowers by short-day photoperiod and is suppressed by hy1, a mutation blocking phytochrome activity. The production of axillary flower by ap2-1 is also suppressed by exogenous gibberellins and by spindly (spy), a mutation that activates basal gibberellin signal transduction in hormone-independent manner. Ectopic axillary flower production and floral branching by ap1 flowers are also suppressed by spy. We conclude that gibberellins promote flower meristem identity and that the inflorescence-like traits of ap2-1 and ap1-1 flowers are due in part to SPY gene activity.

Arabidopsis↗

Growth stimulation of dwarf peas (Pisum sativum L.) through homeopathic potencies of plant growth substances.

BACKGROUND: Efficacy of higher homeopathic potencies is controversial. Universally accepted specific detection assays for homeopathic dilutions do not exist. Basic research has to develop a spectrum of standardized tools to investigate the mode of action and nature of homeopathic potencies. OBJECTIVE: Can the shoot growth reaction of dwarf peas (gibberellin- deficient mutants) be regarded as evidence of treatment with homeopathic potencies of plant growth substances? MATERIALS AND METHODS: Pea seed (Pisum sativum L. cv. Fruher Zwerg) is immersed for 24 hours in homeopathic potency or control solutions for soaking. Plants germinate and grow in a standard cultivation substrate under controlled environmental conditions. Shoot length is measured 14 days after planting. RESULTS: A screening of homeopathic potencies (12x-30x) of four different plant growth substances revealed biological activity of certain potency levels of gibberellin and kinetin (p < 0.05). Growth stimulation through gibberellin 17x (5 x 10(-18 M)) was assessed in six independent replications; results confirmed those of the screening (p < 0.05). The effect of gibberellin 17x seemed to weaken during the course of the experiments. CONCLUSION: The results back the hypothesis that homeopathic potencies of plant growth substances affect pea shoot growth. Dwarf peas might thus be an interesting system model for studying the action of homeopathic potencies. Further work is required to identify all boundary conditions modulating the reactivity of this system.

Adenine↗

Sexual expression in container-grown jack pine seedlings.

We assessed the effects of nitrogen, drought and gibberellin A(4/7) on sexual expression of 2- and 3-year-old jack pine (Pinus banksiana L.) seedlings grown in either a 1/3 peat/sand mix or a 3/1 peat/perlite mix. The seedlings were either watered daily (well-watered treatment) or droughted by withholding water from July 13 to August 24 (drought treatment). Half of the plants in each irrigation treatment were sprayed with 200 mg l(-1)gibberellin A(4/7) at weekly intervals from June 28 to August 24; the remaining plants were not sprayed. Each gibberellin treatment was split into three subplots, and each subplot was supplied with nitrogen in the nutrient solution at 3, 100 or 300 mg l(-1) from June 27 to September 11. The drought treatment increased pollen strobilus production, whereas seed strobilus production was higher in well-watered trees than in drought-treated trees in the first year after treatment, but not in the subsequent year. Gibberellin A(4/7) promoted seed strobilus production but not pollen strobilus production in the first year after treatment but had no effect in the second year. Among the nitrogen treatments, the largest proportion of trees bearing pollen strobili was in the 3 mg l(-1) N treatment. Nitrogen supply did not affect seed strobilus production in the first year after treatment, but in the following year, the proportion of trees with seed strobili was higher in the 3 mg l(-1) N treatment than in the 100 and 300 mg l(-1) N treatments. Growing medium had no detectable effect on pollen strobilus production, but in the year following treatment, more trees produced seed strobili in peat/sand than in peat/perlite.

Journal Article↗

Hormonal Characterization of Transgenic Tobacco Plants Expressing the rolC Gene of Agrobacterium rhizogenes TL-DNA.

Transgenic tobacco (Nicotiana tabacum L. cv Wisconsin 38) plants expressing the Agrobacterium rhizogenes rolC gene under the control of the cauliflower mosaic virus 35S RNA promoter were constructed. These plants displayed several morphological alterations reminiscent of changes in indole-3-acetic acid (IAA), cytokinin, and gibberellin (GA) content. However, investigations showed that neither the IAA pool size nor its rate of turnover were altered significantly in the rolC plants. The biggest difference between rolC and wild-type plants was in the concentrations of the cytokinin, isopentenyladenosine (iPA) and the gibberellin GA19. Radio-immunoassay and liquid chromatography-mass spectrometry measurements revealed a drastic reduction in rolC plants of iPA as well as in several other cytokinins tested, suggesting a possible reduction in the synthesis rate of cytokinins. Furthermore, gas chromatography-mass spectrometry quantifications of GA19 showed a 5- to 6-fold increase in rolC plants compared with wild-type plants, indicating a reduced activity of the GA19 oxidase, a proposed regulatory step in the gibberellin biosynthesis. Thus, we conclude that RolC activity in transgenic plants leads to major alterations in the metabolism of cytokinins and gibberellins.

Journal Article↗

Gibberellic Acid-Promoted Lignification and Phenylalanine Ammonia-lyase Activity in a Dwarf Pea (Pisum sativum).

The effects of gibberellic acid on lignification in seedlings of a dwarf and a tall cultivar of pea (Pisum sativum) grown under red or white light or in the darkness, were studied. Gibberellic acid (10(-6)-10(-4)m) promoted stem elongation in both light and dark and increased the percentage of lignin in the stems of the light-grown dwarf pea. The gibberellin had no effect on the lignin content of the tall pea although high concentrations (10(-4)m) promoted growth of the tall plants. Time course studies indicated that the enhanced lignification in the gibberellin-treated dwarf plants occurred only after a lag period of several days. It was concluded that gibberellic acid-enhanced ligmification had no direct relation to gibberellic acid-promoted growth. The activity of phenylalanine ammonia-lyase (E.C. 4.3.1.5) was higher in gibberellin-treated dwarf plants grown under white or red light than in untreated dwarf plants. Gibberellic acid had no detectable effect on the activity of this enzyme when the plants were grown in darkness, just as it had no effect on lignification under dark conditions. The data suggest that in gibberellin-deficient peas the activity of phenylalanine ammonia-lyase is one of the limiting factors in lignification.

Journal Article↗

Growth regulator induced movement of photosynthetic products into fruits of ;black corinth' grapes.

The effect of exogenous growth regulators on movement of assimilates into flowers and young fruits of ;Black Corinth' grapes was studied. Clusters were treated with growth regulator and after 0.5 hr to 5 days the leaves above the clusters were exposed to (14)CO(2). Control shoots received (14)CO(2) but no growth regulator. At harvest, counting and radioautographic techniques were used to ascertain amount and distribution of activity in clusters. Clusters were dipped in 4-CPA (4-chlorophenoxyacetic acid), GA(3) (gibberellic acid), or BA (benzyladenine). All berries were heavier than controls within 3 days. Total counts in the fruits were increased by 4-CPA, and the distribution of radioactivity among the sugar, organic acid, and amino acid fractions was usually altered by all treatments. In a time series experiment, within 6 hr after treatment of fruits with GA(3) there was almost an 8-fold increase in total counts relative to the control. After 12 hr there was about a 9-fold and 6-fold increase in counts in tartaric and malic acids, respectively, and in gamma-aminobutyric acid, pipecolic acid, and valine increases of 56, 150, and 330%. Radioactivity in fructose was increased 70% in gibberellin-treated clusters over the controls. After 96 hr there were only about 1000 cmp per g fr wt in controls, but there were about 31,000 cpm counts in treated clusters. Treatment of clusters with gibberellin attracted less assimilates into the fruits when shoots had also been sprayed with gibberellin. Dipping portions of clusters in gibberellin increased the movement of (14)C assimilates into the treated portions. Hormonal control of mobilization is discussed.

Journal Article↗

The level of phytohormones in monoecious and gynoecious cucumbers as affected by photoperiod and ethephon.

The endogenous levels of auxin, gibberellin, and inhibitors were followed in monoecious and gynoecious cucumber (Cucumis sativus L.) plants, and in plants treated with the ethylene-releasing compound Ethephon (2-chloroethyl phosphonic acid). Higher auxin inhibitor and lower gibberellin levels were associated with female tendency. The endogenous level of gibberellin and auxin decreased in Ethephon-treated plants. Application of Ethephon induced a rise in abscisic acid. Root application of abscisic acid promoted female tendency of gynoecious cucumbers grown under conditions which increase maleness. High CO(2) levels, which are known to antagonize ethylene, increased maleness of gynoecious cucumbers. The possibility of interrelationship between gibberellin, auxin, ethylene, and abscisic acid on sex expression are discussed.

Journal Article↗

Properties of Kaurene Synthetase from Marah macrocarpus.

The kaurene synthetase from immature seeds of Marah macrocarpus (Greene) Greene was partially purified from cell-free homogenates of endosperm by a combination of QAE-Sephadex A-25 chromatography and hydroxyapatite chromatography and freed of contaminating phosphatase activity. The two catalytic activities associated with kaurene synthetase, the cyclization of geranylgeranyl-pyrophosphate to copalyl-pyrophosphate (activity A) and the cyclization of copalyl-pyrophosphate to ent-kaurene (activity B), were not even partially resolved from one another during these procedures. Both activities had identical elution profiles from a calibrated Sepharose 4B column corresponding to a molecular weight less than that of ovalbumin (45,000).The A and B activities had pH optima of 7.3 and 6.9, respectively. Both activities required millimolar concentrations of the following divalent cations in the order: Mg(2+) > Mn(2+) > Co(2+). Activities A and B were both sensitive to inhibition by Hg(2+), Cu(2+), p-hydroxymercuribenzoate, and N-ethylmaleimide, but activity B was much more sensitive than activity A. The average value of Km' (apparent Km in the absence of substrate inhibition) for geranylgeranyl-pyrophosphate was 1.6 mum. Values of 0.5 and 0.6 mum were obtained for Km' and Km, respectively, for copalyl-pyrophosphate. The Vm' values for the two activities were similar: 12 and 9 pmol/minute.mug protein for activities A and B, respectively.N,N-Dimethylaminoethyl-2,2-diphenylpentanoate (SKF-525A) and N,N-dimethylaminoethyl-2,2-diphenylphentyl ether (SKF-3301A), tributyl-2,4-dichlorobenzylphosphonium chloride (Phosfon D), tributyl-2,4-dichlorobenzylammonium chloride (Phosfon S), 2'-isopropyl-4'-(trimethylammonium chloride)-5'-methylphenyl piperidine-1-carboxylate (Amo-1618), 2-(N,N-dimethyl-N-heptylammonium bromide)-p-methan-1-ol (Q-58), and 2-(N,N-dimethyl-N-octylammonium bromide)-p-methan-1-ol (Q-64), at concentrations from 1 to 5 mum, were effective inhibitors of kaurene synthetase activity A. Acetylcholine chloride and 2-chloroethyl-trimethylammonium chloride were effective inhibitors of activity A only at concentrations of 5 mm or greater. Abscisic acid, indole-3-acetate, gibberellin A(1), gibberellin A(3), a mixture of gibberellins A(4) and A(7), gibberellin A(13), and N,N-dimethylaminosuccinamic acid (B995) were not inhibitory at any of the levels tested. None of these compounds was an effective inhibitor of activity B at concentrations less than 0.5 mm.

Journal Article↗

Hormonal activity in detached lettuce leaves as affected by leaf water content.

The interrelationship between water deficiency and hormonal makeup in plants was investigated in detached leaves of romaine lettuce (Lactuca sativa L. cv. ;Hazera Yellow'). Water stress was imposed by desiccating the leaves for several hours in light or darkness at different air temperatures and relative humidity. In the course of desiccation, a rise in abscisic acid content and a decline in gibberellin and cytokinin activity were observed by gas-liquid chromatography, by both the barley endosperm bioassay and radioimmunoassay and by the soybean callus bioassay. Gibberellin activity began to decline in the stressed leaves before the rise in abscisic acid, the rate of this decline being positively correlated with the rate of increase in leaf water saturation deficit. Recovery from water stress was effected by immersing the leaf petioles in water while exposing the blades to high relative humidity. This resulted in a decrease in leaf water saturation deficit, a reduction in abscisic acid content, and an increase in gibberellin and cytokinin activity.Application of abscisic acid to the leaves caused partial stomatal closure in turgid lettuce leaves, whereas treatment with gibberellic acid and kinetin of such leaves had no effect on the stomatal aperture. In desiccating leaves, however, gibberellic acid and kinetin treatment considerably retarded stomatal closure, thus enhancing the increase in leaf water saturation deficit. These results suggest that the effect of desiccation in changing leaf hormonal make-up, i.e. a rapid increase in abscisic acid and a decrease in both cytokinin and gibberellin activity, is related to a mechanism designed to curtail water loss under conditions inducing water deficiency.

Journal Article↗

Hormonally regulated programmed cell death in barley aleurone cells

Cell death was studied in barley (cv Himalaya) aleurone cells treated with abscisic acid and gibberellin. Aleurone protoplasts incubated in abscisic acid remained viable in culture for at least 3 weeks, but exposure to gibberellin initiated a series of events that resulted in death. Between 4 and 8 days after incubation in gibberellin, >70% of all protoplasts died. Death, which occurred after cells became highly vacuolated, was manifest by an abrupt loss of plasma membrane integrity followed by rapid shrinkage of the cell corpse. Hydrolysis of DNA began before death and occurred as protoplasts ceased production of alpha-amylase. DNA degradation did not result in the accumulation of discrete low molecular weight fragments. DNA degradation and cell death were prevented by LY83583, an inhibitor of gibberellin signaling in barley aleurone. We conclude that cell death in aleurone cells is hormonally regulated and is the final step of a developmental program that promotes successful seedling establishment.

Journal Article↗