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.     

Diffusible and Extractable Gibberellins in Bean Cotyledons in Relation to Dwarfisni

Dwarfism in bean plants (Phaseolus vulgaris L.) is investigated in relation to the diffusible and extractable gibberel-lins in cotyledonary tissues. These gibberellins were partitioned into four parts prior to thin layer chromatograplty: non-acidic and acidic ethyl acetate fractions, and non-acidic and acidic butanol fractions. Cotyledonary segments from a tall plant (cv. Kentucky Wonder) seem to diffuse, preferentially acropetally, more gibberellins in each fraction than those from a dwarf plant (cv. Masterpiece). The diffusion increases with the length of the segments and decreases with period after sowing. From experiments on extraetable gibberellins, however, it is concluded that these phenomena actually result from differences in the gibbereliin contents of the tall and the dwarf plants, from differences in the gibberellin contents of the distal and proximal parts of cotyledons, and from the gibberellin contents before and after imbibition. Ten kinds of gibberellin-Iike substances are detectable in the diffusates, exudates and extracts from the cotyledons of both the plants; two in the non-acidic ethyl acetate, three in the acidic ethyl acetate (GA₁, GA₆ and another), two in the non-acidic butanol, and three in the acidic butanol frac—tion. They are almost identical in quality in the dwarf and tall plants, but in the latter they are more abundant in the cotyledons, particularly in their distal part. With respect to the change in content during the period after sowing, the gibberellin-Iike substances are classified in three groups; unchanging, decreasing (GA₁, GA₆, others) and increasing (butanol soluble glucosyl esters and glucosides of gibberellins)-. The increase of glucose-bound gibberellins and the decrease of free gibberellins during the sowing period suggest the occurrence of conversion. This is obvious only in the tall plants. The gibberellin content in cotyledons is higher in the tall plant than in the dwarf plant. Thus, the marked hypocotyl growth in the tall plants may be dependent on the higher content of gibberellins in their cotyledons and on the higner rate of conversion from free to bound forms.     

On the nature of the hormonal regulation of amylase activity in cotyledons of germinating peas

The effect of the embryo axis and various growth substanceson amylase activity in cotyledons of germinating peas was studied. The embryo axis brought about an elevation in the level of enzymeactivity. Gibberellic acid, kinetin, benzyladenine and zeatinriboside also promoted the activity, but none of these substancesbrought about a full replacement of the axis. Such a replacementwas caused by zeatin or by a simultaneous application of zeatinriboside and gibberellic acid. The results suggest that the regulative effect of the embryoaxis on amylase activity in cotyledons is brought about eitherby zeatin alone or a cooperative action of a cytokinin (or severalcytokinins) and a gibberellin (or several gibberellins). (Received November 30, 1974; )     

Light and molybdenum requirements for substrate induction of nitrate reductase in cotyledons of lettuce,Lactuca sativa L.

Light was required for induction of nitrate reductase (NR, E.C. 1.6.6.1) in intact cotyledons of 2-day old seedlings ofLactuca sativa L. Molybdate strongly enhanced efficiency of induction. Benzyladenine (BA), gibberellin, and succinic acid-2,2-dimethylhydrazide reduced the enzyme activity. BA thrice enhanced incorporation of labelled leucine to the protein fraction. (2-chloroethyl)trimethylammonium chloride did not affect NR activity and markedly inhibited greening and protein synthesis. KNO₃ stimulated protein synthesis as well as growth of the cotyledons.     

The control of seed germination in Trollius ledebouri A model of seed dormancy

Treatment of Trollius ledebouri seeds with gibberellins A₄+A₇ promotes germination. The efficacy of the treatment is dependent upon the duration of imbibition in distilled water prior to GA₄₊₇ application. Presoaking increases both the final percentage germination attained and also its rate of achievement. No presoaking effect is exhibited by seeds induced to germinate by testa removal in the absence of GA₄₊₇. Active washing of Trollius seeds enhances the presoaking effect and the eluent from washed seeds is inhibitory to germination. The results support the hypothesis that the presoaking effect exhibited by Trollius is the result of the leaching of a germination inhibitor from the seeds which is antagonistic to GA₄₊₇. Additionally, treatment of Trollius seeds with the gibberellin-biosynthesis inhibitor (2-chloroethyl)-trimethylammonium chloride (CCC) prior to testa removal retards germination. The inhibitory effect of CCC on germination is overcome by GA₄₊₇. Although CCC inhibits embryo growth during the presoaking of intact seeds, it does not affect the increased sensitivity of presoaked seeds to GA₄₊₇. Therefore, although endogenous gibberellins may be involved in the germination process, they do not contribute to the presoaking phenomenon. The expansion of isolated endosperm tissue is not affected by CCC. However, the chemical markedly inhibits endosperm expansion in intact seeds and implicates the embryo as both the site of production of the germination inhibitor and of gibberellin. These results are discussed in relation to previous studies and a model is presented to account for the characteristics of germination in Trollius.Abbreviations GA gibberellin - CCC (2-chloroethyl)-trimethylammonium chloride     

The effect of amputation of the epicotyl on the level of endogenous gibberellins in the roots of pea seedlings

The content of endogenous gibberellins was determined chromatographically in the roots 14–17 days old pea seedlings cultivated in water cultures in the dark. When the epicotyls are amputated from these plants, the content of endogenous gibberellins increases significantly within 6–12 hours after amputation as compared with the intact controls, then it falls off again considerably up to 24 hours after amputation. The initial increase of the gibberellin level in the roots can be explained by transport inhibition of the endogenous gibberellins from the root to the epicotyl, the later decrease of this level to be interpreted as inhibition of auxin transport from the epicotyl into the root. This is supported by the observation that spreading of a 0.5% paste with IAA over the epicotyl stump immediately after amputation prevents the mentioned decrease of the gibberellin level in the roots, whereas this decrease is intensified by using a paste with TIBA which inhibits the auxin transport. The results of this work support the possibility of direct gibberellin synthesis in the roots.     

Effect of Seed Treatment on Growth and Growth-substance Content of Dwarf French Beans (Phaseolus vulgaris)

The longer the seeds of dwarf French bean (Phaseolus vulgaris)were soaked in water before sowing, the smaller were the seedlingsthey produced. Soaking for 24 hours removed only little drymatter, but detectable amounts of a gibberellin and a betaine.The amounts of these two removed increased with increased timeof soaking. Long soaking of seeds decreased the amounts of gibberelinand auxin in the cotyledons, and of gibberellin, auxin, andbetaine in the primary leaves of seedlings. Aerating, cooling,or increasing the volume of water in which the seeds were soakedalso retarded the growth of the seedlings, but did not affectthe amount of growth substance removed from the seeds or containedin the cotyledons. The deleterious effect of soaking seeds wasnot alleviated by treating the seeds or seedlings with gibberellicacid or glycine betaine. Although larger seedlings were producedby heavy than by light seeds, their cotyledons and primary leavescontained similar concentrations of growth substances.     

Comparison of endogenous gibberellins and response to applied gibberellin of some dwarf and tall wheat cultivars

Summary A number of dwarf wheat cultivars of the Norin 10 type were compared with several tall forms. Applied gibberellic acid markedly stimulated the growth of seedlings of the tall cultivars but not the growth of dwarf seedlings. Several other gibberellins were also inactive when tested with one dwarf cultivar. De-embryonated grains of all cultivars formed -amylase in response to gibberellic acid. Gibberellic acid caused an increase in soluble carbohydrates in the leaves of the tall cultivars but not in those of the dwarfs.Germinating grains, light-grown seedlings and developing stems of the dwarf cultivars contained more endogenous gibberellin-like activity than those of tall cultivars. It is suggested that the dwarf cultivars have a block to the utilisation of gibberellin in the shoot.     

Osmoregulation in hypocotyls of etiolated mung bean seedlings with or without cotyledons in response to water-deficient stress

Effects of water-deficient stress and cotyledon excision on osmoregulation in hypocotyls of dark-grown mung bean seedlings were studied, and following results were obtained. Water-deficient stress inhibited hypocotyl elongation either in intact or decotylized seedlings. The inhibition was more conspicuous in decotylized seedlings than in intact ones. Water-deficient stress decreased osmotic potential in hypocotyls, while cotyledon excision increased it. The concentrations of soluble sugars, free amino acids and potassium ions in hypocotyls of intact or decotylized seedlings increased in response to water-deficient stress. Cotyledon excision reduced the concentration of soluble sugars and free amino acids, but it did not change the concentration of potassium ions, suggesting that a part of soluble sugars and free amino acids is transported from cotyledons. Unlike cotyledon excision, excision of the apex or roots had no influence on osmoregulation in response to water-deficient stress. Segments excised from hypocotyls had the ability to osmoregulate in response to water-deficient stress. Based on these results, the role of cotyledons in osmoregulation in response to water-deficient stress and quantitative relationships between osmotic potential and hypocotyl elongation in etiolated mung bean seedlings are discussed.     

Control of phenylalanine ammonia-lyase and cinnamic acid 4-hydroxylase in gherkin tissues

Blue light mediates a transient increase in the extractable activity of phenylalanine ammonia-lyase from both cotyledons and hypocotyls of etiolated gherkin seedlings, but concurrent changes in extractable cinnamic acid 4-hydroxylase activity only occur in cotyledons. Excision, followed by incubation in the dark, also results in stimulation of the lyase activity in both tissues, but the hydroxylase activity is only stimulated in cotyledons, again concurrently with the lyase. Stimulated levels of hydroxycinnamic acid esters are, however, only formed following light treatment, indicating the presence of another light-sensitive step in their biosynthesis. Treatment of gherkin tissues with 2-aminooxyacetic acid or α-aminooxy-β-phenylpropionic acid inhibits phenylalanine ammonia-lyase activity in situ, reduces the accumulation of hydroxycinnamic acid esters and presumably reduces the endogenous cinnamic acid pool. This treatment increases extractable lyase activity and delays its peak in activity. In cotyledons, these changes in the lyase are associated with concurrent and similar changes in extractable hydroxylase activity, whilst in hypocotyls the hydroxylase is relatively unaffacted. The increase in phenylalanine ammonia-lyase activity following excision of cotyledons and hypocotyls is prevented by cinnamic acid; in cotyledons the hydroxylase is similarly affected, but after a longer lag. Thus whilst cinnamic acid can modify the extractable activity of the lyase, it cannot itself mediate changes in the extractable activity of the hydroxylase.     

Hormonal regulation of germination and early seedling development in Acer pseudoplatanus (L.)

Summary Dormancy of intact sycamore (Acer pseudoplatanus) seeds was broken by chilling (5°C) for several weeks in moist conditions. Treatment of unchilled seeds with kinetin induced some germination, but gibberellin was ineffective. This stimulation by kinetin was not suppressed by the added presence of abscisic acid during incubation.The chilling requirement of intact seeds was eliminated by removal of the testa, and the naked embryos developed with no morphological abnormalities. During early growth of isolated embryos in the light, two distinct developmental processes were recognised. One involved initial elongation of the radicle accompanied by geotropic curvature and was stimulated by kinetin but not by gibberellin, while the other involved unrolling of the cotyledons, which was accelerated by gibberellin but much less by kinetin. Abscisic acid strongly suppressed both developmental processes when applied alone, inhibited cotyledon expansion in the presence of gibberellin, but failed to overcome the promotory effects of kinetin on radicle growth. Experiments with CCC indicated that under natural conditions the unrolling of the cotyledons is dependent upon endogenous gibberellin. Radicle growth of isolated embryos was unimpaired by incubation in the dark, but cotyledon expansion of water incubated embryos was poor, and although it was accelerated by gibberellin, the responses in all treatments were slower than in the corresponding light grown samples.It is suggested that endogenous cytokinins are primary factors in the initiation of radicle growth, while gibberellins are important in cotyledon expansion. Abscisic acid appears to have an inhibitory role in both processes, and the interactions of these regulators in the control of germination and development are discussed.     

Embryonal dormancy in apple seeds is controlled by free and conjugated gibberellin levels in the embryonic axis and cotyledons

Halińska, A., Sińska, I. and Lewak, St. 1987. Embryonal dormancy in apple seeds is controlled by free and conjugated gibberellin levels in the embryonic axis and cotyledons.
Free and conjugated gibberellins (GAs) A₄₊₇ and A₉ were determined in embryonic axes and in cotyledons of seeds of apple ( Malus domestica Borb., cv. Antonó wka) during breaking of dormancy under cold stratification. In both organs, the maximum level of free GA₄₊₇ was found at day 30 of stratification, but the concentration was 700 times higher in axes than in cotyledons. Comparison of changes in free and conjugated GA₄₊₇ levels during stratification allow us to suggest that the accumulation of free hormone in axes is, at the most, to 40% due to release from conjugates already present in the axis; that maximally 20% is derived from hydrolysis of cotyledonary conjugates translocated to axes; and that at least 40% originate from the novo biosynthesis of the hormone. Free and conjugated GA₉ levels were similarly altered in axes and in cotyledons, markedly increasing at the end of afterripening. Both release of the free hormone from conjugates and biosynthesis of GA₉, appeared to be involved in that increase; no translocation of free or bound GA₉, between axes and cotyledons was demonstrated.     

Organ-specificity and inducibility of patatin class I promoter from potato in transgenic arabidopsis plants

Patatin class I promoter (B33 promoter) is a tissue-specific potato (Solanum tuberosum L.) promoter expressing the patatin gene mainly in tubers. However, it can be induced in other organs by sucrose or light. We compared the activity of this promoter fused with the reporter gene during heterological expression in B33::GUS transgenic arabidopsis (Arabidopsis thaliana L.) plants and homological expression of the same DNA construct in potato. Promoter activity was estimated from quantification of β-glucuronidase (GUS) activity. It was shown that, during heterological expression in arabidopsis seedlings, B33 promoter manifested a tissue-specificity and inducibility, although in a different manner than during homological expression in potato. In noninduced arabidopsis seedlings, B33 promoter was most active in the roots, whereas, after induction with sucrose treatment, it became most active in cotyledons. 10 mM sucrose was sufficient for a manifold activation of B33 promoter in intact seedlings. The degree of B33 promoter induction by sucrose in arabidopsis seedlings was strictly organ-specific and increased in the following sequence: root < hypocotyl < cotyledons. 150–200 mM sucrose enhanced B33 promoter activity in cotyledons by 200 to 300 times, i.e., much stronger than in potato organs. Glucose and fructose were less efficient than sucrose. Phytohormones affecting tuber formation in potato (gibberellins, auxins, and cytokinins) did not affect significantly B33 promoter activity in arabidopsis. A lag period of approximately 6 h preceded sucrose-induced B33 promoter activation. This indicates that the patatin promoter is not the primary target for the sucrose signal. The quantitative examination of heterological expression of patatin class I promoter further clarifies its basic functional characteristics and permits a better prognosis of its behavior after transferring into other plant species.     

STUDIES ON THE SOLUBLE CARBOHYDRATES AND CARBOHYDRATE PRECURSORS IN GERMINATING SOYBEAN SEED

The total soluble carbohydrate fraction of the cotyledons and embryo axis of germinating soybean seedlings declined rapidly during the first 3 days of germination. This depletion began earlier in the embryo axis than in the cotyledon. The total carbohydrate content of the cotyledons of plants grown in light and plants grown in dark was approximately the same for the first 7 days of germination. Between day 9 and 13 the total carbohydrate content of the cotyledons of soybean seedlings grown in dark was higher than that of plants grown in light. The reducing sugar content of light-grown soybean cotyledons increased approximately 5-fold during the first 9 days of germination, whereas that of dark-grown soybean cotyledons increased more slowly during this interval. Reducing sugars in the embryo increased during the early stages of germination until they approximately equalled the total carbohydrate. Between day 4 and 13, oil was depleted more rapidly in the cotyledons of seedlings grown in light than those grown in the dark. The reserve carbohydrates of soybean embryos and cotyledons consisted primarily of low molecular weight oligosaccharides, particularly sucrose, stachyose, and raffinose. These compounds decreased rapidly during germination. The isocitritase activity in the cotyledons of germinating soybean seeds increased rapidly for the first 6 days of germination and then decreased for the next 7 days. The isocitritase activity of plants grown in the dark was higher than that of the plants grown in light at all stages of development, particularly between day 7 and 11.     

Specific Effects of Kinetin on the Uptake of Monovalent Cations by Sunflower Cotyledons

Kinetin promoted the uptake of K⁺ and Rb⁺ into detached sunflower cotyledons. This action was concomitant with an acceleration of growth. A slighter promotion of Li⁺ uptake was also noted, but there was no consistent influence on that of Na⁺. A small inhibitory effect on NH₄⁺₄ uptake was apparent when the latter was computed per average weight of sample during the course of incubation. Light also promoted the growth of the cotyledons, but depressed their capacity to absorb potassium. The action of kinetin on cotyledons removed from 5–7 day old seedlings was weaker than on those removed from 2–4 day old seedlings with regard to growth but stronger with regards to K⁺ uptake. When K⁺ uptake by cotyledons taken from 7-day old seedlings was followed with time the kinetin effect was already detectable within a few hours, but it became more pronounced after 10 hours' incubation. Kinetin did not accelerate growth or K⁺ uptake in hypo-cotyl segments. IAA, which was previously shown to promote these processes in hypocotyl segments, inhibited them in cotyledons. A working hypothesis is suggested according to which endogenous auxins and cytokinins regulate the absorption of K⁺ in shoot cells of the intact plant in a manner similar to that in which they act in excised tissues and in this way affect the distribution and redistribution of K⁺ in the shoot; and that they are among the factors which determine the selectivity of ion uptake in the intact plant.     

Gibberellins in Relation to Flowering and Stem Elongation in the Long Day Plant Silene armeria

Two long days induced some flowering and 4 or more long days caused 100% flowering in Silene armeria. On long days microscopically detectable flower primordia were first seen after 6 days, which is at least 1 day before the start of stem elongation. Both gibberellin A₃ and A₇ caused flowering on short days, but the results were variable and flowering was never 100%. Three different gibberellins were detected in Silene extracts. The pattern of gibberellins extracted from plants on short and long days was qualitatively the same, but on long days gibberellin content was up to 100% higher than on short days. Only small amounts of diffusible gibberellins were obtained from Silene shoot tips (including very young leaves) on short days. However, on long days the diffusible gibberellins increased by as much as 10-fold after 4 to 6 long days but then declined somewhat after 10 long days. The gibberellins extracted from the shoot tips at the completion of the diffusion period also increased under long days, although the increase was not as large as for the diffusible gibberellins. An A₅-like gibberellin present in extracts was not detected in diffusates.     

An enzyme to degrade lettuce endosperm cell walls. Appearance of a mannanase following phytochrome- and gibberellin-induced germination

Summary Lettuce seeds (Lactuca sativa L. cv. Grand Rapids) stimulated to germinate by gibberellin and red light produce large amounts of endo--mannanase. This enzyme increases markedly following radicle emergence and is capable of degrading mannose-containing polysaccharides, which are the major components of the endosperm cell wall. Non-germinated seeds contain little enzyme and under conditions where gibberellin- or red light-stimulated germination is prevented (eg. by abscisic acid or prolonged far red light) enzyme levels remain low. Cycloheximide inhibits the increase in enzyme levels when supplied to germinating seeds, but the enzyme once produced is stable in vivo in the presence of this inhibitor for at least 24h. The majority of the extractable mannanase activity is located in the endosperm and we propose that the function of this enzyme is to mobilise the endosperm cell wall polysaccharides as a nutrient source for the growing embryo.Abbreviations ABA abscisic acid - BA benzyladenine - GA gibberellic acid     

The role of endogenous gibberellins in the formation of α-amylase by aleurone layers of germinating barley caryopses

Using sensitive and selective immunological assays we have shown that in germinating caryopses of Hordeum vulgare L. cv. Himalaya, the level of gibberellin A₄ (GA₄) rises approximately 18-to 20-fold shortly (2–4 h) before -amylase activity increases. Gibberellin A₄ is the predominant immunoreactive gibberelin during these developmental stages and reaches a peak amount of approximately 9 pmol per caryopsis about 48 h after imbibition. Isolated aleurone layers produce GA₄ in the presence of an exogenous gibberellin, such as GA₁, which is not a biosynthetic precursor for GA₄. Experiments with inhibitors of gibberellin biosynthesis indicate that gibberellin synthesis is required in this tissue for the induction of -amylase. The inductive effect of exogenously applied GA₁ is indirect and appears to be mediated by GA₄. Embryos form predominantly GA₁; however, very little of this material is released by isolated embryos into the incubation medium. The results presented make it unlikely that the role of the embryo in the process of -amylase induction in aleurone layers is to provide gibberellins or gibberellin precursors.Abbreviations ABA abscisic acid - GA gibberellin - GA₃ gibberellic acid - RIA radioimmunoassay - TLC thin-layer chromatography     

Similarities and differences between the characteristics of gibberellin-binding protein and gibberellin 2-oxidases in adzuki bean (Vigna angularis) seedlings

Gibberellin-binding proteins (GBPs) were purified ca. 230,000 fold. The characteristics of adzuki GBP were examined and compared with those of a recombinant gibberellin 2-oxidase (rVaGA2oxA1) that was fused with glutathione S-transferase (GST). VaGA2oxA1 was most abundantly expressed in etiolated adzuki bean seedlings, and VaGA2oxA1 and GBPs from adzuki bean seedlings showed gibberellin-binding activity when incubated with 2-oxoglutarate and Co2+. Both rVaGA2oxA1 and partially purified GBPs from adzuki bean seedlings showed very similar selectivity to gibberellins in binding assays, where biologically active gibberellins such as GA4, GA3, GA7, and GA1 showed higher binding affinity than biologically inactive gibberellins such as GA8, GA34, and 3-epi-GA4. The polyclonal antibody raised against rVaGA2oxA1 cross-reacted with all rVaGA2oxs (rVaGA2oxA1, rVaGA2oxA2, rVaGA2oxB1, rVaGA2oxB2, and rVaGA2oxB3) whose cDNAs were cloned from adzuki bean seedlings. Treated with the antibody, the recombinants that originally showed gibberellin-binding activity lost both binding activity and enzymatic activity. In contrast to the recombinants, the gibberellin-binding activity of GBPs from adzuki bean seedlings was hardly affected by the antibody treatment. The GBPs showed very weak gibberellin 2-oxidase-like activity, and it was not affected by the antibody treatment either. These observations suggest that a major component that showed GA-binding activity was apparently different from any gibberellin 2-oxidase cloned from the seedlings.