An endogenous RNA-synthesis-promoting oligopeptide from Cucurbita pepto var. patissonina

The effect of CPPTI (Cucurbita pepo patissonina trypsin inhibitor) on the growth of Cucurbita pepo var patissonina (White bush) was examined. Plants treated with CPPTI grew faster than untreated plants and similarly to those treated with gibberellic acid. Isolated cell nuclei from plants treated with CPPTI showed that of the three DNA-dependent RNA polymerases assayed, RNA polymerase I (EC2.7.7.6) activity was preferentially elevated.Abbreviations CPPTI Cucurbita pepo patissonina trypsin inhibitor - GA₃ gibberellic acid - IAA indole-3-acetic acid - kDa kilodalton - RP I DNA-dependent RNA polymerase I     

Effect of gibberellin a(3) on the endoplasmic reticulum and on the formation of glyoxysomes in the endosperm of germinating castor bean

Seedlings of castor bean (Ricinus communis cv. Hale) were exposed to a range of concentrations of gibberellin A₃ (GA₃). Treatments for 20 hours with GA₃ concentrations of 0.5 μM or higher resulted in increased levels of NADH-cytochrome c reductase, phosphorylcholine glyceride transferase, and malate synthase in endoplasmic reticulum (ER) isolated from endosperm on linear sucrose gradients. GA₃ treatment also resulted in increased RNA associated with ER. Malate synthase and catalase in crude homogenates were enhanced by 1 to 100 μM GA₃ concentrations. Isocitrate lyase, citrate synthase, malate synthase, catalase, and glycolate oxidase in isolated glyoxysomes were enhanced by 60, 20, 18, 40, and 28%, respectively, over controls. Treatment with abscisic acid led to decreased levels of glyoxysomal enzymes and reduced glyoxysomal protein. The effect of GA₃ and abscisic acid on the specific activities of glyoxysomes of different densities suggests that GA₃ influences enzyme levels and glyoxysome assembly.     

Modulation by gibberellic acid and adenosine-3′,5′-cyclic monophosphate of starch hydrolysing activity of cowpea seedlings

In cowpea seedlings starch hydrolysing activity increases 35–50 fold on germination for 4 days. This increase in enzyme activity was inhibited by the in vivo addition of 1% glucose but this inhibition was completely overcome by the addition of gibberellic acid (GA₃) (10^?5 M) and adenosine-3′,5′-cyclic monophosphate (cAMP) (10^?5 M). At 5% glucose, GA₃ and cAMP were only partially effective. Structural analogues of cAMP failed to relieve the inhibitory effect of glucose. The inhibition by glucose is not direct but RNA and protein synthesis may be involved. Glucose appears to reduce the internal pool of cAMP which causes inhibition of RNA synthesis and decrease in starch hydrolysing activity. Exogenous application of cAMP may replenish the endogenous pool of cyclic nucleotide and thus overcome inhibition of RNA synthesis and enzyme activity.     

Ribonucleic Acid Synthesis by Cucumber Chromatin: Developmental and Hormone-induced Changes

When intact etiolated 2-day cucumber (Cucumis sativus) embryos were treated with indoleacetic acid (IAA), gibberellin A₇ (GA₇), or kinetin, chromatin derived from the embryonic axes exhibited an increased capacity to support RNA synthesis in either the presence or the absence of bacterial RNA polymerase. An IAA effect on cucumber RNA polymerase activity was evident after 4 hours of hormone treatment; the IAA effect on DNA template activity (bacterial RNA polymerase added) occurred after longer treatments (12 hours). GA₇ also promoted template activity, but again only after a prior stimulation of endogenous chromatin activity. After 12 hours of kinetin treatment, both endogenous chromatin and DNA template activities were substantially above control values, but longer kinetin treatments caused these activities to decline in magnitude. When chromatin was prepared from hypocotyl segments that were floated on a GA₇ solution, a GA-induced increase in endogenous chromatin activity occurred, but only if cotyledon tissue was left attached to the segments during the period of hormone treatment.     

Enzymic glucosylation of gibberellins

Starting from the well-known conversion of exogenously applied free gibberellic acid (GA₃) to its 3(O)-glucoside by intact immature fruits of runner beans (Phaseolus coccineus L.), a protein fraction has been prepared from this plant material possessing glucosylating activity towards GAs. This glucosyltransferase is located in the pericarp only and utilizes preferably UDP-glucose as a sugar donor. The product formed enzymically from GA₃ and UDP-glucose could be identified by derivatization and comparison with the authentic compound to be GA₃-3(O)-glucoside. Among 15 native or chemically modified GAs, the enzyme glucosylates only GA₃ and to a lower extent GA₇ and GA₃₀, indicating a high enzyme specificity with regard to the A ring of gibberellins. The physiological significance of the enzymic GA₃-3(O)-glucoside formation inPhaseolus coccineus is not clear, since this glucoside is not known to be endogenous in this plant. The enzyme preparation did not glucosylate substances of phenolic structure, such as hydroquinone, aesculetin, and quercetin. Glucosylation of GA₃ was achieved also by enzyme preparations fromVigna sinensis and from cell suspension cultures ofDigitalis purpurea. A number of other plant materials showed no activity.     

Control of alpha-amylase mRNA accumulation by gibberellic Acid and calcium in barley aleurone layers

Pulse-labeling of barley (Hordeum vulgare L. cv Himalaya) aleurone layers incubated for 13 hours in 2.5 micromolar gibberellic acid (GA₃) with or without 5 millimolar CaCl₂ shows that α-amylase isozymes 3 and 4 are not synthesized in vivo in the absence of Ca²⁺. A cDNA clone for α-amylase was isolated and used to measure α-amylase mRNA levels in aleurone layers incubated in the presence and absence of Ca²⁺. No difference was observed in α-amylase mRNA levels between layers incubated for 12 hours in 2.5 micromolar GA₃ with 5 millimolar CaCl₂ and layers incubated in GA₃ alone. RNA isolated from layers incubated for 12 hours in GA₃ with and without Ca²⁺ was translated in vitro and was found to produce the same complement of translation products regardless of the presence of Ca²⁺ in the incubation medium. Immunoprecipitation of translation products showed that the RNA for α-amylase synthesized in Ca²⁺-deprived aleurone layers was translatable. Ca²⁺ is required for the synthesis of α-amylase isozymes 3 and 4 at a step after mRNA accumulation and processing.     

An early response to gibberellic Acid not requiring protein synthesis

Cell-free extracts from gibberellic acid-treated barley (Hordeum vulgare L. cv. Himalaya) aleurone layers show phosphorylcholine glyceride transferase activity greater than that from control layers. The increase in activity is not prevented by a mixture of amino acid analogs nor by cordycepin under conditions in which it is demonstrated that the analogs and the cordycepin are entering the cells in effective concentrations. We conclude therefore that the GA₃-dependent increase in phosphorylcholine glyceride transferase activity (which occurs within the first 4 hours of GA₃ treatment) does not require RNA synthesis or protein synthesis.     

Ethylene-induced Leaf Abscission Is Promoted by Gibberellic Acid

Gibberellic acid (GA₃) promoted leaf abscission from cotton (Gossypium hirsutum L.) plants exposed to ethylene. With mature plants, only the rate of abscission was increased, but when vegetative plants were exposed to ethylene for 4 days or less, the amount of abscission was increased markedly. Promotion of abscission occurred at near saturating ethylene levels (10 μl/liter), over a wide range of GA₃ concentrations, and with both GA₃ and GA₇.     

Effects of gibberellic Acid and gold light on germination, enzyme activities, and amino Acid pool size in a dwarf strain of watermelon

Gibberellic acid (GA₃) promotes and continuous gold light inhibits germination of seeds of a dwarf strain (WB-2) of watermelon [Citrullus lanatus (Thunb.) Matsu. and Nakai]. Osmotic inhibition of germination with mannitol in light-grown seeds of WB-2 was only slightly reversed by GA₃ at the concentrations used, whereas, GA₃ substantially relieved osmotic inhibition in dark-grown seeds.

The effects of GA₃ and gold light on development of catalase and invertase activities and on levels of free amino acids in germinating seeds of WB-2 were examined. Light depressed development of catalase and invertase activity. Levels of free amino acids increased more slowly in embryonic axes of light- than dark-incubated seeds, but in cotyledons higher levels of amino acids were maintained in light-grown seeds. GA₃ accelerated the development of catalase activity in whole embryos and invertase activity in embryonic axes, but did not significantly affect invertase activity in cotyledons during germination. GA₃ had little effect on amino acid pools in cotyledons and embryonic axes.

     

Endogenous Gibberellins from Sporophytes of Two Tree Ferns, Cibotium glaucum and Dicksonia antarctica

Gibberellin A₁ (GA₁), 3-epi-GA₁, GA₄, GA₉, 11α-hydroxyGA₁₂, 12α-hydroxyGA₁₂, GA₁₅, GA₁₇, GA₁₉, GA₂₀, GA₂₅, GA₃₇, GA₄₀, GA₅₈, GA₆₉, GA₇₀, and GA₇₁ have been identified from Kovats retention indices and full scan mass spectra by capillary GC-MS analyses of purified extracts from sporophytes of the tree fern, Cibotium glaucum. Abscisic acid, dihydrophaseic acid, an epimer of 4′-dihydrophaseic acid, and the epimeric ent-6α, 7α, 16α, 17-(OH)₄ and ent-6α, 7α, 16β, 17-(OH)₄ derivatives of ent16, 17-dihydrokaurenoic acid, in addition to the epimeric 16α, 17- and 16β, 17-dihydroxy-16, 17-dihydro derivatives of GA₁₂, were also identified in extracts of C. glaucum. An oxodihydrophaseic acid and a hydroxydihydrophaseic acid were also detected. In extracts of sporophytes of Dicksonia antarctica, GA₄, GA₉, 12α- and 12β-hydroxyGA₁₂, GA₁₅, GA₂₅, and GA₃₇ were identified by the same criteria, as well as abscisic acid, phaseic acid, 8′-hydroxymethylabscisic acid and dihydrophaseic acid. This is the first time that GA₄₀ has been identified in a higher plant; it is also the first report of the natural occurrence of the two gibberellins, 11α- and 12β-hydroxyGA₁₂. The total gibberellin (GA) content in C. glaucum (tall) was at least one order of magnitude greater than that of D. antarctica (dwarf) based on total ion current response in GC-MS and bioassay data. Abscisic acid was a major component of D. antarctica and the oxodihydrophaseic acid was a major component of C. glaucum.     

Stimulation of RNA synthesis in cowpea (Vigna sinensis) seedlings by gibberellic acid and adenosine 3′,5′-cyclic monophosphate

There is about 50% stimulation in the incorporation of [³H]uridine into total RNA of cowpea following the application of gibberellic acid (GA₃) and adenosine 3′,5′-cyclic monophosphate (cyclic AMP). Cyclic AMP is very specific in its action. Co-fractionation of ³H- and ¹⁴C-labelled RNA on acrylamide-agarose gels reveal a control by GA₃ and cyclic AMP predominantly on its polydisperse fraction. Both GA₃ and cyclic AMP appear to act through a similar mechanism.     

Hormonal regulation of the development of protease and carboxypeptidase activities in barley aleurone layers

Carboxypeptidase and protease activities of hormone-treated barley (Hordeum vulgare cv Himalaya) aleurone layers were investigated using the substrates N-carbobenzoxy-Ala-Phe and hemoglobin. A differential effect of gibberellic acid (GA₃) on these activities was observed. The carboxypeptidase activity develops in the aleurone layers during imbibition without the addition of hormone, while the release of this enzyme to the incubation medium is enhanced by GA₃. In contrast, GA₃ is required for both the production of protease activity in the aleurone layer and its secretion. The time course for development of protease activity in response to GA₃ is similar to that observed for α-amylase. Treating aleurone layers with both GA₃ and abscisic acid prevents all the GA₃ effects described above. Carboxypeptidase activity is maximal between pH 5 and 6, and is inhibited by diisopropylfluorophosphate and p-hydroxymercuribenzoate. We have observed three protease activities against hemoglobin which differ in charge but are all 37 kilodaltons in size on sodium dodecyl sulfate polyacrylamide gels. The activity of the proteases can be inhibited by sulfhydryl protease inhibitors, such as bromate and leupeptin, yet is enhanced by 2-fold with 2-mercaptoethanol. In addition, these enzymes appear to be active against the wheat and barley storage proteins, gliadin and hordein, respectively. On the basis of these characteristics and the time course of GA₃ response, it is concluded that the proteases represent the GA₃-induced, de novo synthesized proteases that are mainly responsible for the degradation of endosperm storage proteins.     

Developmental and Hormonal Regulation of Gibberellin Biosynthesis and Catabolism in Pea Fruit

In pea (Pisum sativum), normal fruit growth requires the presence of the seeds. The coordination of growth between the seed and ovary tissues involves phytohormones; however, the specific mechanisms remain speculative. This study further explores the roles of the gibberellin (GA) biosynthesis and catabolism genes during pollination and fruit development and in seed and auxin regulation of pericarp growth. Pollination and fertilization events not only increase pericarp PsGA3ox1 message levels (codes for GA 3-oxidase that converts GA₂₀ to bioactive GA₁) but also reduce pericarp PsGA2ox1 mRNA levels (codes for GA 2-oxidase that mainly catabolizes GA₂₀ to GA₂₉), suggesting a concerted regulation to increase levels of bioactive GA₁ following these events. 4-Chloroindole-3-acetic acid (4-Cl-IAA) was found to mimic the seeds in the stimulation of PsGA3ox1 and the repression of PsGA2ox1 mRNA levels as well as the stimulation of PsGA2ox2 mRNA levels (codes for GA 2-oxidase that mainly catabolizes GA₁ to GA₈) in pericarp at 2 to 3 d after anthesis, while the other endogenous pea auxin, IAA, did not. This GA gene expression profile suggests that both seeds and 4-Cl-IAA can stimulate the production, as well as modulate the half-life, of bioactive GA₁, leading to initial fruit set and subsequent growth and development of the ovary. Consistent with these gene expression profiles, deseeded pericarps converted [¹⁴C]GA₁₂ to [¹⁴C]GA₁ only if treated with 4-Cl-IAA. These data further support the hypothesis that 4-Cl-IAA produced in the seeds is transported to the pericarp, where it differentially regulates the expression of pericarp GA biosynthesis and catabolism genes to modulate the level of bioactive GA₁ required for initial fruit set and growth.     

Enzyme formation and release by isolated barley aleurone layers

Aleurone layers, with testa attached, were prepared from degermed, decorticated barley with the aid of a fungal enzyme preparation. The preparations appeared intact under the scanning electron microscope. By using antibiotics only in an early stage preparations were obtained uncontaminated by micro-organisms and which, when incubated under optimal conditions with gibberellic acid, GA₃, produced near-maximal amounts of α-amylase. The enzyme accumulated in the tissue before it was released into the incubation medium. Daily replacement of the incubation medium, containing GA₃, depressed the quantity of α-amylase produced. α-Amylase was also produced in response to gibberellins GA₁, GA₄ and GA₇ and, to a much lesser extent, helminthosporol and helminthosporic acid. A range of other substances, reported elsewhere to induce α-amylase formation, failed to do so in these trials. At some concentrations, glutamine marginally enhanced the quantity of enzyme formed during prolonged incubations. It is confirmed that α-glucosidase occurs in the aleurone layer and embryo of ungerminated barley, and increases in amount during germination. GA₃ is shown to enhance this increase. When embryos arc burnt, to prevent gibberellin formation, no rise in α-glucosidase levels occurs unless GA₃ is supplied to the grains. As the activity of α-glucosidase and other enzymes have been determined as ‘α-amylase’ by some assay methods, their alterations in activity in response to GA₃ necessitates a re-evaluation of the evidence for de novo) synthesis of α-amylase in aleurone tissue.     

Control of protein synthesis in barley aleurone layers by the plant hormones gibberellic acid and abscisic acid

The patterns of protein synthesis in barley aleurone layers treated with gibberellic acid (GA₃) and abscisic acid (ABA) are compared with the patterns observed in wheat germ in vitro translation assays directed by RNA isolated from similarly treated layers. When used alone, GA₃ and ABA both induce the formation of new translatable mRNAs and cause new proteins to be synthesized. The effects of GA₃ are more dramatic than those of ABA. In GA₃-treated tissues, overall protein synthesis is redirected to produce large quantities of α-amylase and a few other GA₃-induced proteins, while other protein synthesis is reduced or stopped. Large amounts of new translatable mRNA for α-amylase are also induced such that the dominant in vitro translation product is α-amylase. These changes are blocked by the simultaneous addition of ABA to the tissue. In GA₃ plus ABA-treated layers, few changes in protein synthesis in vivo are observed when compared to protein synthesis in untreated tissue, although the induction of mRNA for α-amylase and the other GA₃-induced mRNAs does occur. This indicates that ABA does not interfere with GA₃ induction of translatable mRNAs but prevents the translation of these mRNAs in vivo. Thus ABA and potentially GA₃ regulate the translation of proteins in vivo in barley aleurone layers.     

The Role of Auxin and Gibberellin in Controlling Lignin Formation in Primary Phloem Fibers and in Xylem of Coleus blumei Stems

The hypothesis that auxin (IAA) and gibberellic acid (GA₃) control the formation of lignin is confirmed for the primary phloem fibers and for the secondary xylem in the stem of Coleus blumel Benth. Indoleacetic acid alone, or a combination of high IAA/low GA₃ (w/w), induced short phloem fibers with thick secondary walls, that contained lignin rich in syringyl units (high ratio of syringyl/guaiacyl). On the other hand, a combination of high GA₃/low IAA (w/w), which promoted the differentiation of long phloem fibers with thin walls, decreased the relative content of the syringyl units (low syringyl/guaiacyl ratio). In the secondary xylem, these hormonal treatments yielded only slight changes in the noncondensed monomeric guaiacyl units, confirming the relative stability of the guaiacyl lignification pattern in this tissue. In the xylem, indoleacetic acid alone, or a combination of high IAA/low GA₃ induced lignin poor in syringyl units (low syringyl/guaiacyl ratio). A combination of high GA₃/low IAA promoted a relatively slight increase in syringyl yield, indicating greater responsiveness of the syringyl lignification pattern to growth regulators. The possible functional and technological significance of our results is discussed.