Regulation of cell wall synthesis in Avena stem segments by gibberellic Acid

Gibberellic acid induces (a) increased elongation of Avena sativa stem segments, (b) increased formation of cell wall material, measured on the basis of dry weight, and (c) increased incorporation of ¹⁴C-glucose into all fractions of the cell wall material. This increased incorporation of radioactivity correlates well with increased formation of cell wall material and shows a time-course pattern similar to the time course of the elongation response. Approximately one hour after the application of gibberellic acid, the rates both of growth and of incorporation of radioactivity accelerate to about 2-fold over the control rate. Gibberellic acid does not stimulate the incorporation of labeled glucose into the cell wall material simply by increasing the rate of uptake of glucose by internodal cells. The stimulation of the incorporation of ¹⁴C-glucose into cell wall material, which reflects the stimulation of cell wall synthesis, seems to be an important and relatively early effect of gibberellic acid in this system and probably contributes significantly to the elongation response elicited by the hormone.     

A Correlation between a Ribonucleic Acid Fraction Selectively Labeled in the Presence of Gibberellic Acid and Amylase Synthesis in Barley Aleurone Layers

The effects of gibberellic acid on the incorporation of radio-active uridine and adenosine into RNA of barley aleurone layers were investigated using a double labeling method combined with acrylamide gel electrophoresis. After 16 hours of incubation, gibberellic acid stimulated the incorporation of label into all species of RNA, but the effects were very small (0-10%) for ribosomal and transfer RNA and comparatively large (up to 300%) for RNA sedimenting between 5S and 14S. This result was obtained for both isolated aleurone layers and for layers still attached to the endosperm. A similar but less marked pattern occurred in layers incubated for 8 hours, but the effect was not observed after 4 hours. The gibberellic acid-enhanced RNA labeling was not due to micro-organisms. The following evidence was obtained for an association between the gibberellic acid-enhanced RNA synthesis and α-amylase synthesis: (a) synthesis of α-amylase took place in parallel with incorporation of label into gibberellic acid-RNA; (b) actinomycin D inhibited amylase synthesis and gibberellic acid-RNA by similar percentages; (c) 5-fluorouracil halved incorporation of label into ribosomal RNA but had no effect on amylase synthesis and gibberellic acid-RNA; and (d) abscisic acid had little effect on synthesis of RNA in the absence of gibberellic acid, but when it was included with gibberellic acid the synthesis of both enzyme and gibberellic acid-RNA was eliminated. We conclude that large changes in the synthesis of the major RNA species are not necessary for α-amylase synthesis to occur but that α-amylase synthesis does not occur without the production of gibberrellic acid-RNA. Gibberellic acid-RNA is probably less than 1% of the total tissue RNA, is polydisperse on acrylamide gels, and could be messenger species for α-amylase and other hydrolytic enzymes whose synthesis is under gibberellic acid control.     

Growth Regulators Have Rapid Effects on Photosynthate Unloading from Seed Coats of Phaseolus vulgaris L

Of nine plant growth regulators (indoleacetic acid, 1-naphthalene acetic acid, gibberellic acid, giberellin 4/7, 6-benzylaminopurine, 6-furfurylaminopurine, abscisic acid, and 1-aminocyclopropane carboxylic acid) tested, only 6-benzylaminopurine and abscisic acid affected ¹⁴C-photosynthate unloading from excised seed coats of Phaseolus vulgaris L. Unloading, in the presence of KCl, was stimulated by 25 to 40%. Stimulation occurred immediately for 6-benzylaminopurine and for abscisic acid within 10 to 12 minutes of application.     

Obligate methylotroph <Emphasis Type="Italic">Methylobacillus arboreus</Emphasis> Iva<Superscript>T</Superscript> synthesizes a plant hormone,gibberellic acid GA<Subscript>3</Subscript>

An obligate methylotroph Methylobacillus arboreus Iva^Т (VKM B-2590^Т, CCUG 59684^T, DSM 23628T) is the first known aerobic methylotrophic bacterium capable of synthesis of the bioactive gibberellic acid GA₃. Primary separation and identification of gibberellic acid from the culture liquid of methanol-grown culture were carried out using thin-layer chromatography and high-performance liquid chromatography. The concentration and structure of the gibberellic acid GA₃ were determined by liquid chromatography?mass spectrometry (LC/MS). Biological activity of the isolated compound was confirmed by tests on sprouts of lettuce (Laсtuca sativa L.).     

Effects of gibberellic Acid and sucrose on the growth of oat (Avena) stem segments

Gibberellic acid induced growth in Avena (oat) stem segments within 35 minutes after hormone application. The total elongation elicited by gibberellic acid was greater than 15 times the control growth. The sensitivity of the segments to low concentrations of gibberellic acid (1 pmole) and the specificity of the segments to the gibberellin class of hormones suggest that oat stem segments would be a valuable tool for gibberellin bioassays. Both gibberellic acid-induced growth and control growth are temperature-dependent and showed a Q₁₀ of two or greater. Although the most apparent effect of gibberellic acid was to promote the uptake of water into the internode, the hormone also promoted transport of endogenous substrate and the uptake of exogenous substrate into the growing region. The growth promotion was accomplished without an apparent increase in osmotic pressure.     

Phosphoinositides in barley aleurone layers and gibberellic Acid-induced changes in metabolism

Phospholipids of barley (Hordeum vulgare L. cv Himalaya) aleurone layers were labeled with myo-[2-³H]inositol or [³²Pi], extracted, and analyzed by physical (chromatography) and chemical (deacylation) techniques. Three phospholipids were found to incorporate both myo-[2-³H]inositol and [³²Pi]—phosphatidylinositol, phosphatidylinositol-monophosphate, and phosphatidylinositol-bisphosphate. Stimulation of [³H]inositol prelabeled aleurone layers with GA₃ showed enhanced incorporation of label into phosphatidylinositol within 30 seconds and subsequent rapid breakdown. Stimulation of phosphatidylinositol labeling observed in these studies is the earliest response of aleurone cells to gibberellic acid reported.     

Effect of Abscisic Acid on the Activities of Photosynthetic Enzymes and 14CO2 Fixation Products in Leaves of Pennisetum typhoides Seedlings

Abscisic acid inhibited the rate of ¹⁴CO₂ fixation in leaves of Pennisetum typhoides (Burm. f.) Stapf & Hubbard seedlings, but increased the activities of phosphoenol-pyruvate-carboxylase and malic enzyme. The leaves of the seedlings grown in the presence of abscisic acid incorporated, in comparison to the control, more radioactivity in the fraction of organic acids, but less radioactivity was recorded in the amino acid fraction. On the other hand, gibberellic acid which also inhibits photosynthetic ¹⁴CO₂ assimilation and decreases the activities of photosynthetic enzymes, favours greater incorporation in alanine, and reduces that in malate. It is deduced that bio-regulants can greatly influence the flow of ¹⁴C into individual photosynthetic products. As in growth, abscisic and gibberellic acids in combination tended to antagonize each other in their effects on enzyme activity as well as in incorporation of ¹⁴CO₂ into photosynthetic products.     

Cyclization and hydroxylation stereochemistry in the biosynthesis of gibberellic acid

In Gibberella fujikuroi cultures, ent-[3β-³H,17-¹⁴C]kaurene is converted to gibberellic acid with retention of the tritium label at the 3α-position. This evidence for the stereochemistry of 3-hydroxylation also permits the stereochemistry of the ‘proton-initiated’ cyclization step in gibberellic acid biosynthesis to be deduced.     

Movement of Kinetin and Gibberellic Acid in Leaf Petioles during Water Stress-induced Abscission in Cotton

Movement of [¹⁴C]kinetin and [¹⁴C]gibberellic acid was examined in cotton (Gossypium hirsutum L.) cotyledonary petiole sections independent of label uptake or exit from the tissue. Sections 20 millimeters in length were taken from well watered, stressed, and poststressed plants. Transport capacity was determined using a pulse-chase technique. Movement of both kinetin and gibberellic acid was found to be nonpolar with a velocity of 1 millimeter per hour or less, suggesting passive diffusion. Neither water stress nor anaerobic conditions during transport of labeled material affected the transport capacity of the petioles.     

On the nature of the physiological responses of Avena stem segments to gibberellic Acid treatment

Gibberellic acid was found to cause elongation in Avena sativa (oat) stem segments whether it was applied continuously or as a short pulse. The shorter the pulse time became, the higher was the gibberellic acid concentration needed to cause elongation; the segmental growth apparently depends upon the amount of gibberellic acid taken up by the segments. Avena segments showed a decreased growth response to gibberellic acid if the treatments were initiated at increasingly later times after excision from the plant. This decreased responsiveness to gibberellic acid was inhibited by low temperature (0-4 C), but accelerated by anaerobiosis. On the other hand, growth stimulation by a gibberellic acid pulse at the start of incubation was not altered by cold treatment but was nullified by a nitrogen atmosphere. Both the readiness of the segments for growth stimulation by gibberellic acid and its action in promoting growth clearly involve temperature-dependent, aerobic metabolism.     

An autoradiographic study of the effect of the plant hormone abscisic acid on nucleic acid and protein metabolism

Summary Abscisic acid maintains embryos in a state of dormancy and inhibits the incorporation of H³uridine and H³thymidine but not the incorporation of H³leucine. Ribosomes present in imbibed but dormant embryos do not become associated into polysomes until actual germination of the embryos. Protein synthesis still occurs in embryos when RNA synthesis is inhibited and therefore stable m-RNA must be present in dormant embryos. It is concluded that abscisic acid maintains dormancy by inhibiting the production of specific types of m-RNA, and therefore the formation of specific proteins. The activity of abscisic acid is antagonistic to the effect of gibberellic acid in dormancy.     

Trehalose Toxicity in Cuscuta reflexa: Cell Wall Synthesis Is Inhibited upon Trehalose Feeding

α,α-Trehalose induced a rapid blackening of the terminal 2.5-centimeter region of excised Cuscuta reflexa Roxb. vine. The incorporation of radioactivity from [¹⁴C]glucose into alkali-insoluble fraction of shoot tip was markedly inhibited by 12 hours of trehalose feeding to an excised vine. This inhibition was confined to the apical segment of the vine in which cell elongation occurred. The rate of blackening of shoot tip explants was hastened by the addition of gibberellic acid A₃, which promoted elongation growth of isolated Cuscuta shoot tips. The symptom of trehalose toxicity was duplicated by 2-deoxyglucose, which has been shown to be a potent inhibitor of cell wall synthesis in yeast. The observations suggest that trehalose interferes with the synthesis of cell wall polysaccharides, the chief component of which was presumed to be cellulose.     

Regulation of Apoplastic pH in Source Leaves of Vicia faba by Gibberellic Acid

Leaf discs of broad bean (Vicia faba L.), peeled on the spongy mesophyll side, rapidly altered the pH of the surrounding medium (apoplast). Using pH indicator paper appressed against the leaf, immediately after peeling, initial apoplastic pH was estimated to be 4.5. Changes in the apoplastic pH were measured with a microelectrode placed into a 100-microliter drop of an unbuffered solution (2 millimolar KCl, 0.5 millimolar CaCl₂, and 200 millimolar mannitol) on the peeled surface. Discs acidified the medium until the pH stabilized at about 5.0 (about 10 minutes). Acidification was inhibited by 50 micromolar sodium vanadate, an inhibitor of the plasmalemma H⁺-ATPase and attenuated by omitting the osmoticum or potassium ions from the medium. Fusicoccin (10 micromolar) greatly enhanced the rate of acidification. The presence of 0.1 to 1 micromolar gibberellic acid resulted in a slower rate of medium acidification. Gibberellic acid appeared to modulate the activity of the H⁺-translocating ATPase located at the plasma membrane of the mesophyll cells.     

The relationship between the incorporation of 32P into phosphatidic acid and phosphatidylinositol in rat parotid acinar cells

Muscarinic and α-adrenergic stimulation of rat parotid acinar cells increases the turnover of phosphatidylinositol and phosphatidic acid. It is thought that this is initiated by hydrolysis of phosphatidylinositol, which would predict an increase in ³²P incorporation into phosphatidic acid before phosphatidylinositol. We have demonstrated an increase in ³²P incorporation into the former within 1 minute and into the latter by 2 minutes. The initial rapid rate of ³²P incorporation into phosphatidic acid slows, and the ³²P content reaches a steady state after 15 minutes. During the first 2 minutes after the addition of atropine to carbamylcholine stimulated cells, ³²P is lost from phosphatidic acid, and an equal amount is gained by phosphatidylinositol, after which ³²P incorporation equals that of the control. In cells prelabelled with ³²P, carbamylcholine, in the presence of oligomycin stimulated the loss of ³²P from phosphatidylinositol but had no effect on phosphatidic acid.     

Amino Acid incorporation in developing fucus embryos

The incorporation of ¹⁴C-leucine and ¹⁴C-amino acid mixture into protein in unfertilized eggs and developing embryos of the brown alga Fucus vesiculosus L. was studied. Bacterial contamination was initially a problem, but it was found that the addition of 40 μg/ml chloramphenicol to the incubation medium would inhibit bacterial protein synthesis without affecting early development of the Fucus embryos. The kinetics of uptake and incorporation of ¹⁴C-leucine into the trichloroacetic acid-soluble and -insoluble fractions indicated that the exogenous precursor did not equilibrate with the main soluble leucine pool before incorporation into protein. Uptake and incorporation of leucine by embryos 90 to 175 minutes old were proportional to exogenous leucine concentration over the range 5 × 10⁻⁶ m to 5 × 10⁻³ m. Unfertilized eggs will incorporate ¹⁴C-leucine into protein. The rate of this incorporation increases dramatically in newly fertilized eggs with a maximum rate at 3.5 hours, a period of cell wall formation and increasing metabolic rates. Thereafter, the rate of incorporation declines until approximately 15 to 17 hours when it increases again concurrently with the onset of rhizoid initiation and cell division.     

Density labeling of proteins with 13C-labeled amino acids: no accumulation of an inactive alpha-amylase precursor in barley aleurone cells.

Gibberellic acid enhances α-amylase (EC 3.2.1.1) production in isolated barley aleurone layers after a lag period of 4 to 8 h, and most of the enzyme is produced after 12 h of hormone treatment. Amino acids necessary for protein synthesis in barley aleurone layers are derived from the degradation of storage proteins in this tissue. Since bromate is an inhibitor of barley protease, in the presence of bromate the production of α-amylase in aleurone layers becomes dependent on exogenous amino acids. We have incubated aleurone layers with bromate plus ¹³C-labeled amino acids and [³H]leucine from 0 to 24, 0 to 12, and 12 to 24 h after the application of gibberellic acid. The chemical quantity of [³H]leucine was negligible in comparison to that of ¹³C-labeled amino acids. Therefore, any density shift of proteins observed must be due to the incorporation of ¹³C-labeled amino acids. The density shift of α-amylase and that of newly synthesized proteins (radioactivity profile) were determined by isopycnic centrifugation in CsCl density gradients. The density shift of α-amylase isolated from aleurone layers incubated with ¹³C-labeled amino acids from 12 to 24 h after the addition of hormone was much larger than that of α-amylase isolated from aleurone layers incubated with ¹³C-labeled amino acids from 0 to 12 h of hormone treatment. By comparing the density shift of α-amylase with that of newly synthesized proteins, it is apparent that essentially all the amylase molecules are de novo synthesized. We can conclude that there is little or no accumulation of an inactive α-amylase precursor in barley aleurone cells between the time of the application of gibberellic acid and the time of the rapid increase in α-amylase activity.     

Auxin-induced Growth of Helianthus Tuber Tissue V. Role of DNA synthesis in hormonal control of cell growth

The role of DNA synthesis in hormonal control of cell growth was studied, using tissue slices excised from cold- stored Helianthus tubers. Metabolic inhibitors of DNA synthesis such as 5-fluorodeoxyuridine and phenethyl alcohol inhibited the effect of auxin and gibberellie acid on expansion growth, the latter being given during the aging period. The inhibitory effect of 5-fluorodeoxyuridine on expansion growth was alleviated by the simultaneous addition of thymidine but not by that of uridine. Incorporation of ¹⁴C-thymidine into DNA occurred during the aging period and gibberellic acid stimulated the incorporation of ¹⁴C-thymidine into DNA when given during the aging period. Mitomycin C and 2-(chloroethyl)-trimethyl-ammonium chloride (a growth retardant) inhibited incorporation of labelled thymidine into DNA.     

De novo synthesis and hormonal regulation of a dipeptidase in Cucurbita maxima

The following evidence was obtained for the de novo synthesis of dipeptidase in squash (Cucurbita maxima Duch. var. Hubbard) cotyledons during germination: (i) the amount of [¹⁴C]leucine incorporated into the dipeptidase was greater than that found in other proteins; (ii) the enzyme coincided with a peak of radioactivity in DEAE column chromatography; and (iii) the specific radioactivity of the enzyme increased with purification. There was also a positive correlation between the rate of [¹⁴C]leucine incorporation into dipeptidase and the rate of dipeptidase development. Four plant growth regulators, gibberellic acid (GA) benzyladenine (BA), indol-3-acetic acid (IAA), and abscisic acid (ABA) were examined for their effect on the development of dipeptidase activity at 5 × 10^?6 and 5 × 10^?5 M. None of these regulators affected the activity of the isolated dipeptidase per se. In intact see ds, BA and IAA inhibited the development of dipeptidase activity at the higher concentration, ABA reduced the activity at both concentrations; however, GA enhanced its development at the higher concentration. In distal-half cotyledons, BA and GA stimulated enzyme development but they showed no synergistic effect. IAA suppressed the development of enzyme activity at the higher concentration and ABA inhibited development at both levels.     

Effect of compactin on the incorporation of mevalonolactone into gibberellic acid by Gibberella fujikuroi

In Gibberella fujikuroi, strain GF-1a, the effect of the sodium salt of compactin on the incorporation of both radiolabelled acetate and mevalonate into gibberellic acid has been investigated. In each case, a concentration of 40 mg/1. caused a significant reduction in the incorporation.     

A candidate gene OsAPC6 of anaphase-promoting complex of rice identified through T-DNA insertion

A dwarf mutant (Oryza sativa anaphase-promoting complex 6 (OsAPC6)) of rice cultivar Basmati 370 with 50% reduced plant height as compared to the wild type was isolated by Agrobacterium tumefaciens-mediated transformation using Hm^R Ds cassette. This mutant was found to be insensitive to exogenous gibberellic acid (GA₃) application. Homozygous mutant plants showed incomplete penetrance and variable expressivity for plant height and pleiotropic effects including gibberellic acid insensitivity, reduced seed size, panicle length, and female fertility. Single copy insertion of T-DNA and its association with OsAPC6 was confirmed by Southern hybridization, germination on hygromycin, and 3:1 segregation of HPT gene in F₂ from OsAPC6 × Basmati 370 cross. The T-DNA flanking region sequenced through thermal asymmetric interlaced polymerase chain reaction showed a single hit on chromosome 3 of japonica rice cultivar Nipponbare in the second exonic region of a gene which encodes for sixth subunit of anaphase-promoting complex/cyclosome. The candidate gene of 8.6-kb length encodes a 728-amino acid protein containing a conserved tetratricopeptide repeat (TPR) domain and has only a paralog, isopenicillin N-synthase family protein on the same chromosome without the TPR domain. There was no expression of the gene in the mutant while in Basmati 370, it was equal in both roots and shoots. The knockout mutant OsAPC6 interferes with the gibberellic acid signaling pathway leading to reduced height and cell size probably through ubiquitin-mediated proteolysis. Further functional validation of the gene through RNAi is in progress.