Effects of gibberellic Acid on endogenous indole-3-acetic Acid and indoleacetyl aspartic Acid levels in a dwarf pea

Two-week-old dwarf peas (Pisum sativum cv Little Marvel) were sprayed with gibberellic acid (GA₃), and after 3 or 4 days the upper stem and young leaf samples were analyzed for indole-3-acetic acid (IAA) and indole-3-acetyl aspartic acid by an isotope dilution high performance liquid chromatography method. GA₃ increased IAA levels as much as 8-fold and decreased indole-3-acetyl aspartic acid levels.     

Gibberellin-auxin interaction in pea stem elongation

Joint application of gibberellic acid and indole-3-acetic acid to excised stem sections, terminal cuttings, and decapitated plants of a green dwarf pea results in a markedly synergistic growth response to these hormones. Synergism in green tall pea stem sections is comparatively small, although growth is kinetically indistinguishable from similarly treated dwarf sections.

Gibberellin-induced growth does not appear to be mediated through its effect on auxin synthesis, since gibberellin pretreatment of dwarf cuttings fails to elicit an enhanced tryptophan-induced growth response of sections, whereas auxin-induced growth is strongly enhanced. Also, tryptophan-gibberellin synergism is not significant in sections and cuttings of green dwarf peas, while auxin-gibberellin synergism is.

Administration of gibberellic acid prior to indole-3-acetic acid results in greatly increased growth. In reversed order, the application fails to produce any synergistic interaction. This indicates that gibberellin action must precede auxin action in growth regulation.

     

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.     

The Promotion of Indole-3-acetic Acid Oxidation in Pea Buds by Gibberellic Acid and Treatment

Terminal buds of dark-grown pea (Pisum sativum) seedlings have an indole-3-acetic acid oxidase which does not require Mn(2+) and 2,4-dichlorophenol as cofactors. Oxidase activity is at least 50 times higher in buds of tall peas than in dwarf seedlings. Administration of gibberellic acid to dwarf peas stimulates both growth and indoleacetic acid oxidase activity to the same levels as in tall seedlings. By contrast, indoleacetic acid oxidation assayed in the presence of Mn(2+) and 2,4-dichlorophenol proceeds at similar rates regardless of gibberellin application. Treatment of tall peas with the growth retardant AMO-1618 reduces growth and oxidase activity. Such treated seedlings are indistinguishably dwarf. The enzyme does not appear to be polyphenol oxidase, nor do the results suggest that reduced activity in dwarf buds is due to higher levels of a dialyzable inhibitor. The peroxidative nature of the oxidase is probable.     

A new growth inhibitor, pisumin, involved in light inhibition of epicotyl growth of dwarf peas

A new growth inhibitor, tentatively named pisumin, which increased under red light and remained at initial level or decreased when dwarf pea (Pisum sativum L. cv Progress No. 9) seedlings were transferred from red light to dark, has been isolated in the form of a colorless powder from light-exposed epicotyls of dwarf peas, and characterized partially as an aliphatic carboxylic acid (molecular weight 284) by spectrometric analyses.

Exogenous pisumin inhibited the growth of epicotyl segments of dwarf peas at concentrations higher than 0.1 millimolar in the dark.

     

Radioactive gibberellin a(5) and its metabolism in dwarf peas

Radioactive gibberellin A₅ (³H-GA₅) was synthesized from gibberellic acid. When it was applied to dwarf peas grown in the dark, an average of 3% was converted to another acid gibberellin within 48 hours. The biological activity of the metabolite did not account for the response to applied GA₅. GA₅ is therefore assumed to be biologically active per se.³H-GA₅ did not appear to form a stable complex with a macromolecule in pea shoots. When injected into dwarf pea pods, ³H-GA₅ was readily metabolized by maturing seed to more water-soluble substances and to two other acidic compounds. This metabolism continued even throughout germination of the seed without reconversion of the metabolites to GA₅. It is concluded that “bound” GA₅ plays no part in the germination of dwarf pea seeds.     

THE INDEPENDENT ACTION OF MORPHACTINS AND GIBBERELLIC ACID ON HIGHER PLANTS

The comparative activity of three morphactins, n-butyl-9-hydroxyfluorene-9-carboxylate (IT 3233), 9-hydroxy-fluorene-9-carboxylicacid (IT 3235) and methyl-2-chloro-9-hydroxy fluorene-9-carboxylate(IT 3456) on the action of gibberellic acid (GA₃) were examinedutilizing dwarf pea (Progress No. 9), dwarf corn (strain d-5),CCC dwarfed Alaska pea, and embryoless barley half-seeds. When applied either prior to or simultaneously with GA₃ themorphactins were without effect in reducing the response ofthe dwarf peas to the gibberellin. In fact IT 3233 and IT 3235in combination with GA₃ produced taller plants than the GA₃controls. Similar results were observed using CCC dwarfed Alaskapeas. The morphactins did, however, produce several morphologicaleffects on the peas. In addition, they were effective in breakingapical dominance in the peas. The morphactins were also withouteffect on the dwarf corn bioassay and no morphological changeswere observed. In the embryoless barley half-seeds the morphactins did notinhibit the response of the tissue to the GA₃. Also, the morphactinsdid not exhibit gibberellin-like properties. The results suggest that morphactins do not compete with gibberellinfor similar sites of action. ¹Published with the approval of the Director of the MichiganAgricultural Experiment Station as Journal Article Number 3917.     

Grafting and gibberellin effects on the growth of tall and dwarf peas

Tall peas var. Alaska and dwarf peas var. Progress No. 9 were grafted onto their own roots or reciprocally grafted to determine the rootstock effect on the growth of the stem. In all cases the grafted stems grew the same as their ungrafted controls regardless of which rootstock they were grown on. When similarly grafted plants were supplied with gibberellic acid, good graft unions did not inhibit its translocation. This evidence supports the thesis that the mechanism controlling stem growth in peas is located in the stem and that the roots have no direct control over this mechanism.     

Internode length in pisum: do the internode length genes effect growth in dark-grown plants?

Internode length in light-grown peas (Pisum sativum L.) is controlled by the interaction of genes occupying at least five major loci, Le, La, Cry, Na, and Lm. The present work shows that the genes at all of the loci examined (Le, Cry, and Na) also exert an effect on internode length in plants grown in complete darkness. Preliminary results using pure lines were verified using either segregating progenies or near isogenic lines. The major cause of the differences was due to a change in the number of cells per internode rather than to an alteration of the cell length. Since the genes occupying at least two of these loci, Le and Na, have been shown to be directly involved with gibberellin metabolism, it appears that gibberellins are not only essential for elongation in the dark but are limiting for elongation in the nana (extremely short, na), dwarf (Na le), and tall (Na Le) phenotypes. These results are supported by the large inhibitory effects of AMO 1618 treatments on stem elongation in dwarf and tall lines grown in the dark and the fact that applied gibberellic acid could overcome this inhibition and greatly promote elongation in a gibberellin-deficient na line. It is clear that the internode length genes, and in particular the alleles at the Le locus, are not acting by simply controlling the sensitivity of the plant to light.     

The Distribution of Substances similar to Gibberellic Acid in Higher Plants

Gibberellic-acid-like substances have been found in extractsfrom all parts of seedlings of tall and dwarf peas and in matureseeds of wheat, French bean and tall and dwarf peas. They werepresent in amounts equivalent to o‘1–0’3 µg-gibberellicacid in 100 plants (F.W. 100–200 g.). Immature runnerbean seed yielded larger quantities, equivalent to 0.25 µg.gibberellic acid per gram fresh weight, distributed betweentestas, cotyledons, and embryos.     

THE EFFECT OF MALEIC HYDRAZIDE ON THE GROWTH RESPONSE OF PLANTS TO GIBBERELLIC ACID

Maleic hydrazide (MH) and gibberellic acid (GA) were applied alone and in combination at various doses to dwarf and tall varieties of garden pea, and their effect on stem extension measured. Combinations of MH and 3-indolylacetic acid (IAA) were also studied. Stern extension of dwarf peas was accelerated by GA and inhibited by MH. Their effects were not additive, since MH reduced the response to GA at all concentrations of each tested. IAA did not affect stem extension, whether applied alone or in combination with MH. Stem extensions of tall peas was not affected by GA or IAA alone. MH severely inhibited growth and this inhibition was not reduced either by GA or by IAA. At low doses MH broke apical dominance and side branches developed; extension of these was stimulated by GA and IAA and extension of the main axis correspondingly still further reduced. The results show that MH prevents the response to GA of GA-sensitive plants. It is suggested that the rapid growth of tall peas, as compared with that of dwarfs, and their lack of response to GA, are due to a greater capacity to synthesize a 'GA-like hormone'. Growth of tall peas is much more drastically inhibited by MH than that of dwarf peas and the suggestion is made that the inhibition of shoot growth induced by MH is due primarily to blocking the activity of the postulated 'GA-like hormone'.     

Activity of mitochondria isolated and purified from dwarf and tall,light-grown pea seedlings

Mitochondria from dwarf and tall light-grown peas have been separated from contaminating chloroplasts and other organelles on discontinuous sucrose gradients. A comparison between activities of the mitochondria from the two cultivars of light-grown pea seedlings was made. It was observed that both respiratory control (R.C.) indices and phosphate to oxygen (P : O) ratios were superior in mitochondria from the tall cultivar than the dwarf cultivar. The results are discussed in relation to energy status and possible gibberellic acid content in the two cultivars.     

Cooperative binding of the yeast Spt10p activator to the histone upstream activating sequences is mediated through an N-terminal dimerization domain

The yeast Spt10p activator is a putative histone acetyltransferase (HAT) possessing a sequence-specific DNA-binding domain (DBD) which binds to the upstream activation sequences (UAS elements) in the histone gene promoters. Spt10p binds to a pair of histone UAS elements with extreme positive cooperativity. The molecular basis of this cooperativity was addressed. Spt10p (640 residues) is an elongated dimer, but the isolated DBD (residues 283–396) is a monomer and binds non-cooperatively to DNA. A Spt10p fragment comprising the N-terminal domain (NTD), HAT domain and DBD (residues 1–396) binds cooperatively and is a dimer, whereas an overlapping Spt10p fragment comprising the DBD and C-terminal domains (residues 283–640) binds non-cooperatively and is a monomer. These observations imply that cooperative binding requires dimerization. The isolated NTD (residues 1–98) is a dimer and is responsible for dimerization. We propose that cooperativity involves a conformational change in the Spt10p dimer which facilitates the simultaneous recognition of two UAS elements. In vivo, deletion of the NTD results in poor growth, but does not prevent the binding at the HTA1 promoter, suggesting that dimerization is biologically important. Residues 1–396 are sufficient for normal growth, indicating that the critical functions of Spt10p reside in the N-terminal domains.     

Morphogenetic Responses to Gibberellic Acid of a Radiation-induced Mutant Dwarf in Groundsel, Senecio vulgaris L.

A dwarf mutant of Senecio vulgaris L. induced by gamma radiation,which differs from the wild-type by a single recessive gene,grew as tall as the wild-type plant, and almost five times astall as dwarf control plants when treated at weekly intervalsfrom the seedling stage with 10 p.p.m. gibberellic acid. Thewild-type groundsel responded only slightly to similar treatment.Some evidence was obtained that the response to GA was relativilygreater when plants were grown during the late autumn. Dwarfnessin the mutant is due to fewer as well as to shorter internodes;the rate of leaf initiation is reduced, but the onset of thereproductive phase and the duration of the life-cycle are notaffected by the mutation. Gibberellic acid leads to a conspicuouselongation of the internodes intreated dwarf plants, but notalways to a significant increase in their number. An increasein the dry weight of treated plants was observed, and this wasshown to be due to an increase in photosynthetic area and notto increased photosynthetic efficiency. Comparative observationsof shoot morphogenesis in wild-type and dwarf groundsel. andin dwarf treated with gibberellic acid, have shown that thetype of shoot development induced by gibberellic acid in dwarfplants does not correspond exactly to the normal mode of developmentin wild-type plants. Increased mitotic activity in the subapicaltissue of the mutant following gibberellic acid treatment resultedin greater and earlier elongation of the internodes. However,the typical form and dimensions of the wild-type plant werenot wholly restored.     

Preparation of radioactive gibberellin a(1) and its metabolism in dwarf peas

Gibberellin A₁-3,4-³H was prepared by selective catalytic reduction of gibberellic acid with a mixture of tritium and hydrogen. ³H-GA₁ was applied at physiological concentrations to dwarf peas and the metabolism of the hormone was investigated. ³H-GA₁ was converted to an acidic, biologically active compound. Radioactive but biologically inactive compounds were also found in the neutral fraction and could not be converted to acidic gibberellins by hydrolysis. No attachment of gibberellin to any macromolecular fraction was evident.     

Synergism between Gibberellin and Auxin in the Growth of Intact Tomato

Gibberellin A_4&7 was more effective than gibberellic acid in increasing shoot elongation when applied to the apex of intact Lycopersicum esculentum seedlings of Tiny Tim, a dwarf cultivar, and Winsall, a tall cultivar. After 14 days, gibberellic acid and gibberellin A_4&7 stimulated growth of the dwarf more than the tall tomato. In tall tomato the application of indole-3-acetic acid alone (6.1 μg/plant) showed an inhibitory growth effect, but when applied with 17.5 μg per plant of gibberellic acid, it had a synergistic effect at 7 days but not at 14 days. When the auxin concentration was reduced to 0.61 μg per plant a synergistic effect was observed on tall plants at 7 and 14 days between indole-3-acetic acid and gibberellic acid. Application of gibberellin A_4&7 with auxin did not give a synergistic response in tall or dwarf tomato.     

Polygalacturonic acid trans-eliminase of Xanthomonas campestris

Polygalacturonic acid trans-eliminase from the culture fluid of Xanthomonas campestris was purified 66-fold by acetone precipitation, citrate extraction and chromatography on diethylaminoethyl- and carboxymethyl-cellulose. The optimum pH is 9·5 in glycine–sodium hydroxide buffer. Up to 1mm-calcium chloride brings about a remarkable stimulation of the enzyme activity and, at this concentration, no other cations promote or inhibit enzyme action except Ba²⁺ ions, which cause complete inhibition. The enzyme degrades polygalacturonic acid in a random manner; it does not act upon polygalacturonate methyl glycoside, although it can cleave partially (68%) esterified pectin. The end products from polygalacturonic acid at 46% breakdown are unsaturated di- and tri-galacturonic acids, in addition to saturated mono-, di- and tri-galacturonic acids. Pentagalacturonic acid is split preferentially into saturated dimer plus unsaturated trimer, or into saturated trimer plus unsaturated dimer; at a lower rate, it is also split into monomer and unsaturated tetramer. Unsaturated pentamer is split into unsaturated dimer plus unsaturated trimer. Tetragalacturonic acid is split some-what preferentially at the central bond to form dimer and unsaturated dimer, but it is also split into monomer and unsaturated trimer. Unsaturated tetramer is split only at the central bond to yield only unsaturated dimer. Trigalacturonic acid is split into monomer and unsaturated dimer. Unsaturated trimer is cleaved into saturated dimer and probably 4-deoxy-l-5-threo-hexoseulose uronic acid, which has not yet been directly identified. Neither saturated nor unsaturated digalacturonic acid is attacked. The unsaturated digalacturonic acid was isolated and proved to be O-(4-deoxy-β-l-5-threo-hexopyranos-4-enyluronic acid)-(1→4)-d-galacturonic acid.     

Bacillus megaterium, KM beta-galactosidase: purification by affinity chromatography and characterization of the active species

The enzyme β-galactosidase from Bacillus megaterium, strain KM has been purified by affinity chromatography. The enzyme was found to have a dimeric subunit structure, with the monomer having a molecular weight of 120,000. The K_eq of the monomer-dimer equilibrium was strongly shifted towards dissociation in the isolated state. Inclusion of 5% sucrose in the buffer (and maintenance of the temperature at 5 °) minimized this dissociation. Molecularly homogeneous monomer and dimer could be prepared on sucrose gradients. The dimer was determined to have an S_20,w of 8, while the monomer had an S_20,w of 3. The amino acid composition was found to be similar to that of the E. coli β-galactosidase although significant differences occur. The activity of the monomer was studied by both urea-denaturation experiments and by immobilization of the monomer on Sepharose-4B. The monomer, bound to Sepharose-4B, was found to be inactive but still capable of binding the inhibitor thio-methyl galactoside. Activity was reconstituted by adding free monomer, in 8 M urea, to the Sepharose-bound monomer, followed by removal of the urea by dialysis. In addition, free monomers from E. coli β-galactosidase were found to form active hybrids with Sepharose-bound B. megaterium β-galactosidase monomers. We conclude on the basis of these studies that the free monomer is inactive, and that the dimer is the active species, in marked contrast to E. coli β-galactosidase where only the tetrameric form is active.     

Isolation and sequence analysis of sea urchin (Lytechinus pictus) histone H4 messenger RNA

Histone messenger RNAs isolated from early blastula stage Lytechinus pictus sea urchin embryos have been separated into discrete RNA bands on polyacrylamide gels. The most rapidly migrating of these molecules, the putative histone H4 mRNA, has been digested with T₁ ribonuclease to generate oligonucleotides for nucleotide sequence analysis. Many of these sequences are colinear with the highly conserved amino acid sequence of histone H4 protein as determined for both cows and peas.Histone H4 messenger RNA hybridizes in conditions of DNA excess to sea urchin DNA which is repeated approximately 470-fold. Despite this level of repetition the nucleotide sequence of the H4 messenger RNA reflects little evolutionary divergence within the H4 genes of L. pictus as judged by the stoichiometric yield of T₁ oligonucleotides and the hybridization and thermal stability of histone H4 mRNA-DNA hybrids.     

The Effect of Single and Repeated Gibberellic Acid Treatment on Internode Number and Length in Dwarf Peas

In long-term experiments comparing the results of single and repeated treatment of dwarf peas with gibberellic acid, both once-treated and weekly-treated plants formed more internodes than control plants. The initial high rate of internode formation in the once-treated peas dropped rapidly reaching that of control plants three weeks after treatment. Weekly-treated plants maintained a higher rate of internode formation. The ratio of internode lengths of once-treated plants to control plants was high (over 3) for the internodes which were elongating during or shortly after the single treatment but dropped rapidly and approached unity in later-developed internodes. The ratio of internode lengths of weekly-treated plants to control plants continued to be high throughout the experiment.