Richard P. Pharis

Use of insoluble polyvinylpyrrolidone for purification of plant extracts and chromatography of plant hormones

Abstract Polyclar AT, an insoluble form of poly- N -vinylpyrrolidone is highly effective (60-fold reduction in dry weight) in purification of gibberellin-like substances in plant extracts, presumably by selective removal of phenolic compounds and perhaps other organic acids. Biological activity of extracts in several plant bioassays for gibberellins was invariably enhanced by the technique. Quantitative elution of abscisic acid, indoleacetic acid, zeatin, gibberellins exhibiting a wide divergence of polarity and the glucoside of gibberellin A 3 was obtained from columns of Polyclar AT. Separation of a number of gibberellins from each other and from abscisic acid was also possible. The selective properties of Polyclar AT toward these plant hormones are discussed.

Chromatography of 33 gibberellins on a gradient eluted silica gel partition column

Abstract The elution R f of 33 gibberellins (GAs) and abscisic acid from a gradient eluted (increasing amounts of ethyl acetate in hexane) silica gel partition column are given. Certain problems encountered with the use of silica gels for partition chromatography are discussed. An ordering of elution R f is apparent from most of the GAs on the basis of the number and positioning of polar groups in the ent -gibberellane structure. Hydroxylation in rings C and D retards elution to a greater degree than hydroxylation in ring A . The elution of dicarboxylic GAs is not retarded to a significantly greater degree than monocarboxylic GAs, but the elution of tricarboxylic GAs is greatly regarded. Possession of an aldehydic/lactol grouping for a GA retards its elution to a greater degree than its γ-lactone counterpart.

Gibberellin structure and florigenic activity in Lolium temulentum, a long day plant.

Structural requirements for florigenic activity among gibberellins (GAs) and GA derivatives, including several new ones, applied once to leaves of Lolium temulentum, were examined. The compounds were applied to plants kept either in non-inductive short days (SD) or exposed to one inductive long day (LD). Inflorescence initiation and stem-elongation responses were assessed three weeks later. Among the GAs used, the range in effective dose for inflorescence initiation was more than 1000-fold, but substantially less for stem elongation. Some GAs promoted both stem elongation and inflorescence initiation, some promoted one without the other, and some affected neither. The structural features enhancing florigenic activity were often different from those enhancing stem elongation. Except in the case of 2,2-dimethyl GA4, a double bond in the A ring at either C-1,2 or C-2,3 was essential for high florigenic activity, though not for stem elongation. A free carboxy group was needed for both. Inflorescence initiation in Lolium was enhanced by hydroxylation at C-12, −13 and −15, whereas hydroxylation at C-3 reduced the effect on inflorescence initiation but increased that on stem elongation. A 12β-hydroxyl was more effective than the α epimer for inflorescence initiation whereas the reverse was true for stem elongation. Although such differential effectiveness of GAs for inflorescence initiation and for stem elongation could reflect differences in uptake, transport or metabolism, we suggest that it is indicative of specific structural requirements for inflorescence initiation.

Reversed-phase C18 high-performance liquid chromatography of acidic and conjugated gibberellins

Abstract The retention times of gibberellins and their glucosyl esters and glucoside conjugates on C 18 reversed-phase high-performance liquid chromatographic (HPLC) columns were determined using gradient or isocratic elution with methanol—acetic acid 1% . The separation of double-bond gibberellin isomers was accomplished without the need for derivatization or the addition of salts. A combined HPLC—radiocounting with flow-through scintillation spectrometric procedure was suitable for the routine radioassay of acidic and conjugate-like metabolises from [ 3 H] gibberellin feeds. Similarly, HPLC—bioassay detection was suitable for purified plant extracts. The totally volatile methanol-1% acetic acid solvent in the gradient and/or isocratic mode should be capable of separating virtually any of the known gibberellins, their conjugates and their catabolites. However, retention time alone is inadequate and definitive detection techniques must be utilized.

Plant growth substances 1988

Plant Hormones in Lower Plants.- Endogenous Plant Hormones in Rice in Relation to the Regulation of Its Life Cycle.- I Genetic Approaches.- The Benefit of Biosynthesis and Response Mutants to the Study of the Role of Abscisic Acid in Plants.- A Genetic Approach to Auxins and Cytokinins.- Development of Genetic and Analytical Systems for Studies of Auxin Metabolism.- Developmental Studies of Physcomitrella patens Using Auxin and Cytokinin Sensitivity Mutants.- Dwarf Mutants of Maize - Research Tools for the Analysis of Growth.- Gibberellin Synthesis and Sensitivity Mutants in Pisum.- Gibberellin-Insensitive and Overgrowth Mutations in Temperate Cereals.- II Molecular Aspects.- Auxin and Gene Expression.- IAA Perception and Auxin-Regulated Gene-Expression.- Membrane-Bound Auxin Receptors.- Wound-Induced ACC Synthase, an Immunochemical Comparison of the Wound-Induced and Auxin-Induced Enzymes.- Polyphosphoinositide Turnover and Signal Transduction of Auxin on Isolated Membranes of Daucus carota L..- Complexity of the Barley ?-Amylase Gene Families: Determination of Gene Numbers for Each Family and mRNA Levels for Individual Members.- Regulation of Gene Expression by Abscisic Acid in Barley Aleurone Layers.- Novel Affinity Probes for Gibberellin Receptors in Aleurone Protoplasts of Avena Fatua.- Fusicoccin-Binding Sites in Higher Plants.- Cytokinin Mode of Action - Problems and Perspectives.- A Functional Cytokinin-Binding Protein in Photochemical Reactions of Chloroplast.- Structure-Activity Relationship Studies and Development of s-Triazine and Carbamate Anticytokinins.- Construction of a System for Regulated Alterations of Endogenous Cytokinins.- Ethylene Binding Receptors - Is There More Than One?.- III Hormones and Calcium.- Calcium and Second Messengers in Hormonal Regulation.- The Role of Calcium in the Response of Roots to Auxin and Gravity.- The Role of Calcium in Stimulus-Response Coupling.- IV Hormone Synthesis and Metabolism.- Recent Studies of the Metabolism of Abscisic Acid.- Recent Investigations of the Biochemistry of Abscisic Acid.- Tissue-Specific Metabolism of Both Abscisic Acid and Gibberellins in Legume Seeds.- An Overview of Cytokinin Biosynthesis.- Genetic Differences in the Enzymatic Regulation of Zeatine Metabolism in Phaseolus Embryos.- Xylem-Translocated Cytokinin: Metabolism and Function.- Cytokinin Oxidase and the Degradative Metabolism of Cytokinins.- Metabolic Aspects of Ethylene Biosynthesis.- Conjugation of Gibberellins in Zea mays.- Metabolism of Gibberellins A20 and A9 in Plants: Pathways and Enzymology.- The Dioxygenases in Gibberellin Biosynthesis after Gibberellin A12-Aldehyde.- The Action of Plant Growth Retardants at the Biochemical Level.- Measurement of the in Vivo Rate of Indole-3-Acetic Acid Turnover.- Relationship Between Stimuli, IAA and Growth.- Oxidation of Indole-3-Acetylaspartic Acid in Vicia.- Indole-3-Ethanol Metabolism and Its Possible Role in the Regulation of Indole-3-Acetic Acid Biosynthesis.- The Oxindole-3-Acetic Acid Pathway in Zea mays.- Metabolism of Jasmonic Acid.- V Hormones: Physiology and Effects.- How Abscisic Acid Causes Depressions of the Photosynthetic Capacity of Leaves.- The Role of Abscisic Acid in Chilling Resistance.- Dormancy in Cereals - Levels of and Response to Abscisic Acid.- Auxin Regulation of Cell Differentiation in Moss Protonema.- The Second Messenger in Apical Dominance Controlled by Auxin.- Auxin Transport and Its Regulation by Flavonoids.- GC-MS Quantifications of Free and Ester Indol-3-Acetic Acid in Relation to Root Growth and Gravitropism.- Hemmstoff und Wachstum: Growth Inhibitors, Not Auxin, Regulate Phototropism.- Cytokinins as Metabolic Stimulants Which Induce Pod Set.- Gibberellins in Embryo Development.- Gibberellins and Flower Initiation in Herbaceous Angiosperms.- Cytokinins in Flower Initiation.- Hormones and Cuscuta Development: Influence of Hormones on Secondary Xylem Differentiation, Phenylalanine Ammonia Lyase Activity and Lignification.- The Regulation of Tumor Morphology in Crown Gall.- Cytokinin: Evidence for Spatial Control of Signal Transduction.- Plant Wound Signals and Translation.- The Effect of Growth Regulators on the Winter Survival of Winter Wheat.- Hormonal Control of Senescence.- The Possible Role of Protein Kinases in the Plant Cell Response to Phytohormones.- VI Practical Applications and Economic Implications.- New Types of Plant Growth Regulators of Microbial Origin: The Likelihood of Practical Use.- Can PGRs Alleviate the Recently Evolved Cross Resistances to Herbicides?.- Economic Aspects of Plant Growth Regulators.- Practical Considerations in Using Growth Regulators on Turfgrass.- New Aspects for the Practical Use of Ethylene-Releasing Compounds.- Commercial Uses of Gibberellins and Cytokinins and New Areas of Applied Research.- New Types of Plant Growth Retardants: Additional Perspectives for Practical Application in Agriculture and Horticulture.

Gibberellins: A Phytohormonal Basis for Heterosis in Maize

Four commercially important maize parental inbreds and their 12 F1 hybrids were studied to investigate the role of the phytohormone gibberellin (GA) in the regulation of heterosis (hybrid vigor). All hybrids grew faster than any inbred. In contrast, all inbreds showed a greater promotion of shoot growth after the exogenous application of GA3. Concentrations of endogenous GA1, the biological effector for shoot growth in maize, and GA19, a precursor of GA1, were measured in apical meristematic shoot cylinders for three of the inbreds and their hybrids by gas chromatography—mass spectrometry with selected ion monitoring; deuterated GAs were used as quantitative internal standards. In 34 of 36 comparisons, hybrids contained higher concentrations of endogenous GAs than their parental inbreds. Preferential growth acceleration of the inbreds by exogenous GA3 indicates that a deficiency of endogenous GA limits the growth of the inbreds and is thus a cause of inbreeding depression. Conversely, the increased endogenous concentration of GA in the hybrids could provide a phytohormonal basis for heterosis for shoot growth.

The endogenous gibberellins of vegetative and reproductive tissue of G2 peas.

The gibberellins (GAs) of both vegetative (leaves and stems) and reproductive (pods and seeds) tissue of the G2 strain of peas Pisum sativum L. were characterized in purified extracts by a combination of sequential silicic-acid partition column chromatography, and gas chromatography-mass spectrometry. Gibberellins A19, A20, A29 and an A29 catabolite were identified in both types of tissue. Gibberellins A9, A17 and A44 were also found in pods and seeds.

Ethylene, gibberellins, auxin and the apical control of branch angle in a conifer, Cupressus arizonica

Decapitation, gibberellin A3, high light, their combination, and certain levels of indole-3-acetic acid increase ethylene evolution and also induce branch hyponasty (upturning) in seedlings of Cupressus arizonica Greene, the increase in ethylene preceding obvious hyponasty. Exogenous ethylene also causes branch hyponasty and branches of seedlings maintained in an atmosphere scavenged of ethylene by mercuric perchlorate grow downwards. It is concluded that ethylene may play a role in the apical control of branch angle in some conifers. The positive effect of ethylene in increasing branch hyponasty may be direct, or reflect changes in levels of endogenous auxin and/or gibberellin.

The promotion of flowering in forest trees by gibberellin A47 and cultural treatments: A review of the possible mechanisms

Abstract Research papers noting success with gibberellin A 4 7 ( GA 4 7 ) on 19 species representing five genera of Pinaceae family conifers are reviewed. Evidence is presented which suggests that exogenous GA 4 7 plays a direct morphogenic role in the promotion of flowering in these conifers that is independent of nutrient diversion or effects on the vigor and mitotic activity of potentially reproductive primordia. Various cultural treatments (e.g. water stress, root-pruning, girdling, nitrate-N-fertilization and N starvation) which promote flowering, often synergistically with GA 4 7 , can slow the oxidative metabolism of applied GAs and can also result in a build-up of endogenous less-polar GAs (such as GA 4 7 ). This suggest that exogenous GA 4 7 probably promotes flowering in a manner analogous to the natural promotion of flowering. Just how GAs act to promote flowering remains obscure. However, the evidence supports a hypothesis that GAs, endogenous and exogenously applied, are used preferentially for vegetative growth processes, with increased flowering occurring only after a threshold concentration of effector GA is reached.

Promotion of flowering in apple trees with gibberellin A4 and C-3 epi-gibberellin A4

The proportion of spurs flowering on apple trees (Malus domestica Borkh. cv Golden Delicious) displaying a high degree of alternate-year flowering was increased in the “off” year by gibberellin A4 (GA4) and C-3 epi-GA4 applied in the previous year. When applied 4.5 weeks after anthesis amounts of GA4 ranging from 3 to 300 μg per spur and 25 or 50 μg of C-3 epi-GA4 per spur were effective. Treatments with GA4 made seven weeks after anthesis were less effective. A combination of 30 μg GA4 and 30 μg zeatin (6-(4-hydroxy-3-methylbut-trans-2-enylamino)purine) promoted flowering at both treatment times, and tended to be more effective than GA4 alone.