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PubMed1981

Effect of auxin on acropetal auxin transport in roots of corn.

Acropetal [(14)C]indoleacetic acid (IAA) transport was investigated in roots of corn. At least 40 to 50% of this movement is dependent on activities in the root apex. Selective excision of various populations of cells comprising the root apex, e.g. the root cap, quiescent center, or proximal meristem show that the proximal meristem is the critical region in the apex with regard to influencing IAA movement. The quiescent center has no influence and the root cap has only a minor effect. Excision and replacement of the proximal meristem with an exogenous supply of 10(-8) to 10(-9) molar IAA prevents the reduction in acropetal IAA transport which would normally occur in the absence of this meristem. Substituting 10(-9) molar IAA for the excised root cap brings about a significant increase in the amount of IAA moved acropetally, as compared to intact roots with the root cap still in place. From this and previous work, it is concluded that IAA synthesis occurring in the proximal meristem stimulates the movement of IAA from the basal to apical end of the root.

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PubMed1981

Effect of salt on auxin-induced acidification and growth by pea internode sections.

The capacity of excised internode sections of pea to grow and secrete protons in response to indoleacetic acid (IAA) and Ca(2+) and K(+) treatments was examined. By incubating unpeeled and unabraded sections in rapidly flowing solutions, it was shown that acidification of the external medium in the presence or absence of IAA is dependent on the presence of Ca(2+) and K(+). Similar results were obtained when unpeeled and unabraded sections were incubated in dishes with shaking. When peeled or abraded sections were incubated with shaking in IAA, H(+) release was also dependent on the presence of Ca(2+) and K(+). The release of H(+) from sections incubated in Ca(2+) and K(+) is not caused by displacement of H(+) from binding sites in the cell wall. Rather, the release of protons from sections is temperature dependent, and it is concluded that this is a metabolically linked process. Although Ca(2+) and K(+) are essential for the release of H(+) from isolated stem sections of peas, these cations do not influence elongation. Despite the large increase in proton release induced by Ca(2+) and K(+) either in the presence or absence of auxin, growth in the presence of these ions was never greater than it was in their absence. Furthermore, cations do not affect the neutral sugar or uronic acid composition of the solution which can be centrifuged from isolated sections. As is the case for growth, an increase in the neutral sugar and uronide composition of the cell wall solution is dependent only on IAA. It is concluded that IAA-induced growth of pea stem sections is independent of the secretion of protons.

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PubMed1981

In vitro gibberellin a(4) binding to extracts of cucumber hypocotyls.

Cucumber hypocotyls were extracted and the extract centrifuged at 100,000g to yield a supernatant or cytosol fraction. Binding of [(3)H]-gibberellin(4) (GA(4)) to soluble macromolecular components present in the cytosol was demonstrated at 0 C by Sephadex chromatography. Binding assays performed with cytosol that had been preheated or incubated with protease, DNase, RNase, or phospholipase A or C indicated that heat and protease treatments disrupted the binding, which suggests that binding occurred to a protein. Equilibrium dialysis of a protein-enriched fraction prepared by ammonium sulfate precipitation also indicated binding of [(3)H]GA(4) to macromolecular components. [(3)H]GA(4) binding was pH-sensitive, saturable, reversible, and significantly affected by biologically active gibberellins, but not by inactive gibberellins or other plant hormones such as indoleacetic acid, abscisic acid, or kinetin. Thin layer chromatography indicated that [(3)H]GA(4), and not a metabolite, was the species bound. A kinetic analysis indicated that specific binding of [(3)H]GA(4) was due to a single class of binding sites having an estimated K(d) of 10(-7) molar and a concentration of 0.8 x 10(-12) moles gram(-1) fresh weight or 0.4 x 10(-12) moles milligram(-1) soluble protein.

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PubMed1981

Subcellular Localization of IAA Oxidase in Peas.

Indoleacetic acid (IAA) oxidase has been reported to be involved in plant growth because of its alleged role in the control of endogenous IAA levels. This purported role was reevaluated in terms of the properties and subcellular location of the enzyme in etiolated pea (Pisum sativum L. var. Alaska) epicotyls.The enzymic properties of IAA oxidase in the floating pea epicotyl segments are similar to those previously reported in the literature. Other experiments indicate that approximately 70% of the activity is at the cut surfaces of the tissue. In addition, up to 50% of the IAA oxidase activity could be pelleted in a membranous fraction when the released enzyme was centrifuged at 10,000(g). Higher centrifugal forces reduced the proportion of the enzyme in the pellet, suggesting that vesicles containing IAA oxidase rupture at these forces.Subcellular localization of IAA oxidase was accomplished by use of sucrose density gradient centrifugation and fractionation. It was found that the enzyme is associated most closely with Golgi and also to a lesser degree with the lysosomes and endoplasmic reticulum. It is suggested that little if any IAA oxidase is freely soluble in the cytoplasm and is therefore unlikely to have a role in controlling normal growth via IAA levels. The enzyme may, however, have a role in diseased or damaged tissue.

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PubMed1981

Evidence That IAA Conjugates Are Slow-Release Sources of Free IAA in Plant Tissues.

Evidence that indoleacetic acid (IAA) conjugates are metabolized via enzyme-catalyzed hydrolysis to free IAA and that their biological activities are related to the rates at which they are hydrolyzed by the tissue is presented. These conclusions are based on the following observations. Slow but continuous decarboxylation of the IAA moiety of IAA-l-alanine and IAA-glycine occurs when these conjugates are applied to pea (Pisum sativum L. cv. Alaska) stem segments. Inasmuch as IAA conjugates are protected from peroxidase-catalyzed oxidative decarboxylation, the conjugates are probably hydrolyzed and the freed IAA then further metabolized. Free IAA and IAA-l-alanine are converted, by pea stem tissue, into the same metabolites. The metabolism is enzymic, since conjugates of IAA with the d-isomers of the amino acids are inactive. Ethylene production induced by IAA-l-alanine and by IAA-glycine is correlated with their hydrolysis, as indicated by their decarboxylation and with the appearance or nonappearance of IAA metabolites in the tissues.

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PubMed1982

ATP-Dependent Calcium Transport in Plasmalemma Preparations from Soybean Hypocotyls : EFFECT OF HORMONE TREATMENTS.

A plasmalemma-enriched membrane preparation from etiolated soybean (Glycine max L., cv. Wayne) hypocotyls possesses an active ATP-dependent calcium pump which leads to calcium accumulation when assayed by the methods of Gross and Marmé (1978, Proc Natl Acad Sci USA 75: 1232-1236). Two-hour treatment of segments from the elongating zone of the hypocotyl with growth-promoting concentrations of indoleacetic acid gives up to 100 percent increase in the calcium transport activity. Conversely, similar pretreatment with zeatin or other cytokinins is inhibitory. In the meristematic and maturing zones of the hypocotyl, zeatin has the opposite effect of promoting calcium transport activity. One facet of cell-growth regulation may lie with hormonally mediated changes in efflux pumping of calcium.

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PubMed1982

Auxin-induced changes in the population of translatable messenger RNA in elongating sections of soybean hypocotyl.

In vitro translation products of polyadenylated RNA from untreated and auxin-treated elongating sections of soybean (Glycine max var. Wayne) hypocotyl were analyzed by two-dimensional polyacrylamide gel electrophoresis. The levels of translatable messenger RNA for at least ten in vitro translation products are increased by auxin treatment. The induction by auxin occurs rapidly (within 15 minutes), and the amounts of the induced in vitro translation products increase with time of auxin treatment. Indoleacetic acid has the same effect on the population of translatable messenger RNA as 2,4-dichlorophenoxyacetic acid. The auxin-induced in vitro translation products disappear rapidly when Actinomycin D is present during the last two hours of a three-hour auxin treatment.

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PubMed1982

Studies on the Production of Digitalis Cardenolides by Plant Tissue Culture: II. EFFECT OF LIGHT AND PLANT GROWTH SUBSTANCES ON DIGITOXIN FORMATION BY UNDIFFERENTIATED CELLS AND SHOOT-FORMING CULTURES OF DIGITALIS PURPUREA L. GROWN IN LIQUID MEDIA.

Undifferentiated, highly chlorophyllous cell cultures; undifferentiated white cell cultures; green, shoot-forming cultures; and white, shoot-forming cultures of Digitalis purpurea L. were established and subcultured every 3 weeks in liquid media in the light or in the dark. The digitoxin content, the chlorophyll content, and the ribulose bisphosphate carboxylase activity of these cultures were assayed. The light-grown, green, shoot-forming cultures accumulated considerable amounts of digitoxin (about 20 to 40 micrograms per gram dry weight), and the white, shoot-forming cultures without chloroplasts accumulated about one-third that amount of digitoxin. The chlorophyll content and the ribulose bisphosphate carboxylase activity of the undifferentiated green cells were about the same as they were in the green, shoot-forming cultures, but the digitoxin content of the former was extremely low (about 0.05 to 0.2 microgram per gram dry weight), which is about the same as that in undifferentiated white cells without chloroplasts. Thus, it was concluded that the chloroplasts are not essential for the synthesis of digitoxin in Digitalis cells. The optimum concentrations of the tested compounds for accumulation of digitoxin were: benzyladenine, 0.01 to 1 milligram per liter; indoleacetic acid, 0.1 to 1 milligram per liter; alpha-naphthaleneacetic acid; 0.1 milligram per liter; and 2,4-dichlorophenoxyacetic acid, 0.01 milligram per liter.

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PubMed1982

Does ethylene play a role in the release of lateral buds (tillers) from apical dominance in oats?

The growth of lateral buds (tillers), which are undergoing release from apical dominance, was measured in upright and gravistimulated intact Avena sativa L. cv. ;Victory' (oat) shoots as well as in isolated Avena stem segments treated with kinetin and sucrose. During release, the tiller bud initially shows a slow rate of elongation accompanied by swelling. It is followed by a more rapid rate of elongation. Ethylene (C(2)H(4)) production in shoot segments containing a tiller bud was found to occur at the onset of tiller swelling during gravistimulation as well as during inflorescence emergence. Exogenous application of indoleacetic acid or C(2)H(4) inhibits kinetin-induced tiller bud swelling and elongation. However, stem segments pulsed for 24 hours in C(2)H(4) or the C(2)H(4) biosynthesis precursor, 1-amino-cyclopropane-1-carboxylic acid (ACC) and then transferred to kinetin and sucrose, showed a significant increase in swelling elongation as compared with segments maintained under the same conditions but without C(2)H(4) or ACC in the pulse. Segments pulsed for 24 hours with kinetin and sucrose plus the ACC biosynthesis inhibitor, aminoethoxyvinylglycine, or the C(2)H(4) action inhibitor, CO(2), then transferred to kinetin and sucrose medium, showed inhibition of tiller swelling during the pulse and of subsequent elongation. These results indicate that C(2)H(4) plays a role in promoting tiller swelling during the onset of tiller release from apical dominance and may act as a modulator hormone in promoting tiller elongation in the presence of cytokinin.

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PubMed1984

Gravitropism in Higher Plant Shoots : III. Cell Dimensions during Gravitropic Bending; Perception of Gravity.

Cross and longitudinal sections were prepared for light microscopy from vertical control plants (Xanthium strumarium L. Chicago strain), free-bending horizontal stems, plants restrained 48 hours in a horizontal position, and plants restrained 48 hours and then released, bending immediately about 130 degrees . Top cells of free-bending stems shrink or elongate little; bottom cells continue to elongate. In restrained stems, bottom cells elongate some and increase in diameter; top cells elongate about as much but decrease in diameter. Upon release, bottom cells elongate more and decrease in diameter, while top cells shorten and increase in diameter, accounting for the bend. During restraint, bottom cells take up water while tissue pressures increase; top cells fail to take up water although tissue pressures are decreasing.Settling of amyloplasts was observed in cells of the starch sheath.Removal of different amounts of stem (Xanthium; Lycopersicon esculentum Miller, cv Bonny Best; Ricinus communis L. cv Yolo Wonder) showed that perception of gravity occurs in the bending (elongation) zone, although bending of fourth and fifth internodes from the top was less than in uncut controls. Uniform application of 1% indoleacetic acid in lanolin to cut stem surfaces partially restored bending. Reversing the gradient in tension/compression in horizontal stems (top under compression, bottom under tension) did not affect gravitropic bending.

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PubMed1984

Oxidation of indole-3-acetic acid and oxindole-3-acetic acid to 2,3-dihydro-7-hydroxy-2-oxo-1H indole-3-acetic acid-7'-O-beta-D-glucopyranoside in Zea mays seedlings.

Radiolabeled oxindole-3-acetic acid was metabolized by roots, shoots, and caryopses of dark grown Zea mays seedlings to 2,3-dihydro-7-hydroxy-2-oxo-1H indole-3-acetic acid-7'-O-beta-D-glycopyranoside with the simpler name of 7-hydroxyoxindole-3-acetic acid-glucoside. This compound was also formed from labeled indole-3-acetic acid supplied to intact seedlings and root segments. The glucoside of 7-hydroxyoxindole-3-acetic acid was also isolated as an endogenous compound in the caryopses and shoots of 4-day-old seedlings. It accumulates to a level of 4.8 nanomoles per plant in the kernel, more than 10 times the amount of oxindole-3-acetic acid. In the shoot it is present at levels comparable to that of oxindole-3-acetic acid and indole-3-acetic acid (62 picomoles per shoot). We conclude that 7-hydroxyoxindole-3-acetic acid-glucoside is a natural metabolite of indole-3-acetic acid in Z. mays seedlings. From the data presented in this paper and in previous work, we propose the following route as the principal catabolic pathway for indole-3-acetic acid in Zea seedlings: Indole-3-acetic acid --> Oxindole-3-acetic acid --> 7-Hydroxyoxindole-3-acetic acid --> 7-Hydroxyoxindole-3-acetic acid-glucoside.

Hydroxyindoleacetic Acid↗
PubMed1985

Carbohydrates stimulate ethylene production in tobacco leaf discs : I. Interaction with auxin and the relation to auxin metabolism.

Various naturally occurring carbohydrates, applied at a concentration range of 1 to 100 mm, stimulated ethylene production for several days in indoleacetic acid (IAA)-treated or untreated tobacco (Nicotiana tabacum L. cv ;Xanthi') leaf discs. The lag period for this sugar-stimulated ethylene production was 8 to 12 hours after excision in the untreated leaf discs, but less than 2 hours in the IAA-treated ones. Among the tested carbohydrates, 12 were found to increase synergistically ethylene production, with d-galactose, sucrose, and lactose being the most active; mannitol and l-glucose had no effect. The extent and duration of the increased ethylene production was dependent upon the type of sugar applied, the tissue's age, and the existence of both exogenous IAA and sugar in the medium. Sucrose appeared to elicit a continuous IAA effect for 48 hours, as expressed by increased ethylene production, even when IAA was removed from the medium after a 4-hour pulse. Sucrose stimulated both the uptake and decarboxylation of [1-(14)C]IAA, as well as the hydrolysis of the esteric and amide IAA conjugates formed in the tissue after application of free IAA. This gradual hydrolysis was accompanied by a further accumulation of a third IAA metabolite. Moreover, synthetic indole-3-acetyl-l-alanine increased ethylene production mainly with sucrose, and this effect was accompanied by its increased decarboxylation and turnover pattern suggesting that release of free IAA was involved. An esteric IAA conjugate, tentatively identified by GC retention time was found to be the major component (84%) of the naturally occurring IAA conjugates in tobacco leaves. Accordingly the sucrose-stimulated ethylene production in tobacco leaves can be ascribed mainly to the sucrose-stimulated hydrolysis of the esteric IAA conjugate.

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PubMed1985

Auxin and Fusicoccin Enhancement of beta-Glucan Synthase in Peas : An Intracellular Enzyme Activity Apparently Modulated by Proton Extrusion.

Fusicoccin (FC), like indoleacetic acid (IAA), causes Golgi-localized beta-1,4-glucan synthase (GS) activity to increase when applied to pea third internode segments whose GS activity has declined after isolation from the plant. This suggests that GS activity is modulated by H(+) extrusion; in agreement, vanadate and nigericin inhibit the GS response. The GS response is not due to acidification of the cell wall. Treatment of tissue with heavy water, which in effect raises intracellular pH, mimics the IAA/FC GS response. However, various treatments that tend to raise cytoplasmic pH directly, other than IAA- or FC-induced H(+) extrusion, failed to increase GS activity, suggesting that cytoplasmic pH is not the link between H(+) extrusion and increased GS activity. Although FC stimulates H(+) extrusion more strongly than IAA does, FC enhances GS activity at most only as much as, and often somewhat less than, IAA does. This and other observations indicate that GS enhancement is probably not due to membrane hyperpolarization, stimulated sugar uptake, or changes in ATP level, but leave open the possibility that GS is controlled by H(+) transport-driven changes in intracellular concentrations of ions other than H(+).

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PubMed1985

Ethylene production by suspension-cultured pear fruit cells as related to their senescence.

Suspension-cultured pear fruit cells produce low levels of ethylene during growth and division in auxin containing medium. When deprived of auxin, division gradually ceases and ethylene production falls to barely discernible levels. However, notable ethylene production can now be induced by indoleacetic acid, CuCl(2), or 1-aminocyclopropane-1-carboxylic acid. If the auxin-deprived cells are transferred to ;aging' medium that lacks auxin but contains 0.4 molar mannitol, inducible ethylene production increases several-fold reaching levels of 40 to 60 nanoliters/10(6) cells per hour. Maximum inducible ethylene productivity is attained at varying times (1-6 days) after transfer to aging medium and appears to be temporally related to cell survival, i.e. the time of subsequent cell death. It is argued that auxin depletion initiates senescence which, in turn, leads to a transient increase in inducible ethylene production and eventual death. The limitations and potentials of the suspension-cultured pear cells as a system for the study of cellular senescence are discussed.

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PubMed1986

Bound auxin metabolism in cultured crown-gall tissues of tobacco.

Bound auxin metabolism in cultured crown-gall tumor cells and pith callus of tobacco was examined by feeding radiolabeled auxins and auxin conjugates. In all tissues fed [(14)C]indoleacetic acid (IAA), at least one-third of the IAA was decarboxylated, and most of the remaining radiolabel occurred in a compound(s) which did not release IAA with alkaline hydrolysis. In cells transformed by the A6 strain of Agrobacterium tumefaciens, the only detectable IAA conjugate was indole-3-acetylaspartic acid (IAAsp), whereas cells transformed by the gene 2 mutant strain A66 produced an unidentified amide conjugate but no IAAsp. By contrast, cells fed [(14)C]naphthaleneacetic acid (NAA) accumulated several amide and ester conjugates. The major NAA metabolite in A6-transformed cells was naphthaleneacetylaspartic acid (NAAsp), whereas the major metabolites in A66-transformed cells were NAA esters. In addition, A66-transformed cells produced an amide conjugate of NAA which was not found in A6-transformed cells and which showed chromatographic properties similar to the unknown IAA conjugate. Pith callus fed [(14)C] NAA differed from both tumor lines in that it preferentially accumulated amide conjugates other than NAAsp. Differences in the accumulation of IAA and NAA conjugates were attributed in part to the high capacity of tobacco cells to oxidize IAA and in part to the specificity of bound auxin hydrolases. All tissues readily metabolized IAAsp and indole-3-acetyl-myo-inositol, but hydrolyzed NAAsp very slowly. Indirect evidence is provided which suggests that ester conjugates of NAA are poorly hydrolyzed as well. Analysis of tissues fed [(14)C]NAA together with high concentrations of unlabeled IAA or NAA indicates that tissue-specific differences in NAA metabolism were not the result of variation in endogenous auxin levels. Our results support the view that bound auxin hydrolysis is highly specific and an important factor controlling bound auxin accumulation.

Journal Article↗
PubMed1986

Effect of thidiazuron, a cytokinin-active urea derivative, in cytokinin-dependent ethylene production systems.

Cytokinins are known to stimulate ethylene production in mungbean hypocotyls synergistically with indoleacetic acid (IAA), in mungbean hypocotyls synergistically with Ca(2+), and in wilted wheat leaves. Thidiazuron, a substituted urea compound, mimicked the effect of benzyladenine (BA) in all three systems. In the Ca(2+) + cytokinin system and the IAA + cytokinin systems of mungbean hypocotyls, thiadiazuron was slightly more active than BA at equimolar concentration. In mungbean hypocotyls exogenously applied IAA was rapidly conjugated into IAA asparate, and this conjugation process was effectively inhibited by thidiazuron, as by cytokinins. In the wilted wheat leaves system, 10 micromolar thidiazuron exerted stress ethylene production equal to that exerted by 1 millimolar BA, indicating that thidiazuron is more active than BA by two orders. The structure-activity relationship of thidiazuron and its thiadiazolylurea analogs in stimulating Ca(2+)-dependent ethylene production in mungbean hypocotyls was found to agree well with the structure-activity relationship of these derivatives in promoting the growth of callus tissues. These results indicate that thidiazuron and its derivatives are highly active to mimic the adenine-type cytokinin responses in promoting ethylene production and that the structure-activity relationship in promoting the growth of callus and in promoting ethylene production is similar.

Journal Article↗
PubMed1986

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 (14)C-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.

Journal Article↗
PubMed1986

Polar Calcium Flux in Sunflower Hypocotyl Segments : II. The Effect of Segment Orientation, Growth, and Respiration.

Calcium flux in sunflower (Helianthus annuus L. cv Russian mammoth) hypocotyl was measured with a Ca(2+) electrode as the increase or decrease in Ca(2+) in an aqueous solution (10 micromolar CaCl(2)) in contact with either the basal or apical end of 20 millimeter segments. Ca(2+) efflux was significantly higher at the apical end compared with the basal end; this apparent polarity was maintained even when the segments were inverted. No significant difference was observed in the cation exchange capacity of apical and basal cell walls that could explain the difference in Ca(2+) efflux at opposite ends of the hypocotyl segment. The presence of exogenous indoleacetic acid (IAA) in the segment medium resulted in the promotion of both Ca(2+) efflux and segment elongation. However, osmotic inhibition of the IAA-induced elongation did not result in inhibiting the IAA-induced Ca(2+) efflux. Ca(2+) efflux was inhibited by cyanide. Lowering the temperature from 25 degrees C also caused the gradual reduction of Ca(2+) efflux; at 5 degrees C the hypocotyl segments showed a net absorption of Ca(2+) from the segment medium. These findings support the suggestion that: (a) the observed Ca(2+) efflux in hypocotyl segments is probably the manifestation of the system which maintains the transmembrane Ca(2+) gradient at the cellular level. (b) The acropetal polarity of Ca(2+) efflux may be the result of the involvement of Ca(2+) in the basipetal transport of IAA.

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