Natural Auxin Research Articles

Identification of phenylacetic acid as a natural auxin in the shoots of higher plants

Evidence indicating the natural occurrence of the auxin substance, phenylacetic acid (PAA), in a range of crop plants has been obtained from paper chromatography followed by bioassay and from HPLC and GLC analysis of acidic ether extracts from vegetative shoots of these plants. Confirmatory evidence for the presence of PAA in tobacco shoots has been obtained from GC‐MS analysis. Quantitative estimation of the relative amounts of the two auxins, IAA and PAA, in the different shoot extracts was achieved by paper chromatography followed by gas chromatography. The amount of PAA in all six plants was found to be several times greater than that of IAA and calculation of average internal concentrations revealed that PAA is present in vegetative shoots at physiologically active concentrations. Present knowledge of the growth‐regulating activity of this new natural auxin is discussed.

Modulation of the oscillatory system involved in polar transport of auxin by other phytohormones

A wave‐like pattern of the basipetal efflux of natural auxin from the cambial region of a series of consecutive short sections of stems of Larix decidua Mill., Acer pseudoplatanus L. and Picea abies (L.) Karst. has been demonstrated as it was earlier reported for Pinus silvestris L. Apical application of ABA suppressed the IAA‐stimulated increase of the auxin‐wave amplitude, and zeatin or GA3 prevented this repression in stem segments of Pinus silvestris. All the exogenously applied substances were highly effective in physiological concentrations. Already 20‐min of exposure to IAA or ABA at the apical end produced modulations of the auxin‐wave along the whole 6.6 cm long stem segment. Application of 2, 3, 5‐triiodobenzoic acid (TIBA) caused suppression of the wave‐like pattern of auxin efflux similarly as ABA, supporting the association of the modulatory effects of ABA with the phenomena involved in polar transport of auxin. Abscisic acid applied to the basal end of the stem segment also reduced the auxin‐wave amplification caused by simultaneous supply of IAA to the apical end. This finding additionally confirms the hypothesis that: 1) the supracellular auxin‐wave generation is associated with the functioning of a system of oscillators coupled at the cellular level and 2) the auxin‐wave modulations can be propagated acropetally, that is against the main direction of the auxin molecular transport.

In Vitro Promotion by Auxins of Divalent Ion Release from Soybean Membranes

Release of divalent ions from membrane pellets of soybean hypocotyls was promoted by the natural auxin, indole-3-acetic acid, and the synthetic auxin, 2,4-dichlorophenoxyacetic acid. The calcium release occurred at auxin concentrations as low as 1 nanomolar, and maximum release was observed at 1 micromolar. Hormone concentrations greater than 1 micromolar showed reduced effectiveness in releasing membrane-associated calcium. 2,3-Dichlorophenoxyacetic acid, a weak-auxin analog of 2,4-dichlorophenoxyacetic acid, did not promote calcium release. In some experiments, the analog actually promoted calcium association with the membranes. Red blood cells treated in a similar manner to soybean hypocotyl membranes did not release calcium in response to indole-3-acetic acid. The release phenomenon was hormone specific but not ion specific. Auxin released manganese from membranes in a manner similar to that of calcium. The calcium release, following auxin treatment, is accompanied by a decrease in membrane-associated sites for calcium binding.

Specifically deuterated and tritiated auxins

Regiospecific syntheses of monodeuterated and monotritiated natural auxin (indole- 3-acetic acid), a synthetic auxin (naphthalene-1-acetic acid) and a photoaffinity labeling auxin (5-azidoindole-3-acetic acid) are described. These syntheses provide benzene-ring tritiated auxins for use in reversible and covalent binding studies.

Hormonal Modulation of the Oscillatory System Involved in Polar Transport of Auxin

AbstractThe amount of natural auxin collected in agar as a result of basipetal efflux from the cambial region of successive short sections of pine stem varies so that a wave‐like pattern is formed. The wave‐length is several times longer than the cell length in the cambial region, suggesting the existence of a supracellular oscillatory system, which forms a morphogenic field in the stem tissues, The amplitude of the auxin wave is amplified by apical application of IAA to the longer stem sections, particularly at she time of spring initiation of cambial activity. The wave of auxin disappears after simultaneous apical application of IAA and ABA. The modulatory effects of IAA and ABA are translocated along the investigated stem sections faster than known transport velocities of IAA molecules. This fact is considered as evidence of apical control of the morphogenic field by way of influence upon a supracellular system of conjugated oscillators in the tissue.

Hormonal Regulation of Ca2+ Stimulated K+ Influx and Ca2+, K+‐ ATPase in Rice Roots: in vivo and in vitro Effects of Auxins and Reconstitution of the ATPase

AbstractThe role of natural and synthetic auxins in regulation of ion transport and ATPase activity was studied in rice roots (Oryza sativa L. cv. Dunghan Shah). In vivo treatment of seedlings with 2,4‐dichlorophenoxyacetic acid at 2 × 10−6M for a short period enhanced subsequent Ca2+ stimulated K+ influx and ATPase activity, while a longer treatment diminished both K+ influx and ATPase activity. Indoleacetic acid at 10−10–10−8M induced ATPase activity. In in vitro experiments both 2,4‐dichloro phenoxyacetic acid and indoleacetic acid (10−10–10−8M) stimulated Ca2+, K+‐ATPase activity of a plasmalemma rich micro somal fraction from the roots.Acetone extracted ATPase preparations lost their activity. The enzyme regained its activity and its sensitivity towards ions (Ca2++ K+) when reconstituted with phosphatidyl choline. Addition of auxins also indicated that the presence of the lipid was necessary in the interaction between the ATPase and auxins. Auxins and ions probably interact with the intact ATPase lipoprotein complex, which may possess a receptor site for the auxins, possibly as a sub unit.

Effects of auxin on the localization of tobacco mosaic virus in hypersensitively reacting tobacco

In Samsun NN tobacco reacting hypersensitively to tobacco mosaic virus (TMV), the natural auxin indoleacetic acid (IAA) decreased lesion enlargement in a concentration-dependent manner when sprayed on young, expanding leaves between 40 h before and 40 h after inoculation, and again between 60 and 100 h after inoculation. A maximum decrease in lesion size of 30% was routinely obtained by application of 10 −4 m IAA within 12 h of inoculation. The synthetic auxin, a-naphthylacetic acid, likewise reduced lesion size, whereas the less active natural auxin, phenylacetic acid, was effective at higher concentrations only. The auxin-like regulator, 2,4-dichlorophenoxyacetic acid, was atypical in that it reduced lesion size at low, but promoted it at higher concentrations. The non-auxin analogues, β-naphthyl-acetic aced and benzoic acid, were only marginally active. In non-inoculated plants, IAA increased peroxidase activity similarly to TMV infection, but application of the hormone to TMV-inoculated plants counteracted the virus-induced increase in enzyme activity, mainly through partial suppression of the quantitative changes in peroxidase isoenzymes characteristic of the infection. The latter effect is related to the decrease in necrotizing tissue resulting from the reduction in lesion size. Since the effect of the auxin on peroxidase activity was prolonged and systemic, whereas the effect on lesion size was not, the two effects are not directly related. The ability to restrict lesion spread in response to auxin was gradually lost during leaf development or after detachment. It could not be restored by kinetin. However, kinetin selectively inhibited the increase in lesion enlargement due to detachment. Like in mature, fully grown and in old, senescing leaves, auxin did not reduce lesion size in young, systemic resistant leaves further, possibly because of lack of an additional factor. Similar results can be obtained by treatment with ethephon, suggesting that the action of auxin is mediated by auxin-induced ethylene. IAA also inhibited TMV multiplication in young, inoculated leaves of the systemically reacting tobacco cultivar Samsun. Hence, the limiting action of exogenous auxin on lesion spread may depend both on a direct inhibitory effect on virus multiplication in young, hypersensitively reacting leaves, and on a stimulation of factors involved in virus localization.

On the Question of Stem Polarity with Respect to Auxin Transport

AbstractThe effect of section length and number of longitudinally contiguous cells upon polar transport of natural auxin from the pine stem cambial region was investigated with oat coleoptile curvature tests. Basipetal and acropetal efflux of auxin to agar declines with increasing length of the sections, but the polarity quotient varies little and is similar to the polarity of individual cells. An integrated system of cells produces a wave along the stem in the efflux of auxin from consecutive segments. The possible role of such waves in development of polarity gradients and of the morphogenic maps of orientation of cells in the stem cambial region is discussed.

2,4-Dichlorophenoxyacetic Acid Inhibits the Outer Membrane NADH Dehydrogenase of Plant Mitochondria

The NADH dehydrogenase of potato (Solanum tuberosum) and mung bean (Phaseolus aureus) outer mitochondrial membranes is specifically inhibited by both 2,4-dichlorophenoxyacetic and 2,4,5-trichlorophenoxyacetic acids but not by the natural auxin indole-3-acetic acid.

Properties of a Solubilized Microsomal Auxin-binding Protein from Coleoptiles and Primary Leaves of Zea mays

An auxin-binding protein can be solubilized from microsomal membranes of Zea mays using either Triton X-100 extraction of the membranes or buffer extraction of the acetone-precipitated membranes. This paper describes the properties of the binding protein solubilized by these two methods. The binding is assayed by gel filtration chromatography in the presence of naphthalene [2-(14)C]acetic acid. Binding is rapid and reversible with an optimum at pH 5. Both preparations show similar molecular weights by gel filtration (80,000 daltons) at pH 7.6 and 0.1 molar NaCl, and both aggregate at low ionic strength. They appear to be the same active molecular species. The binding activity is destroyed by trypsin, pronase or para-chloromercuribenzoic acid, but not significantly reduced by phospholipase C, DNase, RNase, or dithioerythritol. Since saturating amounts of naphthalene acetic acid protect the molecule from inhibition by para-chloromercuribenzoic acid, it is concluded that the binding protein has a sulfhydryl group at the binding site, or protects such a group in its binding conformation. The dissociation constant of the protein for naphthalene acetic acid is 4.6 x 10(-8) molar with 30 picomoles of sites per gram of tissue fresh weight. Binding constants were estimated for 13 other natural and synthetic auxins by competition with naphthalene[2-(14)C]acetic acid. Their dissociation constants are in general agreement with published values for their binding to intact membranes and their biological activity, although several exceptions were noted. A supernatant factor from the same tissue changes the apparent affinity of the protein for naphthalene acetic acid. This factor may be the same one as has been previously reported to alter the affinity of intact microsomes for auxin.

Stereoselectivity and affinity in molecular pharmacology. III. Structural aspects in the mode of action of natural and synthetic auxins

Analysis of available potency estimates for 35 pairs of enantiomeric arylcarboxylic acids with auxin activity (flax-root-growth inhibition test) revealed extensive correlations between the activity of the more potent and less potent isomers, as well as between the log of the ratio of potencies and the log potency of the more active isomer when structurally similar analogs are compared. 5 structural subgroups were discernible (n, eudismic-affinity quotient (EAQ), r 2); (1) arylpropionic acids (6, −0.36, 0.66); (2) 2-naphthoxy-carboxylic acids (6, +1.07, 0.99); (3) 1-naphthoxycarboxylic acids (3, +1.56, 0.96); (4) ortho-substituted phenoxycarboxylic acids (10, +0.97, 0.96) and (5) ortho-unsubstituted phenoxycarboxylic acids (10, +0.56, 0.70). For achiral lower homologs such as auxin itself 3-indolyl-acetic acid (IAA), phenoxyacetic acid and 1-naphthoxyacetic acid, extrapolated potencies were found to agree well with experimental values. On the basis of these observations an auxin receptor is postulated and binding arrangements are described which explain most of the experimental data available. A 3-point attachment when allowed is the only binding mode compatible with the reported data.

Factors effecting adventitious bud formation of cotyledon culture of Douglas fir

Production of adventitious buds in culture of cotyledon explants derived from young seedlings of Douglas fir ( Pseudotsuga menziesii (Mirb.) Franco) required both cytokinin and auxin. A pronounced influence on morphogenetic responses by variations in physiological condition or genetic complement was observed using the natural auxins, IAA and IBA. However, this influence was nearly eliminated when appropriate concentrations of synthetic auxin, NAA and/or 2,4-D, were used. BAP was more effective in stimulation of organ formation than 2iP or kinetin. A high frequency of organ differentiation was obtained with 5 μM BAP plus either NAA or 2,4-D at 0.5–50 nM.

Auxin Stimulation of Cambial Activity in Pinus silvestris I. The Differential Cambial Response

AbstractIsolated stem segments of Pinus silvestris L. produce new xylem in sterile culture for 5 weeks if sucrose and IAA are present in the medium. The response of cambium varies in the course of the season and along the tree stem. The cambium is more sensitive in spring and in the stem portion closer to tree apex than later in the season and closer to the stem base. Spring initiation of cambial activity in adult pine trees under natural conditions could not be correlated with any consistent concentration gradient of natural auxin extracted from the cambial region. Thus, the relation between concentration of auxin and the activity of cambium is complex and involves changes of cambial responsivity. Interaction with gibberellic acid or kinetin and changing concentration of sucrose were studied during the season, but none of these substances alone appeared to be responsible for the observed variation in cambial response to auxin.

THE PRODUCTION OF HORMONES IN HIGHER PLANTS

THE PRODUCTION OF HORMONES IN HIGHER PLANTS

Correlation between cell enlargement in pea internode segments and decrease in the pH of the medium of incubation: I. Effects of fusicoccin, natural and synthetic auxins and mannitol

In pea internode segments the stimulation of cell enlargement by the auxins indole-3-acetic acid (IAA), naphthaleneacetic acid (NAA), and 2,4-dichlorophenoxyacetic acid (2,4-D) is accompanied by a significant decrease in the pH of the incubation medium. Fusicoccin (FC) which is more effective than these auxins in stimulating growth, is also more effective in decreasing pH in the medium. No effect on pH is observed in the case of the “anti-auxin” p-chlorophenoxyisobutiric acid (4-CPIB). The decrease of pH which parallels auxin or FC-induced cell enlargement does not depend on changes in the rate of “leakage” from the pea segments of substances buffering in the pH range from 4.5 to 6.5. The “pH decrease” effect of FC and of IAA is not suppressed by the addition to the medium of mannitol at concentrations which inhibit growth without affecting the effect of auxin on cell wall extensibility. These results are interpreted as an indication that the growth-promoting activity of IAA and FC is mediated by the capacity of these compounds to induce a decrease of pH in the cell wall and thus an increase of its plastic extensibility.

Mechanism of the growth-promoting action of fusicoccin

Analogies and differences between the growth-promoting action of fusicoccin (FC), the toxic agent produced by Fusicoccum amygdali, and of indole-3-acetic acid (IAA) in etiolated pea internode segments were further investigated. It was confirmed that the effect of FC, at optimal concentration, on growth by cell enlargement is markedly higher (ca. 70%) than that of IAA. The lack of an inhibitory effect of FC at high concentrations corresponds to a lack of capacity of the toxin to induce ethylene synthesis. When FC and IAA are present together at suboptimal concentrations, the effects of the two substances are clearly additive. Growth stimulation by a mixture of FC and IAA at optimal concentrations is equal to that by FC alone. NaF, 2,4-DNP, actinomycin D, and cycloheximide strongly depress FC-induced stimulation of cell enlargement. These data are in agreement with the hypothesis that FC promotes growth through some effect on cell-wall extensibility probably identical to the one mediating auxin-induced growth, while the primary site of action of FC is different from that of the natural and synthetic auxins.

Chromatography of a new natural auxin, 4-chloroindolyl-3-acetic acid and related chloro derivatives

Chromatography of a new natural auxin, 4-chloroindolyl-3-acetic acid and related chloro derivatives

Mechanism of Xylem Differentiation inPinus silvestrisL.

The duration and daily rate of radial growth and secondary-wall formation of all consecutive, radially-formed tracheids throughout the season were investigated in stems of adult trees of Pinus silvestris L. It was found that ( 1 ) the variation in radial diameter of tracheids was probably dependent on seasonal changes in the rate of growth during the phase of radial enlargement, (2) the daily rate of cell-wall formation determined the final cell-wall thickness of tracheids only at the beginning and the end of the season, (3) both rates were affected by temperature, (4) seasonal changes in cell-wall thickness were dependent mostly upon seasonal changes in the duration of the maturation period, (S) the changes in the duration of the maturation period which brought about transition from early to late wood were determined mostly by the delay in onset of autolysis of cytoplasm which terminates the phase of tracheid maturation. This process, unlike the xylem production from cambium and the termination of radial enlargement, was found not to be affected by the seasonal variation of temperature. An attempt to correlate these processes with the activity of natural auxin extracted from the cambial region gave negative results. On the basis of the results obtained, auxin and environmental factors such as precipitation and temperature seem not to be specific for xylem differentiation. They may seriously affect wood differentiation if they become limiting or exceed the limit of tolerance, but probably they do not determine differentiation of the annual ring of conifers into early and late wood.

Multiple 3-Methyleneoxindole Reductases of Peas: DIFFERENTIAL INHIBITION BY SYNTHETIC AUXINS

Abstract The reduction of 3-methyleneoxindole, a biologically active metabolite of natural plant auxin, to the inert 3-methyloxindole is catalyzed by four or five reduced triphosphopyridine nucleotide-linked enzymes. Two of the reductases, which can be separated completely by column chromatography, exhibit differential sensitivity to the synthetic auxins, 1-naphthaleneacetic acid and 2,4-dichlorophenoxyacetic acid. Reductase A is inhibited noncompetitively by 2,4-dichlorophenoxyacetic acid which is a weak competitive inhibitor of reductase B. Reductase B, on the other hand, is inhibited competitively by 1-naphthaleneacetic acid, a weak competitive inhibitor of reductase A. Reductase A is labile, losing activity as well as its pH-dependent sensitivity to 2,4-dichlorophenoxyacetic acid. Reductase B is stable, and its inhibition by the synthetic auxins is pH independent.

Development of the Stem Tuber in Kohlrabi

Stem swelling in kohlrabi (KR) normally occurred between the third and fifth node and followed initiation of meristematic activity in pith parenchyma cells ; this usually began about 7 weeks after germination and was associated with the death of parenchyma cells in the centre of the pith lower down the stem. Cell division began just above this region, at about the third node and zones of dividing cells extended upwards and laterally, giving rise to more centres of active division. Carbohydrate analyses showed that fructose, glucose, starch, and an unidentified sugar accumulated in the stem. Melezitose appeared in the stem and raffinose in the leaves only after visible bulbing had occurred. Sucrose content fluctuated in both leaves and stems and in the latter was positively correlated with hours of sunshine on the day before sampling. The contents of gibberellin-like substances in the leaves of KR were compared at two stages of development with those of marrowstem kale (MSK). Gibberellins were present in both acidic and neutral fractions in KR tissues and the amounts were similar to those in MSK. Stem swelling in KR could not be attributed to a gross deficiency of gibberellins. Inter- and intra-varietal approach-grafts were made with KR and MSK plants. With KR as 'scion' partner, stem elongation of the scion was not greatly affected by the nature of the stock, but stem swelling was much reduced on MSK stocks. Stem swelling in MSK scions was greater on KR stocks than on MSK. Retention of stock leaves and buds above the union tended to inhibit elongation of the scion partner and also inhibited stem swelling in KR scions on MSK stocks. Applica tion of gibberellic acid to leaves of MSK stocks from which the terminal bud had been removed, increased both stem elongation and swelling in KR scions. KR scions top-grafted on defoliated MSK stocks showed vigorous stem elongation without swelling. MSK scions top-grafted on KR stocks showed reduced stem elongation with abnormal stem thickening. It is suggested that high auxin levels may prevent bulbing in KR and that gibberellin-like substances may facilitate both stem elongation and bulbing when auxin levels are low. Applying gibberellic acid (GA3) or indole-3-acetic acid (IAA) alone or in combination, to young intact plants of KR increased stem height and checked stem swelling. Application of benzyladenine (BA) at io-4 M accelerated swelling and checked stem elongation. GA3 applied with BA (io-4 M) increased both stem height and girth, both with or without added IAA. It was concluded that the requisite conditions for stem swelling included the presence of a kinin2 and a low level of auxin. High levels of auxin prevented stem swelling, whether applied as IAA to young intact plants or as natural auxins from leaves and buds of MSK plants grafted to KR scions. Although applied gibberellins usually tended to