Identification of auxins by a chemical genomics approach

Thirteen auxenic compounds were discovered in a screen of 10 000 compounds for auxin-like activity in Arabidopsis roots. One of the most potent substances was 2-(4-chloro-2-methylphenoxy)-N-(4-H-1,2,4-triazol-3-yl)acetamide (WH7) which shares similar structure to the known auxenic herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). A selected set of 20 analogues of WH7 was used to provide detailed information about the structure-activity relationship based on their efficacy at inhibiting and stimulating root and shoot growth, respectively, and at induction of gene expression. It was shown that WH7 acts in a genetically defined auxin pathway. These small molecules will extend the arsenal of substances that can be used to define auxin perception site(s) and to dissect subsequent signalling events.     

Abnormal Stomatal Behavior and Hormonal Imbalance in flacca, a Wilty Mutant of Tomato: II. Auxin- and Abscisic Acid-like Activity

The wilty tomato mutant, flacca, and the control variety, Rheinlands Ruhm, were compared with regard to the endogenous activity and concentration of auxin- and abscisic acid-like substances during ontogeny. The mutant wilts fast under water deficit because of inability to close its stomata. Symptoms characteristic of excessive auxin are evident in the developing mutant. Among these symptoms are branch and leaf epinasty, excessive rooting along the stem, and increased apical dominance. By using a leucine-incorporation assay, spray of whole plants with 2,4-dichlorophenoxyacetic acid, and wheat coleoptile bioassay, indications were found of an excess of activity and concentration of auxin-like substances in shoots of young and mature mutant plants. The wheat coleoptile bioassay also revealed a much lower amount of substances with abscisic acid-like activity in the mutant compared with the normal plant. In contrast to the appearance during ontogeny of morphological symptoms characteristic of auxin excess in the mutant, the absolute amount of auxin-like substances and their activity in incorporation of leucine decreased with age. A parallel decrease of the concentration and activity of auxin-like compounds was also found in the normal plant. The concentration of abscisic acid-like substances increased with age in both genotypes. The disagreement between the increasing morphological symptoms and the decrease of auxin-like activity and concentration is discussed, together with the possibility of a causal relationship between auxin-and abscisic acid-like activity and stomatal behavior.     

The Relationship between Flower Abortion and Endogenous Auxin Content of Rose Shoots

The amount of endogenous growth substances in stem, flowers and leaves of rose plants grown under different temperature and light conditions has been determined. It appeared to be two main growth promoting factors in the acidic fraction of the ether extract. One of them is assumed to be an auxin, probably indol-3yl-acetic acid (IAA); the other is not identified. The level of auxin was much higher in extracts from shoots grown at high temperature than in shoots grown at low temperature. Increasing light intensity also seemed to increase the auxin content of the shoots. Shoots which developed after a high cut back of the rose stem had a higher auxin content than shoots which developed after a low cut back. These findings are discussed in relation to the effect of temperature, light intensity and cut back practise on blind shoot formation in roses. The result of these investigations strongly indicate that abortion in roses is promoted by a low auxin level in the shoots.     

3-Phenyllactic acid is converted to phenylacetic acid and induces auxin-responsive root growth in Arabidopsis plants

Many microorganisms have been reported to produce compounds that promote plant growth and are thought to be involved in the establishment and maintenance of symbiotic relationships. 3-Phenyllactic acid (PLA) produced by lactic acid bacteria was previously shown to promote root growth in adzuki cuttings. However, the mode of action of PLA as a root-promoting substance had not been clarified. The present study therefore investigated the relationship between PLA and auxin. PLA was found to inhibit primary root elongation and to increase lateral root density in wild-type Arabidopsis, but not in an auxin signaling mutant. In addition, PLA induced IAA19 promoter fused β-glucuronidase gene expression, suggesting that PLA exhibits auxin-like activity. The inability of PLA to promote degradation of Auxin/Indole-3-Acetic Acid protein in a yeast heterologous reconstitution system indicated that PLA may not a ligand of auxin receptor. Using of a synthetic PLA labeled with stable isotope showed that exogenously applied PLA was converted to phenylacetic acid (PAA), an endogenous auxin, in both adzuki and Arabidopsis. Taken together, these results suggest that exogenous PLA promotes auxin signaling by conversion to PAA, thereby regulating root growth in plants.     

Indole auxins in spinach plants grown in long and short days

The endogenous auxin-like substances were analyzed in the shoot extracts of young spinach seedlings, exposed to photoperiodic induction. At least eight indole auxins were found. One of them was identified as tryptophan, the other one is most probably IAA. The plants grown in long days had a higher level of ether soluble auxins than the controls in short days. Separate extractions of plants after each of the eight inductive days showed that the auxin content was not constant, but subjected to irregular oscillations. However, parallel oscillations were also found in control plants grown in short days. Staminate plants were found to contain more endogenous auxins than the pistillate ones. It is concluded that the quantitative changes in auxins during the photoperiodic induction are probably not related to flowering, but to some other growth process, common to all plants in that phase of growth. The higher level of auxins in staminate plants may be the cause of their faster elongation before the onset of flowering.     

Determination of metabolic rate-limitations by precursor feeding in Catharanthus roseus hairy root cultures

Precursors from the terpenoid and tryptophan branches were fed to Catharanthus roseus to determine which of the two branches limits metabolic flux to indole alkaloids. The feeding of tryptophan at 17 days of the culture cycle produced auxin-like effects. Addition of low levels of auxin or tryptophan resulted in significant increases in flux to the indole alkaloids. Conversely, feeding higher levels of auxin or tryptophan resulted in increased branching and thickening of the hairy root cultures. A dramatic reduction in flux to the alkaloids was also observed. However, feeding tryptamine or terpenoid precursors had no effect. Therefore, neither pathway tested revealed to be rate-limiting during the late growth phase. Feeding of either geraniol, 10-hydroxygeraniol, or loganin at 21 days each resulted in significant increases in the accumulation of tabersonine. The addition of tryptophan or tryptamine had no effect during the stationary phase of the growth cycle. Thus, during the early stationary phase of growth the terpenoid pathway appears to be rate-limiting. Combined elicitation with jasmonic acid and feeding either loganin or tryptamine did not further enhance the accumulation of indole alkaloids.     

Citrus leaf abscission. Regulatory role of exogenous auxin and ethylene on peroxidases and endogenous growth substances

Abstract The abscission of citrus leaf explants demonstrates the well-known enhancing effect of ethylene and the delaying one of auxin when treatment is started at excision time. Total peroxidase activity increases differently in tissues of the blade, abscission zone, and petiole. The highest activity at zero time is recovered in abscission zone in which also the response to the abscission regulators is the most visible. Isoperoxidase profiles are modified in opposite directions by ethylene and auxin respectively. Both regulators affect the activity of the same cathodic and anodic isoperoxidases without any qualitative changes. By the same time, auxin-like compounds increase in isolated abscission zones at 24 h from excision and decrease at 48 h. The level of one inhibitor complex undergoes an inverse variation. It is suggested that the increase in auxin during the first stage of abscission is necessary for influencing the growth of cells which is required to cause abscission.     

The influence of gamma radiation on the biosynthesis of indoles and gibberellins in barley the action of zinc on the restitution of growth substance level in irradiated plants

Investigations were made on the effect of exposing barley seeds to gamma-radiation (5–40 kR), alone and in combination with the application of zinc (soaking the seeds in solutions containing 5.10^?5–5.10^?1% Zn for 12 hours before sowing) on growth and on the content of tryptophan, indole auxins and gibberellin-like substances in seven-day plants. Radiation decreased both growth and the content of tryptophan (e.g. by about 53% at 30 kR), of indole auxins (by about 60% auxin in the zone of IAA on the chromatogram at 30 kR), and also the content of gibberellin-like substances (by about 67% gibberellin content in the zone of GA₃ on the chromatogram) of plants. The irradiation of standard samples of tryptophan, indolyl-acetic acid and gibberellic acid alone with many times greater doses (up to 1000 kR) did not lead to marked radiochemical degradation of these substances. It can be assumed that radiation damages the enzyme systems “synthesizing” natural growth substances in plants. The damaging effect of radiation on auxins is already displayed in the synthesis of tryptophan, which is inhibited. Zinc interacts with the damaging effect of radiation on growth. Optimum concentrations of zinc (5.10^?3% Zn) counteract the effect of radiation, up to doses of about 12 kR, on the growth in height in 7-day plants so that it is equal to the controls. Normal content of tryptophan and auxin in the position of indolecetic acid on chromatograms can only be reached by the addition of zinc when the dose of radiation was not greater than about 8 kR, which is less than the influence exerted by zinc on the restitution of growth. On the other hand, the biosynthesis of gibberellin-like substances at the position of gibberellic acid on chromatograms can be restored by zinc to their original level to doses of up to 30 kR. The increased biosynthesis of auxins and gibberellins caused by zinc in irradiated plants is explained by the activation of the remaining and non—damaged enzyme systems carrying out this biosynthesis. The activation of the biosynthesis of growth substances by zinc will also contribute to the restitution effect of zinc on the growth of plants from irradiated seeds.     

THE EFFECT OF AUXINS UPON PROTOPLASMIC STREAMING

1. Evidence has accumulated that the action of auxins in promoting growth is exerted not upon the cell wall but upon the cell contents; i.e., the protoplasm. Following indications previously obtained, therefore, the effect of auxins on the rate of protoplasm streaming in the Avena coleoptile was studied. 2. Indole-3-acetic acid, the most active auxin available in pure form, was found to increase the rate of streaming in the epidermal cells of the Avena coleoptile at concentrations between 0.5 and 0.002 mg. per liter, the maximum increase being brought about at 0.01 mg. per liter. This concentration is approximately that which, applied in agar to one side of the decapitated coleoptile, would give a curvature of 1°; i.e., it is well within the range of concentrations active in growth promotion. It is, however, much less than that which produces maximum elongation in immersed sections of Avena coleoptiles. 3. This accelerating effect is readily determined quantitatively by comparison with the streaming in control coleoptiles in pure water, which, if thoroughly aerated, maintain a constant rate for over an hour. The accelerating effect takes place immediately and is over within about 30 minutes. 4. Concentrations of indole-3-acetic acid greater than 0.5 mg.per liter inhibit the streaming, the effect being also over in about 30 minutes, and its extent increasing with increasing auxin concentration. This parallels the effect of high auxin concentrations in inhibiting elongation, although the inhibition of streaming is obtained at much lower concentrations than inhibit elongation. 5. The effects of indole-3-acetic acid on streaming are not specific for that substance, but appear to be common to auxins in general. Thus coumaryl-3-acetic acid and allocinnamic acid, both of which bring about cell enlargement, root formation, and bud inhibition, i.e. are typical auxins, also cause an immediate acceleration of the rate of streaming, and as with indole-acetic add the effect is over in about 30 minutes. The concentrations of these two substances which produce the maximum effect are about ten times that of indole-acetic acid, which approximately corresponds with their relative auxin activities. The curves relating concentrations of these substances to their effects on streaming are very similar to that for indole-acetic acid. 6. On the other hand, certain substances which are known to affect streaming in other materials do not produce any effect comparable to that of auxin. Ethylene chlorhydrin, histidine, and urea in all concentrations were without effect on streaming in the Avena coleoptile within the first 30 minutes of treatment. 7. The effects produced by the auxins were not due to pH. 8. The action on streaming here studied is evidently quite different from the re-starting of streaming after its cessation, studied by Fitting in Vallisneria. Correspondingly histidine, which in Fitting''s experiments showed activity down to 10^–7 M, is inactive here. 9. Per contra, the effect of auxin here studied is on normal streaming. It takes place immediately and at concentrations in the same range as those which produce growth. The curve of effect against concentration parallels that for growth although the actual concentration values differ. It is therefore reasonable to suppose that the effect of auxin on streaming is closely connected with one of the first stages of its effect on the growth process.     

Novel auxin transport inhibitors phenocopy the auxin influx carrier mutation aux1

The hormone auxin is transported in plants through the combined actions of diffusion and specific auxin influx and efflux carriers. In contrast to auxin efflux, for which there are well documented inhibitors, understanding the developmental roles of carrier-mediated auxin influx has been hampered by the absence of specific competitive inhibitors. However, several molecules that inhibit auxin influx in cultured cells have been described recently. The physiological effects of two of these novel influx carrier inhibitors, 1-naphthoxyacetic acid (1-NOA) and 3-chloro-4-hydroxyphenylacetic acid (CHPAA), have been investigated in intact seedlings and tissue segments using classical and new auxin transport bioassays. Both molecules do disrupt root gravitropism, which is a developmental process requiring rapid auxin redistribution. Furthermore, the auxin-insensitive and agravitropic root-growth characteristics of aux1 plants were phenocopied by 1-NOA and CHPAA. Similarly, the agravitropic phenotype of inhibitor-treated seedlings was rescued by the auxin 1-naphthaleneacetic acid, but not by 2,4-dichlorophenoxyacetic acid, again resembling the relative abilities of these two auxins to rescue the phenotype of aux1. Further investigations have shown that none of these compounds block polar auxin transport, and that CHPAA exhibits some auxin-like activity at high concentrations. Whilst results indicate that 1-NOA and CHPAA represent useful tools for physiological studies addressing the role of auxin influx in planta, 1-NOA is likely to prove the more useful of the two compounds.     

Changes in endogenous level of auxins and cytokinins in axillary buds ofpisutn sativum L. in relation to apical dominance

Decapitation of the stem in one-week-old pea seedlings below the first node causes a rapid outgrowth of the two cotyledonary buds. One of them soon becomes dominant, while the other one is inhibited, but can be released from inhibition by cutting off the dominant bud. The level of endogenous auxins and cytokinins was determined in dominant and inhibited buds, as well as in released buds at different time intervals after deinhibition. It was found that the inhibited buds contained very little acidic, ether soluble auxins, a high level of tryptophan and also a high level of cytokinins, in comparison with dominant buds. When the inhibited buda were released from inhibition, their auxin content rose, while that of tryptophan and cytokinins decreased, reaching the level found in dominant buds within six days. Specific changes in content of two undetermined auxin-like substances were found in released buds during de-inhibition. These results are discussed in relation to the current views on the regulation of apical dominance.     

Mutation of E1-CONJUGATING ENZYME-RELATED1 decreases RELATED TO UBIQUITIN conjugation and alters auxin response and development

The ubiquitin-like protein RELATED TO UBIQUITIN (RUB) is conjugated to CULLIN (CUL) proteins to modulate the activity of Skp1-Cullin-F-box (SCF) ubiquitylation complexes. RUB conjugation to specific target proteins is necessary for the development of many organisms, including Arabidopsis (Arabidopsis thaliana). Here, we report the isolation and characterization of e1-conjugating enzyme-related1-1 (ecr1-1), an Arabidopsis mutant compromised in RUB conjugation. The ecr1-1 mutation causes a missense change located two amino acid residues from the catalytic site cysteine, which normally functions to form a thioester bond with activated RUB. A higher ratio of unmodified CUL1 relative to CUL1-RUB is present in ecr1-1 compared to wild type, suggesting that the mutation reduces ECR1 function. The ecr1-1 mutant is resistant to the auxin-like compound indole-3-propionic acid, produces fewer lateral roots than wild type, displays reduced adult height, and stabilizes a reporter fusion protein that is degraded in response to auxin, suggesting reduced auxin signaling in the mutant. In addition, ecr1-1 hypocotyls fail to elongate normally when seedlings are grown in darkness, a phenotype shared with certain other RUB conjugation mutants that is not general to auxin-response mutants. The suite of ecr1-1 molecular and morphological phenotypes reflects roles for RUB conjugation in many aspects of plant growth and development. Certain ecr1-1 elongation defects are restored by treatment with the ethylene-response inhibitor silver nitrate, suggesting that the short ecr1-1 root and hypocotyl result from aberrant ethylene accumulation. Further, silver nitrate supplementation in combination with various auxins and auxin-like compounds reveals that members of this growth regulator family may differentially rely on ethylene signaling to inhibit root growth.     

Genetic Analysis of the Effects of Polar Auxin Transport Inhibitors on Root Growth in Arabidopsis thaliana

Polar auxin transport inhibitors, including N-1-naphthylphthalamicacid (NPA) and 2,3,5-triiodobenzoic acid (TIBA), have variouseffects on physiological and developmental events, such as theelongation and tropism of roots and stems, in higher plants.We isolated NPA-resistant mutants of Arabidopsis thaliana, withmutations designated pir1 and pir2, that were also resistantto TIBA. The mutations specifically affected the root-elongationprocess, and they were shown ultimately to be allelic to aux1and ein2, respectively, which are known as mutations that affectresponses to phytohormones. The mechanism of action of auxintransport inhibitors was investigated with these mutants, inrelation to the effects of ethylene, auxin, and the polar transportof auxin. With respect to the inhibition of root elongationin A. thaliana, we demonstrated that (1) the background levelof ethylene intensifies the effects of auxin transport inhibitors,(2) auxin transport inhibitors might act also via an inhibitorypathway that does not involve ethylene, auxin, or the polartransport of auxin, (3) the hypothesis that the inhibitory effectof NPA on root elongation is due to high-level accumulationof auxin as a result of blockage of auxin transport is not applicableto A. thaliana, and (4) in contrast to NPA, TIBA itself hasa weak auxin-like inhibitory effect. (Received April 12, 1996; Accepted September 2, 1996)     

Changes in root development of Arabidopsis promoted by organic matter from oxisols

The presence of ramified and abundant lateral roots is fundamental to plant growth on highly weathered soils. In this work, the effects of humic acid (HA), fulvic acid and hexanic–methanolic (HM) extract, after alkaline extraction from topsoil of seven different oxisols, on the development of roots of Arabidopsis thaliana was evaluated. Furthermore, we used another emergent plant model, that is, micro‐tom (MT) tomatoes with size similar to that of Arabidopsis to test the effects of HA. It was observed that both humic fractions and the HM extract were able to change the root development, improving the number of lateral roots and their development in comparison to control plants. The promotion of root growth by the three organic matter fractions was higher than that observed by 10^?6 mol L^?1 indole acetic acid. The treatment of MT tomato mutant, less sensitive to auxin, with HA did not promote the emergence of lateral roots, being an additional indication of auxin‐like activities of HA. However, some organic matter fractions exhibited, besides promotion of lateral roots number, increase in the length of principal root, which is not a typical auxin effect, indicating that these substances could contain other physiologically active substances.     

Shoot regeneration from spinach hypocotyl segments by short term treatment with 5,6-dichloro-indole-3-acetic acid

Factors affecting shoot regeneration from hypocotyl segments of spinach (Spinacia oleracea L.) were investigated. When expiants were cultured on medium containing 10 mg/l IAA for 7 weeks, 3 out of 9 cultivars showed relatively high shoot regeneration response (15 – 35%). The other PGRs tested had no effect on shoot regeneration. However, the transfer of explants from auxin-containing medium to auxin-free medium 20 d after culture induced shoot formation from expiants cultured on media containing each of the auxin sources tested individually. By applying this short term auxin treatment, more than 80% shoot regeneration was obtained on medium containing 5–20 mg/l 5,6-Cl₂-IAA, compared to less than 30% with 10–20 mg/l IAA treatment.Abbreviations BAP 6-benzylaminopurine - NAA 1naphthaleneacetic acid - IAA indole-3-acetic acid - 2,4D 2,4-dichlorophenoxyacetic acid - 5,6-Cl₂-IAA 5,6dichloro-indole-3-acetic acid - PGR plant growth regulator - A-PGR auxin-like plant growth regulator     

Rates of uptake and metabolism of indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid by cultured leaf segments at different stages of development in wheat

Immature leaf tissue of Triticum timopheevi Zukh. responded to supplied auxin and showed cell division in culture. The rates of uptake and of metabolism of indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid by such tissues were measured and compared with those of mature auxin-unresponsive tissue. The purpose of these experiments was to determine whether or not the concentration of auxin in cultured mature tissue was a factor limiting the cell division response to auxin. The data indicate that neither alterations in rates of uptake nor alterations in rates of metabolism could explain the loss of responsiveness to auxin which apparently occurs during cell differentiation. The results are discussed in the context of the view that changes in cell sensitivity to growth substances and not only the concentration of these compounds, play an important role in plant growth and development.     

Endogenous auxins in apical buds ofChenopodium rubrum L. after application of (2-chloroethyl) trimethylammonium chloride (CCC)

Apical buds ofChenopodium rubrum from plants treated with CCC contain more endogenous auxins than buds from control plants, the level of these compounds increasing with the application of rising concentrations of the retardant. An especially marked increase was observed in the level of substance “X” which on chromatographic separation runs in the zone of tryptamine or its derivative. Since it has been shown in previous experiments that the inhibitory effect of CCC on flowering ofChenopodium rubrum may be reversed by indole-3-acetic acid (IAA) it is believed that the increase in auxins after application of CCC does not concern biologically active substances immediately available to the plant. It seems more likely that inactive precursors are involved which cannot be converted to the active substance in the presence of CCC, possibly due to blocking of the pertinent enzyme. If we assume that the wheat coleoptile used in the auxin bioassay in our experiments contains the pertinent enzyme it might convert the inactive precursors to active substances and, therefore, exhibit a growth stimulation even though the substances concerned would not necessarily be active in the buds from which they were extracted.     

Content of auxin-inhibitor-and gibberellin-like substances in humic acids

The content of auxin-, inhibitors-and gibberellin-like substances in freeze-dried humic acids was studied. The results indicate the existence of growth-promoting substances in humic acids, possibly corresponding to IAA or its precursors. Inhibitory activity is more marked during germination than during the growth process. The inhibitory effect caused by high concentration of humic acids could be due to an auxin-like action rather than an input per se of growth inhibitors. A significant amount of both free and conjugated gibberellin-like substances were observed.     

THE EFFECT OF AUXINS ON PROTOPLASMIC STREAMING. III

1. A new method is described which gives a continuous record of the absolute rate of protoplasmic streaming in epidermal cells of the Avena coleoptile. 2. With this method a study was made of the influence of malate and iodoacetate on streaming velocity, in order to make correlations with the previously established effects of these substances on growth and respiration. 3. In the presence of optimum concentrations of indole-3-acetic acid in freshly cut sections, malate had no effect on streaming. In the presence of very low concentrations of the auxin, however, malate increased the range of response, so that the threshold of auxin sensitivity was lowered some ten times by the malate. Malate alone had no effect on streaming. 4. In coleoptile sections, soaked overnight in sugar solution or in water, the acceleration of streaming normally caused by auxin almost disappears, but the presence of malate causes large accelerations of streaming by the auxin. 5. Similarly, in sections from old coleoptiles, which no longer show acceleration of streaming by auxin, the acceleration is restored when malate is added together with the auxin. 6. Malate does not enter the cell as rapidly as does auxin, but easily detectable amounts penetrate within 30 minutes. 7. Iodoacetate in the concentration which inhibits growth (5 x 10^–5 M) completely inhibits the acceleration of streaming by auxin. In still lower concentrations iodoacetate slightly accelerates streaming. Higher concentrations, up to 2 x 10^–4 M, did not reduce the rate of streaming below that of controls without auxin. The effect of iodoacetate is therefore to inhibit the acceleration caused by auxin and not to affect the basal streaming rate. 8. It is concluded that, just as for growth and respiration, malate is necessary for the response to auxin shown by acceleration of streaming. This further strengthens the triple parallel between the effects of auxin on streaming, growth, and respiration, all of which are apparently mediated by the 4-carbon acid system.     

A role for the root cap in root branching revealed by the non-auxin probe naxillin

The acquisition of water and nutrients by plant roots is a fundamental aspect of agriculture and strongly depends on root architecture. Root branching and expansion of the root system is achieved through the development of lateral roots and is to a large extent controlled by the plant hormone auxin. However, the pleiotropic effects of auxin or auxin-like molecules on root systems complicate the study of lateral root development. Here we describe a small-molecule screen in Arabidopsis thaliana that identified naxillin as what is to our knowledge the first non-auxin-like molecule that promotes root branching. By using naxillin as a chemical tool, we identified a new function for root cap-specific conversion of the auxin precursor indole-3-butyric acid into the active auxin indole-3-acetic acid and uncovered the involvement of the root cap in root branching. Delivery of an auxin precursor in peripheral tissues such as the root cap might represent an important mechanism shaping root architecture.