Native Auxin Research Articles

Production of IAA by Endophytic Fungi as Phyllanthus emblica Linn.

Indole Acetic Acid (IAA), a phytohormone, has been considered as the most important native auxin. The Amla (Phyllanthus emblica Linn.) serve as an abundance of IAA-producing sources fungus and produce significant quantities of IAA in tryptophan-added medium. Different concentration of L-tryptophan between 1, 2 and 5 mg/ml were chosen for the IAA production. The study shows that the corresponding L-tryptophan concentration was associated with a progressive rise in IAA generation. The impact on IAA production was anticipated to last up to 10 days, with the maximum IAA production being shown in an incubation period of 15 days. Four isolates in total were found to be IAA-producing strains; two of these produced IAA through medium components, and physical conditions were tuned for IAA production. The Amla stem and leaf tissues have the strongest growth-promoting effect in Aspergillus ustus (72 g/20 ml) among the isolates. The positive effects on crop development and yield are attributed to the presence of such growth-promoting fungus.

The DR5 and E8 reporters are suitable systems for studying the application of the <i>Ruby</i> reporter gene in tomato

<sec><title/> Tomato (<italic>Solanum lycopersicum</italic>) fruits can be found in a range of colors, including green, yellow, red, pink and purple. The various colors are determined by the accumulation of molecules such as chlorophyll, carotenoids (including lycopene) and flavonoids. Since the DR5 promoter is induced by auxin, we can infer that the auxin responses differ at different developmental stages in tomato. Fruit-specific promoters can effectively drive gene expression in fruits, and the E8 promoter is one of the most widely used fruit-specific promoters. In this study, DR5::<italic>Ruby</italic> transgenic tomato plants were used to further clarify the auxin content at different developmental stages in tomato. The results showed that the nonuniformity of <italic>Ruby</italic> activity was consistent with the natural distribution characteristics of native auxin, and the results were also consistent with the qRT‒PCR results. Furthermore, the accumulation of endogenous auxin at the immature green stage (IMG) was higher than that at the other stages. </sec><sec><title/> In addition, E8::<italic>Ruby</italic> was transformed into different tomato varieties (red, white and yellow) to generate new tomato varieties with fruit of different colors. The results showed that the E8::<italic>Ruby-</italic>transformed plants exhibited asymmetrical pink coloration in different tomato tissues of plants from different tomato cultivars. In conclusion, all the results showed that DR5 and E8 are suitable systems for studying the application of the <italic>Ruby</italic> reporter gene in tomato. Moreover, the study will provide insight for the improvement of tomato fruit color via biotechnology. </sec>

OsIAGT1 Is a Glucosyltransferase Gene Involved in the Glucose Conjugation of Auxins in Rice

BackgroundIn cereal crop rice, auxin is known as an important class of plant hormone that regulates a plethora of plant growth and development. Glycosylation of auxin is known to be one of the important mechanisms mediating auxin homeostasis. However, the relevant auxin glucosyltransferase (GT) in rice still remains largely unknown.ResultsIn this study, using known auxin glucosyltransferases from other species as queries, twelve putative auxin UDP-glycosyltransferase (UGT) genes were cloned from rice and the one showing highest sequence similarity, named as OsIAGT1, was expressed as recombinant protein. In vitro enzymatic analysis showed that recombinant OsIAGT1 was capable of catalyzing glucosylation of IAA, IBA and other auxin analogs, and that OsIAGT1 is quite tolerant to a broad range of reaction conditions with peak activity at 30 °С and pH 8.0. OsIAGT1 showed favorite activity towards native auxins over artificially synthesized ones. Further study indicated that expression of OsIAGT1 can be upregulated by auxin in rice, and with OsIAGT1 overexpressing lines we confirmed that OsIAGT1 is indeed able to glucosylate IAA in vivo. Consistently, ectopic expression of OsIAGT1 leads to declined endogenous IAA content, as well as upregulated auxin synthesis genes and reduced expression of auxin-responsive genes, which likely leads to the reduced plant stature and root length in OsIAGT1 overexpression lines.ConclusionOur result indicated that OsIAGT1 plays an important role in mediating auxin homeostasis by catalyzing auxin glucosylation, and by which OsIAGT1 regulates growth and development in rice.

A bacterial assay for rapid screening of IAA catabolic enzymes

BackgroundPlants rely on concentration gradients of the native auxin, indole-3-acetic acid (IAA), to modulate plant growth and development. Both metabolic and transport processes participate in the dynamic regulation of IAA homeostasis. Free IAA levels can be reduced by inactivation mechanisms, such as conjugation and degradation. IAA can be conjugated via ester linkage to glucose, or via amide linkage to amino acids, and degraded via oxidation. Members of the UDP glucosyl transferase (UGT) family catalyze the conversion of IAA to indole-3-acetyl-1-glucosyl ester (IAGlc); by contrast, IAA is irreversibly converted to indole-3-acetyl-l-aspartic acid (IAAsp) and indole-3-acetyl glutamic acid (IAGlu) by Group II of the GRETCHEN HAGEN3 (GH3) family of acyl amido synthetases. Dioxygenase for auxin oxidation (DAO) irreversibly oxidizes IAA to oxindole-3-acetic acid (oxIAA) and, in turn, oxIAA can be further glucosylated to oxindole-3-acetyl-1-glucosyl ester (oxIAGlc) by UGTs. These metabolic pathways have been identified based on mutant analyses, in vitro activity measurements, and in planta feeding assays. In vitro assays for studying protein activity are based on producing Arabidopsis enzymes in a recombinant form in bacteria or yeast followed by recombinant protein purification. However, the need to extract and purify the recombinant proteins represents a major obstacle when performing in vitro assays.ResultsIn this work we report a rapid, reproducible and cheap method to screen the enzymatic activity of recombinant proteins that are known to inactivate IAA. The enzymatic reactions are carried out directly in bacteria that produce the recombinant protein. The enzymatic products can be measured by direct injection of a small supernatant fraction from the bacterial culture on ultrahigh-performance liquid chromatography coupled to electrospray ionization tandem spectrometry (UHPLC–ESI-MS/MS). Experimental procedures were optimized for testing the activity of different classes of IAA-modifying enzymes without the need to purify recombinant protein.ConclusionsThis new method represents an alternative to existing in vitro assays. It can be applied to the analysis of IAA metabolites that are produced upon supplementation of substrate to engineered bacterial cultures and can be used for a rapid screening of orthologous candidate genes from non-model species.

An improved auxin-inducible degron system preserves native protein levels and enables rapid and specific protein depletion.

Rapid perturbation of protein function permits the ability to define primary molecular responses while avoiding downstream cumulative effects of protein dysregulation. The auxin-inducible degron (AID) system was developed as a tool to achieve rapid and inducible protein degradation in nonplant systems. However, tagging proteins at their endogenous loci results in chronic auxin-independent degradation by the proteasome. To correct this deficiency, we expressed the auxin response transcription factor (ARF) in an improved inducible degron system. ARF is absent from previously engineered AID systems but is a critical component of native auxin signaling. In plants, ARF directly interacts with AID in the absence of auxin, and we found that expression of the ARF PB1 (Phox and Bem1) domain suppresses constitutive degradation of AID-tagged proteins. Moreover, the rate of auxin-induced AID degradation is substantially faster in the ARF-AID system. To test the ARF-AID system in a quantitative and sensitive manner, we measured genome-wide changes in nascent transcription after rapidly depleting the ZNF143 transcription factor. Transcriptional profiling indicates that ZNF143 activates transcription in cis and regulates promoter-proximal paused RNA polymerase density. Rapidly inducible degradation systems that preserve the target protein's native expression levels and patterns will revolutionize the study of biological systems by enabling specific and temporally defined protein dysregulation.

Evidence for Exaptation of the Marchantia polymorpha M20D Peptidase MpILR1 into the Tracheophyte Auxin Regulatory Pathway.

Auxin homeostasis is tightly regulated by several mechanisms, including conjugation of the hormone to specific moieties, such as amino acids or sugar. The inactive phytohormone conjugate is stored in large pools in plants and hydrolyzed to regain full activity. Many conjugate hydrolases (M20D metallopeptidases) have been identified and characterized throughout the plant kingdom. We have traced this regulatory gene family back to liverwort (Marchantia polymorpha), a member of the most ancient extant land plant lineage, which emerged approximately 475 million years ago. We have isolated and characterized a single hydrolase homologue, dubbed M. polymorpha IAA-Leucine Resistant1 (MpILR1), from liverwort. MpILR1 can hydrolyze two auxin (indole acetic acid [IAA]) substrates (IAA-Leucine and IPA-Alanine) at very low levels of activity, but it cannot hydrolyze the two native auxin conjugates of liverwort (IAA-Glycine and IAA-Valine). We conclude from these results that liverwort likely does not employ active auxin conjugate hydrolysis as a regulatory mechanism and that conjugate homeostasis likely takes place in liverwort by passive background degradation. Furthermore, we present evidence that MpILR1 was probably exapted by tracheophytes over evolutionary time into the auxin regulatory pathway.

Characterization of transmembrane auxin transport in Arabidopsis suspension-cultured cells

Polar auxin transport is a crucial process for control and coordination of plant development. Studies of auxin transport through plant tissues and organs showed that auxin is transported by a combination of phloem flow and the active, carrier-mediated cell-to-cell transport. Since plant organs and even tissues are too complex for determination of the kinetics of carrier-mediated auxin uptake and efflux on the cellular level, simplified models of cell suspension cultures are often used, and several tobacco cell lines have been established for auxin transport assays. However, there are very few data available on the specificity and kinetics of auxin transport across the plasma membrane for Arabidopsis thaliana suspension-cultured cells. In this report, the characteristics of carrier-mediated uptake (influx) and efflux for the native auxin indole-3-acetic acid and synthetic auxins, naphthalene-1-acetic and 2,4-dichlorophenoxyacetic acids (NAA and 2,4-D, respectively) in A. thaliana ecotype Landsberg erecta suspension-cultured cells (LE line) are provided. By auxin competition assays and inhibitor treatments, we show that, similarly to tobacco cells, uptake carriers have high affinity towards 2,4-D and that NAA is a good tool for studies of auxin efflux in LE cells. In contrast to tobacco cells, metabolic profiling showed that only a small proportion of NAA is metabolized in LE cells. These results show that the LE cell line is a useful experimental system for measurements of kinetics of auxin carriers on the cellular level that is complementary to tobacco cells.

Regulation of Auxin Homeostasis and Gradients in Arabidopsis Roots through the Formation of the Indole-3-Acetic Acid Catabolite 2-Oxindole-3-Acetic Acid

The native auxin, indole-3-acetic acid (IAA), is a major regulator of plant growth and development. Its nonuniform distribution between cells and tissues underlies the spatiotemporal coordination of many developmental events and responses to environmental stimuli. The regulation of auxin gradients and the formation of auxin maxima/minima most likely involve the regulation of both metabolic and transport processes. In this article, we have demonstrated that 2-oxindole-3-acetic acid (oxIAA) is a major primary IAA catabolite formed in Arabidopsis thaliana root tissues. OxIAA had little biological activity and was formed rapidly and irreversibly in response to increases in auxin levels. We further showed that there is cell type-specific regulation of oxIAA levels in the Arabidopsis root apex. We propose that oxIAA is an important element in the regulation of output from auxin gradients and, therefore, in the regulation of auxin homeostasis and response mechanisms.

Narciclasine inhibits the responses of Arabidopsis roots to auxin

The plant hormone auxin plays a central role in the regulation of plant growth and development, as well as in responses to environmental stimuli. Narciclasine (NCS, an Amaryllidaceae alkaloid) isolated from Narcissus tazetta bulbs has a broad range of inhibitory effects on plants. In this study, the role of NCS in responses to auxin in Arabidopsis thaliana roots was investigated. We demonstrated the inhibitory effects of NCS on auxin-inducible lateral root formation, root hair formation, primary root growth, and the expression of primary auxin-inducible genes in Arabidopsis roots using DR5::GUS reporter gene, native auxin promoters (IAA12::GUS, IAA13::GUS), and quantitative reverse transcription PCR analysis. Results also showed that NCS did not affect the expression of cytokinin-inducible ARR5::GUS reporter gene. NCS relieved the auxin-enhanced degradation of the Aux/IAA repressor modulated by the SCFTIR1 ubiquitin-proteasome pathway. In addition, NCS did not alter the auxin-stimulated interaction between IAA7/AXR2 (Aux/IAA proteins) and the F-box protein TIR1 activity of the proteasome. Taken together, these results suggest that NCS acts on the auxin signaling pathway upstream of TIR1, which modulates Aux/IAA protein degradation, and thereby affects the auxin-mediated responses in Arabidopsis roots.

Immunophilin-like TWISTED DWARF1 Modulates Auxin Efflux Activities of Arabidopsis P-glycoproteins

The immunophilin-like protein TWISTED DWARF1 (TWD1/FKBP42) has been shown to physically interact with the multidrug resistance/P-glycoprotein (PGP) ATP-binding cassette transporters PGP1 and PGP19 (MDR1). Overlapping phenotypes of pgp1/pgp19 and twd1 mutant plants suggested a positive regulatory role of TWD1 in PGP-mediated export of the plant hormone auxin, which controls plant development. Here, we provide evidence at the cellular and plant levels that TWD1 controls PGP-mediated auxin transport. twd1 and pgp1/pgp19 cells showed greatly reduced export of the native auxin indole-3-acetic acid (IAA). Constitutive overexpression of PGP1 and PGP19, but not TWD1, enhanced auxin export. Coexpression of TWD1 and PGP1 in yeast and mammalian cells verified the specificity of the regulatory effect. Employing an IAA-specific microelectrode demonstrated that IAA influx in the root elongation zone was perturbed and apically shifted in pgp1/pgp19 and twd1 roots. Mature roots of pgp1/pgp19 and twd1 plants revealed elevated levels of free IAA, which seemed to account for agravitropic root behavior. Our data suggest a novel mode of PGP regulation via FK506-binding protein-like immunophilins, implicating possible alternative strategies to overcome multidrug resistance.

Auxins and Cytokinins in Plant Development 2005

The International Symposium ‘‘Auxins and Cytokinins in Plant Development 2005’’ (ACPD 2005), held in Prague, Czech Republic, July 7–12, 2005, was well timed to reflect the current enormous progress in research of the two classes of phytohormones. The symposium was a continuation of a long tradition of conferences devoted to plant growth substances organized by the Institute of Experimental Botany of the Academy of Sciences of the Czech Republic. Five international symposia have already been held in the baroque chateau at Liblice near Prague, and the sixth and last one, ‘‘Auxins and Cytokinins in Plant Development 1999’’ was held in Prague itself. The ACPD 2005 meeting provided a platform for discussions on recent achievements and future directions in and prospects for research on the two hormones for controlling plant development. The meeting fulfilled expectations and highlighted the very recent advances in molecular biology, biochemistry, and physiology of both phytohormones, as well as some aspects of their applications in biotechnology. The scientific program consisted of two opening lectures and seven sessions, which always included both hormones: (1) Biosynthesis and metabolism, (2) Transport, (3) Signaling, (4) Pattern formation and development, (5) Auxin-cytokinin crosstalk and interaction with other hormones, (6) Responses to environment, and (7) Novel methods. The interplay of these processes during plant development is shown in Figure 1. The conference attracted 180 participants from 28 countries in Europe, North America, Asia, Africa, and Australia. Abstracts of oral and poster presentations were published in Biologia Plantarum 49 (Suppl.) 2005 (and are also available on the Web at http://acpd. cas.cz). A comparison between the proportion of contributions to the topics selected for the 1999 conference with that in 2005 revealed that most of the topics selected in 2005 received even attention by the participants, reflecting the attractive program of the meeting (Figure 2). Most noticeable was the increase in contributions in the transport session in 2005 compared to 1999. In the first plenary lecture which he dedicated to Prof. Milan Kutacek, Athanasios Theologis (Plant Gene Expression Center, Albany, NY, USA) focused on the native auxin, indole-3-acetic acid, as a ‘‘simple *Authors are listed alphabetically Received 17 October 2005; accepted 18 October 2005; Online publication 28 February 2006 *Corresponding author ; e-mail: eva.zazim@ueb.cas.cz J Plant Growth Regul (2006) 25:89–97 DOI: 10.1007/s00344-005-0120-0

Asymmetric distribution of auxin correlates with gravitropism and phototropism but not with autostraightening (autotropism) in pea epicotyls.

The relationships between the distribution of the native auxin indole-3-acetic acid (IAA) and tropisms in the epicotyl of red light-grown pea (Pisum sativum L.) seedlings have been investigated. The distribution measurement was made in a defined zone of the third internode, using (3)H-IAA applied from the plumule as a tracer. The tropisms investigated were gravitropism, pulse-induced phototropism, and time-dependent phototropism. The investigation was extended to the phase of autostraightening (autotropism) that followed gravitropic curvature. It was found that IAA is asymmetrically distributed between the two halves of the zone, with a greater IAA level occurring on the convex side, at early stages of gravitropic and phototropic curvatures. This asymmetry was found in epidermal peels and, except for one case (pulse-induced phototropism), no asymmetry was detected in whole tissues. It was concluded, in support of earlier results, that auxin asymmetry mediates gravitropism and phototropism and that the epidermis or peripheral cell layers play an important role in the establishment of auxin asymmetry in pea epicotyls. During autostraightening, which results from a reversal of growth asymmetry, the extent of IAA asymmetry was reduced, but its direction was not reversed. This result demonstrated that autostraightening is not regulated through auxin distribution. In this study, the growth on either side of the investigated zone was also measured. In some cases, the measured IAA distribution could not adequately explain the local growth rate, necessitating further detailed investigation.

Regulation of lycopene formation in cell suspension culture of VFNT tomato(Lycopersicon esculentum)by CPTA, growth regulators, sucrose, and temperature

The onium compound 2-(4-chlorophenylthio)-triethylamine (CPTA) was used to increase lycopene formation to levels approximating those in field-or glasshouse-grown fruit, and then growth regulators, sucrose and temperature were used to regulate lycopene accumulation. It was found that the native auxin indole-3-acetic acid (IAA) was substantially more effective than 2,4-dichlorophenoxyacetic acid (2,4-D) in promoting lycopene formation, sucrose inhibited lycopene formation (cell basis), and temperature produced a pattern similar to that observed in the field with a temperature optimum between 18 and 26 °C. Suggestions for further improvements in technique are included

Role of plasma membrane redox activities in elongation growth in plants

Comparing isolated plasma membrane vesicles and excised hypocotyl segments from etiolated seedlings of soybean [Glycine max (L.) Merr. cv. Williams], certain antiproliferative agents that inhibited growth inhibited plasma membrane redox activities. Additionally, auxins that stimulated growth stimulated plasma membrane redox activities. Hormone stimulation was restricted to NADH oxidase (determined from disappearance of NADH) and was given both by isolated plasma membranes and by a soluhilizedenzyme preparation. Comparing IAA, the native auxin regulator, and 2,4‐D, a synthetic regulator, stimulation was observed, hut the dose‐response curves were different. Yet, the dose‐response relationships of both stimulation of auxin growth and stimulation of NADH oxidase were parallel. Inhibition of auxin‐induced growth by antiproliferative drugs was more complex. Some, like actinomycin D, preferentially inhibited NADH oxidase (EC 1.6.99.2) but inhibited NADH‐ferricya‐nide oxido‐reductase (EC 1.6.99.3) as well. Others, like adriamycin, inhibited primarily the NADH‐ferricyanide oxido‐reductase. Therefore, growth control by auxin appeared to involve NADH oxidase as a rate‐limiting terminal oxidase to link electron flow from NADH to oxygen. This observation may provide a fundamental difference from animal cells. With the latter, impermeant electron acceptors such as diferric transferrin or ferricyanide fulfill such a role. In plants, these impermeant electron acceptors were without effect on growth or were growth inhibitory.

In vitro binding of auxin to particulate fractions from the shoots of dark-grown wheat seedlings

A binding site for auxins was found in the 50,000g pellet from a homogenate of shoots from dark-grown wheat seedlings. The optimum conditions for the binding of native auxin, IAA, were within the range of physiological conditions of growth (pH 5.2, temperature 20° C). The binding site displayed a high affinity to IAA (affinity constant about 107M−1, i.e. dissociation constant about 10−8M) and low capacity, 60 p mol per 1 g of fresh weight. The binding capacity of 3.5-days-old shoots is represented by about 56% and 44% of that of leaves and coleoptiles, respectively. The more rapidly growing leaves also contained more endogenous free IAA (64%) than the coleoptiles from the same seedlings (36%). The binding site was very specific, distinguishing well between strong auxins and structurally related substances which exhibit very weak auxin activity. These physiological properties of this binding site indicate that it may have a certain role in the regulation of physiological processes, such as elongation growth and cell division.

Estimates of free and bound indole-3-acetic acid and zeatin levels in relation to regulation of apical dominance and tiller release in oat shoots

Oat stem segments containing quiescent lateral (tiller) buds during times of strong apical dominance, and growing buds released from this inhibition, were collected for analysis of native auxin and cytokinins. Free IAA and IAA conjugates were determined by a14C-IAA and14C-IBA double isotope dilution assay. Free zeatin (Z), zeatin riboside (Z-r), and their glucoside conjugates were purified from butanol-soluble fractions by means of a cellulose phosphate exchanger and thin-layer chromatography. Hormones were analyzed by gas chromatography and mass spectrometry (GC-MS). Results of these analyses indicate that changes in free and bound IAA within the stem do not correlate well with the release of tiller buds (as brought about by decapitation, gravistimulation, or the emergence of the inflorescence). However, increases in Z-r levels are well correlated with tiller release. The glucoside conjugate of Z-r may act as a storage form of cytokinin in quiescent tiller buds. In light of these results, we find that the auxin-cytokinin ratio in oat stem segments is shifted during tiller

The Rough Fruit Condition of the Shamouti orange—Connections with the Endogenous Hormonal Balance

SummaryThe rough peel disorder is characterized by excessively thick rough peel and large fruit sizes and is found in Shamouti orange fruits growing under less than optimal conditions such as heavy, soils, low relative air humidity, and on sour orange rootstock. Affected fruit is not fit for export.Appreciably more cytokinin-and gibberellin-like activities were found in both external (flavedo) and internal (albedo) peel tissues of rough than of smooth oranges at a very young fruitlet stage (May-June), when peel growth was at its peak (both cell divisions and elongation) and the rough fruit disorder was developing. At this stage no consistent differences could be detected in the level of native auxins or ABA like inhibitors. Higher cytokinin and gibberellin levels were also found in rough mature peel (November) when peel growth of rough peel is resumed. On the basis of these and other data a causal relationship between the endogenous regulator balance and the control of peel roughness is suggested.

Isolation and Identification of Native Auxins in Marine Algae

Isolation and Identification of Native Auxins in Marine Algae

Isolation and Identification of Native Auxins in Marine Algae

Isolation and Identification of Native Auxins in Marine Algae

Regulation of Auxin Levels in Coleus blumei by Ethylene

An investigation of the effects of ethylene pretreatment on several facets of auxin metabolism in Coleus blumei Benth "Scarlet Rainbow" revealed a number of changes presumably induced by the gas. Transport of indoleacetic acid-1-(14)C in excised segments of the uppermost internode was inhibited by about 50%. Decarboxylation of indoleacetic acid-1-(14)C by enzyme breis was not affected by the pretreatment. Levels of extractable native auxin in upper leaf and apical bud tissue of the pretreated plants were approximately one-half of those present in untreated plants. The rate of formation of auxin from tryptophan by enzyme breis from pretreated plants was approximately one-half that occurring in incubation mixtures containing the enzyme system from untreated plants. The conjugation of indoleacetic acid-1-(14)C in a form characterized chromatographically as indoleacetylaspartic acid was increased 2-fold in the upper stem region of plants pretreated with ethylene.