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      Distribution of indole-3-acetic acid in Petunia hybrida shoot tip cuttings and relationship between auxin transport, carbohydrate metabolism and adventitious root formation

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          Abstract

          To determine the contribution of polar auxin transport (PAT) to auxin accumulation and to adventitious root (AR) formation in the stem base of Petunia hybrida shoot tip cuttings, the level of indole-3-acetic acid (IAA) was monitored in non-treated cuttings and cuttings treated with the auxin transport blocker naphthylphthalamic acid (NPA) and was complemented with precise anatomical studies. The temporal course of carbohydrates, amino acids and activities of controlling enzymes was also investigated. Analysis of initial spatial IAA distribution in the cuttings revealed that approximately 40 and 10 % of the total IAA pool was present in the leaves and the stem base as rooting zone, respectively. A negative correlation existed between leaf size and IAA concentration. After excision of cuttings, IAA showed an early increase in the stem base with two peaks at 2 and 24 h post excision and, thereafter, a decline to low levels. This was mirrored by the expression pattern of the auxin-responsive GH3 gene. NPA treatment completely suppressed the 24-h peak of IAA and severely inhibited root formation. It also reduced activities of cell wall and vacuolar invertases in the early phase of AR formation and inhibited the rise of activities of glucose-6-phosphate dehydrogenase and phosphofructokinase during later stages. We propose a model in which spontaneous AR formation in Petunia cuttings is dependent on PAT and on the resulting 24-h peak of IAA in the rooting zone, where it induces early cellular events and also stimulates sink establishment. Subsequent root development stimulates glycolysis and the pentose phosphate pathway.

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          The online version of this article (doi:10.1007/s00425-013-1907-z) contains supplementary material, which is available to authorized users.

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          Most cited references71

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          Characterization of an Arabidopsis enzyme family that conjugates amino acids to indole-3-acetic acid.

          Substantial evidence indicates that amino acid conjugates of indole-3-acetic acid (IAA) function in auxin homeostasis, yet the plant enzymes involved in their biosynthesis have not been identified. We tested whether several Arabidopsis thaliana enzymes that are related to the auxin-induced soybean (Glycine max) GH3 gene product synthesize IAA-amino acid conjugates. In vitro reactions with six recombinant GH3 enzymes produced IAA conjugates with several amino acids, based on thin layer chromatography. The identity of the Ala, Asp, Phe, and Trp conjugates was verified by gas chromatography-mass spectrometry. Insertional mutations in GH3.1, GH3.2, GH3.5, and GH3.17 resulted in modestly increased sensitivity to IAA in seedling root. Overexpression of GH3.6 in the activation-tagged mutant dfl1-D did not significantly alter IAA level but resulted in 3.2- and 4.5-fold more IAA-Asp than in wild-type seedlings and mature leaves, respectively. In addition to IAA, dfl1-D was less sensitive to indole-3-butyric acid and naphthaleneacetic acid, consistent with the fact that GH3.6 was active on each of these auxins. By contrast, GH3.6 and the other five enzymes tested were inactive on halogenated auxins, and dfl1-D was not resistant to these. This evidence establishes that several GH3 genes encode IAA-amido synthetases, which help to maintain auxin homeostasis by conjugating excess IAA to amino acids.
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            Auxin transport inhibitors block PIN1 cycling and vesicle trafficking.

            Polar transport of the phytohormone auxin mediates various processes in plant growth and development, such as apical dominance, tropisms, vascular patterning and axis formation. This view is based largely on the effects of polar auxin transport inhibitors. These compounds disrupt auxin efflux from the cell but their mode of action is unknown. It is thought that polar auxin flux is caused by the asymmetric distribution of efflux carriers acting at the plasma membrane. The polar localization of efflux carrier candidate PIN1 supports this model. Here we show that the seemingly static localization of PIN1 results from rapid actin-dependent cycling between the plasma membrane and endosomal compartments. Auxin transport inhibitors block PIN1 cycling and inhibit trafficking of membrane proteins that are unrelated to auxin transport. Our data suggest that PIN1 cycling is of central importance for auxin transport and that auxin transport inhibitors affect efflux by generally interfering with membrane-trafficking processes. In support of our conclusion, the vesicle-trafficking inhibitor brefeldin A mimics physiological effects of auxin transport inhibitors.
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              Regulation of polar auxin transport by AtPIN1 in Arabidopsis vascular tissue.

              Polar auxin transport controls multiple developmental processes in plants, including the formation of vascular tissue. Mutations affecting the PIN-FORMED (PIN1) gene diminish polar auxin transport in Arabidopsis thaliana inflorescence axes. The AtPIN1gene was found to encode a 67-kilodalton protein with similarity to bacterial and eukaryotic carrier proteins, and the AtPIN1 protein was detected at the basal end of auxin transport-competent cells in vascular tissue. AtPIN1 may act as a transmembrane component of the auxin efflux carrier.
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                Author and article information

                Contributors
                +49-394-825266 , +49-394-825515 , mohammad@ipk-gatersleben.de
                +49-362-01785222 , +49-362-01785250 , druege@erfurt.igzev.de
                Journal
                Planta
                Planta
                Planta
                Springer Berlin Heidelberg (Berlin/Heidelberg )
                0032-0935
                1432-2048
                14 June 2013
                14 June 2013
                2013
                : 238
                : 499-517
                Affiliations
                [ ]Institute of Biological Chemistry (IBC), Washington State University, Pullman, WA 99164-6340 USA
                [ ]Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, Gatersleben, 06466 Seeland, Germany
                [ ]Parque Científico y Tecnológico de la U.P.M, Centro de Biotecnología y Genómica de Plantas U.P.M.-I.N.I.A, Campus de Montegancedo, Pozuelo de Alarcón, 28223 Madrid, Spain
                [ ]Department of Agronomy and Plant Breeding Sciences, College of Abureihan, University of Tehran, Tehran, Iran
                [ ]Australian Centre for Plant Functional Genomics, University of Adelaide, Waite Campus, Hartley Grove Urrbrae, Adelaide, 5064 Australia
                [ ]Leibniz Institute of Vegetable and Ornamental Crops Großbeeren/Erfurt e.V. (IGZ), Kuehnhaeuser Str. 101, 99090 Erfurt, Germany
                Article
                1907
                10.1007/s00425-013-1907-z
                3751230
                23765266
                2a678ece-b598-414d-a8ee-abe0c5670e6d
                © The Author(s) 2013

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.

                History
                : 5 December 2012
                : 28 May 2013
                Categories
                Original Article
                Custom metadata
                © Springer-Verlag Berlin Heidelberg 2013

                Plant science & Botany
                polar auxin transport (pat),iaa,gh3,sink establishment,petunia,root development
                Plant science & Botany
                polar auxin transport (pat), iaa, gh3, sink establishment, petunia, root development

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