INDOLE ACETIC ACID (AUXIN) signal transduction

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CONTENTS Introduction Signal Transduction – What, Why, When & How Factors Affecting Signal Transduction Role of IAA Mechanism of IAA Perception & Signal Transduction Evidence for Receptors Function ( In vivo & In vitro ) Inhibitors of Signal Transduction Conclusion Future Prospects




WHAT IS SIGNAL TRANSDUCTION? Definition : Signal transduction is a process where cells construct responses to a signal which triggers a cascade of events.


WHY AND WHEN SIGNAL TRANSDUCTION? Plant and plant cells continually respond to signals which alters ,Physiology Morphology and Development. Internal Stimuli External Stimuli Biochemical and Physiological responses for growing cells









Signal transduction through hormones is a process of interaction between hormone and its specific receptor which leads changes in the biochemical activities of the cells :

Signal transduction through hormones is a process of interaction between hormone and its specific receptor which leads changes in the biochemical activities of the cells HORMONES SIGNAL TRANSDUCTION

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Site of Synthesis Site of Action Specific action Target Site Transcription Translation Regulation of Gene Expression HORMONE SYSTEM IN PLANTS : IN TERMS OF THREE FUNCTIONAL COMPONENTS 1 2 3

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MAIN EVENTS FOR HORMONE SIGNAL TRANSDUCTION Stimulus Source HORMONE Target Cells RECEPETOR Regulation of Gene Expression Hormonal Response manifest at the Physiological Level HORMONE RECEPETOR ( External and Internal ) PERCEPTION TRANSMISSION DOWN STREAM RESPONSE


WHAT ARE THESE RECEPTORS ? Receptors are (glyco) proteins which specifically and reversibly bind chemical signals but, unlike enzymes do not convert them chemically Receptors upon binding transformed into active state through conformational changes. Conformational change initiates a molecular programme leading to characteristic responses. Thus receptor proteins act as both signal detector and transducer .

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The receptors localized in plasma membrane with hormone-binding moiety facing out side the cell. These receptors function as sensory system for external hormone levels and transduce the signal into intracellular signals, via second messengers like G Protein Followed activation of adenylate cyclase which converts ATP into cyclic AMP and/or activation of phospholipase C. TWO MAJOR PATHWAYS OF HORMONE SIGNAL TRANSDUCTION PLASMA MEMBRANE BOUND RECEPTOR

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ATP cAMP AC cAMP dependent protein kinase Fig 2: G Protein activity cycle with Plasma membrane bound receptor

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Fig 3 : Sequence of transduction events leading form activation of Phospholipase C (PLC) to increase of cytosolic calcium Activation of Na + /H + carrier (Increase cytosolic pH)

Fig 4 : Interaction of intracellular and extracellular Ca+ in cell signaling :

Fig 4 : Interaction of intracellular and extracellular Ca + in cell signaling

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Target cells are equipped with internal receptors which may directly interact with target cell-specific non-histone proteins and DNA sequences of the chromatin. This interaction results in increased rates and/or altered pattern of gene trascription. CYTOPLASMIC / NUCLEAR RECEPTOR

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Nuclear membrane Cytoplasm Hormone Receptor A A A A A A A A A nucleoplasm RNA Poly Gene 1 Gene 2 Amplification Amplification repression activation Nuclear membrane Nuclear membrane Nuclear membrane Cytoplasm Cytoplasm nucleoplasm Secondary response proteins Primary response proteins X Fig 5.

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Fig 6 : Protein kinase cascade model that regulate gene expression

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Fig 7 : Interaction Of Heterotrimeric G Proteins , Adenylcyclate And The Regulation Of Gene Expression


BASED ON THESE MODELS LIBBENGA AND MENNES (1995) PROPOSED THE FOLLOWING WORKING HYPOTHESIS – Plant hormones are detected by specific receptor proteins. The transduction of plant hormones follows at least two of major pathways – Intracellular receptor proteins which are directly involved in gene expression either on transcriptional and/or translational level. Plasma membrane bound receptor proteins which function as sensory system for external hormone levels and which transduce intracellular signals, possibly via the phosphatidyl inositol pathway. The secondary signals control the cells activity via modulation of cytoplasmic Ca 2+ levels and protein kinase activity.


FACTORS AFFECTING SIGNAL TRANSDUCTION The hormone induces the synthesis or release of a second messeger like cAMP, G protein, Inositol Phosphate, Ca ++ , Diacylglycerol etc. The affinity of a hormone for its receptor is weaker than the affinity of the second messenger for its target enzyme. The second messenger interacts with a downstream enzyme or protein


IMPORTANT HORMONES ACTING IN PLANTS Auxin Cytokinenin Gibberlins Ethylene Abscisic Acid


ROLE OF IAA/AUXIN : Auxin is found to regulate Apical dominance, Tropism Shoot elongation, Induction of cambial cell division Root initiation.




AUXIN RESPONSE CATEGORIZED IN TWO MAIN EFFECTS Theologis, 1986 Cell division/ proliferation/ morphogenesis Long term response ( Latent period of hours to days) Cell elongation : Rapid response ( 10 – 25 min ) i.e. one of the fastest hormonal response


MECHANISM OF PERCEPTION AND TRANSDUCTION OF IAA Libbenga and Mannes., 1995 Auxins (IAA), often in combination with other hormones, influence many processes in higher plants. Auxin-receptor research is based on two examples of auxin action: 1. Cell proliferation and regeneration 2. Cell elongation

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PROLIFERATION AND REGENERATION : LONG TERM RESPONSE A classical model system for the study of hormonal control of proliferation and regeneration is cultured stem-pith tissue from Nicotiana tabaccum . In 1957 it was demonstrated for the first time that proliferation and regeneration in this tissue can be controlled by exogenous auxins and cytokinins . Van der Linde et al. (1985) reported high affinity auxin-binding sites in 3-week old callus tissues of tobacco stem-pith. The callus tissue contains three classes of auxin-binding proteins, which can be distinguished by their binding behavior and their location.

Table 1: General Characteristics Of Three Classes Of Auxin Receptors In Tobacco Tissues:

Table 1: General Characteristics Of Three Classes Of Auxin Receptors In Tobacco Tissues General Characteristics Receptors Membrane bound Soluble Auxin recep. Auxin recep. NPA + recep. Presence in tissues : Stem pith + + - Stem pith callus + + + Leaves + + Nd Protoplasts (2 days) + + - Shoot tips Nd Nd + Cell suspension culture +/- + + Location in the cell Plasma lemma Plasma lemma Cytoplasm / Nucleus Optimum temperature for binding ( ˚ C) 25 0 26.0 Optimum pH for binding 5.0 4.0 7.5 Binding time 60 min. rapidly 45 min. Affinity for IAA Low --- Higher Concetration (pM /mg protein) 50 1.6 0 – 0.2 Nd = not determined, NPA = naphtylphthalamic acid (Auxin transport inhibitor) Van der Linde et al . (1985)

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CYTOPLASMIC / NUCLEAR AUXIN-BINDING PROTEINS: Addition of receptor (R) fraction in auxin dependent Mung bean hypocotyl nuclei stimulated RNA polymerase II activity. (Kikuchi et al. (1989)) Van der zaal (1991) reported that 7 cDNA clones of mRNA were specifically transcribed after addition of auxin to auxin-starved cell suspension. The induction of genes corresponding to cDNA clone, showed a good correlation with cell division mRNAs were found to accumulate transiently prior to the cell division response after auxin treatment. Moreover, in vitro experiments with nuclei isolated from auxin-treated cells showed that the 103-genes clearly expressed, but not in nuclei from untreated control cells.

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Endogenous Auxin Exogenous auxin Plasma membrane Membrane bound auxin receptor Cytoplasmic auxin receptor RNA Poly HR Complex Modified RNA Poly Specific gene action mRNA synthesis Proteins FIG 8 : PATHWAY FOR MEDIATION OF AUXIN RECEPTORS IN AUXIN REGULATED GENE EXPRESSION (Kapoor , 1993)

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Endogenous Auxin Exogenous auxin Plasma membrane Membrane bound auxin receptor Cytoplasmic auxin receptor HR Complex Specific gene action mRNA synthesis Proteins Fig 9 : ALTERNATIVE PATHWAY

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MEMBRANE BOUND AUXIN-BINDING PROTEINS: TWO CLASSICAL EXAMPLES OF AUXIN BINDING PROTEIN AND ROOT DIFFERENTIATION Root differentiation in mutant lines (In vivo) Root differentiation in tobacco tissue cultures (In vitro)

1. Correlation between a membrane bound auxin binding protein and root specific peroxidase Nakamura et al., 1988. :

1. Correlation between a membrane bound auxin binding protein and root specific peroxidase Nakamura et al. , 1988. Auxin resistant variant Normal tobacco cell lines Lacking the ability to differentiate root ability to differentiate root Both root specific peroxidase and membrane bound proteins absent Both root specific peroxidase and membrane bound proteins present This indicates good correlation between the abundance of membrane receptors and peroxidase activity for root differentiation

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2. MEMBRANE BOUND AUXIN BINDING PROTEIN IN TOBACCO (Mennes et al ., 1991) Tobacco leaves ( MBR + ) 2,4 D cell suspension (MBR - ) but posses both cytoplasmic and / NPA receptor 2,4 D Callus 4 week old ( MBR – RR - ) 8 week old ( MBR – RR- ) Subculture in NAA/ Kinetin 4 week old ( MBR – RR - ) 8 week old ( MBR + RR - )

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RESULTS : Membrane bound auxin binding protein might have some function in auxin induced root regeneration On the basis of these two studies it was concluded that level of membrane bound auxin binding proteins are having strong correlation with root specific peroxidase activity , which can be strongly used as an biochemical marker.


CELL ELONGATION RAPID RESPONSE Auxin receptor involved in cell elongation has been extensively studied in Zea mays coleoptile. It was found that membrane bound auxin binding proteins (ABP1) were in abundance, localized in endoplasmic reticulum (ER)

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1. Red light inhibition of mesocotyl elongation and auxin binding :

1. Red light inhibition of mesocotyl elongation and auxin binding Walton and Ray. 1981 3 – 4 days old dark grown maize seedling Exposed to Red Light Auxin binding localized on ER of the mesocotyl begins to decrease by 50 – 60 % in 9 hours as compared to dark controls Reason : The auxin binding site are receptor site because light reduce the number of binding site but not their affinity to NAA

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Table 2. Effect of red light on NAA binding and elongation Tissue Specific Binding a (%) NAA Bound Pmol/ mg protein Elongation b (%) Dark Light Dark Light Dark Light Coleoptile 7.9 8.7 1.3 1.4 18 19 Primary leaf 11.0 8.7 0.8 0.8 -- -- Mesocotyle 8.3 4.5 1.9 1.1 66 39 a: Percentage of offered (14 C , NAA specifically bound by crude microsomal particles from 0.5 gm fresh wt per mi assay medium b: percent elongation of segment measured after 16 hours in 10 umol NAA Dark grown maize plants were exposed to light for 30 min then kept in darkness for an additional 10 hours before harvest for binding and growth assay

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RESULTS : Auxin binding activity and elongation response decrease in parallel down the length of mesocotyle when seedlings are given red light CONCLUSION : These observations are consistent with the role of endoplasmic reticulum localized auxin binding sites as receptor for auxin action in cell elongation

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3 mm below coleoptiles tip 5mm segment were cut and collected and fixed in buffer solution 2. Auxin binding protein from coleoptile membrane of corn Localization of a putative auxin receptor Lobler and Klambt. 1985 Maize seedling were grown at 27 ° C in darkness for 3 days Primary leaves of 3 days old etiolated maize seedling were removed from the coleoptiles Immunohistochemical test Growth measurement 10 segments were transferred in incubation media

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The section were cut and transferred to slides after hardening and paraffin embedment Treated with trypsin and washed in PB solution pH 7.4 Blocking of the slide done with goat serum for non specific binding followed by IgG anti-ABP treatment for specific binding Slides were viewed in light microscopy Immunohistochemical test With this IgG anti-ABP the ABP is localized at the plasma lemma of the outer epidermis cells of the coleoptiles , using indirect immunoflurosence labeling

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Growth measurement IgG antiABP + IAA 10 -5 M IAA 10 -5 M IgG + IAA 10 -5 M 0 6 21 h 0 6 21 h 0 6 21 h

3. Auxin dependent cell expansion mediated by overexpressed ABP1 Jones et al., 1998:

3. Auxin dependent cell expansion mediated by overexpressed ABP1 Jones et al ., 1998 Gene for ABP 1 Transgenic tobacco developed with gene encoding ABP1 Auxin induced Auxin analog induced ABP 1 ABP 1 ABP 1 Leaf segment displayed auxin induced leaf curling as a result of cell expansion Failed to induce any response Leaf epinasty depends on a specific interaction of ABP1 and Auxin

ABP1 also mediates cell division Jin Gui Chen et al., 2001 :

ABP1 also mediates cell division Jin Gui Chen et al ., 2001 Arabidopsis embryogenisis was studied Using reverse genetic approach gene for ABP1 was tagged Antisense supression of ABP1 Cell division was low coupled with cell elongation This suggest that ABP1 binds auxin and there by mediates cell elongation and directly or indirectly cell division

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TWO MAJOR THEORIES EXPLAINING AUXIN INDUCED CELL ELONGATION 1. ACID GROWTH THEORY : which suggest that cell enlargement is initiated by proton secretion. 2. GENE ACTIVATION HYPOTHESIS: Proposed that auxin regulates the synthesis of specific mRNAse coding for polypeptides necessary for the growth process ( New concept of this model is AUXIN-INDUCED TRANSCRIPTS )

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Phospholipase A2 (PLA2) excises the fatty acid of a phospholipid, to form a free fatty acid and lysophospholipid (LPC) (with one fatty acid) LPC activates a protein kinase, which in turn activates the ATPase proton pump, and acidifies the cell wall space. Cell wall polymers are extensively cross-linked, which limits expansion. The force of turgor pressure acting on the membrane and cell wall cause the polymers to displace and allow the cell to enlarge The lower pH activates wall-loosening enzymes that cleave the load-bearing bonds. ACID GROWTH THEORY

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Fig 10: A model relating to auxin induced mRNA accumulation , H + secretion and cell elongation Theologis , 1986 mRNA induction H+ secreation cell elongation Cycloheximide


Auxin rapidly and transiently induces accumulation of mainly three families of transcripts which are S MALL A UXIN - U P R NAs ( SAURs ) GH3 -related transcripts AUX IN / I NDOLE-3 - A CETIC A CID ( Aux/IAA ) AUXIN-INDUCED TRANSCRIPTS New concept Woodward and Bartel., 2005


S MALL A UXIN - U P R NAs ( SAURs ) They do not contain introns and give rise to small (0.5 kb) transcripts. In Maize ZmSAUR2 is a small nuclear protein rapidly degrades They have short life , might be due to presence of DST ( Down stream elements in the 3’ untranslated region of the message) They expressed most strongly in epidermal and cortical cells of hypocotyl, epicotyl and stem.

GH3-related transcripts:

GH3 -related transcripts They are part of small multigene family in Soybean and Arabidopsis. Some are found to be IAA- induced, coding for IAA-amino acid conjugate enzymes Several other GH3 are not found to be auxin regulated, but are found to adenylate and conjugate amino acid to molecules other than IAA. They expressed primarily in vascular tissues. GH3 genes are composed of three exons.


AUX IN / I NDOLE-3 - A CETIC A CID ( Aux/IAA ) Aux/IAA family include 28 proteins in Arabdopsis, homologous genes are present in other crops too. Alters ARF transcriptional activity and elicitiing diverse developmental and regulatory consequences. These encoded protein shares extensive sequence identity for 4 conserved domain . Domain I Transcriptional repressor Domain II Critical for Aux/IAA instability confering Auxin resistant mutants Domain III Involved in homo and hetero dimerization with other Aux/IAA proteins and with ARF’s ( Auxin response factors) Domain IV

Including these transcripts many auxin induced genes share a common upstream regulatory regions :

Including these transcripts many auxin induced genes share a common upstream regulatory regions T G T C T C Identified from promoter region of Pea PS-IAA4/5 Aux RE T G T C T C Region including this sequence called Aux RE ( Auxin responsive elements) confers auxin induced gene expression in synthetic constructs

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Aux RE T G T C T C Finding of Aux RE led to the isolation of ARF 1 When ARF bind to Aux RE directly they can either activate or repress target gene transcripts via dimerization with the C terminal domain of III and IV of Aux/IAA Mutation in several Arabidopsis ARF genes confers gene specific developmental defects DBD AD/RD IV III N C ARF 1

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Fig 11. A MODEL FOR EARLY AUXIN REGULATED GENE EXPRESSION AND GENE FUNCTION (Ranjan et al., 2003) Transcriptional Activation of early genes ACS SAUR Aux/IAA GH3 GH2/4 ACC synthase Short lived nuclear protein Intercellular Signaling Regulation of gene expression Stress Response ? Glutathione S - Transferase Detoxicification Cellular Redox State Auxin Binding / Transport Auxin Other inducer Ethylene Aux RE

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Fig 12. Auxin signal in response to jasmonate synthesis and ion channels Trewavas , 2000

Aux/IAA degradation is mediated by the SCFTIR1 ubiquitin ligase Weijers and Jurgens., 2004 :

Aux/IAA degradation is mediated by the SCFTIR1 ubiquitin ligase Weijers and Jurgens., 2004 The Aux/IAA proteins which inhibit auxin response are unstable because they are target of ubiquitin mediated degradation. It is a three step process at E1, E2 and E3 enzyme levels.

Fig 13: Model for auxin action on plant cells :

Fig 13: Model for auxin action on plant cells

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Fig 14: Developmental schedule requires the degradation of the specific protein associated with auxin regulated developmental phase before the next stage can occur

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IDENTIFICATION OF INHIBITORS OF AUXIN TRANSCRIPTIONAL ACTIVATION BY MEANS OF CHEMICAL GENETICS IN ARABIDOPSIS Armstrong et al ., 2004 To elucidate unidentified intermediates in perception and subsequent signaling of IAA, by utilizing small molecules to Perturb a signaling pathway , permitting the identification of relevant gene product at any stage of development Arabidopsis thaliana ecotype Coloumbia-0 for phenotype and microarray analysis through Three artificial constructs viz., BA3, DR5::GUS, ARR5::GUS, HS::AXR3NT-GUS. Chemical genetic screening was done The plates were incubated in NAA, washed and stained for GUS expression About 10,000 structurally diverse small molecules were screened and out of them 30 compounds showed strong inhibition

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The experiment was narrowed on four structurally distinct compounds (A,B,C,D) with activity in low molecular range. Inhibition of auxin signaling obstructed the expression of GUS in root elongation zone A, B and C impaired auxin mediated proteolysis of the Aux / IAA 17 and translational fusion protein AXR3NT::GUS , which implies that an inhibitory mechanism targeting an upstream component in the signaling pathway. Compound A and B inhibited root elongation and induced adventitious roots in dose dependent manner.

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Fig 15: Some of the known interaction in the plant cell signal transduction

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CONCLUSION IAA (Auxin ) perception is a central biochemical events and subsequent signaling event directing gene regulation. The induction of gene expression by IAA , occurs via, protein phosphorylation and post translational modification of preexisting transcriptional factors. The most well characterized component of IAA signaling network are receptors, secondary messenger and transcription factors, these components are linked to the molecular activity of auxin. The continued presence of auxin is necessary for defined development programme, division- expansion-maturation-differentiation Each stage of development is associated with unique set of proteins which subsequently degrade and synthesize into new protein by means of ubiqitination.

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Future Prospects For the signal transduction it is yet to be determined how phosphorylation regulates the transcriptional activity. The exact role of second messenger in IAA signal transduction perhaps remains as puzzle. Though transcripts have been identified responsible for genes regulation, still the role of SAUR & GH 3 are unclear. There is need to identify more mutants to explore the phenomena of signal transduction.

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Subsequently, it was discovered that AXR1 is related to a protein in yeast, AOS1, which also lacks the C-terminal half of E1. In yeast, the AOS1p protein forms a heterodimer with another protein, UBA2p, which is homologous to the C-terminal half of E1. Based on this, Mark Estelle and his colleagues were able to clone an Arabidopsis homolog of the UBA2 gene, called ECR1 . The two proteins AXR1 and ECR1 form an E1-like heterodimer similar to that of the yeast AOS1p–UBA2p heterodimer. However, rather than binding to ubiquitin, the AXR1–ECR1 heterodimer transiently binds to a family of small, ubiquitin-related proteins called RUBs (related to ubiquitin). That breakthrough led to a rapid series of biochemical and genetic studies that have elucidated the mechanism involved in the auxin-induced degradation of the AUX/IAA proteins (reviewed by Hellman and Estelle 2002). Web Figure 19.13.A illustrates a model of the mechanisms involved in ubiqutin-mediated degradation of AUX/IAA proteins recently proposed by Hellman and Estelle (2002). In conjunction with the protein RCE1 and the RBX1 component of the SCFTIR1 (E3 ligase) complex, AXR1–ECR1 activates the SCFTIR1 ubiquitin ligase complex by promoting conjugation of RUB to the SCFTIR1 component CUL1 (see Web Figure 19.13.A). The activated SCFTIR1 complex then transfers ubiquitin from an E2 protein to an AUX/IAA protein substrate, which is then degraded by the 26S proteosome complex. Although the mechanism by which AUX/IAA proteins are rapidly degraded is now largely understood, the mechanism by which auxin stimulates interactions between the AUX/IAA proteins and the SCFTIR1 complex is still unknown

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Model for auxin action on plant cells. (a) Auxin induces cell elongation, possibly through the activity of ABP1, and stimulates the interaction between nuclear Aux/IAA proteins and SCFTIR1 (RBX1 is a RING-domain-containing protein that is associated with the SCF complex [21,41]). This interaction is followed by the ubiquitination and degradation of the Aux/IAA substrate by the 26S proteasome (not depicted). SCF activity requires AXR1/ECR1- and RCE1-dependent modification of AtCUL1. The CSN catalyzes the deconjugation of RUB1/NEDD8 and regulates SCF activity (whereas CAND1 binds to unmodified tCUL1) and thereby influences SCF assembly. Auxin-dependent Aux/IAA degradation can be inhibited by Yokonolide B, PCIB and juglone. The sirtinol target SIR1 may act at the same step. (b) At low auxin concentrations, Aux/IAA proteins (I, II, III and IV represent conserved domains) interact with ARF transcription factors (through the DNA-binding domain [DBD] and the middle region [MR]), presumably at the target DNA site in primary auxin-responsive gene promoters, and counteract ARF activity either by competitive inhibition of homodimerization (left), or by directly influencing the activity of transcriptional complexes (right). (c) Upon Aux/IAA degradation stimulated by high auxin concentrations, ARFs either repress or activate target gene transcription as dimers (left) or as monomers (right). ECR1, E1 C-terminus related 1.

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