Search

Strawberry (Fragaria × ananassa Duch.) was transformed with maize IAA-glucose synthase gene (iaglu) via Agrobacterium tumefaciens using binary vector system. Incorporation of the transgene was confirmed by PCR and Southern blot analysis and its transcription by RT-PCR. Transformation resulted in a significant increase of ester-conjugated IAA level in tissue of all tranegenic plants while free IAA level was significantly lower in two trangenic clones tested and in other two it was comparable to that in wild-type plants. The level of amide-conjugated hormone was not affected by transformation with iaglu. The change in endogenous IAA metabolism affected the growth and development of transgenic strawberry plants but the effect was not fully correlated with changes in endogenous IAA level. In general, the transgenic clones were dwarfish — their leaf laminas were smaller, leaf petioles shorter and the crown diameter smaller in comparison to wild-type ones, although in one clone the difference was significant only for leaf lamina area. Shoots of all transgenic clones formed more roots in vitro than the wild-type plants and in two clones the roots were longer than in the control.

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.

Glucosinolates are a class of secondary metabolites with important roles in plant defense and human nutrition. Here, we characterize a putative UDP-glucose:thiohydroximate S-glucosyltransferase, UGT74B1, to determine its role in the Arabidopsis glucosinolate pathway. Biochemical analyses demonstrate that recombinant UGT74B1 specifically glucosylates the thiohydroximate functional group. Low Km values for phenylacetothiohydroximic acid (approximately 6 microm) and UDP-glucose (approximately 50 microm) strongly suggest that thiohydroximates are in vivo substrates of UGT74B1. Insertional loss-of-function ugt74b1 mutants exhibit significantly decreased, but not abolished, glucosinolate accumulation. In addition, ugt74b1 mutants display phenotypes reminiscent of auxin overproduction, such as epinastic cotyledons, elongated hypocotyls in light-grown plants, excess adventitious rooting and incomplete leaf vascularization. Indeed, during early plant development, mutant ugt74b1 seedlings accumulate nearly threefold more indole-3-acetic acid than the wild type. Other phenotypes, however, such as chlorosis along the leaf veins, are likely caused by thiohydroximate toxicity. Analysis of UGT74B1 promoter activity during plant development reveals expression patterns consistent with glucosinolate metabolism and induction by auxin treatment. The results are discussed in the context of known mutations in glucosinolate pathway genes and their effects on auxin homeostasis. Taken together, our work provides complementary in vitro and in vivo evidence for a primary role of UGT74B1 in glucosinolate biosynthesis.

Auxin response factor (ARF) family genes play a central role in controlling sensitivity to the plant hormone auxin. We characterized the function of ARF8 in Arabidopsis by investigating a T-DNA insertion line (arf8-1) and overexpression lines (ARF8 OX) of ARF8. arf8-1 showed a long-hypocotyl phenotype in either white, blue, red or far-red light conditions, in contrast to ARF8 OX that displayed short hypocotyls in the light. Stronger and weaker apical dominance, and promotion and inhibition of lateral root formation were observed in arf8-1 and ARF8 OX respectively. Sensitivity to auxin was unaltered in arf8-1 hypocotyls with respect to growth inhibition caused by exogenously applied auxin and growth promotion induced by higher temperatures. ARF8 expression was observed constitutively in shoot and root apexes, and was induced in the light condition in hypocotyls. Free IAA contents were approximately 30% reduced in light-grown hypocotyls of ARF8 OX, but were similar between those of arf8-1 and wild type. Expression of the three GH3 genes was reduced in arf8-1 and increased in ARF8 OX, indicating that they are targets of ARF8 transcriptional control. Because the three GH3 proteins may be involved in the conjugation of IAA as suggested by Staswick et al. (2002), and because two of the three GH3 genes are auxin inducible, ARF8 may control the free IAA level in a negative feedback fashion by regulating GH3 gene expression. ARF family genes seem to control both auxin sensitivity and homeostasis in Arabidopsis.

Axis formation occurs in plants, as in animals, during early embryogenesis. However, the underlying mechanism is not known. Here we show that the first manifestation of the apical-basal axis in plants, the asymmetric division of the zygote, produces a basal cell that transports and an apical cell that responds to the signalling molecule auxin. This apical-basal auxin activity gradient triggers the specification of apical embryo structures and is actively maintained by a novel component of auxin efflux, PIN7, which is located apically in the basal cell. Later, the developmentally regulated reversal of PIN7 and onset of PIN1 polar localization reorganize the auxin gradient for specification of the basal root pole. An analysis of pin quadruple mutants identifies PIN-dependent transport as an essential part of the mechanism for embryo axis formation. Our results indicate how the establishment of cell polarity, polar auxin efflux and local auxin response result in apical-basal axis formation of the embryo, and thus determine the axiality of the adult plant.

International audience; Stem cells must balance self-renewal and differentiation; thus, their activities are precisely controlled. In plants, the control circuits that underlie division and differentiation within meristems have been well studied, but those that underlie feedback on meristems from lateral organs remain largely unknown. Here we show that long-distance auxin transport mediates this feedback in a non-cell-autonomous manner. A low-auxin zone is associated with the shoot apical meristem (SAM) organization center, and auxin levels negatively affect SAM size. Using computational model simulations, we show that auxin transport from lateral organs can inhibit auxin transport from the SAM through an auxin transport switch and thus maintain SAM auxin homeostasis and SAM size. Genetic and microsurgical analyses confirmed the model's predictions. In addition, the model explains temporary change in SAM size of yabby mutants. Our study suggests that the canalization-based auxin flux can be widely adapted as a feedback control mechanism in plants.

The IPyA pathway, the major auxin biosynthesis pathway, is transcriptionally regulated through a negative feedback mechanism in response to active auxin levels. The phytohormone auxin plays an important role in plant growth and development, and levels of active free auxin are determined by biosynthesis, conjugation, and polar transport. Unlike conjugation and polar transport, little is known regarding the regulatory mechanism of auxin biosynthesis. We discovered that expression of genes encoding indole-3-pyruvic acid (IPyA) pathway enzymes is regulated by elevated or reduced active auxin levels. Expression levels of TAR2, YUC1, YUC2, YUC4, and YUC6 were downregulated in response to synthetic auxins [1-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D)] exogenously applied to Arabidopsis thaliana L. seedlings. Concomitantly, reduced levels of endogenous indole-3-acetic acid (IAA) were observed. Alternatively, expression of these YUCCA genes was upregulated by the auxin biosynthetic inhibitor kynurenine in Arabidopsis seedlings, accompanied by reduced IAA levels. These results indicate that expression of YUCCA genes is regulated by active auxin levels. Similar results were also observed in auxin-overproduction and auxin-deficient mutants. Exogenous application of IPyA to Arabidopsis seedlings preincubated with kynurenine increased endogenous IAA levels, while preincubation with 2,4-D reduced endogenous IAA levels compared to seedlings exposed only to IPyA. These results suggest that in vivo conversion of IPyA to IAA was enhanced under reduced auxin levels, while IPyA to IAA conversion was depressed in the presence of excess auxin. Based on these results, we propose that the IPyA pathway is transcriptionally regulated through a negative feedback mechanism in response to active auxin levels.

IAA plays a critical role in regulating numerous aspects of plant growth and development. While there is much genetic support for tryptophan-dependent (Trp-D) IAA synthesis pathways, there is little genetic evidence for tryptophan-independent (Trp-I) IAA synthesis pathways. Using Arabidopsis, we identified two mutant alleles of ISS1 (Indole Severe Sensitive) that display indole-dependent IAA overproduction phenotypes including leaf epinasty and adventitious rooting. Stable isotope labeling showed that iss1, but not WT, uses primarily Trp-I IAA synthesis when grown on indole-supplemented medium. In contrast, both iss1 and WT use primarily Trp-D IAA synthesis when grown on unsupplemented medium. iss1 seedlings produce 8-fold higher levels of IAA when grown on indole and surprisingly have a 174-fold increase in Trp. These findings indicate that the iss1 mutant???s increase in Trp-I IAA synthesis is due to a loss of Trp catabolism. ISS1 was identified as At1g80360, a predicted aromatic aminotransferase, and in vitro and in vivo analysis confirmed this activity. At1g80360 was previously shown to primarily carry out the conversion of indole-3-pyruvic acid to Trp as an IAA homeostatic mechanism in young seedlings. Our results suggest that in addition to this activity, in more mature plants ISS1 has a role in Trp catabolism and possibly in the metabolism of other aromatic amino acids. We postulate that this loss of Trp catabolism impacts the use of Trp-D and/or Trp-I IAA synthesis pathways.

Glycosyltransferases play diverse roles in cellular metabolism by modifying the activities of regulatory metabolites. Three stress-regulated UDP-glucosyltransferase-encoding genes have been isolated fromthe stigmas of saffron, UGT85U1, UGT85U2 and UGT85V1, which belong to the UGT85 family that includes members associated with stress responses and cell cycle regulation. Arabidopsis constitutively expressing UGT85U1 exhibited and increased anchor root development. No differences were observed in the timing of root emergence, in leaf, stem and flower morphology or flowering time. However, salt and oxidative stress tolerance was enhanced in these plants. Levels of glycosylated compounds were measured in these plants and showed changes in the composition of several indole-derivatives. Moreover, auxin levels in the roots were higher compared to wild type. The expression of several key genes related to root development and auxin homeostasis, including CDKB2.1, CDKB2.2, PIN2, 3 and 4; TIR1, SHR, and CYCD6, were differentially regulated with an increase of expression level of SHR, CYCD6, CDKB2.1 and PIN2. The obtained results showed that UGT85U1 takes part in root growth regulation via auxin signal alteration and the modified expression of cell cycle-related genes, resulting in significantly improved survival during oxidative and salt stress treatments. Spanish Ministerio de Economia y Competitividad BIO2013-44239-R IBERCAROT network 112RT0445 FPCYTA through the INCRECYT Programme

Growth hormone auxin regulates various cellular processes by altering the expression of diverse genes in plants. Among various auxin-responsive genes, GH3 genes maintain endogenous auxin homeostasis by conjugating excess of auxin with amino acids. GH3 genes have been characterized in many plant species, but not in legumes. In the present work, we identified members of GH3 gene family and analyzed their chromosomal distribution, gene structure, gene duplication and phylogenetic analysis in different legumes, including chickpea, soybean, Medicago, and Lotus. A comprehensive expression analysis in different vegetative and reproductive tissues/stages revealed that many of GH3 genes were expressed in a tissue-specific manner. Notably, chickpea CaGH3-3, soybean GmGH3-8 and -25, and Lotus LjGH3-4, -5, -9 and -18 genes were up-regulated in root, indicating their putative role in root development. In addition, chickpea CaGH3-1 and -7, and Medicago MtGH3-7, -8, and -9 were found to be highly induced under drought and/or salt stresses, suggesting their role in abiotic stress responses. We also observed the examples of differential expression pattern of duplicated GH3 genes in soybean, indicating their functional diversification. Furthermore, analyses of three-dimensional structures, active site residues and ligand preferences provided molecular insights into function of GH3 genes in legumes. The analysis presented here would help in investigation of precise function of GH3 genes in legumes during development and stress conditions.

Auxin is among the key growth regulators that play diverse roles in virtually all aspects of plant growth and development. Intensive investigations during the past two decades have helped in elucidation of auxin perception and signal transduction mechanisms operative in plants. In addition to its primary role in regulating plant development, several studies in recent years have provided unflinching evidence for the involvement of auxin in abiotic stress responses. Functional genomics studies and genome-wide expression analysis have revealed altered expression of auxin-responsive genes, such as Aux/IAA, GH3, SAURs, and ARFs, under abiotic stress conditions. Variations in endogenous levels of auxin at global and local levels under various abiotic stress conditions have been associated with phenotypic changes and provided intriguing evidences regarding its role in response to environmental changes. Modulation of reactive oxygen species (ROS) levels in response to exogenous auxin as well as to drought, salinity, and ABA have indicated towards a complex relationship network between auxin, ROS, and abiotic stresses in plants. The advent of recent functional genomics technologies has led to identification of several candidate genes that may modulate crosstalk between auxin and abiotic stresses. This chapter discusses auxin homeostasis, signal transduction mechanisms, and how these processes are modulated under abiotic stresses, thus emphasizing on the emerging roles of auxin as a key integrator of abiotic stress pathways and plant development.

BAP GEAPSI CT2; International audience; The endosperm plays a pivotal role in the integration between component tissues of molecular signals controlling seed development. It has been shown to participate in the regulation of embryo morphogenesis and ultimately seed size determination. However, the molecular mechanisms that modulate seed size are still poorly understood especially in legumes. DASH (DOF Acting in Seed embryogenesis and Hormone accumulation) is a DOF transcription factor (TF) expressed during embryogenesis in the chalazal endosperm of the Medicago truncatula seed. Phenotypic characterization of three independent dash mutant alleles revealed a role for this TF in the prevention of early seed abortion and the determination of final seed size. Strong loss-of-function alleles cause severe defects in endosperm development and lead to embryo growth arrest at the globular stage. Transcriptomic analysis of dash pods versus wild-type (WT) pods revealed major transcriptional changes and highlighted genes that are involved in auxin transport and perception as mainly under-expressed in dash mutant pods. Interestingly, the exogenous application of auxin alleviated the seedlethal phenotype, whereas hormonal dosage revealed a much higher auxin content in dash pods compared with WT. Together these results suggested that auxin transport/signaling may be affected in the dash mutant and that aberrant auxin distribution may contribute to the defect in embryogenesis resulting in the final seed size phenotype.

Auxin has a central role in the establishment and elaboration of pattern in root meristems. Regulation of root development by auxin begins early in embryogenesis, perhaps even as early as the establishment of polarity in the zygote, and persists throughout the lifetime of a root. Auxin-regulated development depends on a balance of synthesis/import and metabolism/export/sequestration. The overall result of these processes is to establish a state of auxin homeostasis which we hypothesize is required for normal root meristem patterning and development.

The plant hormone auxin regulates many aspects of plant growth and development. Although several auxin biosynthetic pathways have been proposed, none of these pathways has been precisely defined at the molecular level. Here we provide in planta evidence that the twoArabidopsis cytochrome P450s, CYP79B2 and CYP79B3, which convert tryptophan (Trp) to indole-3-acetaldoxime (IAOx) in vitro, are critical enzymes in auxin biosynthesis in vivo. IAOx is thus implicated as an important intermediate in auxin biosynthesis. Plants overexpressing CYP79B2 contain elevated levels of free auxin and display auxin overproduction phenotypes. Conversely, cyp79B2 cyp79B3 double mutants have reduced levels of IAA and show growth defects consistent with partial auxin deficiency. Together with previous work on YUCCA, a flavin monooxygenase also implicated in IAOx production, and nitrilases that convert indole-3-acetonitrile to auxin, this work provides a framework for further dissecting auxin biosynthetic pathways and their regulation.

Auxin controls a myriad of plant developmental processes and plant response to environmental conditions. Precise trafficking of auxin transporters is essential for auxin homeostasis in plants. Here, we report characterization of Arabidopsis CTL1, which controls seedling growth and apical hook development by regulating intracellular trafficking of PIN-type auxin transporters. The CTL1 gene encodes a choline transporter-like protein with an expression pattern highly correlated with auxin distribution and is enriched in shoot and root apical meristems, lateral root primordia, the vascular system, and the concave side of the apical hook. The choline transporter-like 1 (CTL1) protein is localized to the trans-Golgi network (TGN), prevacuolar compartment (PVC), and plasma membrane (PM). Disruption of CTL1 gene expression alters the trafficking of 2 auxin efflux transporters—Arabidopsis PM-located auxin efflux transporter PIN-formed 1 (PIN1) and Arabidopsis PM-located auxin efflux transporter PIN-formed 3 (PIN3)—to the PM, thereby affecting auxin distribution and plant growth and development. We further found that phospholipids, sphingolipids, and other membrane lipids were significantly altered in the ctl1 mutant, linking CTL1 function to lipid homeostasis. We propose that CTL1 regulates protein sorting from the TGN to the PM through its function in lipid homeostasis., Author summary Auxin, a plant hormone, controls many aspects of plant growth and development. The precise transport and distribution of auxin hold the key to its function. A number of transport proteins are known to be involved in auxin translocation, and the PIN proteins, which are an integral part of membranes in plants, play a pivotal role in this process. Several PIN proteins are localized in the plasma membrane to mediate auxin efflux from cells, but their regulation is not well known. In this report, we analyze the role of a choline transport protein, choline transporter-like 1 (CTL1), and find that it controls the trafficking of Arabidopsis PM-located auxin efflux transporter PIN-formed 1 (PIN1) and Arabidopsis PM-located auxin efflux transporter PIN-formed 3 (PIN3) to the plasma membrane, thereby regulating auxin distribution during plant growth and development. In addition, we show that CTL1 has a role in lipid homeostasis in the membrane; thus, these findings provide a mechanistic link between choline transport, lipid homeostasis, and vesicle trafficking in plants. We conclude that CTL1 is a new factor in secretory protein sorting and that this finding contributes to the understanding of not only auxin distribution in plants but also protein trafficking in general.

In this review, we summarize the different biosynthesis-related pathways that contribute to the regulation of endogenous auxin in plants. We demonstrate that all known genes involved in auxin biosynthesis also have a role in root formation, from the initiation of a root meristem during embryogenesis to the generation of a functional root system with a primary root, secondary lateral root branches and adventitious roots. Furthermore, the versatile adaptation of root development in response to environmental challenges is mediated by both local and distant control of auxin biosynthesis. In conclusion, auxin homeostasis mediated by spatial and temporal regulation of auxin biosynthesis plays a central role in determining root architecture.

It was once proposed that there are only two kinds of biology: elegant genetics and sloppy biochemistry (E.C. Pauling, unpublished). For those who study auxin metabolism in Arabidopsis, this geneticist’s view of the different approaches to biological research has particular resonance. Arabidopsis has the advantage of providing a model molecular genetic system in a plant that uses the indole ring to produce diverse compounds, such as the glucosinolate glucobrassicin, the phytoalexin camalexin and the phytohormone indole-3-acetic acid (IAA). This model plant genetic system offers unique opportunities to apply new approaches to answer long-standing questions regarding auxin. However, studies in Arabidopsis can often present us with confounding problems when it comes to careful dissection of the network of indolic pathways in either normal or mutant plants. In this review, we focus our attention on IAA metabolism in Arabidopsis, However, by necessity we have been obliged to draw complementary information from the literature on other species to delineate as completely as possible the most current views on processes responsible for IAA production and its regulation.

Root architecture is developmentally plastic and affected by many intrinsic factors (e.g. plant hormones) and extrinsic factors (e.g. touch, gravity) in order to maximize nutrient and water acquisition. We have recently shown that asymmetrical exposure of cytokinin (CK) at the root tip causes root growth directional changes that is dependent on ethylene signaling and is potentiated by glucose signaling. Auxin homeostasis as maintained by auxin signaling and transport is also involved in CK-induced root cell elongation and differential growth. The signaling pathways eventually converge at actin filament organization since actin filament organization inhibitor latrunculin B (Lat B) can also induce similar growth. We, show that CK can actually alter actin filament organization as seen in actin binding protein 35S::GFP-ABD2-GFP transgenic lines as is also altered by auxin polar transport inhibitor 1-N-naphthylphthalamic acid (NPA) and Lat B in different manners.

Plant architecture attributes such as tillering, plant height and panicle size are important agronomic traits that determine rice (Oryza sativa) productivity. Here, we report that altered auxin content, transport and distribution affect these traits, and hence rice yield. Overexpression of the auxin efflux carrier-like gene OsPIN5b causes pleiotropic effects, mainly reducing plant height, leaf and tiller number, shoot and root biomass, seed-setting rate, panicle length and yield parameters. Conversely, reduced expression of OsPIN5b results in higher tiller number, more vigorous root system, longer panicles and increased yield. We show that OsPIN5b is an endoplasmic reticulum (ER) -localized protein that participates in auxin homeostasis, transport and distribution in vivo. This work describes an example of an auxin-related gene where modulating its expression can simultaneously improve plant architecture and yield potential in rice, and reveals an important effect of hormonal signaling on these traits.

Adventitious root (AR) formation in the stem base of cuttings is the basis for propagation of many plant species and petunia is used as model to study this developmental process. Following AR formation from 2 to 192 hours after excision (hpe) of cuttings, transcriptome analysis by microarray revealed a change of the character of the rooting zone from stem base to root identity. The greatest shift in the number of differentially expressed genes was observed between 24 and 72 hpe, when the categories storage, mineral nutrient acquisition, anti-oxidative and secondary metabolism, and biotic stimuli showed a notable high number of induced genes. Analyses of phytohormone-related genes disclosed multifaceted changes of the auxin transport system, auxin conjugation and the auxin signal perception machinery indicating a reduction in auxin sensitivity and phase-specific responses of particular auxin-regulated genes. Genes involved in ethylene biosynthesis and action showed a more uniform pattern as a high number of respective genes were generally induced during the whole process of AR formation. The important role of ethylene for stimulating AR formation was demonstrated by the application of inhibitors of ethylene biosynthesis and perception as well as of the precursor aminocyclopropane-1-carboxylic acid, all changing the number and length of AR. A model is proposed showing the putative role of polar auxin transport and resulting auxin accumulation in initiation of subsequent changes in auxin homeostasis and signal perception with a particular role of Aux/IAA expression. These changes might in turn guide the entrance into the different phases of AR formation. Ethylene biosynthesis, which is stimulated by wounding and does probably also respond to other stresses and auxin, acts as important stimulator of AR formation probably via the expression of ethylene responsive transcription factor genes, whereas the timing of different phases seems to be controlled by auxin.

We studied the role of Arabidopsis thaliana TCP15, a member of the TEOSINTE BRANCHED1-CYCLOIDEA-PCF (TCP) transcription factor family, in gynoecium development. Plants that express TCP15 from the 35S CaMV promoter (35S:TCP15) develop flowers with defects in carpel fusion and a reduced number of stigmatic papillae. In contrast, the expression of TCP15 fused to a repressor domain from its own promoter causes the development of outgrowths topped with stigmatic papillae from the replum. 35S:TCP15 plants show lower levels of the auxin indoleacetic acid and reduced expression of the auxin reporter DR5 and the auxin biosynthesis genes YUCCA1 and YUCCA4, suggesting that TCP15 is a repressor of auxin biosynthesis. Treatment of plants with cytokinin enhances the developmental effects of expressing TCP15 or its repressor form. In addition, treatment of a knock-out double mutant in TCP15 and the related gene TCP14 with cytokinin causes replum enlargement, increased development of outgrowths, and the induction of the auxin biosynthesis genes YUCCA1 and YUCCA4. A comparison of the phenotypes observed after cytokinin treatment of plants with altered expression levels of TCP15 and auxin biosynthesis genes suggests that TCP15 modulates gynoecium development by influencing auxin homeostasis. We propose that the correct development of the different tissues of the gynoecium requires a balance between auxin levels and cytokinin responses, and that TCP15 participates in a feedback loop that helps to adjust this balance.

© 2014 The Authors. All rights reserved. Arabidopsis phytochrome B (phyB) is the major photoreceptor that senses the ratio of red to far-red light (R:FR) to regulate the shade-avoidance response (SAR). It has been hypothesized that altered homeostasis of phytohormones such as auxin and strigolactone is at least partially responsible for SAR, but the mechanism underlying phyB regulation of the hormonal change is not fully understood. Previously we reported that CONSTANS-LIKE 7 (COL7) enhances branching number under high R:FR but not under low R:FR, implying that COL7 may be involved in the phyB-mediated SAR. In this study, we provide evidence that COL7 reduces auxin levels in a high R:FR-dependent manner. We found that the phyB mutation suppresses the COL7-induced branching proliferation. Moreover, COL7 promotes mRNA expression of SUPERROOT 2 (SUR2), which encodes a suppressor of auxin biosynthesis, in high R:FR but not in low R:FR. Consistently with these results, deficiency of phyB suppresses the elevated transcription of SUR2 in COL7 overexpression plants grown in high R:FR. Taking these results together with data suggesting that photo-excited phyB is required for stabilization of the COL7 protein, we argue that COL7 is a critical factor linking light perception to changes in auxin level in Arabidopsis.

Plant hemoglobins (Hbs) have been identified as master regulators in determining the developmental fate of specific cells during maize embryogenesis. Whether an embryogenic cell lives or undergoes programmed cell death (PCD) is modulated by Hbs, through their tight interactions with nitric oxide (NO) and auxin. During maize embryogenesis, Hb-suppressing cells accumulate NO, are depleted of auxin, and are committed to die. We propose that Hbs control cell fate by regulating NO and auxin homeostasis, and that this type of mechanism may influence other hormonal responses modulating plant behavior during development and stress conditions.

Auxin is a phytohormone involved in cell elongation and division. Levels of indole-3-acetic acid (IAA), the primary auxin, are tightly regulated through biosynthesis, degradation, sequestration, and transport. IAA is sequestered in reversible processes by adding amino acids, polyol or simple alcohols, or sugars, forming IAA conjugates, or through a two-carbon elongation forming indole-3-butyric acid. These sequestered forms of IAA alter hormone activity. To gain a better understanding of how auxin homeostasis is maintained, we have generated Arabidopsis (Arabidopsis thaliana) mutants that combine disruptions in the pathways, converting IAA conjugates and indole-3-butyric acid to free IAA. These mutants show phenotypes indicative of low auxin levels, including delayed germination, abnormal vein patterning, and decreased apical dominance. Root phenotypes include changes in root length, root branching, and root hair growth. IAA levels are reduced in the cotyledon tissue but not meristems or hypocotyls. In the combination mutants, auxin biosynthetic gene expression is increased, particularly in the YUCCA/Tryptophan Aminotransferase of Arabidopsis1 pathway, providing a feedback mechanism that allows the plant to compensate for changes in IAA input pathways and maintain cellular homeostasis.

In high-light environments, plants are exposed to different types of stresses, such as an excess of UV-B, but also drought stress which triggers a common morphogenic adaptive response resulting in a general reduction of plant growth. Here, we report that the Arabidopsis thaliana UV RESISTANCE LOCUS 8 (UVR8) gene, a known regulator of the UV-B morphogenic response, was able to complement a Saccharomyces cerevisiae osmo-sensitive mutant and its expression was induced after osmotic or salt stress in Arabidopsis plants. Under low levels of UV-B, plants overexpressing UVR8 are dwarfed with a reduced root development and accumulate more flavonoids compared to control plants. The growth defects are mainly due to the inhibition of cell expansion. The growth inhibition triggered by UVR8 overexpression in plants under low levels of UV-B was exacerbated by mannitol-induced osmotic stress, but it was not significantly affected by ionic stress. In contrast, uvr8-6 mutant plants do not differ from wild-type plants under standard conditions, but they show an increased shoot growth under high-salt stress. Our data suggest that UVR8-mediated accumulation of flavonoid and possibly changes in auxin homeostasis are the underlying mechanism of the observed growth phenotypes and that UVR8 might have an important role for integrating plant growth and stress signals., This work was supported by the Interuniversity Attraction Poles Programme (IUAP P7/29 'MARS') initiated by the Belgian Science Policy Office, by grants from Ghent University (Bijzonder Onderzoeksfonds Methusalem project no. BOF08/01M00408), and by grants to the MIUR project FIRB Plant-STRESS.

The obligate biotrophic protist Plasmodiophora brassicae causes worldwide devastating losses on Brassica crops. Among these are oilseed rape, vegetable brassicas, and turnips. However, the fact that Arabidopsis thaliana is a good host for P. brassicae, has boosted research on the molecular interaction using the resources available for this model plant. Due to the uncontrolled growth of infected host root tissues the disease has been coined “clubroot.” Consequently, during the last years, alterations in host hormone metabolisms have been described. Influencing the hormonal balance leads to aberrant growth responses in the clubbed roots. The discussion presented in the following will focus on growth promoting hormones, mainly auxins, with the interaction to other growth associated hormonal signaling pathways, such as cytokinins and brassinosteroids.

The phytohormone auxin is important in virtually all aspects of plant growth and development, yet our understanding of auxin homeostasis is far from complete. Plants use several mechanisms to control levels of the active auxin, indole-3-acetic acid (IAA). Plants can synthesize IAA both from tryptophan (Trp-dependent pathways) and from a Trp precursor but bypassing Trp (Trp-independent pathways). Despite progress in identifying enzymes in Trp-dependent IAA biosynthesis, no single IAA biosynthetic pathway is yet defined to the level that all of the relevant genes, enzymes, and intermediates are identified. In addition to de novo synthesis, vascular plants can obtain IAA from the hydrolysis of IAA conjugates. IAA can be conjugated to amino acids, sugars, and peptides; endogenous conjugates that are active in bioassays and hydrolyzed in plants are likely to be important free IAA sources. Conjugation is also used to permanently inactivate excess IAA, and these conjugates may be distinct from the hydrolyzable conjugates. The peroxisomal (3-oxidation of endogenous indole-3-butyric acid (IBA) also can supply plants with IAA, which may account for part of the auxin activity of exogenous IBA. Compartmentalization of enzymes and precursors may contribute to the regulation of auxin metabolism. IAA obtained through de novo synthesis, conjugate hydrolysis, or IBA β-oxidation may have different functions in plant development, and possible roles for the IAA derived from the various pathways are discussed.

The first committed step in the biosynthesis of indole glucosinolates is the conversion of indole-3-acetaldoxime into an indole-3-S-alkyl-thiohydroximate. The initial step in this conversion is catalyzed by CYP83B1 in Arabidopsis (S. Bak, F.E. Tax, K.A. Feldmann, D.A. Galbraith, R. Feyereisen [2001] Plant Cell 13: 101–111). The knockout mutant of the CYP83B1 gene (rnt1-1) shows a strong auxin excess phenotype and are allelic to sur-2. CYP83A1 is the closest relative to CYP83B1 and shares 63% amino acid sequence identity. Although expression of CYP83A1 under control of its endogenous promoter in thernt1-1 background does not prevent the auxin excess and indole glucosinolate deficit phenotype caused by the lack of the CYP83B1 gene, ectopic overexpression of CYP83A1 using a 35S promoter rescues the rnt1-1 phenotype. CYP83A1 and CYP83B1 heterologously expressed in yeast (Saccharomyces cerevisiae) cells show marked differences in their substrate specificity. Both enzymes convert indole-3-acetaldoxime to a thiohydroximate adduct in the presence of NADPH and a nucleophilic thiol donor. However, indole-3-acetaldoxime has a 50-fold higher affinity toward CYP83B1 than toward CYP83A1. Both enzymes also metabolize the phenylalanine- and tyrosine-derived aldoximes. Enzyme kinetic comparisons of CYP83A1 and CYP83B1 show that indole-3-acetaldoxime is the physiological substrate for CYP83B1 but not for CYP83A1. Instead, CYP83A1 catalyzes the initial conversion of aldoximes to thiohydroximates in the synthesis of glucosinolates not derived from tryptophan. The two closely related CYP83 subfamily members therefore are not redundant. The presence of putative auxin responsive cis-acting elements in the CYP83B1 promoter but not in the CYP83A1 promoter supports the suggestion that CYP83B1 has evolved to selectively metabolize a tryptophan-derived aldoxime intermediate shared with the pathway of auxin biosynthesis in Arabidopsis.

Although auxin is known to regulate many processes in plant development and has been studied for over a century, the mechanisms whereby plants produce it have remained elusive. Here we report the characterization of a dominant Arabidopsis mutant, yucca , which contains elevated levels of free auxin. YUCCA encodes a flavin monooxygenase–like enzyme and belongs to a family that includes at least nine other homologous Arabidopsis genes, a subset of which appears to have redundant functions. Results from tryptophan analog feeding experiments and biochemical assays indicate that YUCCA catalyzes hydroxylation of the amino group of tryptamine, a rate-limiting step in tryptophan-dependent auxin biosynthesis.

Genetic screens have been performed to identify mutants with altered auxin homeostasis in Arabidopsis . A tagged allele of the auxin-overproducing mutant sur2 was identified within a transposon mutagenized population. The SUR2 gene was cloned and shown to encode the CYP83B1 protein, which belongs to the large family of the P450-dependent monooxygenases. SUR2 expression is up-regulated in sur1 mutants and induced by exogenous auxin in the wild type. Analysis of indole-3-acetic acid (IAA) synthesis and metabolism in sur2 plants indicates that the mutation causes a conditional increase in the pool size of IAA through up-regulation of IAA synthesis.

Following perception of a pathogenic attack, plants are able to develop a strong response with the corresponding activation of a plethora of defense-related genes. In this study we have characterized the mode of expression of the CEVI-1 gene from tomato plants, which encodes an anionic peroxidase. CEVI-1 expression is induced during the course of compatible viral and subviral infections, like many other defense-related genes, but is induced neither in incompatible interactions nor by signal molecules such as salicylic acid, ethylene, or methyl jasmonate. Additionally, CEVI-1 is induced in detached leaf tissues following a pathway distinct from that related to the classical wound response. We also describe the characterization of the structural CEVI-1 gene and compare the mode of expression in different transgenic plant species harboring a CEVI-1::GUS construct. Furthermore, we have isolated mutants in Arabidopsis, called dth mutants, that are deregulated in the control of expression of this gene. From the initial analysis of some of these mutants it seems that activation of CEVI-1 gene expression correlates with a defect in the perception of auxins by the plant. All these results may suggest that, during systemic infections with viruses, auxin homeostasis is one of the components participating in the regulation of the overall defense response.

Lateral root formation is a critical agronomic trait in plant architecture that determines crop productivity and environmental stress adaptability. It is therefore tightly regulated both by intrinsic developmental cues, such as abscisic acid (ABA) and auxin, and by diverse environmental growth conditions, including water deficit and high salinity in the soil. We have recently reported that an Arabidopsis R2R3-type MYB transcription factor, MYB96, regulates lateral root meristem activation under drought conditions via an ABA-auxin signaling crosstalk. In this signaling scheme, the MYB96-mediated ABA signals are incorporated into an auxin signaling pathway that involves a subset of GH3 gene encoding auxin-conjugating enzymes. The MYB96-overexpressing, activation tagging mutant, which is featured by having dwarfed growth and reduced lateral root formation, exhibits an enhanced drought resistance. In the mutant, expression of the GH3 genes was significantly elevated, which is consistent with the reduced later...

With the aim of investigating the mechanisms that maintain auxin homeostasis in plants, we have monitored the net uptake and metabolism of exogenously supplied indole-3-acetic acid (IAA) and naphthalene-1-acetic acid (NAA) in seedlings of wild type and the IAA-overproducing mutant sur 1 of Arabidopsis thaliana. Tritiated IAA and NAA entered the seedling tissues within minutes and were mostly accumulated as metabolites, probably amino acid and sugar conjugates. The mutant seedlings were marked by a strong increase of [ 3 H]IAA metabolism and a reduction of the accumulation levels of both free [ 3 H]IAA and [ 3 H]NAA. The same characteristics were observed in wild-type seedlings grown on 5 μM picloram. We measured [ 3 H]NAA uptake in the presence of high concentrations of unlabeled NAA or the auxin efflux carrier inhibitor naphthylphthalamic acid (NPA). This abolished the difference in free [ 3 H]NAA accumulation between the mutant or picloram-treated seedlings and wild-type seedlings. These data indicated that active auxin efflux carriers were present in Arabidopsis seedling tissues. Picloram-treated seedlings and seedlings of the IAA-overproducing mutant sur1 displayed increased auxin efflux carrier activity as well as elevated conjugation of IAA. There is previous evidence to suggest that conjugation is a means to remove excess IAA in plant cells. Here, we discuss the possibility of efflux constituting an additional mechanism for regulating free IAA levels in the face of an excess auxin supply.

The metabolism of indole-3-acetic acid (IAA) was investigated in 14-d-old Arabidopsis plants grown in liquid culture. After ruling out metabolites formed as an effect of nonsterile conditions, high-level feeding, and spontaneous interconversions, a simple metabolic pattern emerged. Oxindole-3-acetic acid (OxIAA), OxIAA conjugated to a hexose moiety via the carboxyl group, and the conjugates indole-3-acetyl aspartic acid (IAAsp) and indole-3-acetyl glutamate (IAGlu) were identified by mass spectrometry as primary products of IAA fed to the plants. Refeeding experiments demonstrated that none of these conjugates could be hydrolyzed back to IAA to any measurable extent at this developmental stage. IAAsp was further oxidized, especially when high levels of IAA were fed into the system, yielding OxIAAsp and OH-IAAsp. This contrasted with the metabolic fate of IAGlu, since that conjugate was not further metabolized. At IAA concentrations below 0.5 μm, most of the supplied IAA was metabolized via the OxIAA pathway, whereas only a minor portion was conjugated. However, increasing the IAA concentrations to 5 μm drastically altered the metabolic pattern, with marked induction of conjugation to IAAsp and IAGlu. This investigation used concentrations for feeding experiments that were near endogenous levels, showing that the metabolic pathways controlling the IAA pool size in Arabidopsis are limited and, therefore, make good targets for mutant screens provided that precautions are taken to avoid inducing artificial metabolism.

Auxin concentrations in plants are tightly regulated through both biosynthesis and degradation. In the past few years, much progress was made in the area of auxin metabolism. Genetic and biochemical studies in Arabidopsis unequivocally established a complete tryptophan (Trp)-dependent two-step auxin biosynthesis pathway in which Trp is first converted into indole-3-pyruvate (IPA) by the TAA family of aminotransferases and subsequently indole-3-acetic acid (IAA) is produced from IPA by the YUC family of flavin monooxygenases. The TAA/YUC pathway is highly conserved in the plant kingdom and is probably the main auxin biosynthesis pathway in plants. Recent work also demonstrated that oxidative degradation of auxin plays an essential role in maintaining auxin homeostasis and in regulating plant development. In this chapter, we discuss the recent advancements in auxin biosynthesis and catabolism.

Indole-3-butyric acid (IBA) is an important contributor to auxin homeostasis and blocking IBA conversion to the active auxin indole-3-acetic acid (IAA) results in an array of developmental defects. Similar to IAA, IBA movement within the plant is mediated by carriers, many of which remain unidentified. In this chapter, we discuss roles for IBA in plant development, roles for PLEIOTROPIC DRUG RESISTANCE (PDR) members of the ABCG family in IBA transport, and missing transporters required for IBA movement.

Vegetative propagation of economically important woody, horticultural and agricultural species rely on an efficient adventitious root (AR) formation. The formation of ARs is a complex genetic trait regulated by the interaction of environmental and endogenous factors among which the phytohormone auxin plays an essential role. This article summarizes the current knowledge related to the intricate network through which auxin controls adventitious rooting. How auxin and recently identified auxin-related compounds affect AR formation in different plant species is discussed. Particular attention is addressed to illustrate how auxin has a central role in the hormone cross-talk leading to AR development. In parallel, we describe the molecular players involved in the control of auxin homeostasis, transport and signaling, for a better understanding of the auxin action during adventitious rooting.

The plant cell wall is a highly dynamic structure that changes in response to both environmental and developmental cues. It plays important roles throughout plant growth and development in determining the orientation and extent of cell expansion, providing structural support and acting as a barrier to pathogens. Despite the importance of the cell wall, the signaling pathways regulating its function are not well understood. Two partially redundant leucine-rich-repeat receptor-like kinases (LRR-RLKs), FEI1 and FEI2, regulate cell wall function in Arabidopsis thaliana roots; disruption of the FEIs results in short, swollen roots as a result of decreased cellulose synthesis. We screened for suppressors of this swollen root phenotype and identified two mutations in the putative mitochondrial pyruvate dehydrogenase E1α homolog, IAA-Alanine Resistant 4 (IAR4). Mutations in IAR4 were shown previously to disrupt auxin homeostasis and lead to reduced auxin function. We show that mutations in IAR4 suppress a subset of the fei1 fei2 phenotypes. Consistent with the hypothesis that the suppression of fei1 fei2 by iar4 is the result of reduced auxin function, disruption of the WEI8 and TAR2 genes, which decreases auxin biosynthesis, also suppresses fei1 fei2. In addition, iar4 suppresses the root swelling and accumulation of ectopic lignin phenotypes of other cell wall mutants, including procuste and cobra. Further, iar4 mutants display decreased sensitivity to the cellulose biosynthesis inhibitor isoxaben. These results establish a role for IAR4 in the regulation of cell wall function and provide evidence of crosstalk between the cell wall and auxin during cell expansion in the root.

To study the physiological functions of PIP5K2, a putative knockout mutant pip5k2 (SALK_012487) was identified by searching the Salk Institute T-DNA insertion library database. The T-DNA insertion was confirmed by PCR analysis. Phenotypic observation revealed that the knockout mutant of PIP5K2, pip5k2, has reduced lateral root formation in all the stages of lateral root development and this phenotype could be rescued by exogenous auxin. Transformation rescue of PIP5K2 in pip5k2 background rescues all the phenotype, indicating the defects are indeed caused by PIP5K2 deficiency. Reduced auxin accumulation in pip5k2 is confirmed by GUS activity detection in cross progenies with DR5-GUS marker line. Real-time RT-PCR analysis on auxin biosynthesis and homeostasis genes revealed that auxin biosynthesis-related genes were suppressed while metabolism-related genes that convert auxin to inactive conjugates were stimulated in pip5k2. These results indicate that Arabidopsis PIP5K2 is involved in regulating lateral root formation, probably through modulating auxin accumulation and homeostasis.

The phytohormone auxin is of fundamental importance in plant development. Since the identification of auxin as a plant growth substance, auxin transport has drawn considerable research attention. Intercellular (polar) auxin transport contributes to its graded distribution in cell files or entire organs and allows for dynamic, environmentally controlled rearrangements in auxin accumulation. Insights into polar auxin transport mechanisms have broadened our understanding of the phenotypic flexibility of plants. Besides intercellular auxin transport, auxin is also transported intracellularly across organelle membranes, but its importance in plant development remains nascent. The intracellular sequestration of auxin into cellular compartments, such as peroxisomes and the endoplasmic reticulum (ER), plays important roles in auxin metabolism and could furthermore, in the case of the ER, have a direct impact on auxin signaling events. In this chapter, we review the most recent insights into intracellular auxin transport and its role in cellular auxin homeostasis.

Trafficking of membrane proteins in plants is important for cellular homeostasis, cell–cell communication, and signaling. Identifying plant-specific endosomal components in exocytosis and endocytosis pathways has led to many advances in studies of plant membrane protein trafficking. The Arabidopsis ATP-binding cassette protein subfamily B (ABCB/PGP/MDR) family includes 21 genes and some of them mediate auxin distribution, but little is known about their intracellular trafficking. Among Arabidopsis ABCB proteins, only trafficking of ABCB1, ABCB4, and ABCB19 has been reported. ABCB1, ABCB4, and ABCB19 are localized symmetrically at the plasma membrane (PM) in most tissues and require TWISTED DWARF1 (TWD1) for PM targeting. In addition, the secretory pathway of ABCB19 to the PM is regulated by GNL1 at the Golgi which shows low abundance of ABCB19 in gnl1. ABCB4 and ABCB19 associate strongly in the PM and show non-dynamic and non-recycling features between the PM and early endosomes. ABCB4 slowly endocytoses to the vacuole via SNX1, and the pathway is GNOM independent but actin dependent. The role of ABCB19 and ABCB4 suggests that ABCB19 localization in the detergent resistant membrane contributes to PIN1 stabilization at the PM and that non-dynamic trafficking of ABCB4 may be involved in basal auxin homeostasis in cells.

Auxin levels and the regulation of reversible auxin conjugation as a mechanism of auxin homeostasis has been investigated in Chinese cabagge (Brassica rapa L. ssp. pekinensis) upon biotic stress caused by Cucumber mosaic virus containing satellite RNA (CMVsat) infection and abiotic stress (salt stress, drought) as well as tretments with stress hormones (SA, JA, ABA). To detect the physiological state of plants and their ability to integrate given stress conditions, a set of biochemical markers (glutathione level, antioxidant enzymes activity, carbonyl content) were analysed. Endogenous free and total IAA, and ABA were analysed by GC-MS. The levels of auxin were significantly affected by biotic and abiotic stresses as well as treatments with stress hormones. All stress conditions caused perturbation of IAA levels with a tendency of increasing the IAA conjugate level. Among treatments with stress hormones, JA caused the most significant increase of free and conjugated IAA in comparison to others. Endogenous ABA was significantly higher under both abiotic stress conditions in comparison to the control showing a positive correlation with auxin levels. Results of gene expression experiments (qRT-PCR) showed an increased transcript level of auxin amidohydrolase BrIAR3 upon abiotic stress as well as after treatment with JA. The results implicated an important role of auxin and interactions with other stress hormones in the plant stress response.

1. Introduction Auxin-amidohydrolases (AAHs) from Chinese cabbage (Brassica rapa L.) (BrILL2 and BrIAR3) hydrolase amino acid conjugates (AACs) of plant hormones auxins, thereby releasing free active auxins. The mode of action of free auxins and their accumulation at sites of physiological action are still not well established, but it is evident that AAHs play a role in auxin homeostasis. So far, we established that BrILL2 and BrIA3 preferentially cleave long-chain AACs (IPAala and IBAala), while Arabidopsis AAHs have higher specificity for IAAala. Furthermore, a potential active site of the auxin amidohydrolase BrILL2 was proposed by modelling. AAHs possess two conserved cysteines of which one, Cys 139 is suggested to be a part of the binding pocket that coordinates metal binding. Mn2+ was found to be the only metal ion with cofactor activity, although number and positioning of the cofactor in the binding pocket is still unclear. Herein we investigated the role of Cys residues at positions 139 and 320 in the regulation of the enzyme activity. 2. Results and Discussion The 3D structure of BrILL2 was modeled by using the X-ray structure of A.thaliana IAA-AAH as a template ; the only amidohydrolase crystallized so far with high primary sequence similarity (76%) to BrILL2. The readily recognizable architecture of both AAHs is characterized by two perpendicular domains, with the larger catalytic domain bearing a proposed binuclear metal center, and the smaller ‘‘satellite’’ domain, usually functioning as as a polymerization site. Enzymes, wt and mutants Cys139Ser, Cys320Ser, and Cys139, 320Ser were generated and produced in E. coli as previously described. The preservation of structure of mutants in comparison to wt enzyme was confirmed by circular dichroism. Results of kinetics analysis show that mutant Cys320Ser remains active, but less than wt. Cys139Ser mutation leads to a complete inactivation of the enzyme, probably due to its role in the coordination of one of the Mn2+ ions. Further experiments by isotermal titration calorimetry will give insight into the metal binding and possible role of Cys139.

Plants have evolved sophisticated mechanisms to sense the presence of other plants growing nearby and adjust their growth rate accordingly. The early perception of neighbor proximity depends on the detection of light quality changes. Within a vegetation community, the ratio of red (R) to far-red (FR) light is lowered by the absorption of R light by photosynthetic pigments. This light quality change is perceived through phytochrome (phyB, phyD, and phyE in Arabidopsis) as a signal of the proximity of neighbors and induces a suite of developmental responses (termed the shade avoidance response). In Arabidopsis shade avoidance is regulated by a balance of positive (PIF) and negative (HFR1/SICS1) regulators of gene expression which ensures a fast reshaping of the plant body toward an environment optimal for growth while at the same time avoiding an exaggerated reaction to low R/FR. Persistency of a low R/FR signal enhances the activity of phyA and, in turn, of HY5, a master regulator of seedling de-etiolation. Several hormones, such as gibberellins and brassinosteroids, have been implicated in shade-induced elongation. However, a compelling amount of evidence indicates that low R/FR-induced changes in auxin homeostasis and auxin transport are central in the shade avoidance response. This chapter describes the recent advances in understanding how auxin coordinates plant growth in a low R/FR light environment.

The plant hormones gibberellins (GAs) and auxin display overlapping activities in the regulation of multiple developmental processes, including the differential growth that mediates the response to tropic stimuli and the formation of the apical hook. Several mechanisms have been proposed that explain the interaction between these two hormones, such as the regulation of auxin transport by GAs, and the regulation of GA biosynthesis by auxin. GAs are known to exert their action at the transcriptional level by promoting the degradation of DELLA proteins, which in turn interact with numerous transcription factors and modulate their activity. We have identified INDOLE-3-ACETIC ACID METHYLTRANSFERASE 1 (IAMT1) as one of the earliest target genes upregulated after conditional expression of the DELLA protein GAI in Arabidopsis thaliana. In this Thesis, we have addressed two main issues: (1) the contribution of IAMT1 to auxin homeostasis and its biological relevance; and (2) the molecular mechanism by which DELLAs are able to induce the expression of IAMT1. Using combinations of iamt1 loss-of-function mutants and reporter lines for auxin accumulation and activity, we have found that IAMT1 activity is essential for proper generation and maintenance of the auxin gradients that underlie differential growth. According to our results, the role of IAMT1 would be to restrict polar auxin transport especially during the response to tropic stimuli, preventing excessive auxin accumulation in the responding tissues, and IAMT1 exerts this function, at least in part, by inhibiting the expression of the PIN genes, encoding auxin efflux carriers. Regarding the regulation of IAMT1 expression by DELLAs, dissection of the promoter, in silico analysis of putative DELLA partners, and molecular genetic analysis of reporter lines has allowed us to identify two mechanisms with different relevance depending on the environmental conditions, and through different cis elements. In etiolated seedlings, DELLA proteins are recruited by DORNRÖSCHEN (DRN) to the IAMT1 promoter to induce IAMT1 expression. In the light and in a temperature-dependent manner, DELLA proteins inhibit the DNA-binding activity of PHYTOCHROME-INTERACTING FACTOR4 (PIF4) and BRI1 EMS-SUPPRESSOR1(BES1), which act as repressors of IAMT1 expression. The work presented here highlights how GAs may affect local accumulation of auxin, being particularly relevant in processes that involve differential growth., Abbas, M. (2014). The control of auxin homeostasis through the regulation of IAMT1 by DELLA proteins [Tesis doctoral no publicada]. Universitat Politècnica de València. doi:10.4995/Thesis/10251/39348., Alfresco

A highly active synthetic auxin response element (AuxRE), referred to as DR5, was created by performing site-directed mutations in a natural composite AuxRE found in the soybean GH3 promoter. DR5 consisted of tandem direct repeats of 11 bp that included the auxin-responsive TGTCTC element. The DR5 AuxRE showed greater auxin responsiveness than a natural composite AuxRE and the GH3 promoter when assayed by transient expression in carrot protoplasts or in stably transformed Arabidopsis seedlings, and it provides a useful reporter gene for studying auxin-responsive transcription in wild-type plants and mutants. An auxin response transcription factor, ARF1, bound with specificity to the DR5 AuxRE in vitro and interacted with Aux/IAA proteins in a yeast two-hybrid system. Cotransfection experiments with natural and synthetic AuxRE reporter genes and effector genes encoding Aux/IAA proteins showed that overexpression of Aux/IAA proteins in carrot protoplasts resulted in specific repression of TGTCTC AuxRE reporter gene expression.

The clubroot disease, caused by the obligate biotrophic protist Plasmodiophora brassicae, affects cruciferous crops worldwide. It is characterized by root swellings as symptoms, which are dependent on the alteration of auxin and cytokinin metabolism. Here, we describe that two different classes of auxin receptors, the TIR family and the auxin binding protein 1 (ABP1) in Arabidopsis thaliana are transcriptionally upregulated upon gall formation. Mutations in the TIR family resulted in more susceptible reactions to the root pathogen. As target genes for the different pathways we have investigated the transcriptional regulation of selected transcriptional repressors (Aux/IAA) and transcription factors (ARF). As the TIR pathway controls auxin homeostasis via the upregulation of some auxin conjugate synthetases (GH3), the expression of selected GH3 genes was also investigated, showing in most cases upregulation. A double gh3 mutant showed also slightly higher susceptibility to P. brassicae infection, while all tested single mutants did not show any alteration in the clubroot phenotype. As targets for the ABP1-induced cell elongation the effect of potassium channel blockers on clubroot formation was investigated. Treatment with tetraethylammonium (TEA) resulted in less severe clubroot symptoms. This research provides evidence for the involvement of two auxin signaling pathways in Arabidopsis needed for the establishment of the root galls by P. brassicae.