Auxin Influx Carriers Control Vascular Patterning and Xylem Differentiation in Arabidopsis thaliana

Auxin is an essential hormone for plant growth and development. Auxin influx carriers AUX1/LAX transport auxin into the cell, while auxin efflux carriers PIN pump it out of the cell. It is well established that efflux carriers play an important role in the shoot vascular patterning, yet the contribution of influx carriers to the shoot vasculature remains unknown. Here, we combined theoretical and experimental approaches to decipher the role of auxin influx carriers in the patterning and differentiation of vascular tissues in the Arabidopsis inflorescence stem. Our theoretical analysis predicts that influx carriers facilitate periodic patterning and modulate the periodicity of auxin maxima. In agreement, we observed fewer and more spaced vascular bundles in quadruple mutants plants of the auxin influx carriers aux1lax1lax2lax3. Furthermore, we show AUX1/LAX carriers promote xylem differentiation in both the shoot and the root tissues. Influx carriers increase cytoplasmic auxin signaling, and thereby differentiation. In addition to this cytoplasmic role of auxin, our computational simulations propose a role for extracellular auxin as an inhibitor of xylem differentiation. Altogether, our study shows that auxin influx carriers AUX1/LAX regulate vascular patterning and differentiation in plants.     

The binding of auxin to the Arabidopsis auxin influx transporter AUX1

The cellular import of the hormone auxin is a fundamental requirement for the generation of auxin gradients that control a multitude of plant developmental processes. The AUX/LAX family of auxin importers, exemplified by AUX1 from Arabidopsis (Arabidopsis thaliana), has been shown to mediate auxin import when expressed heterologously. The quantitative nature of the interaction between AUX1 and its transport substrate indole-3-acetic acid (IAA) is incompletely understood, and we sought to address this in the present investigation. We expressed AUX1 to high levels in a baculovirus expression system and prepared membrane fragments from baculovirus-infected insect cells. These membranes proved suitable for determination of the binding of IAA to AUX1 and enabled us to determine a K(d) of 2.6 mum, comparable with estimates for the K(m) for IAA transport. The efficacy of a number of auxin analogues and auxin transport inhibitors to displace IAA binding from AUX1 has also been determined and can be rationalized in terms of their physiological effects. Determination of the parameters describing the initial interaction between a plant transporter and its hormone ligand provides novel quantitative data for modeling auxin fluxes.     

Molecular players of auxin transport systems: advances in genomic and molecular events*

Phytohormone auxin plays an indispensable role in the plethora of plant developmental process starting from the cell division, and cell elongation to morphogenesis. Auxins are transported to different parts of the plant by different sophisticated transporter molecules known as ‘auxin transporters’.There are four auxin transporter families that have been reported so far in the plant kingdom which includes AUX/LAX (AUXIN-RESISTANT1–LIKES), PIN (PIN-FORMED, auxin efflux carriers), ABCB ((ATP-binding cassette-B (ABCB)/P-glycoprotein (PGP)) and PILS (PIN-Likes). Auxin influx and efflux carriers are distributed in a polar fashion in the plasma membrane whereas ABCB and PILS are present in a non-polar fashion. Other than AUX/LAX, other auxin transporters harbor N-and C-terminal conserved domains along with a variable hydrophilic loop in the transmembrane domain. The AUX/LAX, ABCB and PIN transporters mediate long distance auxin transport whereas PILS and PIN5 protein involved in intracellular auxin homeostasis.     

Systems Analysis of Auxin Transport in the Arabidopsis Root Apex

Auxin is a key regulator of plant growth and development. Within the root tip, auxin distribution plays a crucial role specifying developmental zones and coordinating tropic responses. Determining how the organ-scale auxin pattern is regulated at the cellular scale is essential to understanding how these processes are controlled. In this study, we developed an auxin transport model based on actual root cell geometries and carrier subcellular localizations. We tested model predictions using the DII-VENUS auxin sensor in conjunction with state-of-the-art segmentation tools. Our study revealed that auxin efflux carriers alone cannot create the pattern of auxin distribution at the root tip and that AUX1/LAX influx carriers are also required. We observed that AUX1 in lateral root cap (LRC) and elongating epidermal cells greatly enhance auxin’s shootward flux, with this flux being predominantly through the LRC, entering the epidermal cells only as they enter the elongation zone. We conclude that the nonpolar AUX1/LAX influx carriers control which tissues have high auxin levels, whereas the polar PIN carriers control the direction of auxin transport within these tissues.     

The AUX1 LAX family of auxin influx carriers is required for the establishment of embryonic root cell organization in Arabidopsis thaliana

Background and Aims

The root meristem of the Arabidopsis thaliana mature embryo is a highly organized structure in which individual cell shape and size must be regulated in co-ordination with the surrounding cells. The objective of this study was to determine the role of the AUX1 LAX family of auxin import carriers during the establishment of the embryonic root cell pattern.

Methods

The radicle apex of single and multiple aux1 lax mutant mature embryos was used to evaluate the effect of this gene family upon embryonic root organization and root cap size, cell number and cell size.

Key Results

It was demonstrated here that mutations within the AUX1 LAX family are associated with changes in cell pattern establishment in the embryonic quiescent centre and columella. aux1 lax mutants have a larger radicle root cap than the wild type and this is associated with a significant increase in the root-cap cell number, average cell size, or both. Extreme disorganization of the radicle apex was observed among quadruple aux1 lax1 lax2 lax3 mutant embryos, but not in single aux1 null or in lax1, lax2 and lax3 single mutants, indicating redundancy within the AUX1 LAX family.

Conclusions

It was determined that the AUX1 LAX family of auxin influx facilitators participates in the establishment of cell pattern within the apex of the embryonic root in a gene-redundant fashion. It was demonstrated that aux1 lax mutants are affected in cell proliferation and cell growth within the radicle tip. Thus AUX1 LAX auxin importers emerge as new players in morphogenetic processes involved in patterning during embryonic root formation.Key words: AUX1 LAX genes, auxin, Arabidopsis thaliana, embryogenesis, meristem, radicle development, cell pattern establishment     

Analysis of subcellular localization of auxin carriers PIN,AUX/LAX and PGP in Sorghum bicolor

Auxin transport at least correlates to the three gene families: efflux carriers PIN-formed (PIN), p-glycoprotein (PGP), and influx carrier auxin resistant 1/like aux1(AUX/LAX) in Arabidopsis thaliana. In monocotyledon Sorghum bicolor, the biological function of these genes retains unclear. Our previous study reported that the member analysis, organ-specific expression and expression profiles of the auxin transporter PIN, PGP and AUX/LAX gene families in Sorghum bicolor under IAA, brassinosteroid, polar auxin transport inhibitors and abiotic stresses. Here we further supply the prediction of subcellular localization of SbPIN, SbLAX and SbPGP proteins and discuss the potential relationship between the subcellular localization and stress response. The predicted results showed that the most of SbPIN, SbLAX and SbPGP proteins are localized to the plasma membrane, except few localized to vacuolar membrane and endoplasmic reticulum. This data set provides novel information for investigation of auxin transporters in Sorghum bicolor.     

Auxin influx activity is associated with Frankia infection during actinorhizal nodule formation in Casuarina glauca

Plants from the Casuarinaceae family enter symbiosis with the actinomycete Frankia leading to the formation of nitrogen-fixing root nodules. We observed that application of the auxin influx inhibitor 1-naphtoxyacetic acid perturbs actinorhizal nodule formation. This suggests a potential role for auxin influx carriers in the infection process. We therefore isolated and characterized homologs of the auxin influx carrier (AUX1-LAX) genes in Casuarina glauca. Two members of this family were found to share high levels of deduced protein sequence identity with Arabidopsis (Arabidopsis thaliana) AUX-LAX proteins. Complementation of the Arabidopsis aux1 mutant revealed that one of them is functionally equivalent to AUX1 and was named CgAUX1. The spatial and temporal expression pattern of CgAUX1 promoter:beta-glucuronidase reporter was analyzed in Casuarinaceae. We observed that CgAUX1 was expressed in plant cells infected by Frankia throughout the course of actinorhizal nodule formation. Our data suggest that auxin plays an important role during plant cell infection in actinorhizal symbioses.     

Evaluating auxin distribution in tomato (Solanum lycopersicum) through an analysis of the PIN and AUX/LAX gene families

The temporal and spatial control of auxin distribution has a key role in the regulation of plant growth and development, and much has been learnt about the mechanisms that influence auxin pools and gradients in vegetative tissues, particularly in Arabidopsis. For example polar auxin transport, mediated by PIN and AUX/LAX proteins, is central to the control of auxin distribution. In contrast, very little information is known about the dynamics of auxin distribution and the molecular basis of its transport within and between fruit tissues, despite the fact that auxin regulates many aspects of fruit development, which include fruit formation, expansion, ripening and abscission. In addition, functional information regarding the key regulators of auxin fluxes during both vegetative and reproductive development in species other than Arabidopsis is scarce. To address these issues, we have investigated the spatiotemporal distribution of auxin during tomato (Solanum lycopersicum) fruit development and the function of the PIN and AUX/LAX gene families. Differential concentrations of auxin become apparent during early fruit growth, with auxin levels being higher in internal tissues than in the fruit pericarp and the pattern of auxin accumulation depended on polar transport. Ten tomato PIN (SlPIN1 to 10) and five AUX/LAX (SlLAX1 to 5) genes were identified and found to display heterogeneous expression patterns, with tissue and developmental-stage specificity. RNAi-mediated co-silencing of SlPIN4 and SlPIN3 did not affect fruit development, which suggested functional redundancy of PIN proteins, but did lead to a vegetative phenotype, and revealed a role for these genes in the regulation of tomato shoot architecture.     

Auxin transport: providing a sense of direction during plant development

Auxins are key regulators of plant development. Plants employ a specialized delivery system termed polar auxin transport to convey indole-3-acetic acid from source to target tissues. Auxin transport is mediated by the combined activities of specialized influx and efflux carriers. Mutational approaches in the model plant, Arabidopsis thaliana, have led to the molecular genetic characterization of putative auxin influx and efflux carrier components, AUX1 and AtPIN1. Both genes belong to distinct gene families that are being functionally characterized by using a reverse genetic approach in Arabidopsis. AtPIN proteins are asymmetrically localized within plant plasma membranes, providing a molecular mechanism for the characteristic polarity of auxin transport. We outline the epitope tagging strategy being used in our laboratory to immunolocalize AUX1 and discuss the implications of its subcellular localization for auxin redistribution within root apical tissues. Lastly, we describe a novel carrier-based mechanism that plant cells might use to determine their relative position(s) within an auxin gradient, drawing parallels with the mechanism of glucose perception in yeast.     

植物生长素的极性运输载体研究进展

生长素极性运输在植物生长发育中起重要的调控作用.植物细胞间的生长素极性运输主要通过生长素运输载体进行调控.该文对近年来有关生长素极性运输载体,包括输入载体AUX/LAX、输出载体PIN、尤其是新近发现的兼有输入和输出载体功能的MDR/PGP等蛋白家族,以及生长素极性运输中PIN与MDR/PGP蛋白间相互作用关系进行综述.     

Adenosine diphosphate ribosylation factor-GTPase-activating protein stimulates the transport of AUX1 endosome, which relies on actin cytoskeletal organization in rice root development

Polar auxin transport, which depends on polarized subcellular distribution of AUXIN RESISTANT 1/LIKE AUX1 (AUX1/LAX) influx carriers and PIN-FORMED (PIN) efflux carriers, mediates various processes of plant growth and development. Endosomal recycling of PIN1 is mediated by an adenosine diphosphate (ADP)ribosylation factor (ARF)-GTPase exchange factor protein, GNOM. However, the mediation of auxin influx carrier recycling is poorly understood. Here, we report that overexpression of OsAGAP, an ARF-GTPase-activating protein in rice, stimulates vesicle transport from the plasma membrane to the Golgi apparatus in protoplasts and transgenic plants and induces the accumulation of early endosomes and AUX1. AUX1 endosomes could partially colocalize with FM4-64 labeled early endosome after actin disruption. Furthermore, OsAGAP is involved in actin cytoskeletal organization, and its overexpression tends to reduce the thickness and bundling of actin filaments. Fluorescence recovery after photobleaching analysis revealed exocytosis of the AUX1 recycling endosome was not affected in the OsAGAP overexpression cells, and was only slightly promoted when the actin filaments were completely disrupted by Lat B. Thus, we propose that AUX1 accumulation in the OsAGAP overexpression and actin disrupted cells may be due to the fact that endocytosis of the auxin influx carrier AUX1 early endosome was greatly promoted by actin cytoskeleton disruption.     

AUX1 regulates root gravitropism in Arabidopsis by facilitating auxin uptake within root apical tissues

Plants employ a specialized transport system composed of separate influx and efflux carriers to mobilize the plant hormone auxin between its site(s) of synthesis and action. Mutations within the permease-like AUX1 protein significantly reduce the rate of carrier-mediated auxin uptake within Arabidopsis roots, conferring an agravitropic phenotype. We are able to bypass the defect within auxin uptake and restore the gravitropic root phenotype of aux1 by growing mutant seedlings in the presence of the membrane-permeable synthetic auxin, 1-naphthaleneacetic acid. We illustrate that AUX1 expression overlaps that previously described for the auxin efflux carrier, AtPIN2, using transgenic lines expressing an AUX1 promoter::uidA (GUS) gene. Finally, we demonstrate that AUX1 regulates gravitropic curvature by acting in unison with the auxin efflux carrier to co-ordinate the localized redistribution of auxin within the Arabidopsis root apex. Our results provide the first example of a developmental role for the auxin influx carrier within higher plants and supply new insight into the molecular basis of gravitropic signalling.     

Subcellular trafficking of the Arabidopsis auxin influx carrier AUX1 uses a novel pathway distinct from PIN1

The directional flow of the plant hormone auxin mediates multiple developmental processes, including patterning and tropisms. Apical and basal plasma membrane localization of AUXIN-RESISTANT1 (AUX1) and PIN-FORMED1 (PIN1) auxin transport components underpins the directionality of intercellular auxin flow in Arabidopsis thaliana roots. Here, we examined the mechanism of polar trafficking of AUX1. Real-time live cell analysis along with subcellular markers revealed that AUX1 resides at the apical plasma membrane of protophloem cells and at highly dynamic subpopulations of Golgi apparatus and endosomes in all cell types. Plasma membrane and intracellular pools of AUX1 are interconnected by actin-dependent constitutive trafficking, which is not sensitive to the vesicle trafficking inhibitor brefeldin A. AUX1 subcellular dynamics are not influenced by the auxin influx inhibitor NOA but are blocked by the auxin efflux inhibitors TIBA and PBA. Furthermore, auxin transport inhibitors and interference with the sterol composition of membranes disrupt polar AUX1 distribution at the plasma membrane. Compared with PIN1 trafficking, AUX1 dynamics display different sensitivities to trafficking inhibitors and are independent of the endosomal trafficking regulator ARF GEF GNOM. Hence, AUX1 uses a novel trafficking pathway in plants that is distinct from PIN trafficking, providing an additional mechanism for the fine regulation of auxin transport.     

Functional characterization of PaLAX1, a putative auxin permease, in heterologous plant systems

We have isolated the cDNA of the gene PaLAX1 from a wild cherry tree (Prunus avium). The gene and its product are highly similar in sequences to both the cDNAs and the corresponding protein products of AUX/LAX-type genes, coding for putative auxin influx carriers. We have prepared and characterized transformed Nicotiana tabacum and Arabidopsis thaliana plants carrying the gene PaLAX1. We have proved that constitutive overexpression of PaLAX1 is accompanied by changes in the content and distribution of free indole-3-acetic acid, the major endogenous auxin. The increase in free indole-3-acetic acid content in transgenic plants resulted in various phenotype changes, typical for the auxin-overproducing plants. The uptake of synthetic auxin, 2,4-dichlorophenoxyacetic acid, was 3 times higher in transgenic lines compared to the wild-type lines and the treatment with the auxin uptake inhibitor 1-naphthoxyacetic acid reverted the changes caused by the expression of PaLAX1. Moreover, the agravitropic response could be restored by expression of PaLAX1 in the mutant aux1 plants, which are deficient in auxin influx carrier activity. Based on our data, we have concluded that the product of the gene PaLAX1 promotes the uptake of auxin into cells, and, as a putative auxin influx carrier, it affects the content and distribution of free endogenous auxin in transgenic plants.     

Expression studies on AUX1-like genes in Medicago truncatula suggest that auxin is required at two steps in early nodule development

Medicago truncatula contains a family of at least five genes related to AUX1 of Arabidopsis thaliana (termed MtLAX genes for Medicago truncatula-like AUX1 genes). The high sequence similarity between the encoded proteins and AUX1 implies that the MtLAX genes encode auxin import carriers. The MtLAX genes are expressed in roots and other organs, suggesting that they play pleiotropic roles related to auxin uptake. In primary roots, the MtLAX genes are expressed preferentially in the root tips, particularly in the provascular bundles and root caps. During lateral root and nodule development, the genes are expressed in the primordia, particularly in cells that were probably derived from the pericycle. At slightly later stages, the genes are expressed in the regions of the developing organs where the vasculature arises (central position for lateral roots and peripheral region for nodules). These results are consistent with MtLAX being involved in local auxin transport and suggest that auxin is required at two common stages of lateral root and nodule development: development of the primordia and differentiation of the vasculature.     

Local regulation of auxin transport in root-apex transition zone mediates aluminium-induced Arabidopsis root-growth inhibition

Aluminium (Al) stress is a major limiting factor for worldwide crop production in acid soils. In Arabidopsis thaliana, the TAA1-dependent local auxin biosynthesis in the root-apex transition zone (TZ), the major perception site for Al toxicity, is crucial for the Al-induced root-growth inhibition, while the mechanism underlying Al-regulated auxin accumulation in the TZ is not fully understood. In the present study, the role of auxin transport in Al-induced local auxin accumulation in the TZ and root-growth inhibition was investigated. Our results showed that PIN-FORMED (PIN) proteins such as PIN1, PIN3, PIN4 and PIN7 and AUX1/LAX proteins such as AUX1, LAX1 and LAX2 were all ectopically up-regulated in the root-apex TZ in response to Al stress and coordinately regulated local auxin accumulation in the TZ and root-growth inhibition. The ectopic up-regulation of PIN1 in the TZ under Al stress was regulated by both ethylene and auxin, with auxin signalling acting downstream of ethylene. Al-induced PIN1 up-regulation and auxin accumulation in the root-apex TZ was also regulated by the calossin-like protein BIG. Together, our results provide insight into how Al stress induces local auxin accumulation in the TZ and root-growth inhibition through the local regulation of auxin transport.     

Phototropin-mediated perception of light direction in leaves regulates blade flattening

One conserved feature among angiosperms is the development of flat thin leaves. This developmental pattern optimizes light capture and gas exchange. The blue light (BL) receptors phototropins are required for leaf flattening, with the null phot1phot2 mutant showing curled leaves in Arabidopsis (Arabidopsis thaliana). However, key aspects of their function in leaf development remain unknown. Here, we performed a detailed spatiotemporal characterization of phototropin function in Arabidopsis leaves. We found that phototropins perceive light direction in the blade, and, similar to their role in hypocotyls, they control the spatial pattern of auxin signaling, possibly modulating auxin transport, to ultimately regulate cell expansion. Phototropin signaling components in the leaf partially differ from hypocotyls. Moreover, the light response on the upper and lower sides of the leaf blade suggests a partially distinct requirement of phototropin signaling components on each side. In particular, NON PHOTOTROPIC HYPOCOTYL 3 showed an adaxial-specific function. In addition, we show a prominent role of PHYTOCHROME KINASE SUBSTRATE 3 in leaf flattening. Among auxin transporters, PIN-FORMED 3,4,7 and AUXIN RESISTANT 1 (AUX1)/LIKE AUXIN RESISTANT 1 (LAX1) are required for the response while ABCB19 has a regulatory role. Overall, our results show that directional BL perception by phototropins is a key aspect of leaf development, integrating endogenous and exogenous signals.

Phototropins perceive light direction in the leaf and control the auxin signaling pattern to regulate blade flattening.