PIN-FORMED Research Articles

Genome-wide identification and characterization of PIN-FORMED (PIN) and PIN-LIKES (PILS) gene family reveals their role in adventitious root development in tea nodal cutting (Camellia Sinensis)

Auxin affects all aspects of plant growth and development, including morphogenesis and adaptive responses. Auxin transmembrane transport is promoted by PIN formation (PIN) and a structurally similar PIN-like (PILS) gene family, which jointly controls the directional transport of the auxin between plant cells, and the accumulation of intracellular auxin. At present, there is no study investigating the roles of CslPIN and CslPILS gene family in root development in the tea plant (Camellia sinensis). In this study, 8 CslPIN and 10 CslPILS genes were identified in the tea plant, and their evolutionary relationships, physical and chemical properties, conserved motifs, cis-acting elements, chromosome location, collinearity, and expression characteristics were analyzed. The mechanism of CslPIN and CslPILS in the formation of tea adventitious roots (ARs) was studied by the AR induction system. Through functional verification, the regulation of CslPIN3 gene on root growth and development of tea plant was studied by over-expression of CslPIN3 in Arabidopsis thaliana and in situ hybridization in Camellia sinensis. The results confirmed CslPIN3 was involved in the regulation of root growth and development as well as auxin accumulation. This study provides a better insight into the regulatory mechanism of CslPIN and CslPILS gene family on the formation of AR in tea plant.

Genome-Wide Identification of PIN and PILS Gene Families in Areca catechu and the Potential Role of AcPIN6 in Lateral Brace Root Formation.

PIN-FORMED (PIN) and PIN-LIKES (PILS) are two families of auxin transporters that control the directional cell-to-cell transport and intracellular accumulation of auxin, thereby influencing plant growth and development. Most knowledge of PINs and PILSs was obtained from the dicot model plant Arabidopsis thaliana. Here, we focus on the distribution and expression of the PIN and PILS gene families in areca palm (Areca catechu), a monocot tree. The whole genomic dataset of areca palm was used to identify twelve AcPINs and eight AcPILSs, and a phylogenetic tree was constructed of PINS and PILS together with several other palm species, including the date palm (Phoenix dactylifera), oil palm (Elaeis guineensis), and coconut (Cocos nucifera). We further analyzed the expression patterns of AcPIN and AcPILS in areca palm, and found that AcPIN6 displayed an extremely high transcriptional abundance in the brace roots and was extremely stimulated in the lateral root primordium. This result implies that AcPIN6 plays an important role in the growth and formation of brace roots, especially in lateral root initiation. We also overexpressed AcPIN6 and AcPIN6-eGFP in Arabidopsis, and the results revealed that the PIN6 localized on the plasma membrane and affected auxin-related phenomena. Taken together, we analyzed the evolutionary relationships of PINs and PILSs in palm species, and the roles of PIN6 in areca palm root formation. The results will improve the understanding of root system construction in large palm trees.

Histone Acetyltransferase GCN5 Affects Auxin Transport during Root Growth by Modulating Histone Acetylation and Gene Expression of PINs.

General Control Non-Derepressible 5 (GCN5) is a histone acetyltransferase that targets multiple genes and is essential for the acetylation of Lysine residues in the N-terminal tail of histone H3 in Arabidopsis. GCN5 interacts with the transcriptional coactivator Alteration/Deficiency in Activation 2b (ADA2b), which enhances its activity functioning in multiprotein complexes, such as the Spt-Ada-Gcn5-Acetyltransferase complex (SAGA). Mutations in GCN5 and ADA2b result in pleiotropic phenotypes, including alterations in the growth of roots. Auxin is known to regulate root development by modulating gene expression patterns. Auxin moves polarly during plant growth via the Pin-formed (PIN) auxin efflux transport proteins. The effect of GCN5 and ADA2b on auxin distribution at different stages of early root growth (4 to 7 days post-germination) was studied using the reporter lines DR5rev::GFP and PIN1::PIN1-GFP. In wild-type plants, auxin efflux transporter PIN1 expression increases from the fourth to the seventh day of root growth. The PIN1 expression was reduced in the roots of gcn5-1 and ada2b-1 compared to the wild type. The expression of PIN1 in ada2b-1 mutants is confined only to the meristematic zone, specifically in the stele cells, whereas it is almost abolished in the elongation zone. Gene expression analysis showed that genes associated with auxin transport, PIN1, PIN3 and PIN4, are downregulated in gcn5-1 and ada2b-1 mutants relative to the wild type. As a result, auxin accumulation was also reduced in gcn5-1 and ada2b-1 compared to wild-type roots. Furthermore, acetylation of Lysine 14 of histone H3 (H3K14) was also affected in the promoter and coding region of PIN1, PIN3 and PIN4 genes during root growth of Arabidopsis in gcn5 mutants. In conclusion, GCN5 acts as a positive regulator of auxin distribution in early root growth by modulating histone H3 acetylation and the expression of auxin efflux transport genes.

Mitochondrial GPAT-derived LPA controls auxin-dependent embryonic and postembryonic development

Membrane properties are emerging as important cues for the spatiotemporal regulation of hormone signaling. Lysophosphatidic acid (LPA) evokes multiple biological responses by activating G protein-coupled receptors in mammals. In this study, we demonstrated that LPA derived from the mitochondrial glycerol-3-phosphate acyltransferases GPAT1 and GPAT2 is a critical lipid-based cue for auxin-controlled embryogenesis and plant growth in Arabidopsis thaliana. LPA levels decreased, and the polarity of the auxin efflux carrier PIN-FORMED1 (PIN1) at the plasma membrane (PM) was defective in the gpat1 gpat2 mutant. As a consequence of distribution defects, instructive auxin gradients and embryonic and postembryonic development are severely compromised. Further cellular and genetic analyses revealed that LPA binds directly to PIN1, facilitating the vesicular trafficking of PIN1 and polar auxin transport. Our data support a model in which LPA provides a lipid landmark that specifies membrane identity and cell polarity, revealing an unrecognized aspect of phospholipid patterns connecting hormone signaling with development.

Molecular mechanism of leaf adaxial upward curling caused by BpPIN3 suppression in Betula pendula.

Leaves are one of the vegetative organs of plants that are essential for plant growth and development. PIN-FORMED (PINs) gene is an indoleacetic acid (IAA) transporter that plays a critical role in leaf development. To determine the function of BpPIN3 in leaf polarity formation in Betula pendula, the transgenic lines with BpPIN3 overexpression (OE) and BpPIN3-reduced expression (RE) were analyzed using the Agrobacterium-mediated method. The RE lines displayed the characteristics of leaf margin adaxial upward curling, with lower expression of BpPIN3 resulting in greater rolling. Tissue localization of IAA in the auxin GUS reporter system proved that auxin in the RE was mainly distributed in the secondary veins, palisade tissues, and epidermal cells in the leaf margin area. The auxin content in the leaf margin area was significantly greater than that in the main vein tissue. The cell density of the palisade tissue and the ratio of palisade tissue to spongy tissue in the curled leaf margin of the RE lines were found to be significantly decreased. RNA-seq analysis revealed that the RE hormone-signaling pathway genes were significantly enriched compared with those of the OE and WT lines; in particular, the auxin response-related genes SAURs (i.e., SAUR23, SAUR24, SAUR28, and SAUR50) and GH3.10 were found to be significantly upregulated. qRT-PCR analysis indicated that BpPIN3 expression at the leaf margin was significantly lower than that near the main vein in the RE lines. In contrast, the expression levels of SAURs and GH3.10 were significantly higher than those near the midrib. In conclusion, BpPIN3 regulates the expression of auxin response-related genes and the polar transport of auxin to change the polar form of the proximal and distal axes of birch leaves.

Mapping of the Classical Mutation rosette Highlights a Role for Calcium in Wound-Induced Rooting.

Removal of the root system induces the formation of new roots from the remaining shoot. This process is primarily controlled by the phytohormone auxin, which interacts with other signals in a yet unresolved manner. Here, we study the classical tomato mutation rosette (ro), which lacks shoot-borne roots. ro mutants were severely inhibited in formation of wound-induced roots (WiRs) and had reduced auxin transport rates. We mapped ro to the tomato ortholog of the Arabidopsis thaliana BIG and the mammalians UBR4/p600. RO/BIG is a large protein of unknown biochemical function. In A. thaliana, BIG was implicated in regulating auxin transport and calcium homeostasis. We show that exogenous calcium inhibits WiR formation in tomato and A. thaliana ro/big mutants. Exogenous calcium antagonized the root-promoting effects of the auxin indole-3-acetic-acid but not of 2,4-dichlorophenoxyacetic acid, an auxin analog that is not recognized by the polar transport machinery, and accumulation of the auxin transporter PIN-FORMED1 (PIN1) was sensitive to calcium levels in the ro/big mutants. Consistent with a role for calcium in mediating auxin transport, both ro/big mutants and calcium-treated wild-type plants were hypersensitive to treatment with polar auxin transport inhibitors. Subcellular localization of BIG suggests that, like its mammalian ortholog, it is associated with the endoplasmic reticulum. Analysis of subcellular morphology revealed that ro/big mutants exhibited disruption in cytoplasmic streaming. We suggest that RO/BIG maintains auxin flow by stabilizing PIN membrane localization, possibly by attenuating the inhibitory effect of Ca2+ on cytoplasmic streaming.

Specification of female germline by microRNA orchestrated auxin signaling in Arabidopsis

Germline determination is essential for species survival and evolution in multicellular organisms. In most flowering plants, formation of the female germline is initiated with specification of one megaspore mother cell (MMC) in each ovule; however, the molecular mechanism underlying this key event remains unclear. Here we report that spatially restricted auxin signaling promotes MMC fate in Arabidopsis. Our results show that the microRNA160 (miR160) targeted gene ARF17 (AUXIN RESPONSE FACTOR17) is required for promoting MMC specification by genetically interacting with the SPL/NZZ (SPOROCYTELESS/NOZZLE) gene. Alterations of auxin signaling cause formation of supernumerary MMCs in an ARF17- and SPL/NZZ-dependent manner. Furthermore, miR160 and ARF17 are indispensable for attaining a normal auxin maximum at the ovule apex via modulating the expression domain of PIN1 (PIN-FORMED1) auxin transporter. Our findings elucidate the mechanism by which auxin signaling promotes the acquisition of female germline cell fate in plants.

PIN1 regulates epidermal cells development under drought and salt stress using single-cell analysis.

Over the course of evolution, plants have developed plasticity to acclimate to environmental stresses such as drought and salt stress. These plant adaptation measures involve the activation of cascades of molecular networks involved in stress perception, signal transduction and the expression of stress related genes. Here, we investigated the role of the plasma membrane-localized transporter of auxin PINFORMED1 (PIN1) in the regulation of pavement cells (PCs) and guard cells (GCs) development under drought and salt stress conditions. The results showed that drought and salt stress treatment affected the development of PCs and GCs. Further analysis identified the different regulation mechanisms of PIN1 in regulating the developmental patterns of PCs and GCs under drought and salt stress conditions. Drought and salt stress also regulated the expression dynamics of PIN1 in pif1/3/4/5 quadruple mutants. Collectively, we revealed that PIN1 plays a crucial role in regulating plant epidermal cells development under drought and salt stress conditions, thus contributing to developmental rebustness and plasticity.

Fumonisin B1 as a Tool to Explore Sphingolipid Roles in Arabidopsis Primary Root Development.

Fumonisin B1 is a mycotoxin that is structurally analogous to sphinganine and sphingosine and inhibits the biosynthesis of complex sphingolipids by repressing ceramide synthase. Based on the connection between FB1 and sphingolipid metabolism, FB1 has been widely used as a tool to explore the multiple functions of sphingolipids in mammalian and plant cells. The aim of this work was to determine the effect of sphingolipids on primary root development by exposing Arabidopsis (Arabidopsis thaliana) seedlings to FB1. We show that FB1 decreases the expression levels of several PIN-FORMED (PIN) genes and the key stem cell niche (SCN)-defining transcription factor genes WUSCHEL-LIKE HOMEOBOX5 (WOX5) and PLETHORAs (PLTs), resulting in the loss of quiescent center (QC) identity and SCN maintenance, as well as stunted root growth. In addition, FB1 induces cell death at the root apical meristem in a non-cell-type-specific manner. We propose that sphingolipids play a key role in primary root growth through the maintenance of the root SCN and the amelioration of cell death in Arabidopsis.

Expression analysis of PIN family genes in Chinese hickory reveals their potential roles during grafting and salt stress.

Grafting is an effective way to improve Chinese hickory while salt stress has caused great damage to the Chinese hickory industry. Grafting and salt stress have been regarded as the main abiotic stress types for Chinese hickory. However, how Chinese hickory responds to grafting and salt stress is less studied. Auxin has been proved to play an essential role in the stress response through its re-distribution regulation mediated by polar auxin transporters, including PIN-formed (PIN) proteins. In this study, the PIN gene family in Chinese hickory (CcPINs) was identified and structurally characterized for the first time. The expression profiles of the genes in response to grafting and salt stress were determined. A total of 11 CcPINs with the open reading frames (ORFs) of 1,026–1,983 bp were identified. Transient transformation in tobacco leaves demonstrated that CcPIN1a, CcPIN3, and CcPIN4 were localized in the plasma membrane. There were varying phylogenetic relationships between CcPINs and homologous genes in different species, but the closest relationships were with those in Carya illinoinensis and Juglans regia. Conserved N- and C-terminal transmembrane regions as well as sites controlling the functions of CcPINs were detected in CcPINs. Five types of cis-acting elements, including hormone- and stress-responsive elements, were detected on the promoters of CcPINs. CcPINs exhibited different expression profiles in different tissues, indicating their varied roles during growth and development. The 11 CcPINs responded differently to grafting and salt stress treatment. CcPIN1a might be involved in the regulation of the grafting process, while CcPIN1a and CcPIN8a were related to the regulation of salt stress in Chinese hickory. Our results will lay the foundation for understanding the potential regulatory functions of CcPIN genes during grafting and under salt stress treatment in Chinese hickory.

The unconventional prefoldin RPB5 interactor mediates the gravitropic response by modulating cytoskeleton organization and auxin transport in Arabidopsis.

Gravity-induced root curvature involves the asymmetric distribution of the phytohormone auxin. This response depends on the concerted activities of the auxin transporters such as PIN-FORMED (PIN) proteins for auxin efflux and AUXIN RESISTANT 1 (AUX1) for auxin influx. However, how the auxin gradient is established remains elusive. Here we identified a new mutant with a short root, strong auxin distribution in the lateral root cap and an impaired gravitropic response. The causal gene encoded an Arabidopsis homolog of the human unconventional prefoldin RPB5 interactor (URI). AtURI interacted with prefoldin 2 (PFD2) and PFD6, two β-type PFD members that modulate actin and tubulin patterning in roots. The auxin reporter DR5rev :GFP showed that asymmetric auxin redistribution after gravistimulation is disordered in aturi-1 root tips. Treatment with the endomembrane protein trafficking inhibitor brefeldin A indicated that recycling of the auxin transporter PIN2 is disrupted in aturi-1 roots as well as in pfd mutants. We propose that AtURI cooperates with PFDs to recycle PIN2 and modulate auxin distribution.

Tonoplast proton pumps regulate nuclear spacing of female gametophytes via mediating polar auxin transport in Arabidopsis.

The vacuole is an important organelle with multiple functions in plants, and the tonoplast that wraps the vacuole also plays essential roles in intracellular trafficking and ion homeostasis. Previous studies found that tonoplast proton pumps regulate embryo development and morphogenesis through their effects on vacuole biogenesis and distribution, as well as polar auxin transport and concomitant auxin gradient. However, the precise roles of the tonoplast proton pumps in gametophyte development remain unclear. Here we demonstrated that the lack of two types of tonoplast proton pumps or the absence of V-ATPase alone leads to abnormal development and nuclear localization of female gametophyte (FG), and slowed endosperm nuclei division after fertilization of the central cell. We further revealed that V-ATPase regulates auxin levels in ovules through coordinating the content and localization of PIN-FORMED 1 (PIN1) protein, hence influencing nuclear spacing between centra cell and egg cell, and subsequent endosperm development. Collectively, our findings revealed a crucial role of V-ATPase in auxin-mediated FG development in Arabidopsis and expanded our understanding of the functions of tonoplast proton pumps in seed plants reproductive development.

Genome-Wide Characterization of PIN Auxin Efflux Carrier Gene Family in Mikania micrantha.

Mikania micrantha, recognized as one of the world’s top 10 pernicious weeds, is a rapidly spreading tropical vine that has invaded the coastal areas of South China, causing serious economic losses and environmental damage. Rapid stem growth is an important feature of M. micrantha which may be related to its greater number of genes involved in auxin signaling and transport pathways and its ability to synthesize more auxin under adverse conditions to promote or maintain stem growth. Plant growth and development is closely connected to the regulation of endogenous hormones, especially the polar transport and asymmetric distribution of auxin. The PIN-FORMED (PIN) auxin efflux carrier gene family plays a key role in the polar transport of auxin and then regulates the growth of different plant tissues, which could indicate that the rapid growth of M. micrantha is closely related to this PIN-dependent auxin regulation. In this study, 11 PIN genes were identified and the phylogenetic relationship and structural compositions of the gene family in M. micrantha were analyzed by employing multiple bioinformatic methods. The phylogenetic analysis indicated that the PIN proteins could be divided into five distinct clades. The structural analysis revealed that three putative types of PIN (canonical, noncanonical and semi-canonical) exist among the proteins according to the length and the composition of the hydrophilic domain. The majority of the PINs were involved in the process of axillary bud differentiation and stem response under abiotic stress, indicating that M. micrantha may regulate its growth, development and stress response by regulating PIN expression in the axillary bud and stem, which may help explain its strong growth ability and environmental adaptability. Our study emphasized the structural features and stress response patterns of the PIN gene family and provided useful insights for further study into the molecular mechanism of auxin-regulated growth and control in M. micrantha.

PIN-FORMED1 polarity in the plant shoot epidermis is insensitive to the polarity of neighboring cells

SummaryAt the Arabidopsis shoot apex, epidermal cells are planar-polarized along an axis marked by the asymmetric localization patterns of several proteins including PIN-FORMED1 (PIN1), which facilitates the directional efflux of the plant hormone auxin to pattern phyllotaxis. While PIN1 polarity is known to be regulated non-cell autonomously via the MONOPTEROS (MP) transcription factor, how this occurs has not been determined. Here, we use mosaic expression of the serine threonine kinase PINOID (PID) to test whether PIN1 polarizes according to the polarity of neighboring cells. Our findings reveal that PIN1 is insensitive to the polarity of PIN1 in neighboring cells arguing against auxin flux or extracellular auxin concentrations acting as a polarity cue, in contrast to previous model proposals.

WAVY GROWTH Arabidopsis E3 ubiquitin ligases affect apical PIN sorting decisions

Directionality in the intercellular transport of the plant hormone auxin is determined by polar plasma membrane localization of PIN-FORMED (PIN) auxin transport proteins. However, apart from PIN phosphorylation at conserved motifs, no further determinants explicitly controlling polar PIN sorting decisions have been identified. Here we present Arabidopsis WAVY GROWTH 3 (WAV3) and closely related RING-finger E3 ubiquitin ligases, whose loss-of-function mutants show a striking apical-to-basal polarity switch in PIN2 localization in root meristem cells. WAV3 E3 ligases function as essential determinants for PIN polarity, acting independently from PINOID/WAG-dependent PIN phosphorylation. They antagonize ectopic deposition of de novo synthesized PIN proteins already immediately following completion of cell division, presumably via preventing PIN sorting into basal, ARF GEF-mediated trafficking. Our findings reveal an involvement of E3 ligases in the selective targeting of apically localized PINs in higher plants.

Genome-Wide Identification and Expression Analysis of the PIN Auxin Transporter Gene Family in Zanthoxylum armatum DC

PIN-formed (PIN) proteins are important auxin carriers that participate in the polar distribution of auxin in plants. In this study, 16 ZaPIN genes were identified from the whole genome of Zanthoxylum armatum DC. The physicochemical properties and structure of PIN proteins were determined, and the gene sequences and promoter regions were analyzed to identify cis-acting elements and conserved motifs. The transcript profiles of ZaPIN genes in different tissues and in response to auxin and gibberellin treatments were also analyzed. A phylogenetic analysis separated the 16 ZaPIN genes into four groups. The ZaPIN genes showed the closest evolutionary relationship to those of Citrus sinensis and the most distant evolutionary relationship to those of Oryza sativa. A cis-element analysis revealed a large number of cis elements in ZaPIN promoter regions related to plant hormones, plant growth and development, and stress stimuli, suggesting that ZaPINs have a wide range of biological activities. Additionally, gene expression profiling revealed that ZaPINs had different expression patterns in nine tissues. Further qRT-PCR analyses revealed that most ZaPINs were upregulated by auxin and gibberellin in young leaves. Our results provide useful information for further structural and functional analyses of the ZaPIN gene family in Z. armatum.

PIN and PILS family genes analyses in Chrysanthemum seticuspe reveal their potential functions in flower bud development and drought stress

Auxin affects almost all plant growth and developmental processes. The PIN-FORMED (PIN) and PIN-LIKES (PILS) family genes determine the direction and distribution gradient of auxin flow by polar localization on the cell membrane. However, there are no systematic studies on PIN and PILS family genes in chrysanthemum. Here, 18 PIN and 13 PILS genes were identified in Chrysanthemum seticuspe. The evolutionary relationships, physicochemical properties, conserved motifs, cis-acting elements, chromosome localization, collinearity, and expression characteristics of these genes were analyzed. CsPIN10a, CsPIN10b, and CsPIN10c are unique PIN genes in C. seticuspe. Expression pattern analysis showed that these genes had different tissue specificities, and the expression levels of CsPIN8, CsPINS1, CsPILS6, and CsPILS10 were linearly related to the developmental period of the flower buds. In situ hybridization assay showed that CsPIN1a, CsPIN1b, and CsPILS8 were expressed in floret primordia and petal tips, and CsPIN1a was specifically expressed in the middle of the bract primordia, which might regulate lateral expansion of the bracts. CsPIN and CsPILS family genes are also involved in drought stress responses. This study provides theoretical support for the cultivation of new varieties with attractive flower forms and high drought tolerance.

Mutation of OsPIN1b by CRISPR/Cas9 Reveals a Role for Auxin Transport in Modulating Rice Architecture and Root Gravitropism.

The distribution and content of auxin within plant tissues affect a variety of important growth and developmental processes. Polar auxin transport (PAT), mainly mediated by auxin influx and efflux transporters, plays a vital role in determining auxin maxima and gradients in plants. The auxin efflux carrier PIN-FORMED (PIN) family is one of the major protein families involved in PAT. Rice (Oryza sativa L.) genome possesses 12 OsPIN genes. However, the detailed functions of OsPIN genes involved in regulating the rice architecture and gravity response are less well understood. In the present study, OsPIN1b was disrupted by CRISPR/Cas9 technology, and its roles in modulating rice architecture and root gravitropism were investigated. Tissue-specific analysis showed that OsPIN1b was mainly expressed in roots, stems and sheaths at the seedling stage, and the transcript abundance was progressively decreased during the seedling stages. Expression of OsPIN1b could be quickly and greatly induced by NAA, indicating that OsPIN1b played a vital role in PAT. IAA homeostasis was disturbed in ospin1b mutants, as evidenced by the changed sensitivity of shoot and root to NAA and NPA treatment, respectively. Mutation of OsPIN1b resulted in pleiotropic phenotypes, including decreased growth of shoots and primary roots, reduced adventitious root number in rice seedlings, as well as shorter and narrower leaves, increased leaf angle, more tiller number and decreased plant height and panicle length at the late developmental stage. Moreover, ospin1b mutants displayed a curly root phenotype cultured with tap water regardless of lighting conditions, while nutrient solution culture could partially rescue the curly root phenotype in light and almost completely abolish this phenotype in darkness, indicating the involvement of the integration of light and nutrient signals in root gravitropism regulation. Additionally, amyloplast sedimentation was impaired in the peripheral tiers of the ospin1b root cap columella cell, while it was not the main contributor to the abnormal root gravitropism. These data suggest that OsPIN1b not only plays a vital role in regulating rice architecture but also functions in regulating root gravitropism by the integration of light and nutrient signals.

Structural insights into auxin recognition and efflux by Arabidopsis PIN1

Polar auxin transport is unique to plants and coordinates their growth and development1,2. The PIN-FORMED (PIN) auxin transporters exhibit highly asymmetrical localizations at the plasma membrane and drive polar auxin transport3,4; however, their structures and transport mechanisms remain largely unknown. Here, we report three inward-facing conformation structures of Arabidopsis thaliana PIN1: the apo state, bound to the natural auxin indole-3-acetic acid (IAA), and in complex with the polar auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). The transmembrane domain of PIN1 shares a conserved NhaA fold5. In the substrate-bound structure, IAA is coordinated by both hydrophobic stacking and hydrogen bonding. NPA competes with IAA for the same site at the intracellular pocket, but with a much higher affinity. These findings inform our understanding of the substrate recognition and transport mechanisms of PINs and set up a framework for future research on directional auxin transport, one of the most crucial processes underlying plant development.

Structures and mechanisms of the Arabidopsis auxin transporter PIN3.

The PIN-FORMED (PIN) protein family of auxin transporters mediates polar auxin transport and has crucial roles in plant growth and development1,2. Here we present cryo-electron microscopy structures of PIN3 from Arabidopsis thaliana in the apo state and in complex with its substrate indole-3-acetic acid and the inhibitor N-1-naphthylphthalamic acid (NPA). A. thalianaPIN3 exists as a homodimer, and its transmembrane helices 1, 2 and 7 in the scaffold domain are involved in dimerization. The dimeric PIN3 forms a large, joint extracellular-facing cavity at the dimer interface while each subunit adopts an inward-facing conformation. The structural and functional analyses, along with computational studies, reveal the structural basis for the recognition of indole-3-acetic acid and NPA and elucidate the molecular mechanism of NPA inhibition on PIN-mediated auxin transport. The PIN3 structures support an elevator-like model for the transport of auxin, whereby the transport domains undergo up-down rigid-body motions and the dimerized scaffold domains remain static.