PIN-FORMED Polarity Research Articles

Divergence of trafficking and polarization mechanisms for PIN auxin transporters during land plant evolution

The phytohormone auxin, and its directional transport through tissues, plays a fundamental role in the development of higher plants. This polar auxin transport predominantly relies on PIN-FORMED (PIN) auxin exporters. Hence, PIN polarization is crucial for development, but its evolution during the rise of morphological complexity in land plants remains unclear. Here, we performed a cross-species investigation by observing the trafficking and localization of endogenous and exogenous PINs in two bryophytes, Physcomitrium patens and Marchantia polymorpha, and in the flowering plant Arabidopsis thaliana. We confirmed that the GFP fusion did not compromise the auxin export function of all examined PINs by using a radioactive auxin export assay and by observing the phenotypic changes in transgenic bryophytes. Endogenous PINs polarize to filamentous apices, while exogenous Arabidopsis PINs distribute symmetrically on the membrane in both bryophytes. In the Arabidopsis root epidermis, bryophytic PINs have no defined polarity. Pharmacological interference revealed a strong cytoskeletal dependence of bryophytic but not Arabidopsis PIN polarization. The divergence of PIN polarization and trafficking is also observed within the bryophyte clade and between tissues of individual species. These results collectively reveal the divergence of PIN trafficking and polarity mechanisms throughout land plant evolution and the co-evolution of PIN sequence-based and cell-based polarity mechanisms.

Regulation of PIN-FORMED Protein Degradation.

Auxin action largely depends on the establishment of auxin concentration gradient within plant organs, where PIN-formed (PIN) auxin transporter-mediated directional auxin movement plays an important role. Accumulating studies have revealed the need of polar plasma membrane (PM) localization of PIN proteins as well as regulation of PIN polarity in response to developmental cues and environmental stimuli, amongst which a typical example is regulation of PIN phosphorylation by AGCVIII protein kinases and type A regulatory subunits of PP2A phosphatases. Recent findings, however, highlight the importance of PIN degradation in reestablishing auxin gradient. Although the underlying mechanism is poorly understood, these findings provide a novel aspect to broaden the current knowledge on regulation of polar auxin transport. In this review, we summarize the current understanding on controlling PIN degradation by endosome-mediated vacuolar targeting, autophagy, ubiquitin modification and the related E3 ubiquitin ligases, cytoskeletons, plant hormones, environmental stimuli, and other regulators, and discuss the possible mechanisms according to recent studies.

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.

Subcellular trafficking and post-translational modification regulate PIN polarity in plants.

Auxin regulates plant growth and tropism responses. As a phytohormone, auxin is transported between its synthesis sites and action sites. Most natural auxin moves between cells via a polar transport system that is mediated by PIN-FORMED (PIN) auxin exporters. The asymmetrically localized PINs usually determine the directionality of intercellular auxin flow. Different internal cues and external stimuli modulate PIN polar distribution and activity at multiple levels, including transcription, protein stability, subcellular trafficking, and post-translational modification, and thereby regulate auxin-distribution-dependent development. Thus, the different regulation levels of PIN polarity constitute a complex network. For example, the post-translational modification of PINs can affect the subcellular trafficking of PINs. In this review, we focus on subcellular trafficking and post-translational modification of PINs to summarize recent progress in understanding PIN polarity.

Initiation of aboveground organ primordia depends on combined action of auxin, ERECTA family genes, and PINOID.

Leaves and flowers are produced by the shoot apical meristem (SAM) at a certain distance from its center, a process that requires the hormone auxin. The amount of auxin and the pattern of its distribution in the initiation zone determine the size and spatial arrangement of organ primordia. Auxin gradients in the SAM are formed by PIN-FORMED (PIN) auxin efflux carriers whose polar localization in the plasma membrane depends on the protein kinase PINOID (PID). Previous work determined that ERECTA (ER) family genes (ERfs) control initiation of leaves. ERfs are plasma membrane receptors that enable cell-to-cell communication by sensing extracellular small proteins from the EPIDERMAL PATTERNING FACTOR/EPF-LIKE (EPF/EPFL) family. Here, we investigated whether ERfs regulate initiation of organs by altering auxin distribution or signaling in Arabidopsis (Arabidopsis thaliana). Genetic and pharmacological data suggested that ERfs do not regulate organogenesis through PINs while transcriptomics data showed that ERfs do not alter primary transcriptional responses to auxin. Our results indicated that in the absence of ERf signaling the peripheral zone cells inefficiently initiate leaves in response to auxin signals and that increased accumulation of auxin in the er erecta-like1 (erl1) erl2 SAM can partially rescue organ initiation defects. We propose that both auxin and ERfs are essential for leaf initiation and that they have common downstream targets. Genetic data also indicated that the role of PID in initiation of cotyledons and leaves cannot be attributed solely to regulation of PIN polarity and PID is likely to have other functions in addition to regulation of auxin distribution.

Calcium-dependent protein kinase 29 modulates PIN-FORMED polarity and Arabidopsis development via its own phosphorylation code.

PIN-FORMED (PIN)-mediated polar auxin transport (PAT) is involved in key developmental processes in plants. Various internal and external cues influence plant development via the modulation of intracellular PIN polarity and, thus, the direction of PAT, but the mechanisms underlying these processes remain largely unknown. PIN proteins harbor a hydrophilic loop (HL) that has important regulatory functions; here, we used the HL as bait in protein pulldown screening for modulators of intracellular PIN trafficking in Arabidopsis thaliana. Calcium-dependent protein kinase 29 (CPK29), a Ca2+-dependent protein kinase, was identified and shown to phosphorylate specific target residues on the PIN-HL that were not phosphorylated by other kinases. Furthermore, loss of CPK29 or mutations of the phospho-target residues in PIN-HLs significantly compromised intracellular PIN trafficking and polarity, causing defects in PIN-mediated auxin redistribution and biological processes such as lateral root formation, root twisting, hypocotyl gravitropism, phyllotaxis, and reproductive development. These findings indicate that CPK29 directly interprets Ca2+ signals from internal and external triggers, resulting in the modulation of PIN trafficking and auxin responses.

Plant cell biology: PIN polarity maintained

PIN-FORMED (PIN) polar protein localization directs transport of the growth and developmental regulator auxin in plants. Once established after cytokinesis, PIN polarity requires maintenance. Now, direct interactions between PIN, MAB4/MEL and PID proteins suggest self-reinforced maintenance of PIN polarity through limiting lateral diffusion.

Strigolactones inhibit auxin feedback on PIN-dependent auxin transport canalization

Directional transport of the phytohormone auxin is a versatile, plant-specific mechanism regulating many aspects of plant development. The recently identified plant hormones, strigolactones (SLs), are implicated in many plant traits; among others, they modify the phenotypic output of PIN-FORMED (PIN) auxin transporters for fine-tuning of growth and developmental responses. Here, we show in pea and Arabidopsis that SLs target processes dependent on the canalization of auxin flow, which involves auxin feedback on PIN subcellular distribution. D14 receptor- and MAX2 F-box-mediated SL signaling inhibits the formation of auxin-conducting channels after wounding or from artificial auxin sources, during vasculature de novo formation and regeneration. At the cellular level, SLs interfere with auxin effects on PIN polar targeting, constitutive PIN trafficking as well as clathrin-mediated endocytosis. Our results identify a non-transcriptional mechanism of SL action, uncoupling auxin feedback on PIN polarity and trafficking, thereby regulating vascular tissue formation and regeneration.

Early Endosomal Trafficking Component BEN2/VPS45 Plays a Crucial Role in Internal Tissues in Regulating Root Growth and Meristem Size in Arabidopsis.

Polar auxin transport is involved in multiple aspects of plant development, including root growth, lateral root branching, embryogenesis, and vasculature development. PIN-FORMED (PIN) auxin efflux proteins exhibit asymmetric distribution at the plasma membrane (PM) and collectively play pivotal roles in generating local auxin accumulation, which underlies various auxin-dependent developmental processes. In previous research, it has been revealed that endosomal trafficking components BEN1/BIG5 (ARF GEF) and BEN2/VPS45 (Sec1/Munc 18 protein) function in intracellular trafficking of PIN proteins in Arabidopsis. Mutations in both BEN1 and BEN2 resulted in defects in polar PIN localization, auxin response gradients, and in root architecture. In this study, we have attempted to gain insight into the developmental roles of these trafficking components. We showed that while genetic or pharmacological disturbances of auxin distribution reduced dividing cells in the root tips and resulted in reduced root growth, the same manipulations had only moderate impact on ben1; ben2 double mutants. In addition, we established transgenic lines in which BEN2/VPS45 is expressed under control of tissue-specific promoters and demonstrated that BEN2/VPS45 regulates the intracellular traffic of PIN proteins in cell-autonomous manner, at least in stele and epidermal cells. Furthermore, BEN2/VPS45 rescued the root architecture defects when expressed in internal tissues of ben1; ben2 double mutants. These results corroborate the roles of the endosomal trafficking component BEN2/VPS45 in regulation of auxin-dependent developmental processes, and suggest that BEN2/VPS45 is required for sustainable root growth, most likely through regulation of tip-ward auxin transport through the internal tissues of root.

A Conditional Mutation in SCD1 Reveals Linkage Between PIN Protein Trafficking, Auxin Transport, Gravitropism, and Lateral Root Initiation.

Auxin is transported in plants with distinct polarity, defined by transport proteins of the PIN-formed (PIN) family. Components of the complex trafficking machinery responsible for polar PIN protein localization have been identified by genetic approaches, but severe developmental phenotypes of trafficking mutants complicate dissection of this pathway. We utilized a temperature sensitive allele of Arabidopsis thaliana SCD1 (stomatal cytokinesis defective1) that encodes a RAB-guanine nucleotide exchange factor. Auxin transport, lateral root initiation, asymmetric auxin-induced gene expression after gravitropic reorientation, and differential gravitropic growth were reduced in the roots of the scd1-1 mutant relative to wild type at the restrictive temperature of 25°C, but not at the permissive temperature of 18°C. In scd1-1 at 25°C, PIN1- and PIN2-GFP accumulated in endomembrane bodies. Transition of seedlings from 18 to 25°C for as little as 20 min resulted in the accumulation of PIN2-GFP in endomembranes, while gravitropism and root developmental defects were not detected until hours after transition to the non-permissive temperature. The endomembrane compartments that accumulated PIN2-GFP in scd1-1 exhibited FM4-64 signal colocalized with ARA7 and ARA6 fluorescent marker proteins, consistent with PIN2 accumulation in the late or multivesicular endosome. These experiments illustrate the power of using a temperature sensitive mutation in the gene encoding SCD1 to study the trafficking of PIN2 between the endosome and the plasma membrane. Using the conditional feature of this mutation, we show that altered trafficking of PIN2 precedes altered auxin transport and defects in gravitropism and lateral root development in this mutant upon transition to the restrictive temperature.

A role for the auxin precursor anthranilic acid in root gravitropism via regulation of PIN-FORMED protein polarity and relocalisation in Arabidopsis.

distribution of auxin within plant tissues is of great importance for developmental plasticity, including root gravitropic growth. Auxin flow is directed by the subcellular polar distribution and dynamic relocalisation of auxin transporters such as the PIN-FORMED (PIN) efflux carriers, which can be influenced by the main natural plant auxin indole-3-acetic acid (IAA). Anthranilic acid (AA) is an important early precursor of IAA and previously published studies with AA analogues have suggested that AA may also regulate PIN localisation. Using Arabidopsis thaliana as a model species, we studied an AA-deficient mutant displaying agravitropic root growth, treated seedlings with AA and AA analogues and transformed lines to over-produce AA while inhibiting its conversion to downstream IAA precursors. We showed that AA rescues root gravitropic growth in the AA-deficient mutant at concentrations that do not rescue IAA levels. Overproduction of AA affects root gravitropism without affecting IAA levels. Treatments with, or deficiency in, AA result in defects in PIN polarity and gravistimulus-induced PIN relocalisation in root cells. Our results revealed a previously unknown role for AA in the regulation of PIN subcellular localisation and dynamics involved in root gravitropism, which is independent of its better known role in IAA biosynthesis.

PIN2 Polarity Establishment in Arabidopsis in the Absence of an Intact Cytoskeleton.

Cell polarity is crucial for the coordinated development of all multicellular organisms. In plants, this is exemplified by the PIN-FORMED (PIN) efflux carriers of the phytohormone auxin: The polar subcellular localization of the PINs is instructive to the directional intercellular auxin transport, and thus to a plethora of auxin-regulated growth and developmental processes. Despite its importance, the regulation of PIN polar subcellular localization remains poorly understood. Here, we have employed advanced live-cell imaging techniques to study the roles of microtubules and actin microfilaments in the establishment of apical polar localization of PIN2 in the epidermis of the Arabidopsis root meristem. We report that apical PIN2 polarity requires neither intact actin microfilaments nor microtubules, suggesting that the primary spatial cue for polar PIN distribution is likely independent of cytoskeleton-guided endomembrane trafficking.

Expression of BpPIN is associated with IAA levels and the formation of lobed leaves in Betula pendula ‘Dalecartica’

Auxin polar transport genes PIN (PIN-FORMED) determine the concentration gradient of auxin in plants. To understand the relationship between the development of different tissues in Betula pendula ‘Dalecartica’, BpPIN gene expression and indole-3-acetic acid (IAA) content were analyzed using qRT-PCR, ELISA, and GUS staining. Gene expression of BpPIN genes and IAA levels in the leaves, buds, stems, xylem, and roots of B. pendula ‘Dalecartica’ and B. pendula as a control were measured. BpPIN1, BpPIN5 and BpPIN6 were upregulated during development in both species, suggesting a dominant role in the development of B. pendula ‘Dalecartica’ leaves. Moreover, BpPIN1 gene expression was positively associated with IAA levels during leaf, vein and petiole development in B. pendula ‘Dalecartica’ only. The correlation coefficient of the first three leaves was 0.69 (P = 0.04), while that of the first three petioles was 0.85 (P = 0.001). In addition, GUS staining of the pro-DR5::GUS transgenic line of cultivar was correlated with the results of BpPIN1 expression. Overall, these findings suggest that BpPIN1 is associated with the formation of lobed leaves in B. pendula ‘Dalecartica’.

PID/WAG-mediated phosphorylation of the Arabidopsis PIN3 auxin transporter mediates polarity switches during gravitropism

Intercellular distribution of the plant hormone auxin largely depends on the polar subcellular distribution of the plasma membrane PIN-FORMED (PIN) auxin transporters. PIN polarity switches in response to different developmental and environmental signals have been shown to redirect auxin fluxes mediating certain developmental responses. PIN phosphorylation at different sites and by different kinases is crucial for PIN function. Here we investigate the role of PIN phosphorylation during gravitropic response. Loss- and gain-of-function mutants in PINOID and related kinases but not in D6PK kinase as well as mutations mimicking constitutive dephosphorylated or phosphorylated status of two clusters of predicted phosphorylation sites partially disrupted PIN3 phosphorylation and caused defects in gravitropic bending in roots and hypocotyls. In particular, they impacted PIN3 polarity rearrangements in response to gravity and during feed-back regulation by auxin itself. Thus PIN phosphorylation, besides regulating transport activity and apical-basal targeting, is also important for the rapid polarity switches in response to environmental and endogenous signals.

Activation and Polarity Control of PIN-FORMED Auxin Transporters by Phosphorylation.

Auxin controls almost every aspect of plant development. Auxin is distributed within the plant by passive diffusion and active cell-to-cell transport. PIN-FORMED (PIN) auxin efflux transporters are polarly distributed in the plasma membranes of many cells, and knowledge about their distribution can predict auxin transport and explain auxin distribution patterns, even in complex tissues. Recent studies have revealed that phosphorylation is essential for PIN activation, suggesting that PIN phosphorylation needs to be taken into account in understanding auxin transport. These findings also ask for a re-examination of previously proposed mechanisms for phosphorylation-dependent PIN polarity control. We provide a comprehensive summary of the current knowledge on PIN regulation by phosphorylation, and discuss possible mechanisms of PIN polarity control in the context of recent findings.

Mutations in CsPID encoding a Ser/Thr protein kinase are responsible for round leaf shape in cucumber (Cucumis sativus L.).

Two round-leaf mutants, rl-1 and rl-2, were identified from EMS-induced mutagenesis. High throughput sequencing and map-based cloning suggested CsPID encoding a Ser/Thr protein kinase as the most possible candidate for rl-1. Rl-2 was allelic to Rl-1. Leaf shape is an important plant architecture trait that is affected by plant hormones, especially auxin. In Arabidopsis, PINOID (PID), a regulator for the auxin polar transporter PIN (PIN-FORMED) affects leaf shape formation, but this function of PID in crop plants has not been well studied. From an EMS mutagenesis population, we identified two round-leaf (rl) mutants, C356 and C949. Segregation analysis suggested that both mutations were controlled by single recessive genes, rl-1 and rl-2, respectively. With map-based cloning, we show that CsPID as the candidate gene of rl-1; a non-synonymous SNP in the second exon of CsPID resulted in an amino acid substitution and the round leaf phenotype. As compared in the wild type plant, CsPID had significantly lower expression in the root, leaf and female flowers in C356, which may result in the less developed roots, round leaves and abnormal female flowers, respectively in the rl-1 mutant. Among the three copies of PID genes, CsPID, CsPID2 and CSPID2L (CsPID2-like) in the cucumber genome, CsPID was the only one with significantly differential expression in adult leaves between WT and C356 suggesting CsPID plays a main role in leaf shape formation. The rl-2 mutation in C949 was also cloned, which was due to another SNP in a nearby location of rl-1 in the same CsPID gene. The two round leaf mutants and the work presented herein provide a good foundation for understanding the molecular mechanisms of CsPID in cucumber leaf development.

ROTUNDA3 function in plant development by phosphatase 2A-mediated regulation of auxin transporter recycling

The shaping of organs in plants depends on the intercellular flow of the phytohormone auxin, of which the directional signaling is determined by the polar subcellular localization of PIN-FORMED (PIN) auxin transport proteins. Phosphorylation dynamics of PIN proteins are affected by the protein phosphatase 2A (PP2A) and the PINOID kinase, which act antagonistically to mediate their apical-basal polar delivery. Here, we identified the ROTUNDA3 (RON3) protein as a regulator of the PP2A phosphatase activity in Arabidopsis thaliana. The RON3 gene was map-based cloned starting from the ron3-1 leaf mutant and found to be a unique, plant-specific gene coding for a protein with high and dispersed proline content. The ron3-1 and ron3-2 mutant phenotypes [i.e., reduced apical dominance, primary root length, lateral root emergence, and growth; increased ectopic stages II, IV, and V lateral root primordia; decreased auxin maxima in indole-3-acetic acid (IAA)-treated root apical meristems; hypergravitropic root growth and response; increased IAA levels in shoot apices; and reduced auxin accumulation in root meristems] support a role for RON3 in auxin biology. The affinity-purified PP2A complex with RON3 as bait suggested that RON3 might act in PIN transporter trafficking. Indeed, pharmacological interference with vesicle trafficking processes revealed that single ron3-2 and double ron3-2 rcn1 mutants have altered PIN polarity and endocytosis in specific cells. Our data indicate that RON3 contributes to auxin-mediated development by playing a role in PIN recycling and polarity establishment through regulation of the PP2A complex activity.

Pin1At regulates PIN1 polar localization and root gravitropism.

Root gravitropism allows plants to establish root systems and its regulation depends on polar auxin transport mediated by PIN-FORMED (PIN) auxin transporters. PINOID (PID) and PROTEIN PHOSPHATASE 2A (PP2A) act antagonistically on reversible phosphorylation of PINs. This regulates polar PIN distribution and auxin transport. Here we show that a peptidyl-prolyl cis/trans isomerase Pin1At regulates root gravitropism. Downregulation of Pin1At suppresses root agravitropic phenotypes of pp2aa and 35S:PID, while overexpression of Pin1At affects root gravitropic responses and enhances the pp2aa agravitropic phenotype. Pin1At also affects auxin transport and polar localization of PIN1 in stele cells, which is mediated by PID and PP2A. Furthermore, Pin1At catalyses the conformational change of the phosphorylated Ser/Thr-Pro motifs of PIN1. Thus, Pin1At mediates the conformational dynamics of PIN1 and affects PID- and PP2A-mediated regulation of PIN1 polar localization, which correlates with the regulation of root gravitropism.

Cell Polarity and Patterning by PIN Trafficking through Early Endosomal Compartments in Arabidopsis thaliana

PIN-FORMED (PIN) proteins localize asymmetrically at the plasma membrane and mediate intercellular polar transport of the plant hormone auxin that is crucial for a multitude of developmental processes in plants. PIN localization is under extensive control by environmental or developmental cues, but mechanisms regulating PIN localization are not fully understood. Here we show that early endosomal components ARF GEF BEN1 and newly identified Sec1/Munc18 family protein BEN2 are involved in distinct steps of early endosomal trafficking. BEN1 and BEN2 are collectively required for polar PIN localization, for their dynamic repolarization, and consequently for auxin activity gradient formation and auxin-related developmental processes including embryonic patterning, organogenesis, and vasculature venation patterning. These results show that early endosomal trafficking is crucial for cell polarity and auxin-dependent regulation of plant architecture.

Origin and evolution of PIN auxin transporters in the green lineage

Polarized auxin transport is crucial for many developmental processes in flowering plants and requires the PIN-FORMED (PIN) family of auxin efflux carriers. However, the impact of polar auxin transport and PIN proteins on the development of non-seed plant species and green algal lineages is largely unknown. Using recently available sequence information from streptophyte algae and other non-seed plant species, we have constructed a preliminary phylogenetic framework and present several hypotheses for PIN protein evolution. We postulate that PIN proteins originated in streptophyte algae at the endoplasmic reticulum (ER) and that plasma membrane localization was acquired during land plant evolution. We also suggest that PIN proteins are evolutionarily distinct from another family of auxin transporters at the ER, the PIN-LIKES (PILS) proteins.