PIN-FORMED Research Articles

The auxin signaling pathway to its PIN transporters: insights based on a meta-analysis of auxin-induced transcriptomes.

Active polar transport of the plant hormone auxin carried out by its PIN transporters is a key link in the formation and maintenance of auxin distribution, which, in turn, determines plant morphogenesis. The plasticity of auxin distribution is largely realized through the molecular genetic regulation of the expression of its transporters belonging to the PIN-FORMED (PIN) protein family. Regulation of auxin-response genes occurs through the ARF-Aux/ IAA signaling pathway. However, it is not known which ARF-Aux/IAA proteins are involved in the regulation of PIN gene expression by auxin. In Arabidopsis thaliana, the PIN, ARF, and Aux/IAA families contain a larger number of members; their various combinations are possible in realization of the signaling pathway, and this is a challenge for understanding the mechanisms of this process. The use of high-throughput sequencing data on auxin-induced transcriptomes makes it possible to identify candidate genes involved in the regulation of PIN expression. To address this problem, we created an approach for the meta-analysis of auxin-induced transcriptomes, which helped us select genes that change their expression during the auxin response together with PIN1, PIN3, PIN4 and PIN7. Possible regulators of ARF-Aux/ IAA signaling pathway for each of the PINs under study were identif ied, and so were the aspects of their regulatory circuits both common for groups of PIN genes and specif ic for each PIN gene. Reconstruction of gene networks and their analysis predicted possible interactions between genes and served as an additional conf irmation of the pathways obtained in the meta-analysis. The approach developed can be used in the search for gene expression regulators in other genomewide data.

Roots hunt nutrients

Plant Science Soil nutrients such as nitrogen sources are not uniformly distributed. Neither are plant roots. Nonetheless, roots of Arabidopsis seedlings seek out pockets of soil ammonium and nitrate. Otvos et al. clarify how root growth and branching are regulated to optimize nitrogen uptake. The auxin efflux carrier PIN-FORMED 2 (PIN2) is regulated posttranscriptionally by phosphorylation at its serine-439 in response to shifts in ammonium or nitrate availability. Phosphorylation in turn triggers changes in PIN2 subcellular localization, which drives changes in auxin distribution between root cortex and epidermis, causing altered root growth rate or direction. EMBO J. e106862 (2020).

LAZY Gene Family in Plant Gravitropism

Adapting to the omnipresent gravitational field was a fundamental basis driving the flourishing of terrestrial plants on the Earth. Plants have evolved a remarkable capability that not only allows them to live and develop within the Earth’s gravity field, but it also enables them to use the gravity vector to guide the growth of roots and shoots, in a process known as gravitropism. Triggered by gravistimulation, plant gravitropism is a highly complex, multistep process that requires many organelles and players to function in an intricate coordinated way. Although this process has been studied for several 100 years, much remains unclear, particularly the early events that trigger the relocation of the auxin efflux carrier PIN-FORMED (PIN) proteins, which presumably leads to the asymmetrical redistribution of auxin. In the past decade, the LAZY gene family has been identified as a crucial player that ensures the proper redistribution of auxin and a normal tropic response for both roots and shoots upon gravistimulation. LAZY proteins appear to be participating in the early steps of gravity signaling, as the mutation of LAZY genes consistently leads to altered auxin redistribution in multiple plant species. The identification and characterization of the LAZY gene family have significantly advanced our understanding of plant gravitropism, and opened new frontiers of investigation into the novel molecular details of the early events of gravitropism. Here we review current knowledge of the LAZY gene family and the mechanism modulated by LAZY proteins for controlling both roots and shoots gravitropism. We also discuss the evolutionary significance and conservation of the LAZY gene family in plants.

Expression of a plastid-localized sugar transporter in the suspensor is critical to embryogenesis.

Plant growth and development rely on sugar transport between source and sink cells and between different organelles. The plastid-localized sugar transporter GLUCOSE-6-PHOSPHATE TRANSLOCATER1 (GPT1) is an essential gene in Arabidopsis (Arabidopsis thaliana). Using a partially rescued gpt1 mutant and cell-specific RNAi suppression of GPT1, we demonstrated that GPT1 is essential to the function of the embryo suspensor and the development of the embryo. GPT1 showed a dynamic expression/accumulation pattern during embryogenesis. Inhibition of GPT1 accumulation via RNAi using a suspensor-specific promoter resulted in embryos and seedlings with defects similar to auxin mutants. Loss of function of GPT1 in the suspensor also led to abnormal/ectopic cell division in the lower part of the suspensor, which gave rise to an ectopic embryo, resulting in twin embryos in some seeds. Furthermore, loss of function of GPT1 resulted in vacuolar localization of PIN-FORMED1 (PIN1) and altered DR5 auxin activity. Proper localization of PIN1 on the plasma membrane is essential to polar auxin transport and distribution, a key determinant of pattern formation during embryogenesis. Our findings suggest that the function of GPT1 in the embryo suspensor is linked to sugar and/or hormone distribution between the embryo proper and the maternal tissues, and is important for maintenance of suspensor identity and function during embryogenesis.

Genome-wide identification of polar auxin transporter gene families reveals a possible new polar auxin flow in inverted cuttings of Populus yunnanensis.

Inverted cuttings of Populus yunnanensis are characterized by enlarged stems and dwarfed new shoots, and phytohormones play a crucial role in the response to inversion. The polar auxin transport (PAT) system is distinct from the transport systems of other hormones and is controlled by three major transporter gene families: pin-formed (PIN), auxin-resistant/like aux (AUX/LAX) and ATP-binding cassette transporters of the B class (ABCB). Here, we identified these three families in P. trichocarpa, P. euphratica and P. yunnanensis through a genome-wide analysis. The Populus PIN, AUX/LAX and ABCB gene families comprised 15, 8 and 31 members, respectively. Most PAT genes in Populus and Arabidopsis were identified as clear sister pairs, and some had unique motifs. Transcriptome profiling revealed that the expression of most PAT genes was unrelated to cutting inversion and that only several genes showed altered expression when cuttings were inverted. The auxin content difference at positions was opposite in upright and inverted cutting bodies during rooting, which obeyed the original plant polarity. However, during plant growth, the two direction types exhibited similar auxin movements in the cutting bodies, and the opposite auxin changes were observed in new shoots. Four PAT genes with a positive response to cutting inversion, PyuPIN10, PyuPIN11, PyuLAX6 and PyuABCB27, showed diverse expression patterns between upright and inverted cuttings during rooting and plant growth. Furthermore, PAT gene expression retained its polarity, which differs from the results found for auxin flow during plant growth. The inconformity indicated that a new downward auxin flow in addition to the old upward flow might be established during the growth of inverted cuttings. Some highly polar PAT genes were involved in the maintenance of original auxin polarity, which might cause the enlarged stems of inverted cuttings. This work lays a foundation for understanding the roles of auxin transport in plant responses to inversion.

Functional innovations of PIN auxin transporters mark crucial evolutionary transitions during rise of flowering plants.

Flowering plants display the highest diversity among plant species and have notably shaped terrestrial landscapes. Nonetheless, the evolutionary origin of their unprecedented morphological complexity remains largely an enigma. Here, we show that the coevolution of cis-regulatory and coding regions of PIN-FORMED (PIN) auxin transporters confined their expression to certain cell types and directed their subcellular localization to particular cell sides, which together enabled dynamic auxin gradients across tissues critical to the complex architecture of flowering plants. Extensive intraspecies and interspecies genetic complementation experiments with PINs from green alga up to flowering plant lineages showed that PIN genes underwent three subsequent, critical evolutionary innovations and thus acquired a triple function to regulate the development of three essential components of the flowering plant Arabidopsis: shoot/root, inflorescence, and floral organ. Our work highlights the critical role of functional innovations within the PIN gene family as essential prerequisites for the origin of flowering plants.

MtPIN1 and MtPIN3 Play Dual Roles in Regulation of Shade Avoidance Response under Different Environments in Medicago truncatula.

Polar auxin transport mediated by PIN-FORMED (PIN) proteins is critical for plant growth and development. As an environmental cue, shade stimulates hypocotyls, petiole, and stem elongation by inducing auxin synthesis and asymmetric distributions, which is modulated by PIN3,4,7 in Arabidopsis. Here, we characterize the MtPIN1 and MtPIN3, which are the orthologs of PIN3,4,7, in model legume species Medicago truncatula. Under the low Red:Far-Red (R:FR) ratio light, the expression of MtPIN1 and MtPIN3 is induced, and shadeavoidance response is disrupted in mtpin1 mtpin3 double mutant, indicating that MtPIN1 and MtPIN3 have a conserved function in shade response. Surprisingly, under the normal growth condition, mtpin1 mtpin3 displayed the constitutive shade avoidance responses, such as the elongated petiole, smaller leaf, and increased auxin and chlorophyll content. Therefore, MtPIN1 and MtPIN3 play dual roles in regulation of shadeavoidance response under different environments. Furthermore, these data suggest that PIN3,4,7 and its orthologs have evolved conserved and specific functions among species.

Auxin-Regulated Reversible Inhibition of TMK1 Signaling by MAKR2 Modulates the Dynamics of Root Gravitropism

SummaryPlants are able to orient their growth according to gravity, which ultimately controls both shoot and root architecture.1 Gravitropism is a dynamic process whereby gravistimulation induces the asymmetric distribution of the plant hormone auxin, leading to asymmetric growth, organ bending, and subsequent reset of auxin distribution back to the original pre-gravistimulation situation.1, 2, 3 Differential auxin accumulation during the gravitropic response depends on the activity of polarly localized PIN-FORMED (PIN) auxin-efflux carriers.1, 2, 3, 4 In particular, the timing of this dynamic response is regulated by PIN2,5,6 but the underlying molecular mechanisms are poorly understood. Here, we show that MEMBRANE ASSOCIATED KINASE REGULATOR2 (MAKR2) controls the pace of the root gravitropic response. We found that MAKR2 is required for the PIN2 asymmetry during gravitropism by acting as a negative regulator of the cell-surface signaling mediated by the receptor-like kinase TRANSMEMBRANE KINASE1 (TMK1).2,7, 8, 9, 10 Furthermore, we show that the MAKR2 inhibitory effect on TMK1 signaling is antagonized by auxin itself, which triggers rapid MAKR2 membrane dissociation in a TMK1-dependent manner. Our findings suggest that the timing of the root gravitropic response is orchestrated by the reversible inhibition of the TMK1 signaling pathway at the cell surface.

Role of Arabidopsis RAB5 GEF vps9a in maintaining potassium levels under sodium chloride stress.

Salt stress is one of the major factors impacting crop productivity worldwide. Through a variety of effector and signaling pathways, plants achieve survival under salinity stress by maintaining high cytosolic potassium/sodium ion (K+/Na+) ratios, preventing Na+ cytotoxicity, and retaining osmotic balance. Ras‐related protein 5 (Rab5) members are involved in the trafficking of endosomes to the vacuole or plasma membrane (PM). The vacuolar protein sorting‐ associated protein 9 (vps9a) encodes the single guanine nucleotide exchange factor (GEF) that activates all three known Rab5 proteins in Arabidopsis thaliana. Previous work from our group has reported the critical function of vps9a for the operation of salt‐induced endocytic pathway, as well as the expansion of endomembrane compartments under saline stress conditions. Here we show an additional role of vps9a in plant response to salt stress via maintenance of K+ status of the cell rather than Na+ homeostasis. Our results show that roots from vps9a‐2 mutant, subjected to 100 mM NaCl, display alterations in transcript levels of genes involved in the K+ homeostasis pathway. Concurrent with the observed sensitivity of vps9a‐2 mutant under NaCl stress, exposure to low K+ environments resulted in growth retardation, and reduced rate of endocytosis. Furthermore, vps9a‐2 mutant displays reduced expression of auxin reporter, Direct Repeat‐5 (DR5), and alterations in polarity and abundance of auxin efflux carrier PIN‐ FORMED2 (PIN2). Imposition of NaCl stress was found to be restrictive to the elongation capacity of cells in the root elongation zone of vps9a‐2 mutant. Together our results indicate that alterations in K+ homeostasis and associated cellular changes causing increased cell wall pH, contribute to diminished root growth and compromised survival of vps9a‐2 mutant under salt stress conditions.

FERONIA mediates root nutating growth.

Root nutation indicates the behavior that roots grow in a waving and skewing way due to unequal growth rates on different sides. Although a few developmental and environmental factors have been reported, genetic pathways mediating this process are obscure. We report here that the Arabidopsis CrRLK1L family member FERONIA (FER) is critical for root nutation. Functional loss of FER resulted in enhanced root waviness on tilted plates or roots forming anti-clockwise coils on horizontal plates. Suppressing polar auxin transport, either by pharmacological treatment or by introducing mutations at PIN-FORMED2 (PIN2) or AUXIN RESISTANT1 (AUX1), suppressed the asymmetric root growth (ARG) in fer-4, a null mutant of FER, indicating that FER suppression of ARG depends on polar auxin transport. We further showed by pharmacological treatments that dynamic microtubule organization and Ca2+ signaling are both critical for FER-mediated ARG. Results presented here demonstrate a key role of FER in mediating root nutating growth, through PIN2- and AUX1-mediated auxin transport, through dynamic microtubule organization, and through Ca2+ signaling.

Cellular requirements for PIN polar cargo clustering in Arabidopsis thaliana.

Summary Cell and tissue polarization is fundamental for plant growth and morphogenesis. The polar, cellular localization of Arabidopsis PIN‐FORMED (PIN) proteins is crucial for their function in directional auxin transport. The clustering of PIN polar cargoes within the plasma membrane has been proposed to be important for the maintenance of their polar distribution. However, the more detailed features of PIN clusters and the cellular requirements of cargo clustering remain unclear.Here, we characterized PIN clusters in detail by means of multiple advanced microscopy and quantification methods, such as 3D quantitative imaging or freeze‐fracture replica labeling. The size and aggregation types of PIN clusters were determined by electron microscopy at the nanometer level at different polar domains and at different developmental stages, revealing a strong preference for clustering at the polar domains.Pharmacological and genetic studies revealed that PIN clusters depend on phosphoinositol pathways, cytoskeletal structures and specific cell‐wall components as well as connections between the cell wall and the plasma membrane.This study identifies the role of different cellular processes and structures in polar cargo clustering and provides initial mechanistic insight into the maintenance of polarity in plants and other systems.

The volatile organic compounds of Floccularia luteovirens modulate plant growth and metabolism in Arabidopsis thaliana

The volatile organic compounds (VOCs) produced by soil microbes modulated plant growth and development. Floccularia luteovirens, an edible mushroom, is beneficial to the growth of alpine meadow plants on the Qinghai-Tibet Plateau. We aimed to elucidate the physiological and molecular mechanisms underlying the mushroom fungal VOC-mediated plant growth and development. Here, we investigated the effects of VOCs produced by F. luteovirens on the root system development and seedling growth by integrating physiology, genetics, transcriptome and metabolome analysis using 1/2 MS medium-grown Arabidopsis thaliana seedlings. Treatment with F. luteovirens VOCs reduce primary root growth by aggravating auxin accumulation through the repression of the abundance of auxin efflux carrier PIN-FORMED 2 (PIN2) protein, whereas it increases the lateral root number of A. thaliana seedlings. In addition to modulating root system architecture, treatment with F. luteovirens VOCs markedly increased aboveground growth. The transcriptome and metabolome analyses further supported the notion that F. luteovirens VOCs modulate plant growth and development through the induction of carbon/nitrogen metabolism and antioxidant defense while repressing several secondary metabolism and amino acid catabolism pathways. These results suggested that application of F. luteovirens VOCs promote growth by inducing changes in root system architecture through auxin pathway and regulating metabolism in plants.

Auxin regulation and MdPIN expression during adventitious root initiation in apple cuttings

Adventitious root (AR) formation is critical for the efficient propagation of elite horticultural and forestry crops. Despite decades of research, the cellular processes and molecular mechanisms underlying AR induction in woody plants remain obscure. We examined the details of AR formation in apple (Malus domestica) M.9 rootstock, the most widely used dwarf rootstock for intensive production, and investigated the role of polar auxin transport in postembryonic organogenesis. AR formation begins with a series of founder cell divisions and elongation of the interfascicular cambium adjacent to vascular tissues. This process is associated with a relatively high indole acetic acid (IAA) content and hydrolysis of starch grains. Exogenous auxin treatment promoted this cell division, as well as the proliferation and reorganization of the endoplasmic reticulum and Golgi membrane. In contrast, treatment with the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) inhibited cell division in the basal region of the cuttings and resulted in abnormal cell divisions during the early stage of AR formation. In addition, PIN-FORMED (PIN) transcripts were differentially expressed throughout the whole AR development process. We also detected upregulation of MdPIN8 and MdPIN10 during induction; upregulation of MdPIN4, MdPIN5, and MdPIN8 during extension; and upregulation of all MdPINs during AR initiation. This research provides an improved understanding of the cellular and molecular underpinnings of the AR process in woody plants.

Indeterminate domain 3 negatively regulates plant erectness and the resistance of rice to sheath blight by controlling PIN-FORMED gene expressions

ABSTRACT Plant architecture and disease resistance are the key factors that control the production of yield. However, the mechanism behind these factors is largely unknown. In this study, we identified that indeterminate domain 3 (IDD3) was obviously induced by inoculation of Rhizoctonia solani AG1-IA. Plants that overexpressed IDD3 (IDD3 OX) were more susceptible, while idd3 mutants showed a similar response to sheath blight disease compared with wild-type plants. Interestingly, IDD3 OX plants developed a wider tiller angle and exhibited altered shoot gravitropism, while idd3 knock-out mutants showed no visible morphological differences compared with the wild-type plants. IDD3 is ubiquitously expressed in different tissues and stages, and the IDD3 transcript was induced by exogenously applied auxin. Expression of the PIN-FORMED (PIN) and Aux/IAA genes was altered in IDD3 OX compared with wild-type plants. Furthermore, IDD3 OX plants are sensitive to auxin and the polar auxin transporter inhibitor N-1-naphthylphalamic acid (NPA). Further yeast-one hybrid, chromatin immunoprecipitation (ChIP) and transient assays revealed that IDD3 directly represses PIN1b via promoter binding. Inoculation with R. solani indicated that PIN1b RNAi plants are more susceptible to sheath blight disease (ShB) compared with the wild-type. Taken together, our analyses suggest that IDD3 controls plant architecture and the resistance of rice to ShB via the regulation of PIN auxin transporter genes.

Genome-Wide Analysis of the PIN Auxin Efflux Carrier Gene Family in Coffee.

Coffee is one of the most popular beverages around the world, which is mainly produced from the allopolyploid Coffea arabica. The genomes of C. arabica and its two ancestors C. canephora and C. eugenioides have been released due to the development of next generation sequencing. However, few studies on C. arabica are related to the PIN-FORMED (PIN) auxin efflux transporter despite its importance in auxin-mediated plant growth and development. In the present study, we conducted a genome-wide analysis of the PIN gene family in the three coffee species. Totals of 17, 9 and 10 of the PIN members were characterized in C. Arabica, C. canephora and C. eugenioides, respectively. Phylogenetic analysis revealed gene loss of PIN1 and PIN2 homologs in C. arabica, as well as gene duplication of PIN5 homologs during the fractionation process after tetraploidy. Furthermore, we conducted expression analysis of PIN genes in C. arabica by in silico and qRT-PCR. The results revealed the existence of gene expression dominance in allopolyploid coffee and illustrated several PIN candidates in regulating auxin transport and homeostasis under leaf rust fungus inoculation and the tissue-specific expression pattern of C. arabica. Together, this study provides the basis and guideline for future functional characterization of the PIN gene family.

Endosidin 2 accelerates PIN2 endocytosis and disturbs intracellular trafficking of PIN2, PIN3, and PIN4 but not of SYT1.

We established that Endosidin2 (ES2) affected the trafficking routes of both newly synthesized and endocytic pools of PIN-FORMED2 (PIN2) in Arabidopsis root epidermal cells. PIN2 populations accumulated in separated patches, which gradually merged into large and compact ES2 aggregates (ES2As). FM4-64 endocytic tracer labeled ES2As as well. Both PIN2 pools also appeared in vacuoles. Accelerated endocytosis of PIN2, its aggregation in the cytoplasm, and redirection of PIN2 flows to vacuoles led to a substantial reduction of the abundance of this protein in the plasma membrane. Whereas PIN-FORMED3 and PIN-FORMED4 also aggregated in the cytoplasm, SYT1 was not sensitive to ES2 treatment and did not appear either in the cytoplasmic aggregates or vacuoles. Ultrastructural analysis revealed that ES2 affects the Golgi apparatus so that stacks acquired cup-shape and even circular shape surrounded by several vesicles. Abnormally shaped Golgi stacks, stack remnants, multi-lamellar structures, separated Golgi cisterna rings, tubular structures, and vesicles formed discrete clusters.

Covalent Self-Labeling of Tagged Proteins with Chemical Fluorescent Dyes in BY-2 Cells and Arabidopsis Seedlings.

Synthetic chemical fluorescent dyes promise to be useful for many applications in biology. Covalent, targeted labeling, such as with a SNAP-tag, uses synthetic dyes to label specific proteins in vivo for studying processes such as endocytosis or for imaging via super-resolution microscopy. Despite its potential, such chemical tagging has not been used effectively in plants. A major drawback has been the limited knowledge regarding cell wall and membrane permeability of the available synthetic dyes. Of 31 synthetic dyes tested here, 23 were taken up into BY-2 cells, while eight were not. This creates sets of dyes that can serve to measure endocytosis. Three of the dyes that were able to enter the cells, SNAP-tag ligands of diethylaminocoumarin, tetramethylrhodamine, and silicon-rhodamine 647, were used to SNAP-tag α-tubulin. Successful tagging was verified by live cell imaging and visualization of microtubule arrays in interphase and during mitosis in Arabidopsis (Arabidopsis thaliana) seedlings. Fluorescence activation-coupled protein labeling with DRBG-488 was used to observe PIN-FORMED2 (PIN2) endocytosis and delivery to the vacuole as well as preferential delivery of newly synthesized PIN2 to the actively forming cell plate during mitosis. Together, the data demonstrate that specific self-labeling of proteins can be used effectively in plants to study a wide variety of cellular and biological processes.

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.