Phosphoinositide-dependent regulation of VAN3 ARF-GAP localization and activity essential for vascular tissue continuity in plants

ACAP-type ARF GTPase activating proteins (ARF-GAPs) regulate multiple cellular processes, including endocytosis, secretion, phagocytosis, cell adhesion and cell migration. However, the regulation of ACAP functions by other cellular proteins is poorly understood. We have reported previously that a plant ACAP, VAN3, plays a pivotal role in plant venation continuity. Here, we report on newly identified VAN3 regulators: the CVP2 (cotyledon vascular pattern 2) 5 PTase, which is considered to degrade IP3 and also to produce PtdIns(4) P from PtdIns(4,5) P-2; and a PH domain-containing protein, VAB (VAN3 binding protein). Combinational mutations of both CVP2 and its closest homologue CVL1 (CVP2 like 1) phenocopied the strong allele of van3 mutants, showing severe vascular continuity. The phenotype of double mutants between van3, cvp2 and vab suggested that VAN3, CVP2 and VAB function in vascular pattern formation in the same pathway. Localization analysis revealed that both CVP2 and VAB colocalize with VAN3 in the trans-Golgi network (TGN), supporting their functions in the same pathway. The subcellular localization of VAN3 was dependent on its PH domain, and mislocalization of VAN3 was induced in cvp2 or vab mutants. These results suggest that CVP2 and VAB cooperatively regulate the subcellular localization of VAN3 through the interaction between its PH domain and phosphoinositides and/or inositol phosphates. In addition, PtdIns(4) P, to which VAN3 binds preferentially, enhanced the ARF-GAP activity of VAN3, whereas IP3 inhibited it. These results suggest the existence of PtdIns(4) P and/or IP3-dependent subcellular targeting and regulation of VAN3 ACAP activity that governs plant vascular tissue continuity

Cellular mechanisms for cargo delivery and polarity maintenance at different polar domains in plant cells

The asymmetric localization of proteins in the plasma membrane domains of eukaryotic cells is a fundamental manifestation of cell polarity that is central to multicellular organization and developmental patterning. In plants, the mechanisms underlying the polar localization of cargo proteins are still largely unknown and appear to be fundamentally distinct from those operating in mammals. Here, we present a systematic, quantitative comparative analysis of the polar delivery and subcellular localization of proteins that characterize distinct polar plasma membrane domains in plant cells. The combination of microscopic analyses and computational modeling revealed a mechanistic framework common to diverse polar cargos and underlying the establishment and maintenance of apical, basal, and lateral polar domains in plant cells. This mechanism depends on the polar secretion, constitutive endocytic recycling, and restricted lateral diffusion of cargos within the plasma membrane. Moreover, our observations suggest that polar cargo distribution involves the individual protein potential to form clusters within the plasma membrane and interact with the extracellular matrix. Our observations provide insights into the shared cellular mechanisms of polar cargo delivery and polarity maintenance in plant cells

The PIN-FORMED (PIN) protein family of auxin transporters

The PIN-FORMED (PIN) proteins are secondary transporters acting in the efflux of the plant signal molecule auxin from cells. They are asymmetrically localized within cells and their polarity determines the directionality of intercellular auxin flow. PIN genes are found exclusively in the genomes of multicellular plants and play an important role in regulating asymmetric auxin distribution in multiple developmental processes, including embryogenesis, organogenesis, tissue differentiation and tropic responses. All PIN proteins have a similar structure with amino- and carboxy-terminal hydrophobic, membrane-spanning domains separated by a central hydrophilic domain. The structure of the hydrophobic domains is well conserved. The hydrophilic domain is more divergent and it determines eight groups within the protein family. The activity of PIN proteins is regulated at multiple levels, including transcription, protein stability, subcellular localization and transport activity. Different endogenous and environmental signals can modulate PIN activity and thus modulate auxin-distribution-dependent development. A large group of PIN proteins, including the most ancient members known from mosses, localize to the endoplasmic reticulum and they regulate the subcellular compartmentalization of auxin and thus auxin metabolism. Further work is needed to establish the physiological importance of this unexpected mode of auxin homeostasis regulation. Furthermore, the evolution of PIN-based transport, PIN protein structure and more detailed biochemical characterization of the transport function are important topics for further studies

Gibberellin DELLA signaling targets the retromer complex to redirect protein trafficking to the plasma membrane

The plant hormone gibberellic acid (GA) is a crucial regulator of growth and development. The main paradigm of GA signaling puts forward transcriptional regulation via the degradation of DELLA transcriptional repressors. GA has also been shown to regulate tropic responses by modulation of the plasma membrane incidence of PIN auxin transporters by an unclear mechanism. Here we uncovered the cellular and molecular mechanisms by which GA redirects protein trafficking and thus regulates cell surface functionality. Photoconvertible reporters revealed that GA balances the protein traffic between the vacuole degradation route and recycling back to the cell surface. Low GA levels promote vacuolar delivery and degradation of multiple cargos, including PIN proteins, whereas high GA levels promote their recycling to the plasma membrane. This GA effect requires components of the retromer complex, such as Sorting Nexin 1 (SNX1) and its interacting, microtubule (MT)-associated protein, the Cytoplasmic Linker-Associated Protein (CLASP1). Accordingly, GA regulates the subcellular distribution of SNX1 and CLASP1, and the intact MT cytoskeleton is essential for the GA effect on trafficking. This GA cellular action occurs through DELLA proteins that regulate the MT and retromer presumably via their interaction partners Prefoldins (PFDs). Our study identified a branching of the GA signaling pathway at the level of DELLA proteins, which, in parallel to regulating transcription, also target by a nontranscriptional mechanism the retromer complex acting at the intersection of the degradation and recycling trafficking routes. By this mechanism, GA can redirect receptors and transporters to the cell surface, thus coregulating multiple processes, including PIN-dependent auxin fluxes during tropic responses

PIN auxin efflux carrier polarity is regulated by PINOID kinase-mediated recruitment into GNOM-independent trafficking in Arabidopsis

The phytohormone auxin plays a major role in embryonic and postembryonic plant development. The temporal and spatial distribution of auxin largely depends on the subcellular polar localization of members of the PIN-FORMED (PIN) auxin efflux carrier family. The Ser/Thr protein kinase PINOID (PID) catalyzes PIN phosphorylation and crucially contributes to the regulation of apical-basal PIN polarity. The GTP exchange factor on ADP-ribosylation factors (ARF-GEF), GNOM preferentially mediates PIN recycling at the basal side of the cell. Interference with GNOM activity leads to dynamic PIN transcytosis between different sides of the cell. Our genetic, pharmacological, and cell biological approaches illustrate that PID and GNOM influence PIN polarity and plant development in an antagonistic manner and that the PID-dependent PIN phosphorylation results in GNOM-independent polar PIN targeting. The data suggest that PID and the protein phosphatase 2A not only regulate the static PIN polarity, but also act antagonistically on the rate of GNOM-dependent polar PIN transcytosis. We propose a model that includes PID-dependent PIN phosphorylation at the plasma membrane and the subsequent sorting of PIN proteins to a GNOM-independent pathway for polarity alterations during developmental processes, such as lateral root formation and leaf vasculature development

Auxin-dependent cell cycle reactivation through transcriptional regulation of Arabidopsis E2Fa by lateral organ boundary proteins

Multicellular organisms depend on cell production, cell fate specification, and correct patterning to shape their adult body. In plants, auxin plays a prominent role in the timely coordination of these different cellular processes. A well-studied example is lateral root initiation, in which auxin triggers founder cell specification and cell cycle activation of xylem pole-positioned pericycle cells. Here, we report that the E2Fa transcription factor of Arabidopsis thaliana is an essential component that regulates the asymmetric cell division marking lateral root initiation. Moreover, we demonstrate that E2Fa expression is regulated by the LATERAL ORGAN BOUNDARY DOMAIN18/LATERAL ORGAN BOUNDARY DOMAIN33 (LBD18/LBD33) dimer that is, in turn, regulated by the auxin signaling pathway. LBD18/LBD33 mediates lateral root organogenesis through E2Fa transcriptional activation, whereas E2Fa expression under control of the LBD18 promoter eliminates the need for LBD18. Besides lateral root initiation, vascular patterning is disrupted in E2Fa knockout plants, similarly as it is affected in auxin signaling and lbd mutants, indicating that the transcriptional induction of E2Fa through LBDs represents a general mechanism for auxin-dependent cell cycle activation. Our data illustrate how a conserved mechanism driving cell cycle entry has been adapted evolutionarily to connect auxin signaling with control of processes determining plant architecture

Emergence of tissue polarization from synergy of intracellular and extracellular auxin signaling

Here, we provide a novel mechanistic framework for cell polarization during auxin-driven plant development that combines intracellular auxin signaling for regulation of expression of PINFORMED (PIN) auxin efflux transporters and the theoretical assumption of extracellular auxin signaling for regulation of PIN subcellular dynamics.The competitive utilization of auxin signaling component in the apoplast might account for the elusive mechanism for cell-to-cell communication for tissue polarization.Computer model simulations faithfully and robustly recapitulate experimentally observed patterns of tissue polarity and asymmetric auxin distribution during formation and regeneration of vascular systems, and during the competitive regulation of shoot branching by apical dominance.Our model generated new predictions that could be experimentally validated, highlighting a mechanistically conceivable explanation for the PIN polarization and canalization of the auxin flow in plants

Recycling, clustering, and endocytosis jointly maintain PIN auxin carrier polarity at the plasma membrane

A combination of super-resolution microscopy in live cells and computational modeling provides new insights into the dynamic and interwoven mechanism that maintains the polar distribution of an important plant cargo

Cellular requirements for PIN polar cargo clustering in Arabidopsis thaliana

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