Mechanistic Framework for Establishment, Maintenance, and Alteration of Cell Polarity in Plants

Cell polarity establishment, maintenance, and alteration are central to the developmental and response programs of nearly all organisms and are often implicated in abnormalities ranging from patterning defects to cancer. By residing at the distinct plasma membrane domains polar cargoes mark the identities of those domains, and execute localized functions. Polar cargoes are recruited to the specialized membrane domains by directional secretion and/or directional endocytic recycling. In plants, auxin efflux carrier PIN proteins display polar localizations in various cell types and play major roles in directional cell-to-cell transport of signaling molecule auxin that is vital for plant patterning and response programs. Recent advanced microscopy studies applied to single cells in intact plants reveal subcellular PIN dynamics. They uncover the PIN polarity generation mechanism and identified important roles of AGC kinases for polar PIN localization. AGC kinase family members PINOID, WAG1, and WAG2, belonging to the AGC-3 subclass predominantly influence the polar localization of PINs. The emerging mechanism for AGC-3 kinases action suggests that kinases phosphorylate PINs mainly at the plasma membrane after initial symmetric PIN secretion for eventual PIN internalization and PIN sorting into distinct ARF-GEF-regulated polar recycling pathways. Thus phosphorylation status directs PIN translocation to different cell sides. Based on these findings a mechanistic framework evolves that suggests existence of cell side-specific recycling pathways in plants and implicates AGC3 kinases for differential PIN recruitment among them for eventual PIN polarity establishment, maintenance, and alteration

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

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

Microtubule plus-ends reveal essential links between intracellular polarization and localized modulation of endocytosis during division-plane establishment in plant cells

BACKGROUND: A key event in plant morphogenesis is the establishment of a division plane. A plant-specific microtubular preprophase band (PPB) accurately predicts the line of cell division, whereas the phragmoplast, another plant-specific array, executes cell division by maintaining this predicted line. Although establishment of these specific arrays apparently involves intracellular repolarization events that focus cellular resources to a division site, it still remains unclear how microtubules position the cell division planes. Here we study GFP-AtEB1 decorated microtubule plus-ends to dissect events at the division plane. RESULTS: Early mitotic events included guided growth of endoplasmic microtubules (EMTs) towards the PPB site and their coincident localization with endocytic vesicles. Consequently, an endosomal belt lay in close proximity to the microtubular PPB at its maturation and was maintained during spindle formation. During cytokinesis, EMTs radiated from the former spindle poles in a geometrical conformation correlating with cell-plate navigation and tilt-correction. Naphthylphtalamic acid (NPA), an inhibitor of polar auxin efflux, caused abnormal PPBs and shifted division planes. CONCLUSION: Our observations reveal a spatio-temporal link between microtubules and intracellular polarization essential for localized endocytosis and precise establishment of the division plane in plants. Additionally, they implicate the growth regulator, auxin, in this important cellular event

The process of setting micronutrient recommendations: a cross-European comparison of nutrition-related scientific advisory bodies

Copyright @ The Authors 2010Objective: To examine the workings of the nutrition-related scientific advisory bodies in Europe, paying particular attention to the internal and external contexts within which they operate. Design: Desk research based on two data collection strategies: a questionnaire completed by key informants in the field of micronutrient recommendations and a case study that focused on mandatory folic acid (FA) fortification. Setting: Questionnaire-based data were collected across thirty-five European countries. The FA fortification case study was conducted in the UK, Norway, Denmark, Germany, Spain, Czech Republic and Hungary. Results: Varied bodies are responsible for setting micronutrient recommendations, each with different statutory and legal models of operation. Transparency is highest where there are standing scientific advisory committees (SAC). Where the standing SAC is created, the range of expertise and the terms of reference for the SAC are determined by the government. Where there is no dedicated SAC, the impetus for the development of micronutrient recommendations and the associated policies comes from interested specialists in the area. This is typically linked with an ad hoc selection of a problem area to consider, lack of openness and transparency in the decisions and over-reliance on international recommendations. Conclusions: Even when there is consensus about the science behind micronutrient recommendations, there is a range of other influences that will affect decisions about the policy approaches to nutrition-related public health. This indicates the need to document the evidence that is drawn upon in the decisions about nutrition policy related to micronutrient intake.This work has been carried out within the EURRECA Network of Excellence (www.eurreca.org) which is financially supported by the Commission of the European Communities, specific Research, Technology and Development (RTD) Programme Quality of Life and Management of Living Resources, within the Sixth Framework Programme, contract no. 036196

Auxin influx inhibitors 1-NOA, 2-NOA, and CHPAA interfere with membrane dynamics in tobacco cells

The phytohormone auxin is transported through the plant body either via vascular pathways or from cell to cell by specialized polar transport machinery. This machinery consists of a balanced system of passive diffusion combined with the activities of auxin influx and efflux carriers. Synthetic auxins that differ in the mechanisms of their transport across the plasma membrane together with polar auxin transport inhibitors have been used in many studies on particular auxin carriers and their role in plant development. However, the exact mechanism of action of auxin efflux and influx inhibitors has not been fully elucidated. In this report, the mechanism of action of the auxin influx inhibitors (1-naphthoxyacetic acid (1-NOA), 2-naphthoxyacetic acid (2-NOA), and 3-chloro-4-hydroxyphenylacetic acid (CHPAA)) is examined by direct measurements of auxin accumulation, cellular phenotypic analysis, as well as by localization studies of Arabidopsis thaliana L. auxin carriers heterologously expressed in Nicotiana tabacum L., cv. Bright Yellow cell suspensions. The mode of action of 1-NOA, 2-NOA, and CHPAA has been shown to be linked with the dynamics of the plasma membrane. The most potent inhibitor, 1-NOA, blocked the activities of both auxin influx and efflux carriers, whereas 2-NOA and CHPAA at the same concentration preferentially inhibited auxin influx. The results suggest that these, previously unknown, activities of putative auxin influx inhibitors regulate overall auxin transport across the plasma membrane depending on the dynamics of particular membrane vesicles

Nutri-RecQuest: a web-based search engine on current micronutrient recommendations

Background: The EURRECA (EURopean micronutrient RECommendations Aligned) Network of Excellence collated current micronutrient recommendations. A user-friendly tool, Nutri-RecQuest, was developed to allow access to the collated data and to create a database source for use in other nutritional software tools. Methods: Recommendations, that is, intakes of micronutrients sufficient to meet the requirements of the majority of healthy individuals of that population, from 37 European countries/organizations and eight key non-European countries/regions comprising 29 micronutrients were entered into a database. General information on the source of the recommendations, as well scientific background information, was added. Results: A user-friendly web-based interface was developed to provide efficient search, comparison, display, print and export functions. Conclusion: Easy access to existing recommendations through the web-based tool may be valuable for bodies responsible for setting recommendations, as well as for users of recommendations including scientists, policy makers, health professionals and industry. Adding related dietary reference values such as average nutrient requirements and upper limits may extend the utility of the tool. European Journal of Clinical Nutrition (2010) 64, S43-S47; doi:10.1038/ejcn.2010.6

Collision induced spatial organization of microtubules

The dynamic behavior of microtubules in solution can be strongly modified by interactions with walls or other structures. We examine here a microtubule growth model where the increase in size of the plus-end is perturbed by collisions with other microtubules. We show that such a simple mechanism of constrained growth can induce ordered structures and patterns from an initially isotropic and homogeneous suspension. First, microtubules self-organize locally in randomly oriented domains that grow and compete with each other. By imposing even a weak orientation bias, external forces like gravity or cellular boundaries may bias the domain distribution eventually leading to a macroscopic sample orientation.Comment: Submitted to Biophysical Journa

Trafficking modulator TENin1 inhibits endocytosis, causes endomembrane protein accumulation at the pre-vacuolar compartment and impairs gravitropic response in Arabidopsis thaliana

Auxin gradients are established and maintained by polarized distribution of auxin transporters that undergo constitutive endocytic recycling from the PM (plasma membrane) and are essential for the gravitropic response in plants. The present study characterizes an inhibitor of endomembrane protein trafficking, TE1 (trafficking and endocytosis inhibitor 1/TENin1) that reduces gravitropic root bending in Arabidopsis thaliana seedlings. Short-term TE1 treatment causes accumulation of PM proteins, including the BR (brassinosteroid) receptor BRI1 (BR insensitive 1), PIP2a (PM intrinsic protein 2a) and the auxin transporter PIN2 (PIN-FORMED 2) in a PVC (pre-vacuolar related compartment), which is sensitive to BFA (Brefeldin A). This compound inhibits endocytosis from the PM and promotes trafficking to the vacuole, consistent with inhibition of retrieval of proteins to the TGN (trans-Golgi network) from the PVC and the PM. However, trafficking of newly synthesized proteins to the PM is unaffected. The short-term protein trafficking inhibition and long-term effect on plant growth and survival caused by TE1 were fully reversible upon drug washout. Structure-activity relationship studies revealed that only minor modifications were possible without loss of biological activity. Diversity in Arabidopsis ecotypes was also exploited to identify two Arabidopsis accessions that display reduced sensitivity to TE1. This compound and the resistant Arabidopsis accessions may be used as a resource in future studies to better understand endomembrane trafficking in plants

A ROP GTPase-Dependent Auxin Signaling Pathway Regulates the Subcellular Distribution of PIN2 in Arabidopsis Roots

SummaryPIN-FORMED (PIN) protein-mediated auxin polar transport is critically important for development, pattern formation, and morphogenesis in plants. Auxin has been implicated in the regulation of polar auxin transport by inhibiting PIN endocytosis [1, 2], but how auxin regulates this process is poorly understood. Our genetic screen identified the Arabidopsis SPIKE1 (SPK1) gene whose loss-of-function mutations increased lateral root density and retarded gravitropic responses, as do pin2 knockout mutations [3]. SPK1 belongs to the conserved DHR2-Dock family of Rho guanine nucleotide exchange factors [4–6]. The spk1 mutations induced PIN2 internalization that was not suppressed by auxin, as did the loss-of-function mutations for Rho-like GTPase from Plants 6 (ROP6)-GTPase or its effector RIC1. Furthermore, SPK1 was required for auxin induction of ROP6 activation. Our results have established a Rho GTPase-based auxin signaling pathway that maintains PIN2 polar distribution to the plasma membrane via inhibition of its internalization in Arabidopsis roots. Our findings provide new insights into signaling mechanisms that underlie the regulation of the dynamic trafficking of PINs required for long-distance auxin transport and that link auxin signaling to PIN-mediated pattern formation and morphogenesis