Daria Bloch

A Novel ROP/RAC Effector Links Cell Polarity, Root-Meristem Maintenance, and Vesicle Trafficking

ROP/RAC GTPases are master regulators of cell polarity in plants, implicated in the regulation of diverse signaling cascades including cytoskeleton organization, vesicle trafficking, and Ca(2+) gradients [1-8]. The involvement of ROPs in differentiation processes is yet unknown. Here we show the identification of a novel ROP/RAC effector, designated interactor of constitutive active ROPs 1 (ICR1), that interacts with GTP-bound ROPs. ICR1 knockdown or silencing leads to cell deformation and loss of root stem-cell population. Ectopic expression of ICR1 phenocopies activated ROPs, inducing cell deformation of leaf-epidermis-pavement and root-hair cells [3, 5, 6, 9]. ICR1 is comprised of coiled-coil domains and forms complexes with itself and the exocyst vesicle-tethering complex subunit SEC3 [10-13]. The ICR1-SEC3 complexes can interact with ROPs in vivo. Plants overexpressing a ROP- and SEC3-noninteracting ICR1 mutant have a wild-type phenotype. Taken together, our results show that ICR1 is a scaffold-mediating formation of protein complexes that are required for cell polarity, linking ROP/RAC GTPases with vesicle trafficking and differentiation.

Regulation of membrane trafficking, cytoskeleton dynamics, and cell polarity by ROP/RAC GTPases.

Rho of plants (ROP) proteins, also known as RAC proteins, are Rho-related GTPases that function as molecular switches in a multitude of signaling cascades involved in the regulation of the actin and microtubule cytoskeleton, of vesicle trafficking, and of plant responses to hormones, stresses, or

A Rho Scaffold Integrates the Secretory System with Feedback Mechanisms in Regulation of Auxin Distribution

In plants, auxin distribution and tissue patterning are coordinated via a feedback loop involving the auxin-regulated cell polarity factor ICR1 and the secretory machinery.

Protein lipid modifications in signaling and subcellular targeting

Classically perceived as means for recruiting proteins to the membranes, protein lipid modifications are known today to play diverse roles in subcellular targeting, protein-protein interactions and signaling. This review focuses on three protein lipid modifications: prenylation, S-acylation and N-myristoylation and attempts to provide an up-to-date view of their function by focusing on several model proteins.

Nitrogen source interacts with ROP signalling in root hair tip‐growth

Root hairs elongate in a highly polarized manner known as tip growth. Overexpression of constitutively active Rho of Plant (ROP)/RAC GTPases mutants induces swelling of root hairs. Here, we demonstrate that Atrop11(CA)-induced swelling of root hairs depends on the composition of the growth medium. Depletion of ammonium allowed normal root hair elongation in Atrop11(CA) plants, induced the development of longer root hairs in wild-type plants and suppressed the effect of Atrop11(CA) expression on actin organization and reactive oxygen species distribution, whereas membrane localization of the protein was not affected. Ammonium at concentrations higher than 1 mM and the presence of nitrate were required for induction of swelling. Oscillations in wall and cytoplasmic pH are known to accompany tip growth in root hairs, and buffering of the growth medium decreased Atrop11(CA)-induced swelling. Fluorescence ratio imaging experiments revealed that in wild-type root hairs, the addition of NH₄NO₃ to the growth medium induced an increase in the amplitude of extracellular and intracellular pH oscillations and an overall decrease in cytoplasmic pH at the cell apex. Based on these results, we suggest a model in which ROP GTPases and nitrogen-dependent pH oscillations function in parallel pathways, creating a positive feedback loop during root hair growth.

Cell polarity signaling

Cell polarity is a fundamental entity of living organisms. Cells must receive accurate decisions where to divide and along which plane, along which axis to grow, where to grow structures like flagellum or filopodium and how to differentially respond to external stimuli. In multicellular organisms cell polarity also regulates cell-cell communication, pattern formation and cell identity. In eukaryotes the RHO family of small G proteins have emerged as central regulators of cell polarity signaling. It is by now well established that ROPs, the plant specific RHO subfamily members, affect cell polarization. Work carried out over the last several years is beginning to reveal how ROPs are activated, how their activity is spatially regulated, through which effectors they regulate cell polarity and how they interact with hormonal signaling and other polarity determinants. The emerging picture is that while the mechanisms of cell polarity signaling are often unique to plants, the principles that govern cell polarization signaling can be similar. In this review, we provide an updated view of polarity signaling in plants, primarily focusing on the function of ROPs and how they interact with and coordinate different polarity determinants.

Exocyst SEC3 and Phosphoinositides Define Sites of Exocytosis in Pollen Tube Initiation and Growth

The SEC3 exocyst subunit serves as a landmark for secretory vesicles, and its localization and differential regulation by membrane lipids determine pollen tube initiation and polar growth. Polarized exocytosis is critical for pollen tube growth, but its localization and function are still under debate. The exocyst vesicle-tethering complex functions in polarized exocytosis. Here, we show that a sec3a exocyst subunit null mutant cannot be transmitted through the male gametophyte due to a defect in pollen tube growth. The green fluorescent protein (GFP)-SEC3a fusion protein is functional and accumulates at or proximal to the pollen tube tip plasma membrane. Partial complementation of sec3a resulted in the development of pollen with multiple tips, indicating that SEC3 is required to determine the site of pollen germination pore formation. Time-lapse imaging demonstrated that SEC3a and SEC8 were highly dynamic and that SEC3a localization on the apical plasma membrane predicts the direction of growth. At the tip, polar SEC3a domains coincided with cell wall deposition. Labeling of GFP-SEC3a-expressing pollen with the endocytic marker FM4-64 revealed the presence of subdomains on the apical membrane characterized by extensive exocytosis. In steady-state growing tobacco (Nicotiana tabacum) pollen tubes, SEC3a displayed amino-terminal Pleckstrin homology-like domain (SEC3a-N)-dependent subapical membrane localization. In agreement, SEC3a-N interacted with phosphoinositides in vitro and colocalized with a phosphatidylinositol 4,5-bisphosphate (PIP2) marker in pollen tubes. Correspondingly, molecular dynamics simulations indicated that SEC3a-N associates with the membrane by interacting with PIP2. However, the interaction with PIP2 is not required for polar localization and the function of SEC3a in Arabidopsis (Arabidopsis thaliana). Taken together, our findings indicate that SEC3a is a critical determinant of polar exocytosis during tip growth and suggest differential regulation of the exocytotic machinery depending on pollen tube growth modes.

Co-regulation of root hair tip growth by ROP GTPases and nitrogen source modulated pH fluctuations

Growth of plant cells involves tight regulation of the cytoskeleton and vesicle trafficking by processes including the action of the ROP small G proteins together with pH-modulated cell wall modifications. Yet, little is known on how these systems are coordinated. In a paper recently published in Plant Cell and Environment1 we show that ROPs/RACs function synergistically with NH4NO3-modulated pH fluctuations to regulate root hair growth. Root hairs expand exclusively at their apical end in a strictly polarized manner by a process known as tip growth. The highly polarized secretion at the apex is maintained by a complex network of factors including the spatial organization of the actin cytoskeleton, tip-focused ion gradients and by small G proteins. Expression of constitutively active ROP mutants disrupts polar growth, inducing the formation of swollen root hairs. Root hairs are also known to elongate in an oscillating manner, which is correlated with oscillatory H+ fluxes at the tip. Our analysis shows that root hair elongation in wild type plants and swelling in transgenic plants expressing a constitutively active ROP11 (rop11CA) is sensitive to the presence of NH4+ at concentrations higher than 1 mM and on NO3-. The NH4+ and NO3- ions did not affect the localization of ROP in the membrane but modulated pH fluctuations at the root hair tip. Actin organization and reactive oxygen species distribution were abnormal in rop11CA root hairs but were similar to wild type root hairs when seedlings were grown on medium lacking NH4+ and / or NO3-. These observations suggest that the nitrogen source-modulated pH fluctuations may function synergistically with ROP regulated signaling during root hair tip growth. Interestingly, under certain growth conditions, expression of rop11CA suppressed ammonium toxicity, similar to auxin resistant mutants. In this Addendum article we discuss these findings and their implications.

A novel ROP/RAC GTPase effector integrates plant cell form and pattern formation

ROPs/RACs are the only known signaling Ras superfamily small GTPases in plants. As such they have been suggested to function as central regulators of diverse signaling cascades. The ROP/RAC signaling networks are largely unknown, however, because only few of their effector proteins have identified. In a paper that was published in the June 5, 2007 issue of Current Biology we described the identification of a novel ROP/RAC effector designated ICR1 (Interactor of Constitutive active ROPs 1). We demonstrated that ICR1 functions as a scaffold that interacts with diverse but specific group of proteins including SEC3 subunit of the exocyst vesicle tethering complex. ICR1-SEC3 complexes can interact with ROPs in vivo and are thereby recruited to the plasma membrane. ICR1 knockdown or silencing leads to cell deformation and loss of the root stem cells population and ectopic expression of ICR1 phenocopies activated ROPs/RACs. ICR1 presents a new paradigm in ROP/RAC signaling and integrates mechanisms regulating cell form and pattern formation at the whole plant level.