Microtubule-associated ROP interactors affect microtubule dynamics and modulate cell wall patterning and root hair growth.

Rho of plant (ROP) proteins and the interactor of constitutively active ROP (ICR) family member ICR5/MIDD1 have been implicated to function as signaling modules that regulate metaxylem secondary cell wall patterning. Yet, loss-of-function mutants of ICR5 and its closest homologs have not been studied and, hence, the functions of these ICR family members are not fully established. Here, we studied the functions of ICR2 and its homolog ICR5. We show that ICR2 is a microtubule-associated protein that affects microtubule dynamics. Secondary cell wall pits in the metaxylem of Arabidopsis icr2 and icr5 single mutants and icr2 icr5 double mutants are smaller than those in wild-type Col-0 seedlings; however, they are remarkably denser, implying a complex function of ICRs in secondary cell wall patterning. ICR5 has a unique function in protoxylem secondary cell wall patterning, whereas icr2, but not icr5, mutants develop split root hairs, demonstrating functional diversification. Taken together, our results show that ICR2 and ICR5 have unique and cooperative functions as microtubule-associated proteins and as ROP effectors.

Formation of self-organizing functionally distinct Rho of plants domains involves a reduced mobile population.

Rho family proteins are central to the regulation of cell polarity in eukaryotes. Rho of Plants-Guanyl nucleotide Exchange Factor (ROPGEF) can form self-organizing polar domains following co-expression with an Rho of Plants (ROP) and an ROP GTPase-Activating Protein (ROPGAP). Localization of ROPs in these domains has not been demonstrated, and the mechanisms underlying domain formation and function are not well understood. Here we show that six different ROPs form self-organizing domains when co-expressed with ROPGEF3 and GAP1 in Nicotiana benthamiana or Arabidopsis (Arabidopsis thaliana). Domain formation was associated with ROP-ROPGEF3 association, reduced ROP mobility, as revealed by time-lapse imaging and Fluorescence Recovery After Photobleaching beam size analysis, and was independent of Rho GTP Dissociation Inhibitor mediated recycling. The domain formation depended on the ROPs' activation/inactivation cycles and interaction with anionic lipids via a C-terminal polybasic domain. Coexpression with the microtubule-associated protein ROP effector INTERACTOR OF CONSTITUTIVELY ACTIVE ROP 1 (ICR1) revealed differential function of the ROP domains in the ability to recruit ICR1. Taken together, the results reveal mechanisms underlying self-organizing ROP domain formation and function.

A novel Ca2+-binding protein that can rapidly transduce auxin responses during root growth.

Signaling cross talks between auxin, a regulator of plant development, and Ca2+, a universal second messenger, have been proposed to modulate developmental plasticity in plants. However, the underlying molecular mechanisms are largely unknown. Here, we report that in Arabidopsis roots, auxin elicits specific Ca2+ signaling patterns that spatially coincide with the expression pattern of auxin-regulated genes. We have identified the single EF-hand Ca2+-binding protein Ca2+-dependent modulator of ICR1 (CMI1) as an interactor of the Rho of plants (ROP) effector interactor of constitutively active ROP (ICR1). CMI1 expression is directly up-regulated by auxin, whereas the loss of function of CMI1 associates with the repression of auxin-induced Ca2+ increases in the lateral root cap and vasculature, indicating that CMI1 represses early auxin responses. In agreement, cmi1 mutants display an increased auxin response including shorter primary roots, longer root hairs, longer hypocotyls, and altered lateral root formation. Binding to ICR1 affects subcellular localization of CMI1 and its function. The interaction between CMI1 and ICR1 is Ca2+-dependent and involves a conserved hydrophobic pocket in CMI1 and calmodulin binding-like domain in ICR1. Remarkably, CMI1 is monomeric in solution and in vitro changes its secondary structure at cellular resting Ca2+ concentrations ranging between 10-9 and 10-8 M. Hence, CMI1 is a Ca2+-dependent transducer of auxin-regulated gene expression, which can function in a cell-specific fashion at steady-state as well as at elevated cellular Ca2+ levels to regulate auxin responses.

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

A rho scaffold integrates the secretory system with feedback mechanisms in regulation of auxin distribution.

Development in multicellular organisms depends on the ability of individual cells to coordinate their behavior by means of small signaling molecules to form correctly patterned tissues. In plants, a unique mechanism of directional transport of the signaling molecule auxin between cells connects cell polarity and tissue patterning and thus is required for many aspects of plant development. Direction of auxin flow is determined by polar subcellular localization of PIN auxin efflux transporters. Dynamic PIN polar localization results from the constitutive endocytic cycling to and from the plasma membrane, but it is not well understood how this mechanism connects to regulators of cell polarity. The Rho family small GTPases ROPs/RACs are master regulators of cell polarity, however their role in regulating polar protein trafficking and polar auxin transport has not been established. Here, by analysis of mutants and transgenic plants, we show that the ROP interactor and polarity regulator scaffold protein ICR1 is required for recruitment of PIN proteins to the polar domains at the plasma membrane. icr1 mutant embryos and plants display an a array of severe developmental aberrations that are caused by compromised differential auxin distribution. ICR1 functions at the plasma membrane where it is required for exocytosis but does not recycle together with PINs. ICR1 expression is quickly induced by auxin but is suppressed at the positions of stable auxin maxima in the hypophysis and later in the embryonic and mature root meristems. Our results imply that ICR1 is part of an auxin regulated positive feedback loop realized by a unique integration of auxin-dependent transcriptional regulation into ROP-mediated modulation of cell polarity. Thus, ICR1 forms an auxin-modulated link between cell polarity, exocytosis, and auxin transport-dependent tissue patterning.

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.

Activation status-coupled transient S acylation determines membrane partitioning of a plant Rho-related GTPase.

ROPs or RACs are plant Rho-related GTPases implicated in the regulation of a multitude of signaling pathways that function at the plasma membrane by virtue of posttranslational lipid modifications. The relationship between ROP activation status and membrane localization has not been established. Here we demonstrate that endogenous ROPs, as well as a transgenic His(6)-green fluorescent protein (GFP)-AtROP6 fusion protein, were partitioned between Triton X-100-soluble and -insoluble membranes. In contrast, an activated His(6)-GFP-Atrop6(CA) mutant protein accumulated exclusively in detergent-resistant membranes. GDP induced accumulation of ROPs in Triton-soluble membranes, whereas GTPgammaS induced accumulation of ROPs in detergent-resistant membranes. Recombinant wild-type and constitutively active AtROP6 isoforms were purified from Arabidopsis plants, and their lipids were cleaved and analyzed by gas chromatography-coupled mass spectrometry. In Triton-soluble membranes, wild-type AtROP6 was only prenylated, primarily by geranylgeranyl. The activated AtROP6 that accumulated in detergent-resistant membranes was modified by prenyl and acyl lipids. The acyl lipids were identified as palmitic and stearic acids. In agreement, activated His(6)-GFP-Atrop6(CA)mS(156) in which cysteine(156) was mutated into serine accumulated in Triton-soluble membranes. These findings show that upon GTP binding and activation, AtROP6 and possibly other ROPs are transiently S acylated, which induces their partitioning into detergent-resistant membranes.

Corrected and Republished from: Activation Status-Coupled Transient S-Acylation Determines Membrane Partitioning of a Plant Rho-Related GTPase.

ROPs or RACs are plant Rho-related GTPases implicated in the regulation of a multitude of signaling pathways that function at the plasma membrane via posttranslational lipid modifications. The relationships between ROP activation status and membrane localization has not been established. Here, we show that endogenous ROPs, as well as a transgenic His6-green fluorescent protein (GFP)-Arabidopsis thaliana ROP6 (AtROP6) fusion protein, were partitioned between Triton X-100-soluble and -insoluble membranes. In contrast, the His6-GFP-Atrop6CA activated mutant accumulated exclusively in detergent-resistant membranes. GDP induced accumulation of ROPs in Triton-soluble membranes, whereas GTPγS induced accumulation of ROPs in detergent-resistant membranes. Recombinant wild-type and constitutively active AtROP6 proteins were purified from Arabidopsis plants, and in turn, their lipids were cleaved and analyzed by gas chromatography-coupled mass spectrometry. In Triton-soluble membranes, the wild-type AtROP6 was only prenylated, primarily by geranylgeranyl. The activated AtROP6 that accumulated in detergent-resistant membranes was modified by prenyl and acyl lipids, identified as palmitic and stearic acids. Consistently, activated His6-GFP-Atrop6CAmS156, in which C156 was mutated into serine, accumulated in Triton-soluble membranes. These findings show that upon GTP binding and activation, AtROP6, and possibly other ROPs, are transiently S-acylated, inducing their partitioning into detergent-resistant membranes.

A cell-specific, prenylation-independent mechanism regulates targeting of type II RACs.

The RHO proteins, which regulate numerous signaling cascades, undergo prenylation, facilitating their interaction with membranes and with proteins called RHO.GDP dissociation inhibitors. It has been suggested that prenylation is required for RHO function. Eleven RHO-related proteins were identified in Arabidopsis. Eight of them are putatively prenylated. We show that targeting of the remaining three proteins, AtRAC7, AtRAC8, and AtRAC10, is prenylation independent, requires palmitoylation, and occurs by a cell-specific mechanism. AtRAC8 and AtRAC10 could not be prenylated by either farnesyltransferase or geranylgeranyltransferase I, whereas AtRAC7 could be prenylated by both enzymes in yeast. The association of AtRAC7 with the plasma membrane in plants did not require farnesyltransferase or a functional CaaX box. Recombinant AtRAC8 was palmitoylated in vitro, and inhibition of protein palmitoylation relieved the association of all three proteins with the plasma membrane. Interestingly, AtRAC8 and a constitutively active mutant, Atrac7mV(15), were not associated with the plasma membrane in root hair cells, whose elongation requires the localization of prenylated RHOs in the plasma membrane at the cell tip. Moreover, Atrac7mV(15) did not induce root hair deformation, unlike its prenylated homologs. Thus, AtRAC7, AtRAC8, and AtRAC10 may represent a group of proteins that have evolved to fulfill unique functions.

Enlarged meristems and delayed growth in plp mutants result from lack of CaaX prenyltransferases.

Meristems require a myriad of intercellular signaling pathways for coordination of cell division within and between functional zones and clonal cell layers. This control of cell division ensures a constant availability of stem cells throughout the life span of the meristem while limiting overproliferation of meristematic cells and maintaining the meristem structure. We have undertaken a genetic screen to identify additional components of meristem signaling pathways. We identified pluripetala (plp) mutants based on their dramatically larger meristems and increased floral organ number. PLURIPETALA encodes the alpha-subunit shared between protein farnesyltransferase and protein geranylgeranyltransferase-I. plp mutants also have altered abscisic acid responses and overall much slower growth rate. plp is epistatic to mutations in the beta-subunit of farnesyltransferase and shows a synergistic interaction with clavata3 mutants. plp mutants lead to insights into the mechanism of meristem homeostasis and provide a unique in vivo system for studying the functional role of prenylation in eukaryotes.