Abiotic stress modulates root patterning via ABA-regulated microRNA expression in the endodermis initials.

Patterning of the root xylem into protoxylem (PX) and metaxylem is regulated by auxin-cytokinin signaling and microRNA miR165a/166b-mediated suppression of genes encoding Class III HOMEODOMAIN LEU-ZIPPER (HD-ZIPIII) proteins. We found that, in Arabidopsis, osmotic stress via core abscisic acid (ABA) signaling in meristematic endodermal cells induces differentiation of PX in radial and longitudinal axes in association with increased VND7 expression. Similarly, in tomato, ABA enhanced PX differentiation longitudinally and radially, indicating an evolutionarily conserved mechanism. ABA increased expression of miR165a/166b and reduced expression of the miR165a/166b repressor ZWILLE (ZLL) (also known as ARGONAUTE10), resulting in reduced levels of all five HD-ZIPIII RNAs. ABA treatments failed to induce additional PX files in a miR165a/166b-resistant PHB mutant, phb1-d, and in scr and shr mutants, in which miR165a/166b expression is strongly reduced. Thus, ABA regulates xylem patterning and maturation via miR165a/166b-regulated expression of HD-ZIPIII mRNAs and associated VND7 levels. In lateral root initials, ABA induced an increase in miR165a levels in endodermal precursors and inhibited their reduction in the future quiescent center specifically at pre-emergence stage. Hence, ABA-induced inhibition of lateral root is associated with reduced HD-ZIPIII levels.

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.

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.

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.

Ectopic expression of an activated RAC in Arabidopsis disrupts membrane cycling.

Rho GTPases regulate the actin cytoskeleton, exocytosis, endocytosis, and other signaling cascades. Rhos are subdivided into four subfamilies designated Rho, Racs, Cdc42, and a plant-specific group designated RACs/Rops. This research demonstrates that ectopic expression of a constitutive active Arabidopsis RAC, AtRAC10, disrupts actin cytoskeleton organization and membrane cycling. We created transgenic plants expressing either wild-type or constitutive active AtRAC10 fused to the green fluorescent protein. The activated AtRAC10 induced deformation of root hairs and leaf epidermal cells and was primarily localized in Triton X-100-insoluble fractions of the plasma membrane. Actin cytoskeleton reorganization was revealed by creating double transgenic plants expressing activated AtRAC10 and the actin marker YFP-Talin. Plants were further analyzed by membrane staining with N-[3-triethylammoniumpropyl]-4-[p-diethylaminophenylhexatrienyl] pyridinium dibromide (FM4-64) under different treatments, including the protein trafficking inhibitor brefeldin A or the actin-depolymeryzing agents latrunculin-B (Lat-B) and cytochalasin-D (CD). After drug treatments, activated AtRAC10 did not accumulate in brefeldin A compartments, but rather reduced their number and colocalized with FM4-64-labeled membranes in large intracellular vesicles. Furthermore, endocytosis was compromised in root hairs of activated AtRAC10 transgenic plants. FM4-64 was endocytosed in nontransgenic root hairs treated with the actin-stabilizing drug jasplakinolide. These findings suggest complex regulation of membrane cycling by plant RACs.

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.

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 Environment 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 (rop11(CA)) 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 rop11 (CA) suppressed ammonium toxicity, similar to auxin resistant mutants. In this Addendum article we discuss these findings and their implications.

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.

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 been 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.

Catalase expression in pancreatic alpha cells of diabetic and non-diabetic mice.

The pancreatic islet beta cells are very sensitive to oxidative stress, probably due to the extremely low level of anti-oxidant enzymes, particularly catalase. In contrast to beta cells, pancreatic alpha cells are significantly more resistant to diabetogenic toxins. However, whether alpha cells express a different level of catalase is not known. The aim of this study was to evaluate catalase expression in alpha cells of diabetic and non-diabetic mice. Diabetes was induced by a single injection of streptozotocin. After 3 weeks of persistent hyperglycemia, pancreatic tissues were collected. Catalase localization in alpha cells was identified by a dual-immunofluorescence staining with anti-glucagon and anti-catalase antibodies. In intact mice, intensive catalase and glucagon immunostaining was found in the peripheral area of islets. Merged images of glucagon and catalase show their localization in the same cell type, namely, alpha cells. Confocal microscopy indicated that the glucagon and catalase staining was distributed throughout the cytoplasm. Similar co-expression of catalase and glucagon was found in the alpha cells of diabetic animals. The results of this study show the intensive catalase expression in alpha cells of diabetic and non-diabetic mice. This knowledge may be useful to better understand the defense mechanisms of pancreatic alpha cells against oxidative stress.