Plants contain a novel unique subfamily of Rho GTPases, vital components of cellular signalling networks. Here we report a general role for some members of this family in polarized plant growth processes. We show that Arabidopsis AtRop4 and AtRop6 encode functional GTPases with similar intrinsic GTP hydrolysis rates. We localized AtRop proteins in root meristem cells to the cross-wall and cell plate membranes. Polar localization of AtRops in trichoblasts specifies the growth sites for emerging root hairs. These sites were visible before budding and elongation of the Arabidopsis root hair when AtRops accumulated at their tips. Expression of constitutively active AtRop4 and AtRop6 mutant proteins in root hairs of transgenic Arabidopsis plants abolished polarized growth and delocalized the tip-focused Ca2+ gradient. Polar localization of AtRops was inhibited by brefeldin A, but not by other drugs such as latrunculin B, cytochalasin D or caffeine. Our results demonstrate a general function of AtRop GTPases in tip growth and in polar diffuse growth.

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GTP-binding proteins are found in all organisms. They are important switches that cycle between an active and an inactive state, ensuring vectorial flow of information on the expense of guanosine triphosphate (GTP). In this review, we discuss current progress in the molecular characterization and functional analysis of plant genes encoding heterotrimeric and small GTPases. An up-to-date list including all cloned plant GTPase genes is given and a systematic classification is proposed.

SummaryIn plants, Rop/Rac GTPases have emerged as central regulators of diverse signalling pathways in plant growth and pathogen defence. When active, they interact with a wide range of downstream effectors. Using yeast two-hybrid screening we have found three previously uncharacterized receptor-like protein kinases to be Rop GTPase-interacting molecules: a cysteine-rich receptor kinase, named NCRK, and two receptor-like cytosolic kinases from the Arabidopsis RLCK-VIb family, named RBK1 and RBK2. Uniquely for Rho-family small GTPases, plant Rop GTPases were found to interact directly with the protein kinase domains. Rop4 bound NCRK preferentially in the GTP-bound conformation as determined by flow cytometric fluorescence resonance energy transfer measurements in insect cells. The kinase RBK1 did not phosphorylate Rop4 in vitro, suggesting that the protein kinases are targets for Rop signalling. Bimolecular fluorescence complementation assays demonstrated that Rop4 interacted in vivo with NCRK and RBK1 at the plant plasma membrane. In Arabidopsis protoplasts, NCRK was hyperphosphorylated and partially co-localized with the small GTPase RabF2a in endosomes. Gene expression analysis indicated that the single-copy NCRK gene was relatively upregulated in vasculature, especially in developing tracheary elements. The seven Arabidopsis RLCK-VIb genes are ubiquitously expressed in plant development, and highly so in pollen, as in case of RBK2. We show that the developmental context of RBK1 gene expression is predominantly associated with vasculature and is also locally upregulated in leaves exposed to Phytophthora infestans and Botrytis cinerea pathogens. Our data indicate the existence of cross-talk between Rop GTPases and specific receptor-like kinases through direct molecular interaction.

Domain conservation in several volvocalean cell wall proteinsWoessner, J.P.; Molendijk, A.J.; van Egmond, P.; Klis, F.M.; Goodenough, U.W.; Haring, M.A. Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: http://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. Key words: cell wall, HRGPs, domain conservation, exon shuffling, protein evolution, Chlamydomonas AbstractBased on our previous work demonstrating that (SerPro)x epitopes are common to extensin-like cell wall proteins in Chlamydomonas reinhardtii, we looked for similar proteins in the distantly related species C. eugametos. Using a polyclonal antiserum against a (SerPro)l 0 oligopeptide, we found distinct sets of stage-specific polypeptides immunoprecipitated from in vitro translations of C. eugarnetos RNA. Screening of a C. eugametos cDNA expression library with the antiserum led to the isolation of a cDNA (WP6) encoding a (SerPro)×-rich multidomain wall protein. Analysis of a similarly selected cDNA (VSP-3) from a C. reinhardtii cDNA expression library revealed that it also coded for a (SerPro)x-rich multidomain wall protein. The C-terminal rod domains of VSP-3 and WP6 are highly homologous, while the N-terminal domains are dissimilar; however, the N-terminal domain of VSP-3 is homologous to the globular domain of a cell wall protein from VoIvox carteri. Exon shuffling might be responsible for this example of domain conservation over 350 million years of volvocalean cell wall protein evolution.

The small GTPases of the Rho family play a key role in actin cytoskeletal organization. In plants, a novel Rho subfamily, called ROP (Rho of plants), has been found. In Arabidopsis, 12 ROP GTPases have been identified which differ mainly at their C-termini. To test the localization of two members of this subfamily (AtROP4 and AtROP6), we have generated translational fusions with the green fluorescent protein (GFP). Microscopic analysis of transiently transfected BY2 cells revealed a predominant localization of AtROP4 in the perinuclear region, while AtROP6 was localized almost exclusively to the plasma membrane. Swapping of the AtROP4 and AtROP6 C-termini produced a change in localization. As RhoGDIs are known to bind to the C-terminus of GTPases of the Rho family, we searched for Arabidopsis RhoGDI genes. We identified the AtRhoGDI1 gene and mapped it to chromosome 3. AtRhoGDI1 encodes a 22.5 kDa protein which contains highly conserved amino acids in the isoprene binding pocket and exhibits 29% to 37% similarity to known mammalian RhoGDI homologues. The AtRhoGDI1 gene was expressed in all tissues studied. Using the yeast two-hybrid system, we showed specific interaction of AtRhoGDI1 with both AtROP4 and AtROP6 as well as with their GTP-locked mutants, but not with a GTPase of the RAB family. Recombinant GST-AtRhoGDI1 could bind GFP-AtROP4 from transgenic tobacco BY2 cell extracts, confirming the interaction observed with the two-hybrid system.

SUMMARYBacterial protein toxins which modify Rho GTPase are useful for the analysis of Rho signalling in animal cells, but these toxins cannot be taken up by plant cells. We demonstrate in vitro deamidation of Arabidopsis Rop4 by Escherichia coli Cytotoxic Necrotizing Factor 1 (CNF1) and glucosylation by Clostridium difficile toxin B. Expression of the catalytic domain of CNF1 caused modification and activation of co-expressed Arabidopsis Rop4 GTPase in tobacco leaves, resulting in hypersensitive-like cell death. By contrast, the catalytic domain of toxin B modified and inactivated co-expressed constitutively active Rop4, blocking the hypersensitive response caused by over-expression of active Rops. In transgenic Arabidopsis, both CNF1 and toxin B inhibited Rop-dependent polar morphogenesis of leaf epidermal cells. Toxin B expression also inhibited Rop-dependent morphogenesis of root hairs and trichome branching, and resulted in root meristem enlargement and dwarf growth. Our results show that CNF1 and toxin B transgenes are effective tools in Rop GTPase signalling studies.

The cell cycle of Chlamydomonas eugmetos was synchronized at low cell densities by growing cells under conditions of alternating light and dark periods. Synchronization of the cell cycle was demonstrated by determining cell size, cytokinesis, and release of daughter cells. The microscopic data were in agreement with the accumulation patterns of mRNAs for ribulose bisphosphate carboxylase/oxygenase small subunit, histone H4 and P-tubulin. Cells of C. eugametos secrete a sticky material which enables cells to adhere to a solid substrate during cell division. The secretion of this material was also cell-cycle-regulated and preceded cytokinesis. Recently, it was reported that in synchronous exponential-phase cultures of high cell densities, young daughter cells temporarily behave like gametes during the cell cycle. However, under our growth conditions this was not observed. First, daughter cells did not agglutinate with normal gametes of the opposite mating type. Second, extraction of growing cells did not release any agglutinin activity as measured both by an in uitro agglutination assay and by an agglutinin-specific monoclonal antibody. We conclude therefore that gametogenesis in C. eugametos is not necessarily a normal phase of the cell cycle.