Genetic, molecular and functional studies of RAC GTPases and the WAVE-like regulatory protein complex in Arabidopsis thaliana.

Abstract

Small GTP-binding proteins are molecular switches that serve as important regulators of numerous cellular processes. In animal and plant cells, the Rho family of small GTPases participate in e.g. organisation of the actin cytoskeleton, production of reactive oxygen species through the NADPH oxidase complex, regulation of gene expression. The three most extensively studied subgroups of the Rho GTPase family are Cdc42, Rho and Rac. One of the mechanisms by which animal Rac and Cdc42 GTPases regulate actin filament organisation is through activation of the ARP2/3 complex, a multimeric protein complex which induces branching and nucleation/elongation/polymerisation of actin filaments. Activation of the ARP2/3 complex by Rac and Cdc42 is mediated through the proteins WAVE and WASP, respectively.

In a search for Ras-like GTPases in Arabidopsis, we identified a family of genes with similarity to Rac GTPases. Screens of cDNA and genomic libraries resulted in the finding of 11 genes named ARACs/AtRACs. Genes encoding Rho, Cdc42 or Ras homologues were not identified. Expression analysis of AtRAC1 to AtRAC5 indicated that AtRAC1, AtRAC3, AtRAC4 and AtRAC5 are expressed in all parts of the plant, whereas AtRAC2 is preferentially expressed in root, hypocotyl and stem.

The AtRAC gene family can be divided into two main groups based on sequence similarity, gene structure and post-translational modification. AtRAC group II genes contain an additional exon, caused by the insertion of an intron which disrupts the C-terminal geranylgeranylation motif. Instead, group II AtRACs contain a putative motif for palmitoylation. Phylogenetic analyses indicated that the division of plant RACs into group I and group II occurred before the split of monocotyledonous and dicotyledonous plants. Analyses of the genes neighbouring AtRAC genes revealed that several of the plant RAC genes have been created through duplications.

The restricted/tissue-specific expression pattern of AtRAC2 led us to do a more detailed expression analysis of this gene. A 1.3 kb fragment of the upstream (regulatory) sequence of AtRAC2 directed expression of GUS or GFP to developing primary xylem in root, hypocotyl, leaves and stem. In root tips, the onset GUS staining or GFP fluorescence regulated by the AtRAC2 promoter slighty preceded the appearance of secondary cell walls. In stems, GUS staining coincided with thickening of xylem cell walls. Transgenic plants expressing constitutively active AtRAC2 displayed defects in the polar growth of leaf epidermal cells, indicating that AtRAC2 may be able to regulate the actin cytoskeleton. Surprisingly, an AtRAC2 T-DNA insertion mutant did not show any observable phenotypes. GFP fusion proteins of wild type and constitutively active AtRAC2 were both localised to the plasma membrane. The data suggest that AtRAC2 is involved in development of xylem vessels, likely through regulation of the actin cytoskeleton or NADPH oxidase.

The role of RAC GTPases in regulation of the actin cytoskeleton in plants is well documented. However, although the ARP2/3 complex had been identified in plants/Arabidopsis, the mechanisms regulating this complex were unknown. Through database searches, we identified three Arabidopsis genes, AtBRK1, AtNAP and AtPIR, which encoded proteins with similarity to subunits of a protein complex shown to regulate the activity of WAVE1 in mammalian cells. T-DNA inactivation mutants of AtNAP and AtPIR displayed morphological defects on epidermal cells undergoing polar expansion, such as trichomes and leaf pavement cells. The phenotypes were similar to those observed for ARP2/3 complex mutants, suggesting that AtNAP and AtPIR act in the same pathway as the ARP2/3 complex in plants. The actin cytoskeleton in atnap and atpir mutants was less branched than in wild type plants; instead, actin filaments aggregated in thick actin bundles.

Finally, we have recently discovered a small gene family encoding putative WAVE homologues. In mammalian cells, Rac activates WAVE1 through binding to PIR121 or Sra1 (the mammalian homologues of AtPIR). The discovery of a putative WAVE regulatory complex as well as putative WAVE homologues in Arabidopsis suggests that plant RAC GTPases regulate organisation of the actin cytoskeleton during polar growth at least partly through the ARP2/3 complex, using an evolutionarily conserved mechanism.