Monomeric guanosine 5Ј-triphosphate (GTP)-binding proteins are involved in regulating the essential functions of eukaryotic cells, such as cell differentiation, intracellular vesicle transport, and cytoskeleton organization.1) Based on amino acid homology and deduced functions, these low molecular weight GTP-binding proteins are classified into several subfamilies.1-3) Among them, the Rac/Rop GTPase family of plants corresponds to Rho family in animal cells. 2,3)These proteins have been shown to regulate various cellular processes of plants, such as growth, hormone signaling and defense responses. 4,5) Rac/Rop GTPase genes are usually organized as a multigene family in plant cells; seven and 11 Rac/Rop genes were reported for rice and Arabidopsis thaliana, respectively. 4,5) It has been assumed that each member of the Rap/Rop subfamily is functionally distinct and plays specific roles, respectively. [6][7][8][9] It is well known 10) that jasmonic acid and its ester, methyl jasmonate (MJ), functions as the messenger molecule in various plant defense responses such as phytoalexin production and hypersensitive cell death. We previously demonstrated 11,12) that the activation of Ca 2ϩ -cascade in plant cells is an essential event to induce defensive secondary metabolism, and calmodulin (CAM), a well known Ca 2ϩ -modulator protein, functions as the key component in these processes. CAM and Rac/Rop GTPases may therefore be expected to play important roles in the defense-related signal transduction processes triggered by MJ. As in Rac/Rop GTPases, plant CAMs are encoded by more than 10 genes, and selective expression of the specific gene is sometimes induced upon contact of plant cells with various stimuli. 13,14) We showed previously 15) that several CAM-dependent processes, including biosynthesis of diterpene compounds, in Scoparia dulcis were markedly enhanced when the plant was stimulated by MJ. However, we have recently found 16) that, unlike in many other plants, CAM in S. dulcis is encoded as a copy gene in the genome, and therefore, it should be interesting to examine how this plant responds to numerous stimuli with only 1 CAM gene.The primary aim of the present study was the elucidation of the genomic organization and possible transcriptional regulation of Rac/Rop small GTPase genes as candidates for the modulation for proper responses of S. dulcis, a unique "sole CAM gene" plant. An attempt was made to isolate the Rac/Rop gene from S. dulcis, and its genomic organization was examined by Southern blot analysis. Possible change in the expression level of the Rac/Rop GTPase gene toward various stimuli was also tested. MATERIALS AND METHODSCloning of Sd-racrop from S. dulcis S. dulcis was grown in a greenhouse of the Experimental Station for Medicinal Plant Research in the University of Toyama, and total RNA was isolated from leaf tissues of the plant using the RNeasy plant mini kit (Qiagen). The RNA obtained (approximately 1 mg) was subjected to rapid amplification of cDNA ends (RACE) methods, using the GeneRace...

Translocation of two Rac/Rop guanosine 5 -triphosphate-binding proteins from Scoparia dulcis, Sdrac-1 and Sdrac-2, was examined employing transformed belladonna which overproduces these proteins as glutathione-S-transferase-tagged forms. The transferase activities of the fused proteins in microsomal fraction of belladonna markedly increased by the incubation with methyl jasmonate either in Sdrac-1 or Sdrac-2 transformant, while low and constant activities were observed in the untreated control. Recombinant Sdrac-2 protein was found to bind to prenyl chain in the presence of cell extracts prepared from methyl jasmonate-treated S. dulcis, however, Sdrac-1 was palmitoylated by the addition of the cell extracts. These results suggest that both Sdrac-1 and Sdrac-2 translocate to plant membranes by the stimulation with methyl jasmonate, however, targeting of these proteins is triggered by the independent modification mechanisms, palmitoylation for Sdrac-1 and prenylation for Sdrac-2.