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Transmembrane signaling mediated by heterotrimeric GTP-binding proteins is one of the most conservative mechanisms in eukaryote cells, and these G proteins transmit the numerous external stimuli to the activation of appropriate intracellular functional proteins called effectors. 1,2) It has been well known [1][2][3] that G proteins are consisted of three subunits, a, b, g, and a subunit is dissociated from the heterotrimeric complex upon the activation by G proteincoupled receptor. The dissociation of a subunit results in the activation of several functional proteins called effector, including adenylyl cyclase, guanylyl cyclase, phosphodiesterase and phospholipase, to evoke various cellular responses. Multiple a subunits of GTP-binding proteins have been reported from mammalian cells, and these Ga subunits are appropriately classified into several subfamilies such as Gs, Gi, Gq and Go according to their target effector proteins and cell physiological functions.3)It has been demonstrated that GTP-binding proteins of higher plants also play important roles in various cellular processes including blue or red light-mediated responses, 4) plant hormone signaling, 5) regulation of ion channels 6) and pathogen-induced resistance mechanisms. 7) However, only very limited information is available about the properties of G proteins and their target effector molecules in plant cells. Several cDNAs encoding Ga subunit have been isolated from some 'model plants' such as rice, 8) potato, 9) Arabidopsis thaliana, 10) however, unlike in mammalians, the possibility has been suggested that plant G protein a subunits might be encoded by very small numbers of genes. [8][9][10] We have previously shown that GTP-binding protein(s) mediates an inducible plant defense response, phytoalexin biosynthesis, in carrot cells triggered by elicitor-active chemicals.11) In order to understand the properties and functions of a subunit of GTP-binding protein in the induced plant defense response, in the present experiments, we attempted to isolate cDNA that encodes a subunit of the mediator protein from carrot. The properties of the isolated Ga gene, such as transcriptional regulation and genomic organization, were also examined. MATERIALS AND METHODSDcga Genes from Carrot Carrot seedlings were grown in a green house of Experimental Station for Medicinal Plant Research in University of Toyama, and total RNA was isolated from the plant with RNeasy Plant Mini Kit (Qiagen). The RNA obtained (approximately 1 mg) was subjected to the rapid amplification of cDNA end (RACE) methods using the GeneRacer Kit (Invitrogen) after the generation of cDNA templates by reverse-transcription (RT) reaction with AMV-RT. The 3Ј-RACE was performed with GeneRacer Oligo dT as the reverse primer, and 5Ј-TTY ACN GTN TGG GAY GTN GGN GGN CAR GA-3Ј, 5Ј-GCC AGG ACA AGA TCC GTC CTT T-3Ј and 5Ј-GGG TAA TGA ACT CCA AGT TCC TGA CTG C-3Ј as the gene-specific forward primers for polymerase chain reaction (PCR) amplification of the DNA fragments. The 5Ј-RACE was carried out with Gen...

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Monomeric GTP-binding proteins are involved in regulating numerous essential functions of eukaryotic cells, such as cell differentiation, intracellular vesicle transport, and cytoskeleton organization. [1][2][3][4][5] Based on the amino acid homology and the deduced functions, the low molecular weight GTP-binding proteins are generally classified into several families. 2,5) It has been well known 3,4) that activation of these small GTP-binding proteins is strictly controlled by the two regulatory proteins, GDP/GTP exchanging factor (GEF) and the GTPase-activating protein (GAP). GEP interacts with inactive small G-proteins to covert them to GTP-bound active form, while small G-protein-GTP complex is changed to the inactive form by the hydrolysis of GTP to GDP by the action of GTPase-activating protein (GAP). ADP-ribosylation factor (ARF) is a member of the Arf/Sar family of small G-proteins, 1,4) and several lines of evidence suggest that these proteins function in vesicular transport from endoplasmic reticulum to the plasma membrane via the Golgi apparatus.6,7) As well as other small G-proteins, the functions of this class of are regulated by ARF-specific GEF and GAP. It has been demonstrated 8) that over-expression of ARF-specific GEF restores the inhibitor-induced defect of secretory activity. On the other hand, it has been recently shown 9) that over-expression of ARF-specific GAP appreciably inhibits the proteintrafficking processes from the endoplasmic reticulum to Golgi apparatus. Therefore, the effects of unphysiologically high concentrations of the two ARF-regulator proteins GEF and GAP have been examined in detail. In contrast, however, only very limited information is available on the biochemical properties of the transformed cells over-expressing ARF protein itself.We have reported previously 10) that genes encoding ARF proteins of carrot are organized as a small or multi-gene family in the genome, and arf-001 (GeneBank accession no. AY874441) has been isolated as a carrot ARF gene. The primary amino acid sequence of arf-001 appears to show significant homology to ARF proteins from various biological sources. In order to understand the physiological functions and effects of over-accumulation of the products of ARF genes in higher plant cells, in the present experiments, we attempted to prepare the transformed cells of Atropa belladonna over-expressing carrot arf-001. The newly developed vectors, pBCR82 and pBCR90, have been employed for over-expression of arf-001 and as the empty vector control, respectively, 11) and the transformed cells were obtained as hairy root tissues by co-expression of rol cluster in these vectors. MATERIALS AND METHODS MaterialsSeeds of A. belladonna L. were surface-sterilized in 70% (v/v) ethanol and 2% (v/v) sodium hypochlorite, successively, and, after several washings with autoclaved water, they were placed on Murashige and Skoog's agar medium 12) for germination. The sterilized seeds were then incubated at 26°C under constant illumination for germination, and the leaf se...