Cloning and Expression of Dcga Gene Encoding .ALPHA. Subunit of GTP-Binding Protein in Carrot Seedlings

Sharma

Elucidation of Abiotic Stress Signaling in Plants

2015

The sessile nature of plants has enabled them to develop a higher level of plasticity in adaptation against the hostile environmental conditions such as biotic and abiotic stresses. The intricate molecular machineries in plants, consisting of signal sensors, signal transducers, and generation of response, are highly similar with animal's signal transduction components; however, a large number of pathways also show a high level of divergence. G proteins are a part of signal transduction components, which play pivotal role in regulating a large number of physiological and developmental processes in both plants and animals. G proteins or guanine nucleotide-binding proteins serve as molecular switches, existing in two forms: an active conformation, when bound to GTP, and an inactive conformation, when bound to GDP. In active conformation, G proteins interact with downstream effector molecules to initiate various cellular responses. Based on their structure, they have been divided into heterotrimeric and monomeric G proteins, also called large and small G proteins, respectively, where the former is composed of three subunits (α, β, and γ) and latter is composed of single subunit (α). Plant growth and development is partially regulated by the combined activity of large and small G proteins. Besides, identifi cation of profuse number of stress-related genes interacting with monomeric and heterotrimeric G-protein complex in both Arabidopsis and rice suggests their involvement in modulation of abiotic stress responses. Further, based on the functional analysis of several G proteins (large and small) regulating cell growth, cell proliferation, seed germination, sugar sensing, and hormonal responses indicate these proteins are equally essential for plant development. Due to increased demand of food crops to sustain growing population in adverse environmental conditions, the role of G proteins and modulation of GTPase signaling has been studied in plants to produce genetically modifi ed crop plants with increased stress tolerance and productivity.

Section: Abiotic Stressmentioning

confidence: 84%

Small and Large G Proteins in Biotic and Abiotic Stress Responses in Plants

Sharma

Elucidation of Abiotic Stress Signaling in Plants

2015

Biological & Pharmaceutical Bulletin

“…11,15,21) The deduced amino acid sequence of Sdga translate was further compared with other Ga-proteins from various biological sources by molecular phylogenetic tree analysis ( Fig. 2A).…”

Section: Resultsmentioning

confidence: 99%

“…10) This structural property of putative ADP-ribosylation sites, presence of cholera toxinbinding site and absence of pertussis toxin-binding site, is also observed in Ga-subunits from various plant sources. 11,15,21) The deduced amino acid sequence of Sdga translate was further compared with other Ga-proteins from various biological sources by molecular phylogenetic tree analysis ( Fig. 2A).…”

Section: Resultsmentioning

confidence: 99%

“…8) It is well known that a-subunits of G-protein complex in mammalian cells are generally composed of more than 20 isoforms 9,10) ; however, in contrast, it has been demonstrated that Ga proteins in plants are usually encoded by only one or two genes in the genomes. [11][12][13][14][15] We showed previously 16) that the biosynthetic activity of a series of tetracyclic diterpenoids, such as scopadulcic acid, scopadulciol, and scopadulin, in Scoparia dulcis is markedly increased when the plant was treated with MJ. We have also demonstrated that the activation of Ca-cascade in S. dulcis is an essential event for MJ-induced enhancement of the diterpenoids production, and calmodulin (CAM), a well known Ca-modulator protein of eukaryote cells, functions as a key component in the elevation of expression levels of several genes encoding biosynthetic enzymes of theses compounds.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Cloning and Characterization of Sdga Gene Encoding .ALPHA.-Subunit of Heterotrimeric Guanosine 5'-Triphosphate-Binding Protein Complex in Scoparia dulcis

Shite

Yamamura

Hayashi

et al. 2008

Self Cite

A homology-based cloning strategy yielded Sdga, a cDNA clone presumably encoding a a-subunit of heterotrimeric guanosine 5Ј-triphosphate-binding protein complex, from leaf tissues of Scoparia dulcis. Phylogenetic tree analysis of G-protein a a-subunits from various biological sources suggested that, unlike in animal cells, classification of Ga a-proteins into specific subfamilies could not be applicable to the proteins from higher plants. Restriction digests of genomic DNA of S. dulcis showed a single hybridized signal in Southern blot analysis, suggesting that Sdga is a sole gene encoding Ga a-subunit in this plant. The expression level of Sdga appeared to be maintained at almost constant level after exposure of the leaves to methyl jasmonate as analyzed by reverse-transcription polymerase chain reaction. These results suggest that Sdga plays roles in methyl jasmonate-induced responses of S. dulcis without a notable change in the transcriptional level.Key words a-subunit; guanosine 5Ј-triphosphate-binding protein; heterotrimer complex; Scoparia dulcis Biol. Pharm. Bull. 31(11) 2150-2153 (2008) © 2008 Pharmaceutical Society of Japan * To whom correspondence should be addressed. e-mail: kurosaki@pha.u-toyama.ac.jp (G-Biosciences) according to the instruction manuals, and the restriction digests were prepared using XbaI, XhoI, EcoRI, or EcoRV (Takara). They were electrophoresed on a 0.8% agarose gel, and the separated DNA fragments were transferred onto an Immobilon-NY ϩ (Millipore). The translatable region of Sdga including stop codon (1152 nucleotides) was amplified by PCR and was directly labeled with AlkPhos Direct Labeling and Detection System (GE Healthcare Bio-Science) to be used as the probe. The membrane was hybridized with the probe at 55°C for 24 h in a solution containing 6ϫSSC, and, after appropriate washings, the filters were dried and exposed to an X-ray film at room temperature for 3 h. Expression of Sdga GeneThe expression level of Sdga was analyzed semi-quantitatively using RT-PCR. The leaves of S. dulcis were detached from the middle part of the stem, and incubated with MJ (100 mmol l Ϫ1 water, Wako Pure Chemicals) in Petri dishes at 26°C under constant illumination according to the method described previously. 20) Control treatments received only water instead of MJ. At regular intervals, the treated leaves were harvested and total RNA was isolated according to the method described above. Aliquots of RNA solutions (approximately 0.5 mg RNA equivalent) were subjected to RT reaction using Transcriptor First Strand cDNA Synthesis Kit (Roche), and then, PCR was carried out with the primer pair, 5Ј-ATG CGG TGG ATG CTA ATC November 2008 2151 Fig. 1. Alignment of Amino Acids Sequences of a-Subunits of GTP-Binding Protein Complex from Various Biological SourcesA cDNA clone encoding the a-subunit of GTP-binding protein, Sdga, was isolated from S. dulcis, and the deduced amino acid sequence was compared with the proteins from various biological sources (Arab, Arabidopsis thaliana; Rice, Oryza sativa...

Biological & Pharmaceutical Bulletin

“…16,17) Control treatments received only water instead of these chemicals. In some experiments, leaf tissues were incubated at 42°C or in 400 mM NaCl solution.…”

mentioning

confidence: 99%

Cloning and Characterization of a Gene Encoding Rac/Rop-Like Monomeric Guanosine 5'-Triphosphate-Binding Protein from Scoparia dulcis

Mitamura

Shite

Yamamura

et al. 2009

Self Cite

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...