Gene Os01g0678500， namely OsTPC1,meaning rice two-pore channel 1,can rescue the Ca2+ uptake activity and growth rate of a yeast mutant cch1
Os01g0678500 encodes voltage-dependent calcium channel protein TPC1(OsTCP1),which functions as major voltage-gated Ca2+ channel across the plasma membrane .As cytosolic free Ca2+ serves as an important second messenger participating in signal transduction of various environmental stresses and OsTCP1 is involved in the regulation of cytosolic Ca2+ channel,the OsTCP1 plays a important role in regulating the plant growth,acts as the major ROS-responsive Ca2+ channel and is the possible target of Al-dependent inhibition.Also OsTPC1 functions as one of the key regulation factor in elicitor induced disease resistance reaction, the activation of MAPK cascade and hypersensitive cell death. By generating Ostpc1 knock-out and apoaequorin-expressing Ostpc1 cells, Haruyasu Hamada et al indicates that OsTPC1 has a role in the regulation of TvX-induced sustained increase in cytosolic Ca2+.
1.The OsTPC1overexpressors showed remarkably slowed growth and reduced fertility in adult plants[Fig-1 C,D].These phenotypes were exhibited in all six independent T1 transgenic lines and the expression level of the OsTPC1transcripts in the 35S::OsTPC1 plants well correlated with the severity of the phenotypes[Fig-1B,C]. OsTPC1 overexpressing lines showing severe phenotype also displayed a dwarf phenotype with dark green leaves in adult plants, and green colored roots in the seedling stage under light condition[Fig-1E] 
Fig-1:Phenotypes of plants overexpressing OsTPC1cDNA. (A) The OsTPC1expression vector for rice transformation. 35S P, CaMV 35S promoter; OsTPC1, ORF regions (2,274 bp in size) for OsTPC1cDNA; nos T, terminator sequence of the gene for nopaline synthase. 35S::GUSwas used as vector controls. (B) RT-PCR analysis of OsTPC1in the plants with sense OsTPC1. ActincDNA was used as a control. (C) Phenotype of overexpressing OsTPC1lines (T1) at adult stage. Plants were grown for 60 d in the greenhouse. Right: plant overexpressing OsTPC1mRNA and left: control plant overexpressing GUSgene. White bars indicate 10 cm. (D) Phenotype of overexpressing OsTPC1lines (T1) at seedling stage. Seedlings were grown for 10 d in the MS medium (16 h light/8 h darkness, 28℃). White bars indicate 10 cm. (E) Root greening observed in overexpressing OsTPC1lines (T1). Seedlings were grown for 14 d in the MS medium (continuous light, 28℃). Red bars indicate 1 mm. Control lines:C1-1 and C2-5, OsTPC1overexpressors: S2-3, S2-4 and S2-5.
2.By generating Tos17 insertional mutant of the OsTPC1[Fig-2A] and confirming the mutant OsTPC1 lose the complement function in yeast[Fig-2D] ,Takamitsu Kurusu et al shows adult plants of Ostpc1mutant showed slightly reduced growth rate under normal growth condition[Fig-3A] and less sensitivity to Ca2+ concentration[Fig-3B],These results suggest that OsTPC1 translocates substantial amount of Ca2+ across the plasmamembrane, and is one of the major factors affecting Ca2+ sensitivity at least in suspension-cultured cells.
Fig-2:Isolation of Tos17insertional mutant of the OsTPC1. (A) The structure of the OsTPC1gene and insertion site of Tos17in the OsTPC1 gene of the mutant line (NF1041). The exon and intron regions of OsTPC1are presented as closed and open bars, respectively. (B) A schematic representation of OsTPC1mRNA and the location of the deletion in the Ostpc1mutant. The approximate locations of primer sequences are shown by arrows. (C) PCR analysis of OsTPC1mRNA in Ostpc1and wild type. PCR products generated by using primers exon11P and P2, which amplify the mRNA region derived fromthe splicing of exon 11 to exon 21. (D) Yeast complementation analysis using Ostpc1cDNA. Square, K927 (cch1)/pYES2; circle, K927 (cch1)/pYES2-OsTPC1; triangle, K927 (cch1)/pYES2-Ostpc1. Vertical bars represent SE (n= 3) 
3.By performing elicitor sensitivity in both transgenic cultured cells overexpressing OsTPC1, it indicates that the elicitor-induced browning of the cells was much more evident in OsTPC1-overexpressing cells than in b-glucuronidase (GUS)-expressing control cells, and theirdarkness corresponded to theOsTPC1expression level in35S:OsTPC1 transgenic cells[Fig-5 a,b]. However in the Tos17 insertional mutant of the OsTPC1 cells,cell browning was remarkably reduced and the mitochondrial reductase activity is not induced, even at concentrations of TvX up to 120ug/ml[Fig6 a,c]. 
Expression of the OsTPC1mRNA is detected at a similar level ubiquitously in calli, mature leaves and shoots except for roots where its expression was weaker.[Fig-4] 
To study the intracellular localization of the OsTPC1 protein, Takamitsu Kurusu et al introduced the green fluorescent protein (GFP) construct fused to the N-terminus of OsTPC1 into onion epidermal cells and examined its intracellular localization by confocal laser scanning microscopy. When GFP alone was expressed, it localized to the nucleus and the cytoplasm[Fig-7 a-c],In contrast, the GFP-OsTPC1fusion protein was specifically targeted to the plasma membrane in patches (Figure-7 d–f), suggesting that OsTPC1 functions at the plasma membrane.
The OsTCP1 contains 757 amino acids and three conserved domains[From NCBI][Fig-8].It shows that OsTCP1 belongs to Ion transport protein and this family contains Sodium, Potassium, Calcium ion channels. This family is 6 transmembrane helices in which the last two helices flank a loop which determines ion selectivity. In some sub-families (e.g. Na channels) the domain is repeated four times, whereas in others (e.g. K channels) the protein forms as a tetramer in the membrane. A bacterial structure of the protein is known for the last two helices but is not the Pfam family due to it lacking the first four helices.] Also OsTCP1 contains a EF-hand which is a calcium binding motif.A diverse superfamily of calcium sensors and calcium signal modulators; most examples in this alignment model have 2 active canonical EF hands. Ca2+ binding induces a conformational change in the EF-hand motif, leading to the activation or inactivation of target proteins. EF-hands tend to occur in pairs or higher copy numbers.
Fig-8: OsTCP1'S predict domian
Labs working on this gene
- Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Tokyo, 184-8588 Japan
- Medical R & D Center, The Tokyo Metropolitan Institute of Medical Science, Honkomagome 3-18-22, Bunkyo-ku, Tokyo, 113-8613 Japan
- Department of Biology, Tokyo Gakugei University, Tokyo, 184-8501 Japan
- CREST, Japan Science and Technology Agency, Saitama, 332-0012 Japan
- Department of Applied Biological Science, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan
- Department of Molecular Genetics, National Institute of Agrobiological Sciences, Kannondai, Tsukuba, Ibaraki 305-8602,Japan
- Genome & Drug Research Center, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan
- Graduate School of Natural Science and Technology, Okayama University, Okayama,700-8530, Japan
- Biotechnology Research Center, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
- Food Science Research Laboratories, Meiji Co., Ltd., 5-3-1, Chiyoda, Sakado, Saitama 350-0289, Japan
- Environmental Biology Division, National Institute for Environmental Studies, Tsukuba, Ibaraki, 305-8506 Japan
- Division of Genome and Biodiversity Research, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan
- Kurusu T, Sakurai Y, Miyao A, Hirochika H, Kuchitsu K. Identification of a putative voltage-gated Ca2+ -permeable channel (OsTPC1) involved in Ca2+ influx and regulation of growth and development in rice. Plant Cell Physiol. 2004 Jun;45(6):693-702.
- Kurusu T, Yagala T, Miyao A, Hirochika H, Kuchitsu K. Identification of a putative voltage-gated Ca2+ channel as a key regulator of elicitor-induced hypersensitive cell death and mitogen-activated protein kinase activation in rice. Plant J. 2005 Jun;42(6):798-809.
- Hashimoto K, Saito M, Matsuoka H, Iida K, Iida H. Functional analysis of a rice putative voltage-dependent Ca2+ channel, OsTPC1, expressed in yeast cells lackingits homologous gene CCH1.Plant Cell Physiol. 2004 Apr;45(4):496-500.
- Hamada H, Kurusu T, Okuma E, Nokajima H, Kiyoduka M, Koyano T, Sugiyama Y, Okada K, Koga J, Saji H, Miyao A, Hirochika H, Yamane H, Murata Y, Kuchitsu K. Regulation of a proteinaceous elicitor-induced Ca2+ influx and production of phytoalexins by a putative voltage-gated cation channel, OsTPC1, in cultured rice cells. J Biol Chem. 2012 Mar 23;287(13):9931-9.
- Marchler-Bauer A et al. (2013), "CDD: conserved domains and protein three-dimensional structure.", Nucleic Acids Res. 41(D1):D384-52.