In-silico analysis of Camelina sativa triacylglycerol pathway gene, CsPDCT1
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Saeedeh Alidoust,1,*
1. University of Mohaghegh Ardabili
- Introduction: Camelina sativa is an oil seed that can grow in a variety of climatic and soil conditions and requires less water, fertilizer, and pesticides than other oil seeds. It can be a new source for fuels and edible oils (Moser, 2010; Budin et al., 1995; Zubr, 1997). Camelina oil is rich in polyunsaturated fatty acids (PUFA) (approximately 50%) and contains linoleic acid (18: 2) and linolenic acid (18: 3 ω3) in its oil composition (Eidhin et al., 2003). These unsaturated fatty acids are a source of unhealthy trans fats in the processing of many foods (Zimmermann et al. 2004; Dahlqvist et al., 2000; Cases et al., 2001). In oil seed cells, 18: 1 is unsaturated by 2 desaturase enzymes from the endoplasmic reticulum, FAD2 and FAD3, to 18: 2 and 18: 3 (Arondel et al., 1992; Okuley et al., 1994). Prior to unsaturation, 18: 1 must be added to phosphatidylcholine (PC), the only substrate known for the FAD2 and FAD3 desaturases (Stymne et al., 1978). Phosphatidylcholine: diacyl glycerol choline phosphotransferase enzyme (PDCT) is encoded by the ROD1 gene (At3g15820) in Arabidopsis and catalyzes transfer of the phosphocholine headgroup from PC to diacylglycerol, and mutation of rod1 reduces 18:2 and 18:3 accumulation in seed TAG by 40%. (Lu et al., 2009). In this study we have analyzed Camelina sativa genome to find homologues of AtROD1, which may have a crucial role on the component and quality of Camelina sativa oil and contribute to the control of poly unsaturated fatty acid synthesis in seeds.
- Methods: A blast search was performed in the NCBI database using BLASTp to find protein sequences similar to AtROD1 (gene accession number At3g15820). Multiple sequence alignment of AtROD1 protein sequence and CsPDCT1 isoforms was performed using MUSLE program. The evolutionary analyses of selected sequences were conducted in MEGA7. Phylogenetic tree was generated on MEGA7 with maximum likelihood method. Using ROD1 Arabidopsis gene ID (At3g15820), information was obtained about the Camelina sativa PDCT1 gene expression from eFP (bar.utoronto.ca/efp_camelina/cgi-bin/efpWeb.cgi). The expression pattern of AtROD1 gene was investigated using microarray data available at Genevestigator (www.genevestigator.com). Conserved domains, regulatory elements, isoelectric points (Pi) and molecular weight (Mw) of PDCT1 were also evaluated
- Results: By homology searches for protein sequences similar to Arabidopsis ROD1 at the NCBI database, three gene loci with relatively high homology to AtROD1 were identified on 3 different chromosomes of C. sativa. The first identified sequence with 86% amino acid similarity to AtROD1, called phosphatidylcholine-like diacyl phosphotransferase 1, is located on chromosome 19 of Camelina. This protein was abbreviated as CsPDCT1_1. The two other isoforms of this protein, CsPDCT1_2 and CsPDCT1_3 were detected on chromosome 1 and 15 of Camelina respectively. Identification of three copies of the CsPDCT1 gene in C. sativa is consistent with the results of previous research on the Camelina plant. The C. sativa genome appears to be organized into three distinct versions (Hutcheon et al., 2010). PAP2_3 conserved domains of the PAP2 superfamily, the AtROD1 transmembrane conserved region and signal peptide are conserved domains found in CsPDCT1 sequences as well.
Phylogenetic studies show a close relationship of ROD1 with phosphatidylcholine: ceramide choline phosphotransferases SMS1 and SMS2 from the LPT family in Homo sapiens. These enzymes catalyze the transfer of the phosphocoline head group from the phosphatidylcholine to the alcohol ceramide head group. SMS1 and SMS2 function as bidirectional lipid cholinephosphotransferases capable of converting phosphatidylcholine (PC) and ceramide to sphingomyelin (SM) and diacyl glycerol (DAG) and vice versa (Huitema et al., 2004).
Analysis of the CsPDCT1 conserved domains indicates the existence of another domain of the PAP2 family called PAP2_C (Pfam profile PF14360) with a higher E-value (4.89e-03), which is located at the C terminal of the PAP2 domain. This domain is found in SMS and other proteins (Huitema et al., 2004). Phylogenetic tree analysis indicates a close relationship of three CsPDCT1 protein isoforms with their homologue in Capsella rubella. This analysis also shows that CsPDCT1 had a common ancestor with AtROD1 during a period of evolution. Using microarray data at the Genevestigator, the expression pattern of ROD1 gene at different developmental stages were evaluated. The ROD1 showed the highest expression at the stage of flowering and pod formation, and its expression decreased as the pods began to ripen. Data from the Arabidopsis gene expression atlas (Schmid et al., 2005) show that AtROD1 transcript levels are highest in seeds in which triacylglycerol accumulates. A search of the eFP site revealed that most of the changes in CsPDCT1 gene expression in Camelina are in the early to middle stages of seed development. Analysis of 1,500 bp upstream of PDCT1 genes at the PlantCare revealed that there are different cis regulatory elements in the promoter region of CsPDCT1 gene that can be classified in different groups such as stress, physiology, light and hormonal responses of the plant according to their role. Examination of gene expression under different conditions using microarray data in Genevestigator showed that ROD1 gene has the highest expression (4.5 fold) in the experiment of transferring etiolated Arabidopsis leaves from dark to light. Therefore, this gene seems to play an effective role in responding to light.
The PDCT1L promoter of Arabidopsis plants (ROD1) and Camelina sativa have specific elements responsive to the abscisic acid and gibberellin. The promoter region of the Camelina CsPDCTB isoform has ABA-responsive ABRE elements. Water loss appears to induce ABA production, which in turn induces different genes (Shinozaki and Yamaguchi-Shinozaki, 2000). DRE and MBS were other drought responsive elements found in the promoter region of CsPDCT1. Interestingly, AtROD1 lacks MBS element. Drought response elements (DRE) are involved in various types of abiotic stress responses through ABA-dependent and non-ABA-dependent pathways. Microarray studies in Genevestigator also show a reaction of AtROD1 to ABA and a 3.63-fold decrease in its expression. Other elements include BOX-W1, HSE and TC-rich repeats and other elements that play a role in adjustment in stress and defense.
- Conclusion: The study of Cis elements suggest the possible roles of CsPDCT1 proteins in biotic and abiotic stresses, especially drought stress. According to the regulatory elements in the promoter region of CsPDCT1L isoforms, these proteins seem to respond more effectively than its Arabidopsis homologue under drought stress. The existence of 3 copies of CsPDCT1 in C. sativa genome is an opportunity and a challenge for modifying oil content of this plant. Further studies are needed to determine the functional characteristics of CsPDCT1, .
- Keywords: Camelina sativa, fatty acid, ROD1,, phosphatidylcholine diacylglycerol choline phosphotransferas
