Biologia plantarum 65:273-282, 2021 | DOI: 10.32615/bp.2021.033
Transcriptome analysis of developing castor bean seeds and identification of ricinoleic acid biosynthesis genes
- 1 Applied Biotechnology Center, Wuhan University of Bioengineering, Wuhan 430415, P.R. China
- 2 School of Food and Biological Engineering Hubei University of Technology, Wuhan 430064, P.R. China
Ricinoleic acid is a kind of unsaturated fatty acids in oil of castor bean (Ricinus communis) seeds with wide application value. However, there is little transcriptomic information on genes related to ricinoleic acid biosynthesis in castor beans. To better understand the regulation mechanism of ricinoleic acid biosynthesis, immature seeds at three developmental stages (S1, S2, and S3 corresponding to 15, 30, and 45 d after pollination) were collected. The results indicated that the accumulation of castor bean oil and ricinoleic acid increased gradually during seed development, and reached the maximum value at the late stages of seed development (45 d after pollination). Furthermore, RNA sequencing was conducted to analyze the transcriptome of the developing seeds at three developmental stages. Totals of 9 875 differentially expressed genes (DEGs) were identified among the three time points. Based on the annotation information, 49 DEGs related to lipid biosynthesis were screened among all DEGs. Through cluster analysis of the 49 DEGs, ten genes with increasing FPKM values from seed development stages S1 to S3 were selected as candidate key enzymes, since they showed similar patterns of increase with castor bean oil accumulation and ricinoleic acid biosynthesis during seed development. The transcriptomic data of the 10 candidate key enzyme genes was further validated by RT-qPCR. Ultimately, a putative model of key genes correlated with ricinoleic acid accumulation was built. Our study identified a series of key genes and revealed the proposed molecular mechanism of ricinoleic acid accumulation in castor bean seeds through the transcriptional analysis. It broadens our knowledge of ricinoleic acid biosynthesis and castor bean oil accumulation and also provides a theoretical foundation for the genetic engineering key genes that can improve the ricinoleic acid production in castor beans as well as in other plants.
Keywords: castor bean oil, cluster analysis, differentially expressed genes, ricinoleic acid, Ricinus communis, transcriptome sequencing.
Received: February 3, 2021; Revised: May 13, 2021; Accepted: May 28, 2021; Published online: September 29, 2021 Show citation
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References
- Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J.: Gapped BLAST and PSIBLAST: a new generation of protein database search programs. - Nucl. Acids Res. 25: 3389-3402, 1997.
Go to original source... - Badami, R.C., Kudari, S.M.: Analysis of Hiptage madablota seed oil. - J. Sci. Food Agr. 21: 248-249, 1970.
Go to original source... - Bafor, M., Smith, M.A., Jonsson, L., Stobart, K., Stymne, S.: Ricinoleic acid biosynthesis and triacylglycerol assembly in microsomal preparations from developing castor bean (Ricinus communis) endosperm. - Biochem. J. 280: 507-514, 1991.
Go to original source... - Bates, P.D.: Understanding the control of acyl flux through the lipid metabolic network of plant oil biosynthesis. - Biochim. Biophys. Acta 1861: 1214-1225, 2016.
Go to original source... - Bates, P.D., Browse, J.: The pathway of triacylglycerol synthesis through phosphatidylcholine in Arabidopsis produces a bottleneck for the accumulation of unusual fatty acids in transgenic seeds. - Plant J. 68: 387-399, 2011.
Go to original source... - Broun, R., Somerville, C.: Accumulation of ricinoleic, lesquerolic, and densipolic acids in seeds of transgenic in Arabidopsis plants that express a fatty acyl hydroxylase cDNA from castor bean. - Plant Physiol. 113: 933-942, 1997.
Go to original source... - Cagliari, A., Margis-Pinheiro, M., Loss, G., Mastroberti, A.A., Mariath, J.E.A., Margis, R.: Identification and expression analysis of castor bean (Ricinus communis) genes encoding enzymes from the triacylglycerol biosynthesis pathway. - Plant Sci. 179: 499-500. 2010.
Go to original source... - Chan, A.P., Crabtree, J., Zhao, Q., Lorenzi, H., Orvis, J., Puiu, D., Melake-Berhan, A., Jones, K.M., Redman, J., Chen, G., Cahoon, E.B., Gedil, M., Stanke, M., Haas, B.J., Wortman, J.R., Fraser-Liggett, C.M., Ravel, J., Rabinowicz, P.D.: Draft genome sequence of the oilseed species Ricinus communis. - Nat. Biotechnol. 28: 951-956, 2010.
Go to original source... - Chandrasekaran, U., Liu, A.Z.: Seed filling and fatty acid changes in developing seeds of castor bean (Ricinus communis L.). - Aust. J. Crop Sci. 11: 1761-1765, 2013.
- Chandrasekaran, U., Xu, W., Liu, A.Z.: Transcriptome profiling identifies ABA mediated regulatory changes towards storage filling in developing seeds of castor bean (Ricinus communis L.). - Cell Biosci. 4: 33, 2014.
Go to original source... - Chen, G., Woodfield, H., Pan, X., Harwood, J.L., Weselake, R.J.: Acyl‑trafficking during plant oil accumulation. - Lipids 50: 1057-1068, 2015.
Go to original source... - Cingolani, P., Platts, A., Wang, L.L., Coon, M., Nguyen, T., Wang, L., Land, S.J., Lu, X., Ruden, D.M.: A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff. - Fly 6: 80-92, 2012.
Go to original source... - Du, H., Yu, Y., Ma, Y., Gao, Q., Cao, Y., Chen, Z., Ma, B., Qi, M., Li, Y., Zhao, X., Wang, J., Liu, K., Qin, P., Yang, X., Zhu, L., Li, S., Liang, C.: Sequencing and de novo assembly of a near complete indica rice genome. - Nat. Commun. 8: 15324, 2017.
Go to original source... - Hanano, A., Burcklen, M., Flenet, M., Ivancich, A., Louwagie, M., Garin, J., Blée, E.: Plant seed peroxygenase is an original heme-oxygenase with an EF-hand calcium binding motif. - J. biol. Chem. 281: 33140-33151, 2006.
Go to original source... - Horn, P.J., Liu, J., Cocuron, J.C., McGlew, K., Thrower, N.A., Larson, M., Lu, C., Alonso, A., Ohlrogge, J.: Identification of multiple lipid genes with modifications in expression and sequence associated with the evolution of hydroxy fatty acid accumulation in Physaria fendleri. - Plant J. 86: 322-348, 2016.
Go to original source... - Hu, Z., Ren, Z., Lu, C.: The phosphatidylcholine diacylglycerolcholinephosphotransferase is required for efficient hydroxy fatty acid accumulation in transgenic Arabidopsis. - Plant Physiol. 158: 1944-1954, 2012.
Go to original source... - Huang, A.H.C.: Plant lipid droplets and their associated proteins: potential for rapid advances. - Plant Physiol. 176: 1894-1918, 2018.
Go to original source... - Langmead, B., Salzberg, S.L.: Fast gapped-read alignment with bowtie 2. - Nat. Methods 9: 357, 2012.
Go to original source... - Lin, J., Arcinas, A.: Regiospecific analysis of diricinoleoylacylglycerols in castor (Ricinus communis L.) oil by electrospray ionization-mass spectrometry. - J. Agr. Food Chem. 55: 2209-2216, 2007.
Go to original source... - Lindqvist, Y., Huang, W., Schneider G., Shanklin J.: Crystal structure of delta9 stearoyl-acyl carrier protein desaturase from castor seed and its relationship to other di-iron proteins. - EMBO J. 15: 4081-4092, 1996.
Go to original source... - Lu, C., Xin, Z., Ren, Z., Miquel, M., Browse, J.: An enzyme regulating triacylglycerol composition is encoded by the ROD1 gene of Arabidopsis. - Proc. nat. Acad. Sci. USA 106: 18837-18842, 2009.
Go to original source... - Lung, S.C., Chye, M.L.: The binding versatility of plant acyl-CoA-binding proteins and their significance in lipid metabolism. - Biochim. biophys. Acta 1861: 1409-1421, 2016.
Go to original source... - McKenna, A., Hanna, M., Banks, E., Sivachenko, A., Cibulskis, K., Kernytsky, A., Garimella, K., Altshuler, D., Gabriel, S., Daly, M., Depristo, M.A.: The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. - Genome Res. 20: 1297-1303, 2018.
Go to original source... - Pan, X., Siloto, R.M., Wickramarathna, A.D., Mietkiewska, E., Weselake, R.J.: Identification of a pair of phospholipid:diacylglycerol acyltransferases from developing flax (Linum usitatissimum L.) seed catalyzing the selective production of trilinolenin. - J. biol. Chem. 288: 24173-24188, 2013.
Go to original source... - Parthibane, V., Rajakumari, S., Venkateshwari, V., Iyappan, R., Rajasekharan, R.: Oleosin is bifunctional enzyme that has both monoacylglycerol acyltransferase and phospholipase activities. - J. biol. Chem. 287: 1946-1954, 2012.
Go to original source... - Puyaubert, J., Dieryck, W., Costaglioli, P., Chevalier, S., Breton, A., Lessire, R.: Temporal gene expression of 3-ketoacyl-CoA reduetase is different in high and in low erucic acid. Brassica napus cultivars during seed development. - Biochim. biophys. Acta 1687: 152-163, 2005.
Go to original source... - Roesler, K., Shintani, D., Savage, L., Boddupalli, S., Ohlrogge, J.: Targeting of the Arabidopsis homomeric acetyl-coenzyme A carboxylase to plastids of rapeseeds. - Plant Physiol. 113: 75-81, 1997.
Go to original source... - Saito, J., Yamada, M., Watanabe, T., Iida, M., Kitagawa, H., Takahata, S., Lida, M., Kitagawa, H., Takahata, S., Ozawa, T., Takeuchi, Y., Ohsawa, F.: Crystal structure of enoyl-acyl carrier protein reductase (FabK) from Streptococcus pneumoniae reveals the binding mode of an inhibitor. - Protein Sci. 17: 691-699, 2008.
Go to original source... - Sasaki, Y., Nagano, Y.: Plant acetyl-CoA carboxylase: structure, biosynthesis, regulation, and gene manipulation for plant breeding. - Biosci. Biotech. Biochem. 68: 1l75-1184, 2004.
Go to original source... - Schmittgen, T.D., Livak, K.J.: Analyzing real-time PCR data by the comparative CT method. - Nat. Protoc. 3: 1101-1108, 2008.
Go to original source... - Slabas, A.R., White, A., O'hara, P., Fawcett, T.: Investigations into the regulation of lipid biosynthesis in Brassica napus using antisense down-regulation. - Biochem. Soc. Trans. 30: 1056-1059, 2002.
Go to original source... - Tian, B., Lu, T., Xu, Y., Wang, R., Chen, G.: Identification of genes associated with ricinoleic acid accumulation in Hiptage benghalensis via transcriptome analysis. - Biotechnol. Biofuels 12: 16, 2019.
Go to original source... - Trapnell, C., Pachter, L., Salzberg, S.L.: TopHat: discovering splice junctions with RNA-Seq. - Bioinformatics 25: 1105-1111, 2009.
Go to original source... - Trapnell, C., Roberts, A., Goff, L., Pertea, G., Kim, D., Kelley, D.R., Pimentel, H., Salzberg, S.L., Rinn, J.L., Pachter, L.: Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and cufflinks. - Nat. Protoc. 7: 562-578, 2012.
Go to original source... - Troncoso-Ponce, M.A., Kilaru, A., Cao, X., Durrett, T.P., Fan, J., Jensen, J.K, Thrower, N.A., Pauly, M., Wilkerson, C., Ohlrogge, J.B.: Comparative deep transcriptional profiling of four developing oilseeds. - Plant J. 68: 1014-1027, 2011.
Go to original source... - Van Erp, H., Bates, P.D., Burgal, J., Shockey, J., Browse, J.: Castor phospholipid:diacylglycerol acyltransferase facilitates efficient metabolism of hydroxyl fatty acids in transgenic Arabidopsis. - Plant Physiol. 15: 683-693, 2011.
Go to original source... - Wang, L., Jiang, X., Wang, L., Wang, W., Fu, C., Yan, X., Geng, X.: A survey of transcriptome complexity using PacBio single-molecule real-time analysis combined with Illumina RNA sequencing for a better understanding of ricinoleic acid biosynthesis in Ricinus communis. - BMC Genomics 20: 456, 2019.
Go to original source... - Wang, X., Wang, L., Yan, X., Wang, L., Tan, M., Geng, X., Wei, W.: Transcriptome analysis of the germinated seeds identifies low-temperature responsive genes involved in germination process in Ricinus communis. - Acta Physiol. Plant. 38: 6, 2016.
Go to original source... - Weiss, S.B., Kennedy, E.P., Kiyasu, J.Y.: The enzymatic synthesis of triglycerides. - J. biol. Chem. 235: 40-44, 1960.
Go to original source... - Weng, J., Gu, S., Wan, X., Gao, H., Guo, T., Su, N., Lei, C., Zhang, X., Cheng, Z., Guo, X., Wang, J., Jiang, L., Zhai, H., Wan, J.: Isolation and initial characterization of GW5, a major QTL associated with rice grain width and weight. - Cell Res. 18: 1199-1209, 2008.
Go to original source... - Xu, Y., Caldo, K.M.P., Pal-Nath, D., Ozga, J., Lemieux, M.J., Weselake, R.J., Chen, G.: Properties and biotechnological applications of acyl-CoA: diacylglycerol acyltransferase and phospholipid: diacylglycerol acyltransferase from terrestrial plants and microalgae. - Lipids 53: 663-688, 2018b.
Go to original source... - Xu, Y., Holic, R., Li, D., Pan, X., Weselake, R.J.: Substrate preferences of long-chain acyl-CoA synthetase and diacylglycerol acyltransferase contribute to enrichment of flax seed oil with α-linolenic acid. - Biochem. J. 475: 1473-1489, 2018a.
Go to original source... - Zhou, P., Florova, Q., Reynolds, K.A.: Polyketide synthase acyl carrier protein (ACP) as a substrate and a catalyst for malonyl ACP biosynthesis. - Chem. Biol. 6: 577-584, 1999.
Go to original source... - Zhou, X.R., Singh, S.P., Green, A.G.: Characterization of the FAD2 gene family from Hiptage benghalensis: a ricinoleic acid accumulating plant. - Phytochemistry 92: 42-48, 2013.
Go to original source... - Zhu, C., Li, X., Zheng, J.: Transcriptome profiling using Illumina- and SMRT based RNA-seq of hot pepper for in-depth understanding of genes involved in CMV infection. - Gene 666: 123-133, 2018.
Go to original source...



