Abstract
Sinapine is the most prominent antinutritive compound in the seeds of oilseed rape (Brassica napus L.). A reduction in sinapine content could improve the quality of rapeseed meal as an animal feed and in food industry. We had selected loss-of-function mutations of two sinapine biosynthesis genes BnSGT and BnREF1 and crossed them to produce double mutants. We measured their expression and enzyme activities in developing seeds as well as sinapoyl ester accumulation in mature seeds in three segregating F2 populations. Significant depletion of SGT enzyme activity in developing seeds proved loss of function of both gene copies and ruled out background effects. REF1 enzyme activities showed minor reductions and pointed at different substrate specificities of the paralogs and the presence of unspecific aldehyde dehydrogenases. Sinapine contents in the double mutants dropped dramatically by up to 71 %. F3 seeds with two stop-codon mutations in BnREF1 genes had the lowest sinapine contents (2.4 mg/g) as compared to the EMS control (7.5 mg/g). A BnREF1 splice site mutation did not result in a decrease in seed sinapine content probably due to incomplete splicing. We demonstrate that only the combination of different knockdown mutations drastically alters the composition of a major secondary metabolite. The results cast new light on the activities of gene paralogs in a polyploid species. The selected double mutants will be of major importance to further improve the quality of rapeseed.
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Acknowledgments
We thank Gislind Bräcker and Monika Bruisch for technical assistance in the laboratory and in the greenhouse, and Prof. Dr. Wolfgang Bilger and Jens Hermann from the Institute of Botany, University of Kiel for performing HPLC analyses. We thank the Zentrum für Molekulare Biowissenschaften (ZBM), University of Kiel for providing the facilities for DNA isolation and normalization. We are also grateful to the Institute for Clinical Molecular Biology, University Kiel for Sanger sequencing. This project was funded by the seed companies Norddeutsche Pflanzenzucht Hans-Georg Lembke KG (Hohenlieth, Germany), Deutsche Saatveredelung AG (Lippstadt, Germany), KWS SAAT AG (Einbeck, Germany), the German Federal Ministry for Education, Research and Technology BMBF (GABI Future Grant No. 0315052C), and the German Research foundation (DFG) Grant No. Ju205/14-1.
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Nazgol Emrani and Hans-Joachim Harloff have contributed equally to this work.
Accession numbers: BnaA.SGT.a (FM872285), BnaC.SGT.a (FM872284), BnaC.REF1.a (FN995990), and BnaA.REF1.a (FN995991).
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Emrani, N., Harloff, HJ., Gudi, O. et al. Reduction in sinapine content in rapeseed (Brassica napus L.) by induced mutations in sinapine biosynthesis genes. Mol Breeding 35, 37 (2015). https://doi.org/10.1007/s11032-015-0236-2
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DOI: https://doi.org/10.1007/s11032-015-0236-2