Overexpression of Sinapine Esterase BnSCE3 in Oilseed Rape Seeds Triggers Global Changes in Seed Metabolism

Department of Secondary Metabolism, Leibniz Institute of Plant Biochemistry, D-06120 Halle, Germany.
Plant physiology (Impact Factor: 6.84). 03/2011; 155(3):1127-45. DOI: 10.1104/pp.110.169821
Source: PubMed


Sinapine (O-sinapoylcholine) is the predominant phenolic compound in a complex group of sinapate esters in seeds of oilseed rape (Brassica napus). Sinapine has antinutritive activity and prevents the use of seed protein for food and feed. A strategy was developed to lower its content in seeds by expressing an enzyme that hydrolyzes sinapine in developing rape seeds. During early stages of seedling development, a sinapine esterase (BnSCE3) hydrolyzes sinapine, releasing choline and sinapate. A portion of choline enters the phospholipid metabolism, and sinapate is routed via 1-O-sinapoyl-β-glucose into sinapoylmalate. Transgenic oilseed rape lines were generated expressing BnSCE3 under the control of a seed-specific promoter. Two distinct single-copy transgene insertion lines were isolated and propagated to generate homozygous lines, which were subjected to comprehensive phenotyping. Sinapine levels of transgenic seeds were less than 5% of wild-type levels, whereas choline levels were increased. Weight, size, and water content of transgenic seeds were significantly higher than those of wild-type seeds. Seed quality parameters, such as fiber and glucosinolate levels, and agronomically important traits, such as oil and protein contents, differed only slightly, except that amounts of hemicellulose and cellulose were about 30% higher in transgenic compared with wild-type seeds. Electron microscopic examination revealed that a fraction of the transgenic seeds had morphological alterations, characterized by large cavities near the embryonic tissue. Transgenic seedlings were larger than wild-type seedlings, and young seedlings exhibited longer hypocotyls. Examination of metabolic profiles of transgenic seeds indicated that besides suppression of sinapine accumulation, there were other dramatic differences in primary and secondary metabolism. Mapping of these changes onto metabolic pathways revealed global effects of the transgenic BnSCE3 expression on seed metabolism.

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    • "M64633) encoding a seed storage protein of the napin type. Seed specificity of this promoter has been demonstrated (Kridl et al., 1991), and the promoter has been used frequently for seed-specific transgenic approaches in oilseed rape (Hüsken et al., 2005; Clauss et al., 2011). Assembly of these elements was done in plasmid pBluescript II KS2, from which the whole suppression construct was cut as SpeI-HindIII fragment and inserted into the binary vector pLH7000 (Hausmann and Töpfer, 1999) to give the seedspecific BnREF1 suppression plasmid pLH-BnREF1i. "
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    ABSTRACT: ABSTRACT As a result of the phenylpropanoid pathway, many Brassicaceae produce considerable amounts of soluble hydroxycinnamate (HCA) conjugates, mainly sinapate esters. From Brassica napus (oilseed rape; Canola) we cloned two orthologs of the Arabidopsis gene REDUCED EPIDERMAL FLUORESCENCE1 (REF1) encoding a coniferaldehyde/sinapaldehyde dehydrogenase (CALDH/SALDH). The enzyme is involved in the formation of ferulate and sinapate from the corresponding aldehydes, thereby linking lignin and HCA biosynthesis as a potential branch point enzyme. We used RNA interference to silence REF1 genes in seeds of B. napus. Non-targeted metabolite profiling showed that BnREF1-suppressing seeds produced a novel chemotype characterized by reduced levels of sinapate esters, the appearance of conjugated mono-, di- and trilignols, altered accumulation patterns of kaempferol glycosides and changes in minor conjugates of caffeate, ferulate and 5-hydroxyferulate. BnREF1 suppression affected the level of minor sinapate conjugates more severely than that of the major component sinapine. Mapping of the changed metabolites onto the phenylpropanoid metabolic network revealed partial redirection of metabolic sequences as a major impact of BnREF1-suppression.
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