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

Abstract

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-b-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.Brassicaceous plants, such as oilseed rape (Brassica napus) and Arabidopsis (Arabidopsis thaliana), are characterized by the pronounced formation of soluble sinapate esters (Bouchereau et al., 1992). Seeds of this plant family accumulate sinapine, the choline ester of sinapate (O-sinapoylcholine; Henry and Garot, 1825; Gadamer, 1897), as the major component of complex patterns of sinapate esters, including various sinapoylated Glcs, gentiobioses, and kaempferol glycosides (Baumert et al., 2005;Wolfram et al., 2010). When oilseed rape seeds are processed, a high-quality vegetable oil is produced, and most of the sinapine is found in the residual protein meal. However, this compound is unpalatable and antinutritive (Ismail et al., 1981), making the meal unsuitable for human or animal consumption. Oilseed rape could be a valuable source of protein as well as oil if sinapine and related sinapate esters were reduced or eliminated from the seeds.The enzymes involved in the brassicaceous sinapate ester metabolism include UDP-Glc:sinapate glucosyltransferase (EC 2.4...

Differential Costs of Two Distinct Resistance Mechanisms Induced by Different Herbivore Species in Arabidopsis

“…Moreover, Kim et al (2015) reported that the level of indole-3-acetaldoxime, a precursor in indole GLS, auxin, and camalexin biosynthesis, negatively affected SM level in Arabidopsis. Based on available information on the SM biosynthetic pathway (Clauss et al, 2011) and our results, we speculate that SM biosynthesis is regulated through a novel molecular mechanism that may involve reactive oxygen species. Future studies using an integrative analysis of transcriptomics and metabolomics are required to identify the regulatory network of SM biosynthesis from signaling to metabolite biosynthesis.…”

Reprogramming the Phenylpropanoid Metabolism in Seeds of Oilseed Rape by Suppressing the Orthologs ofREDUCED EPIDERMAL FLUORESCENCE1

“…BnSCE3-overexpressing seeds were nearly devoid of sinapine. However, in contrast to the silencing of BnREF1 and UGT84A9, the fraction of related sinapate esters was not affected by the induced sinapine esterase activity (Clauss et al, 2011). With regard to conventional breeding, BnREF1 genes appear as the favored molecular targets.…”

“…On the other hand, an increase was observed in the amounts of nonacylated kaempferol 3-O-sophoroside (42) and its sinapoylated derivative (43). The overaccumulation of compounds 42 and 43 was also found in the oilseed rape transformant suppressing UDP-Glc:sinapate glucosyltransferase (UGT84A9), the enzyme converting sinapate to 1-O-sinapoylglucose (Wolfram et al, 2010) and in the transgenic overexpressor of sinapoylcholine esterase (BnSCE3) involved in sinapine hydrolysis (Clauss et al, 2011). Hence, this metabolic change should be related to a perturbed sinapate ester metabolism.…”

“…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.…”

“…subjected to nontargeted LC/MS-based metabolite profiling. To enable a comprehensive analysis of semipolar seed metabolites, a previously developed two-step extraction/fractionation procedure was applied for sample preparation (Clauss et al, 2011). Low concentrated methanolic total extracts (2.5 mg fresh weight mL 21 ) were prepared from which the abundant phenolic choline esters and related compounds were profiled by ultra-performance liquid chromatography-coupled electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC/ESI-QTOFMS) in positive ion mode.…”