Over-expression 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: 7.39). 03/2011; 155(3):1127-45. DOI: 10.1104/pp.110.169821
Source: PubMed

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-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.

Download full-text


Available from: Carsten Milkowski, Aug 20, 2015
1 Follower
  • Source
    • "Hydroxycinnamoyl esters also cross-link with lignocellulosic polymers, thereby affecting cell-wall strength and biomass utilization (Ralph et al., 2004). Consistent with their multiple roles in phenylpropanoid metabolism, genetic perturbations affecting hydroxycinnamates or hydroxycinnamate conjugates have wide-ranging effects on phenylpropanoid carbon allocation between different branch pathways (Sinlapadech et al., 2007; Lanot et al., 2008; Clauß et al., 2011; Mittasch et al., 2013). Hydroxycinnamate glucose esters represent the most common form of hydroxycinnamate conjugates in plants (Corner and Swain, 1965). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The diversity of phenylpropanoids offers a rich inventory of bioactive chemicals that can be exploited for plant improvement and human health. Recent evidence suggests that glycosylation may play a role in the partitioning of phenylpropanoid precursors for a variety of downstream uses. This work reports the functional characterization of a stress-responsive glycosyltransferase, GT1-316 in Populus. GT1-316 belongs to the UGT84A subfamily of plant glycosyltransferase family 1 and is designated UGT84A17. Recombinant protein analysis showed that UGT84A17 is a hydroxycinnamate glycosyltransferase and able to accept a range of unsubstituted and substituted cinnamic and benzoic acids as substrates in vitro. Overexpression of GT1-316 in transgenic Populus led to plant-wide increases of hydroxycinnamoyl-glucose esters, which were further elevated under N-limiting conditions. Levels of the two most abundant flavonoid glycosides, rutin and kaempferol-3-O-rutinoside, decreased, while levels of other less abundant flavonoid and phenylpropanoid conjugates increased in leaves of the GT1-316-overexpressing plants. Transcript levels of representative phenylpropanoid pathway genes were unchanged in transgenic plants, supporting a glycosylation-mediated redirection of phenylpropanoid carbon flow as opposed to enhanced phenylpropanoid pathway flux. The metabolic response of N-replete transgenic plants overlapped with that of N-stressed wild types, as the majority of phenylpropanoid derivatives significantly affected by GT1-316 overexpression were also significantly changed by N stress in the wild types. These results suggest that UGT84A17 plays an important role in phenylpropanoid metabolism by modulating biosynthesis of hydroxycinnamoyl-glucose esters and their derivatives in response to developmental and environmental cues.
    Journal of Experimental Botany 08/2014; 65:4191-4200. DOI:10.1093/jxb/eru192 · 5.79 Impact Factor
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    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.
    Plant physiology 02/2013; DOI:10.1104/pp.113.215491 · 7.39 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Lipase is one of the lipid hydrolyzing enzymes, distributed broadly throughout plants, animal and microorganisms. GDSL-lipases is one of the lipases that exhibit a GDSL motif GxSxxxxG, in which the active site Serine is located near the N-terminus and display a Gly-Asp-Ser-(Leu) [GDS(L)] motif in conserved block I. However, the knowledge about their roles in developmental processes and response to various stimuli are still very limited in rice. A systematic analysis revealed the presence of at least 113 GDSL lipase (GLIP) genes in the rice genome. The tandem gene duplications have contributed a major role in expansion of this gene family. Phylogenetic analysis classed proteins into three groups; OsGLIP group B contained 56 genes, 50 in group A and only 2 genes in group C. The organization of putative motifs indicated potential diverse functions of GLIP gene family members in rice. Microarray data analysis revealed tissue and developmental stage-specific expression patterns of several OsGLIP genes. 38 OsGLIP genes were especially expressed in stigma and seed germination, several genes expressed exclusively in root and 17 OsGLIP genes were induced by any of three stresses. Our analysis also suggests differential accumulation of cluster genes during these processes. Our analyses indicated OsGLIP genes may have potential roles in rice development and abiotic stresses.
Show more