Sinapate esters in brassicaceous plants: biochemistry, molecular biology, evolution and metabolic engineering

Brassicaceous plants are characterized by a pronounced metabolic flux toward sinapate, produced by the shikimate/phenylpropanoid pathway, which is converted into a broad spectrum of O-ester conjugates. The abundant sinapate esters in Brassica napus and Arabidopsis thaliana reflect a well-known metabolic network, including UDP-glucose:sinapate glucosyltransferase (SGT), sinapoylglucose:choline sinapoyltransferase (SCT), sinapoylglucose:l-malate sinapoyltransferase (SMT) and sinapoylcholine (sinapine) esterase (SCE). 1-O-Sinapoylglucose, produced by SGT during seed development, is converted to sinapine by SCT and hydrolyzed by SCE in germinating seeds. The released sinapate feeds via sinapoylglucose into the biosynthesis of sinapoylmalate in the seedlings catalyzed by SMT. Sinapoylmalate is involved in protecting the leaves against the deleterious effects of UV-B radiation. Sinapine might function as storage vehicle for ready supply of choline for phosphatidylcholine biosynthesis in young seedlings. The antinutritive character of sinapine and related sinapate esters hamper the use of the valuable seed protein of the oilseed crop B. napus for animal feed and human nutrition. Due to limited variation in seed sinapine content within the assortment of B. napus cultivars, low sinapine lines cannot be generated by conventional breeding giving rise to genetic engineering of sinapate ester metabolism as a promising means. In this article we review the progress made throughout the last decade in identification of genes involved in sinapate ester metabolism and characterization of the encoded enzymes. Based on gene structures and enzyme recruitment, evolution of sinapate ester metabolism is discussed. Strategies of targeted metabolic engineering, designed to generate low-sinapate ester lines of B. napus, are evaluated.     

Genomic microstructure and differential expression of the genes encoding UDP-glucose:sinapate glucosyltransferase (UGT84A9) in oilseed rape (Brassica napus)

In oilseed rape (Brassica napus), the glucosyltransferase UGT84A9 catalyzes the formation of 1-O-sinapoyl-β-glucose, which feeds as acyl donor into a broad range of accumulating sinapate esters, including the major antinutritive seed component sinapoylcholine (sinapine). Since down-regulation of UGT84A9 was highly efficient in decreasing the sinapate ester content, the genes encoding this enzyme were considered as potential targets for molecular breeding of low sinapine oilseed rape. B. napus harbors two distinguishable sequence types of the UGT84A9 gene designated as UGT84A9-1 and UGT84A9-2. UGT84A9-1 is the predominantly expressed variant, which is significantly up-regulated during the seed filling phase, when sinapate ester biosynthesis exhibits strongest activity. In the allotetraploid genome of B. napus, UGT84A9-1 is represented by two loci, one derived from the Brassica C-genome (UGT84A9a) and one from the Brassica A-genome (UGT84A9b). Likewise, for UGT84A9-2 two loci were identified in B. napus originating from both diploid ancestor genomes (UGT84A9c, Brassica C-genome; UGT84A9d, Brassica A-genome). The distinct UGT84A9 loci were genetically mapped to linkage groups N15 (UGT84A9a), N05 (UGT84A9b), N11 (UGT84A9c) and N01 (UGT84A9d). All four UGT84A9 genomic loci from B. napus display a remarkably low micro-collinearity with the homologous genomic region of Arabidopsis thaliana chromosome III, but exhibit a high density of transposon-derived sequence elements. Expression patterns indicate that the orthologous genes UGT84A9a and UGT84A9b should be considered for mutagenesis inactivation to introduce the low sinapine trait into oilseed rape.     

Overexpression of sinapine esterase BnSCE3 in oilseed rape seeds triggers global changes in seed metabolism

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.     

The role of UDP-glucose:hydroxycinnamate glucosyltransferases in phenylpropanoid metabolism and the response to UV-B radiation in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Arabidopsis harbors four UDP-glycosyltransferases that convert hydroxycinnamates (HCAs) to 1-O-β-glucose esters, UGT84A1 (encoded by At4g15480), UGT84A2 (At3g21560), UGT84A3 (At4g15490), and UGT84A4 (At4g15500). To elucidate the role of the individual UGT84A enzymes in planta we analyzed gene expression, UGT activities and accumulation of phenylpropanoids in Arabidopsis wild type plants, ugt mutants and overexpressing lines. Individual ugt84A null alleles did not significantly reduce the gross metabolic flux to the accumulating compounds sinapoylcholine (sinapine) in seeds and sinapoylmalate in leaves. For the ugt84A2 mutant, LC/MS analysis revealed minor qualitative and quantitative changes of several HCA choline esters and of disinapoylspermidine in seeds. Overexpression of individual UGT84A genes caused increased enzyme activities but failed to produce significant changes in the pattern of accumulating HCA esters. For UGT84A3, our data tentatively suggest an impact on cell wall-associated 4-coumarate. Exposure of plants to enhanced UV-B radiation induced the UGT84A-encoding genes and led to a transient increase in sinapoylglucose and sinapoylmalate concentrations. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Reduction of Sinapate Ester Content in Transgenic Oilseed Rape (<Emphasis Type="Italic">Brassica napus</Emphasis>) by dsRNAi-based Suppression of <Emphasis Type="Italic">BnSGT1</Emphasis> Gene Expression

Seeds of oilseed rape (Brassica napus) accumulate high amounts of antinutritive sinapate esters (SE) with sinapoylcholine (sinapine) as major component, accompanied by sinapoylglucose. These phenolic compounds compromise the use of the protein-rich valuable seed meal. Hence, a substantial reduction of the SE content is considered essential for establishing rape as a protein crop. The present work focuses on the suppression of sinapine synthesis in rape. Therefore, rape (spring cultivar Drakkar) was transformed with a dsRNAi construct designed to silence seed-specifically the BnSGT1 gene encoding UDP-glucose:sinapate glucosyltransferase (SGT1). This resulted in a substantial decrease of SE content in T2 seeds with a reduction reaching 61%. In T2 seeds a high and significant correlation between the contents of sinapoylglucose and all other sinapate esters has been observed. Among transgenic plants, no significant difference in other important agronomic traits, such as oil, protein, fatty acid and glucosinolate content in comparison to the control plants was observed. Maximal reduction of total SE content by 76% was observed in seeds of one homozygous T2 plant (T3 seeds) carrying the BnSGT1 suppression cassette as a single copy insert. In conclusion, this study is an initial proof of principle that suppression of sinapoylglucose formation leads to a strong reduction of SE in rape seeds and is thus a promising approach in establishing rape, currently an important oil crop, as a protein crop as well.     

Formation of a complex pattern of sinapate esters in Brassica napus seeds, catalyzed by enzymes of a serine carboxypeptidase-like acyltransferase family?

Members of the Brassicaceae accumulate complex patterns of sinapate esters, as shown in this communication with seeds of oilseed rape (Brassica napus). Fifteen seed constituents were isolated and identified by a combination of high-field NMR spectroscopy and high resolution electrospray ionisation mass spectrometry. These include glucose, gentiobiose and kaempferol glycoside esters as well as sinapine (sinapoylcholine), sinapoylmalate and an unusual cyclic spermidine amide. One of the glucose esters (1,6-di-O-sinapoylglucose), two gentiobiose esters (1-O-caffeoylgentiobiose and 1,2,6'-tri-O-sinapoylgentiobiose) and two kaempferol conjugates [4'-(6-O-sinapoylglucoside)-3,7-di-O-glucoside and 3-O-sophoroside-7-O-(2-O-sinapoylglucoside)] seem to be new plant products. Serine carboxypeptidase-like (SCPL) acyltransferases catalyze the formation of sinapine and sinapoylmalate accepting 1-O-beta-acetal esters (1-O-beta-glucose esters) as acyl donors. To address the question whether the formation of other components of the complex pattern of the sinapate esters in B. napus seeds is catalyzed via 1-O-sinapoyl-beta-glucose, we performed a seed-specific dsRNAi-based suppression of the sinapate glucosyltransferase gene (BnSGT1) expression. In seeds of BnSGT1-suppressing plants the amount of sinapoylglucose decreased below the HPLC detection limit resulting in turn in the disappearance or marked decrease of all the other sinapate esters, indicating that formation of the complex pattern of these esters in B. napus seeds is dependent on sinapoylglucose. This gives rise to the assumption that enzymes of an SCPL acyltransferase family catalyze the appropriate transfer reactions to synthesize the accumulating esters.     

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

As a result of the phenylpropanoid pathway, many Brassicaceae produce considerable amounts of soluble hydroxycinnamate conjugates, mainly sinapate esters. From oilseed rape (Brassica napus), we cloned two orthologs of the Arabidopsis (Arabidopsis thaliana) gene REDUCED EPIDERMAL FLUORESCENCE1 (REF1) encoding a coniferaldehyde/sinapaldehyde dehydrogenase. The enzyme is involved in the formation of ferulate and sinapate from the corresponding aldehydes, thereby linking lignin and hydroxycinnamate biosynthesis as a potential branch-point enzyme. We used RNA interference to silence REF1 genes in seeds of oilseed rape. Nontargeted metabolite profiling showed that BnREF1-suppressing seeds produced a novel chemotype characterized by reduced levels of sinapate esters, the appearance of conjugated monolignols, dilignols, 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.Phenylpropanoid metabolism provides plants with a vast array of phenolic compounds that contribute to nearly all aspects of plant life (Vogt, 2010). In species of the Brassicaceae family, soluble hydroxycinnamate conjugates, mainly sinapate esters, constitute an abundant metabolite fraction produced from a branch of the phenylpropanoid pathway (Fraser and Chapple, 2011). As major compounds, sinapoylmalate accumulates in leaves (Hause et al., 2002) and sinapoylcholine (sinapine) in seeds (Bouchereau et al., 1991; Fig. 1).Open in a separate windowFigure 1.Biosynthesis of sinapate esters emphasizing the crucial role of the bifunctional hydroxycinnamaldehyde dehydrogenase CALDH/SALDH encoded by the gene REF1. Dashed arrows symbolize multistep biosyntheses. Abbreviations for enzymes are as follows: CAD, (hydroxy)cinnamyl alcohol dehydrogenase; SAD, sinapyl alcohol dehydrogenase; SCT, 1-O-sinapoylglucose:choline sinapoyltransferase; SGT, UDP-Glc:sinapate glucosyltransferase; SMT, 1-O-sinapoylglucose:malate sinapoyltransferase.In Arabidopsis (Arabidopsis thaliana), the disturbed accumulation of sinapoylmalate caused the mutant phenotype reduced epidermal fluorescence (ref; Ruegger and Chapple, 2001). Molecular characterization of the ref1 mutant led to the identification of the gene At3g24503 (REF1) encoding coniferaldehyde dehydrogenase/sinapaldehyde dehydrogenase (CALDH/SALDH; EC 1.2.1.68; Nair et al., 2004). The bifunctional enzyme CALDH/SALDH was shown to catalyze the NADP⁺-dependent oxidation of coniferaldehyde and sinapaldehyde to yield the corresponding hydroxycinnamates ferulate and sinapate. As a potential branching enzyme, the enzymatic activity of the REF1-encoded CALDH/SALDH might be crucial for the partition ratio of metabolites between lignin and hydroxycinnamate biosynthesis (Fig. 1). Therefore, manipulation of REF1 expression coupled to a comprehensive metabolite analysis of transgenic plants appeared as an interesting strategy to gain a deeper understanding of the plant phenylpropanoid metabolic network. Moreover, in crop plants like oilseed rape (Brassica napus), where high levels of sinapate esters contribute to antinutritive features, the suppression of REF1 orthologs might cause increased quality.This work describes the isolation of REF1 orthologous genes from oilseed rape (BnREF1). By RNA interference, transgenic lines of oilseed rape were generated that suppress BnREF1 gene expression during seed development. Homozygous transgenic progeny were developed and used in a comprehensive liquid chromatography-mass spectrometry (LC/MS)-based metabolite profiling to investigate the impact of BnREF1 silencing in seeds. Mapping of changed metabolite patterns onto the phenylpropanoid metabolic network revealed insight into the molecular mechanisms by which silencing of BnREF1 produced a novel seed chemotype in oilseed rape.     

Resveratrol glucoside (Piceid) synthesis in seeds of transgenic oilseed rape (Brassica napus L.)

Resveratrol is a phytoalexin produced in various plants like wine, peanut or pine in response to fungal infection or UV irradiation, but it is absent in members of the Brassicaceae. Moreover, resveratrol and its glucoside (piceid) are considered to have beneficial effects on human health, known to reduce heart disease, arteriosclerosis and cancer mortality. Therefore, the introduction of the gene encoding stilbene synthase for resveratrol production in rapeseed is a tempting approach to improve the quality of rapeseed products. The stilbene synthase gene isolated from grapevine (Vitis vinifera L.) was cloned under control of the seed-specific napin promotor and introduced into rapeseed (Brassica napus L.) by Agrobacterium-mediated co-transformation together with a ds-RNA-interference construct deduced from the sequence of the key enzyme for sinapate ester biosynthesis, UDP-glucose:sinapate glucosyltransferase (BnSGT1), assuming that the suppression of the sinapate ester biosynthesis may increase the resveratrol production in seeds through the increased availability of the precursor 4-coumarate. Resveratrol glucoside (piceid) was produced at levels up to 361 μg/g in the seeds of the primary transformants. This value exceeded by far piceid amounts reported from B. napus expressing VST1 in the wild type sinapine background. There was no significant difference in other important agronomic traits, like oil, protein, fatty acid and glucosinolate content in comparison to the control plants. In the third seed generation, up to 616 μg/g piceid was found in the seeds of a homozygous T3-plant with a single transgene copy integrated. The sinapate ester content in this homozygous T3-plant was reduced from 7.43 to 2.40 mg/g. These results demonstrate how the creation of a novel metabolic sink could divert the synthesis towards the production of piceid rather than sinapate ester, thereby increasing the value of oilseed products.     

Targeted modulation of sinapine biosynthesis pathway for seed quality improvement in Brassica napus

Arabidopsis thaliana and other members of the Brassicaceae accumulate the hydroxycinnamic acid esters sinapoylmalate in leaves and sinapoylcholine in seeds. Our recent understanding of the phenylpropanoid pathway although complex has enabled us to perturb the sinapine biosynthesis pathway in plants. Sinapine (sinapoylcholine) is the most abundant antinutritional phenolic compound in seeds of cruciferous species and therefore is a target for elimination in canola (Brassica napus) meal. We analysed A. thaliana mutants with specific blocks in the phenylpropanoid pathway and identified mutant lines with significantly altered sinapine content. Knowledge gained from A. thaliana was extended to B. napus and the corresponding phenylpropanoid pathway genes were manipulated to disrupt sinapine biosynthesis in B. napus. Based on our understanding of the A. thaliana genetics, we have successfully developed transgenic B. napus lines with ferulic acid 5-hydroxylase (FAH) and sinapoylglucose:choline sinapoyltransferase (SCT)-antisense. These lines with concomitant downregulation of FAH and SCT showed up to 90% reduction in sinapine. In addition to reduced sinapine content, we detected higher levels of free choline accumulation in the seeds. These results indicate that it is possible to develop plants with low sinapine and higher choline by manipulating specific steps in the biosynthetic pathway. These improvements are important to add value to canola meal for livestock feed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

QTL for phytosterol and sinapate ester content in <Emphasis Type="Italic">Brassica napus</Emphasis> L. collocate with the two erucic acid genes

Improving oil and protein quality for food and feed purposes is an important goal in rapeseed (Brassica napus L.) breeding programs. Rapeseed contains phytosterols, used to enrich food products, and sinapate esters, which are limiting the utilization of rapeseed proteins in the feed industry. Increasing the phytosterol content of oil and lowering sinapate ester content of meal could increase the value of the oilseed rape crop. The objective of the present study was to identify quantitative trait loci (QTL) for phytosterol and sinapate ester content in a winter rapeseed population of 148 doubled haploid lines, previously found to have a large variation for these two traits. This population also segregated for the two erucic acid genes. A close negative correlation was found between erucic acid and phytosterol content (Spearman’s rank correlation, r _s = −0.80^**). For total phytosterol content, three QTL were detected, explaining 60% of the genetic variance. The two QTL with the strongest additive effects were mapped on linkage groups N8 and N13 within the confidence intervals of the two erucic acid genes. For sinapate ester content four QTL were detected, explaining 53% of the genetic variance. Again, a close negative correlation was found between erucic acid and sinapate ester content (r _s = −0.66^**) and the QTL with the strongest additive effects mapped on linkage groups N8 and N13 within the confidence intervals of the two erucic acid genes. The results suggests, that there is a pleiotropic effect of the two erucic acid genes on phytosterol and sinapate ester content; the effect of the alleles for low erucic acid content is to increase phytosterol and sinapate ester content. Possible reasons for this are discussed based on known biosynthetic pathways. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Pleiotropic changes in Arabidopsis <Emphasis Type="Italic">f5h</Emphasis> and <Emphasis Type="Italic">sct</Emphasis> mutants revealed by large-scale gene expression and metabolite analysis

Hydrocinnamic acid esters, lignin, flavonoids, glucosinolates, and salicylic acid protect plants against UV exposure, oxidative stress, diseases, and herbivores. Through the phenylpropanoid pathway, certain Brassicaceae family members, including Arabidopsis thaliana and Brassica napus, accumulate large amounts of the anti-nutritive sinapoylcholine (sinapine) in the seed. We successfully down-regulated activities of key enzymes in the pathway including F5H and SCT and achieved reduction of sinapine and lignin in B. napus seeds. Despite this success, it was unclear how multiple agronomic traits were affected in the transgenic plants. Here, we report altered large-scale gene expression of new alleles of f5h and sct mutants of A. thaliana and resultant accumulation of sinapoylglucose, disinapoylglucose, quercetin-3-O-rhamnoside, salicylic acid glucoside, and total indolyl glucosinolates in the two mutants. Expression of several flowering genes was altered in these mutants when grown under drought and NaCl treatments. Furthermore, both mutants were more susceptible to fungal infection than the wild type. Microarray experiments identified distinctive spatial and temporal expression patterns of gene clusters involved in silique/seed developmental processes and metabolite biosynthesis in these mutants. Taken together, these findings suggest that both f5h and sct mutants exhibit major differences in accumulation of diverse metabolites in the seed and profound changes in global large-scale gene expression, resulting in differential pleiotropic responses to environmental cues.     

Enhancement of syringin contents in soybean seeds with seed-specific expression of a chimeric <Emphasis Type="Italic">UGT72E3</Emphasis>/<Emphasis Type="Italic">E2</Emphasis> gene

Syringin, sinapyl alcohol 4-O-glucoside, is well known as a plant-derived bioactive monolignol glucoside. In Arabidopsis, recombinant chimeric protein UGT72E3/2 has been previously reported to lead to significantly higher syringin production than the parental enzymes UGT72E2 and UGT72E3. To enhance syringin content in Korean soybean (Glycine max L. ‘Kwangan’), we cloned the UGT72E3/2 gene under the control of the β-conglycinin or CaMV-35S promoter to generate β-UGT72E3/2 and 35S-UGT72E3/2 constructs, respectively, and then transformed them into soybean to obtain transgenic plants using the modified half-seed method. Real-time semi-quantitative PCR (RT-PCR) analysis showed that the UGT72E3/2 gene was expressed in the leaves of the β-UGT72E3/2 and 35S-UGT72E3/2 transgenic lines. HPLC analysis of the seeds and mature tissues of the T2 generation plants revealed that the β-UGT72E3/2 transgenic seeds accumulated 0.15 µmol/g DW of total syringin and 0.29 µmol/g DW of total coniferin, whereas coniferin and syringin were not detected in non-transgenic seeds. Moreover, coniferin and syringin also accumulated at high levels in non-seed tissues, particularly the leaves of β-UGT72E3/2 transgenic lines. In contrast, 35S-UGT72E3/2 lines showed no differences in the contents of coniferin and syringin between transgenic and non-transgenic soybean plants. Thus, the seed-specific β-conglycinin promoter might be an effective tool to apply to the nutritional enhancement of soybean crops through increased syringin production.     

Sinapate esters provide greater UV-B attenuation than flavonoids in Arabidopsis thaliana (Brassicaceae)

Mutants affected in flavonoid (tt4) or sinapate ester (fah1) biosynthesis were used to assess the relative importance of these phenolic UV photoprotectants in Arabidopsis. Flavonoid and sinapate ester absorption was more specific for UV-B than major nonphenolic chromophores in crude extracts. A new method of evaluating phenolic UV-B attenuation was developed using fluorescence analysis. When excited by UV-B, sinapate ester containing leaves and cotyledons had enhanced sinapate ester fluorescence and reduced chlorophyll fluorescence relative to those without sinapate esters. Although fluorescence analysis gave no evidence of UV-B attenuation by flavonoids, enhanced chlorophyll and protein loss were observed upon UV-B exposure in flavonoid-deficient leaves, suggesting they have another mechanism of UV-B protection. The hydroxycinnamates have been largely ignored as UV-B attenuating pigments, and the results indicate that greater attention should be paid to their role in attenuating UV-B.     

Efficacy of karanjin and phorbol ester fraction against termites (Odontotermes obesus)

The non-edible oil seeds of Jatropha curcas (physic nut) and Pongamia pinnata (karanja) contain some toxic components (phorbol esters in J. curcas and karanjin in P. pinnata), which may be used as biopesticides. In this study, the active components of J. curcas and P. pinnata oil were extracted and their efficacy against the termites Odontotermes obesus (Rambur), was tested. The phorbol ester fraction of J. curcas and karanjin of P. pinnata oil were found to be effective against termites. A mortality rate of 100% was achieved in 6 h with karanjin and in 12 h with phorbol ester fraction. The LC₅₀ levels of karanjin and phorbol esters fractions were 0.038 and 0.071 g ml⁻¹, respectively, after 24 h at a 95% (0.05) confidence limit.     

Overexpression of phyA and appA Genes Improves Soil Organic Phosphorus Utilisation and Seed Phytase Activity in Brassica napus

Phytate is the major storage form of organic phosphorus in soils and plant seeds, and phosphorus (P) in this form is unavailable to plants or monogastric animals. In the present study, the phytase genes phyA and appA were introduced into Brassica napus cv Westar with a signal peptide sequence and CaMV 35S promoter, respectively. Three independent transgenic lines, P3 and P11 from phyA and a18 from appA, were selected. The three transgenic lines exhibited significantly higher exuded phytase activity when compared to wild-type (WT) controls. A quartz sand culture experiment demonstrated that transgenic Brassica napus had significantly improved P uptake and plant biomass. A soil culture experiment revealed that seed yields of transgenic lines P11 and a18 increased by 20.9% and 59.9%, respectively, when compared to WT. When phytate was used as the sole P source, P accumulation in seeds increased by 20.6% and 46.9% with respect to WT in P11 and a18, respectively. The P3 line accumulated markedly more P in seeds than WT, while no significant difference was observed in seed yields when phytate was used as the sole P source. Phytase activities in transgenic canola seeds ranged from 1,138 to 1,605 U kg^–1 seeds, while no phytase activity was detected in WT seeds. Moreover, phytic acid content in P11 and a18 seeds was significantly lower than in WT. These results introduce an opportunity for improvement of soil and seed phytate-P bioavailability through genetic manipulation of oilseed rape, thereby increasing plant production and P nutrition for monogastric animals.     

Cloning and heterologous expression of a rape cDNA encoding UDP-glucose:sinapate glucosyltransferase

A cDNA encoding a UDP-glucose:sinapate glucosyltransferase (SGT) that catalyzes the formation of 1-O-sinapoylglucose, was isolated from cDNA libraries constructed from immature seeds and young seedlings of rape (Brassica napus L.). The open reading frame encoded a protein of 497 amino acids with a calculated molecular mass of 55,970 Da and an isoelectric point of 6.36. The enzyme, functionally expressed in Escherichia coli, exhibited broad substrate specificity, glucosylating sinapate, cinnamate, ferulate, 4-coumarate and caffeate. Indole-3-acetate, 4-hydroxybenzoate and salicylate were not conjugated. The amino acid sequence of the SGT exhibited a distinct sequence identity to putative indole-3-acetate glucosyltransferases from Arabidopsis thaliana and a limonoid glucosyltransferase from Citrus unshiu, indicating that SGT belongs to a distinct subgroup of glucosyltransferases that catalyze the formation of 1-O-acylglucosides (β-acetal esters). Received: 14 July 2000 / Accepted: 8 August 2000     

Tissue-Specific Distribution of Secondary Metabolites in Rapeseed (Brassica napus L.)

Four different parts, hypocotyl and radicle (HR), inner cotyledon (IC), outer cotyledon (OC), seed coat and endosperm (SE), were sampled from mature rapeseed (Brassica napus L.) by laser microdissection. Subsequently, major secondary metabolites, glucosinolates and sinapine, as well as three minor ones, a cyclic spermidine conjugate and two flavonoids, representing different compound categories, were qualified and quantified in dissected samples by high-performance liquid chromatography with diode array detection and mass spectrometry. No qualitative and quantitative difference of glucosinolates and sinapine was detected in embryo tissues (HR, IC and OC). On the other hand, the three minor compounds were observed to be distributed unevenly in different rapeseed tissues. The hypothetic biological functions of the distribution patterns of different secondary metabolites in rapeseed are discussed.