Sinapine Content Research Articles

Genetic Variation and Association Analysis of Phenolic Compounds in Rapeseed (Brassica napus L.) Mutant Lines Using Genotyping-by-Sequencing (GBS)

The concentration of phenolic compounds in rapeseed is important because they are either anti-nutritional compounds or directly related to antioxidant activity. In this study, single nucleotide polymorphisms (SNPs) were identified using genotyping-by-sequencing (GBS), and an association study was conducted to investigate phenolic content in 95 rapeseed mutant lines derived from gamma rays and their original cultivar. A total of 3,196,318 SNPs were detected, resulting in the identification of a set of 70,208 union SNPs used to perform association studies. Seven compounds were identified, sinapine being the major phenolic compound (91.2–99.2%) in all genotypes. An association study was conducted for seven compounds and total phenolic content (TPC). It identified 241 SNPs that were significantly associated with these compounds, total sinapine content (TSC), and TPC. Based on the SNP markers detected, BnaC02g20420D was associated with dihexose, BnaC08g30570D with progoitrin, BnaA01g06890D with methyl sinapate, BnaA09g16810D with sinapine(4-O-8′)guaiacyl, BnaCnng39930D with trans-sinapine 1, BnaA07g31720D with trans-sinapine 2, BnaC03g31950D with sinapoyl malate, and BnaAnng27700D with TPC. These were selected as candidate genes that may play a key role in rapeseed. The SNP markers associated with these key phenolic compounds can be used as targets in breeding programs to reduce anti-nutritional components.

Analysis of the effects of sulfamethoxazole on the secondary metabolites and antioxidants in oilseed rape (Brassica napus L.) and the underlying mechanisms

The secondary metabolism of plants is key for mediating responses to environmental stress, but few studies have examined how the relationship between secondary metabolism and the stress response of plants is affected by exposure to antibiotics. Here, we studied the effects of sulfamethoxazole (SMZ) on the secondary metabolism and antioxidant activity of oilseed rape (Brassica napus L.). SMZ significantly affected the growth of rape seedlings. Low and high concentrations of SMZ induced the production of a large number of reactive oxygen species (ROS) in rape seedlings, which damaged cells. SMZ stress altered the activity of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), as well as the content of malondialdehyde (MDA). SMZ promoted the activities of phenylalanine ammonia lyase (PAL), tyrosine ammonia lyase (TAL), cinnamic acid-4-hydroxylase (C4H), and 4-coumaric acid: coenzyme A ligase (4CL) by activating the phenylpropanoid pathway. The content of secondary metabolites changed. The content of phenolic acids and flavonoids increased, and the content of sinapine and anthocyanins was altered to cope with the oxidative damage induced by antibiotics. Transcriptomic and metabolomic analysis showed that differentially expressed genes and differentially expressed metabolites were mainly involved in Phenylpropanoid biosynthesis. SMZ alters the secondary metabolites of rapeseed, which mitigates the deleterious effects of stress, by modulating upstream secondary metabolism pathways and the production of plant hormones involved in signal transduction. In sum, these results provide a new perspective on the effects of SMZ on plants relative to secondary metabolites and improve our understanding of the toxicity of SMZ.

Diverting phenylpropanoid pathway flux from sinapine to produce industrially useful 4-vinyl derivatives of hydroxycinnamic acids in Brassicaceous oilseeds

Sinapine (sinapoylcholine) is an antinutritive phenolic compound that can account for up to 2% of seed weight in brassicaceous oilseed crops and reduces the suitability of their protein-rich seed meal for use as animal feed. Sinapine biosynthesis draws on hydroxycinnamic acid precursors produced by the phenylpropanoid pathway. The 4-vinyl derivatives of several hydroxycinnamic acids have industrial applications. For example, 4-vinyl phenol (4-hydroxystyrene) is a building block for a range of synthetic polymers applied in resins, inks, elastomers, and coatings. Here we have expressed a modified bacterial phenolic acid decarboxylase (PAD) in developing seed of Camelina sativa to redirect phenylpropanoid pathway flux from sinapine biosynthesis to the production of 4-vinyl phenols. PAD expression led to a ∼95% reduction in sinapine content in seeds of both glasshouse and field grown C. sativa and to an accumulation of 4-vinyl derivatives of hydroxycinnamic acids, primarily as glycosides. The most prevalent aglycone was 4-vinyl phenol, but 4-vinyl guaiacol, 6-hydroxy-4-vinyl guaiacol and 4-vinylsyringol (Canolol) were also detected. The molar quantity of 4-vinyl phenol glycosides was more than twice that of sinapine in wild type seeds. PAD expression was not associated with an adverse effect on seed yield, harvest index, seed morphology, storage oil content or germination in either glasshouse or field experiments. Our data show that expression of PAD in brassicaceous oilseeds can supress sinapine accumulation, diverting phenylpropanoid pathway flux into 4-vinyl phenol derivatives, thereby also providing a non-petrochemical source of this class of industrial chemicals.

Influence of extraction technology on rapeseed oil functional quality: a study on rapeseed polyphenols.

Extraction technology can influence the vegetable oil functional quality. Polyphenols in rapeseed oil have been proved to be beneficial for cardiovascular health. In this study, we evaluated the effect of extraction methods on the functional quality of rapeseed oil from the perspective of phenolic compounds. The results showed that hot pressing produces the highest amount of phenolic compounds in rapeseed oil. Its most abundant phenolic compound, sinapine (9.18 μg g-1), showed the highest activity in inhibiting anaerobic choline metabolism with an EC50 value of 1.9 mM, whose downstream products are related to cardiovascular diseases. Molecular docking and molecular dynamics (MD) simulations revealed that sinapine exhibits good binding affinity toward CutC, and CutC-sinapine is a stable complex with fewer conformational fluctuations and similar tightness. Taken together, hot pressing can be considered the best extraction method for rapeseed oil from the perspective of phenolic compounds.

Assessment of Antinutritional Compounds and Chemotaxonomic Relationships between Camelina sativa and Its Wild Relatives.

We compared the secondary metabolite composition in seeds of Camelina sativa and its wild relatives to identify potential germplasm with reduced levels of antinutritional compounds. Twenty Camelina accessions, from five different species, were analyzed by liquid chromatography mass spectrometry and subjected to principal component analysis, which revealed that Camelina spp. separated into distinct chemotaxonomic groups. Three major glucosinolates (GSs) were identified in our study, namely, 9-methylsulfinylnonyl GS (GS9), 10-methylsulfinyldecyl GS (GS10), and 11-methylsulfinylundecylGS (GS11). While there were differences in total GS levels, species-specific patterns for GS9 and GS11 were noted. Sinapine content ranged between 1.4 and 5.6 mg/g FW, with the lowest levels observed in C. laxa and C. sativa. Lignin levels were also lowest in C. sativa, with most accessions containing less than 6 mg/g FW. Our results show that wild Camelina spp. have distinct metabolomes, and based on their levels of major antinutritionals, some could be incorporated into breeding programs with C. sativa.

Changes in Glucosinolates, Sinapine and Flavonols during Seed Development of Camelina sativa (L.) Crantz

Camelina sativa (L.) Crantz is an oilseed crop whose oil is used as biofuel and the meal as animal feed. While the seed composition is widely documented, little is known about variations in the concentration of some compounds during seed development of camelina. The aim of this study was to characterize changes in the content of glucosinolates, sinapine, sinapic acid and flavonols (quercetin and koempferol) at different stages of seed maturation. Glucosinolates are accumulated in camelina seed in the first 4 weeks after pollination, while the other substances were transiently accumulated during development and may play a role in seed development.

Development of transgenic Brassica juncea lines for reduced seed sinapine content by perturbing phenylpropanoid pathway genes.

Sinapine is a major anti-nutritive compound that accumulates in the seeds of Brassica species. When ingested, sinapine imparts gritty flavuor in meat and milk of animals and fishy odor to eggs of brown egg layers, thereby compromising the potential use of the valuable protein rich seed meal. Sinapine content in Brassica juncea germplasm ranges from 6.7 to 15.1 mg/g of dry seed weight (DSW) which is significantly higher than the prescribed permissible level of 3.0 mg/g of DSW. Due to limited natural genetic variability, conventional plant breeding approach for reducing the sinapine content has largely been unsuccessful. Hence, transgenic approach for gene silencing was adopted by targeting two genes—SGT and SCT, encoding enzymes UDP- glucose: sinapate glucosyltransferase and sinapoylglucose: choline sinapoyltransferase, respectively, involved in the final two steps of sinapine biosynthetic pathway. These two genes were isolated from B. juncea and eight silencing constructs were developed using three different RNA silencing approaches viz. antisense RNA, RNAi and artificial microRNA. Transgenics in B. juncea were developed following Agrobacterium-mediated transformation. From a total of 1232 independent T0 transgenic events obtained using eight silencing constructs, 25 homozygous lines showing single gene inheritance were identified in the T2 generation. Reduction of seed sinapine content in these lines ranged from 15.8% to 67.2%; the line with maximum reduction had sinapine content of 3.79 mg/g of DSW. The study also revealed that RNAi method was more efficient than the other two methods used in this study.

Glucosinolates and Sinapine in Camelina Meal

Forty seven accessions of camelina (Camelina sativa L. Crantz) were analyzed for glucosinolates (GSLs) and sinapine in defatted meal. These antinutritional compounds are undesirable in camelina meal for use in animal feeding and therefore we show their variability to identify the best varieties for future breeding programs. Total GSLs ranged from 19.6 to 40.3 mmol Kg-1 dry weight (DW) with an average of 30.3 mmol kg-1 DW. Great variability has also been observed in the levels of individual GSLs (GSL1, GSL2 and GSL3), so that the content of GSL1 and GSL3 were not correlated to each other in the accessions of camelina. Five out of six winter forms of camelina showed low content of GSLs. Sinapine ranged from 1.09 to 4.75 g Kg-1 DW with an average of 2.57 g kg-1 DW. The sinapine content was not correlated with that of GSLs. The use of camelina meal is only limited by the presence of GSLs while sinapine content can be ignored in camelina varieties.

Reduction in sinapine content in rapeseed (Brassica napus L.) by induced mutations in sinapine biosynthesis genes

Sinapine is the most prominent antinutri- tive 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 composi- tion 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.

Endogenous Phenolics in Hulls and Cotyledons of Mustard and Canola: A Comparative Study on Its Sinapates and Antioxidant Capacity

Endogenous sinapic acid (SA), sinapine (SP), sinapoyl glucose (SG) and canolol (CAN) of canola and mustard seeds are the potent antioxidants in various lipid-containing systems. The study investigated these phenolic antioxidants using different fractions of canola and mustard seeds. Phenolic compounds were extracted from whole seeds and their fractions: hulls and cotyledons, using 70% methanol by the ultrasonication method and quantified using HPLC-DAD. The major phenolics from both hulls and cotyledons extracts were SP, with small amounts of SG, and SA with a significant difference of phenolic contents between the two seed fractions. Cotyledons showed relatively high content of SP, SA, SG and total phenolics in comparison to hulls (p < 0.001). The concentration of SP in different fractions ranged from 1.15 ± 0.07 to 12.20 ± 1.16 mg/g and followed a decreasing trend- canola cotyledons > mustard cotyledons > mustard seeds > canola seeds > mustard hulls > canola hulls. UPLC-tandem Mass Spectrometry confirmed the presence of sinapates and its fragmentation in these extracts. Further, a high degree of correlation (r = 0.93) was noted between DPPH scavenging activity and total phenolic content.

Influence of Microwaves Treatment of Rapeseed on Phenolic Compounds and Canolol Content

Rapeseeds were treated with microwaves under 800 W for 0, 1, 2, 3, 4, 5, 6, 7, and 8 min at a frequency of 2450 MHz, and oil was extracted with a press to investigate the influence on phenolic compounds, including sinapine, the main free phenolic acids, and canolol content in the rapeseeds and oil from them. The results indicated that sinapine and sinapic acid was the main phenolic compound and free phenolic acid in the rapeseed, respectively, and canolol was the main phenolic compound in the oil from rapeseed by cold press. Microwave treatment significantly influenced phenolic compounds content in the rapeseeds and oil from them. The sinapine, sinapic acid, and canolol content in rapeseed first increased and then decreased depending on the period of microwave radiation (p < 0.05). The canolol content of 7 min microwave pretreatment rapeseed increased to the maximum and was approximately six times greater than that of the unroasted rapeseed. The amount of canolol formed was significantly correlated with the content of sinapic acid and sinapine (for sinapic acid, r = -0.950, p < 0.001, for sinapine, r = -0.828, p < 0.05) and also the loss of sinapic acid and sinapine (for sinapic acid, r = 0.997, p < 0.001, for sinapine, r = 0.952, p < 0.05) during roasting. There were differences in the transfer rate of difference phenolic compounds to the oil extracted by press. Almost all of the sinapine remained in the cold-pressed cake and only 1.4-2.7% of the sinapic acid, whereas approximately 56-83% of the canolol was transferred to the oil. The transfer ratio of canolol significantly increased with microwave radiation time (p < 0.001). Microwave pretreatment of rapeseed benefited improving the oxidative stability of oil.

Evaluation of Camelina sativa (L.) Crantz meal as an alternative protein source in ruminant rations

Camelina sativa (CS) is an oilseed crop used for biofuel production. By-products from oil extraction are high in protein and can be used in ruminant rations; more information about their nutritive value is required also considering the antinutrional factor content of the by-products. The aim of this study was to evaluate the nutritive value of CS meal genotypes in comparison with canola. Ten CS genotypes and one canola cultivar were evaluated. Meals were obtained from seeds after solvent oil extraction. CS average crude protein (CP) content (g kg⁻¹ dry matter) was 457. Numerical differences in lysine and sulfur amino acid content were observed among CS genotypes. Glucosinolate (mmol kg⁻¹) content was higher for CS (23.1) than canola (7.2). Sinapine content (g kg⁻¹) was lower for CS (2.79) than for canola (4.32). Differences were observed among CS genotypes for rumen undegraded protein (RUP). Average RUP (g kg⁻¹ CP) was 316 for CS and 275 for canola. CS meal has potential for use in ruminant rations as a high-quality protein source. In vivo studies are needed to compare CS with other protein sources used in cattle rations. Implementation of breeding programs for improved meal quality is recommend.

Glucosinolates and other seed quality traits of resynthesized Brassica napus L. derived from domesticated and wild Brassica taxa

Resynthesized Brassica napus L. genotypes can be used to increase the variation in the rapeseed genepool. We evaluated seed quality parameters of resynthesized lines that originated from crosses of oilseed B. rapa L. with domesticated (vegetable) B. oleracea L., and resynthesized lines that were obtained from crosses of B. rapa oilseed cultivars with wild B. oleracea ssp. oleracea and with seven Brassica species from the B. oleracea group (B. bourgeaui Kuntze, B. cretica Lam., B. incana Ten., B. hilarionis Post, B. montana Pourret, B. rupestris Raf., B. villosa Bivona-Bernardi). Twenty-three resynthesized lines and ten cultivars were grown in 1 year and six locations in Germany and the United Kingdom. The mean seed oil, phytosterole, and sinapine contents of the resynthesized lines were lower than in the cultivars, while protein, glucosinolate, and erucic acid contents were higher, respectively. Nearly all resynthesized lines derived from wild Brassica taxa showed a glucosinolate pattern very different from all cultivars and from nearly all resynthesized lines obtained from crosses with domesticated B. oleracea. The newly developed wild Brassica based resynthesized lines represent an immense source of genetic variation that has not been used to full capacity up to now.

Antioxidant Extraction from Mustard (Brassica juncea) Seed Meal Using High‐Intensity Ultrasound

Brassicaceae oilseeds provide feedstocks for the biofuels industry, but value-added coproducts are necessary to supply financial incentives for increased production. Our objective was to use high-intensity ultrasound to optimize extraction of antioxidants from mustard (Brassica juncea) seed meal. The ultrasound-assisted extraction (UAE) variables included temperature, solvent-to-material ratio, sonication duration, and EtOH concentration. Extracts were analyzed for total phenolics content (TPC), antioxidant activity, and sinapine content. Conventional extraction using water and 70% EtOH (v/v) at 80°C for 3×30min yielded 7.83 ± 0.07 and 8.81 ± 0.17 mg sinapic acid equivalents (SAE)/g meal, respectively. UAE extraction at 40°C for 30min yielded similar phenolics content (8.85 ± 0.33 mg SAE/g meal) as conventional hot ethanolic extraction, but required less time and lower temperature. The highest TPC (13.79 ± 0.38 mg SAE/g meal) was in the 7-d aqueous extracts. Sonicated solutions of pure sinapine and sinapic acid showed 1st-order reaction kinetics with greater degradation of isolated compounds than those present in extracts. Sinapine contained in extracts showed insignificant (P< 0.05) degradation after 30min of sonication. Our research indicates that ultrasound treatment can assist the extraction of antioxidants from B. juncea meal by reducing both the temperature and time requirement without significant degradation of the primary antioxidants present.

Effect of Microwave Treatment on Sinapic Acid Derivatives in Rapeseed and Rapeseed Meal

AbstractMicrowave treatment is a new method for rapeseed processing. To better understand the benefits of microwave pretreatment on rapeseed processing, qualitative and quantitative analysis of sinapic acid derivatives is needed. In this study, the effect of microwave treatment on the content of sinapic acid derivatives in rapeseed and the effect of cold‐press and solvent extraction on the content of sinapic acid derivatives in rapeseed meal were evaluated using ultrahigh pressure liquid chromatography. It was found that during the microwave treatment, the content of sinapine and sinapic acid in the rapeseed increased slightly at the outset of treatment and significantly decreased thereafter. Following 7 min of microwave treatment, the content of sinapine in rapeseed exhibited its largest decrease of 16.7 %. By contrast, microwave heating of the rapeseed increased the canolol content significantly. We employed two sample paired t tests as a means to analyze the difference between meal and defatted cold‐pressed cake. The results indicated that the content of sinapine and sinapic acid in defatted cake was significantly higher than that in meal, while the different extraction methods (solvent extraction and cold‐press) did not appear to have a significant influence on the content of canolol in meal (p ≤ 0.05).

Antinutritive Compounds in Twelve &amp;lt;i&amp;gt;Camelina sativa &amp;lt;/i&amp;gt;Genotypes

Camelina sativa is an oilseed crop becoming important in North America and Europe for biodiesel production. The use of Camelina flours in animal diet may be limited by antinutritive compounds. The content of glucosinolates, phytic acid, sinapine and condensed tannins was evaluated in twelve accessions of Camelina sativa. All compounds showed significant differences among genotypes. Only the concentration of glucosinolates in the flour deserves attention, while the content of phytic acid, sinapine and condensed tannins are to acceptable levels. Camelina showed the presence of three different glucosinolates (GSL1, GSL2 and GSL3) in the flour, with differences among genotypes regarding the relative abundance of each glucosinolate. The content of glucosinolates is inversely correlated with that of sinapine. The glucosinolate content in Camelina flour has to be reduced to increase the use of this flour in animal diet, but avoiding altering the sinapine content.

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.

Determination of Sinapic Acid Derivatives in Canola Extracts Using High‐Performance Liquid Chromatography

A high-performance liquid chromatographic (HPLC) method with diode array detection (DAD) was used to determine the total phenolics, including sinapic acid derivatives in canola. Ten Western Canadian canola seeds, six other commodity canola seeds, their corresponding press cakes and meals were analyzed. Seeds of European 00 rapeseed and Brassica Juncea (Indian mustard) were included for comparison. Phenolic compounds were separated using a gradient elution system of water–methanol-ο-phosphoric acid solution with a flow rate of 0.8 ml/min. In addition to sinapine (SP) and sinapic acid (SA), sinapoyl glucose (SG) is reported in the methanolic extracts. The detection and quantification limits of these compounds were 0.20–0.40 and 0.50–0.80 μg/ml, respectively with recovery values over 98.0%. The content of total phenolics, SP, SA and SG in canola extracts ranged from 9.16 to 16.13, 6.39 to 12.28, 0.11 to 0.59 and 1.36 to 7.50 mg/g, respectively with significant differences among varieties.

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.

Manipulation of sinapine, choline and betaine accumulation in Arabidopsis seed: Towards improving the nutritional value of the meal and enhancing the seedling performance under environmental stresses in oilseed crops

Sinapoylcholine (sinapine) is the most abundant antinutritional phenolic compound in cruciferous seeds. The quaternary ammonium compounds, choline, betaine and N,N-dimethylglycine, reside along a biosynthetic pathway linked to the synthesis of membrane phospholipids and neurotransmitters with various biological functions. In chicken, choline intake is required for optimal egg-laying performance and a choline supplement in diet is positively correlated with weight gains. A key step in sinapine biosynthesis is catalyzed by sinapoylglucose: choline sinapoyltransferase (SCT; EC 2.3.1.91) to form an ester linkage with sinapoylglucose and choline. The objective of this work was to reduce the sinapine content and simultaneously enhance free choline levels in cruciferous seeds. We report here the characterization of an Arabidopsis T-DNA insertion mutant lacking SCT activity in the seed. The sct mutant seeds contain less than 1% of sinapine and a more than 2-fold increase in free choline compared with wild type. We further expressed a choline oxidase (COX; EC 1.1.3.17) gene from Arthrobacter pascens in the Arabidopsis sct mutant and wild-type background using a napin gene promoter to convert free choline into betaine, an effective stress-alleviating compound in plants. Betaine was not detected in WT or sct mutant seeds. The sct+COX seeds contain nearly 2-fold greater levels of betaine relative to WT+COX seeds, demonstrating a positive correlation between endogenous choline and betaine production. In contrast, stable comparable levels of free choline were detected between sct+COX and WT+COX plants suggesting choline homeostasis likely prevent high levels of betaine production in the seed of transgenic COX plants.