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

Abstract

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.

Pleiotropic changes in Arabidopsis f5h and sct mutants revealed by large-scale gene expression and metabolite analysis

“…NMR-based conWrmation of sinapoylglucoside, salicylic acid glucoside, and quercetin-3-O-rhamnoside Given the importance of understanding pleiotropic eVects of mutations on secondary metabolism, we previously used HPLC to compare diVerences in accumulation of phenolic compounds in leaves and seeds (Huang et al 2008;Bhinu et al 2009a) rather than performing a complete metabolome analysis. However, as the use of HPLC and MS limits the identiWcation of complex phenolic mixtures that have a rapid turnover (Sakakibara et al 2003), we used NMR to identify variations in levels of sinapoylglucoside, salicylic acid glucoside, and quercetin-3-O-rhamnoside in the mutants.…”

“…SCT is the last enzyme of the pathway responsible for the formation of sinapoylcholine (sinapine), a compound unique to cruciferous seeds (Shirley et al 2001;Milkowski et al 2004). Of several key enzymes of the phenylpropanoid pathway, down regulation of F5H alone (Nair et al 2000) or in combination with SCT (Bhinu et al 2009a) resulted in 40-90% sinapine reduction in transgenic B. napus seed.…”

“…While mutations in F5H and SCT genes lead to reduced sinapine content in members of Brassicaceae (Nair et al 2000;Shirley et al 2001;Bhinu et al 2009a), there are no deWnitive studies on the impact of these mutations on agronomic traits. In addition, given the common pathway and precursor shared between the sinapine, Xavonoid, lignin, and other branches, it is interesting to investigate how mutations in F5H and SCT genes aVect the accumulation of other secondary metabolites such as Xavonoids in plants.…”

The Phenylpropanoid Pathway in Arabidopsis

“…Because of the antinutritive properties of sinapoylcholine, engineering sinapate ester metabolism to reduce its biosynthesis in the seeds of B. napus could improve the nutritive quality of canola meal Milkowski and Strack, 2010). Two SCT genes (BnSCT1 and BnSCT2) have been identified in B. napus since the characterization of the Arabidopsis SCT, and metabolic engineering projects are currently underway to eliminate the biosynthesis of choline in B. napus seeds (Milkowski et al, 2004;Huang et al, 2008;Weier et al, 2008;Bhinu et al, 2009;Milkowski and Strack, 2010).…”

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

“…Another suppression strategy aimed at silencing the gene encoding sinapoylglucose:choline sinapoyltransferase, the final enzyme in sinapine biosynthesis, led to a significant reduction in the level of sinapine. Unfortunately, the decrease in sinapine content was counterbalanced by an increase in the level of sinapoylglucose, the metabolic precursor of sinapine (Weier et al, 2008;Bhinu et al, 2009). Metabolites were analyzed via routine GC-MS-based metabolite profiling and are presented as fold change of genetically modified over wild-type samples (n = 8 pools of 20 seeds).…”