An Expanded Role for WRINKLED1 Metabolic Control Based on Combined Phylogenetic and Biochemical Analyses

During triacylglycerol biosynthesis in developing oilseeds of , fatty acid production is regulated by the seed-specific transcription factor WRINKLED1 (WRI1). WRI1 is known to directly stimulate the expression of fatty acid biosynthetic enzymes and a few targets in glycolysis. However, it remains unclear to what extent and how the conversion of sugars into fatty acid biosynthetic precursors in seeds is controlled by WRI1. Based on a previously reported DNA binding motif for WRI1, the ASML1/WRI1 (AW)- box, we developed a comparative genomics approach to search for conserved binding motifs in upstream regions of protein-encoding genes and orthologous regions of 11 other Brassicaceae species. The AW-box was over-represented across orthologs for 915 genes. Among these, 73 genes with functions in the biosynthesis of fatty acids and triacylglycerols and in glycolysis were enriched. For 90 AW-box sequences associated with these target genes, binding affinity to heterologously expressed WRI1 protein was determined using Microscale Thermophoresis. Sites with low dissociation constants are preferentially located close to the transcriptional start site and are highly conserved between the 12 Brassicaceae species. Most of the associated genes were found to be co-expressed with WRI1 during seed development. When 46 automatically and manually curated genes containing conserved AW-sites with high binding affinity are mapped to central metabolism, a conserved regulatory blueprint emerges that infers concerted control of contiguous pathway sections in fatty acid biosynthesis and glycolysis. Among unexpectedly identified putative targets of WRI1 are plastidic fructokinase, phosphoglucose isomerase and several transcription factors.