Kuczynski Cathleen, McCorkle Sean, Keereetaweep Jantana, Shanklin John, Schwender Jorg
Biology Department, Brookhaven National Laboratory, Upton, NY, United States.
Front Plant Sci. 2022 Aug 5;13:955589. doi: 10.3389/fpls.2022.955589. eCollection 2022.
The transcription factor () is known as a master regulator of fatty acid synthesis in developing oilseeds of and other species. is known to directly stimulate the expression of many fatty acid biosynthetic enzymes and a few targets in the lower part of the glycolytic pathway. However, it remains unclear to what extent and how the conversion of sugars into fatty acid biosynthetic precursors is controlled by 1. To shortlist possible gene targets for future experimental validation, here we present a strategy that combines phylogenetic foot printing of cis-regulatory elements with additional layers of evidence. Upstream regions of protein-encoding genes in were searched for the previously described DNA-binding consensus for WRI1, the ASML1/WRI1 (AW)-box. For about 900 genes, AW-box sites were found to be conserved across orthologous upstream regions in 11 related species of the crucifer family. For 145 select potential target genes identified this way, affinity of upstream AW-box sequences to WRI1 was assayed by Microscale Thermophoresis. This allowed definition of a refined WRI1 DNA-binding consensus. We find that known WRI1 gene targets are predictable with good confidence when upstream AW-sites are phylogenetically conserved, specifically binding WRI1 in the assay, positioned in proximity to the transcriptional start site, and if the gene is co-expressed with WRI1 during seed development. When targets predicted in this way are mapped to central metabolism, a conserved regulatory blueprint emerges that infers concerted control of contiguous pathway sections in glycolysis and fatty acid biosynthesis by WRI1. Several of the newly predicted targets are in the upper glycolysis pathway and the pentose phosphate pathway. Of these, plastidic isoforms of fructokinase (3) and of phosphoglucose isomerase (1) are particularly corroborated by previously reported seed phenotypes of respective null mutations.
转录因子（）被认为是油菜及其他物种发育中的油籽中脂肪酸合成的主要调节因子。已知它能直接刺激许多脂肪酸生物合成酶的表达以及糖酵解途径下游的一些靶点。然而，尚不清楚糖转化为脂肪酸生物合成前体在多大程度上以及如何受到1的控制。为了筛选出未来实验验证可能的基因靶点，我们在此提出一种策略，该策略将顺式调控元件的系统发育足迹与其他证据层相结合。在油菜中编码蛋白质的基因上游区域搜索先前描述的WRI1的DNA结合共有序列，即ASML1/WRI1（AW）框。在十字花科11个相关物种的直系同源上游区域中，发现约900个基因的AW框位点是保守的。对于以此方式鉴定出的145个选定潜在靶基因，通过微量热泳测定上游AW框序列与WRI1的亲和力。这使得能够定义一个更精确的WRI1 DNA结合共有序列。我们发现，当上游AW位点在系统发育上保守、在实验中特异性结合WRI1、位于转录起始位点附近且该基因在种子发育过程中与WRI1共表达时，已知的WRI1基因靶点可以很有把握地被预测到。当以这种方式预测的靶点映射到中心代谢时，会出现一个保守的调控蓝图，推断WRI1对糖酵解和脂肪酸生物合成中相邻途径部分的协同控制。几个新预测的靶点位于糖酵解上游途径和磷酸戊糖途径中。其中，果糖激酶（3种）和磷酸葡萄糖异构酶（1种）的质体同工型特别得到了先前报道的各自无效突变体种子表型的证实。
