Reuben Sheela, Rai Amit, Pillai Bhinu V S, Rodrigues Amrith, Swarup Sanjay
Department of Biological Sciences, National University of Singapore, 14, Science Drive 4, Singapore 117543.
J Exp Bot. 2013 Nov;64(16):5183-94. doi: 10.1093/jxb/ert310. Epub 2013 Oct 1.
Metabolic perturbations by a gain-of-function approach provide a means to alter steady states of metabolites and query network properties, while keeping enzyme complexes intact. A combination of genetic and targeted metabolomics approach was used to understand the network properties of phenylpropanoid secondary metabolism pathways. A novel quercetin oxidoreductase, QuoA, from Pseudomonas putida, which converts quercetin to naringenin, thus effectively reversing the biosynthesis of quercetin through a de novo pathway, was expressed in Arabidopsis thaliana. QuoA transgenic lines selected for low, medium, and high expression levels of QuoA RNA had corresponding levels of QuoA activity and hypocotyl coloration resulting from increased anthocyanin accumulation. Stems of all three QuoA lines had increased tensile strength resulting from increased lignification. Sixteen metabolic intermediates from anthocyanin, lignin, and shikimate pathways had increased accumulation, of which 11 paralleled QuoA expression levels in the transgenic lines. The concomitant upregulation of the above pathways was explained by a significant downregulation of the phenolamide pathway and its precursor, spermidine. In a tt6 mutant line, lignifications as well as levels of the lignin pathway metabolites were much lower than those of QuoA transgenic lines. Unlike QuoA lines, phenolamides and spermidine were not affected in the tt6 line. Taken together, these results suggest that phenolamide pathway plays a major role in directing metabolic intermediates into the lignin pathway. Metabolic perturbations were accompanied by downregulation of five genes associated with branch-point enzymes and upregulation of their corresponding products. These results suggest that gene-metabolite pairs are likely to be co-ordinately regulated at critical branch points. Thus, these perturbations by a gain-of-function approach have uncovered novel properties of the phenylpropanoid metabolic network.
通过功能获得方法进行的代谢扰动提供了一种改变代谢物稳态并探究网络特性的手段，同时保持酶复合物的完整性。采用遗传和靶向代谢组学方法相结合来了解苯丙烷类次生代谢途径的网络特性。来自恶臭假单胞菌的一种新型槲皮素氧化还原酶QuoA，可将槲皮素转化为柚皮素，从而通过从头合成途径有效地逆转槲皮素的生物合成，该酶在拟南芥中表达。选择QuoA RNA低、中、高表达水平的QuoA转基因系，其具有相应水平的QuoA活性以及因花青素积累增加而导致的下胚轴着色。所有三个QuoA系的茎由于木质化增加而具有更高的拉伸强度。花青素、木质素和莽草酸途径的16种代谢中间体积累增加，其中11种与转基因系中QuoA的表达水平平行。上述途径的同时上调是由酚酰胺途径及其前体亚精胺的显著下调所解释的。在tt6突变系中，木质化以及木质素途径代谢物的水平远低于QuoA转基因系。与QuoA系不同，酚酰胺和亚精胺在tt6系中不受影响。综上所述，这些结果表明酚酰胺途径在引导代谢中间体进入木质素途径中起主要作用。代谢扰动伴随着与分支点酶相关的五个基因的下调及其相应产物的上调。这些结果表明基因 - 代谢物对可能在关键分支点处受到协同调节。因此，通过功能获得方法进行的这些扰动揭示了苯丙烷类代谢网络的新特性。
