Department of Pharmacognosy, College of Pharmacy, Second Military Medical University, Shanghai 200433, China.
Chemical Experiment Teaching Center, College of Pharmacy, Second Military Medical University, Shanghai 200433, China.
Molecules. 2019 Mar 21;24(6):1128. doi: 10.3390/molecules24061128.
Flavonoids with various structures play a vital role in plant acclimatization to varying environments as well as in plant growth, development, and reproduction. Exogenous applications of ethylene and 1-aminocyclopropane carboxylic acid (ACC), could affect the accumulation of flavonoids. Very few attempts have been made to investigate the effect of 1-aminocyclopropane carboxylic acid oxidase (ACO), a unique enzyme that catalyzes ACC to ethylene, on genes and metabolites in the flavonoid biosynthetic pathway. In this study, two ACOs in safflower (CtACOs) were cloned, and then transgenic safflower with overexpressed CtACO1 was generated through the Agrobacterium-mediated floral dipping method.
CtACO1 and CtACO2 were both characterized by the 2-oxoglutarate binding domain RxS and the ferrous iron binding site HxDxnH as ACOs from other plants. However, the transcript levels of CtACO1 in flowers at stages I, II, III, and IV were all higher than those of CtACO2. At the cellular level, by using electroporation transformation, CtACO1 was found to be localized at the cytomembrane in onion epidermal cells. CtACO1 overexpression had varying effects on genes involved in the ethylene and flavonoid biosynthetic pathways. The metabolites analysis showed that CtACO1 overexpression lines had a higher accumulation of quercetin and its glycosylated derivatives (quercetin 3-β-d-glucoside and rutin). In contrast, the accumulation of quinochalcones (hydroxysafflor yellow A and carthamin), kaempferol glycosylated derivatives (kaempferol-3--β-rutinoside and kaempferol-3--β-d-glucoside), apigenin, and luteolin in CtACO1 overexpression lines were decreased.
This study confirmed the feasibility of applying the floral dipping method to safflower and showed a novel regulatory effect of CtACO1 in the flavonoid biosynthetic pathway. It provides hypothetical and practical groundwork for further research on regulating the overall metabolic flux of flavonoids in safflower, particularly hydroxysafflor yellow A and other quinochalcones, by using appropriate genetic engineering strategies.
具有各种结构的类黄酮在植物适应不同环境以及生长、发育和繁殖中起着至关重要的作用。外源施用乙烯和 1-氨基环丙烷羧酸（ACC）会影响类黄酮的积累。很少有人尝试研究 1-氨基环丙烷羧酸氧化酶（ACO），一种催化 ACC 转化为乙烯的独特酶，对类黄酮生物合成途径中的基因和代谢物的影响。在这项研究中，克隆了两种红花中的 ACO（CtACO1 和 CtACO2），然后通过农杆菌介导的花浸法生成了过表达 CtACO1 的转基因红花。
CtACO1 和 CtACO2 都具有 2-氧戊二酸结合域 RxS 和亚铁结合位点 HxDxnH，这是其他植物中 ACO 的特征。然而，在 I、II、III 和 IV 期花中 CtACO1 的转录水平均高于 CtACO2。在细胞水平上，通过电穿孔转化发现 CtACO1 定位于洋葱表皮细胞的细胞质膜上。CtACO1 的过表达对参与乙烯和类黄酮生物合成途径的基因有不同的影响。代谢物分析表明，CtACO1 过表达系中槲皮素及其糖基化衍生物（槲皮素 3-β-d-葡萄糖苷和芦丁）的积累更高。相比之下，CtACO1 过表达系中 quinochalcones（羟基红花黄色 A 和红花黄素）、kaempferol glycosylated derivatives（kaempferol-3--β-rutinoside 和 kaempferol-3--β-d-glucoside）、apigenin 和 luteolin 的积累减少。
本研究证实了采用花浸法在红花中的可行性，并展示了 CtACO1 在类黄酮生物合成途径中的新的调控作用。它为进一步研究通过适当的基因工程策略调节红花中类黄酮的整体代谢通量，特别是羟基红花黄色 A 和其他 quinochalcones，提供了假设和实践基础。
