Sunflower (Helianthus annuus L.) is an important oil crop, the secondary metabolites of it include many compounds such as flavonoids and lignin. However, the research on the biosynthesis of phenolic compounds in sunflowers is still scarce. Cinnamate 4-hydroxylase (C4H) belongs to the cytochrome P450-dependent monooxygenase family and is involved in the synthesis of many phenolic compounds, but C4H in sunflowers has not yet been cloned and functionally characterized. In this study, we screened three C4H genes from the sunflower transcriptome and genomic databases, named HaC4H1, HaC4H2, and, HaC4H3, respectively. In heterologous expression experiments, we had improved a method from previous studies by the addition of restriction sites to make it easier to express multiple C4H functions and suitable for in vitro activity verification. HaC4Hs without the N-terminal membrane anchor region was fused with a redox partner of Arabidopsis thaliana cytochrome P450 enzyme (CYP450) by the method and functionally expressed in E. coli and the results showed that these three enzymes catalyzed the formation of p-coumaric acid. To further investigate whether our fusion protein approach is applicable to other C4Hs, we used this method to explore the functions of C4H from Peucedanum praeruptorum and Angelica decursiva, and they can also convert trans-cinnamic acid to p-coumaric acid. The gene expression profile showed that all three HaC4H genes showed the highest transcription levels in the roots and might be up-regulated by MeJA. In summary, these results reveal the function of HaC4Hs in sunflower and provide a simpler way to explore C4H and even other cytochrome P450 enzymes in prokaryotic expression systems.

向日葵（Helianthus annuus L.）是重要的油料作物，其次生代谢产物包括许多化合物，如黄酮类化合物和木质素。但是，关于向日葵中酚类化合物的生物合成的研究仍然很少。肉桂酸4-羟化酶（C4H）属于细胞色素P450依赖的单加氧酶家族，参与许多酚类化合物的合成，但是向日葵中的C4H尚未被克隆和功能表征。在这项研究中，我们从向日葵转录组和基因组数据库中筛选了三个C4H基因，分别命名为HaC4H1，HaC4H2和HaC4H3， 分别。在异源表达实验中，我们通过添加限制性酶切位点改进了以前的研究方法，使其更易于表达多种C4H功能，并适合进行体外活性验证。通过该方法，将不具有N端膜锚定区的HaC4Hs与拟南芥细胞色素P450酶（CYP450）的氧化还原伴侣融合，并在大肠杆菌中进行功能表达，结果表明这三种酶催化了对香豆酸的形成。。为了进一步研究我们的融合蛋白方法是否适用于其他C4H，我们使用了这种方法来探索白uce（Peucedanum praeruptorum）中C4H的功能。和当归，它们还可以将反式肉桂酸转化为对香豆酸。基因表达谱表明，所有三个HaC4H基因在根中均显示最高的转录水平，并且可能被MeJA上调。总之，这些结果揭示了HaC4Hs在向日葵中的功能，并提供了一种简单的方法来探索原核表达系统中的C4H甚至其他细胞色素P450酶。