Elsevier

Gene

Volume 758, 20 October 2020, 144950
Gene

Research paper
Functional characterization of cinnamate 4-hydroxylase from Helianthus annuus Linn using a fusion protein method

https://doi.org/10.1016/j.gene.2020.144950Get rights and content

Highlights

Abstract

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.

Introduction

Sunflower (Helianthus annuus L.) is an annual, bee-pollinated plant domesticated around 4000 BP in North America (Putt, 1997). As an essential oil crop and dried fruit, sunflower has an important economic and ornamental value. Sunflower is both a symbol of society and a major research focus of scientists. The researches on the chemical constituents of sunflowers started very early. A variety of compounds with complex structures isolated from sunflowers mainly included flavonoids, as well as some lignin, terpenoids and coumarin. As a wide class of plant polyphenols, flavonoids gradually draw the investigator's attention to the account of various biological activities (Sarbu et al., 2019). Flavonoids, which act as signal molecules, phytoalexins, allelochemicals, antibacterial defense compounds, and antidote, can protect plants from different biotic and abiotic stresses, such as UV damage and mechanical injury (Havsteen, 2002). Recent work that has provided some genomic information for sunflowers makes it easier to study the polyphenols biosynthesis process (Badouin et al., 2017). In short, based on the important role of secondary metabolites of the phenylpropanoid pathway, such as phenolic compounds in plant physiology, it is important to study the phenylpropanoid pathway related to the synthetic pathway of phenolic compounds in sunflower.

Cinnamate 4-hydroxylase (C4H) is the second key enzyme in the phenylpropanoid pathway. The key reactions in the phenylpropanoid pathway produce important secondary metabolites involved in plant development, disease resistance, and defense responses (Dixon and Paiva, 1995). The phenylpropanoid pathway involves three enzymes: firstly, L-phenylalanine is deaminated to produce trans-cinnamic acid (t-CA) by the action of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) (Lois et al., 1989). Subsequently, the formation of p-coumaric acid under the action of cinnamic acid 4-hydroxylase (C4H, EC 1.14.13.11) (Hubner et al., 2003). Finally, the conversion of p-coumaric acid to 4-Coumaroyl-CoA catalyzed by 4-coumarate: coenzyme A ligase (4CL, EC 6.2.1.12) (Vogt, 2010). 4-Coumaroyl-CoA is the precursor for many phenylpropanoid compounds including flavonoids, coumarins, and lignin (Jürgen Ehlting, 2006) (Fig. 1). C4H is a member of the CYP73 family that belongs to cytochrome P450 monooxygenases (CYP450s) (Chapple, 1998), which was discovered early in plants and was one of the typical P450s identified in plants (Nair and Vining, 1965, Russell and Conn, 1967). So, this enzyme is the most fully studied in plant cytochrome P450s and shows high specificity of its substrate trans-cinnamic acid (Kumar et al., 2013, Park et al., 2010, Tuan et al., 2010, Xia et al., 2017, Xu et al., 2010). The conversion of other structurally closely related compounds cannot occur or have low conversion efficiency. In general, trans-conformation and the presence of the carboxy group are essential for efficient catalysis (Werck-Reichhart, 1995).

Since the functional expression of CYP450s directly in the prokaryotic expression system was difficult. In the initial experiments, the proteins were mainly expressed in the modified yeast cells overexpressing CYP450 reductase (CPR, EC 1.6.2.4) (Pierrel et al., 1994, Urban et al., 1997, Urban et al., 1994). When the cytochrome P450 enzymes catalyze a lot of oxidation reactions it needs a CPR as a redox partner for supplying two electrons from NADPH through the FAD and FMN cofactors (Vermilion et al., 1981). CPR is confirmed to be present in most organisms, such as yeasts, plants, and animals. The function of CPR is to provide electrons to CYP450s to support P450-catalyzed oxidation reactions (Lee et al., 2014, Ro et al., 2002, Yang et al., 2010). In addition, the CYP450s and CPR are supposed to commonly localized at the cytoplasmic side of the endoplasmic reticulum (ER) membrane (Achnine et al., 2004). The N-terminus of both CYP450s and CPR contains a hydrophobic membrane-binding domain, which makes the expression of these proteins in prokaryotic microbes difficult because of low solubility. In short, the functional expression of CYP450s is difficult in prokaryotic microbes such as E. coli which lacks necessary organelles and CPR to support the function of plant CYP450s (Leonard et al., 2006). However, if the hydrophobic membrane-binding domain of CYP450s and CPR is removed and the fusion protein is expressed in E. coli to transfer electrons from NADPH, this problem can be solved (Leonard et al., 2006).

Although some studies have shown that some CYP450s, especially some C4H, can be correctly functionalized in prokaryotic microbes (Kim et al., 2009, Leonard et al., 2006, Li et al., 2018). However, it was difficult to express large fusion proteins in prokaryotic expression systems. Therefore, in some studies, only in vivo activity verification was performed (Li et al., 2018). So, it was still necessary to improve generic fusion protein methods for expressing various P450s in E. coli, due to the complexity of functional expression in yeast, and the difficulty of expressing large fusion proteins in prokaryotic expression systems and the difficulty of in vitro activity exploration in prokaryotic expression systems. Sunflower as an important plant, using the improved E. coli expression system to verify its C4H activity, and even using this method to explore the activity of other P450s related to important secondary metabolites of sunflower is very valuable.

In this study, we have screened and cloned three C4H genes in the current online access to the H. annuus (common sunflower) transcriptome and genomic database. We named it HaC4H1, HaC4H2, HaC4H3 and analyzed their expression profiles in different organs of sunflower and under various abiotic stresses. To validate the function of HaCHs, we improved an expression system containing modified Arabidopsis thaliana CYP450 redox partner ATR2 (belongs to CPR) by adding restriction sites and increasing linker. High-Performance Liquid Chromatography-Electrospray ionization mass spectrometry (HPLC-ESI-MS) analysis showed these three enzymes catalyzed the formation of p-coumaric acid. Meanwhile, the in vitro activity was successfully verified on the basis of concentration and purification. Subsequently, to clarify whether this fusion protein method can be applicable to the expression of C4H from other plants in E. coli, we used this expression vector to verify the activity of PpC4H (C4H from Peucedanum praeruptorum) and AdC4H (C4H from Angelica decursiva) and they can also convert trans-cinnamic acid to p-coumaric acid.

Section snippets

Plant materials and chemicals

The seeds of The H. annuus are freshly harvested from the Xinjiang province of China. Sunflowers were grown in a light incubator with a long light cycle of 16 h light and 8  h dark every 24 h, the temperature was maintained at 30 ℃, the relative humidity was 50–65% and the light intensity was 3000 lx. Two months after plant germination, two-month-old seedlings were used for subsequent experiments.

Clone of HaC4Hs, PpC4H, and AdC4H

For the functional characterization of HaC4Hs, total RNA was extracted from H. annuus leaf powder

Identification and cloning of the cDNA encoding C4H in H. annuus

Using C4H sequences reported previously and the genomic sequence (accession no. HanXRQr1.0) and the transcriptome database of H. annuus (download from https://www.sunflowergenome.org/transcriptome/), a BLAST search was conducted. According to the E-value of alignments and after removing redundancies, three C4Hs were identified and cloned. We named them HaC4H1, HaC4H2 and HaC4H3. The ORF lengths of HaC4H1 and HaC4H3 were 1518 bp (encoding 506 residues) but HaC4H2 were 1521 bp (encoding 507

Discussion and conclusion

Sunflower has a good meaning as an important cash crop and an ornamental plant. Therefore, studying the key enzymes in metabolic pathways in sunflower is very important to understand and even modify plant metabolic pathways. C4H is a key enzyme in the core phenylpropanoid pathway and is involved in the synthesis and regulation of downstream flavonoids, lignins, and coumarins (Werck-Reichhart, 1995). But in the initial experiments, the proteins were mainly expressed in the modified yeast (Jürgen

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was financially supported by the “Double First-Class” University project (CPU2018GY08, China), the 111 Project from Ministry of Education of China and the State Administration of Foreign Export Affairs of China (B18056), and the Drug Innovation Major Project (2018ZX09711-001-007)

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