Integrated metabolomics and transcriptomics reveal flavonoids glycosylation difference in two Citrus peels

HIGHLIGHTS

• Citrus HJH peel has 1.5-fold total flavonoid than SHHJ peel.

• Of 203 differentially accumulated metabolites (DAMs) are identified.

• Of 3517 differentially expressed genes are identified.

• Flavonoids (51.2%) contribute to the major DAMs.

• Seven GT enzymes might devote to the high level of glycosylated flavonoid in HJH.

Abstract

Citrus reticulata ‘Huajuhong’ (HJH) and C. reticulata ‘Sanhuhongju’ (SHHJ) have distinct level of bioactive ingredients including flavonoid derivatives in peels meanwhile its underlying mechanism remain unclear. Integrated analysis of metabolome and transcriptome was comparatively investigated differences of HJH and SHHJ peels. The total flavonoid in HJH peels was significantly higher than that in SHHJ. Of 203 differentially accumulated metabolites (DAMs) and 3,517 differentially expressed genes (DEGs) were identified. KEGG enrichment analysis indicated that flavonoid pathway was overrepresented. Among the DAMs, the major components were flavonoids (104, 51.2%) followed by phenolic acids (30, 14.7%). Seven of top 10 flavonoid glycosides in HJH were highly accumulated and positively correlated with 10 glucosyltransferase genes. Collectively, higher content of flavonoid glycosides in HJH peels than in SHHJ peels might contribute to the distinct health-promoting effect, which provides a novel insight into improving the fruit quality of citrus as a health-promoting food resource.

Introduction

The fruits in Citrus L. genus of Rutaceae family have a high reputation for edible and medicinal characteristics (Lv et al., 2015). According to the pharmacopeia of the People's Republic of China, several citrus varieties, including Pericarpium Citri Reticulatae, have been regarded as the source of common Chinese medicines due to richness in bioactive components such as flavonoids. Previous studies documented that flavonoids were abundantly distributed in flesh and peel of citrus (Tripoli et al., 2007), and that the specific bioactive substances in citrus peels were mainly polyphenolic compounds (Gómez-Mejía et al., 2019). Noticeably, the types of bioactive compounds in citrus peels were usually higher than those in fleshes and seeds (Manners, 2007; Manthey and Grohmann, 2001). As the largest secondary metabolites in plants, flavonoids play a vital role in human health (Koolaji et al., 2020; Nan et al., 2016). Previous studies have proved that those compounds in Citrus have significant effect on resolving phlegm and relieving cough, anti-inflammation, anti-oxidation, immune regulation, prevention and treatment of diabetes, anti-tumor, etc. (Peng et al., 2019; X et al., 2020). In particular, the dried epicarp of Chinese herb Exocarpium Citri grandis not only contains pharmacologically active compound naringin, but also has many clinical applications in the prevention and treatment of multiple respiratory diseases, including coronavirus disease 2019 (Su et al.,2020). Currently, the flavonoid biosynthesis in citrus peel remains unclear, especially for medicinal varieties, which seriously impede the salvage of citrus peel.

Flavonoid can be modified by glycosylation, methylation and acylation leading to a high degree of chemical diversity, resulting in different biological activities (Ito et al., 2017; Yang et al., 2016). Glycosylation is one of the most common modification processes, including two main types of O-glycosylation and C-glycosylation, which could improve the physiological properties such as solubility and stability of flavonoids. There were three identified flavone glycosides (sudachiins B, sudachiins C and sudachiins D) from the green peels of C. suduchi (Horie et al., 1986). Moreover, many studies have shown that the natural flavonoids almost exist as O- or C-glycosides forms in plants, such as vitexin, isovitexin, orientin, isoorientin and their relative glycosides (Nan et al., 2016). In addition, the dietary flavonoid C-glycosides have hypoglycemic and hypolipidemic effects on type 2 diabetes through inhibiting age and digestive enzymes, stimulating glycogen storage and activating insulin signals (Xiao et al., 2016). In most cases, flavonoid C-glycosides have a higher antioxidant capacity than their corresponding flavonoid O-glycosides and aglycones (Yang et al., 2018). Consequently, the glycosylated flavonoid might contribute to a large extent to the medicinal characteristics of citrus peel.

To date, the study on flavonoid in citrus peels mainly focused on content determination, fruit coloration and pharmacological action (Xi et al., 2014). Studies on the biosynthetic mechanism of main active ingredients in citrus varieties are largely delayed. For instance, a highly accumulated unique flavonoid (Tricin 4′ -O-syringyl alcohol) was recently identified in ‘Dahongpao’ and could be used as a marker to distinguish the other citrus varieties (Wang et al., 2019). In addition, the peel of C. reticulata cv. ‘Shatangju’ infected with HLB accumulated less flavonoid than control, which contributed to less pigmentation and antifungal activity of mandarin fruit peel (Wang et al., 2020). Due to the fast development of transcriptomics and metabolomics, many active compounds and related metabolic pathways have been rapidly identified in non-model medicinal plants (He et al., 2013), such as Mirabilis himalaica (Gu et al., 2018), Fagopyrum tataricum (Li et al., 2019), Ziziphora bungeana (He et al., 2020).

C. reticulata ‘Huajuhong’ (HJH), referred to as ‘Huajuhong’ in Chinese, is a kind of important medical citrus and mainly distributes in Guangdong, Guangxi, Hunan, Jiangxi and Yunnan provinces, which has found in the Southern Song Dynasty with effect on resolving phlegm and relieving cough. While C. reticulata ‘Sanhuhongju’ (SHHJ), is a variety of citrus with superior quality and originated from Xingan County, Jiangxi Province, China. Recent study shows that the composition and content of flavonoid in dried C. reticulata peels positively associate with antioxidant and anti-inflammatory activities (Chen et al., 2017). Although both HJH and SHHJ varieties are used medically, their chemical compositions and pharmacological effects are largely distinct.

Due to the announcement of whole Citrus genome (Xu et al., 2013), it provides a good chance to globally investigate the underlying regulatory mechanism at transcription and metabolic level of bioactive medicinal compounds in citrus peel. In this study, the total flavonoid content, transcriptome and metabolome in the peels of HJH and SHHJ were comparatively analyzed. Our finding indicated that some differentially expressed genes and corresponding candidate metabolites were identified, which may function as important medical bioactive ingredients. The biosynthesis of glycosylated flavonoid in citrus peel was discussed.