*Article* **Di**ff**erential Regulation of Anthocyanins in Green and Purple Turnips Revealed by Combined De Novo Transcriptome and Metabolome Analysis**

**Hongmei Zhuang <sup>1</sup> , Qian Lou <sup>2</sup> , Huifang Liu <sup>1</sup> , Hongwei Han <sup>1</sup> , Qiang Wang <sup>1</sup> , Zhonghua Tang 3,4, Yanming Ma <sup>4</sup> and Hao Wang 1,\***


Received: 17 August 2019; Accepted: 31 August 2019; Published: 6 September 2019

**Abstract:** Purple turnip *Brassica rapa* ssp. rapa is highly appreciated by consumers but the metabolites and molecular mechanisms underlying the root skin pigmentation remain open to study. Herein, we analyzed the anthocyanin composition in purple turnip (PT) and green turnip (GT) at five developmental stages. A total of 21 anthocyanins were detected and classified into the six major anthocynanin aglycones. Distinctly, PT contains 20 times higher levels of anthocyanins than GT, which explain the difference in the root skin pigmentation. We further sequenced the transcriptomes and analyzed the differentially expressed genes between the two turnips. We found that PT essentially diverts dihydroflavonols to the biosynthesis of anthocyanins over flavonols biosynthesis by strongly down-regulating one flavonol synthase gene, while strikingly up-regulating dihydroflavonol 4-reductase (DFR), anthocyanidin synthase and UDP-glucose: flavonoid-3-*O*-glucosyltransferase genes as compared to GT. Moreover, a nonsense mutation identified in the coding sequence of the DFR gene may lead to a nonfunctional protein, adding another hurdle to the accumulation of anthocyanin in GT. We also uncovered several key members of MYB, bHLH and WRKY families as the putative main drivers of transcriptional changes between the two turnips. Overall, this study provides new tools for modifying anthocyanin content and improving turnip nutritional quality.

**Keywords:** pigment; turnip; gene expression; antioxidant; nutritional quality

## **1. Introduction**

Turnip (*Brassica rapa* ssp. rapa), belongs to the Cruciferae family and represents one of the most important leaf and root vegetable crops for human consumption and animal fodder in China and throughout East Asia. Turnip vegetables provide dietary fiber, vitamin C, high amounts of glucosinolates [1–3], and are also an important source of dietary phenolic and other bioactive compounds [4,5]. There are several turnip varieties with purple colored root skin, which are highly appreciated by consumers. Similar to turnip, there are various *B. rapa* subspecies enriched with purple pigments previously characterized as anthocyanins [6].

Anthocyanins are secondary metabolites with health-promoting virtues, such as anti-oxidation, anti-mutation, prevention of cardiovascular disease, liver protection, and inhibiting the metastasis of tumor cells [7–12]. Besides, they also play important fundamental physiological functions in plants, including UV protection, pigmentation of flowers and fruits to attract pollinators and for seed dispersal, and responses to biotic and environmental stresses [13–20]. Therefore, the identification, analysis and

genetic manipulation of anthocyanin metabolites have become an important topic in plant secondary metabolite research [21].

The biosynthesis and accumulation of anthocyanins are determined by metabolic networks correlated with the expression of several genes and regulatory factors [22]. During the past decades, extensive studies have been conducted to elucidate the biosynthetic pathway of anthocyanins in plants. Progressively, it has become evident that the anthocyanin biosynthetic pathway is a very well conserved network in plant species [23]. It starts with the chalcone synthase (CHS) mediated synthesis of naringenin chalcone from 4-coumaroyl-CoA and malonyl-CoA. Then, naringenin chalcone is isomerized by chalcone isomerase (CHI) to naringenin. Flavanone 3-hydroxylase (F3H) converts naringenin into dihydrokaempferol which can be further hydroxylated by flavonoid 30 -hydroxylase (F30H) or flavonoid 3 0 ,50 -hydroxylase (F305 0H) into two other dihydroflavonols, dihydroquercetin and dihydrotricetin, respectively. Then, the three dihydroflavonols are converted into colorless leucoanthocyanidins by dihydroflavonol 4-reductase (DFR) and subsequently to colored anthocyanidins by anthocyanidin synthase (ANS). Anthocyanidins are glycolsylated to facilitate their accumulation in cells by the enzyme flavonoid 3-*O*-glucosyltransferase (UFGT) and might be further acylated with aromatic acyl groups by acyltransferases [18,22]. Although a well conserved biosynthetic pathway in plants, several studies have shown the species-specific peculiarity of anthocyanin regulation. For example, the numbers of structural genes (CHS, F3H, F30H, CHI, DFR, ANS, UFGT, F305 0H, etc.) vary considerably across species as do their expression levels [24–27]. Also, genetic mutations, microRNAs, transcription factors such as MYB, bHLH, WD40, WRKY, NAC, etc., have been linked to the regulation of anthocyanin biosynthetic structural genes through varying complex mechanisms among plants [14,26–41]. Therefore, in order to pinpoint the major players associated with quantitative and qualitative variations of anthocyanins in plant, a thorough investigation is necessary.

Herein, we investigated the anthocyanin compositions at different developmental stages in root skin of two turnip varieties (purple turnip and green turnip) widely grown in Xinjiang (China). In addition, we generated extensive transcriptome data and profiled the key genes involved in the differential pigmentation. The goal of this work was to elucidate the molecular and metabolic mechanisms underlying the differential pigmentation in turnips, as a foundation for the development of turnip varieties that are rich in anthocyanin compounds to meet the increasing demand for health-promoting components in our daily diet.

## **2. Results**
