**1. Introduction**

*Rhododendron* is the largest genus belonging to the family Ericaceae, which is known for colorful flowers [1]. In recent years, varieties of *Rhododendron* have been grown as ornamental plants, making them become the most popular evergreen shrubs all over the world [2]. As one of the important characteristics of *Rhododendron*, color has long been a concern of breeders and consumers. The author (N.Z.) and her team found that there were a few white and pink flowers in the purple *Rhododendron pulchrum* Sweet community. Petals are an extremely essential part of numerous ornamental plants, and differences in petal color, tones, and intensity directly influence the ornamental value of a plant [3]. Previous studies have shown that flower color is attributed to specific pigments in petal cells, including flavonoids, carotenoids, and alkaloids [4,5]. In addition, flavonoids are major pigments in flowers that are responsible for the coloration of plant petals [6,7]. Flavonoids are naturally occurring polyphenols in plants. According to their structural characteristics, flavonoids are usually classified into anthocyanins and flavonoid alcohols [8,9]. Among them, anthocyanins are the main water-soluble pigments in flowers that constitute the color of plant petals. They are mainly accumulated in the vacuoles of petal epidermal cells and impart to the petals a colorful appearance from light pink to purple [10]. Color modification of flowers can be achieved by enhancing the accumulation of anthocyanins [11]. Flavonoids, on the other hand, have auxiliary effects on anthocyanins. The petals of plants with a higher concentration of flavonoids usually show bright colors [12]. In general, anthocyanins are

**Citation:** Zhu, N.; Zhou, C. Transcriptomic Analysis Reveals the Regulatory Mechanism of Color Diversity in *Rhododendron pulchrum* Sweet (Ericaceae). *Plants* **2023**, *12*, 2656. https://doi.org/10.3390/ plants12142656

Academic Editors: Aiping Song and Yu Chen

Received: 12 April 2023 Revised: 12 July 2023 Accepted: 13 July 2023 Published: 15 July 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

synthesized through the secondary metabolic pathway, which occurs in a wide range of plants, such as *Gerbera hybrida* and *Triticum aestivum* [13,14]. However, the molecular mechanisms regulating anthocyanin synthesis have not been elucidated in *R. pulchrum* due to the structural diversity of anthocyanins among the different plant species [15].

Anthocyanins are derived from the branches of the flavonoid biosynthesis pathway, which also leads to the production of isoflavonoids and flavonols. The anthocyanin biosynthesis pathway has been extensively studied in a range of plant species and involves several enzymes encoded by different structural genes [16,17]. First, phenylalanine ammonia-Lyase (PAL) catalyzes the deamination of phenylalanine to cinnamic acid in the initial step of the flavonoid pathway [18], while chalcone synthase (CHS) catalyzes the synthesis of naringenin chalcone with 4-coumaroyl CoA and malonyl CoA as substrates in the first committed step of flavonoid biosynthesis [19]. Subsequently, chalcone isomerase (CHI) catalyzes the conversion of naringenin chalcone to naringenin [20,21]. The naringenin is then catalyzed to dihydrokaempferol (DHK) via flavanone-3-hydroxylase (F3H). Moreover, DHK can be further hydroxylated by flavonoid 3 -hydroxylase (F3 H) to produce dihydroquercetin (DHQ). In the third stage, all obtained DHQ are reduced to leucocyanidin by dihydroflavonol 4-reductase (DFR) and then are further converted into anthocyanidins by anthocyanidin synthase (ANS). Of the main key enzymes discovered, CHS catalyzes the first step of flavonoid biosynthesis, and DFR is the first committed enzyme of anthocyanin biosynthesis [22].

It is known that the MBW complexes (MYB-*bHLH*-WD40) consist of the MYB transcription factors (TFs), *bHLH* TFs, and one WD-40 repeat factor (WDR) [23], which play important roles in regulation of the expression of the genes involved in the anthocyanin biosynthetic pathway at the transcriptional level [24]. The MYB regulator is a large family of proteins with diverse functions, and most of the MYB genes in plants belong to *R2R3-MYB* TFs. Several studies have shown that *R2R3-MYB* TFs control the transcriptional regulation of anthocyanin structural genes [25]. Another crucial TF regulating anthocyanin biosynthesis is the *bHLH* protein, which is critical for the activity of *R2R3-MYB*. For example, the *bHLH* TF in *Arabidopsis* interacts with the *R2R3-MYB* protein to regulate DFR gene expression in anthocyanin biosynthesis [26]. It was considered that the WDR proteins could interact with different *R2R3-MYB* and *bHLH* to form transcription complexes, which play vital roles in anthocyanin accumulation in vegetative tissues [27].

Currently, to our best knowledge, research on flower coloration in *R. pulchrum* is very limited, and the molecular mechanism regulating the flower coloration remains unknown. It would help to clarify the mechanisms controlling anthocyanin accumulation through the analysis of the expression pattern of the key genes related to color formation in *Rhododendron*. In recent years, with the development of next-generation sequencing, transcriptome sequencing (RNA-seq) has been widely used to identify differentially expressed genes (DEGs) in many plants [28,29]. Therefore, transcriptome sequencing of *R. pulchrum* flowers will provide meaningful knowledge to unravel the molecular mechanism of color formation. In this current study, four varieties with different petal colors were used as the experimental materials to determine the genotypic difference at the transcriptional level using RNA-seq technology. Our findings will provide a useful resource for further analyzing the molecular mechanism of color formation and intensity of *R. pulchrum*.
