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Qianqian Shi, Long Li, Lin Zhou and Yan Wang

Paeonia delavayi is a species endemic to Southwest China and an important genetic resource for flower color breeding of tree peonies. The mechanisms underlying the flower coloration of this plant have not been fully elucidated. In this article, the petals of yellow-colored individual (Pl) and purple–red-colored individual (Pd) of P. delavayi were studied. And anatomical observations revealed that a large amount of yellow protoplasts and a small amount of colorless protoplasts were located in the yellow-colored Pl petals, whereas a mixture of purple, red, and pink protoplasts were observed in the purple–red-colored Pd petals. The Pl cells were subrotund and flat, whereas the Pd cells were irregularly polygon-shaped and bulging. Chemical analyses were performed, and the results indicated that significant differences occurred between the cell sap pH of the Pl and Pd flowers and large differences occurred in the contents of Fe and Al between Pl and Pd. Cyanidin- and peonidin-based anthocyanins with flavones and flavonols as copigments determined the Pd flower color, whereas chalcone 2 ′G with apigenin 7-O-neohesperidoside and chrysoeriol 7-O-glucoside as copigments determined the yellow color of Pl. Correspondingly, the genes dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS) were significantly highly expressed in Pd, whereas chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavone synthase (FNS), flavonol synthase (FLS), flavonoid 7-O-glycosyltransferase (7GT), and 2′4′6′4-tetrahydroxychalcone 2′-glucosyltransferase (THC) had high transcript levels in Pl relative to Pd. The results indicate that the color variation of P. delavayi petals may be related to a delicately controlled balance of the aforementioned factors.

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Lin Zhou, Qianqian Shi, Yan Wang, Kui Li, Baoqiang Zheng and Kun Miao

Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) is a sensitive and widely used technique for gene expression analysis that depends on stability of the reference genes used for data normalization. Tree peony (Paeonia suffruticosa), known as one of the most famous traditional ornamental plants in China, is very popular in both domestic and international markets for its showy and colorful flowers. To date, no systematic studies on reference genes have been performed in tree peony with different flower colors. In this study, we evaluated the expression stability of 12 candidate reference genes in different tissues and five flower developmental stages of tree peony with six different colors by three algorithms: geNorm, NormFinder, and BestKeeper. The results showed that protein phosphatase 2A (PP2A), ubiquitin protein ligase (UPL), and ubiquitin (UBQ) were the most stable genes across all samples. Helicase, alpha-tubulin (TUA), and eukaryotic translation initiation factor 5A (EIF5A) also exhibited high expression stability in different tissues, in samples with different colors, and at different flower developmental stages. According to the geNorm analysis, the combination of two most stable reference genes was optimal for normalization in all tested sample sets in this study. To further validate the suitability of the reference genes identified in this study, the expression patterns of two putative homologs of chalcone synthase gene (PsCHS1) and chalcone isomerase gene (PsCHI1) were studied at different developmental stages of white flowers. The results provide information for transcriptional analyses in future studies of gene expression on tree peony flower development and pigmentation.

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Qianqian Shi, Xiaoxiao Zhang, Xiang Li, Lijuan Zhai, Xiaoning Luo, Jianrang Luo, Lixia He, Yanlong Zhang and Long Li

Tree peony (Paeonia sp.) is a popular traditional ornamental plant in China. Among the nine wild species, Paeonia rockii displays wide-ranging, deep purple variegation at the base of the petals, whereas Paeonia ostii exhibits purely white petals. Overall, the posttranscriptional regulation involved in tree peony flower opening and pigmentation remains unclear. To identify potential microRNAs (miRNAs) involved in flower variegation, six small RNA libraries of P. ostii and P. rockii petals at three different opening stages were constructed and sequenced. Using Illumina-based sequencing, 22 conserved miRNAs and 27 novel miRNAs were identified in P. rockii and P. ostii petals. Seventeen miRNAs were differentially expressed during flower development, and several putative target genes of these miRNAs belonged to transcription factor families, such as Myb domain (MYB), and basic helix-loop-helix (bHLH) transcription factors. Furthermore, an integrative analysis of the expression profiles of miRNAs and their corresponding target genes revealed that variegation formation might be regulated by miR159c, miR168, miR396a, and novel_miR_05, which target the MYB transcription factors, chalcone synthase (CHS), and ABC transporter. Our preliminary study is the first report of miRNAs involved in Paeonia flower pigmentation. It provides insight regarding the molecular mechanisms underlying the regulation of flower pigmentation in tree peony.