Tree peony (Paeonia suffruticosa Andr.) is a popular ornamental plant in China due to its showy and colorful flowers. However, yellow-colored flowers are rare in both wild species and domesticated cultivars. The molecular mechanisms underlying yellow pigmentation remain poorly understood. Here, petal tissues of two tree peony cultivars, “High Noon” (yellow flowers) and “Roufurong” (purple–red flowers), were sampled at five developmental stages (S1–S5) from early flower buds to full blooms. Five petal color indices (brightness, redness, yellowness, chroma, and hue angle) and the contents of ten different flavonoids were determined. Compared to “Roufurong,” which accumulated abundant anthocyanins at S3–S5, the yellow-colored “High Noon” displayed relatively higher contents of tetrahydroxychalcone (THC), flavones, and flavonols but no anthocyanin production. The contents of THC, flavones, and flavonols in “High Noon” peaked at S3 and dropped gradually as the flower bloomed, consistent with the color index patterns. Furthermore, RNA-seq analyses at S3 showed that structural genes such as PsC4Hs, PsDFRs, and PsUFGTs in the flavonoid biosynthesis pathway were downregulated in “High Noon,” whereas most PsFLSs, PsF3Hs, and PsF3’Hs were upregulated. Five transcription factor (TF) genes related to flavonoid biosynthesis were also upregulated in “High Noon.” One of these TFs, PsMYB111, was overexpressed in tobacco, which led to increased flavonols but decreased anthocyanins. Dual-luciferase assays further confirmed that PsMYB111 upregulated PsFLS. These results improve our understanding of yellow pigmentation in tree peony and provide a guide for future molecular-assisted breeding experiments in tree peony with novel flower colors.

Real-time reverse transcription quantitative PCR has become a common method for studying gene expression, however, the optimal selection of stable reference genes is a prerequisite for obtaining accurate quantification of transcript abundance. Suitable reference genes for RT-qPCR have not yet been identified for Chinese prickly ash (Zanthoxylum bungeanum Maxim.). Chinese prickly ash is the source of an important food seasoning in China. In recent years, Chinese prickly ash has also been developed as a medicinal plant. The expression stabilities of ten genes (18S, 28S, EF, UBA, UBQ, TIF, NTB, TUA, RPS, and TIF5A) were evaluated in roots, stems, leaves, flowers and fruits at five developmental stages and also under stress from cold, drought, and salt. To do this we used three different statistical algorithms: geNorm, NormFinder and BestKeeper. Among the genes investigated, UBA and UBQ were found to be most stable for the different cultivars and different tissues examined, UBQ and TIF for fruit developmental stage. Meanwhile, EF and TUA were most stable under cold treatment, EF and UBQ under drought treatment and NTB and RPS under salt treatment. UBA and UBQ for all samples evaluated were most stably expressed, but 18S, TUA and RPS were found to be generally unreliable as reference genes. Our results provide a basis for the future selection of reference genes for biological research with Chinese prickly ash, under a variety of conditions.

Tree peony (Paeonia suffruticosa Andrews) is an important traditional flower in China. Besides its beautiful flower, the leaf of tree peony has also good ornamental value owing to its leaf color change in spring. So far, the molecular mechanism of leaf color change in tree peony is unclear. In this study, the pigment level and transcriptome of three different color stages of tree peony leaf were analyzed. The purplish red leaf was rich in anthocyanin, while yellowish green leaf was rich in chlorophyll and carotenoid. Transcriptome analysis revealed that 4302 differentially expressed genes (DEGs) were upregulated, and 4225 were downregulated in the purplish red leaf vs. yellowish green leaf. Among these DEGs, eight genes were predicted to participate in anthocyanin biosynthesis, eight genes were predicted involved in porphyrin and chlorophyll metabolism, and 10 genes were predicted to participate in carotenoid metabolism. In addition, 27 MYBs, 20 bHLHs, 36 WD40 genes were also identified from DEGs. Anthocyanidin synthase (ANS) is the key gene that controls the anthocyanin level in tree peony leaf. Protochlorophyllide oxido-reductase (POR) is the key gene which regulated the chlorophyll content in tree peony leaf.

Paeonia qiui is a wild species of tree peony. P. qiui has good ornamental value owing to its leaf color change in spring. So far, the molecular mechanism of leaf color change in P. qiui is unclear. This study analyzes the anthocyanin level and transcriptome of two different color stages in P. qiui leaf. The purplish-red leaf stage is rich in anthocyanin, while the green leaf has very low levels of anthocyanin. Transcriptome analysis reveals that 6678 differentially-expressed genes (DEGs) are up-regulated, and 14,667 are down-regulated in the purplish-red leaf. Among these DEGs, 40 MYB (v-myb avian myeloblastosis viral oncogene homolog) genes, 40 bHLH (MYC-like basic helix–loop–helix) genes, and 15 WD40 (WD-repeat protein) genes were found. Based on phylogenetic and alignment analysis with the deduced amino acid sequences with known transcription factors, Unigene0024459 (MYB1) is likely the R2R3-MYB that promotes anthocyanin biosynthesis; Unigene0050761 (MYB2) is likely the R2R3-MYB that represses anthocyanin biosynthesis; Unigene0005081 (bHLH1) and Unigene0006146 (WD40-1) are likely the bHLH and WD40 that participate in regulating anthocyanin biosynthesis. Additionally, quantitative RT-PCR results confirmed the transcriptome analyses for key genes.

Eucommia ulmoides Oliver is widely distributed in China. This species has been used mainly in medicine due to the high concentration of chlorogenic acid (CGA), flavonoids, lignans, and other compounds in the leaves and barks. However, the categories of metabolites, dynamic changes in metabolite accumulation and overall molecular mechanisms involved in metabolite biosynthesis during E. ulmoides leaf growth and development remain unknown. Here, a total of 515 analytes, including 127 flavonoids, 46 organic acids, 44 amino acid derivatives, 9 phenolamides, and 16 vitamins, were identified from four E. ulmoides samples using ultraperformance liquid chromatography–mass spectrometry (UPLC-MS) (for widely targeted metabolites). The accumulation of most flavonoids peaked in growing leaves, followed by old leaves. UPLC-MS analysis indicated that CGA accumulation increased steadily to a high concentration during leaf growth and development, and rutin showed a high accumulation level in leaf buds and growing leaves. Based on single-molecule long-read sequencing technology, 69,020 transcripts and 2880 novel loci were identified in E. ulmoides. Expression analysis indicated that isoforms in the flavonoid biosynthetic pathway and flavonoid metabolic pathway were highly expressed in growing leaves and old leaves. Co-expression network analysis suggested a potential direct link between the flavonoid and phenylpropanoid biosynthetic pathways via the regulation of transcription factors, including MYB (v-myb avian myeloblastosis viral oncogene homolog) and bHLH (basic/helix-loop-helix). Our study predicts dynamic metabolic models during leaf growth and development and will support further molecular biological studies of metabolite biosynthesis in E. ulmoides. In addition, our results significantly improve the annotation of the E. ulmoides genome.