A tree peony RING-H2 finger protein, PsATL33, plays an essential role in cold-induced bud dormancy release by regulating gibberellin content.

Bud dormancy is crucial for woody perennial plants to resist low-temperature stress in winter. However, the molecular regulatory mechanisms underlying bud dormancy release are largely unclear. Here, a tree peony (Paeonia suffruticosa) transcript ARABIDOPSIS TOXICOS EN LEVADURA 33 (PsATL33), encoding a RING-H2 finger protein, was selected from previously generated RNA sequencing data of chilling-treated buds. The objective of this study is to investigate the role of PsATL33 in the regulation of cold-induced bud dormancy release. Subcellular localization assay revealed that PsATL33 was localized to the nucleus and plasma membrane. Reverse transcription-quantitative PCR analysis showed that PsATL33 was dramatically upregulated during cold-triggered bud dormancy release. Exogenous treatments with gibberellin (GA3) increased, but abscisic acid (ABA) inhibited the transcription of PsATL33. Ectopic transformation assay indicated that overexpression of PsATL33 in petunia promoted seed germination, plant growth, and axillary bud break. Silencing of PsATL33 in tree peony through virus-induced gene silencing assay delayed bud dormancy release. tobacco rattle virus (TRV)-PsATL33-infected buds exhibited reduced expression levels of dormancy break-related genes EARLY BUD-BREAK 1 (PsEBB1) and CARBOXYLESTERASE 15 (PsCXE15). Silencing of PsATL33 decreased the accumulation of bioactive GAs, GA1 and GA3, rather than ABA. Transcript levels of several genes involved in GA biosynthesis and signaling, including GA20-OXIDASE 1 (PsGA20ox1), GA3-OXIDASE 1 (PsGA3ox1), PsGA3ox3, GA2-OXIDASE 1 (PsGA2ox1), and GA-INSENSITIVE 1A (PsGAI1A), were changed by PsATL33 silencing. Taken together, our data suggest that PsATL33 functions as a positive regulator of cold-induced bud dormancy release by modulating GA production.

Transcriptome and volatile compounds analyses of floral development provide insight into floral scent formation in Paeonia lactiflora 'Wu Hua Long Yu'.

Herbaceous peony (Paeonia lactiflora) is a well-known ornamental plant in China, celebrated for its beautiful flowers that can emit fragrances. However, exact molecular mechanisms governing synthesis of floral volatiles within herbaceous peony remain unclear. To address this gap in knowledge, our study focused on analyzing the transcriptome and the levels of floral volatile compounds in P. lactiflora 'Wu Hua Long Yu' at different stages of flower development. Using gas chromatography-mass spectrometry (GC-MS), we obtained eighteen major volatile compounds, with monoterpenes being the dominant components among them. Our transcriptome analysis, based on pooled sequencing data, revealed the most differentially expressed genes (DEGs) existed between stages S1 and S3 of flower development. Among these DEGs, we identified 89 functional genes associated with the synthesis of volatile monoterpenes, with 28 of these genes showing a positive correlation with the release of monoterpenes. Specifically, key regulators of monoterpene synthesis in herbaceous peony appear to be 1-deoxy-D-xylulose 5-phosphate synthase (DXS), geranyl pyrophosphate synthase (GPPS), and terpene synthase (TPS). Additionally, our study identified some transcription factors (TFs) that may be involved in the biosynthesis of monoterpenes. These discoveries offer invaluable illumination into the intricate molecular underpinnings orchestrating the generation of floral fragrances in herbaceous peonies, and they offer a foundation for further research to identify and utilize candidate gene resources for this purpose.

Volatile Composition and Classification of Paeonia lactiflora Flower Aroma Types and Identification of the Fragrance-Related Genes.

Flower scent is one of the main ornamental characteristics of herbaceous peony, and the improvement of flower fragrance is a vital objective of herbaceous peony breeding. In this study, 87 herbaceous peony cultivars were divided into three groups (no/light fragrance, medium fragrance, and strong fragrance) based on their sensory evaluation scores, and 16 strong fragrance cultivars and one no fragrance cultivar were selected for subsequent analysis. Sixty-eight volatile components were detected in these 17 cultivars based on solid-phase microextraction (SPME) and gas chromatography/mass spectrometry (GC/MS), and 26 types were identified as important scent components. They were composed of terpenoids, benzenoids/phenylpropanoids, and fatty acid derivatives. According to the content and odor threshold of these main aroma components, the characteristic aroma substances of herbaceous peony were identified, including linalool, geraniol, citronellol, and phenylethyl alcohol (2-PE). The cultivars of strong scented herbaceous peony were divided into three types: rose scent, lily scent, and mixed scent. We explored the possible key genes of characteristic aroma substances in herbaceous peony petals with different odors through the qRT-PCR. The key genes encoding monoterpene biosynthesis were found to be PlDXS2, PlDXR1, PlMDS1, PlHDR1, PlGPPS3, and PlGPPS4. In addition, the linalool synthase (LIS) gene and the geraniol synthase (GES) gene were also found. PlAADC1, PlPAR1, and PlMAO1, related to the biosynthesis of 2-PE were detected, and the synthetic pathway of 2-PE was speculated. In conclusion, these findings revealed that the difference in gene expression of monoterpene and 2-PE synthesis pathway was related to the difference in the fragrance of herbaceous peony. This study explored the releasing pathway of herbaceous peony characteristic aroma substances and provided key genetic resources for fragrance improvement.

The Paeonia qiui R2R3-MYB Transcription Factor PqMYBF1 Positively Regulates Flavonol Accumulation.

Tree peony is a "spring colored-leaf" plant which has red leaves in early spring, and the red color of the leaves usually fades in late spring. Flavonols are one subgroup of flavonoids, and they affect the plant organs' color as co-pigments of anthocyanins. To investigate the color variation mechanism of leaves in tree peony, PqMYBF1, one flavonol biosynthesis-related MYB gene was isolated from Paeonia qiui and characterized. PqMYBF1 contained the SG7 and SG7-2 motifs which are unique in flavonol-specific MYB regulators. Subcellular localization and transactivation assay showed that PqMYBF1 localized to the nucleus and acted as a transcriptional activator. The ectopic expression of PqMYBF1 in transgenic tobacco caused an observable increase in flavonol level and the anthocyanin accumulation was decreased significantly, resulting in pale pink flowers. Dual-luciferase reporter assays showed that PqMYBF1 could activate the promoters of PqCHS, PqF3H, and PqFLS. These results suggested that PqMYBF1 could promote flavonol biosynthesis by activating PqCHS, PqF3H, and PqFLS expression, which leads metabolic flux from anthocyanin to flavonol pathway, resulting in more flavonol accumulation. These findings provide a new train of thought for the molecular mechanism of leaf color variation in tree peony in spring, which will be helpful for the molecular breeding of tree peony with colored foliage.

Diacylglycerol Acyltransferase 3(DGAT3) Is Responsible for the Biosynthesis of Unsaturated Fatty Acids in Vegetative Organs of Paeonia rockii.

'Diacylglycerol acyltransferase (DGAT)' acts as a key rate-limiting enzyme that catalyzes the final step of the de novo biosynthesis of triacylglycerol (TAG). The study was to characterize the function of the DGAT3 gene in Paeonia rockii, which is known for its accumulation of high levels of unsaturated fatty acids (UFAs). We identified a DGAT3 gene which encodes a soluble protein that is located within the chloroplasts of P. rockii. Functional complementarity experiments in yeast demonstrated that PrDGAT3 restored TAG synthesis. Linoleic acid (LA, C18:2) and α-linolenic acid (ALA, C18:3) are essential unsaturated fatty acids that cannot be synthesized by the human body. Through the yeast lipotoxicity test, we found that the yeast cell density was largely increased by adding exogenous LA and, especially, ALA to the yeast medium. Further ectopic transient overexpression in Nicotiana benthamiana leaf tissue and stable overexpression in Arabidopsis thaliana indicated that PrDGAT3 significantly enhanced the accumulation of the TAG and UFAs. In contrast, we observed a significant decrease in the total fatty acid content and in several major fatty acids in PrDGAT3-silenced tree peony leaves. Overall, PrDGAT3 is important in catalyzing TAG synthesis, with a substrate preference for UFAs, especially LA and ALA. These results suggest that PrDGAT3 may have practical applications in improving plant lipid nutrition and increasing oil production in plants.

The Paeonia qiui R2R3-MYB Transcription Factor PqMYB113 Positively Regulates Anthocyanin Accumulation in Arabidopsis thaliana and Tobacco.

Paeonia qiui is a wild species of tree peony native to China. Its leaves are purplish red from the bud germination to the flowering stage, and anthocyanin is the main pigment in purplish red leaves. However, the anthocyanin synthesis regulation mechanism in tree peony leaves remains unclear. In this study, an R2R3-MYB, PqMYB113 was identified from the leaves of P. qiui. Phylogenetic analysis revealed that PqMYB113 clustered with Liquidambar LfMYB113 and grape VvMYBA6. Subcellular location analysis showed that PqMYB113 was located in the cell nucleus. The transient reporter assay suggested that PqMYB113 was a transcriptional activator. The overexpression of PqMYB113 in Arabidopsis thaliana and tobacco (Nicotiana tabacum) resulted in increased anthocyanin accumulation and the upregulation of CHS, F3H, F3'H, DFR, and ANS. The dual luciferase reporter assay showed that PqMYB113 could activate the promoters of PqDFR and PqANS. Bimolecular fluorescence complementation assays and yeast two-hybrid assays suggested that PqMYB113 could form a ternary MBW complex with PqbHLH1 and PqWD40 cofactors. These results provide insight into the regulation of anthocyanin biosynthesis in tree peony leaves.

A Novel R2R3-MYB Transcription Factor PqMYB4 Inhibited Anthocyanin Biosynthesis in Paeonia qiui.

Paeonia qiui is a wild tree peony native to China. Its leaves show a clear purple-red color from the germination to the flowering stage, and it has high leaf-viewing value. A MYB transcription factor gene, designated as PqMYB4, was isolated from leaves of P. qiui based on transcriptome datas. The full-length cDNA of PqMYB4 was 693 bp, encoding 230 amino acids. Sequence alignment and phylogenetic analysis revealed that PqMYB4 was a R2R3-MYB transcription factor clustered with AtMYB4 in Arabidopsis thaliana. Moreover, it contained a C1 motif, an EAR repression motif and a TLLLFR motif in the C-terminal domains, which were unique in transcription repression MYB. Subcellular location analysis showed that PqMYB4 was located in the cell nucleus. PqMYB4 was highly expressed in the late stage of leaf development, and was negatively correlated with the anthocyanin content. The petiole of wild-type Arabidopsis seedlings was deeper in color than the transgenic lines of PqMYB4 and showed a little purple-red color. The seed coat color of Arabidopsis seeds that overexpressed PqMYB4 gene was significantly lighter than that of wild-type seeds. In transgenic Arabidopsis, the expression level of AtCHS, AtCHI, AtDFR and AtANS were down-regulated significantly. These results showed that PqMYB4 was involved in the negative regulation of anthocyanin biosynthesis in tree peony leaves, which can control the anthocyanin pathway genes. Together, these findings provide a valuable resource with which to further study the regulatory mechanism of anthocyanin biosynthesis in the leaf of P. qiui. They also benefit the molecular breeding of tree peony cultivars with colored leaf.

Biochemical and Comparative Transcriptomic Analyses Identify Candidate Genes Related to Variegation Formation in Paeonia rockii.

Paeonia rockii is a wild tree peony species with large and dark purple variegations at the base of its petals. It is the genetic resource for various variegation patterns in tree peony cultivars, which is in contrast to the pure white petals of Paeonia ostii. However, the molecular mechanism underlying the formation of variegation in this plant is still unknown. Here, we conducted Illumina transcriptome sequencing for P. rockii, P. ostii (with pure white petals) and their F1 individuals (with purple-red variegation). A total of 181,866 unigenes were generated, including a variety of unigenes involved in anthocyanin biosynthesis and sequestration and the regulation of anthocyanin biosynthesis. The dark purple or purple-red variegation patterns mainly occurred due to the proportions of cyanidin (Cy)- and peonidin (Pn)-based anthocyanins. The variegations of P. rockii exhibited a "Cy > Pn" phenotype, whereas the F1 progeny showed a "Pn > Cy" phenotype. The CHS, DFR, ANS, and GST genes might play key roles in variegation pigmentation in P. rockii according to gene expression and interaction network analysis. Two R2R3-MYB transcription factors (c131300.graph_c0 and c133735.graph_c0) regulated variegation formation by controlling CHS, ANS and GST genes. Our results indicated that the various variegation patterns were caused by transcriptional regulation of anthocyanin biosynthesis genes, and the transcription profiles of the R2R3-MYBs provided clues to elucidate the mechanisms underlying this trait. The petal transcriptome data produced in this study will provide a valuable resource for future association investigations of the genetic regulation of various variegation patterns in tree peonies.

Transcriptomic Analysis of Leaf in Tree Peony Reveals Differentially Expressed Pigments Genes.

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.

Transcriptomic Analysis Reveals Transcription Factors Related to Leaf Anthocyanin Biosynthesis in Paeonia qiui.

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.