Synergistic actions of three MYB transcription factors underpin blotch formation in tree peony.

Blotches in floral organs attract pollinators and promote pollination success. Tree peony (Paeonia suffruticosa Andr.) is an internationally renowned cut flower with extremely high ornamental and economic value. Blotch formation on P. suffruticosa petals is predominantly attributed to anthocyanin accumulation. However, the endogenous regulation of blotch formation in P. suffruticosa remains elusive. Here, we identified the regulatory modules governing anthocyanin-mediated blotch formation in P. suffruticosa petals, which involves the transcription factors PsMYB308, PsMYBPA2, and PsMYB21. PsMYBPA2 activated PsF3H expression to provide sufficient precursor substrate for anthocyanin biosynthesis. PsMYB21 activated both PsF3H and PsFLS expression and promoted flavonol biosynthesis. The significantly high expression of PsMYB21 in non-blotch regions inhibited blotch formation by competing for anthocyanin biosynthesis substrates, while conversely, its low expression in the blotch region promoted blotch formation. PsMYB308 inhibited PsDFR and PsMYBPA2 expression to directly prevent anthocyanin-mediated blotch formation. Notably, a smaller blotch area, decreased anthocyanin content, and inhibition of anthocyanin structural gene expression were observed in PsMYBPA2-silenced petals, while the opposite phenotypes were observed in PsMYB308-silenced and PsMYB21-silenced petals. Additionally, PsMYBPA2 and PsMYB308 interacted with PsbHLH1-3, and their regulatory intensity on target genes was synergistically regulated by the PsMYBPA2-PsbHLH1-3 and PsMYB308-PsbHLH1-3 complexes. PsMYB308 also competitively bound to PsbHLH1-3 with PsMYBPA2 to fine-tune the regulatory network to prevent overaccumulation of anthocyanin in blotch regions. Overall, our study uncovers a complex R2R3-MYB transcriptional regulatory network that governs anthocyanin-mediated blotch formation in P. suffruticosa petals, providing insights into the molecular mechanisms underlying blotch formation in P. suffruticosa.

PsSOC1 is involved in the gibberellin pathway to trigger cell proliferation and budburst during endodormancy release in tree peony.

Tree peony (Paeonia suffruticosa) undergoes bud endodormancy, and gibberellin (GA) pathway plays a crucial role in dormancy regulation. Recently, a key DELLA protein PsRGL1 has been identified as a negative regulator of bud dormancy release. However, the mechanism of GA signal to break bud dormancy remains unknown. In this study, yeast two-hybrid screened PsSOC1 interacting with PsRGL1 through its MADS domain, and interaction was identified using pull-down and luciferase complementation imaging assays Transformation in tree peony and hybrid poplar confirmed that PsSOC1 facilitated bud dormancy release. Transcriptome analysis of PsSOC1-overexpressed buds indicated PsCYCD3.3 and PsEBB3 were its potential downstream targets combining with promoter survey, and they also accelerated bud dormancy release verified by genetic analysis. Yeast one-hybrid, electrophoretic mobility shifts assays, chromatin immunoprecipitation quantitative PCR, and dual luciferase assays confirmed that PsSOC1 could directly bind to the CArG motif of PsCYCD3.3 and PsEBB3 promoters via its MADS domain. PsRGL1-PsSOC1 interaction inhibited the DNA-binding activity of PsSOC1. Additionally, PsCYCD3.3 promoted bud dormancy release by rebooting cell proliferation. These findings elucidated a novel GA pathway, GA-PsRGL1-PsSOC1-PsCYCDs, which expanded our understanding of the GA pathway in bud dormancy release.

Tree peony seed oil alleviates hyperlipidemia and hyperglycemia by modulating gut microbiota and metabolites in high-fat diet mice.

With the changes of people's lifestyle, hyperlipidemia and hyperglycemia which were induced from a diet high in both fat and sugar have become serious health concerns. Tree peony seed oil (PSO) is a novel kind of edible oil that shows great potential in the food industry because of its high constituent of unsaturated fatty acids. Based 16S rRNA and gut untargeted metabolomics, this study elucidated that the mechanism of PSO regulating blood glucose (Glu) and lipids. The impact of PSO on gut microbiota balance and gut metabolites of mice with a high-fat diet (HFD) was evaluated. The findings indicated that PSO decreased HFD mice's body weight and fat accumulation, ameliorating the levels of blood lipid, reduced liver fat vacuole levels. What's more PSO modulated the proportion of gut microbiota in HFD mice and enhanced the abundance of probiotics. Furthermore, untargeted metabolomic analysis revealed that PSO not only impacted the generation of short-chain fatty acids (SCFAs) by gut microorganism and altered metabolic pathway but exerted influence on secondary bile acids (BA), amino acid metabolism, and various other metabolites. These results suggested that PSO has the potential function for mitigating HFD-induced hyperlipidemia and hyperglycemia by regulating gut microbiota and host metabolism.

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.

Functional Characterization of PoEP1 in Regulating the Flowering Stage of Tree Peony.

The tree peony, a traditional flower in China, has a short and concentrated flowering period, restricting the development of the tree peony industry. To explore the molecular mechanism of tree peony flowering-stage regulation, PoEP1, which regulated the flowering period, was identified and cloned based on the transcriptome and degradome data of the early-flowering mutant Paeonia ostii 'Fengdan' (MU) and Paeonia ostii 'Fengdan' (FD). Through bioinformatics analysis, expression pattern analysis, and transgene function verification, the role of PoEP1 in the regulation of tree peony flowering was explored. The open-reading frame of PoEP1 is 1161 bp, encoding 386 amino acids, containing two conserved domains. PoEP1 was homologous to the EP1 of other species. Subcellular localization results showed that the protein was localized in the cell wall and that PoEP1 expression was highest in the initial decay stage of the tree peony. The overexpression of PoEP1 in transgenic plants advanced and shortened the flowering time, indicating that PoEP1 overexpression promotes flowering and senescence and shorten the flowering time of plants. The results of this study provide a theoretical basis for exploring the role of PoEP1 in the regulation of tree peony flowering.

Identification of ARF Genes and Elucidation of the Regulatory Effects of PsARF16a on the Dormancy of Tree Peony Plantlets.

The low survival rate of transplanted plantlets, which has limited the utility of tissue-culture-based methods for the rapid propagation of tree peonies, is due to plantlet dormancy after rooting. We previously determined that the auxin response factor PsARF may be a key regulator of tree peony dormancy. To clarify the mechanism mediating tree peony plantlet dormancy, PsARF genes were systematically identified and analyzed. Additionally, PsARF16a was transiently expressed in the leaves of tree peony plantlets to examine its regulatory effects on a downstream gene network. Nineteen PsARF genes were identified and divided into four classes. All PsARF genes encoded proteins with conserved B3 and ARF domains. The number of motifs, exons, and introns varied between PsARF genes in different classes. The overexpression of PsARF16a altered the expression of NCED, ZEP, PYL, GA2ox1, GID1, and other key genes in abscisic acid (ABA) and gibberellin (GA) signal transduction pathways, thereby promoting ABA synthesis and decreasing GA synthesis. Significant changes to the expression of some key genes contributing to starch and sugar metabolism (e.g., AMY2A, BAM3, BGLU, STP, and SUS2) may be associated with the gradual conversion of sugar into starch. This study provides important insights into PsARF functions in tree peonies.

Auxin efflux carrier PsPIN4 identified through genome-wide analysis as vital factor of petal abscission.

PIN-FORMED (PIN) proteins, which function as efflux transporters, play many crucial roles in the polar transportation of auxin within plants. In this study, the exogenous applications of auxin IAA and TIBA were found to significantly prolong and shorten the florescence of tree peony (Paeonia suffruticosa Andr.) flowers. This finding suggests that auxin has some regulatory influence in petal senescence and abscission. Further analysis revealed a total of 8 PsPINs distributed across three chromosomes, which could be categorized into two classes based on phylogenetic and structural analysis. PsPIN1, PsPIN2a-b, and PsPIN4 were separated into the "long" PIN category, while PsPIN5, PsPIN6a-b, and PsPIN8 belonged to the "short" one. Additionally, the cis-regulatory elements of PsPIN promoters were associated with plant development, phytohormones, and environmental stress. These genes displayed tissue-specific expression, and phosphorylation sites were abundant throughout the protein family. Notably, PsPIN4 displayed distinct and elevated expression levels in roots, leaves, and flower organs. Expression patterns among the abscission zone (AZ) and adjacent areas during various flowering stages and IAA treatment indicate that PsPIN4 likely influences the initiation of peony petal abscission. The PsPIN4 protein was observed to be co-localized on both the plasma membrane and the cell nucleus. The ectopic expression of PsPIN4 reversed the premature flower organs abscission in the Atpin4 and significantly protracted florescence when introduced to Col Arabidopsis. Our findings established a strong basis for further investigation of PIN gene biological functions, particularly concerning intrinsic relationship between PIN-mediated auxin polar.

A Set of Artificial Pollination Technical Measures: Improved Seed Yields and Active Ingredients of Seeds in Oil Tree Peonies.

The tree peony, a novel woody oil crop extensively cultivated in China, necessitates further investigation into artificial pollination technology to enhance seed yield. In this study, we conducted artificial pollination experiments with 6-year-old Paeonia ostii 'Feng Dan' seedings for suitable pollen sources, pollen concentration, pollination timing, and pollination frequency. By evaluating seed yields, active ingredients, and oil quality, we derived the following significant conclusions. Firstly, compared to natural pollination, artificial pollination could significantly increase the fruit diameter by 13.94-27.58%, seed yields by 35.17-58.99%, and oil content by 6.45-7.52% in tree peonies. In active ingredients, seeds produced by pollen from Hantai County significantly enhanced starch content (by 48.64%), total phenols (by 41.18%) and antioxidant capacity (by 54.39%). In oil quality, seeds produced by pollen from Heyang County exhibited the highest α-linolenic acid and total fatty acid content with enhancements of 1.68%, 7.41%, and 8.48%. Secondly, hand pollination with pure pollen significantly increased seed yield by 58.99%, total phenol content by 40.97%, antioxidant capacity by 54.39%, and oil content by 1.53% compared to natural pollination. Thirdly, pollination at 2/3 bloom range significantly increased seed number by 63.08% and yield by 45.61% compared to natural pollination. Finally, the effect of one, two, and three pollination events had no difference in seed yield. So, to summarize, applying a 100% concentration of allochthonous pollen once is recommended when the bloom range is more than two thirds.

Inhibitory activities of five fungicides on Alternaria suffruticosae and their field control efficacy against tree peony black spot.

Tree peony black spot (TPBS), mainly caused by Alternaria suffruticosae, is a common leaf disease on the ornamental peony, which posed a great threat on the flower buds in the current year and the flowering quality in the next year. However, there was only one fungicide registered for the control of the disease, difenoconazole. In order to avoid the severe problem of pathogen resistance caused by long-term use of difenoconazole, it is necessary to screen more chemical fungicides for the prevention and control of TPBS. In the paper, the biological activities of flutolanil, phenamacril, pyraclostrobin, and boscalid on mycelial growth, conidial germination, germ tube elongation and sporulation quantity of A. suffruticosae were determined, and field control efficacy were conducted to evaluate the preventive and therapeutic activities. Difenoconazole, was used as a control simultaneously. The results showed that pyraclostrobin had the strongest inhibitory effects on the conidial germination, mycelium growth, germ tube elongation and sporulation quantity, with the average EC50 of 0.0517, 0.5343, 0.0008 and 0.8068 µg/mL respectively. The inhibitory activity of flutolanil on the four developmental stages of A. suffruticosae was weaker than the other three fungicides. Compared with flutolanil, boscalid, the other succinate dehydrogenase inhibitors, had more srtong inhibitory effects on the mycelial growth and sporulation quantity, with the average EC50 of 3.8603 and 1.4760 µg/mL respectively. Phenamacril had a moderate inhibitory level, which had more inhibitory activity on conidial germination and germ tube elongation, with the average EC50 of 31.5349 and 5.2597 µg/mL. All of the four fungicides had no significant effects on the shape of spores and germ tubes. The control fungicide difenoconazole had the strongest inhibitory activity on mycelial growth, and the average EC50 was only 0.3297 µg/ml. However, its inhibitory activity on the other three growth stages was not high. In the field trials, pyraclostrobin had high control efficacy on TPBS even at low concentrations, reaching a minimum of 62.6293%, which was higher than that of difenoconazole. The other three fungicides had higher control efficacy at high concentrations, but decreased significantly at low concentrations. Considering the dosage and control efficacy, pyraclostrobin was the first choice for the control of TPBS. Pyraclostrobin is the preferred alternative fungicide of difenoconazole for the prevention and control of TPBS in production.

Calcium signaling facilitates chilling- and GA- induced dormancy release in tree peony.

Calcium plays a crucial role in plant growth and development, yet little is known about its function in endodormancy regulation. Tree peony (Paeonia suffruticosa), characterized by compound buds and large flowers, is well-known for its ornamental and medicinal value. To break bud dormancy release is a prerequisite of flowering and forcing culture, particularly during the Spring Festival. In this study, the Ca2+ chelator EGTA and Ca2+ channel blocker LaCl3 were applied, resulting in a significant delay in budburst during both chilling- and gibberellin (GA)- induced dormancy release in a dosage-dependent manner. As expected, the retardation of bud break was recovered by the supplementation of 30 mM CaCl2, indicating a facilitating role of calcium in dormancy release. Accordingly, several calcium-sensor-encoding genes including Calmodulin (CaM) and Ca2+-dependent protein kinases (CDPKs) were significantly up-regulated by prolonged chilling and exogenous GAs. Ultrastructure observations revealed a decline in starch grains and the reopening of transport corridors following prolonged chilling. Calcium deposits were abundant in the cell walls and intercellular spaces at the early dormant stage but were enriched in the cytosol and nucleus before dormancy release. Additionally, several genes associated with dormancy release, including EBB1, EBB3, SVP, GA20ox, RGL1, BG6, and BG9, were differentially expressed after calcium blocking and recovery treatments, indicating that calcium might partially modulate dormancy release through GA and ABA pathways. Our findings provide novel insights into the mechanism of dormancy release and offer potential benefits for improving and perfecting forcing culture technology in tree peonies.