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.

The tree peony nuclear factor Y transcription factor PrNF-YC2 promotes seed oil accumulation.

Seed oil not only provides energy for seed postgermination development but also provides essential nutrients and raw materials for human products. However, the transcriptional regulatory mechanism controlling seed oil accumulation remains largely unknown. Tree peony (Paeonia rockii) is an emerging woody oilseed crop in China that is known for its high-quality seed oil. Here, we revealed that a tree peony nuclear factor Y transcription factor, PrNF-YC2, is expressed predominantly in developing seeds and functions as an essential positive regulator of seed oil accumulation. PrNF-YC2 promoted oil accumulation in both transient ectopic overexpression Nicotiana benthamiana leaves and stable transgenic Arabidopsis thaliana seeds, globally upregulating the expression of genes involved in oil accumulation. In contrast, PrNF-YC2-silenced tree peony leaves using a virus-induced gene silencing system showed reduced oil content and expression of oil synthesis-related genes, including four master positive regulators contributing to oil accumulation, namely, LEAFY COTYLEDON1 (LEC1), ABSCISIC ACID INSENSITIVE3 (ABI3), FUSCA3 (FUS3), and WRINKLED1 (WRI1). We demonstrated that PrNF-YC2 directly activates PrLEC1 and PrABI3 alone and indirectly activates PrFUS3 and PrWRI1 by interacting with PrLEC1. Moreover, interaction with PrLEC1 also enhances the activation capacity of PrNF-YC2. The activation of these four master positive regulators by PrNF-YC2 triggered the upregulation of numerous oil synthesis-related genes, thus promoting oil accumulation. These findings provide new insights into the regulatory mechanism of seed oil accumulation and manipulation of PrNF-YC2 may be beneficial for enhancing oil yield in tree peony and other oilseed crops.

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.

Exploring the effect of active components in oil tree peony seed meal on swine disease resistance and its potential mechanisms based on network pharmacology and molecular docking.

This study aims to explore the feasibility of using network pharmacology and molecular docking technology to predict the effects of active components from oil tree peony seed meal (PSM) on swine diseases. Ten active components of PSM were screened through literature search. The results showed that the average number of cross genes between the potential target genes of PSM active components and each swine disease target gene accounted for 7.64% of the total number of swine disease target genes. The GO enrichment analysis indicates that the assumed target is widely present in swine. The KEGG enrichment analysis results showed that these putative genes were involved in various cancer progression pathways, signaling pathways, and hormone regulatory pathways. A total of 8 core targets were obtained through protein-protein interaction networks analysis. It was found that three pathways are not only associated with kinds of swine disease, but also with multiple core targets of PSM active components. In addition, the molecular docking results indicate that the core ingredients have strong affinity with hub genes. The research suggests that the active components of PSM may intervene in swine diseases through multiple components, targets, and pathways.

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 poses a great threat to the flower buds in the current year and the flowering quality in the next year. However, there is only one fungicide registered for the control of this 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 this study, 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 the field control efficacy was tested 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 values 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 that of the other three fungicides. Compared with flutolanil, boscalid, the other succinate dehydrogenase inhibitor, had more strong inhibitory effects on the mycelial growth and sporulation quantity, with the average EC50 values of 3.8603 and 1.4760 µg/ml, respectively. Phenamacril had a moderate inhibitory level and had more inhibitory activity on conidial germination and germ tube elongation, with the average EC50 values of 31.5349 and 5.2597 µg/ml, respectively. 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 value 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 to difenoconazole for the prevention and control of TPBS in production.

Molecular mechanism of somatic embryogenesis in paeonia ostii 'Fengdan' based on transcriptome analysis combined histomorphological observation and metabolite determination.

BACKGROUND: Tree peony (Paeonia sect. Moutan DC.) is a famous flower native to China with high ornamental, medicinal, and oil value. However, the low regeneration rate of callus is one of the main constraints for the establishment of a genetic transformation system in tree peony. By histomorphological observation, transcriptomic analysis and metabolite determination, we investigated the molecular mechanism of somatic embryogenesis after the establishment of a culture system and the induction of somatic embryo(SE) formation. RESULTS: We found that SE formation was successfully induced when cotyledons were used as explants. A total of 3185 differentially expressed genes were screened by comparative transcriptomic analysis of embryogenic callus (EC), SE, and non-embryogenic callus (NEC). Compared to NEC, the auxin synthesis-related genes GH3.6 and PCO2 were up-regulated, whereas cytokinin dehydrogenase (CKX6) and CYP450 family genes were down-regulated in somatic embryogenesis. In SE, the auxin content was significantly higher than the cytokinin content. The methyltransferase-related gene S-adenosylmethionine synthase (SAMS) and the flavonoid biosynthesis-related gene (ANS and F3'5'H) were down-regulated in somatic embryogenesis. The determination of flavonoids showed that rhoifolin and hyperoside had the highest content in SE. The results of transcriptome analysis were consistent with the relative expression of 8 candidate genes by quantitative polymerase chain reaction analysis. CONCLUSION: The results revealed that auxin and cytokinin may play a key role in 'Fengdan' somatic embryogenesis. The genes related to somatic embryogenesis were revealed, which has partly elucidated the molecular mechanism of somatic embryogenesis in 'Fengdan'.

Tree peony PsMYB44 negatively regulates petal blotch distribution by inhibiting dihydroflavonol-4-reductase gene expression.

BACKGROUND AND AIMS: The tree peony (Paeonia suffruticosa Andr.) has been widely cultivated as a field plant, and petal blotch is one of its important traits, which not only promotes proliferation but also confers high ornamental value. However, the regulatory network controlling blotch formation remains elusive owing to the functional differences and limited conservation of transcriptional regulators in dicots. METHODS: We performed phylogenetic analysis to identify MYB44-like transcription factors in P. suffruticosa blotched cultivar 'High noon' petals. A candidate MYB44-like transcription factor, PsMYB44, was analysed via expression pattern analysis, subcellular localization, target gene identification, gene silencing in P. suffruticosa petals and heterologous overexpression in tobacco. KEY RESULTS: A blotch formation-related MYB44-like transcription factor, PsMYB44, was cloned. The C-terminal of the PsMYB44 amino acid sequence had a complete C2 motif that affects anthocyanin biosynthesis, and PsMYB44 was clustered in the MYB44-like transcriptional repressor branch. PsMYB44 was located in the nucleus, and its spatial and temporal expression patterns were negatively correlated with blotch formation. Furthermore, a yeast one-hybrid assay showed that PsMYB44 could target the promoter of the late anthocyanin biosynthesis-related dihydroflavonol-4-reductase (DFR) gene, and a dual-luciferase assay demonstrated that PsMYB44 could repress PsDFR promoter activity. On the one hand, overexpression of PsMYB44 significantly faded the red colour of tobacco flowers and decreased the anthocyanin content by 42.3 % by downregulating the expression level of the tobacco NtDFR gene. On the other hand, PsMYB44-silenced P. suffruticosa petals had a redder blotch colour, which was attributed to the fact that silencing PsMYB44 redirected metabolic flux to the anthocyanin biosynthesis branch, thereby promoting more anthocyanin accumulation in the petal base. CONCLUSION: These results demonstrated that PsMYB44 negatively regulated the biosynthesis of anthocyanin by directly binding to the PsDFR promoter and subsequently inhibiting blotch formation, which helped to elucidate the molecular regulatory network of anthocyanin-mediated blotch formation in plants.

Comprehensive Metabolite Profile Uncovers the Bioactive Components, Antioxidant and Antibacterial Activities in Wild Tree Peony Leaves.

Tree peonies (Paeonia Section Moutan)-including nine wild species, which belong to subsections Vaginatae and Delavayanae-are economically important plants with ornamental, nutritional, and medicinal applications. In this study, for the first time, we determined the bioactive components and antioxidant activities and antibacterial activities of the newly grown leaves of nine wild tree peony species (WTPS). A total of 276 bioactive components were identified through non-targeted metabolomics; more than 80% of the 276 metabolites identified are terpenoids and flavonoids. A total of 42 differential metabolites were quantitatively determined. The main differential metabolites were Paeoniflorin, Luteoloside, Hyperin, Apigenin-7-glucoside, Rhoifolin, and Cantharidin. Such a high terpenoid and flavonoid content of the leaf extracts renders them as species with strong antibacterial capacities, and most of the bacteria tested showed greater sensitivity derived from the members of subsection Vaginatae than those of subsection Delavayanae. All WTPS have significant antioxidant activity; this activity is attributed to high levels of the total phenolic content (TPC) and total flavonoid content (TFC), of which, among the nine WTPS, P. lutea has the strongest antioxidant capacity. Our results provided a theoretical basis for the in-deep application of tree peony leaves for food, medical, and pharmaceutical industries.

Tree Peony R2R3-MYB Transcription Factor PsMYB30 Promotes Petal Blotch Formation by Activating the Transcription of the Anthocyanin Synthase Gene.

Petal blotches are commonly observed in many angiosperm families and not only influence plant-pollinator interactions but also confer high ornamental value. Tree peony (Paeonia suffruticosa Andr.) is an important cut flower worldwide, but few studies have focused on its blotch formation. In this study, anthocyanins were found to be the pigment basis for blotch formation of P. suffruticosa, and peonidin-3,5-di-O-glucoside (Pn3G5G) was the most important component of anthocyanins, while the dihydroflavonol-4-reductase gene was the key factor contributing to blotch formation. Then, the R2R3-myeloblastosis (MYB) transcription factor PsMYB30 belonging to subgroup 1 was proven as a positive anthocyanin regulator with transcriptional activation and nuclear expression. Furthermore, silencing PsMYB30 in P. suffruticosa petals reduced blotch size by 37.9%, faded blotch color and decreased anthocyanin and Pn3G5G content by 23.6% and 32.9%, respectively. Overexpressing PsMYB30 increased anthocyanin content by 14.5-fold in tobacco petals. In addition, yeast one-hybrid assays, dual-luciferase assays and electrophoretic mobility shift assays confirmed that PsMYB30 could bind to the promoter of the anthocyanin synthase (ANS) gene and enhance its expression. Altogether, a novel MYB transcription factor, PsMYB30, was identified to promote petal blotch formation by activating the expression of PsANS involved in anthocyanin biosynthesis, which provide new insights for petal blotch formation in plants.

Protective effects of tree peony seed protein hydrolysate on Cd-induced oxidative damage, inflammation and apoptosis in zebrafish embryos.

The objective of this study was to investigate protective effects of tree peony seed protein hydrolysate by Alcalase (AL-TPSPH) on oxidative damage, inflammation and apoptosis using Cd-induced zebrafish embryos. Zebrafish embryos were treated with either Cd (2 µg/L) or AL-TPSPH (25, 50 and 75 µg/mL) alone or in combination of both from 4 to 144 h post fertilization (hpf). The effects of these treatments on developments, antioxidant parameters and mRNA expression of genes related to oxidative damage, inflammation and apoptosis were examined. The results showed that co-treatment with Cd and AL-TPSPH significantly increased hatching and survival rates and decreased malformation rates of zebrafish embryos compared with Cd treatment alone group (P < 0.05). Cd-induced increase of MDA content, decreases of T-AOC content, GSH/GSSG ratio and activities of SOD, CAT and GPx in zebrafish embryos were modified upon treatment with AL-TPSPH. AL-TPSPH treatment significantly suppressed Cd-induced down-regulations of the antioxidant gene expressions (Mn-sod, Cat and GPx1a) in zebrafish embryos (P < 0.05). AL-TPSPH also prevented Cd-induced up-regulations of pro-inflammatory cytokine (TNF-α, IL-1ß and IFN-γ) expressions. Moreover, AL-TPSPH inhibited Cd-induced up-regulations of pro-apoptotic genes (C-jun, Caspase-3 and Caspase-9) in zebrafish embryos. Collectively, these results indicated that AL-TPSPH could reduce Cd-induced oxidative damage, inflammation and apoptosis in zebrafish embryos, suggesting its future applications as functional food or pharmaceutical ingredient.

Abortion occurs during double fertilization and ovule development in Paeonia ludlowii.

Paeonia ludlowii (Stern & Taylor) D.Y.Hong, an endangered species, is indigenous to Tibet, China and propagated only by seed under natural conditions. Its natural reproduction is constrained by low fecundity. Excess seed abortion is a key factor restricting its natural reproduction, cultivation, introduction, and protection. Understanding the specific origin and occurrence of aborted ovules is important for the protection of offspring. Using serial sectioning analysis, we studied the process of pollination and fertilization of P. ludlowii and examined the characteristics of aborted ovules, developmental differences after flowering of normal and aborted ovules, and their ratios at different positions in P. ludlowii ovaries. During pollination, fertilization, and seed development, ovule abortion was frequent, with a random abortion position. There were three types of abortion, namely, abnormal pistil, sterile ovules, and embryo and endosperm abortions. Of these, embryo and endosperm abortions could be divided into early abortion and middle abortion. The early aborted ovules stopped growing on day 12, the endoblast and endosperm in the embryo sac aborted gradually. Furthermore, the shape of the embryo sac cavity changed. The volume of aborted ovules was significantly different from that of fertile ovules. At ripening, the external morphology of different types of aborted seeds was significantly different. The possible reasons for the abortion of the ovules are also discussed.

First Report of Colletotrichum aenigma Causing Anthracnose of tree peony in China.

Tree peonies (Paeonia suffruticosa Andr. and hybrids) are well-known ornamental and medicinal plants cultivated in temperate and subtropical regions around the world. From June to September 2021, severe leaf spot disease was observed on 3 tree peony cultivars ('Luoyanghong', 'Moyushenghui', 'Roufurong') in Xinxiang (35º29´N, 113º95´E) and Luoyang (34º64´N, 112º49´E), Henan Province, China. Leaf spot incidence was as high as 28% ('Luoyanghong'), 45% ('Moyushenghui') and 67% ('Roufurong'), respectively. Symptoms appeared initially as small purple spots less than 1 mm in diameter in the middle and upper parts of the leaves, and then evolved to coalescent lesions, causing brown scorch ultimately. From each cultivar, 5 diseased leaves were collected. Leaflet tissues (3-4 mm2) cut from spot margins were surface sterilized in 75% alcohol for 45 s, washed 5 times with sterile distilled water, and then cultivated on potato dextrose agar (PDA) medium at 28 °C in the dark. Eleven isolates were obtained, and colonies grown from single conidia on PDA were 80-85 mm in diameter after 10 d, with scattered small, dark-based spikes on the surface of the colonies. The aerial mycelium was cottony, dense, and dark gray near the center on the reverse side. Conidia were cylindrical to clavate, with rounded apex and rounded base, and the conidia contents were granular, 8.44-14.06×3.60-4.31 µm (mean=11.28×3.69 µm, n=40). Appressoria were mostly subglobose or with a few broad lobes, pale to medium brown, 3.36-6.72×3.35-5.60 µm (mean=5.02×4.55 µm, n=20). Based on the culture representation and conidial morphology, the isolates were characterized as Colletotrichum gloeosporioides species complex (Weir et al. 2012; Fu et al. 2019). To further identity the species, the actin (ACT), calmodulin (CAL), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the ribosomal internal transcribed spacers (ITS) loci of isolates PSW0002, PSW0008 and PSW0009 were amplified using ACT-512F/ACT-783R, CL1C/CL2C, CHS-79F/CHS-345R, GDF/GDR, and ITS1/ITS4, primers (Weir et al. 2012; Schena et al; 2014; Kim et al. 2021; Li et al. 2021). Fifteen sequences were deposited in GenBank (ACT for OP225605, OP225606, and OP225607, CAL for OP225608, OP225609 and OP225610, CHS for OP225611, OP225612 and OP225613, GAPDH for ON321897, OP225614, and OP225615, and ITS for ON323473, OP214349 and OP214350 ), which showed 100% sequence similarity to Colletotrichum aenigma (JX009443 and JX009519 for ACT, JX009683 and JX009684 for CAL, JX009774 and JX009903 for CHS-1, JX010244 and JX009913 for GAPDH, JX010243 and JX010148 for ITS). Three isolates clustered with C. aenigma (ex-holotype culture ICMP 18608) in the multi-locus phylogenetic tree with a bootstrap value of 100%. To achieve Koch's postulates, pathogenicity was tested on 5-year-old healthy potted plants ('Luoyanghong'). Thirty leaves were inoculated with 10 µL conidial suspension (isolate PSW0002, 1×106 conidia/mL) and the controls were inoculated with sterile water. Plants were placed in a greenhouse at 28°C under conditions with 12 h photoperiod and 90% relative humidity. After 5 to 10 days, distinct spots were observed on the inoculated leaves, while the control leaves showed no symptoms. C. aenigma was reisolated from all inoculated leaves, but not from the control. C. aenigma has been reported to cause anthracnose on Pyrus pyrifolia (Weir et al. 2012), Camellia sasanqua (Chen et al. 2019), Juglans regia (Wang et al. 2020), Paeonia ostii (Ren et al. 2020), and Capsicum annuum (Sharma et al. 2022). A previous study reported C. gloeosporioides as a pathogen of anthracnose in tree peonies in China (Xuan et al. 2017), the typical symptoms were big necrotic lesions (5-10 mm diam) on leaves，which were significantly different from those caused by C. aenigma. To our knowledge, this is the first report of C. aenigma causing anthracnose in tree peonies in China. This finding may help to take effective control of anthracnose in tree peonies.

First Report of Epicoccum sorghinum Causing Leaf Spot on Paeonia suffruticosa in China.

Tree peony (Paeonia suffruticosa Andr.; Paeoniaceae) is highly valued in Chinese culture for their ornamental and medicinal benefits in anti-inflammation, anti-arrhythmia, activating the immune system and protecting the cardiovascular system (Zhai et al. 2020). In May from 2019 to 2021, leaf spot symptoms were observed on tree peony in an area of 0.8 hm2 in Bozhou, Anhui Province, China; with a disease incidence of 35%. Initially, black-brown spots (0.5-2.4 mm) appeared on the leaves, as the disease developed, the spots enlarged to form round, black lesions. Small pieces of tissue taken from the margins of lesions were surface-disinfected by 75% ethanol for 20 s followed by 0.1% mercuric chloride for 3 min, rinsed with distilled sterile water for three times, placed on potato dextrose agar (PDA) at 25 ºC for 7 days in the dark.Fifteen single spore isolates were obtained as described (Dong 2009). When cultured on PDA for 10 days, colonies were white, villose, then became gray-brown, with the reverse side becoming reddish-brown; Chlamydospores globose or irregular shaped, dark brown, unicellular or multicellular (4.3 to 12.2 × 13.1 to 33.7 µm) (n = 200). Pycnidia were black-brown, mostly spheroid, and 86 to 128 × 110 to 197 µm (n = 50); Conidia were hyaline, ellipsoidal, unicellular, aseptate, and 4.1 to 5.2 × 1.8 to 2.4 µm (n = 200) . The DNA of a representative isolate AF21-1-2 was extracted using the CTAB protocol (Doyle and Doyle 1987). The rDNA internal transcribed spacer (ITS), ß-tubulin (TUB) and 28s nrDNA (LSU) genes were amplified and sequenced using the primers ITS1/ITS4 (White et al. 1990), Bt2a/Bt2b (Glass et al. 1995), and LR5/LR0R (Vilgalys and Hester, 1990), respectively (OK485136, OK631973, and ON358331). BLASTn analysis showed that the ITS, TUB, and LSU sequences had 99% (497/501 bp), 98% (530/539 bp), and 99% (878/882 bp) identity with the Epicoccum sorghinum isolates ESCZO20 (MN944541), CZ01 (MT254851) and Dl16-338 (LN907481), respectively. Maximum likelihood phylogenetic tree revealed that AF21-1-2 clustered with E. sorghinum isolates. For pathogenicity test, leaves of 3-year-old healthy tree peony plant in the field were inoculated with 105 conidia/ml spore suspension (prepared with 30-day-old cultures on PDA), added with 0.1% (v/v) Tween-80. Ten surface-sterilized leaves per plant were sprayed with 30 ml suspension until runoff, a total of 3 plants were inoculated. Leaves sprayed with sterilized water serve as a negative control. Each plant was covered with a plastic bag to maintain high relative humidity at 15 to 23°C and was monitored daily for symptom development. Typical symptoms appeared on more than 80% of the leaves after 7 days of inoculation, while control leaves remained symptomless. Cultures isolated from inoculated leaves had the same morphological and molecular traits as those that were previously used to test Koch's postulates. Based on the morphological and molecular characteristics, the causal agent on tree peony was identified as E. sorghinum (Aveskamp et al. 2010). To our knowledge, this is the first report of E. sorghinum causing leaf spot on tree peony in China. The disease will cause serious economic loss for tree peony if it is not managed properly. This report may provide the basis for diagnosis and control of the disease.

Characteristics of PoVIN3, a Key Gene of Vernalization Pathway, Affects Flowering Time.

The tree peony (Paeonia section Moutan DC.) is the candidate flower in China, with abundant germplasm resources and high ornamental value. However, the short and concentrated flowering period severely restricted the improvement of the economic value of tree peonies. Based on the full-length transcriptome database of tree peonies, the PoVIN3 (GenBank ID: OP341879), involved in the flowering regulation of tree peonies were identified and cloned for the first time. The PoVIN3 was also characterized by bioinformatics methods, quantitative real-time PCR (qRT-PCR), and the establishment of a transgenic system. The expression levels of PoVIN3 in seven different petals developmental stages were the highest at the initial flowering stage of the variant cultivar of Paeonia ostii 'Fengdan,' the initial decay stage of the normal flowering Paeonia ostii 'Fengdan,' and the half opening stage of the late flowering Paeonia suffruticosa 'Lianhe.' Tissue-specific expression analysis showed that the relative expression levels of PoVIN3 were the highest in sepals of both normal flowering Paeonia ostii 'Fengdan' and the late flowering Paeonia suffruticosa 'Lianhe,' and the highest expression was in stamens of early flowering mutant Paeonia ostii 'Fengdan.' In addition, the flowering time of pCAMBIA2300-PoVIN3 transgenic plants was significantly earlier than that of the wild-type, indicating that PoVIN3 could promote plant flowering. The results provide a theoretical basis for exploring the role of PoVIN3 in the regulation of flowering in tree peonies.

A Comparative Transcriptome Analysis Reveals the Molecular Mechanisms That Underlie Somatic Embryogenesis in Peaonia ostii 'Fengdan'.

Low propagation rate is the primary problem that limits industry development of tree peony. In this study, a highly efficient regeneration system for tree peony using somatic embryogenesis (SE) was established. The transcriptomes of zygotic embryo explants (S0), non-embryonic callus (S1), embryonic callus (S2), somatic embryos (S3), and regenerated shoots (S4) were analyzed to determine the regulatory mechanisms that underlie SE in tree peony. The differentially expressed genes (DEGs) were identified in the pairwise comparisons of S1-vs-S2 and S1-vs-S3, respectively. The enriched DEGs were primarily involved in hormone signal transduction, stress response and the nucleus (epigenetic modifications). The results indicated that cell division, particularly asymmetric cell division, was enhanced in S3. Moreover, the genes implicated in cell fate determination played central roles in S3. Hormone signal pathways work in concert with epigenetic modifications and stress responses to regulate SE. SERK, WOX9, BBM, FUS3, CUC, and WUS were characterized as the molecular markers for tree peony SE. To our knowledge, this is the first study of the SE of tree peony using transcriptome sequencing. These results will improve our understanding of the molecular mechanisms that underly SE in tree peony and will benefit the propagation and genetic engineering of this plant.

Transcriptomic analysis of α-linolenic acid content and biosynthesis in Paeonia ostii fruits and seeds.

BACKGROUND: Paeonia ostii is a potentially important oilseed crop because its seed yield is high, and the seeds are rich in α-linolenic acid (ALA). However, the molecular mechanisms underlying ALA biosynthesis during seed kernel, seed testa, and fruit pericarp development in this plant are unclear. We used transcriptome data to address this knowledge gap. RESULTS: Gas chromatograph-mass spectrometry indicated that ALA content was highest in the kernel, moderate in the testa, and lowest in the pericarp. Therefore, we used RNA-sequencing to compare ALA synthesis among these three tissues. We identified 227,837 unigenes, with an average length of 755 bp. Of these, 1371 unigenes were associated with lipid metabolism. The fatty acid (FA) biosynthesis and metabolism pathways were significantly enriched during the early stages of oil accumulation in the kernel. ALA biosynthesis was significantly enriched in parallel with increasing ALA content in the testa, but these metabolic pathways were not significantly enriched during pericarp development. By comparing unigene transcription profiles with patterns of ALA accumulation, specific unigenes encoding crucial enzymes and transcription factors (TFs) involved in de novo FA biosynthesis and oil accumulation were identified. Specifically, the bell-shaped expression patterns of genes encoding SAD, FAD2, FAD3, PDCT, PDAT, OLE, CLE, and SLE in the kernel were similar to the patterns of ALA accumulation in this tissue. Genes encoding BCCP, BC, KAS I- III, and FATA were also upregulated during the early stages of oil accumulation in the kernel. In the testa, the upregulation of the genes encoding SAD, FAD2, and FAD3 was followed by a sharp increase in the concentrations of ALA. In contrast, these genes were minimally expressed (and ALA content was low) throughout pericarp development. CONCLUSIONS: We used three tissues with high, moderate, and low ALA concentrations as an exemplar system in which to investigate tissue-specific ALA accumulation mechanisms in P. ostii. The genes and TFs identified herein might be useful targets for future studies of ALA accumulation in the tree peony. This study also provides a framework for future studies of FA biosynthesis in other oilseed plants.

GA3 is superior to GA4 in promoting bud endodormancy release in tree peony (Paeonia suffruticosa) and their potential working mechanism.

BACKGROUND: Sufficient low temperature accumulation is the key strategy to break bud dormancy and promote subsequent flowering in tree peony anti-season culturing production. Exogenous gibberellins (GAs) could partially replace chilling to accelerate dormancy release, and different kinds of GAs showed inconsistent effects in various plants. To understand the effects of exogenous GA3 and GA4 on dormancy release and subsequent growth, the morphological changes were observed after exogenous GAs applications, the differentially expressed genes (DEGs) were identified, and the contents of endogenous phytohormones, starch and sugar were measured, respectively. RESULTS: Morphological observation and photosynthesis measurements indicated that both GA3 and GA4 applications accelerated bud dormancy release, but GA3 feeding induced faster bud burst, higher shoot and more flowers per plant. Full-length transcriptome of dormant bud was used as the reference genome. Totally 124 110 459, 124 015 148 and 126 239 836 reads by illumina transcriptome sequencing were obtained in mock, GA3 and GA4 groups, respectively. Compared with the mock, there were 879 DEGs and 2 595 DEGs in GA3 and GA4 group, 1 179 DEGs in GA3 vs GA4, and 849 DEGs were common in these comparison groups. The significant enrichment KEGG pathways of 849 DEGs highlighted plant hormone signal transduction, starch and sucrose metabolism, cell cycle, DNA replication, etc. Interestingly, the contents of endogenous GA1, GA3, GA4, GA7 and IAA significantly increased, ABA decreased after GA3 and GA4 treatments by LC-MS/MS. Additionally, the soluble glucose, fructose and trehalose increased after exogenous GAs applications. Compared to GA4 treatment, GA3 induced higher GA1, GA3 and IAA level, more starch degradation to generate more monosaccharide for use, and promoted cell cycle and photosynthesis. Higher expression levels of dormancy-related genes, TFL, FT, EBB1, EBB3 and CYCD, and lower of SVP by GA3 treatment implied more efficiency of GA3. CONCLUSIONS: Exogenous GA3 and GA4 significantly accelerated bud dormancy release and subsequent growth by increasing the contents of endogenous bioactive GAs, IAA, and soluble glucose such as fructose and trehalose, and accelerated cell cycle process, accompanied by decreasing ABA contents. GA3 was superior to GA4 in tree peony forcing culture, which might because tree peony was more sensitive to GA3 than GA4, and GA3 had a more effective ability to induce cell division and starch hydrolysis. These results provided the value data for understanding the mechanism of dormancy release in tree peony.

Metabolomics analysis reveals Embden Meyerhof Parnas pathway activation and flavonoids accumulation during dormancy transition in tree peony.

BACKGROUND: Bud dormancy is a sophisticated strategy which plants evolve to survive in tough environments. Endodormancy is a key obstacle for anti-season culture of tree peony, and sufficient chilling exposure is an effective method to promote dormancy release in perennial plants including tree peony. However, the mechanism of dormancy release is still poorly understood, and there are few systematic studies on the metabolomics during chilling induced dormancy transition. RESULTS: The tree peony buds were treated with artificial chilling, and the metabolmics analysis was employed at five time points after 0-4 °C treatment for 0, 7, 14, 21 and 28 d, respectively. A total of 535 metabolites were obtained and devided into 11 groups including flavonoids, amino acid and its derivatives, lipids, organic acids and its derivates, nucleotide and its derivates, alkaloids, hydroxycinnamoyl derivatives, carbohydrates and alcohols, phytohormones, coumarins and vitamins. Totally, 118 differential metabolites (VIP ≥ 1, P < 0.05) during chilling treatment process were detected, and their KEGG pathways involved in several metabolic pathways related to dormancy. Sucrose was the most abundant carbohydrate in peony bud. Starch was degradation and Embden Meyerhof Parnas (EMP) activity were increased during the dormancy release process, according to the variations of sugar contents, related enzyme activities and key genes expression. Flavonoids synthesis and accumulation were also promoted by prolonged chilling. Moreover, the variations of phytohormones (salicylic acid, jasmonic acid, abscisic acid, and indole-3-acetic acid) indicated they played different roles in dormancy transition. CONCLUSION: Our study suggested that starch degradation, EMP activation, and flavonoids accumulation were crucial and associated with bud dormancy transition in tree peony.

Tree peony variegated flowers show a small insertion in the F3'H gene of the acyanic flower parts.

BACKGROUND: The tree peony (Paeonia suffruticosa Andr.) cultivar 'Er Qiao' is appreciated for its unstable variegated flower coloration, with cyanic and acyanic flowers appearing on different branches of the same plant and occasionally in a single flower or petal. However, the variegation mechanism is still unclear. RESULTS: In this study, we found significantly higher contents and more diverse sets of anthocyanins in the cyanic petals than in the acyanic petals. Comparative transcriptome analysis between the two flower types revealed 477 differentially expressed genes (DEGs). Quantitative real-time PCR results verified that the transcript levels of the flavonol synthase (FLS) gene were significantly increased in the acyanic petals. Furthermore, we found that a GCGGCG insertion at 246 bp in the flavonoid 3'-hydroxylase (F3'H) gene-coding region constitutes a duplication of the 241-245 bp section and was consistently found only in acyanic flowers. Sequence alignment of the F3'H gene from different plant species indicated that only the acyanic petals of 'Er Qiao' contained the GCGGCG insertion. The transformation of Arabidopsis tt7-1 lines demonstrated that the ectopic expression of F3'H-cyanic, but not F3'H-acyanic, could complement the colors in the hypocotyl and seed coat. CONCLUSION: In summary, we found that an indel in F3'H and the upregulation of FLS drastically reduced the anthocyanin content in acyanic petals. Our results provide molecular candidates for a better understanding of the variegation mechanisms in tree peony.

Functional identification of PsMYB57 involved in anthocyanin regulation of tree peony.

BACKGROUND: R2R3 myeloblastosis (MYB) genes are widely distributed in plants and comprise one of the largest transcription factor gene families. They play important roles in the regulatory networks controlling development, metabolism, and stress responses. Researches on functional genes in tree peony are still in its infancy. To date, few MYB genes have thus far been reported. RESULTS: In this study, we constructed a comprehensive reference gene set by transcriptome sequencing to obtain R2R3 MYB genes. The transcriptomes of eight different tissues were sequenced, and 92,837 unigenes were obtained with an N50 of 1662 nt. A total of 48,435 unigenes (77.98%) were functionally annotated in public databases. Based on the assembly, we identified 57 R2R3 MYB genes containing full-length open reading frames, which clustered into 35 clades by phylogenetic analysis. PsMYB57 clustered with anthocyanin regulation genes in Arabidopsis and was mainly transcribed in the buds and young leaves. The overexpression of PsMYB57 induced anthocyanin accumulation in tobacco, and four detected anthocyanin structural genes, including NtCHS, NtF3'H, NtDFR, and NtANS, were upregulated. The two endogenous bHLH genes NtAn1a and NtAn1b were also upregulated and may work in combination with PsMYB57 in regulating anthocyanin structural genes. CONCLUSIONS: Our study offers a useful reference to the selection of candidate MYB genes for further functional studies in tree peony. Function analysis of PsMYB57 is helpful to understand the color accumulation in vegetative organs of tree peony. PsMYB57 is also a promising resource to improve plant color in molecular breeding.