Comprehensive analyses of the proteome and ubiquitome revealed mechanism of high temperature accelerating petal abscission in tree peony

Tree peony(Paeonia suffruticosa Andrews)is a well-known ornamental plant with high economic value,but the short fluorescence is a key obstacle to its ornamental value and industry development.High temperature accelerates flower senescence and abscission,but the associated mechanisms are poorly understood.In this study,the tandem mass tag(TMT)proteome and label-free quantitative ubiquitome from tree peony cut flowers treated with 20℃for 0 h(RT0),20℃or 28℃for 60 h(RT60 or HT60)were examined based on morphological observation,respectively.Totally,6970 proteins and 1545 lysine ubiquitinated(Kub)sites in 844 proteins were identified.Hydrophilic residues(such as glutamate and aspartate)neighboring the Kub sites were in preference,and 36.01%of the Kub sites were located on the protein surface.The differentially expressed proteins(DEPs)and Kub-DEPs in HT60 vs RT60 were mainly enriched in ribosomal protein,protein biosynthesis,secondary metabolites biosynthesis,flavonoid metabolism,carbohydrate catabolism,and auxin biosynthesis and signaling revealed by GO and KEGG analysis,accompanying the increase of endogenous abscisic acid(ABA)accumulation and decrease of endogenous indoleacetic acid(IAA)level.Additionally,the expression patterns of six enzymes(SAMS,ACO,YUC,CHS,ANS and PFK)putatively with Kub modifications were analyzed by proteome and real-time quantitative RT-PCR.The cell-free degradation assays showed PsSAMS and PsACO proteins could be degraded via the 26 S proteasome system in tree peony flowers.Finally,a working model was proposed for the acceleration of flower senescence and abscission by high temperature.In summary,all results contributed to understanding the mechanism of flower senescence induced by high temperature and prolonging fluorescence in tree peony.

Changes in the root system of the herbaceous peony and soil properties under different years of continuous planting and replanting

The herbaceous peony(Paeonia lactiflora Pall.)has high ornamental value.Replanting problems occur when seedlings are replanted into previous holes.We studied the root system and soil environment of the'Dongjingnvlang'variety under a continuous planting regime of one,four,and seven years,and a replanting regime of one and four years.Under the condition of continuous planting,with the increase of number of years,pH,ammonium nitrogen,and nitrate nitrogen decreased in the rhizosphere and non-rhizosphere soils,whereas organic matter,available phosphorus and potassium,enzyme activities,and the number of bacteria,fungi,and actinomycetes increased.Under the condition of replanting,with the increase of number of years,fungi and actinomycetes in both soils increased,while pH,organic matter,nutrients,enzyme activities,and bacterial number decreased.pH,organic matter,nutrient content,enzyme activity and the number of bacterial were lower in soil replanted for four years,whereas the abundance of fungi and actinomycetes was higher,altering the soil from“bacterial high-fertility”to“fungal low-fertility”with increasing years of replanting.The activity of antioxidant enzymes and MDA content in roots of peony in replanting were higher than those in continuous planting,while the content of osmotic regulatory substances in replanting was lower than that in continuous planting.The results showed that there were no obvious adverse factors in soil during seven years of continuous planting,and herbaceous peony could maintain normal growth and development.However,soils after four years of replanting were not suitable for herbaceous peony growth.Benzoic acid increased with years of replanting,which potentially caused replanting problems.This study provides a theoretical basis for understanding the mechanism of replanting problems in the herbaceous peony.

Osmotic Regulation,Antioxidant Enzyme Activities and Photosynthetic Characteristics of Tree Peony(Paeonia suffruticosa Andr.)in Response to High-Temperature Stress

Tree peony(Paeonia suffruticosa Andr.)is a traditional Chinese flower,which prefers cool weather.However,high temperature in summer in the middle and lower reaches of the Yangtze River restricts its growth and development.In this study,osmotic regulation,antioxidant enzyme activities,and photosynthetic characteristics of tree peony in response to high-temperature stress were investigated.The results showed that high-temperature stress had destroyed the cell membrane,manifested as the increased relative electrical conductivity and malondialdehyde content.Moreover,high-temperature stress led to excessive accumulation of reactive oxygen species,thereby,activating antioxidant enzyme activities.Also,photosynthetic parameters and chlorophyll fluorescence parameters directly reflected the damage to the photosystem II reflection center under high-temperature stress.In addition,high-temperature stress led to stomatal closure and chloroplast damage.This study revealed the physiological responses of tree peony to high-temperature stress,laying a foundation for the promotion of tree peony in high-temperature areas and the improvement of high-temperature resistance.

Selection of Stable Reference Genes for Quantitative Real-Time PCR on Herbaceous Peony (Paeonia lactiflora Pall.) in Response to Drought Stress

Herbaceous peony(Paeonia lactiflora Pall.),as a high-end cut flower in the international market,has high ornamental and medicinal values.But in Northern China,drought is a major environmental factor influencing the growth and development of P.lactiflora.Quantitative real-time polymerase chain reaction(qRT-PCR)can evaluate gene expression levels under different stress conditions,and stable internal reference is the key for qRT-PCR.At present,there is no systematic screening of internal reference for correcting gene expressions of P.lactiflora in response to drought stress.In this study,10 candidate genes[ubiquitin(UBQ2),UBQ1,elongation factor 1-α(EF-1α),Histidine(His),eukaryotic initiation factor(eIF),tubulin(TUB),actin(ACT),UBQ3,ACT2,RNA polymerase II(RNA Pol II)]were chosen,and 4 analysis methods were used to compare the stabilities for these 10 genes coping with drought stress.Due to the difference of operation methods,the results of different analysis were distinct,and the final comprehensive analysis indicated that EF-1αwas a relatively stable internal reference gene for P.lactiflora under drought stress.Also,UBQ1 and UBQ2 were the best reference gene combination according to GeNorm analysis.This study will lay a foundation for screening the key genes of P.lactiflora in response to drought stress.

Research on the Development Status of Oil-use Peony Industry in Heze City

The peony industry is a characteristic industry in Heze City,and oil-use peony is an emerging industry.Heze City has planned oil-use peony as a key support and development industry in the local area.The oil-use peony industry is full of treasures,but there are still many urgent problems to be solved for its sustained and healthy development:cultivation and planting,variety selection,product processing,and enterprise development,which have hindered the development of the oil-use peony industry.This paper delved into the comprehensive benefits of oil-use peony and its industrial development,providing some reference for the further development of the oil-use peony industry in Heze.

Research on Sustainable Development Strategies for Oil-use Peony in Heze City

Oil-use peony is a treasure with broad prospects.Heze City plans to develop oil-use peony as a local key industry.However,there are still many urgent problems that need to be solved for the sustained and healthy development of the oil-use peony industry.This paper delved into the comprehensive benefits of oil-use peony and its industrial development from the aspects of cultivating high-quality germplasm resources,technological innovation,policy support,developing leading enterprises,improving industrialization system,comprehensive promotion,and strengthening storage and transportation technology,aiming to provide some reference for further development of Heze oil-use peony industry.

Effect of exogenous GA3 on flowering quality, endogenous hormones, and hormone-and flowering-associated gene expression in forcing-cultured tree peony（Paeonia suffruticosa）

Gibberellins(GAs)promote flowering in the forcing-cultured tree peony(Paeonia suffruticosa),however,the mechanism of regulating flowering is not fully understood.In this study,exogenous GA3 was applied to five-year-old Luoyang Hong plants to explore responses in terms of endogenous hormones,flowering quality,and the hormone-and flowering-associated gene expression.Exogenous GA3 application significantly promoted flower bud development and new branch growth,as well as improved flowering quality.Exogenous GA3 application also stimulated the synthesis of endogenous GA3 and indole-3-acetic acid(IAA)but reduced abscisic acid(ABA)levels.To further elucidate the regulatory mechanism,eight genes for GA biosynthesis and signaling,including PsCPS,PsKS,PsGA3ox,PsGA2ox,PsGID1b,PsGID1c,PsDELLA,and PsGID2 were cloned for the first time,and sequence analysis was also performed.The results suggested that all the cloned genes have conserved structure as each homologous gene reported in the other species.Phylogenetic trees constructed by the each cloned gene showed that the phylogenetic evolutionary relationship of P.suffruticosa was closely related to Vitis vinifera.The expression patterns of the above genes,and genes for ABA and IAA biosynthetic and signaling,and the flowering time were also investigated.Most of the above genes showed higher expression in the control buds than those in the GA3 treated buds at six developmental stages,whereas the expression levels of PsSOC1 and PsSPL9 were up-regulated by GA3 treatment.The results also showed that the GA-biosynthetic and signaling pathways are conserved in tree peony,and the PsCPS,PsGA3ox,PsGA2ox,PsGID1,PsDELLA,and PsGID2 genes are necessary for feedback regulation of GAs.Furthermore,hormone changes promoted PsSOC1 and PsSPL9 expression,and repressed PsSVP expression,which contributed to the improvement flowering quality in tree peony of forcing culture.

Light shading improves the yield and quality of seed in oil-seed peony(Paeonia ostii Feng Dan)

Tree peony seed is unique for its super-high content of unsaturated fatty acid and is thus considered as an important source of woody oil. However, photosynthetic production is greatly reduced under high light intensity and air temperature during the seed filling period, which negatively affects seed yield and quality. The objective of this study was to determine if appropriate shading improves yield and quality of seed in oilseed peony. In this study, oilseed peony trees were shaded by different density polyethylene nets from four weeks after flowering to harvest stages to form light, moderate, and severe shadings, equivalent to about 80, 40, and 20% of full solar exposure, respectively. The effects of different shadings on some physiological parameters, yield and yield components, and nutritional composition of seed were examined. Averaged across two years, light shading increased the actual net photosynthetic rate（P_n） by 16.8%, the maximum net photosynthetic rate（P_（max）） by 81.4%, chlorophyll（Chl） content by 52.8%, auxin（IAA） content by 38.1%, and gibberellic acid（GA_3） content in leaves by 6.3%; it decreased the accumulation of H_2O_2 in leaves by 24.8%, malondialdehyde（MDA） by 22%, and endogenous abscisic acid（ABA） by 8.8%, indicating that leaf senescence in late season was considerably delayed. Light shading increased seed yield, and contents of crude fat and unsaturated fatty acids by 9.7, 5.6, and 9.6%, respectively, while moderate or severe shading significantly reduced all the three parameters. Light shading increased seed weight, but moderate or severe shading reduced seed weight or follicle density. The improved seed yield under light shading was mainly due to increased seed weight, while the reduced seed yield under moderate or severe shading was mainly attributed to reduce follicle density and seed weight. The improved seed weight and content of unsaturated fatty acids under light shading was possibly due to the delayed leaf senescence. The overall results indicated that light shading is beneficial to yield and quality parameters of seed in oilseed peony. Cultivating oilseed peony under a light shading environments such as partially closed forests would better increase total output and income per unit land area than that under full solar exposure.

RAPD-PCR Analysis on Genetic Relationships Between Cultivars of Tree Peony

Random amplified polymorphic DNA (RAPD) was used to analyze genetic polymophism of 35 Tree Peony cultivars with 7 different color groups. Thirty four primers amplified 418 DNA fragments and 337 polymorphic bands (80.6%), including specific DNA markers for 18 cultivars that could be used to differentiate cultivars. The UPCMA method was used to analyze the genetic relationship among cultivars. The results showed that 35 Peony cultivars could be divided into 2 cluster groups when using similarity criteria of 1.5, and into 4 cluster groups when using similarity criteria of 1.0. The result confirmed that the flower color has no relation to the genetic clusters and the Tree Peony cultivars originated from the same area has close genetic relationship. Therefore, genetic background has no large effect on the genetic relationship. The sequence based on polymorphic rate from high to low was Blue groups > Yellow groups > Bark red groups > Blake groups > White groups>Green groups>Red groups.

Molecular characterization of chalcone isomerase (CHI) regulating flower color in herbaceous peony (Paeonia lactiflora Pall.)

Chalcone isomerase （CHI） is a key enzyme that converts yellow chalcone to colorless naringenin, playing an important regulatory role in color formation of ornamental flowers. We determined the coding sequence of CHI in herbaceous peony using rapid-amplification of cDNAends （RACE） technology, and subsequently detected the expression pattern of CHI in the inner and outer petals at different developmental stages using qRT-PCR. We cloned the upstream promoter sequences of CHI using genome walking technology and predicted the location of CpG islands and 5＇ truncation. In addition, we con- structed five dual-luciferase reporter gene carriers and detected the promoter activities of different fragments. Our results showed that the full-length cDNA sequence of CHI was 898 bp, and the 5＂-upstream core promoter was located at -1 651 to -2050 bp region, where contained one CpG island （-1 897 to -2010 bp） and several important binding sites of transcription factor, such as Spl, serum response factor （SRF）, activating protein （AP）-2alpha and CCAAT/enhancer binding protein （C/ EBP）alpha. Expression results showed that the expression of CHI at different developmental stages was generally higher in inner petals than those in outer petals, and the maximum at the bud stage （S1）. Thus, this study will provide theoretical basis for an in-depth study of CHI gene function and expression regulation.

Optimum harvesting time of herbaceous peony buds for cutting flowers

The herbaceous peony is one of the cut flowers that has become increasingly popular in the international market in recent years. In the study, 11 cultivars of herbaceous peonies suitable for cutting flowers were selected; different harvesting stages （three or four stages） were identified according to bud development observation （bud firmness, bud diameter, sepal angle, petal angle, and color showing）. Moreover, flower development and vase life were also recorded in the experiment of vase. Bud development observation shows that there are great differences in optimum harvesting stage between the culti- vars, especially between the hybrid peony group and lactiflora group. This implies that bud diameter could not be the only indicator for optimum harvesting stage. Bud description, including sepal opening angle, sepal color, petal color showing, and degree of firmness, is necessary for the determination of the harvesting stage. Among the 11 cultivars,‘Pink Hawaiian Coral＇, ‘Red Charm＇, ‘Edulis Superba＇, ‘Red Magic＇, and ‘Sarah Bernhardt＇ should be harvested at stage 1. ‘Duchesse de Nemours＇, ‘Taff＇, ‘Sorbet＇ and ‘Monsieur Jules Elie＇ should be harvested at stage 2. ‘Kansas＇ should be at stage 3 and ‘Karl Rosenfield＇ at stage 4. The determination of optimum harvesting stage for each cultivar is an important element in cut flower production.

Genetic diversity and relatedness analysis of nine wild species of tree peony based on simple sequence repeats markers

Tree peony has nine wild species,but the evolutionary relationship of them is still unclear.Here,a total of 274 specimens from 22 natural populations of nine wild species were collected,and their genetic diversity and similarity was analyzed based on Simple Sequence Repeats(SSR)molecular markers.A total of 106 alleles were generated based on 20 primers and with an average of 5.3 alleles per primer.Shannon’s information index(I)ranged from 0.6333 to 1.7842,and the average of Nei’s genetic diversity coefficient(H)was 0.5771.Polymorphism Information Content(PIC)value varied from 0.29 to 0.77,ten of these primers had high polymorphism(PIC≥0.50).All the above genetic parameters of primers reflect more rich genetic diversity information compared with other researches using SSR molecular markers to study the genetic diversity of tree peony wild species.At the population level,the lowest and highest degree of genetic diversity occurred in Paeonia ludlowii-P1 and P.delavayi-P3 population,respectively.Whereas at species level,the genetic diversity of 9 wild peony species was as follows:P.lutea>P.delavayi>P.rockii>P.qiui>P.ostii>P.decomposita>P.potaninii>P.spontanea>P.ludlowii.Furthermore,cluster analysis at species level divided the nine wild tree peony species into two branches.In branch I,the closest phylogenetic relationship was found between P.ostii and P.rockii,followed by P.spontanea,P.qiui,and P.decomposita.In branch II,the closest relationship occurred between P.lutea and P.delavayi,followed by P.potaninii and P.ludlowii.Clustering results supported the division of tree peonies into two subsects(Delavayanae and Vaginatae),it also supported P.potaninii and P.ludlowii as independent species.The results provided novel insight into the genetic diversity and phylogenetic relationship of nine wild tree peony species.It will help formulate comprehensive protection measures of wild germplasm resources and select proper parents for distant hybridization in the future.

Identification of differentially expressed genes associated with bud dormancy release in tree peony(Paeonia suffruticosa) by suppression subtractive hybridization

A subtractive cDNA library was developed to study genes associated with bud dormancy release in tree peonies. In order to identify genes that are highly expressed in buds released from dormancy, 588 clones were examined by differential screening. Of these, 185 clones were selected to be sequenced. A total of 37 unique sequences were obtained of which only 31 sequences have matches in the NCBI database or the Arabidopsis thaliana protein database. Semi-quantitative RT-PCR was used to confirm further the expression profiles for 12 transcripts identified within the subtractive cDNA library. Gene ontology analyses indicated that many of the different genes identified have unknown or hypothetical functions while it is speculated that other genes play different mo- lecular roles. In our study, genes involved in bud dormancy release were growth-related or stress-responsive, while low-temperature-induced ribosomal proteins may also play a role in bud dormancy release. Our results provide interesting information for further understanding of the molecular mechanism of bud dormancy release in tree peonies.

Development and application of microsatellite markers within transcription factors in flare tree peony(Paeonia rockii)based on next-generation and single-molecule long-read RNA-seq

Tree peonies native to China are a precious crop with ornamental,medicinal and edible oil properties,of which flare tree peony(Paeonia rockii)is one of the most significant germplasms in Paeonia.The development and application of expressed sequence tag-simple sequence repeat(EST-SSR)markers are very valuable for genetic and breeding applications,but EST-SSR resources for the genus Paeonia are still limited.In this study,we first reported the development of SSRs within transcription factors(TFs)in P.rockii based on next-generation sequencing(NGS)and single-molecule long-read sequencing(SMLRS).A total of 166 EST-SSRs containing six nucleotide repeat types were identified from 959 candidate TFs associated with yield,with an average of one SSR per 5.83 unigenes.In total,102(61.45%)pairs of primers produced amplification products in the two RNA-seq cultivars.Among them,58(56.86%)pairs of primers from 18 gene families(AP2,b HLH,HSF,etc.)were identified to be polymorphic both in the parents of a linkage mapping population and in eight randomly selected accessions of P.rockii.Further,the 58 EST-SSRs indicated a high level of informativeness with PIC values ranging from 0.32 to 0.91(mean 0.70)after assessment in 37 tree peony accessions.Transferability studies indicated that the amplification ratio of the 58 pairs of primers ranged from 89.66 to 100%across seven species of Paeonia.In addition,a genetic relationship study was performed in 62 accessions.Cluster analysis using the neighbour-joining(NJ)tree demonstrated that major clusters corresponded to the known pedigree trees.Taken together,these newly developed EST-SSRs have a potential use in the conservation of tree peony germplasm and marker-assisted selection(MAS)breeding.

Flavonoid content and expression analysis of flavonoid biosynthetic genes in herbaceous peony(Paeonia lactiflora Pall.)with double colors

Herbaceous peony（Paeonia lactiflora Pall.） is a famous flower with medicinal values, and its flowers have a number of medicinal constituents, especially flavonoids. In this study, a P. lactiflora cultivar with double colors including white outer-petal and yellow inner-petal was used as the experimental materials to perform the qualitative and quantitative analysis of flavonoids by high-performance liquid chromatograph-electrospray ionization-mass spectrometry（HPLC-ESI-MSn） and investigate the expression patterns of flavonoid biosynthetic genes using real-time quantitative polymerase chain reaction（Q-PCR）. The results showed that the colors of both petals gradually weakened with flower development. Moreover, one main anthocyanin composition（peonidin 3,5-di-O-glucoside） and five main anthoxanthin compositions（kaempferol di-hexoside, kaempferol-3-O-malonylglucoside-7-O-glucoside, quercetin-3-O-galactoside, luteolin-7-O-glucoside and isorhamnetin-3-O-glucoside） were found in the both, differing significantly in their peak areas only. Total anthocyanin, anthoxanthin and flavonoid contents in white outer-petal and yellow inner-petal gradually decreased during flower development, and were consistently higher in white outer-petal. Furthermore, the expression patterns of nine structural genes in P. lactiflora flavonoid biosynthetic pathway showed that the expression levels of phenylalanine ammonialyase gene（Pl PAL）, chalcone synthase gene（PlC HS）, flavanone 3-hydroxylase gene（PlF 3H）, anthocyanidin synthase gene（PlA NS） and UDP-glucoside： flavonoid 5-O-glucosyltransferase gene（Pl F5GT） in two petals basically presented declined tendencies, and transcription levels of Pl PAL, Pl CHS, Pl ANS, Pl F3 GT and Pl F5 GT also tended to be higher in white outer-petal, which was correlated with their flavonoid contents. These results would lay a solid foundation for the exploration and utilization of flavonoid resources in P. lactiflora flowers.

Effects of Waterlogging Stress on the Physiological Characteristics and Secondary Metabolites of Herbaceous Peony (<i>Paeonia lactiflora</i>Pall.)

Herbaceous peony is an ornamental plant with medicinal properties. Waterlogging can affect its yield and quality as it grows and matures. In this study, we subjected “Taohuafeixue”, “Yangfeichuyu” and “Hongxiuqiu” herbaceous peony varieties to a simulated waterlogging stress treatment and investigated the effects of waterlogging on their physiological characteristics and the secondary metabolite contents in their leaves and roots. Short-term waterlogging caused the leaves to turn yellow or red and the roots to turn black. The stele and the cell wall of the endothelial cells thickened, and the cortical cells enlarged. Waterlogging did not significantly change plant height, leaf length, and leaf area;however, it significantly decreased the root-shoot ratio of “Yangfeichuyu” and “Hongxiuqiu” varieties. The activity of antioxidant enzymes and the content of osmotic regulators increased under waterlogging. After short-term waterlogging stress treatment, the content of paeoniflorin and albiflorin increased in the roots of “Taohuafeixue” and “Yangfeichuyu”, and the content of benzoylpaeoniflorin increased in the root of “Hongxiuqiu”. The content of gallic acid and total flavonoids increased in the leaves of “Taohuafeixue” and “Yangfeichuyu”. After the waterlogging, paeoniflorin and benzoylpaeoniflorin increased in the autumn root of “Hongxiuqiu”. This study expands our knowledge about the medicinal properties of herbaceous peony and informs about its production and cultivation under waterlogged conditions.

A comparison of pharmacokinetic interaction of paeoniflorin, albiflorin and total peony glucosides

Aim Paeoniflorin （PF） and albiflorin （AF） are the main components of total peony glucosides （TPG）, which has been widely used as the valuable monoterpene glycosides in Paeouia lactiflora Pall and have many biological activities such as anti-inflammatory, anti-hypertension and antioxidation effects. In this study, the pharmacokinetic interaction and possible interaction mechanism among Pie and A1e and TGP were conducted and in- vestigated. A sensitive and specific ultra high-performance liquid chromatography - tandem mass spectrometry method （UPLC-MS） was successfully developed and validated for simultaneous quantification of Pie and AF in rat plasma after intragastric administration and the comparative study of pharmacokinetics for PF and A1e in different combinations. The combinations of Pie （ 80 mg· kg^- 1 ）, Ale （ 14 mg ·kg^- 1 ）, co-administration of Pie （ 80 mg · kg^-1） and Ale （14 mg· kg^-1）, TPG （Pie 80 rag. kg^-1） and TPG （Alel4 mg.·1kg^-1） were orally administrated to rats respectively. Chromatographic separation was performed on a ZORBAX Eclipse plus Cls column using 0.2% formic acid-methanol （79：21, V/V） as mobile phase. The calibration curves were linear in ranges of 0.5 - 1000 μg · L^-1. The results indicated that the main pharmacokinetic parameters including AUC, MRT and tl/2 between the single ingredient （ Pie and Ale） and TPG had significant difference （ P 〈 0.05 or P 〈 0.01 ）. The different phar- macokinetics data implied that the multiple active components of TPG resulting some potential drug-drug interac- tions, which may promote the absorption and decease the elimination of Pie and Ale. Our results could be helpful for further investigation of the interaction mechanism of Pie and Ale.

Transcriptome Profiling of Flower Development Reveals Key Genes Mediating Yellow Formation in Tree Peony

Tree peony cultivars with yellow flowers are rare and valuable,but the molecular mechanism of pigment accumulation is not clear.In this study,the petal transcriptome of three developmental stages were sequenced to determine the differentially expressed genes(DEGs)regulating yellow flowers color.The results showed that 10,842 and 12,022 DEGs were screened in stage 1 vs.stage 2 and in stage 2 vs.stage 3,respectively.Through analysis of flavonoid structural genes(FSGs),we found that the transcription level of DFR was very low in the three developmental stages.In a small group of cultivars,the DFR transcription level of red flowers was 862 times higher than that of yellow flowers.These data suggested that the flavonoid pathway is interrupted in the later stage due to the low transcriptional level of DFR,which limits the biosynthesis of anthocyanins in yellow flowers.The transcription levels of F3’H and FLS were upregulated from stage 1 to stage 2,while those of CHI and FLS were downregulated from stage 2 to stage 3.In addition,67 MYBs and 44 bHLHs showed similar transcription profiles with different members of FSGs.The results deepen our understanding of the molecular mechanism of yellow pigment accumulation in tree peony.

Age-Related Alterations in DNA Methylation and APETALA2(AP2)Levels in Herbaceous Peony(Paeonia lactiflora Pall.)

The ornamental and commercial values of herbaceous peony(Paeonia lactiflora Pall.)are directly related to its flower pattern.However,the molecular mechanisms underlying the type formation of P.lactiflora flowers have not been studied in great detail.Previous studies identified,using integrated multipleomics analysis,revealed that APETALA2(AP2)is an important candidate gene that modulates type formation of P.lactiflora flowers.To further reveal the expression mechanism of AP2 in P.lactiflora petals,we examined the profile of AP2 expression in the inner and outer petals of‘ZiFengyu’at various developmental stages using qRT-PCR and BSP+Miseq methylation analysis.Based on our data,the AP2 levels in the outer petals were obviously increased,relative to the inner petals.In addition,the S3 levels at the bloom stage were significantly higher than at the flower-bud stage S1,thereby promoting bloom stage S2,while declining stage S4.Using chromosome walking,the 2000 bp of the 5′-end upstream promoter region was achieved.This region harbored a CpG island(−665∼−872 bp),with multiple essential transcription factor binding sites(TFBS)such as NF-kappa B,GATA-1,Sp1,and C/EBP.Methylation sequencing revealed 7 methylated CpG sites in the CpG island region of the AP2 promoter,thereinto,the methylation ratio of the CpG-3 site in the inner petals was significantly higher than in the outer petals.Correlation analysis revealed a negative association between the level of methylation(CpG-3,CpG-6),and AP2 mRNA expression.CpG-3 was located on the Sp1 transcription factor binding site.Thus,we speculated that the CpG-3 methylation may inhibit transcription factor Sp1 binding to the gene promoter,thereby regulating AP2 expression.Herein,we examined the role of AP2 in the determination of flower patterns in P.lactiflora.Our conclusion will provide theoretical guidance for the molecular breeding of the flower pattern in P.lactiflora.

Genetic Diversity Analysis of Morphological Traits among 22 Peony Cultivars

[ Objective] This study analyzed genetic diversity of 22 representative peony cultivars to investigate genetic differences between peony cultivars and ex- plore excellent germplasm resources, aiming at providing scientific basis for analysis of genetic relationships between different peony cultivars and germplasm innova- tion. [ Method ] Flower color, flower type, plant height, plant width, flower diameter, leaf length, leaf width, plant type and leaf type of 22 peony cultivars were surveyed. The genetic distances between peony cuhivars were calculated with Euclidean distance method. The genetic diversity of 22 peony cultivars was analyzed by UPGMA cluster analysis. [ Result] Based on the overall average of total genetic distance, UPGMA cluster analysis results were classified. Thus, 22 peony culti- vars were divided into five categories. Category [ consisted of Yinhongqiaodui, Jizhuahong, Babaoxiang, Chongloudiancui, Congzhongxiao, Weizi, Yanlongzi and Qiejiazi ; category II consisted of Huhong; category III consisted of Xiaohuhong, Baiyulan, Yingsuhong, Fengdan, Dajinfeng, Jingyu, Panzhongquguo, Qingshan- guanxue, Mojiatrrong, Dahongyipin and Lanhuakui; category IV consisted of Haihuang; category V consisted of Sihelian. The classification results were consistent with pedigree analysis. Peony cuhivars with unknown pedigree were assigned to corresponding heterosis group. [ Conclusion] The diversity analysis of morphologi- cal traits is an effective method for investigating genetic differences between different cultivars, which may lay the theoretic foundation for breeding, industrial devel- opment and application of new peony cultivars.