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.

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.

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.

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.

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.

Distribution of Metabolites in Root Barks of Seven Tree Peony Cultivars for Quality Assessment Using NMR-based Metabolomics

Objective To determine the distribution of metabolites in the root barks of different tree peony cultivars for quality assessment. Methods Seven tree peony phenotypic cultivars with different colors were systematically analyzed using NMR-based metabolomics. Results A total of 16 metabolites from their methanol extracts were simultaneously identified and quantified, including one primary metabolite （sucrose） and 15 secondary ones （acetophenones, phenolics, monoterpene glycosides, flavonoids, and unsaturated fatty acids）. The quantitative data indicated that sucrose （90-180 mg/g） and acetophenones （15-100 mg/g）, and non-phenolics, monoterpene glycosides, flavonoids, and unsaturated fatty acids （2-15 mg/g） were the major metabolites in these tree peony cultivars. The significantly increasing levels of paeonoside with bioactivity were observed in ＂Xiangyu＂, ＂Wujinyaohui＂, ＂Roufurong＂, ＂Yaohuang＂, ＂Zhaofen＂, ＂Doulu＂, and ＂Yingrihong＂ in order. Opposite trends in the levels of paeonoside and paeonol were observed in ＂Xiangyu＂ and ＂Yingrihong＂, suggesting that the changes of the secondary metabolites in plants were influenced by primary metabolites, such as sucrose/glucose, and the different physiological processes occurred in different tree peony cultivars. Conclusion ＂Yingrihong＂ with red flower has the highest medicine quality whereas ＂Xiangyu＂ with white flower has the worst one based on the content of paeonoside.

Molecular Cloning and Expression Analysis of Cryptochrome Gene Ps CRY2in Tree Peony

Cryptochromes are blue/ultraviolet-A(UV-A) light receptors involved in regulating various aspects of plant growth and development.Investigations of the structure and functions of cryptochromes in plants have largely focused on herbaceous plants. However, few data on the function of CRY2 are available in woody plants. In this study, a cryptochrome 2(CRY2) gene was isolated from Paeonia suffruticosa by Reverse Transcription Polymerase Chain Reaction(RT-PCR). Sequence alignment and motif analysis showed that the deduced amino acids contained a PHR domain near the amino terminus and a CCT domain near the carboxy terminus. Ps CRY2 showed high identity with At CRY2 of Arabidopsis.Phylogenetic analysis indicated that it was closely related to Citrus sinensis. Gene expression analysis revealed that the highest expression levels of Ps CRY2 occurred in the bud and seed embryo of P. suffruticosa, followed by the roots, stems, and leaves. Ps CRY2 was upregulated during the entire process of bud differentiation, whereas this was downregulated during the early stage of bud development and upregulated in the middle and late stages. The highest level of Ps CRY2 expression was observed in the big bell-like flower buds. These results suggested that Ps CRY2 plays an important role in both bud differentiation and bud development. The expression patterns of Ps CRY2 in the buds of ‘Luoyanghong' and ‘Qiufa1' were similar, whereas that in the buds of ‘Qiufa 1' was significantly higher than in the buds of ‘Luoyanghong'. The buds of plants subjected to different photoperiod treatments exhibited variations in Ps CRY2 expression patterns. The expression of Ps CRY2 decreased during bud sprouting and in the small bell-like flower buds that were subjected to short-day photoperiod compared to that observed under long-day photoperiod.

Effects of Foliage Boron-Spraying on Seed Yields and Fatty Acid Composition of Tree Peony(Paeonia ostii ‘Feng Dan’)

The influence of the foliage boron（B）-spaying concentration on the yield and fatty acid composition of tree peony（Paeonia ostii ＇Feng Dan＇） was explored in the experiment of 2014-2015. In this research, a statistically significant correlation was found between the mass concentration of foliage boron-spraying and tree peony seed yield. Maximum yield increment of tree peony seeds was obtained when the boron-spraying concentration was 4 g/L. The composition of fatty acid was analyzed by gas chromatography-mass spectrometry（GC-MS）, and the main components of the fatty acid composition were quantified by internal standard method. The increase in the oil extraction rate after foliage boron-spraying nutrition of different concentration was significant and ranged between 0.04% and 11.43%. Seed oil production and oil extraction rate were also increased due to the increase of seed yields. Furthermore, foliage boron-spraying had a significant effect on the content of linolenic acid and linoleic acid in seed oil.

Evaluation on the fresh eating quality of tree peony flowers

Tree peony(Paeonia suffruticosa)is an ornamental and medicinal plant in China.The fresh eating of flower petals has gradually become a new idea for further development.This study analyzed the sensory indexes,nutritional indexes and antioxidant indexes of fresh flowers of five cultivars,namely,‘Fengdan’(P.ostia‘Fengdan’),‘Xiangyu’(P.suffruticosa‘Xiangyu’),‘Zhaofen’(P.suffruticosa‘Zhanfen’),‘Luoyanghong’(P.suffruticosa‘Luoyanghong’)and‘High Noon’(P.‘High Noon’),to provide a theoretical basis for eating fresh tree peony flowers.The aroma volatiles of petals mainly comprised terpenes,alcohols and alkanes.‘Luoyanghong’and‘High Noon’have brighter colors.The taste test showed that‘Luoyanghong’,‘High Noon’and‘Xiangyu’were the most palatable.‘High Noon’contained the highest soluble sugar content,‘Luoyanghong’contained the highest starch and vitamin C(VC)levels,and‘Zhaofen’contained the highest soluble protein content.The contents of bioactive compounds were the highest in‘Luoyanghong’,and superoxide dismutase(SOD)activity was the highest in‘Xiangyu’.The petals were rich in essential amino acids,with the highest amount in‘Xiangyu’.‘Luoyanghong’and‘High Noon’showed strong antioxidant capacity.Therefore,tree peony flowers have excellent sensory qualities,abundant nutrients and strong antioxidant activity and could be further developed as a food to be eaten fresh.The results showed that tree peony flowers had great edible potential.Flowers of red and yellow cultivars can be considered for fresh eating on the basis of the traditional consumption of white and pink tree peony flowers.

Regulation of phytohormones on root primordium initiation and adventitious root formation in the etiolated shoots of Paeonia suffruticosa'Yinfen Jinlin'

Tree peony is well known and sought after for its large, colorful flowers. Its propagation is via vegetative methods. Mech- nisms of the adventitious rooting and the regulation of rooting processes are the principles and techniques of plant propagation and improvement. Microstructures and fluctuations of phytohormones in the adventitious rooting were studied with the etiolated soft- wood shoots of Paeonia suffkuticosa ＇Yinfen Jinlin＇. There are no pre-primordia in the shoots of the cultivar. Adventitious roots are produced in five stages： shoot selection, primordium initiation, primordium growth, conducting tissue differentiation and root protru- sion. Primordia initiated in the cortex. The contents of the endogenous hormones, IAA, ABA and GA, were 5.842, 0.873 and 1.043 nmol·g^-1 FW on the bases of shoots, respectively. CTKs which included isopentenyl adenine （iPA）, zeatin riboside （ZR） and dihy- drozeatin riboside （DHZR） were 0.949, 0.695 and 2.034 nmol·g^-1 FW, respectively. DHZR is active among CTKs. The ratio of IAA to GA, CTK and ABA clearly increased at the stage of primordium initiation, while they showed low levels at the stages of primor- dium growth. The ratios were restored at the shoot levels at the stage of root protrusion. IBA provoked primordia initiation in the cortex, the vascular cambium, the pith and even in the callus induced on the base of shoots. ]AA levels in the treated shoots increased gradually to its highest level （three times of control） at the stage of conducting tissue differentiation. The ratios of IAA to GA, CTK and ABA clearly decreased at the stage of primordium initiation. The ratio of IAA to ABA is regulated at 10：1.

Establishment of highly efficient plant regeneration of Paeonia ostii‘Fengdan’through optimization of callus,adventitious shoot,and rooting induction

Tree peony is a famous ornamental plant,while the low propagation rate is the main hurdles hindering the industry development.Till now,the highly efficient regeneration system for tree peony is not established.In this study,using Paeonia ostii’Fengdan’mature embryos,the effects of variations in inoculation method,initiating culture,adventitious shoot induction,rooting media,plant growth regulators(PGRs),and a nonconventional PGR(plant extracts)on regeneration from explants were evaluated.In embryo cultures,embryonic callus induction rate of 1/4 embryos was the highest among those of embryos with other three technical treatments(whole embryos,1/2 embryos,and pieces of embryos).The woody plant medium(WPM)containing 1.0 mg·L^(-1)6-BA,0.5 mg·L^(-1)GA3,30.0 g·L^(-1)sucrose,and 3.0 g·L^(-1)phytagel significantly improved shoot induction and multiplication.3.0 mg·L^(-1)plant extracts promoted hypocotyl germination,rooting,and root growth,in direct embryo culture,and a combination of 3.0 mg·L^(-1)plant extracts+2.0 mg·L^(-1)IBA+1.5 mg·L^(-1)IAA produced optimal rooting induction rate for multiple shoots in direct embryo culture and indirect somatic embryogenesis.For the three in vitro micropropagation methods,the highest shoot proliferation coefficient(5.4±0.2)was obtained with indirect somatic embryogenesis.Fortunately,the propagation ability of shoots remains high,even when culture propagation was continued for more than two years.Thus,a reliable system for plant regeneration from mature embryos derived from P.ostii’Fengdan’callus and two direct embryo culture systems have been established.The novel regeneration system could facilitate uniform seedling production.

Transcriptome Analysis and Genes Function Verification of Root Development of Paeonia suffruticosa under Sandy Loam Cultivation

Relatively poor in vitro rooting has limited the large-scale commercial production of tree peony.In this study,on the basis of transcriptome sequencing,differentially expressed genes and the associated metabolic pathways were identified in tree peony roots at different stages of root formation under sandy loam cultivation.A total of 31.63 Gb raw data were generated and 120,188 unigenes(mean length of 911.98 bp)were annotated according to six databases(NR,NT,GO,KEGG,COG,and Swiss-Prot).Analyses of the ungerminated root primordium period,induced root primordium period,and root formation period detected 8,232,6,907,and 10,687 differentially expressed genes related to 133,132,and 133 metabolic pathways,respectively.Two significantly differentially expressed genes(Unigene13430_All and CL10096.Contig1_All)were associated with the auxin pathway.The full-length Unigene13430_All coding sequence(843 bp)encoded 280 amino acids,whereas the full-length CL10096.Contig1_All coding sequence(1,470 bp)encoded 489 amino acids.Unigene13430_All and CL10096.Contig1_All were identified as IAA gene family members and were respectively named PsIAA27 and PsARF19.The qRT-PCR analysis and functional verification indicated that the expressions of PsARF19 and PsIAA27 in tree peony seedlings,cuttings and grafted seedlings were significant different.PsARF19 promoted root development,it might be a regulatory gene related to the formation of tree peony roots,while PsIAA27 inhibited lateral root development,and it might be involved in controlling auxin sensitivity during root formation.The results of this study may form the basis of future investigations on the mechanism mediating peony root formation.The transcriptome data will be an excellent resource for researchers interested in characterizing the rooting-related tree peony genes.

安徽药用牡丹炭疽病病原菌鉴定

牡丹(Paeonia suffruticosa Andr.)是芍药科植物,为多年生落叶灌木,具有很高的观赏和药用价值。因其风姿华丽、色彩艳丽、品种繁多、深受大家喜爱,是中国传统名花和世界著名花卉之一。其根皮也被称为丹皮,具有抗炎、抑制血小板、止痛、清热等作用,是六味地黄丸等著名中成药的主要配方,也是饮片中的常用品[1-2]。近年来,随着牡丹种植面积的不断扩大,造成牡丹病害的大面积发生,已给牡丹生产带来严重影响。