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.

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.

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.

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.

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.

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.

Study on the Response and Recovery Characteristics of Different Herbaceous Peony (<i>Paeonia lactiflora</i>Pall.) Varieties to Waterlogging Stress

The root of herbaceous peony (Paeonia lactiflora Pall.) is fleshy, and different varieties have different tolerance to waterlogging stress. In order to explore its response and recovery characteristics to waterlogging stress, six varieties of herbaceous peony with strong, medium and weak waterlogging tolerance and high ornamental value were selected as experimental materials. After the vegetative growth of each variety was completed, the field simulated waterlogging stress experiment was carried out by the semi-flooded (the water surface is half the height of the flowerpot) pot method. Changes in photosynthetic parameters, chlorophyll fluorescence parameters, relative water content (RWC), relative conductivity (REC), chlorophyll content, lutein cycle, and leaf microstructure were analyzed during recovery from waterlogging stress and stress relief. The results showed that the time of reaching the most significant difference between CK and tested varieties was different. From the beginning of stress to 60% of the leaves with symptoms, the varieties with strong and moderate waterlogging tolerance experienced longer time;the proportion of palisade tissue in leaves was larger;the maximum photochemical quantum yield (Fv/Fm) was reduced less;it could increase xanthophyll cycle and heat dissipation (NPQ) to consume excess light energy, and maintain a higher net photosynthetic rate (Pn) for normal growth of plants in a short period of time. The REC in leaves of varieties with weak waterlogging tolerance increased more, and the damage of cell membrane was more serious. After the stress was removed, all indexes recovered to different degrees. Based on our comprehensive analysis, the comprehensive waterlogging resistance of the experimental materials followed the pattern: “Lihong” > “Yangfeichuyu” > “Taohuafeixue” > “Dafugui” > “Qihualushuang” > “Hongxiuqiu”. It is suggested that the variety “Lihong” and “Yangfeichuyu”, with strong comprehensive waterlogging tolerance, can be selected for propagation and cultivation in areas prone to waterlogging. In the later stage, it is still necessary to further expand the number and scale of varieties, combined with the in-depth study of waterlogging-resistance genes, so as to provide a theoretical reference for the cultivation and production of new waterlogging-resistant varieties.

Composition and antioxidant activity of Paeonia lactiflora petal flavonoid extract and underlying mechanisms of the protective effect on H_(2)O_(2)-induced oxidative damage in BRL3A cells

Flavonoids have attracted considerable attention due to their health benefits. This study aimed to investigate the flavonoid profiles and antioxidant activity of Paeonia lactiflora petal flavonoid extract(PPF). The UHPLC-ESI-Q-Exactive HF MS/MS method was established for characterization, and 21 predominant flavonoid compounds were tentatively identified in PPF. Among them, isoscutellarein-7-(6’-acetylallosyl-(1->2)-glucoside) and scutellarin methylester were discovered in PPF for the first time. Pretreatment with PPF significantly reduced H2O2-induced cell damage, ROS accumulation, and malondialdehyde content and increased the activity of SOD, CAT, and GSH-Px in buffalo rat liver 3A(BRL3A) cells. Moreover, the expression of nuclear Factor E2-related factor(Nrf2) was upregulated by PPF, whose expression trend was consistent with that of heme oxygenase-1(HO-1), glutamate-cysteine ligase catalytic subunit(GCLC), and NAD(P)H quinone oxidoreductase-1(NQO1). These findings suggested that herbaceous peony flavonoids can be used as a natural bioactive agent to prevent oxidative stress.