Lactiflora Cultivars Research Articles

Widely targeted metabolomics reveals differences in metabolites of Paeonia lactiflora cultivars.

Paeonia lactiflora contains diverse active constituents and exhibits various pharmacological activities. However, only partial identification of biologically active substances from P. lactiflora has been achieved using low-throughput techniques. Here, the roots of P. lactiflora, namely, Fenyunu (CK), Dafugui (DFG), and Red Charm (HSML), were studied. The primary and secondary metabolites were investigated using ultrahigh-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESIMS/MS). The chemical compounds and categories were detected using broadly targeted UPLC-MS/MS. Principal component analysis (PCA), orthogonal partial least-squares discriminant analysis (OPLS-DA), and hierarchical clustering analysis (HCA) were carried out for metabolites of different varieties of P. lactiflora. A total of 1237 compounds were detected and classified into 11 categories. HCA, PCA, and OPLS-DA of these metabolites indicated that each variety of P. lactiflora was clearly separated from the other groups. Differential accumulated metabolite analysis revealed that the three P. lactiflora varieties contained 116 differentially activated metabolites (DAMs) involved in flavonoid, flavone, and flavonol metabolism. KEGG pathway analysis revealed that, in 65 pathways, 336 differentially abundant metabolites (DMs) were enriched in the CK and DFG groups; moreover, the type and content of terpenoids were greater in the CK group than in the DFG group. The CK and HSML groups contained 457 DMs enriched in 61 pathways; the type and amount of flavonoids, terpenoids, and tannins were greater in the CK group than in the HSML group. The DFG and HSML groups contained 497 DMs enriched in 65 pathways; terpenoids and alkaloids were more abundant in the HSML variety than in the DFG variety. A total of 1237 compounds were detected, and the results revealed significant differences among the three P. lactiflora varieties. Among the three P. lactiflora varieties, phenolic acids and flavonoids composed the largest and most diverse category of metabolites, and their contents varied greatly. Therefore, CK is suitable for medicinal plant varieties, and DFG and HSML are suitable for ornamental plant varieties. Twelve proanthocyanidin metabolites likely determined the differences in color among the three varieties.

The Composition and Function of the Rhizosphere Bacterial Community of Paeonia lactiflora Varies with the Cultivar.

The composition and diversity of the rhizosphere microbial community maintain the stability of the root microclimate, and several studies have focused on this aspect of rhizosphere microorganisms. However, how these communities vary with cultivars of a species is not completely understood. Paeonia lactiflora-a perennial herb species of the family Paeoniaceae-includes a wide variety of cultivars, with rich rhizosphere microbial resources. Hence, we studied the differences in rhizosphere bacterial communities associated with eight P. lactiflora cultivars. We noted that Actinobacteria, Proteobacteria, Acidobacteria, Bacteroidetes, Firmicutes, Verrucomicrobia, Planctomycetes and Chloroflexi were the dominant phyla associated with the cultivars. The composition of rhizosphere bacterial community of different cultivars was highly similar at taxonomic levels, but there were slightly differences in the relative abundance. LEfSe analysis showed that the cultivars "Sheng Tao Hua" and "Zi Lou Xian Jin" exhibited the most biomarkers. Differential ASV analysis revealed the maximum difference in ASV abundance between "Lian Tai" and "Zi Hong Zheng Hui", as well as between "Sheng Tao Hua" and "Tao Hua Fei Xue", and the maximum similarity between "Duo Ye Zi" and "Xue Feng". Co-occurrence network analysis revealed that rhizosphere bacteria in most cultivars maintain homeostasis by cooperation, wherein Actinobacteria and Proteobacteria played a vital role. In addition, microbial resources related to cultivars like bioremediation, organic degradation and resistance to diseases are found. This study revealed the structures of the rhizosphere bacterial communities associated with different cultivars of P. lactiflora and explored their stress resistance potential, which can be used to guide future agricultural practices.

Comprehensive Microbiome and Metabolome Analyses Reveal the Medicinal Components of Paeonia lactiflora.

Paeonia lactiflora Pall. is not only a traditional ornamental plant, but also an important medicinal plant. Currently, some P. lactiflora cultivars are used for ornamental purposes, but their potential medicinal value is ignored. To explore the medicinal potential of the ornamental varieties, the medicinal cultivar 'Hangbaishao' (HS) and the ornamental cultivar 'Zifengyu' (ZFY) were selected, and microbiome and metabolome analyses were performed to compare the composition of the endophytes and metabolites in the roots. The diversity and abundance of bacteria were not significantly different between HS and ZFY; however, the diversity and abundance of endophytic fungi in the ornamental cultivar ZFY were much higher than those in the medicinal cultivar HS. The flavonoids and phenolic acid contents of the ornamental cultivar ZFY were significantly higher than those of the medicinal cultivar HS, indicating that ZFY has medicinal value. The differences in root endophytes between HS and ZFY may lead to differences in phenolic acids and flavonoids. To explore the relationship between endophytes and the accumulation of phenolic acids and flavonoids, a joint analyses of the microbiome and metabolome were performed. The key bacterium, Ruminococcaceae bacterium GD7, led to the accumulation of phenolic acids and flavonoids in the ZFY. This study contributes to future research on the potential medicinal value of ornamental P. lactiflora and provides a new approach for realizing the 'dual use of medicine and appreciation' of P. lactiflora.

Xylem development and phloem conductivity in relation to the stem mechanical strength of Paeonia lactiflora

The quality of cut Paeonia lactiflora flowers is limited by their low stem mechanical strength, but the underlying mechanism of this low strength is poorly understood. In this study, two P. lactiflora cultivars with distinct stem mechanical strengths (Chui Touhong with low stem mechanical strength and Da Fugui with high stem mechanical strength) were used as test materials. The xylem development was examined at the cellular level, and the phloem conductivity was analyzed by evaluating phloem geometry. The results showed that the secondary cell wall formation of the xylem of Chui Touhong was affected primarily in fiber cells but was affected little in vessel cells. The formation of the secondary cell walls in the xylem fiber cells of Chui Touhong was delayed, resulting in longer and thinner fiber cells with a lack of cellulose and S-lignin in the secondary cell walls. Moreover, the phloem conductivity of Chui Touhong was lower than that of Da Fugui, and more callose was accumulated in the lateral walls of the phloem sieve elements of Chui Touhong. Consequently, the delayed deposition of the secondary cell walls of the xylem fiber cells was the main factor leading to the low stem mechanical strength of Chui Touhong, and the low stem mechanical strength was closely related to the low conductivity of sieve tubes and extensive callose accumulation in the phloem. These findings provide a new perspective on enhancing P. lactiflora stem mechanical strength by targeting single cell level, and lay the foundation for future works on the correlation between phloem long-distance transport and stem mechanical strength.

Proteomic Analysis of the Inflorescence Stem Mechanical Strength Difference in Herbaceous Peonies (Paeonia lactiflora Pall.).

The herbaceous peony (Paeonia lactiflora Pall.) is a traditional rare flower in China, and production of its cut flowers has developed gradually in many places of the world. However, the inflorescence stems of some P. lactiflora cultivars have such low mechanical strength that the cut flower production was severely restricted. To better understand the causes of this problem from a protein expression level, two P. lactiflora cultivars with different inflorescence stem mechanical strengths were analyzed by two-dimensional electrophoresis and MALDI-TOF/MS. More than 1700 clear protein spots were detected, 53 of which varied significantly. Moreover, 23 of the differentially expressed proteins were identified and confirmed and are involved in various biological processes such as metabolism, protein biosynthesis and transport, signal transduction, and defensive response. Especially, cinnamyl alcohol dehydrogenase (CAD) and xyloglucan endotransglucosylase/hydrolase (XTH) were strongly connected to the inflorescence stem mechanical strength in P. lactiflora.

Melatonin enhances stem strength by increasing lignin content and secondary cell wall thickness in herbaceous peony.

Cut flower quality is severely restrained by stem bending due to low stem strength. Melatonin has been shown to function in many aspects of plant growth and development, yet whether it can enhance stem strength, and the corresponding underlying mechanisms remain unclear. We investigated the role of melatonin in enhancement of stem strength in herbaceous peony (Paeonia lactiflora Pall.) by applying exogenous melatonin and changing endogenous melatonin biosynthesis. Endogenous melatonin content positively correlated with lignin content and stem strength in various P. lactiflora cultivars. Supplementation with exogenous melatonin significantly enhanced stem strength by increasing lignin content and the S/G lignin compositional ratio, up-regulating lignin biosynthetic gene expression. Moreover, overexpression of TRYPTOPHAN DECARBOXYLASE GENE (TDC) responsible for the first committed step of melatonin biosynthesis in tobacco, significantly increased endogenous melatonin, which further increased the S/G ratio and stem strength. In contrast, silencing PlTDC in P. lactiflora decreased endogenous melatonin, the S/G ratio and stem strength. Finally, manipulating the expression of CAFFEIC ACID O-METHYLTRANSFERASE GENE (COMT1), which is involved in both melatonin and lignin biosynthesis, showed even greater effects on melatonin, the S/G ratio and stem strength. Our results suggest that melatonin has a positive regulatory effect on P. lactiflora stem strength.

Development of SCAR Markers to Identify Medicinal Cultivars of Paeonia lactiflora.

Paeoniae Radix, the dried root of Paeonia lactiflora, is one of the most important ingredients in Kampo medicine. It is known that Paeoniae Radix is derived from various P. lactiflora cultivars, including medicinal and horticultural cultivars, and that cultivar identification by DNA analysis has been unsuccessful. We attempted to develop sequence characterized amplified region (SCAR) markers as useful DNA markers for the identification and herbal medicine authentication of two cultivars developed in Japan, 'Bonten' and 'Kitasaisho,' which are two superior medicinal strains of P. lactiflora. Sequence-related amplified polymorphism (SRAP) analysis was conducted on fourteen P. lactiflora cultivars, and polymorphic fragments specific to 'Bonten' or 'Kitasaisho' were detected. Then, SCAR markers for 'Bonten' and 'Kitasaisho' were developed from the sequence information of these polymorphic fragments. Thirty cultivars of P. lactiflora and five herbal medicine samples were used to validate the specificity of the developed SCAR markers. As a result, we confirmed that our SCAR markers can identify 'Bonten' or 'Kitasaisho' from the plant samples and the herbal medicine samples. Thus, we have successfully designed two highly specific DNA markers and established an easy, rapid, and cost-efficient method to identify specific cultivars of P. lactiflora. Our SCAR markers are expected to contribute to the maintenance of P. lactiflora cultivars such as 'Bonten' as superior medicinal strains, the development of more elite cultivars in the future, and the deterrence of outflow of original cultivars to foreign countries.

Isolation and Screening of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase producing PGPR from Paeonia lactiflora rhizosphere and enhancement of plant growth

Plant growth-promoting rhizobacteria (PGPR) work in a variety of ways to promote plant growth and increase crop yield. They've recently gotten a lot of attention in crops, but not so much in ornamental plants. In this study, 13 PGPR strains containing 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase named YDSY1-13 were isolated from the rhizosphere soil of four Paeonia lactiflora cultivars using the medium with ACC as the sole nitrogen source. The ACC deaminase activity, IAA synthesis ability, phosphorus-solubilizing ability, and siderophile production ability of these strains were significantly different, YDSY11 has the highest ACC deaminase activity and YDSY8 has the highest ability to synthesize IAA. The pot experiment showed that inoculation with YDSY8 and YDSY11 dramatically enhanced the photosynthetic capacity, nutrient accumulation, and antioxidant enzyme activity of Paeonia lactiflora. The Petri-dish growth-promoting experiment revealed that PGPR strains boosted Arabidopsis thaliana development, with different strains having different impacts on root morphology, including main root length, number of root tips and lateral roots, and root hair density. Among them, YDSY1, YDSY2, YDSY4, and YDSY8 have the greatest effect on increasing A. thaliana biomass, and the increase in aboveground biomass was positively correlated with that of underground biomass, with the exception of YDSY8. Different strains have diverse growth-promoting abilities based on their growth-promoting characteristics, and this ability is mostly controlled by the combined effect of different growth-promoting characteristics. In addition, we found that the same PGPR may have different growth-promoting mechanisms for different plants. This study provides primary data for the development and utilization of rhizosphere growth-promoting bacteria of P. lactiflora and lays a foundation for further research on the growth-promoting mechanism of PGPR and provides a solution for alleviating fertilizer pollution to the environment.

Silicon enhances stem strength by promoting lignin accumulation in herbaceous peony (Paeonia lactiflora Pall.)

Herbaceous peony (Paeonia lactiflora Pall.) is a popular high-end cut flower, but stem bending caused by low stem strength severely decreases its quality. To enhance stem strength, the regulatory effects of exogenous silicon were investigated in P. lactiflora. The results showed that silicon application enhanced stem strength by increasing the thickness of secondary cell walls and the layers of thickened secondary cells. Moreover, more lignin accumulated, particularly G-lignin and S-lignin, and the activities of lignin biosynthetic enzymes increased with silicon application. In addition, based on transcriptome analysis, silicon application induced the expression of genes participating in lignin biosynthesis pathway. Among them, hydroxycinnamoyl-CoA: shikimate hydroxycinnamoyl transferase gene (HCT1) was isolated from P. lactiflora and found to be mainly localized in the cytoplasm of cells. Overexpression of PlHCT1 increased the layers of thickened secondary cells and lignin accumulation in tobacco, resulting in enhanced stem strength and demonstrably straight stems. Finally, silicon content, lignin content and PlHCT1 expression in P. lactiflora cultivars with high stem strengths were totally higher than those in cultivars with low stem strengths. These results indicated that silicon application enhanced stem strength by promoting lignin accumulation in P. lactiflora, which has prospects for stem quality improvement in general.

Polymorphism analysis of TCP gene region to intraspecific analysis of Paeonia lactiflora, and application for authentication of Paeoniae Radix.

We attempted to conduct an intraspecific analysis of 30 peony cultivars in Japan and to authenticate five herbal medicine samples derived from Paeoniae Radix by polymorphism analysis of the TEOSINTE BRANCHED1, CYCLOIDEA, and PCF (TCP) gene region. We focused on cultivar-dependent differences in leaf margin undulation and analyzed the sequence of the related TCP gene region. As a result, we found that the nucleotide sequences of 29 cultivars of Paeonia lactiflora except 'America' exhibit interspecific variations compared with the nucleotide sequences of Paeonia suffruticosa and Paeonia tenuifolia. Therefore, in the dendrogram constructed on the basis of the sequence similarity in the TCP gene region, the 29 cultivars of P. lactiflora were separated from P. suffruticosa, P. tenuifolia, and 'America', and clustered into three subgroups. There were 16 variations containing heterogenous DNA sequences within P. lactiflora species, and two characteristic variations in subgroup I. Some P. lactiflora cultivars showed the same nucleotide sequence in the TCP gene region, whereas the five herbal medicine samples showed different sequences, although all of them could be authenticated. The results suggest that Paeoniae Radix in the Japanese crude drug market can be authenticated by analysis of the TCP gene region.

Lignin provides mechanical support to herbaceous peony (Paeonia lactiflora Pall.) stems

Stem bending caused by mechanical failure is a major constraint for high-quality herbaceous peony (Paeonia lactiflora Pall.) cut flowers, but little is known about the underlying factors. In this study, two P. lactiflora cultivars, Xixia Yingxue (bending) and Hong Feng (upright), were used to investigate differences in stem bending. The results showed that the stem mechanical strength of Hong Feng was significantly higher than that of Xixia Yingxue, and the thickening of the secondary cell wall and the number of thickened secondary cell wall layers in Hong Feng were significantly higher than those in Xixia Yingxue. Moreover, compared with Xixia Yingxue, Hong Feng showed greater lignification of the cell wall and lignin deposition in the cell walls of the sclerenchyma, vascular bundle sheath and duct. All three types of lignin monomers were detected. The S-lignin, G-lignin, and total lignin contents and the activities of several lignin biosynthesis-related enzymes were higher in Hong Feng than in the other cultivar, and the S-lignin content was closely correlated with stem mechanical strength. In addition, 113,974 full-length isoforms with an average read length of 2106 bp were obtained from the full-length transcriptome of P. lactiflora stems, and differential expression analysis was performed based on the comparative transcriptomes of these two cultivars. Ten lignin biosynthesis-related genes, including 26 members that were closely associated with lignin content, were identified, and multiple upregulated and downregulated transcription factors were found to positively or negatively regulate lignin biosynthesis. Consequently, lignin was shown to provide mechanical support to P. lactiflora stems, providing useful information for understanding the formation of P. lactiflora stem strength.

HpeNet: Co-expression Network Database for de novo Transcriptome Assembly of Paeonia lactiflora Pall.

The herbaceous peony (Paeonia lactiflora Pall.) is a well-known ornamental flowering and pharmaceutical plant found in China. Its high medicinal value has long been recognized by traditional Chinese medicine (as Radix paeoniae Alba and Radix paeoniae Rubra), and it has become economically valued for its oilseed in recent years; like other Paeonia species, it has been identified as a novel resource for the α-linolenic acid used in seed oil production. However, its genome has not yet been sequenced, and little transcriptome data on Paeonia lactiflora are available. To obtain a comprehensive transcriptome for Paeonia lactiflora, RNAs from 10 tissues of the Paeonia lactiflora Pall. cv Shaoyou17C were used for de novo assembly, and 416,062 unigenes were obtained. Using a homology search, it was found that 236,222 (approximately 57%) unigenes had at least one BLAST hit in one or more public data resources. The construction of co-expression networks is a feasible means for improving unigene annotation. Using in-house transcriptome data, we obtained a co-expression network covering 95.13% of the unigenes. Then we integrated co-expression network analyses and lipid-related pathway genes to study lipid metabolism in Paeonia lactiflora cultivars. Finally, we constructed the online database HpeNet (http://bioinformatics.cau.edu.cn/HpeNet) to integrate transcriptome data, gene information, the co-expression network, and so forth. The database can also be searched for gene details, gene functions, orthologous matches, and other data. Our online database may help the research community identify functional genes and perform research on Paeonia lactiflora more conveniently. We hope that de novo transcriptome assembly, combined with co-expression networks, can provide a feasible means to predict the gene function of species that do not have a reference genome.

NUTRITIONAL EVALUATION OF HERBACEOUS PEONY (PAEONIA LACTIFLORA PALL.) PETALS

Herbaceous peony (Paeonia lactiflora Pall.) is a kind of plant with ornamental, edible and medicinal values, and few studies were concerned to edible aspect of herbaceous peony petals. The aim of this research is to establish edible quality evaluation system of herbaceous peony flowers. Petals of 46 P. lactiflora cultivars at full bloom period were used to determine the content of soluble sugar, organic acid, protein, Vc (Vitamin C), total phenolics, total flavonoids, mineral elements and SOD (superoxide dismutase) activity and screen out evaluation index for edible herbaceous peony. The results showed that flower petals of herbaceous peony contained lots of nutrients and the contents varied with different cultivars. The soluble sugar content was 66.55-177.28 mg/g FW, organic acid 2.19-6.90 mg/g FW, soluble protein 6.53-121.56 mg/g FW, Vc 9.77-30.24 mg/100 g FW, total phenolics 9.41-33.01 mg/g DW, total flavonoids 3.50-17.56 mg/g DW, SOD activity 305.62-520.42 U/g FW, total amino acids 6.43-11.99 g/100 g DW. The average content of Na, Mg, K, Ca, Mn, Fe, Ni, Zn, Mo, Cr were 55.88±14.90 µg/g DW, 1218.22±349.60 µg/g DW, 11252.23±2477.54 µg/g DW, 1975.40±706.58 µg/g DW, 8.30±6.55 µg/g DW, 103.56±182.72 µg/g DW, 10.73±37.94 µg/g DW, 22.80±16.68 µg/g DW, 1.84±5.89 µg/g DW and 17.36±44.89 µg/g DW, respectively. Based on principal component analysis and cluster analysis, we found ‘Dielian Qihua’, ‘Zhushapan’, ‘Xueyuan Honghua’, ‘Wulong Jisheng’, ‘Honglou’, ‘Bingshan’, ‘Hongyan Yushuang’, ‘Zituo Ronghua’, ‘Zifengyu’, ‘Fenlou Dianchun’ had better edible quality. The results will provide some information for the comprehensive utilization of herbaceous peony petals and the breeding of edible herbaceous peony cultivars.

Evaluation of ITS2 for intraspecific identification of Paeonia lactiflora cultivars

Herbaceous peony (Paeonia lactiflora Pall.) is an important ornamental and medicinal plant. DNA barcodes can reveal species identity via the nucleotide diversity of short DNA segments. In this study, two main candidate DNA barcodes (ITS2 and psbA-trnH) were tested to identify twenty-one cutting cultivars of P. lactiflora and their wild species. The efficacy of the candidate DNA barcodes was assessed by PCR amplification, sequence quality, sequence diversity, rate of correct identification, and phylogenetic analysis. ITS2 was easy to be amplified and sequenced among the samples. The identification by Blastn and phylogenetic analysis was 95.4% and 63.6%, respectively. For psbA-trnH, the presence of poly A-T led to sequencing failure which limited its use as DNA barcode candidate. Moreover, the authentic efficiency of psbA-trnH was lower than ITS2. The results showed that ITS2 is suitable as a candidate DNA barcode for the intraspecific identification of P. lactiflora cultivars.

Rescue and in vitro culture of herbaceous peony immature embryos by organogenesis

Herbaceous peony (Paeonia lactiflora Pall.) is a landscaping plant that has considerable development prospects worldwide. However, its embryo dysplasia severely limits the breeding of high-quality P. lactiflora cultivars by the cross-breeding method. In this study, the immature natural hybrid embryos of P. lactiflora ‘Zi Fengyu’ with double flower were used to study its rescue and in vitro culture by organogenesis. The results showed that the best medium for adventitious bud induction was modified Murashige and Skoog (MS) medium supplemented with 1.0mg/L thidiazuron (TDZ), 600mg/L acidic hydrolysis casein (ACH), 10% coconut water and 100g/L sucrose. The best medium for adventitious bud multiplication was modified MS medium supplemented with 4.0mg/L TDZ, 15% coconut water, 60g/L sucrose and 0.1% activated charcoal (AC). Modified MS medium containing 0.5mg/L gibberellic acid (GA3), 100g/L sucrose and 0.1% AC was the more suitable medium for adventitious bud growth. For root regeneration, indole-3-acetic acid (IAA) was a more effective plant growth regulator (PGR), and 1/2 MS medium supplemented with 0.5mg/L IAA, 30g/L sucrose and 0.1% AC was the more suitable medium, resulting in a rooting rate of up to 92.12%, and the survival rate was 70.8%. Additionally, histological observation confirmed that the rescue and in vitro culture of P. lactiflora immature embryos by organogenesis was successful. These results provide useful technical support for breeding high-quality P. lactiflora cultivars by the cross-breeding method.

Transcriptome and digital gene expression analysis of herbaceous peony (Paeonia lactiflora Pall.) to screen thermo-tolerant related differently expressed genes

Herbaceous peony (Paeonia lactiflora Pall.) is easily injured by heat stress (HS), which greatly restricts its application and promotion. In this study, the thermo-tolerance of three representative P. lactiflora cultivars had been firstly assessed. ‘Zifengyu’ was identified as the thermo-tolerant cultivar with relatively lower values and smaller variations in malondialdehyde, hydrogen peroxide (H2O2) and proline contents under HS. Subsequently, their transcriptomes were sequenced by RNA sequencing (RNA-seq) technology to construct a complete database. 81,599 unigenes were obtained, and 34,940 unigenes had been annotated. Moreover, through digital gene expression analysis of thermo-tolerant ‘Zifengyu’ and moderately thermo-tolerant ‘Hongyanzhenghui’, 161 heat stress response genes had been screened involving heat shock protein genes, plant hormone signal transduction related genes, fatty acid synthesis genes, reactive oxygen species-scavenging genes and secondary metabolites related genes. And the effectively and timely response of these genes to HS could endow thermo-tolerance to ‘Zifengyu’. Among these genes, 11 key thermo-tolerant related genes whose expressions were all significantly up-regulated in ‘Zifengyu’ and ‘Hongyanzhenghui’ during development and the former possessed higher levels could be regarded as the candidate genes, including isoprene synthase gene, 2 peroxidase genes, 3-oxoacyl-acyl carrier protein reductase gene (FabG), 3 transcription factor genes (bHLH, NAC and WRKY), HSP20 and 3 HSP70. These results could provide a better understanding of heat stress response in P. lactiflora, and pave for the breeding of thermo-tolerant cultivars.

Biochemical and molecular responses of herbaceous peony to high temperature stress

To clarify the theoretical basis of the differences in high temperature stress tolerance among herbaceous peony (Paeonia lactiflora Pall.), we investigated the heat injury index of twelve P. lactiflora cultivars. Of these, heat-tolerant ‘Zifengyu’ and moderately heat-tolerant ‘Hongyanzhenghui’ were selected to study the biochemical and molecular responses to high temperature stress. ‘Zifengyu’ had reduced malondialdehyde (MDA) content, increased soluble sugar, chlorophyll (Chl) a, Chl b, Chl a + b. and carotenoid contents, as well as elevated antioxidant enzymes activities, photosynthetic rate (Pn), transpiration rate (Tr) and relatively intact cellular structures compared with ‘Hongyanzhenghui’, especially when the temperature was the highest. Additionally, we isolated partial cDNAs of two heat shock protein genes (HSP60 and HSP90) from P. lactiflora, which were 880-bp and 1077-bp nucleotides in length, respectively. The expression levels of PlHSP60, PlHSP70 and PlHSP90 were lower in ‘Zifengyu’ than in ‘Hongyanzhenghui’ for the first three of four developmental stages examined. These results indicated that heat-tolerant P. lactiflora cultivar could effectively scavenge reactive oxygen species (ROS), protect cellular structures, reduce thermal damage and delay the death of plants by enhancing antioxidant enzymes activities and HSP expression under high temperature stress. These findings provide a theoretical basis for breeding heat-tolerant P. lactiflora cultivars.

Anatomical and biochemical analysis reveal the role of anthocyanins in flower coloration of herbaceous peony

Herbaceous peony (Paeonia lactiflora Pall.) is particularly appreciated because of its elegant and gorgeous flower color, but little is known about the underlying mechanisms of flower coloration. In this study, three P. lactiflora cultivars ‘Xuefeng’, ‘Fenyulou’ and ‘Dahonglou’ with white, pink and red flower were selected as the materials. Their anatomical structures, cell sap pH and metal elements were investigated, and the colored pigment mainly distributed in palisade mesophyll was only found in ‘Fenyulou’ and ‘Dahonglou’, and their shape of epidermal cells, cell sap pH and metal elements were not the key factors deciding phenotype color. Moreover, the qualitative and quantitative analysis of flavonoids were performed, their total anthocyanin, anthoxanthin and flavonoid contents were decreased during flower development, and only anthocyanin content in ‘Dahonglou’ was always higher than that in ‘Xuefeng’ and ‘Fenyulou’. Subsequently, three anthocyanin compositions were found, and peonidin 3,5-di-O-glucoside (Pn3G5G) was identified as the main anthocyanin composition. In addition, the full-length of flavonol synthase gene (FLS) was isolated with the GenBank accession number KM259902, and the expression patterns of eight flavonoid biosynthetic genes showed that only PlDFR and PlANS basically had the highest levels in ‘Dahonglou’ and the lowest levels in ‘Xuefeng’, and they basically displayed a descended trend during flower development especially PlDFR, suggesting that these two genes might play a key role in the anthocyanin biosynthesis which resulted in the shift from white to pink and red in flowers. These results would contribute to understand the underlying molecular mechanisms of flower coloration in P. lactiflora.

Identification of miRNAs Responsive to Botrytis cinerea in Herbaceous Peony (Paeonia lactiflora Pall.) by High-Throughput Sequencing.

Herbaceous peony (Paeonia lactiflora Pall.), one of the world’s most important ornamental plants, is highly susceptible to Botrytis cinerea, and improving resistance to this pathogenic fungus is a problem yet to be solved. MicroRNAs (miRNAs) play an essential role in resistance to B. cinerea, but until now, no studies have been reported concerning miRNAs induction in P. lactiflora. Here, we constructed and sequenced two small RNA (sRNA) libraries from two B. cinerea-infected P. lactiflora cultivars (“Zifengyu” and “Dafugui”) with significantly different levels of resistance to B. cinerea, using the Illumina HiSeq 2000 platform. From the raw reads generated, 4,592,881 and 5,809,796 sRNAs were obtained, and 280 and 306 miRNAs were identified from “Zifengyu” and “Dafugui”, respectively. A total of 237 conserved and 7 novel sequences of miRNAs were differentially expressed between the two cultivars, and we predicted and annotated their potential target genes. Subsequently, 7 differentially expressed candidate miRNAs were screened according to their target genes annotated in KEGG pathways, and the expression patterns of miRNAs and corresponding target genes were elucidated. We found that miR5254, miR165a-3p, miR3897-3p and miR6450a might be involved in the P. lactiflora response to B. cinerea infection. These results provide insight into the molecular mechanisms responsible for resistance to B. cinerea in P. lactiflora.

Identification of ages and determination of paeoniflorin in roots of Paeonia lactiflora Pall. From four producing areas based on growth rings

Growth rings were used to determine the root age of medicinal Paeonia lactiflora from four producing areas, and their corresponding paeoniflorin content were measured based on the identification of ages. Different P. lactiflora root samples of different ages were collected from the four major growing areas in China: Bozhou, Anhui Province; Pan'an, Zhejiang Province; Zhongjiang, Sichuan Province; and Heze, Shandong Province. The relationship between the number of growth rings and age was analyzed using hand sections and paraffin sections. The paeoniflorin content in the roots of different P. lactiflora cultivars from different growing areas was measured using high-performance liquid chromatography (HPLC). The growth rings in the P. lactiflora roots were consistent with the age of the plant from Heze, Zhongjiang, Pan'an, whereas that for the P. lactiflora from Bozhou was one less than the age of the plant. The HPLC results show that the paeoniflorin content was highest in P. lactiflora 'Baihuachuanshaoyao,' followed by 'Baihuahangshaoyao,' 'Honghuachuanshaoyao,' and 'Honghuahangshaoyao,' 'Bozhoushaoyao' had the lowest levels of paeoniflorin. With increasing age, the paeoniflorin in the roots of the different P.lactiflora cultivars slowly declined or remained the same. In summary, the age of the roots of P. lactiflora from different growing areas can be determined using growth rings. The paeoniflorin content in the roots of P. lactiflora is correlated with cultivar and it was slowly declined with increasing age.