[Selection and validation of reference genes for quantitative real-time PCR analysis in Paeonia veitchii].

Paeonia veitchii and P. lactiflora are both original plants of the famous Chinese medicinal drug Paeoniae Radix Rubra in the Chinese Pharmacopoeia. They have important medicinal value and great potential in the flower market. The selection of stable and reliable reference genes is a necessary prerequisite for molecular research on P. veitchii. In this study, two reference genes, Actin and GAPDH, were selected as candidate genes from the transcriptome data of P. veitchii. The expression levels of the two candidate genes in different tissues(phloem, xylem, stem, leaf, petiole, and ovary) and different growth stages(bud stage, flowering stage, and dormant stage) of P. veitchii were detected using real-time fluorescence quantitative technology(qRT-PCR). Then, the stability of the expression of the two reference genes was comprehensively analyzed using geNorm, NormFinder, BestKeeper, ΔCT, and RefFinder. The results showed that the expression patterns of Actin and GAPDH were stable in different tissues and growth stages of P. veitchii. Furthermore, the expression levels of eight genes(Pv-TPS01, Pv-TPS02, Pv-CYP01, Pv-CYP02, Pv-CYP03, Pv-BAHD01, Pv-UGT01, and Pv-UGT02) in different tissues were further detected based on the transcriptome data of P. veitchii. The results showed that when Actin and GAPDH were used as reference genes, the expression trends of the eight genes in different tissues of P. veitchii were consistent, validating the reliability of Actin and GAPDH as reference genes for P. veitchii. In conclusion, this study finds that Actin and GAPDH can be used as reference genes for studying gene expression levels in different tissues and growth stages of P. veitchii.

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

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

Tree peony transcription factor PrWRI1 enhances seed oil accumulation.

BACKGROUND: WRINKLED1 (WRI1) encodes a transcription factor, belonging to the APETALA2 (AP2) family, and plays a key role in regulating plant oil biosynthesis. As a newly woody oil crop, tree peony (Paeonia rockii) was notable for the abundant unsaturated fatty acids in its seed oil. However, the role of WRI1 during the accumulation of P. rockii seeds oil remains largely unknown. RESULTS: In this study, a new member of the WRI1 family was isolated from P. rockii and was named PrWRI1. The ORF of PrWRI1 consisted of 1269 nucleotides, encoding a putative protein of 422 amino acids, and was highly expressed in immature seeds. Subcellular localization analysis in onion inner epidermal cells showed that PrWRI1 was located at the nucleolus. Ectopic overexpression of PrWRI1 could significantly increase the total fatty acid content in Nicotiana benthamiana leaf tissue and even PUFAs in transgenic Arabidopsis thaliana seeds. Furthermore, the transcript levels of most genes related to fatty acids (FA) synthesis and triacylglycerol (TAG) assembly were also up-regulated in transgenic Arabidopsis seeds. CONCLUSIONS: Together, PrWRI1 could push carbon flow to FA biosynthesis and further enhance the TAG amount in seeds with a high proportion of PUFAs.

[Identification of genes involved in biosynthesis of paeoniflorin in Paeonia lactiflora based on transcriptome analysis].

Paeoniflorin, a representative pinane monoterpene glycoside, is the main active component and quality index of Paeoniae Radix Alba and Paeoniae Radix Rubra.The possible biosynthesis of paeoniflorin is as follows: GPP is derived from mevalonate(MVA) and/or 2-C-methyl-D-erythritol 4-phosphate(MEP) pathway(s) followed by the catalysis with terpene synthase, cytochrome P450(CYP450), UDP-glucuronosyltransferase(UGT), and acyltransferase(AT), respectively.This study aims to explore the genes rela-ted to the biosynthesis of paeoniflorin.To be specific, the cDNA libraries for flowers, leaves, and roots of Paeonia lactiflora were established and sequenced.A total of 30 609 open reading frames(ORFs) were yielded.Through functional annotation and expression analysis of all CYP450 genes in the transcriptome, 11 CYP450 genes belonging to CYP71 A and CYP71 D subfamilies and showing expression trend consistent with monoterpene synthase PlPIN that may be involved in paeoniflorin biosynthesis were screened out.Subsequently, 7 UGT genes and 9 AT genes demonstrating the expression trend consistent with PlPIN which were possibly involved in paeoniflorin biosynthesis were further screened by functional annotation analysis, full-length sequence analysis, expression analysis, and phylogeny analysis.This study provided a systematic screening method with smaller number of candidate genes, thus reducing the workload of functional gene verification.The result laid a foundation for analyzing the biosynthesis pathway of paeoniflorin and the formation mechanism.

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.

Identification of differentially expressed miRNAs and their target genes in response to brassinolide treatment on flowering of tree peony (Paeonia ostii).

Tree peony is a famous flower plant in China, but the short and concentrated flowering period limits its ornamental value and economic value. Brassinolide (BR) plays an important role in plant growth and development including flowering. There have been a large number of reports on the molecular aspects of the flowering process, but the genetic mechanism that was responsible for miRNA-guided regulation of tree peony is almost unclear. In this study, the leaves of tree peony cultivar, 'Feng Dan', were sprayed with different concentrations of BR, and the obvious bloom delay was found at the treatment with BR 50 µg/L. The small RNA sequencing and transcriptome sequencing were performed on the petals of tree peony under an untreated control (CK) and the treatment with BR 50 µg/L during four consecutive flowering development stages. A total of 22 known miRNAs belonging to 12 families were identified and 84 novel miRNAs were predicted. Combined with transcriptome data, a total of 376 target genes were predicted for the 18 differentially expressed known miRNAs and 177 target genes were predicted for the 23 differentially expressed novel miRNAs. Additionally, the potential miRNAs and their target genes were identified, including miR156b targeting SPL, miR172a_4 targeting AP2 and four novel miRNAs targeting SPA1, and revealed that they might affect the flowering time in tree peony. Collectively, these results would provide a theoretical basis for further analysis of miRNA-guided regulation on flowering period in tree peony.

Changes of pollen viability after preservation of Paeonia lactiflora in different provenances.

Pollen contains all the haploid genetic information of species and is of great significance to preserve germplasm resources safely and effectively. The acquisition of high quality materials is a very important step in germplasm preservation. This study compared the viability and physiological condition of Paeonia lactiflora pollen from several provenances after preservation, to explore the effect of provenance difference on pollen viability and physiological responses after preservation. The results showed that: the pollen viability of two cultivars were significantly different in provenances after preserved at -20 °C or liquid nitrogen (LN) for 3 months, the pollen viability of 'Fen Yu Nu' showed Lanzhou > Beijing > Luoyang > Heze, while the pollen viability of 'Zi Feng Chao Yang' showed Luoyang > Beijing > Heze. Similarly, the oxidative stress levels of the Paeonia lactiflora pollen after preservation with LN or -20 °C were also significantly different among the provenances, and there was a relationship between the viability and the oxidative stress levels produced by the provenances differences. Reactive oxygen species (ROS) content, malondialdehyde (MDA) content, superoxide dismutase (SOD) activity and glutamate reductase (GR) activity in pollen from different provenances were contrary to the changes of viability; while catalase (CAT), ascorbic acid peroxidase (APX), ascorbic acid (AsA) and glutathione (GSH) were consistent with the changes of viability. The results indicated that the responses of antioxidant systems of two cultivars pollen to preservation with LN or -20 °C were different in provenances, and this difference was one of the reasons for the different viability of pollen after preservation with LN or -20 °C.

Characterization of genes encoding ω-6 desaturase PoFAD2 and PoFAD6, and ω-3 desaturase PoFAD3 for ALA accumulation in developing seeds of oil crop Paeonia ostii var. lishizhenii.

Paeonia ostii var. lishizhenii has emerged as a valuable oil-producing crop with splendid characteristic of high α-linolenic acid (C18:3, ALA) content in its seed oil for healthy food supplement, but the molecular mechanism for seed ALA accumulation remains enigmatic. In our previous report, a PoSAD gene encoding stearoyl-ACP desaturase had been cloned and functional charactered for the first desaturation procedure involved in ALA biosynthesis pathway in P. ostii var. lishizhenii endosperms, while other participants have not been identified to date. In this study, full-length cDNAs of PoFAD2 (1489 bp), PoFAD6 (1638 bp), and PoFAD3 (1709 bp) were isolated based on our recent transcriptome sequencing data. Bioinformatic analyses revealed that the PoFADs were closest to their counterparts from Paeoniaceae species P. ludlowii, P. rockii, and P. suffruticosa in phylogenetic tree, which shared highly conserved histidine boxes (HXXXH, HXXHH, and HXXHH), exhibiting typical characters of membrane-bound desaturases in higher plants. Additionally, the PoFAD2 and PoFAD3 were specifically expressed and highly associated with LA and ALA accumulation in developing endosperms, whereas PoFAD6 expression has no significantly difference during whole seed developing stages. The catalytic function of these PoFADs were further analyzed by heterologous expression in Saccharomyces cerevisiae and Arabidopsis thaliana. The results showed that PoFAD2 and PoFAD6 could catalyze linoleic acid (C18:2) synthesis, while PoFAD3 had ability to produce ALA. This study functional identified three PoFAD genes, which indicates their critical roles in ALA biosynthesis pathway in P. ostii var. lishizhenii, and is of great theoretical and practical meaning on breeding and cultivating new tree peony varieties to promote human health and nutrition supplement.

Cryopreserved-pollen viability is regulated by NO-induced programmed cell death.

KEY MESSAGE: After cryopreservation, the NO content in pollen increased, inducing programmed cell death as a key reason for reduced viability. Low recovery of biomaterials after cryopreservation is a bottleneck that limits the application of this technology. At present, the mechanism of viability decline after cryopreservation is not fully understood. In this study, the effects of nitric oxide (NO) on programmed cell death (PCD) and its relationship with viability were investigated, using Paeonia lactiflora 'Fen Yu Nu' pollen with significantly decreased viability after cryopreservation. The results showed that: the activity of caspase-3-like and caspase-9-like protease and the apoptosis rate of pollen cells were significantly increased, the expression level of the promoting PCD (pro-PCD) genes was up-regulated, while the expression level of the inhibiting PCD (anti-PCD) genes was down-regulated after preservation in liquid nitrogen (LN); the NO content in pollen cells increased significantly after LN exposure. The correlation analysis showed that NO was significantly correlated with pollen viability and all indicators of PCD. The addition of a NO carrier SNP after LN storage reduced pollen viability, increased endogenous NO content, decreased mitochondrial membrane potential level, activated caspase-3-like and caspase-9-like protease in pollen cells, and increased cell apoptosis rate. The expression levels of pro-PCD genes PDCD2 and ATG8CL were significantly up-regulated, while the expression levels of anti-PCD genes DAD1, BI-1 and LSD1 were significantly down-regulated. The addition of NO scavenger c-PTIO improved pollen viability, and produced the opposite effect of sodium nitroferricyanide (III) dihydrate (SNP), but did not change the mitochondrial membrane potential. These results suggest that NO induced PCD during the cryopreservation of pollen, which was one of the reasons for the significant decrease of pollen viability after cryopreservation.

Analysis and Functional Verification of PoWRI1 Gene Associated with Oil Accumulation Process in Paeonia ostii.

The plant transcription factor WRINKLED1 (WRI1), a member of AP2/EREBP, is involved in the regulation of glycolysis and the expression of genes related to the de novo synthesis of fatty acids in plastids. In this study, the key regulator of seed oil synthesis and accumulation transcription factor gene PoWRI1 was identified and cloned, having a complete open reading frame of 1269 bp and encoding 422 amino acids. Subcellular localization analysis showed that PoWRI1 is located at the nucleus. After the expression vector of PoWRI1 was constructed and transformed into wild-type Arabidopsis thaliana, it was found that the overexpression of PoWRI1 increased the expression level of downstream target genes such as BCCP2, KAS1, and PKP-ß1. As a result, the seeds of transgenic plants became larger, the oil content increased significantly, and the unsaturated fatty acid content increased, which provide a scientific theoretical basis for the subsequent use of genetic engineering methods to improve the fatty acid composition and content of plant seeds.

Comparative and phylogenetic analyses of the chloroplast genomes of species of Paeoniaceae.

Plants belonging to family Paeoniaceae are not only economically important ornamental plants but also medicinal plants used as an important source of traditional Chinese medicine. Owing to the complex network evolution and polyploidy evolution of this family, its systematics and taxonomy are controversial and require a detailed investigation. In this study, three complete chloroplast genomes of sect. Paeonia, one of the sections of Paeonia, were sequenced and then analysed together with 16 other published chloroplast genomes of Paeoniaceae species. The total lengths of the chloroplast genomes of these species were 152,153-154,405 bp. A total of 82-87 protein-coding genes, 31-40 tRNA genes and 8 rRNA genes were annotated. Bioinformatics analysis revealed 61-74 simple sequence repeats (SSRs) in the chloroplast genomes, most of which have A/T base preference. Codon usage analysis showed that A/U-ending codons were more positive than C/G-ending codons, and a slight bias in codon usage was observed in these species. A comparative analysis of these 19 species of Paeoniaceae was then conducted. Fourteen highly variable regions were selected for species relationship study. Phylogenetic analysis revealed that the species of sect. Paeonia gathered in one branch and then divided into different small branches. P. lactiflora, P. anomala, P. anomala subsp. veitchii and P. mairei clustered together. P. intermedia was related to P. obovata and P. obovata subsp. willmottiae. P. emodi was the sister to all other species in the sect. Paeonia.

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.

Expression profiles of genes involved in fatty acid and lipid biosynthesis in developing seeds of Paeonia ostii.

BACKGROUND: Paeonia ostii seeds were identified as novel sources of edible plant oil with a high proportion of α-linolenic acid, a type of n-3 fatty acid with many health benefits. Due to the unreliability of seed oil content and quality, it is necessary to discover the mechanism underlying lipid biosynthesis in Paeonia ostii seeds. OBJECTIVES: This study aimed to identify the key genes involved in lipid biosynthesis in Paeonia ostii seeds by analyzing the relationship among the seed characteristics and the expression patterns of lipid genes in Paeonia ostii during seed development. METHODS: Preliminary research on Paeonia ostii seed development was carried out from 10 days after pollination until maturity, focusing on phenology, oil content and lipid profiles. In addition, we investigated the spatiotemporal expression of 36 lipid biosynthetic genes in Paeonia ostii by using quantitative real-time PCR. RESULTS: The results suggested that the development of Paeonia ostii seeds from pollination to maturity could be divided into three periods. The 36 lipid genes showed various spatiotemporal expression patterns and five gene groups with distinct temporal patterns during seed development were identified by clustering analysis of expression data. Furthermore, the relationships between gene expression and lipid/fatty acid accumulation and some candidate key lipid genes were discussed. CONCLUSIONS: This study provided the global patterns of fatty acid and lipid biosynthesis-related gene expression, which are critical to understanding the molecular basis of lipid biosynthesis and identifying the lipid accumulation rate-limiting genes during seed development.

Identification of genes associated with the biosynthesis of unsaturated fatty acid and oil accumulation in herbaceous peony 'Hangshao' (Paeonia lactiflora 'Hangshao') seeds based on transcriptome analysis.

BACKGROUND: Paeonia lactiflora 'Hangshao' is widely cultivated in China as a traditional Chinese medicine 'Radix Paeoniae Alba'. Due to the abundant unsaturated fatty acids in its seed, it can also be regarded as a new oilseed plant. However, the process of the biosynthesis of unsaturated fatty acids in it has remained unknown. Therefore, transcriptome analysis is helpful to better understand the underlying molecular mechanisms. RESULTS: Five main fatty acids were detected, including stearic acid, palmitic acid, oleic acid, linoleic acid and α-linolenic acid, and their absolute contents first increased and then decreased during seed development. A total of 150,156 unigenes were obtained by transcriptome sequencing. There were 15,005 unigenes annotated in the seven functional databases, including NR, NT, GO, KOG, KEGG, Swiss-Prot and InterPro. Based on the KEGG database, 1766 unigenes were annotated in the lipid metabolism. There were 4635, 12,304, and 18,291 DEGs in Group I (60 vs 30 DAF), Group II (90 vs 60 DAF) and Group III (90 vs 30 DAF), respectively. A total of 1480 DEGs were detected in the intersection of the three groups. In 14 KEGG pathways of lipid metabolism, 503 DEGs were found, belonging to 111 enzymes. We screened out 123 DEGs involved in fatty acid biosynthesis (39 DEGs), fatty acid elongation (33 DEGs), biosynthesis of unsaturated fatty acid (24 DEGs), TAG assembly (17 DEGs) and lipid storage (10 DEGs). Furthermore, qRT-PCR was used to analyze the expression patterns of 16 genes, including BBCP, BC, MCAT, KASIII, KASII, FATA, FATB, KCR, SAD, FAD2, FAD3, FAD7, GPAT, DGAT, OLE and CLO, most of which showed the highest expression at 45 DAF, except for DGAT, OLE and CLO, which showed the highest expression at 75 DAF. CONCLUSIONS: We predicted that MCAT, KASIII, FATA, SAD, FAD2, FAD3, DGAT and OLE were the key genes in the unsaturated fatty acid biosynthesis and oil accumulation in herbaceous peony seed. This study provides the first comprehensive genomic resources characterizing herbaceous peony seed gene expression at the transcriptional level. These data lay the foundation for elucidating the molecular mechanisms of fatty acid biosynthesis and oil accumulation for herbaceous peony.

Plant hormonal changes and differential expression profiling reveal seed dormancy removal process in double dormant plant-herbaceous peony.

Herbaceous peony (Paeonia lactiflora Pall.) is a popular ornamental and medicinal plant. Taking approximately six to seven months, the seeds germination under natural conditions experiences dual dormancies, which seriously affects horticultural cultivation. Few studies have been conducted on exploring both biological and molecular mechanism that regulates dormancy removal process in hypocotyls double dormant plants. Here, we first measured ABA and GA3 content changes at four key dormancy break stages, and then performed transcriptomic analyses to identify the differentially expressed genes (DEGs) using RNA-seq. We subsequently carried out Quantitative real-time PCR (qRT-PCR) to validate RNA-seq data. ABA content decreased during the whole dormancy removal process and GA3 content exhibited decreasing slightly and then increasing trend. RNA sequencing de novo assembly generated a total of 99,577 unigenes. 20,344 unigenes were differentially expressed in the whole dormancy release process. The qPCR results of 54 selected unigenes were consistent with the FPKM values obtained from RNA-seq. Our results summarize a valuable collection of gene expression profiles characterizing the dormancy release process. The DEGs are candidates for functional analyses of genes affecting the dormancy release, which is a precious resource for the on-going physiological and molecular investigation of seeds dormancy removal in other perennial plants.

Development of SSR markers in Paeonia based on De Novo transcriptomic assemblies.

Peony is a famous ornamental and medicinal plant in China, and peony hybrid breeding is an important means of germplasm innovation. However, research on the genome of this species is limited, thereby hindering the genetic and breeding research on peony. In the present study, simple sequence repeat (SSR) locus analysis was performed on expressed sequence tags obtained by the transcriptome sequencing of Paeonia using Microsatellite software. Primers with polymorphism were obtained via polymerase chain reaction amplification and electrophoresis. As a result, a total of 86,195 unigenes were obtained by assembling the transcriptome data of Paeonia. Functional annotations were obtained in seven functional databases including 49,172 (Non-Redundant Protein Sequence Database: 57.05%), 38,352 (Nucleotide Sequence Database: 44.49%), 36,477 (Swiss Prot: 42.32%), 38,905 (Clusters of Orthologous Groups for Eukaryotic Complete Genomes: 45.14%), 37,993 (Kyoto Encyclopedia of Genes and Genomes: 44.08%), 26,832 (Gene Ontology: 31.13%) and 37,758 (Pfam: 43.81%) unigenes. Meanwhile, 21,998 SSR loci were distributed in 17,567 unigenes containing SSR sequences, and the SSR distribution frequency was 25.52%, with an average of one SSR sequence per 4.66 kb. Mononucleotide, dinucleotide, and trinucleotide were the main repeat types, accounting for 55.74%, 25.58%, and 13.21% of the total repeat times, respectively. Forty-five pairs of the 100 pairs of primers selected randomly could amplify clear polymorphic bands. The polymorphic primers of these 45 pairs were used to cluster and analyze 16 species of peony. The new SSR molecular markers can be useful for the study of genetic diversity and marker-assisted breeding of peony.

Age-associated methylation change of CHI promoter in herbaceous peony (Paeonia lactiflora Pall).

Chalcone isomerase gene (CHI) is a key gene that regulates the formation of yellow traits in petals. To reveal transcriptional regulatory mechanisms of CHI gene in petals of Paeonia lactiflora, we investigated the CHI expression using qPCR, the pigment content by HPLC, and methylation levels using BSP+Miseq sequencing in 'Huangjinlun' variety during different developmental stages including flower-bud stage (S1), initiating bloom (S2), bloom stage (S3), and withering stage (S4). Results showed that the expression level of CHI gene at S2 stage was significantly higher than that at other stages (P<0.05), and at S4 stage was extremely significantly lower than other stages (P<0.01). Besides, total anthocyanin, anthoxanthin, and flavonoid contents in petals presented a similar trend with CHI expression during developmental stages. A total of 16 CpG sites varying methylation levels were detected in CHI gene core promoter region, of which the methylation levels at mC-4 and mC-16 sites were extremely significantly negatively correlated with CHI mRNA expression (P<0.01). mC-16 site is located in the binding region of C/EBPα transcription factor, suggesting that methylation at the mC-16 site may inhibit the binding of C/EBPα to CHI promoter DNA, thereby regulating the tissue-specific expression of CHI gene. Our study revealed the expression pattern of CHI gene in petal tissues of P. lactiflora at different developmental stages, which is related to promoter methylation. Moreover, the important transcription regulation element-C/EBPα was identified, providing theoretical reference for in-depth study on the function of CHI gene in P. lactiflora.

Characterization of the glycerol-3-phosphate acyltransferase gene and its real-time expression under cold stress in Paeonia lactiflora Pall.

Elucidating the cold tolerance mechanism of Paeonia lactiflora, which is one of the most valuable ornamental and medicinal plants in Asia, fundamentally impacts its breeding and production. The glycerol-3-phosphate acyltransferase (GPAT) gene plays a pivotal role in cold resistance in a variety of plant species. Here, we cloned the P. lactiflora GPAT gene, determined its expression pattern, and tested its role in cold resistance. We obtained the full-length P. lactiflora GPAT gene using tissue-cultured seedlings and real-time polymerase chain reaction and rapid amplification of cDNA ends analyses. We named this gene PlGPAT in P. lactiflora. Phylogenetic analysis indicates that the PlGPAT gene is closely related with the GPAT genes in core eudicots. The phylogenetic tree containing 31 angiosperm species based on GPAT protein sequences is largely consistent with the known phylogeny in flowering plants. We conducted a time-course PlGPAT expression analysis and demonstrated that PlGPAT expression is correlated with low-temperature stress. Our results suggest that the PlGPAT gene plays an important role in regulating cold resistance in P. lactiflora.

Oil biosynthesis and transcriptome profiles in developing endosperm and oil characteristic analyses in Paeonia ostii var. lishizhenii.

Paeonia ostii var. lishizhenii, a well-known medicinal and horticultural plant, is indigenous to China. Recent studies have shown that its seed has a high oil content, and it was approved as a novel resource of edible oil with a high level of α-linolenic acid by the Chinese Government. This study measured the seed oil contents and fatty acid components of P. ostii var. lishizhenii and six other peonies, P. suffruticosa, P. ludlowii, P. decomposita, P. rockii, and P. lactiflora Pall. 'Heze' and 'Gansu'. The results show that P. ostii var. lishizhenii exhibits the average oil characteristics of tested peonies, with an oil content of 21.3%, α-linolenic acid 43.8%, and unsaturated fatty acids around 92.1%. Hygiene indicators for the seven peony seed oils met the Chinese national food standards. P. ostii var. lishizhenii seeds were used to analyze transcriptome gene regulation networks on endosperm development and oil biosynthesis. In total, 124,117 transcripts were obtained from six endosperm developing stages (S0-S5). The significant changes in differential expression genes (DEGs) clarify three peony endosperm developmental phases: the endosperm cell mitotic phase (S0-S1), the TAG biosynthesis phase (S1-S4), and the mature phase (S5). The DEGs in plant hormone signal transduction, DNA replication, cell division, differentiation, transcription factors, and seed dormancy pathways regulate the endosperm development process. Another 199 functional DEGs participate in glycolysis, pentose phosphate pathway, citrate cycle, FA biosynthesis, TAG assembly, and other pathways. A key transcription factor (WRI1) and some important target genes (ACCase, FATA, LPCAT, FADs, and DGAT etc.) were found in the comprehensive genetic networks of oil biosynthesis.

Identification and characterization of microRNAs in tree peony during chilling induced dormancy release by high-throughput sequencing.

Tree peony, one of the most valuable horticultural and medicinal plants in the world, has to go through winter to break dormancy. Growing studies from molecular aspects on dormancy release process have been reported, but inadequate study has been done on miRNA-guided regulation in tree peony. In this study, high-throughput sequencing was employed to identify and characterize miRNAs in three libraries (6 d, 18 d and 24 d chilling treatments). There were 7,122, 10,076 and 9,097 unique miRNA sequences belonging to 52, 87 and 68 miRNA families, respectively. A total of 32 conserved miRNAs and 17 putative novel miRNAs were identified during dormancy release. There were 771 unigenes as potential targets of 62 miRNA families. Total 112 known miRNAs were differentially expressed, of which 55 miRNAs were shared among three libraries and 28 miRNAs were only found in 18 d chilling duration library. The expression patterns of 15 conserved miRNAs were validated and classified into four types by RT-qPCR. Combining with our microarray data under same treatments, five miRNAs (miR156k, miR159a, miR167a, miR169a and miR172a) were inversely correlated to those of their target genes. Our results would provide new molecular basis about dormancy release in tree peony.