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MicroRNA (miRNA) belongs to a class of non-coding small RNAs that post-transcriptionally regulate gene expression. In this study, we employed transcriptome, small RNA, and degradome sequencing to s...

Oil peony (Paeonia spp.) is a new type of woody oil crop in China with a large cultivation area. Inadequate pollination is one of the main reasons for low seed yield. A pollination net room was built at an oil tree peony base, the numbers of honeybees (Apis mellifera L.) and ground bumblebees (Bombus terrestris L.) were artificially increased and the foraging behaviors and daily activities of the two bees on the plants were observed. Four different pollination methods (honeybee pollination, ground bumblebee pollination, natural field pollination and pollination without insects) were applied. The visit interval, single-flower visit time, number of single-flower visits, number of flowers visited per minute and number of stigma contacts were compared. Meanwhile, the effects of honeybee and bumblebee pollination on the oil yield and quality of peony seeds were compared. There were noticeable differences in daily activity between honeybees and ground bumblebees. Significant differences in the single-flower visit time, visit interval and visit frequency were also observed; honeybee and ground bumblebee pollination increased the seed yield of oil tree peony by 78.74% and 31.88%, respectively. Therefore, both honeybees and ground bumblebees are effective pollinators of oil tree peony. These results provide a theoretical basis for further utilization of bee resources for oil tree peony pollination.

Paeonia ostii T. Hong & J.X. Zhang, an emerging oil crop, was intercropped with 3-year-old plantations of pawpaw [Chaenomeles sinensis (Thouin.) Koehne], Chinese toon (Toona sinensis Roem.), and walnut (Juglans regia L.). In order to achieve a better production system, we studied the effects of intercropping on the photosynthesis, growth, and yield of P. ostii. The results showed that different pattern of agroforestry systems changed microclimatic and growing site conditions in comparison to the control treatment. The correlation analysis demonstrated that both similarities and differences were found in relationship between net photosynthetic rate (PN) and main ecophysiological factors at different treatments. Agroforestry systems significantly reduced transpiration rate and increased water-use efficiency (WUE), maximal quantum yield of PSII photochemistry, chlorophyll (Chl) a and total Chl contents, whereas there were no differences between PN, intercellular CO2 concentration, photochemical efficiency of PSII in the light, and plant height in these systems. The obviously exponential relationship between PN and PAR during a day were observed in Chinese toon and walnut treatments. The highest effective quantum yield of PSII photochemistry, electron transport rate, and photochemical quenching coefficient were observed in walnut treatment. The higher WUE and SPAD value, the thinnest stem, the biggest crown, the lowest stomatal conductance, and Chl a/b ratio as well as the fewest pods and harvest seed yield were observed in pawpaw treatment. In addition, there was a significant correlation between SPAD value and Chl (a+b) of P. ostii in agroforestry system. This study could be an important contribution for the science of land management in oil peony and other understory crops.

Background The tree peony (Paeonia section Moutan DC), one of the traditional famous flowers with both ornamental and medicinal value, was widely used in China. Surprisingly little is known about the full-length transcriptome sequencing in tree peony, limiting the research on its gene function and molecular mechanism. The trehalose phosphate phosphatase (TPS) family genes has been found to affect plant growth and development and the function of TPS genes in Paeonia ostii is unknown. Methods In our study, we performed single molecule, full-length transcript sequencing in P. ostii. 10 TPS family members were identified from PacBio sequencing for bioinformatics analysis and transcriptional expression analysis. Results A total of 230,736 reads of insert (ROI) sequences and 114,215 full-Length non-chimeric reads (FLNC) were obtained for further ORFs and transcription factors prediction, SSR analysis and lncRNA identification. NR, Swissprot, GO, COG, KOG, Pfam and KEGG databases were used to obtain annotation information of transcripts. 10 TPS family members were identified with molecular weights between 48.0 to 108.5 kD and isoelectric point between 5.61 to 6.37. Furthermore, we found that TPS family members contain conserved TPP or TPS domain. Based on phylogenetic tree analysis, PoTPS1 protein was highly similar to AtTPS1 protein in Arabidopsis. Finally, we analyzed the expression levels of all TPS genes in P. ostii and found PoTPS5 expressed at the highest level. In conclusion, this study combined the results of the transcriptome to systematically analyze the 10 TPS family members, and sets a framework for further research of this important gene family in development of tree peony.

Tree peony (Paeonia section Moutan DC.) is an excellent ornamental plant, of which Paeonia ostii (P. ostii) has a high oil value. It is widely cultivated in China, but severe drought affects its growth. In this study, the effects of exogenous calcium on drought-induced damage of P. ostii were studied. The results showed that under drought stress, leaf water content showed a downward trend, while reactive oxygen species (ROS), relative electrical conductivity (REC), proline (Pro) content, and related antioxidant enzymes increased significantly. Spraying CaCl2 could effectively slow leaf wilting and water loss, induced an increase in enzyme activity of the antioxidant enzyme system, and reduced the accumulation of ROS caused by drought stress. Simultaneously, REC and Pro content could be alleviated, and the degree of cell membrane damage could be reduced. In addition, CaCl2 improved photosynthetic characteristics and chlorophyll fluorescence parameters. These results indicated that CaCl2 reduced the harmful effects of drought stress on the growth of P. ostii by regulating infiltration, activating photosynthesis, and enhancing the antioxidant system. These findings suggested that CaCl2 can be used to manage drought stress in P. ostii cultivation.

Two tree peony species, Paeonia ostii and Paeonia rockii, from eight different provenances in China (four provenances for each species) were collected for their photosynthetic characteristics. Their diurnal variations of photosynthesis, the light- and CO2-response curves and chlorophyll content were determined. Among all eight provenances, Ziwuling P. rockii had a significantly higher average daily net photosynthetic rate, showing a superior photosynthetic capacity, followed by Fengxiang P. ostii and Xunyang P. ostii. In addition, Ziwuling P. rockii had the highest maximum net photosynthetic rates, thus, presented a strong photosynthetic potential. Furthermore, Xunyang P. ostii, Fengxiang P. ostii, and Heze P. ostii also exhibited a great photosynthetic capacity and light adaptability, which was consistent with their high chlorophyll a/b values. Taken together, Ziwuling P. rockii had the strongest photosynthetic capacity among all tested sources, followed by Fengxiang P. ostii, and Xunyang P. ostii, which are recommended to be used as seed sources for oil peony cultivation and cross-breeding.

Paeonia ostii is one of the important woody oleaginous plants, and is cultivated on a large scale in China. The main purpose of this study is to evaluate the characteristic difference of P. ostii seed and oil sourced different geographical regions in China. The thousand seed weight (TSW), oil content and kernel content of the P. ostii seed, the composition and content of the main fatty acids, and the physicochemical characteristics of the seed oil of 62 P. ostii seed samples from different cultivation regions were monitored and evaluated in this study. The results showed that there were extremely significant difference among the TSW and the contents of the main fatty acids in P. ostii seed and oil. The geographical regions were related to the variability in TSW, the contents of the main fatty acids (FAs) and the total content of the main fatty acids (TCMFA), perhaps due to the influence of environmental factors, and the water, fertilizer application, crop management, or other factors. P. ostii would become a kind of woody oleaginous plant and an industrial crop with broad prospects for its high content of alpha-linolenic acid (ALA) and the high oil content of the seeds.

Seed yield and nutritional quality are dependent on seed development in oil crops. In a previous study, we showed that light shading improved seed yield and quality in oilseed peony, but the underlying mechanisms driving light shading impacts on seed yield and nutritional quality as mediated through seed development remain unclear. To develop an understanding of these mechanisms, oilseed peony trees were shaded by different density polyethylene nets starting at 28 days after pollination (DAP) and continuing until harvest. The seed volume, weight, and nutritional quality parameters, as well as the expression of genes related to the synthesis of unsaturated fatty acids (UFAs), were examined at 14-day intervals. Results showed that hundred-seed volume (Vh), fresh weight (Wf), and dry weight (Wd) increased by 8.2, 15.2, and 33.0 times, respectively, from 14 to 70 DAP. During the same period, the contents of starch, soluble protein, crude oil, and UFAs increased by 2.8, 3.7, 20.4, and 21.1 times, respectively. From 71 to 98 DAP, Vh, Wf, and Wd increased by 4.1, 5.4, and 20.8%, respectively. During this period, the contents of soluble protein, crude oil, and UFAs increased by 36.8, 51.4, and 49.5%, respectively. From 99 to 112 DAP, Vh, Wf, and Wd slightly increased, and the content of soluble protein increased by 25.2%. In contrast, the contents of soluble sugar, starch, and crude oil decreased by 28.3, 15.1, and 6.3%, respectively. Thus, seed development of oilseed peony can be divided into three periods of rapid growth (before 70 DAP), slow growth (71–98 DAP), and maturation (99–112 DAP). At 98 DAP, light shading increased the content of soluble sugar by 8.3%. At 112 DAP, light shading increased Vh, Wf, Wd, the content of crude oil, and UFAs by 6.9, 8.3, 8.3, 6.6, and 9.6%, respectively. The expression level of FAD2 in peony seed was up-regulated by 87.1 and 219.3% at 98 and 112 DAP, respectively. The expression level of FAD8 was up-regulated by 10.8, 71.7, 162.1, and 371.3% at 70, 84, 98, and 112 DAP, respectively. The expression level of SAD was up-regulated by 53.5 and 150.3% at 98 and 112 DAP, respectively. It is thus concluded that light shading significantly improved seed yield and nutritional quality parameters via increased seed growth and the expression of fatty acid desaturase genes during the slow growth and the maturation periods of seed development.

Paeonia ostii (Paeoniaceae), a famous traditional flowering plant, is an edible perennial shrub having medicinal uses. In recent decades, growing market demands and unprecedented damage to forests has caused severe degeneration of its natural habitat. As a result, land managers involved in conserving and restoring its habitat need to identify the environmental factors that shape its distribution. The present study predicted the potential distribution of this species and evaluated habitat suitable for it. The main environmental factors affecting this species were determined using 145 occurrence records, 30 environmental variables, MaxEnt modeling, and an analysis using ArcGIS 10.2 software. The results showed that the moderately and highly suitable habitat for P. ostii encompasses ca. 8.23 × 105 km2; the areas with a high suitability index (>0.6) for the distribution of this species were in southern Anhui, Henan (Kaifeng, Luoyang, Pingdingshan, Xuchang, and Zhengzhou), Hubei (Huanggang and Huangshi), and Shanxi (Yan'an) provinces. Annual mean temperature, mean UV-B of the lightest month, vapor pressure, and annual precipitation were the main environmental factors affecting the distribution of P. ostii. The results of this study can allow land managers to avoid wasted human effort and materials as well as the exhaustion of wild P. ostii resources that could result from the blind introduction of this species into unsuitable habitat while improving both the quality and yield of P. 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-β1. 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.

Lipid components in the developing kernel of Paeonia ostii were determined, and the fatty acid (FA) distributions in triacylglycerol and phospholipids were characterized. The lipids in the kernel were mainly phospholipids (43%), neutral glycerides (24%), fatty acyls (26%), and sphingolipids (4.5%). The dominant neutral glycerides were TAG and diacylglycerol. The PL components included phosphatidic acid, phosphatidyl glycerol, phosphatidyl choline, phosphatidyl serine, phosphatidyl inositol, and phosphatidyl ethanolamine. As the kernel developed, the profiles of the molecular species comprising TAG and PL changed, especially during the earlier phases of oil accumulation. During rapid oil accumulation, the abundances of sphingosine-1-phosphate, pyruvic acid, stearic acid, and alpha-linolenic acid changed significantly; the sphingolipid metabolism and unsaturated FAs biosynthesis pathways were significantly enriched in these differentially abundant metabolites. Our results improve our understanding of lipid accumulation in tree peony seeds, and provide a framework for the analysis of lipid metabolisms in other oil crops.

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. 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. 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. 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. 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.

Chalcone synthase (CHS) is the key enzyme in the flavonoid biosynthetic pathway and has been studied in many plants, but the function of the CHS gene has not been well characterized in Paeonia ostii. In this study, we obtained a CHS homologue gene from P. ostii, which possessed the putative conserved amino acids of chalcone synthase by multiple alignment analysis and demonstrated the highest expression in developing seeds. In vitro assays of the recombinant PoCHS protein confirmed enzymatic activity using malonyl-CoA and 4-coumaroyl-CoA as substrates, and the optimal pH and reaction temperature were 7.5 and 40°C, respectively. Furthermore, ectopic over-expression of PoCHS in Arabidopsis up-regulated the expression levels of genes involved in seed development (ABI), glycolysis (PKp2, PDH-E1a, and SUS2/3), and especially fatty acid biosynthesis (BCCP2, CAC2, CDS2, FatA and FAD3). This resulted in an increased unsaturated fatty acid content, especially α-linolenic acid, in transgenic Arabidopsis seeds. In this study, we examined the functions of CHS homologue in P. ostii and demonstrated its new function in seed fatty acid biosynthesis. This article is protected by copyright. All rights reserved.

Tree peony seeds are rich in α-linolenic acid (ALA), and the peony seed oil is now being produced in China. Paeonia ostii is the most widely used tree peony species for oil extraction, which is commercially called Fengdan and treated as a single cultivar. Here, 50 P. ostii individuals from the same population in northern China were randomly selected for fatty acids (FAs) analysis. Thirteen FAs were isolated, and the most abundant five were palmitic acid (5.31–6.99%), stearic acid (1.22–2.76%), oleic acid (18.78–28.15%), linoleic acid (11.86–26.10%), and ALA (41.11–57.51%). There were significant individual differences of plants in FA quality and quantity and the linoleic acid content in Plant No. 48 even exceeded the scope of 1–99%. Further statistical analysis indicated that most of the individual FAs, saturated FAs, unsaturated FAs, and total FAs levels showed significant positive correlations to each other, whereas the seed yield per plant was independent and not correlated to the factors mentioned above. Ward's hierarchical clustering results grouped the 50 plants into four clusters based on FA contents and seed yield, and the seven plants in Cluster IV were identified as good candidates for oil production. Our results confirmed that the individual differences did occur in P. ostii and Fengdan cannot be simply treated as one uniform cultivar. Also, these results may help simplify the selection of plants for oil peony breeding and accelerate the development of the oil peony industry.

Tree peony seed is unique for its super-high content of unsaturated fatty acid and is thus considered as an important source of woody oil. However, photosynthetic production is greatly reduced under high light intensity and air temperature during the seed filling period, which negatively affects seed yield and quality. The objective of this study was to determine if appropriate shading improves yield and quality of seed in oilseed peony. In this study, oilseed peony trees were shaded by different density polyethylene nets from four weeks after flowering to harvest stages to form light, moderate, and severe shadings, equivalent to about 80, 40, and 20% of full solar exposure, respectively. The effects of different shadings on some physiological parameters, yield and yield components, and nutritional composition of seed were examined. Averaged across two years, light shading increased the actual net photosynthetic rate ( P n ) by 16.8%, the maximum net photosynthetic rate ( P max ) by 81.4%, chlorophyll (Chl) content by 52.8%, auxin (IAA) content by 38.1%, and gibberellic acid (GA 3 ) content in leaves by 6.3%; it decreased the accumulation of H 2 O 2 in leaves by 24.8%, malondialdehyde (MDA) by 22%, and endogenous abscisic acid (ABA) by 8.8%, indicating that leaf senescence in late season was considerably delayed. Light shading increased seed yield, and contents of crude fat and unsaturated fatty acids by 9.7, 5.6, and 9.6%, respectively, while moderate or severe shading significantly reduced all the three parameters. Light shading increased seed weight, but moderate or severe shading reduced seed weight or follicle density. The improved seed yield under light shading was mainly due to increased seed weight, while the reduced seed yield under moderate or severe shading was mainly attributed to reduce follicle density and seed weight. The improved seed weight and content of unsaturated fatty acids under light shading was possibly due to the delayed leaf senescence. The overall results indicated that light shading is beneficial to yield and quality parameters of seed in oilseed peony. Cultivating oilseed peony under a light shading environments such as partially closed forests would better increase total output and income per unit land area than that under full solar exposure.

Seed germination is a critical process that is influenced by various factors. In the present study, the effect of low temperature (4 °C) on tree peony seed germination was investigated. Compared to seeds maintained at 25 °C, germination was inhibited when seeds were kept at 4 °C. Furthermore, low-temperature exposure of seeds resulted in a delay in water uptake, starch degradation, and soluble sugar consumption and a subsequent increase in soluble protein levels. Two-dimensional gel electrophoresis (2-DE) proteomic analysis identified 100 protein spots. Comparative analysis indicated that low-temperature exposure apparently mainly affected glycolysis and the tricarboxylic acid (TCA) cycle, while also significantly affecting proteometabolism-related factors. Moreover, low-temperature exposure led to the induction of abscisic acid, whereas the gibberellin pathway was not affected. Further comparison of the two temperature conditions showed that low-temperature exposure delays carbohydrate metabolism, adenosine triphosphate (ATP) production, respiration, and proteolysis and increases defense response factors. To further examine the obtained proteomic findings, four genes were evaluated by quantitative polymerase chain reaction (qPCR). The obtained transcriptional results for the GAPC gene coincided with the translational results, thus further suggesting that the delay in glycolysis may play a key role in low-temperature-induced inhibition of seed germination. However, the other three genes examined, which included FPP synthase, PCNT115, and endochitinase, showed non-correlative transcriptional and translational profiles. Our results suggest that the exposure of tree peony seeds to low temperature results in a delay in the degradation of starch and other metabolites, which in turn affects glycolysis and some other processes, thereby ultimately inhibiting seed germination.

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.

The effects of different pollen sources on fruit and seed characteristics of Paeonia ostii ‘Feng Dan’ were investigated using analysis of three different pollination treatments and pollen sources from fifteen cultivars in two successive years. The results showed that self-pollination decreased fruit setting, the number of seeds per fruit, seed volume, seed and kernel weights, and the linoleic acid (LA) concentration in the seed oil, but increased the concentration of oleic acid (OA) compared with cross-pollination. Among those pollen donors, P. suffruticosa ‘Yinhong Qiaodui’ produced the highest fruit set (87.5%); the lowest fruit set was obtained with P. suffruticosa ‘Mo Run Jue Lun’ (44.33%). The most seeds per fruit were achieved by P. suffruticosa ‘Mochi Jin Hui’. P. suffruticosa ‘Dahong Baozhu’ produced the largest fruit, which contained larger and heaviest seeds. The oil extraction ratio (26% to 31.6%) and the concentration of three major unsaturated fatty acids (UFAs) in seed oil also significantly differed among pollen sources. The content of OA, LA, and α-linolenic acid (ALA) ranged from 13.82 to 24.79, 12.09 to 21.84, and 23.50 to 38.64 g/100 g crude oil, respectively. Overall, pollen source has clear effects on seed yield and even on fatty acid (FA) composition of seed oil in tree peony.

The accumulation of chemical constituents of some medicinal plants, such as Paeonia ostii T. Hong et J. X. Zhang, Houpo&euml, a officinalis (Rehder and E. H. Wilson) N. H. Xia and C. Y. Wu. and Atractylodes lancea (Thunb.) DC, can precipitate on the surface and form frosts after natural or artificial intervention. The characteristics of these three medicinal plants and their frosts were analyzed by light microscope, polarizing microscope, stereomicroscope, and metalloscope. The results of ordinary Raman of P. ostii and H. officinalis showed that the frosts of P. ostii matched paeonol, while that of H. officinalis matched magnolol and honokiol. In P. ostii and its frost, 19 peaks were identified by UPLC-Q/TOF-MS, and the main component was paeonol. Eleven components were identified in H. officinalis and its frosts, and the main components were magnolol and honokiol. A. lancea and its frosts were analyzed by gas chromatography-mass spectrometry (GC-MS), 21 were identified, and its main components were hinesol and &beta, eudesmol. These three medicinal plants accumulate compounds and precipitate frosts on the surface. The results show that the components of the frosts provide a basis for quality evaluation and research on similar medicinal plants, and reveals the scientific connotation of &ldquo, taking the medicinal materials&rsquo, precipitated frosts as the best&rdquo, of P. ostii, H. officinalis, and A. lancea, to some extent.

Tree peony seed is a novel resource of edible oil with high α-linolenic acid (ALA) content (> 40%). To assess whether pollen sources influence the seed oil quality, fatty acids of hybrids from the combination of Paeonia ostii ‘Feng Dan’ and 30 cultivars were monitored and evaluated in this study. The results indicated that the content of total fatty acid and ALA ranged from 21.23 to 29.68% and from 46.75 to 64.71%, respectively. Thirty combinations were classified into four clusters. Cluster IV including ‘Tian Xiang Jin’, ‘Zhou Ban Hong’, ‘Xiang Yang Hong’ and ‘Yu Jie’ were good pollen donors because their hybrid seeds possessed the highest contents of ALA and unsaturated fatty acids, with an average of 159.39 mg g−1 and 263.76 mg g−1, respectively. This study provides a good base for breeding new cultivars with excellent edible oil and essential fatty acid contents for food.

To solve the issue of insufficient pollinating of insects for the oil tree peony &lsquo, Feng Dan&rsquo, (Paeonia ostii T. Hong et J.X. Zhang) and improve its seed set and yield, we conducted observations from 2017 to 2018 to investigate the relationship between honey bee (Apis mellifera L.) foraging behavior and diurnal activity. We compared the single-fruit seed set ratio among three flower types on the same plants of the oil tree peony, which flowered simultaneously, in three pollination areas (bee pollination, natural field pollination, and controlled pollination by pollinators) and in a net room under self-pollination, wind pollination and bee pollination. Apis mellifera exhibited short single visitations, long visitations to a single flower and repeated visits to flowers of the oil tree peony. The number of flower visits of A. mellifera was significantly and positively yet weakly correlated with the number of stigma visits (2017: r = 0.045, p &lt, 0.05, 2018: r = 0.195, p &lt, 0.01). The seed set of oil tree peony follicles in the A. mellifera pollination area was significantly higher than that in the natural pollination field area and the control net rooms. On the same oil tree peony plant with synchronous flowering, the percent seed set of follicles pollinated by A. mellifera at a high density was significantly higher than that resulting from wind pollination and self-pollination.

Paeonia ostii is an emerging woody oil crop, but drought severely inhibits its growth and promotion in arid or semiarid areas, and little is known about the mechanism governing this inhibition. In this study, the full-length cDNA of a caffeoyl-CoA O-methyltransferase gene (CCoAOMT) from P. ostii was isolated, and determined to be comprised of 987 bp. PoCCoAOMT encoded a 247-amino acid protein, which was located in the nucleus and cytosol. Significantly higher PoCCoAOMT transcription was detected in P. ostii treated with drought stress. Subsequently, the constitutive overexpression of PoCCoAOMT in tobacco significantly conferred drought stress tolerance. Under drought stress, transgenic lines exhibited lower reactive oxygen species (ROS) accumulation, and higher antioxidant enzyme activities and photosynthesis. Moreover, the expression levels of senescence-associated genes were significantly downregulated, whereas the expression levels of lignin biosynthetic genes and PoCCoAOMT were significantly upregulated in transgenic lines. Similarly, transgenic lines produced significantly higher lignin, especially guaiacyl-lignin. These results suggest that PoCCoAOMT is a vital gene in promoting lignin synthesis and ROS scavenging to confer drought stress tolerance in P. ostii.

Paeonia ostii var. lishizhenii has been approved as a woody oil crop with the outstanding characteristic of abundant α-linolenic acid (C18:3, ALA) in its seed oil. The stearoyl-ACP desaturase gene (SAD) regulates the first key step from stearic acid (C18:0, SA) to oleic acid (C18:1, OA) in the ALA biosynthetic pathway, but its functional characterization in P. ostii var. lishizhenii is absent to date. In this study, a key PoSAD gene (1719 bp in length) was acquired from endosperm of P. ostii var. lishizhenii by transcriptome sequencing analysis and the RACE (rapid-amplification of cDNA ends) method. Bioinformatic analysis of the PoSAD protein showed high homology (ranging from 90.4% to 94.4%) and similar physical and chemical properties to SAD from other higher plants, indicating that it encodes a putative stearoyl-ACP desaturase. Analysis of cis-acting elements found several endosperm tissue-specific motifs; i.e., one Prolamin box, thirteen DOFCOREs and one RY repeat in its promoter. The results of the qRT-PCR experiments verified that PoSAD was most highly expressed in developing endosperm at 59 days after pollination (53.7 times that in shoots) compared with that in roots (1.4 times), stems (2.5 times), leaves (3.1 times), petals (13.1 times) and stamens (46.0 times). Meanwhile, the fatty acid contents in P. ostii var. lishizhenii endosperm at seven growth stages were compared with variation in PoSAD expression. Heterologous expression of PoSAD significantly decreased SA and increased OA content, which effectively reduced the ratios of SA to OA in Saccharomyces cerevisiae and Arabidopsis thaliana. However, contents and ratios of palmitic acid (C16:0) and palmitoleic acid (C16:1) were stable in transgenic yeast, and palmitoleic acid remained absent in transgenic A. thaliana seeds. These results illustrate that PoSAD plays an essential role in endosperm development of P. ostii var. lishizhenii, strictly in catalysis of SA desaturation and OA biosynthesis but without functioning in PA desaturation. The results contribute to our understanding of the characterization of PoSAD in OA biosynthesis in P. ostii var. lishizhenii.

Stamen from Paeonia ostii 'Fengdan Bai' and Paeonia rockii is rich in phenolic compounds and popularly used as tea materials with various pharmaceutical functions. In order to investigate whether stamen from other tree peony cultivars could be used as a natural antioxidant, the quality of stamen from thirty-five cultivars collected from the same garden was evaluated based on their phenolic composition and content by high-performance liquid chromatography analysis and in vitro antioxidant properties coupled with comprehensive chemometrics analysis. The results revealed that phenolic contents and antioxidant capacities of tree peony stamen were unique and cultivar dependent. Stamen from 'Zi Erqiao' exhibited the highest total phenolic and flavonol content, and strongest antioxidant activities, while that of 'Fengdan Bai' and P. rockii were at below-average levels among test samples. Further, thirty-seven cultivars of tree peony were divided into three major groups with a significant difference in total metabolites content and antioxidant properties, which were mainly contributed by six phytochemical compounds. Among these, naringin and benzoylpaeoniflorin were found to be critical chemical markers for the identification of tree peony stamen with high quality by chemometric analysis. Moreover, correlation analysis suggested that stamen from the earlier flowering cultivars with the hidden pistil, double petal, shorter thrum, more carpel and volume were possibly of higher quality. Together, cultivars with stamen enriched in phenolics and antioxidants properties, and their relevant critical phenotypic and phytochemical traits were screened out. This study would benefit the rapid identification of tree peony stamen with high quality and provide a valuable reference for its development and utilization as functional foods and pharmaceutical resources.

As a residue of oil processing, research about Paeonia ostii cake has not been reported. We used six enzymes to hydrolyze the Paeonia ostii cake and obtained six enzymatic polypeptides. The enzymatic polypeptide yields of the six methods were between 10.0 and 25.0 % and average molecular weight was between 3.0 and 10.0 kDa. The average particle size of six enzymatic polypeptides was less than 1000.0 nm. The enzymatic hydrolysis method had a significant effect on the surface morphology of the enzymatic residues. The enzymatic polypeptide from alkaline protease method had better thermal stability and stronger infrared absorption. The eight essential amino acid contents of six enzymatic polypeptides from pepsin, papain, neutral protease, protemax, trypsase and alkaline protease method were 14.7, 13.3, 14.2, 12.8, 13.8 and 15.0 %, and hydrophobic amino acid contents were 14.7, 14.2, 14.2, 13.9, 13.7 and 15.1 %, respectively. Among the 12 non-essential amino acids, only glutamine and asparagine were absent. In addition, the different amino acid increment in six enzymatic polypeptides was significantly different. Among them, cysteine had the largest increment, followed by glycine and glutamic acid. By 5-axe cobweb chart method, the enzymatic polypeptide from alkaline protease method was proved to have the strongest comprehensive antioxidant activity. The enzymatic polypeptides of alkaline protease and protemax method had stronger inhibition activity of α-glucosidase and α-amylase. In general, the enzymatic polypeptide from alkaline protease method had good antioxidant activity and physiological activity. Our research will provide basic data support for new potential applications of enzymatic polypeptide from peony cake and increase the added value of Paeonia ostii processing enterprises.

Interspecific hybridisation is the main method for improvement and breeding of tree peony (Paeonia ostii T.Hong & J.X.Zhang), but cross-incompatibility as the major factor restricting the rapid development of interspecific hybridisation. To better understand the molecular mechanisms involved in cross-incompatibility between tree peony (Paeonia ostii cv. Fengdanbai) and herbaceous peony (Paeonia lactiflora Pall. cv. Fenyunu), a quantitative proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ) technology was performed on the stigma 24h after pollination. Of the 2900 proteins whose levels were quantitated, 685 proteins were differentially expressed in the stigma after hybrid pollination, in contrast to self-pollination. Functional annotation analysis showed that dysregulated proteins involved in RNA degradation, the Ca signalling pathway, the phosphatidylinositol signalling system and the mitogen-activated protein kinase (MAPK) signalling pathway may have made contributions to cross-incompatibility. The downregulated expression of enolase, DnaK (Heat Shock Proteins, HSP70), GroEL (Heat Shock Proteins, HSP60), calmodulin and glyoxalase I, and the upregulated expression of adenine nucleotide translocator indicated that the energy synthesis required by pollen tube growth, the signal pathway and the metabolic pathway related to the growth polarity of the pollen tube were blocked after hybrid pollination. Eight genes were selected to confirm their expression by quantitative real-time PCR. Compared with the STRING database, a protein–protein interaction network of the chosen proteins was constructed. These results provide fundamental and important information for research into the molecular mechanisms of cross-incompatibility in peony and should facilitate interspecific hybridisation in agricultural practice.

The FAD8 gene catalyzes the conversion of diene fatty acids to triene fatty acids and is a key enzyme that determines the synthesis of alpha-linolenic acid. In this study, the full-length cDNAs of FAD8-1, FAD8-2, and FAD8-3 are cloned from Paeonia ostii T. Hong & J. X. Zhang and named as PoFAD8-1, PoFAD8-2, and PoFAD8-3. Their open reading frame is 1203 bp, 1152 bp, and 1353 bp which encoded 400, 371, and 450 amino acids. The molecular weights of the amino acids are 46 kDa, 43 kDa, and 51 kDa while the isoelectric points are 7.34, 8.74, and 9.23, respectively. Bioinformatics analysis shows that all three genes are hydrophobic-hydrophobic, PoFAD8-1 has three transmembrane domains, and PoFAD8-2 and PoFAD8-3 have two transmembrane domains. Multiple series alignment and phylogenetic analysis revealed that PoFAD8-1 and PoFAD8-2 are closely related while PoFAD8-3 is more closely related to Paeonia delavayi. Subcellular localization results showed that PoFAD8-1 was located on the ER membrane and PoFAD8-2 and PoFAD8-3 were located on the chloroplast membrane. The relative expression level of PoFAD8-1 in seeds is very high. PoFAD8-2 expressed more in the ovary than the other two genes. PoFAD8-3 was highly expressed in roots, stems, leaves, petals, and ovaries.

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.

Paeonia rockii is a wild tree peony species with large and dark purple variegations at the base of its petals. It is the genetic resource for various variegation patterns in tree peony cultivars, which is in contrast to the pure white petals of Paeonia ostii. However, the molecular mechanism underlying the formation of variegation in this plant is still unknown. Here, we conducted Illumina transcriptome sequencing for P. rockii, P. ostii (with pure white petals) and their F1 individuals (with purple-red variegation). A total of 181,866 unigenes were generated, including a variety of unigenes involved in anthocyanin biosynthesis and sequestration and the regulation of anthocyanin biosynthesis. The dark purple or purple-red variegation patterns mainly occurred due to the proportions of cyanidin (Cy)- and peonidin (Pn)-based anthocyanins. The variegations of P. rockii exhibited a “Cy > Pn” phenotype, whereas the F1 progeny showed a “Pn > Cy” phenotype. The CHS, DFR, ANS, and GST genes might play key roles in variegation pigmentation in P. rockii according to gene expression and interaction network analysis. Two R2R3-MYB transcription factors (c131300.graph_c0 and c133735.graph_c0) regulated variegation formation by controlling CHS, ANS and GST genes. Our results indicated that the various variegation patterns were caused by transcriptional regulation of anthocyanin biosynthesis genes, and the transcription profiles of the R2R3-MYBs provided clues to elucidate the mechanisms underlying this trait. The petal transcriptome data produced in this study will provide a valuable resource for future association investigations of the genetic regulation of various variegation patterns in tree peonies.

Paeonia ostii, a member of tree peony, is an emerging oil crop with important medical and oil uses and widely cultivated in China. Dissolving the genetic diversity and domestication history of this species is important for further genetic improvements and deployments. We firstly selected 29 simple sequence repeats (SSRs) via transcriptome mining, segregation analyses and polymorphism characterizations; then, 901 individuals from the range-wide samples were genotyped using well-characterized SSR markers. We observed moderate genetic diversity among individuals, and Shaanxi Province was identified as the center of genetic diversity for our cultivated plants. Five well-separated gene pools were detected by STRUCTURE analyses, and the results suggested that multiple independent domestication origins occurred in Shaanxi Province and Tongling City (Anhui Province). Taken together, the genetic evidence and the historical records suggest multiple long-distance introductions after the plant was domesticated in Shandong, Henan and Hunan provinces. The present study provides the first genetic evaluation of the domestication history of P. ostii, and our results provide an important reference for further genetic improvements and deployments of this important crop.

Paeonia ostii is an important woody oil plant cultivated in China on a large scale. Its seed oil is enriched with unsaturated fatty acids and a high content of alpha-linolenic acid (ALA), which are beneficial to human health. The aim of this research is to determine the qualitative traits characteristic of P. ostii seed from various production areas in China. In this study, seed quality traits were evaluated on the basis of proximate composition, content of fatty acids, tocopherol, secondary metabolites, and the antioxidant activity of seed coat (PSC) and kernel (PSK). A high content of total fatty acids (298.89&ndash, 399.34 mg g&minus, 1), crude protein (16.91%&ndash, 22.73%), and total tocopherols (167.83&ndash, 276.70 &mu, g g&minus, 1) were obtained from PSK. Significant differences were found in the content of palmitic acids (11.31&ndash, 14.27 mg g&minus, 1), stearic acids (2.42&ndash, 4.24 mg g&minus, 1), oleic acids (111.25&ndash, 157.63 mg g&minus, 1), linoleic acids (54.39&ndash, 83.59 mg g&minus, 1), and ALA (99.85&ndash, 144.71 mg g&minus, 1) in the 11 main production areas. Eight and seventeen compounds were detected in PSC and PSK, respectively. A significantly higher content of total phenols was observed in PSC (139.49 mg g&minus, 1) compared with PSK (3.04 mg g&minus, 1), which was positively related to antioxidant activity. This study indicates that seeds of P. ostii would be a good source of valuable oil and provides a basis for seed quality evaluation for the production of edible oil and potential ALA supplements from the promising woody oil plant.

Keywords: Paeonia ostii &lsquo, Fengdan&rsquo, tree peony, somatic embryogenesis, development

Tree peony is a perennial deciduous shrub with great ornamental and medicinal value. A limitation of its current functional genomic research is the lack of effective molecular genetic tools. Here, the first application of a Tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) in the tree peony species Paeonia ostii is presented. Two different approaches, leaf syringe-infiltration and seedling vacuum-infiltration, were utilized for Agrobacterium-mediated inoculation. The vacuum-infiltration was shown to result in a more complete Agrobacterium penetration than syringe-infiltration, and thereby determined as an appropriate inoculation method. The silencing of reporter gene PoPDS encoding phytoene desaturase was achieved in TRV-PoPDS-infected triennial tree peony plantlets, with a typical photobleaching phenotype shown in uppermost newly-sprouted leaves. The endogenous PoPDS transcripts were remarkably down-regulated in VIGS photobleached leaves. Moreover, the green fluorescent protein (GFP) fluorescence was detected in leaves and roots of plants inoculated with TRV-GFP, suggesting the capability of TRV to silence genes in various tissues. Taken together, the data demonstrated that the TRV-based VIGS technique could be adapted for high-throughput functional characterization of genes in tree peony.

Tree peonies are a group of traditional ornamental plants, especially in East Asia, with Paeonia ostii as one of the most important ancestral species. P. ostii has flowers with varying colors, ranging from nearly white, light pink to deep pink. However, few studies have been done to unravel the molecular mechanisms underlying the flower color intensity variation in plants. Based on comparative analyses of the pigment composition and transcriptomes of P. ostii with different flower color intensities, we found that the anthocyanin concentration was significantly correlated with the flower color intensity in P. ostii. Transcriptome analysis by RNA-Sequencing revealed 7187 genes that were differentially expressed between flowers with different color intensities. Functional enrichment analysis of differentially expressed genes revealed multiple pathways possibly responsible for color intensity variation in P. ostii, including flavonoid biosynthesis, fatty acid oxidation, carbohydrate metabolism, and hormone-mediated signaling. Particularly, while anthocyanin biosynthesis genes showing positive correlations between their expression and anthocyanin concentration in flowers, two transcription factors, PoMYB2 and PoSPL1, seem to negatively regulate anthocyanin accumulation by affecting the activation capacity of the MYB-bHLH-WDR complex, exhibiting an inverse relationship between their expression and anthocyanin accumulation. Our results showed that, although anthocyanin biosynthesis had a direct effect on the pigmentation of the P. ostii flower, other metabolic and hormone-mediated signaling pathways were also contributed to the flower color intensity variation in P. ostii, suggesting complex coordinated changes in the transcriptional network. Differential expression of genes encoding anthocyanin repressors seems to be the major factor responsible for the intensity variation in anthocyanin pigmentation in P. ostii.

Paeonia ostii, an important ornamental and medicinal plant, grows normally on copper (Cu) mines with widespread Cu contamination of soils, and it has the ability to lower Cu contents in the Cu-contaminated soils. However, very little molecular information concerned with Cu resistance of P. ostii is available. In this study, high-throughput de novo transcriptome sequencing was carried out for P. ostii with and without Cu treatment using Illumina HiSeq 2000 platform. A total of 77,704 All-unigenes were obtained with a mean length of 710 bp. Of these unigenes, 47,461 were annotated with public databases based on sequence similarities. Comparative transcript profiling allowed the discovery of 4324 differentially expressed genes (DEGs), with 2207 up-regulated and 2117 down-regulated unigenes in Cu-treated library as compared to the control counterpart. Based on these DEGs, Gene Ontology (GO) enrichment analysis indicated Cu stress-relevant terms, such as 'membrane' and 'antioxidant activity'. Meanwhile, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis uncovered some important pathways, including 'biosynthesis of secondary metabolites' and 'metabolic pathways'. In addition, expression patterns of 12 selected DEGs derived from quantitative real-time polymerase chain reaction (qRT-PCR) were consistent with their transcript abundance changes obtained by transcriptomic analyses, suggesting that all the 12 genes were authentically involved in Cu tolerance in P. ostii. This is the first report to identify genes related to Cu stress responses in P. ostii, which could offer valuable information on the molecular mechanisms of Cu resistance, and provide a basis for further genomics research on this and related ornamental species for phytoremediation.