Integrated analysis of metabolome, transcriptome, and bioclimatic factors of Acer truncatum seeds reveals key candidate genes related to unsaturated fatty acid biosynthesis, and potentially optimal production area.

BACKGROUND: Lipids found in plant seeds are essential for controlling seed dormancy, dispersal, and defenses against biotic and abiotic stress. Additionally, these lipids provide nutrition and energy and are therefore important to the human diet as edible oils. Acer truncatum, which belongs to the Aceaceae family, is widely cultivated around the world for its ornamental value. Further because its seed oil is rich in unsaturated fatty acids (UFAs)- i.e. α-linolenic acid (ALA) and nervonic acid (NA)- and because it has been validated as a new food resource in China, the importance of A. truncatum has greatly risen. However, it remains unknown how UFAs are biosynthesized during the growth season, to what extent environmental factors impact their content, and what areas are potentially optimal for their production. RESULTS: In this study, transcriptome and metabolome of A. truncatum seeds at three representative developmental stages was used to find the accumulation patterns of all major FAs. Cumulatively, 966 metabolites and 87,343 unigenes were detected; the differential expressed unigenes and metabolites were compared between stages as follows: stage 1 vs. 2, stage 1 vs. 3, and stage 2 vs. 3 seeds, respectively. Moreover, 13 fatty acid desaturases (FADs) and 20 ß-ketoacyl-CoA synthases (KCSs) were identified, among which the expression level of FAD3 (Cluster-7222.41455) and KCS20 (Cluster-7222.40643) were consistent with the metabolic results of ALA and NA, respectively. Upon analysis of the geographical origin-affected diversity from 17 various locations, we found significant variation in phenotypes and UFA content. Notably, in this study we found that 7 bioclimatic variables showed considerable influence on FAs contents in A. truncatum seeds oil, suggesting their significance as critical environmental parameters. Ultimately, we developed a model for potentially ecological suitable regions in China. CONCLUSION: This study provides a comprehensive understanding of the relationship between metabolome and transcriptome in A. truncatum at various developmental stages of seeds and a new strategy to enhance seed FA content, especially ALA and NA. This is particularly significant in meeting the increasing demands for high-quality edible oil for human consumption. The study offers a scientific basis for A. truncatum's novel utilization as a woody vegetable oil rather than an ornamental plant, potentially expanding its cultivation worldwide.

Histology, physiology, and transcriptomic and metabolomic profiling reveal the developmental dynamics of annual shoots in tree peonies (Paeonia suffruticosa Andr.).

The development of tree peony annual shoots is characterized by "withering", which is related to whether there are bud points in the leaf axillaries of annual shoots. However, the mechanism of "withering" in tree peony is still unclear. In this study, Paeonia ostii 'Fengdan' and P. suffruticosa 'Luoyanghong' were used to investigate dynamic changes of annual shoots through anatomy, physiology, transcriptome, and metabolome. The results demonstrated that the developmental dynamics of annual shoots of the two cultivars were comparable. The withering degree of P. suffruticosa 'Luoyanghong' was higher than that of P. ostii 'Fengdan', and their upper internodes of annual flowering shoots had a lower degree of lignin deposition, cellulose, C/N ratio, showing no obvious sclerenchyma, than the bottom ones and the whole internodes of vegetative shoot, which resulted in the "withering" of upper internodes. A total of 36 phytohormone metabolites were detected, of which 33 and 31 were detected in P. ostii 'Fengdan' and P. suffruticosa 'Luoyanghong', respectively. In addition, 302 and 240 differentially expressed genes related to lignin biosynthesis, carbon and nitrogen metabolism, plant hormone signal transduction, and zeatin biosynthesis were screened from the two cultivars. Furtherly, 36 structural genes and 40 transcription factors associated with the development of annual shoots were highly co-expressed, and eight hub genes involved in this developmental process were identified. Consequently, this study explained the developmental dynamic on the varied annual shoots through multi-omics, providing a theoretical foundation for germplasm innovation and the mechanized harvesting of tree peony annual shoots.

Combined genome-wide association studies and expression quantitative trait locus analysis uncovers a genetic regulatory network of floral organ number in a tree peony (Paeonia suffruticosa Andrews) breeding population.

Great progress has been made in our understanding of floral organ identity determination and its regulatory network in many species; however, the quantitative genetic basis of floral organ number variation is far less well understood for species-specific traits from the perspective of population variation. Here, using a tree peony (Paeonia suffruticosa Andrews, Paeoniaceae) cultivar population as a model, the phenotypic polymorphism and genetic variation based on genome-wide association studies (GWAS) and expression quantitative trait locus (eQTL) analysis were analyzed. Based on 24 phenotypic traits of 271 representative cultivars, the transcript profiles of 119 cultivars were obtained, which indicated abundant genetic variation in tree peony. In total, 86 GWAS-related cis-eQTLs and 3188 trans-eQTL gene pairs were found to be associated with the numbers of petals, stamens, and carpels. In addition, 19 floral organ number-related hub genes with 121 cis-eQTLs were obtained by weighted gene co-expression network analysis, among which five hub genes belonging to the ABCE genes of the MADS-box family and their spatial-temporal co-expression and regulatory network were constructed. These results not only help our understanding of the genetic basis of floral organ number variation during domestication, but also pave the way to studying the quantitative genetics and evolution of flower organ number and their regulatory network within populations.

PhUGT78A22, a novel glycosyltransferase in Paeonia 'He Xie', can catalyze the transfer of glucose to glucosylated anthocyanins during petal blotch formation.

BACKGROUND: Flower color patterns play an important role in the evolution and subsequent diversification of flowers by attracting animal pollinators. This interaction can drive the diversity observed in angiosperms today in many plant families such as Liliaceae, Paeoniaceae, and Orchidaceae, and increased their ornamental values. However, the molecular mechanism underlying the differential distribution of anthocyanins within petals remains unclear in Paeonia. RESULTS: In this study, we used an intersectional hybrid between the section Moutan and Paeonia, hereafter named Paeonia 'He Xie', which has purple flowers with dark purple blotches. After Ultra-high performance liquid chromatography-diode array detector (UPLC-DAD) analysis of blotched and non-blotched parts of petals, we found the anthocyanin content in the blotched part was always higher than that in the non-blotched part. Four kinds of anthocyanins, namely cyanidin-3-O-glucoside (Cy3G), cyanidin-3,5-O-glucoside (Cy3G5G), peonidin-3-O-glucoside (Pn3G), and peonidin-3,5-O-glucoside (Pn3G5G) were detected in the blotched parts, while only Cy3G5G and Pn3G5G were detected in the non-blotched parts. This suggests that glucosyltransferases may play a vital role in the four kinds of glucosylated anthocyanins in the blotched parts. Moreover, 2433 differentially expressed genes (DEGs) were obtained from transcriptome analysis of blotched and non-blotched parts, and a key UDP-glycosyltransferase named PhUGT78A22 was identified, which could use Cy3G and Pn3G as substrates to produce Cy3G5G and Pn3G5G, respectively, in vitro. Furthermore, silencing of PhUGT78A22 reduced the content of anthocyanidin 3,5-O-diglucoside in P. 'He Xie'. CONCLUSIONS: A UDP-glycosyltransferase, PhUGT78A22, was identified in P. 'He Xie', and the molecular mechanism underlying differential distribution of anthocyanins within petals was elucidated. This study provides new insights on the biosynthesis of different kinds of anthocyanins within colorful petals, and helps to explain petal blotch formation, which will facilitate the cultivar breeding with respect to increasing ornamental value. Additionally, it provides a reference for understanding the molecular mechanisms responsible for precise regulation of anthocyanin biosynthesis and distribution patterns.

Optimization technology and kinetic studies of Acer truncatum seed oil saponification and crystallization separation of nervonic acid.

Acer truncatum seed oil (ATSO) contains abundant unsaturated fatty acids, with significant quantities of nervonic acid (NA, > 5%), which was authenticated as a new food resource in China. For the sake of minimizing animal consumption and the importance of NA for human health, extraction of NA from plants has become a research hotspot. In the present study, three extraction factors were determined to significantly influence the saponification reaction based on single-factor experiments: NaOH dosage, reaction time, and reaction temperature. These three factors were used to further optimize the saponification process through the response surface methodology, and the highest yield of mixed fatty acids was 83.12%. Moreover, the activation energy (40.8228 kJ/mol), the pre-exponential factor [2.568 × 106 m3 /(kmol·min)], and the kinetic equation [rA = kcA cB = 2.568 × 106 ·exp(- 4970 . 1 T ) $\frac{{{\rm{4970}}{\rm{.1}}}}{{\rm{T}}})$ cA cB ] of the ATSO saponification reaction were determined by combining the chemical reaction rate equation of the elementary reaction, the Arrhenius equation, and the NaOH concentration in the substrate. Finally, the mixed fatty acids of ATSO were crystallized by triple-stage low-temperature crystallization, and we achieved 25.05% purity for NA. This study provides a technological basis and strategy for specific fatty acid production from ASTO, as well as other vegetable oils important in the field of food and health supplement products. PRACTICAL APPLICATION: Nervonic acid (NA) is an essential component of neural cells and neural tissue, and it is vital for maintaining the normal work of nerve tissues in organisms and promotes neurodevelopment. NA has traditionally been mainly obtained from shark hunting, which is now restricted due to an international ban on shark fishing. The alternative way to produce NA cheaply and in large quantities is from plant sources. The techniques utilized in this study provide an effective method of NA separation from Acer truncatum seed oil for industrial production.

Modulating Effect of Paeonol on Piglets With Ulcerative Colitis.

Piglet enteritis is a major problem that needs to be solved urgently in modern pig production. Paeonol (Pae) has been used as a novel treatment option due to its good medicinal value. This study purported to elucidate the regulatory mechanism of Pae on dextran sodium sulfate (DSS)-induced ulcerative colitis (UC) in weaned piglets. A total of 36 crossbred (Duroc × Landrace × Yorkshire) weaned piglets were stochastically split into six groups: the control group, DSS group, 0.2% Pae group, 0.4% Pae group, 0.8% Pae group, and mesalazine group. The control and DSS groups were fed with a basic diet, the three Pae and mesalazine groups were fed with 0.2, 0.4, 0.8%, and 2 g mesalazine per kilogram of basic diet throughout the study. On the 15th day of the test period, the control group was gavaged with 10 ml of normal saline, while the remaining five groups were gavaged with 10 ml 5% DSS solution for 13 days. The study lasted for 27 days. The results showed that the 0.8% Pae group significantly increased the average daily feed intake (ADFI) and Occludin mRNA expression in the colon of piglets (P < 0.05). The 0.2% Pae group markedly increased the average daily gain (ADG) and zonula occludens-1 (ZO-1) mRNA expression (P < 0.05). In the 0.2% and 0.4% Pae groups, the feed-to-gain ratio (F/G) was significantly reduced and the mRNA expression levels of Caspase-8, respectively, markedly enhanced the mRNA expression levels of transforming growth factor-ß (TGF-ß) and interleukins-4 (IL-4) (P < 0.05). In the 0.8% Pae group, the relative abundance of Campilobacterota was significantly reduced (P < 0.05). In the 0.4% Pae group, the relative abundance of Firmicutes was notably increased (P < 0.05). In the 0.2 and 0.8% Pae groups, the relative abundance of Prevotella was markedly increased (P < 0.05). In the 0.2% Pae group, the contents of propionic acid, butyric acid, and valerate acid were markedly higher (P < 0.05). Thus, it is speculated that Pae may regulate the balance of anti-inflammatory/pro-inflammatory factors, improve intestinal tight junction expression, reduce apoptosis, and improve intestinal microflora structure and growth performance of piglets, thereby restoring intestinal barrier function and alleviating DSS-induced UC in piglets.

Inhibitory Effect of Acer truncatum Bunge Seed Coat Extract on Fatty Acid Synthase, Differentiation and Lipid Accumulation in 3T3-L1 Adipocytes.

Acer truncatum Bunge is now widely cultivated throughout the world. Fatty acid synthase (FAS) is a potential target in the treatment of both obesity and cancer. Only a few FAS inhibitors have been reported. In this study, the inhibitory effect of A. truncatum seed coat (ESA) on FAS and the inhibition mechanisms were investigated using a FAS activity assay and an enzyme kinetics study. The main chemicals of ESA were analyzed with UPLC-MS/MS. The effects of ESA on 3T3-L1 adipocyte differentiation and lipid accumulation were investigated using Oil red O staining. We first identified seven main compounds (quinic acid, malic acid, gentisic acid, procyanidin dimer, procyanidin trimer, catechin, and quercetin) from 50% ethanol extracts of seed coats of A. truncatum (ESAs), which were then found to inhibit 3T3-L1 adipocyte differentiation at the concentration of 50 µg/mL. ESA obviously reduced the visible triglyceride droplets accumulation, and dramatically decreased the number of the adipocytes at a comparatively high concentration. It is suggested that the effects are due to the inhibition of FAS by ESA; FAS activity is inhibited by ESA at a half inhibition concentration (IC50) of 0.57 µg/mL, which is lower than that of classically known FAS inhibitors. Meanwhile, ESA displayed different inhibition kinetics and reacting sites for FAS. These results provide new clues for the development of novel products for obesity treatment and a scientific basis for the full use of byproducts for future industrial production of vegetable oil.

ABSCISIC ACID-INSENSITIVE 5-ω3 FATTY ACID DESATURASE3 module regulates unsaturated fatty acids biosynthesis in Paeonia ostii.

Paeonia ostii is an authorized novel vegetable oil crop due to its seeds rich in unsaturated fatty acids (UFAs) especially α-linolenic acid (ALA), which overweight the current available edible oil. However, little is known on the regulation mechanism of UFAs biosynthesis during its seed development. Here, we used transcriptome and proteome data combining phytochemistry means to uncover the relationship between abscisic acid (ABA) signaling and UFAs biosynthesis during P. ostii seed development. Based on transcriptome and proteome analysis, two desaturases of omega-6 and omega-3 fatty acid, named as PoFAD2 and PoFAD3 responsible for ALA biosynthesis were identified. Then, an ABSCISIC ACID-INSENSITIVE 5 (ABI5) proteins was identified as an upstream transcriptional factor, which activated the expression of PoFAD3 instead of PoFAD2. Moreover, silencing of PoABI5 repressed the response of PoFAD3 to ABA. This study provides the first view on the connection between the function of ABA signaling factors and ALA biosynthesis in the P. ostii seed, which lays the foundation for studies on the regulatory mechanism of ABA signaling involved in the UFAs synthesis during seeds development, meanwhile, it will shed light on manipulation of ALA content for satisfying human demands on high quality of edible oil or healthy supplement.

Quantitative analysis of resveratrol derivatives in the seed coats of tree peonies and their hypoglycemic activities in vitro/vivo.

Tree peonies are well-known horticultural and medicinal plants. The tree peony seeds, as emerging woody oil crops, recently have attracted great attention for their metabolites and bioactivities. In this study, the phytochemicals isolated from tree peony seed coats were systematically investigated. Seven polyphenolics were separated and prepared, mainly belonging to resveratrol derivatives. There was a great variation in the seed coat polyphenolic content among eight Paeonia species, and the contents of the resveratrol trimers and dimers were significantly higher in the seed coats of Paeonia ostii than other species. Based on the HPLC fingerprint characteristics and chemometric analysis, a clear discrimination among Paeonia plants was found, including the composition patterns and contents of the constituents. Moreover, the characteristic phytochemicals (vateriferol and trans-ε-viniferin) could significantly reduce the starch-mediated levels of postprandial blood glucose in diabetic/normal mice. In addition, in vitro enzyme tests showed that the two compounds could effectively and competitively inhibit α-glucosidase, with the IC50 values of 3.01 and 7.75 µM, respectively, indicating that vateriferol and trans-ε-viniferin could be therapeutic potential agents for hyperglycemia and diabetes mellitus.

Phytochemical profiles and the hypoglycemic effects of tree peony seed coats.

As emerging woody oil crops, the tree peony seeds recently have been attracting great attention for their metabolites and bioactivities. In this research, the phytochemical profiles of the seed coats of tree peonies from different production regions were investigated systematically. Twelve phytochemicals were separated and prepared, mainly belonging to stilbenes. A great variation in stilbene content was detected in the three Paeonia plants, and Paeonia ostii seed coats (POSC) had significantly higher contents of the stilbene compounds than other species. There were nineteen significant correlations between ecogeographical factors and the predominant compounds. A clear discrimination among the species was observed in their HPLC fingerprint and chemometric analysis. Furthermore, POSC extracts could significantly reduce the starch mediated PBG (postprandial blood glucose) levels in normal/diabetic mice. Meanwhile, in vitro enzyme tests revealed that the predominant compounds, suffruticosol B and ampelopsin D, could effectively and competitively inhibit α-glucosidase, indicating that POSC could be a natural source of hypoglycemics in the food and drug fields.

Dietary Moutan Cortex Radicis Improves Serum Antioxidant Capacity and Intestinal Immunity and Alters Colonic Microbiota in Weaned Piglets.

Background: Moutan cortex radicis (MCR), as a common traditional Chinese medicine, has been widely used as an antipyretic, antiseptic, and anti-inflammatory agent in China. Objectives: This study aimed to investigate the effects of dietary MCR supplementation on the antioxidant capacity and intestinal health of the pigs and to explore whether MCR exerts positive effects on intestinal health via regulating nuclear factor kappa-B (NF-κB) signaling pathway and intestinal microbiota. Methods: MCR powder was identified by LC-MS analysis. Selected 32 weaned piglets (21 d of age, 6.37 ± 0.10 kg average BW) were assigned (8 pens/diet, 1 pig/pen) to 4 groups and fed with a corn-soybean basal diet supplemented with 0, 2,000, 4,000, and 8,000 mg/kg MCR for 21 d. After the piglets were sacrificed, antioxidant indices, histomorphology examination, and inflammatory signaling pathway expression were assessed. The 16s RNA sequencing was used to analyze the effects of MCR on the intestinal microbiota structure of piglets. Results: Supplemental 4,000 mg/kg MCR significantly increased (P < 0.05) the average daily weight gain (ADG), average daily feed intake (ADFI), total antioxidative capability, colonic short-chain fatty acids (SCFA) concentrations, and the crypt depth in the jejunum but decreased (P < 0.05) the mRNA expression levels of interferon γ, tumor necrosis factor-α, interleukin-1ß, inhibiting kappa-B kinase ß (IKKß), inhibiting nuclear factor kappa-B (IκBα), and NF-κB in the jejunum and ileum. Microbiota sequencing identified that MCR supplementation significantly increased the microbial richness indices (Chao1, ACE, and observed species, P < 0.05) and the relative abundances of Firmicutes and Lactobacillus (P < 0.05), decreased the relative abundances of Bacteroides, Parabacteroides, unidentified_Lachnospiraceae, and Enterococcus (P < 0.05) and had no significant effects on the diversity indices (Shannon and Simpson, P > 0.05). Microbial metabolic phenotypes analysis also showed that the richness of aerobic bacteria and facultative anaerobic bacteria, oxidative stress tolerance, and biofilm forming were significantly increased (P < 0.05), and the richness of anaerobic bacteria and pathogenic potential of gut microbiota were reduced (P < 0.05) by MCR treatment. Regression analysis showed that the optimal MCR supplemental level for growth performance, serum antioxidant capacity, and intestinal health of weaned piglets was 3,420 ~ 4,237 mg/kg. Conclusions: MCR supplementation improved growth performance and serum antioxidant capacity, and alleviated intestinal inflammation by inhibiting the IKKß/IκBα/NF-κB signaling pathway and affecting intestinal microbiota in weaned piglets.

Comparative transcriptomic analysis of genes involved in stem lignin biosynthesis in woody and herbaceous Paeonia species.

Paeonia is recognized globally due to its ornamental value. However, the mechanisms behind the formation of distinct levels of lignification in Paeonia stems remain largely unknown. In this study, we selected three representative Paeonia species, namely P. ostii (shrub), P. lactiflora (herb), and P. × 'Hexie' (semi-shrub), to evaluate and contrast their respective anatomical structure, phytochemical composition and transcriptomic profile. Our results showed that the degree of lignin deposition on the cell wall, along with the total amount of lignin and its monomers (especially G-lignin) were higher in P. ostii stems compared to the other two species at almost all development stages except 80 days after flowering. Furthermore, we estimated a total number of unigenes of 60,238 in P. ostii, 43,563 in P. × 'Hexie', and 40,212 in P. lactiflora from stem transcriptome. We then built a co-expression network of 25 transcription factors and 21 enzyme genes involved in lignin biosynthesis and identified nine key candidate genes. The expression patterns of these genes were positively correlated with the transcription levels of PAL, C4H, 4CL2, CCR, and COMT, as well as lignin content. Moreover, the highest relative expression levels of CCR, 4CL2, and C4H were found in P. ostii. This study provides an explanation for the observed differences in lignification between woody and herbaceous Paeonia stems, and constitutes a novel reference for molecular studies of stem-specific lignification process and lignin biosynthesis that can impact the ornamental industry.

A comprehensive study of three species of Paeonia stem and leaf phytochemicals, and their antioxidant activities.

ETHNOPHARMACOLOGICAL RELEVANCE: Paeonia plants have been widely used as traditional Chinese medicinal materials for more than 2,000 years in the treatment of cardiovascular, extravasated blood and female genital diseases; paeoniflorin and paeonol have been implicated as the plants' primary active ingredients. AIM OF THE STUDY: Previous studies have been singularly focused on the chemical constituents and content variation of the Paeonia roots in the advancement of traditional Chinese medicine, with the plants' stems and leaves considered useless. This study aims to explore the chemical constituents, content variation, and antioxidant capacity in Paeonia stems and leaves for the future utilization of traditional Chinese medicine, given that current practices of digging and trade endanger Paeonia in the wild. MATERIALS AND METHODS: Herein, secondary metabolites from the stems and leaves from six developmental stages of the annual growth cycle of Paeonia ostii T. Hong & J. X. Zhang, P. 'Hexie', and P. lactiflora Pall. were qualitatively and quantitatively analyzed via high-performance liquid chromatography with a diode array detector (HPLC-DAD) and high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF-MS). Antioxidant capacity at each stage was also evaluated by various free radical scavenging assays. RESULTS: A total of 24 metabolites were detected and identified, including 5 monoterpene glycosides, 4 tannins, 5 phenols, 9 flavonoids, and paeonol. Excepting paeonol and the phenols, the levels of each metabolite category were significantly higher in the leaves than the stems during all developmental stages. The paeoniflorin content in the P. ostii leaves was the highest during the first developmental stage and higher than the standards of the Chinese Pharmacopoeia, suggesting it to be the optimal harvesting stage for medicinal uses. Notably, the antioxidant capacity of the leaves was significantly greater than in the stems, particularly for the leaves of P. 'Hexie'. CONCLUSION: Our study indicates that the leaves of P. 'Hexie' have the potential to be a worthy medicinal substitute to Paeonia roots due to their high monoterpene glycosides, phenols, and flavonoids as well as their strong antioxidant capacity. Further, this study provides a theoretical basis for the development and utilization of non-root Paeonia plant sections as medicinal plant resources.

Ecotopic over-expression of PoCHS from Paeonia ostii altered the fatty acids composition and content in Arabidopsis thaliana.

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 homolog 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 homolog of P. ostii and demonstrated its new function in seed fatty acid biosynthesis.

In Vitro Evaluation of a Fluorescent Microemulsion as an Oral Delivery Carrier and its Potential Application in Tracking Bioactive Compounds Label-Free.

In this work, a microemulsion emitting fluorescence was fabricated as a potential oral delivery system for bioactive compounds. In simulated oral administration, the microemulsion was characterized for its microstructure by 1hydrogen-nuclear magnetic resonance (1H-NMR). Results showed that microemulsions not only have good resistance to oral and gastric phases, but also lay a solid foundation for the release of bioactive compounds in the intestine. Fluorescence stability tests showed that microemulsions exhibit a remarkable fluorescence intensity in the digestive environment, indicating feasibility as a label-free delivery carrier. Moreover, in vitro release tests of bioactive compounds confirmed that an α-linolenic acid (ALA)-loaded microemulsion mainly released in the intestine, thereby achieving the aim of controlling the release of bioactive compounds. These results suggest that the synthesized fluorescent microemulsion, combining the favorable features of nontoxicity, antidigestive stability, remarkable fluorescence intensity, and controllable release, can be regarded as a promising label-free delivery carrier for oral administration.

Chalcone synthase is ubiquitinated and degraded via interactions with a RING-H2 protein in petals of Paeonia 'He Xie'.

Flavonoids are secondary metabolites widely distributed among angiosperms, where they play diverse roles in plant growth, development, and evolution. The regulation of flavonoid biosynthesis in plants has been extensively studied at the transcriptional level, but post-transcriptional, translational, and post-translational control of flavonoid biosynthesis remain poorly understood. In this study, we analysed post-translational regulation of flavonoid biosynthesis in the ornamental plant Paeonia, using proteome and ubiquitylome profiling, in conjunction with transcriptome data. Three enzymes involved in flavonoid biosynthesis were identified as being putative targets of ubiquitin-mediated degradation. Among these, chalcone synthase (PhCHS) was shown to have the greatest number of ubiquitination sites. We examined PhCHS abundance in petals using PhCHS-specific antibody and found that its accumulation decreased at later developmental stages, resulting from 26S proteasome-mediated degradation. We further identified a ring domain-containing protein (PhRING-H2) that physically interacts with PhCHS and demonstrated that PhRING-H2 is required for PhCHS ubiquitination. Taken together, our results suggest that PhRING-H2-mediates PhCHS ubiquitination and degradation is an important mechanism of post-translational regulation of flavonoid biosynthesis in Paeonia, providing a theoretical basis for the manipulation of flavonoid biosynthesis in plants.

A Novel R2R3-MYB Transcription Factor Contributes to Petal Blotch Formation by Regulating Organ-Specific Expression of PsCHS in Tree Peony (Paeonia suffruticosa).

Flower color patterns play critical roles in plant-pollinator interactions and represent one of the most common adaptations during angiosperm evolution. However, the molecular mechanisms underlying flower color pattern formation are less understood in non-model organisms. The aim of this study was to identify genes involved in the formation of petal blotches in tree peony (Paeonia suffruticosa) through transcriptome profiling and functional experiments. We identified an R2R3-MYB gene, PsMYB12, representing a distinct R2R3-MYB subgroup, with a spatiotemporal expression pattern tightly associated with petal blotch development. We further demonstrated that PsMYB12 interacts with a basic helix-loop-helix (bHLH) and a WD40 protein in a regulatory complex that directly activates PsCHS expression, which is also specific to the petal blotches. Together, these findings advance our understanding of the molecular mechanisms of pigment pattern formation beyond model plants. They also benefit molecular breeding of tree peony cultivars with novel color patterns and promote germplasm innovation.

Fatty Acid Composition, Phytochemistry, Antioxidant Activity on Seed Coat and Kernel of Paeonia ostii from Main Geographic Production Areas.

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 (298.89-399.34 mg g-1), crude protein (16.91%-22.73%), and total tocopherols (167.83-276.70 µg g-1) were obtained from PSK. Significant differences were found in the content of palmitic acids (11.31-14.27 mg g-1), stearic acids (2.42-4.24 mg g-1), oleic acids (111.25-157.63 mg g-1), linoleic acids (54.39-83.59 mg g-1), and ALA (99.85-144.71 mg g-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-1) compared with PSK (3.04 mg g-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.

Fatty acid desaturase 3 (PsFAD3) from Paeonia suffruticosa reveals high α-linolenic acid accumulation.

α-linolenic acid (ALA) deficiency and a skewed ω6: ω3 fatty acid ratio in the diet are thought to be a major cause for the high incidence of cardiovascular, inflammatory, and autoimmune diseases. Recent years, tree peony (Paeonia suffruticosa Andr.) with the high proportion of ALA (more than 45% in seed oil) is widely concerned. However, the underlying accumulation mechanism of the ALA in tree peony seeds remains unknown. In this study, comparative transcriptome analysis was performed between two cultivars ('Saiguifei' and 'Jingshenhuanfa') with different ALA contents. The analysis of the metabolic enzymes associated with ALA biosynthesis and temporal accumulation patterns of unsaturated fatty acids demonstrated the importance of microsomal ω-3 fatty acid desaturase 3 (FAD3). Moreover, PsFAD3 gene was identified from tree peony seeds, which was located in endoplasmic reticulum and the expression levels of PsFAD3 were consistent with ALA accumulation patterns in seeds. Heterologous expression in Saccharomyces cerevisiae and Arabidopsis thaliana confirmed that the isolated PsFAD3 protein could catalyze ALA synthesis. These results indicated that PsFAD3 was involved in the synthesis of ALA in seeds and could be exploited by the genetic breeding of new cultivars with high ALA content in tree peony as well as other potential crops.

Identification of microRNAs and long non-coding RNAs involved in fatty acid biosynthesis in tree peony seeds.

MicroRNAs (miRNAs) and long noncoding RNAs (lncRNAs) act as important molecular regulators in a wide range of biological processes during plant development and seed formation, including oil production. Tree peony seeds contain >90% unsaturated fatty acids (UFAs) and high proportions of α-linolenic acid (ALA, > 40%). To dissect the non-coding RNAs (ncRNAs) pathway involved in fatty acids synthesis in tree peony seeds, we construct six small RNA libraries and six transcriptome libraries from developing seeds of two cultivars (J and S) containing different content of fatty acid compositions. After deep sequencing the RNA libraries, the ncRNA expression profiles of tree peony seeds in two cultivars were systematically and comparatively analyzed. A total of 318 known and 153 new miRNAs and 22,430 lncRNAs were identified, among which 106 conserved and 9 novel miRNAs and 2785 lncRNAs were differentially expressed between the two cultivars. In addition, potential target genes of the microRNA and lncRNAs were also predicted and annotated. Among them, 9 miRNAs and 39 lncRNAs were predicted to target lipid related genes. Results showed that all of miR414, miR156b, miR2673b, miR7826, novel-m0027-5p, TR24651|c0_g1, TR24544|c0_g15, and TR27305|c0_g1 were up-regulated and expressed at a higher level in high-ALA cultivar J when compared to low-ALA cultivar S, suggesting that these ncRNAs and target genes are possibly involved in different fatty acid synthesis and lipid metabolism through post-transcriptional regulation. These results provide a better understanding of the roles of ncRNAs during fatty acid biosynthesis and metabolism in tree peony seeds.