Acquired EML4-ALK fusion and EGFR C797S in cis mutation as resistance mechanisms to osimertinib in a non-small cell lung cancer patient with EGFR L858R/T790M.

The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) dramatically improve the clinical outcomes of non-small cell lung cancer (NSCLC) patients harboring EGFR -sensitive mutations. Despite the remarkable efficacy of first-and second-generation EGFR TKIs, disease relapse is inevitable. EGFR T790M mutation is a primary contributor to the acquired resistance to first- and second-generation EGFR TKIs. Osimertinib, which is an irreversible third-generation EGFR TKI, was designed for EGFR -activating mutations as well as the EGFR T790M mutation in patients with advanced NSCLC and has demonstrated a convincing efficacy. However, acquired resistance to osimertinib after treatment inevitably occurs. The acquired resistance mechanisms to osimertinib are highly complicated and not fully understood, encompassing EGFR -dependent as well as EGFR -independent mechanisms. Treatment approaches for patients progressing from osimertinib have not been established. We present a case of a stage IV lung adenocarcinoma patient harboring EGFR L858R, acquired T790M after treatment with first-line gefitinib. She then acquired a new EML4-ALK gene fusion after treatment with osimertinib. A combination targeted therapy of osimertinib plus alectinib was initiated, with a progression-free survival of 5 months without any serious adverse reaction. After disease progression, EGFR C797S in cis was detected with a loss of the EML4-ALK fusion by targeted next-generation sequencing. Then therapy was changed to pemetrexed combined with bevacizumab plus camrelizumab, but no obvious effect was observed. The patient had achieved an overall survival of 31 months. As far as we know, this was the first reported case that an EGFR -mutant NSCLC patient-acquired ALK fusion mediating resistance to osimertinib, and sequential EGFR C797S mutation mediating resistance to combined targeted therapy with osimertinib and alectinib. Our case shows that EML4-ALK fusion is a rare but critical resistance mechanism to osimertinib, and C797S mutation in cis may be an underlying mechanism of acquired resistance mutation in double TKIs therapy. Furthermore, molecular detection and rebiopsy play important roles in the selection of therapeutic strategies when the disease progresses.

Enzymatic basis for stepwise C-glycosylation in the formation of flavonoid di-C-glycosides in sacred lotus (Nelumbo nucifera Gaertn.).

Lotus plumule, the embryo of the seed of the sacred lotus (Nelumbo nucifera), contains a high accumulation of secondary metabolites including flavonoids and possesses important pharmaceutical value. Flavonoid C-glycosides, which accumulate exclusively in lotus plumule, have attracted considerable attention in recent decades due to their unique chemical structure and special bioactivities. As well as mono-C-glycosides, lotus plumule also accumulates various kinds of di-C-glycosides by mechanisms which are as yet unclear. In this study we identified two C-glycosyltransferase (CGT) genes by mining sacred lotus genome data and provide in vitro and in planta evidence that these two enzymes (NnCGT1 and NnCGT2, also designated as UGT708N1 and UGT708N2, respectively) exhibit CGT activity. Recombinant UGT708N1 and UGT708N2 can C-glycosylate 2-hydroxyflavanones and 2-hydroxynaringenin C-glucoside, forming flavone mono-C-glycosides and di-C-glycosides, respectively, after dehydration. In addition, the above reactions were successfully catalysed by cell-free extracts from tobacco leaves transiently expressing NnCGT1 or NnCGT2. Finally, enzyme assays using cell-free extracts of lotus plumule suggested that flavone di-C-glycosides (vicenin-1, vicenin-3, schaftoside and isoschaftoside) are biosynthesized through sequentially C-glucosylating and C-arabinosylating/C-xylosylating 2-hydroxynaringenin. Taken together, our results provide novel insights into the biosynthesis of flavonoid di-C-glycosides by proposing a new biosynthetic pathway for flavone C-glycosides in N. nucifera and identifying a novel uridine diphosphate-glycosyltransferase (UGT708N2) that specifically catalyses the second glycsosylation, C-arabinosylating and C-xylosylating 2-hydroxynaringenin C-glucoside.

Analysis of Chemical Components in the Roots of Eight Intersubgeneric Hybrids of Paeonia.

Paeonia cultivars are famous ornamental plants, and some of them are also traditional Chinese medicinal resources. Intersubgeneric hybrids of Paeonia (IHPs) are formed by the hybridization of herbaceous peony (Paeonia lactiflora) and tree peony (Paeonia×suffruticosa or lutea hybrid tree peony). The phenotypic characteristics of IHPs are similar to those of herbaceous peony, and their root systems are large and vigorous. However, their medicinal value has not been reported yet. In this study, the roots of eight IHP samples were analyzed by high performance liquid chromatography quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF-MS/MS). A total of 18 compounds were identified, including phenols, paeonols, monoterpene glycosides, and tannins. The contents of monoterpene glycosides and tannins in IHPs were higher than herbaceous peony and tree peony, exceeding 44.76 mg/g DW and 11.50 mg/g DW, respectively. Three IHPs, 'Prairie Charm', 'Garden Treasure', and 'Yellow Emperor', with more types and a higher content of medicinal compounds, were screened out by cluster analysis. These IHPs have considerable potential for the development of medicinal resources.

Identification of flavonoids and chlorogenic acids in elm fruits from the genus Ulmus and their antioxidant activity.

Elm fruits were once an important food source in the years of famine. Research on the functional compounds in elm fruits was almost unavailable. In this study, we established an efficient high-performance liquid chromatography method for the simultaneous separation of eight chlorogenic acids and 28 flavonoids in elm fruits for the first time. Total flavonoid contents ranged from 286 mg/100 g (Ulmus laciniata) to 1228 mg/100 g (U. pumila). High concentrations of rutin, quercetin 3-O-glucoside, and kaempferol derivatives were present in U. laevis, U. castaneifolia, and U. pumila, respectively. Furthermore, the fruit extracts of U. americana, U. castaneifolia, U. davidiana, and U. pumila showed higher antioxidant activity. These results suggest that fruits of these species can be used as bioresources for the extraction of the corresponding functional compounds. This work provides informative data and can be an important reference for future research on elm fruits as a renewed food resource.

Transcriptome sequencing and metabolite analysis for revealing the blue flower formation in waterlily.

BACKGROUND: Waterlily (Nymphaea spp.), a perennial herbaceous aquatic plant, is divided into two ecological groups: hardy waterlily and tropical waterlily. Although the hardy waterlily has no attractive blue flower cultivar, its adaptability is stronger than tropical waterlily because it can survive a cold winter. Thus, breeding hardy waterlily with real blue flowers has become an important target for breeders. Molecular breeding may be a useful way. However, molecular studies on waterlily are limited due to the lack of sequence data. RESULTS: In this study, six cDNA libraries generated from the petals of two different coloring stages of blue tropical waterlily cultivar Nymphaea 'King of Siam' were sequenced using the Illumina HiSeq™ 2500 platform. Each library produced no less than 5.65 Gb clean reads. Subsequently, de novo assembly generated 112,485 unigenes, including 26,206 unigenes annotated to seven public protein databases. Then, 127 unigenes could be identified as putative homologues of color-related genes in other species, including 28 up-regulated and 5 down-regulated unigenes. In petals, 16 flavonoids (4 anthocyanins and 12 flavonols) were detected in different contents during the color development due to the different expression levels of color-related genes, and four flavonols were detected in waterlily for the first time. Furthermore, UA3GTs were selected as the most important candidates involved in the flavonoid metabolic pathway, UA3GTs induced blue petal color formation in Nymphaea 'King of Siam'. CONCLUSIONS: This study will improve our understanding of the molecular mechanism of blue flowers in waterlily and provide the basis for molecular breeding of blue hardy waterlily cultivars.

Fatty acid composition of developing tree peony (Paeonia section Moutan DC.) seeds and transcriptome analysis during seed development.

BACKGROUND: Tree peony (Paeonia section Moutan DC.) is known for its excellent ornamental and medicinal values. In 2011, seeds from P. ostii have been identified as novel resource of α-linolenic acid (ALA) for seed oil production and development in China. However, the molecular mechanism on biosynthesis of unsaturated fatty acids in tree peony seeds remains unknown. Therefore, transcriptome data is needed to better understand the underlying mechanisms. RESULTS: In this study, lipid accumulation contents were measured using GC-MS methods across developing tree peony seeds, which exhibited an extraordinary ALA content (49.3%) in P. ostii mature seeds. Transcriptome analysis was performed using Illumina sequencing platform. A total of 144 million 100-bp paired-end reads were generated from six libraries, which identified 175,874 contigs. In the KEGG Orthology enrichment of differentially expressed genes, lipid metabolism pathways were highly represented categories. Using this data we identified 388 unigenes that may be involved in de novo fatty acid and triacylglycerol biosynthesis. In particular, three unigenes (SAD, FAD2 and FAD8) encoding fatty acid desaturase with high expression levels in the fast oil accumulation stage compared with the initial stage of seed development were identified. CONCLUSIONS: This study provides the first comprehensive genomic resources characterizing tree peony seeds gene expression at the transcriptional level. These data lay the foundation for further understanding of molecular mechanism responsible for lipid biosynthesis and the high unsaturated fatty acids (especially ALA) accumulation. Meanwhile, it provides theoretical base for potential oilseed application in the respect of n-6 to n-3 ratio for human diets and future regulation of target healthy components of oils.

Methylation mediated by an anthocyanin, O-methyltransferase, is involved in purple flower coloration in Paeonia.

Anthocyanins are major pigments in plants. Methylation plays a role in the diversity and stability of anthocyanins. However, the contribution of anthocyanin methylation to flower coloration is still unclear. We identified two homologous anthocyanin O-methyltransferase (AOMT) genes from purple-flowered (PsAOMT) and red-flowered (PtAOMT) Paeonia plants, and we performed functional analyses of the two genes in vitro and in vivo. The critical amino acids for AOMT catalytic activity were studied by site-directed mutagenesis. We showed that the recombinant proteins, PsAOMT and PtAOMT, had identical substrate preferences towards anthocyanins. The methylation activity of PsAOMT was 60 times higher than that of PtAOMT in vitro. Interestingly, this vast difference in catalytic activity appeared to result from a single amino acid residue substitution at position 87 (arginine to leucine). There were significant differences between the 35S::PsAOMT transgenic tobacco and control flowers in relation to their chromatic parameters, which further confirmed the function of PsAOMT in vivo. The expression levels of the two homologous AOMT genes were consistent with anthocyanin accumulation in petals. We conclude that AOMTs are responsible for the methylation of cyanidin glycosides in Paeonia plants and play an important role in purple coloration in Paeonia spp.

Rapid separation and identification of anthocyanins from flowers of Viola yedoensis and V. prionantha by high-performance liquid chromatography-photodiode array detection-electrospray ionisation mass spectrometry.

INTRODUCTION: Anthocyanins are important plant secondary metabolites. They show strong antioxidant activities and have potential as anti-cancer agents. Viola yedoensis and V. prionantha are traditional Chinese medicines and ornamental plants. However, the anthocyanin compositions of these two species are still unresolved. OBJECTIVE: To develop a rapid and reliable high-performance liquid chromatography (HPLC) method for the separation and identification of anthocyanins from V. yedoensis and V. prionantha. METHODOLOGY: Samples were extracted in methanol-water-formic acid-TFA (70:27:2:1, v/v). HPLC analysis was done on a C(18) column (TSK-GEL ODS-80Ts: 150 × 4.6 mm i.d.). Four solvent systems were tested to optimise the separation of anthocyanins using different gradient separation systems. HPLC-photodiode array detection (DAD) coupled to electrospray ionisation mass spectrometry (ESI-MS) was used to carry out the comprehensive characterisation of anthocyanins. RESULTS: Fourteen anthocyanins were characterised within 40 min with satisfactory peak resolution by a gradient composed of 10% aqueous formic acid and formic acid-acetonitrile-water (10:40:50, v/v). The calibration curve showed an excellent linear regression (r(2) = 0.9995) and low intra- and inter-day variations (RSD < 3.67%). The detected anthocyanins derived from Dp, Cy, Pt, Mv and Pn, could be divided into three groups: non-acylated glycosides, acetylglycosides and coumaroylglycosides. Anthocyanins distribution exhibited remarkable differences in aglycone levels and acylation patterns. CONCLUSION: The optimised method was successfully applied for the analysis of 14 anthocyanins from V. yedoensis and V. prionantha. The identification of anthocyanin constitutions is valuable for breeding and will open up new prospects for their medicinal application.

[Analysis of lumbosacral sagittal balance parameter variation in minimally invasive transforaminal lumbar interbody fusion with real-time 3D navigation techniques].

OBJECTIVE: To explore the dynamic changes of lumbosacral sagittal parameters after real-time three-dimensional navigation assisted minimally invasive transforaminal lumbar interbody fusion (MIS-TLIF) and traditional open TLIF for treatment of lumbar degenerative disease. METHODS: The clinical data of 61 patients with lumbar degenerative disease underwent single-segment surgery from September 2017 to September 2019 were retrospectively analyzed. Among them, 31 cases underwent MIS-TLIF with 3D navigation techniques (MIS-TLIF group) and another 30 cases underwent conventional open TLIF (traditional open TLIF group). The basic information, operative time and intraoperative blood loss were collected. The sagittal radiologic parameters were measured before surgery and 3 months after surgery, including lumbar lordosis (LL), segmental lordosis (SL), pelvic incidence (PI), pelvic tilt (PT), sacral slope (SS), anterior disc height (ADH), posterior disc height(PDH).And the average disc height(DH) and pelvic incidence to lumbar lordosis mismatch (PI-LL) were calculated. RESULTS: Operative time and intraoperative blood loss in MIS-TLIF group were significantly less than in traditional open TLIF group(P<0.05). In MIS-TLIF group, LL, SL, PI-LL, and DH were significantly improved at 3 months after surgery (P<0.05), while PI, PT, and SS were not statistically different from those before surgery (P>0.05). LL, PI-LL, and DH of patients in the traditional open TLIF group were significantly improved at 3 months after surgery (P<0.05), while the PI, PT, SS, and SL were not statistically different from those before surgery (P>0.05). LL change showed a significant correlation with SL change (r= 0.433, P<0.001). Change in SL closely correlated to change in ADH (r=0.621, P<0.05) and PDH(r=0.527, P<0.05). CONCLUSION: Real-time navigation-assisted MIS-TLIF and traditional open TLIF can recover DH in a short term for lumbar degenerative diseases, improve LL and PI-LL, and make the arrangement of the sagittal plane of the lumbosacral region more coordinated after surgery. But only the navigation assisted MIS -TLIF can significantly improve SL. Compared with traditional open TLIF, real-time navigation assisted MIS-TLIF in the treatment of degenerative lumbar diseases has the advantages of short operation time and less intraoperative bleeding.

Characterization of phytochemicals in the roots of wild herbaceous peonies from China and screening for medicinal resources.

Paeonia Radix Rubra (PRR) is a very common traditional Chinese medicine (TCM). The roots of Paeonia lactiflora and Paeonia anomala subsp. veitchii are used for the production of PRR. However, other species of section Paeonia in China are also used to produce PRR. The roots of section Paeonia from 20 populations of seven species and two subspecies in China were analyzed by high performance liquid chromatography quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF- MS). A total of 21 metabolites were identified, including nine monoterpene glycosides, seven tannins, three phenols, one paeonol and one flavonoid. There were significant differences in the composition and content of metabolites among different populations. The relative contents of monoterpene glycosides and tannins were generally higher in most samples. Cluster analysis showed that 20 populations could be divided into four groups. Among them, the populations of P. lactiflora and Paeonia mairei were clustered together in one group. The content of paeoniflorin in P. lactiflora was high (>22.20 mg g-1, dry weight, the same below), and the content of lactiflorin in P. mairei (>17.50 mg g-1) was significantly higher than in other species (<2.30 mg g-1). The monoterpene glycoside content in P. mairei (>51.60 mg g-1) was significantly higher than in other species (<43.40 mg g-1), suggesting that it could be useful medicinal germplasm for the development of monoterpene glycosides. In addition, some populations of Paeonia emodi, Paeonia sterniana and P. mairei may also be used as potential germplasm resources for use in PRR. Genetic and environmental factors resulted in differences in the composition and content of metabolites in different populations of the same species. Therefore, it is necessary to carefully consider the origin of Paeonia germplasm when selecting medicinal materials to be used as resources for the production of PRR.

Simultaneous analysis of anthocyanins and flavonols in petals of lotus (Nelumbo) cultivars by high-performance liquid chromatography-photodiode array detection/electrospray ionization mass spectrometry.

A fast and reliable HPLC method for the simultaneous separation of anthocyanins and flavonols in lotus petals was developed based on the study of four candidate solvent systems. Fifteen flavonoids were identified by high-performance liquid chromatography with photodiode array detection/mass spectrometry. Among them, two anthocyanins and nine flavonols were discovered in lotus petals for the first time. This work is valuable for both the hybrid breeding on lotus oriented to flower color and the utilization of lotus petals as functional food materials.

Studies on Paeonia cultivars and hybrids identification based on SRAP analysis.

Plants of Paeonia are valuable for their ornamental and medicinal values. Genetic relations and hybrids identification among different sections of Paeonia were studied using sequence related amplified polymorphism (SRAP) markers. A total of 29 cultivars including 2 intersectional hybrids, 13 sect. Moutan and 14 from sect. Paeonia were used. A total of 197 bands were produced using 24 primer combinations, among which 187 bands showed polymorphism. From the bands amplified, we can identify the peony cultivars using unique SRAP markers and specific primer combinations. Fourteen peony cultivars were distinguished among each other by using totally 35 SRAP markers, which were generated by 16 primer pairs. Two specific primer pairs of Me8/Em8 and Me8/Em1 can be used to identify cultivars from different sections. The mean genetic similarity coefficient (GS), the gene diversity (GD), and the Shannon's information index of peony cultivars were 0.45, 0.19 and 0.32, respectively. Both UPGMA (unweighted pair-group method of arithmetic average) dendrogram and PCA (principle component analysis) analysis showed clear genetic relationships among the 29 peony cultivars, and within section and its intersectional hybrids. The above results are valuable for estimating and analyzing genetic background of Paeonia, parent selection in crossing breeding programs, molecular marker assisted selection (MAS) breeding for further germplasm innovation programs.

Determination of anthocyanins and exploration of relationship between their composition and petal coloration in crape myrtle (Lagerstroemia hybrid).

Petal coloration and pigment components in 12 American crape myrtle cultivars (Lagerstroemia indicaxLagerstroemia fauriei) and five Chinese crape myrtle cultivars (L. indica hybrids) were studied. Color was measured by CIEL*a*b* scale and anthocyanin composition of crape myrtle was determined using high-performance liquid chromatography coupled to photodiode array detection and electrospray ionization mass spectrometry. The presence of the previously reported delphinidin 3-O-glucoside, petunidin 3-O-glucoside and malvidin 3-O-glucoside were confirmed. Cyanidin 3-O-glucoside was identified in crape myrtle for the first time. We explored the relationship between petal color and anthocyanin contents by multiple linear regression analyses. The results indicated that total flavones and flavonols were important variables and contributed to blue-enhancing in crape myrtle. Based on anthocyanins and co-pigments analysis, flower color breeding in crape myrtle towards true-red and blue were discussed.

[Comparison of the content of effective components between tree peony wild species and main cultivars].

OBJECTIVE: To determine the content of paeonol and paeoniflorin in wild tree peony species and main medicinal cultivars in order to provide scientific foundations for the industrialization of tree peony cortex. METHODS: HPLC was used to compare the effective content of different resources. RESULTS: (1) The content range of paeonol and paeoniflorin of 7 wild species was 0.10%-0.61% and 2.22%-5.57%, respectively; (2) The content range of paeonol and paeoniflorin of all cultivars from different producing area was 0.33%-1.43% and 1.60%-2.85%; (3) The content range of paeonol and paeoniflorin of different cultivars in the same place (Changping, Beijing) was 0.27%-0.75% and 1.87%-3.96%; (4) The content range of paeonol of cultivars from the same area was 0.34%-1.10%. There was no significant difference of relative content of paeoniflorin from the same areas. CONCLUSION: The relative content of main medicinal components of wild species was not higher than cultivar; the differences were significant among cultivars; the relative content of paeonol of 'JSF' was high; more attention of choosing cultivars and producing areas should be paid in tree peony cortex producing.

Phytochemical variation among the traditional Chinese medicine Mu Dan Pi from Paeonia suffruticosa (tree peony).

Mu Dan Pi is a traditional Chinese medicine used to treat inflammation, cancer, allergies, diabetes, angiocardiopathy, and neurodegenerative diseases. In this study, the metabolome variation within Mu Dan Pi collected from 372 tree peony cultivars was systematically investigated. In total, 42 metabolites were identified, comprising of 14 monoterpene glucosides, 11 tannins, 8 paeonols, 6 flavonoids, and 3 phenols. All cultivars revealed similar metabolite profiles, however, they were further classified into seven groups on the basis of their varying metabolite contents by hierarchical cluster analysis. Traditional cultivars for Mu Dan Pi were found to have very low metabolite contents, falling into clusters I and II. Cultivars with the highest amounts of metabolites were grouped in clusters VI and VII. Five potential cultivars, namely, 'Bai Yuan Qi Guan', 'Cao Zhou Hong', 'Da Zong Zi', 'Sheng Dan Lu', and 'Cheng Xin', with high contents of monoterpene glycosides, tannins, and paeonols, were further screened. Interestingly, the majority of investigated cultivars had relatively higher metabolite contents compared to the traditional medicinal tree peony cultivars.

Determination of xanthones and flavonoids of methanol extracts obtained from different parts of the plants of three Gentianaceae species.

Gentianopsis barbata, Halenia corniculata, and Gentianella acuta were widely distributed throughout China and commonly used in traditional Chinese medicine. However, owing to similar living environments and morphological features, locals often had trouble distinguishing between these three species. In this present study, chromatograms at 350 nm were obtained and the composition and content of their chemical compounds determined using HPLC-DAD/ESI-MS2. In total, 35 chemical compounds were detected, 32 of which were identified, 25 of which were xanthones, 6 flavonoids, and 1 chlorogenic acid. The 350 nm chromatograms of these three species displayed evident differences. The individual compounds and their occurrence and content in different parts of the plant within different species were included in our results. This basic data will be useful for future pharmacological study. The total compositions of flavonoids and xanthones were approximately comparable in G. barbata and H. corniculata. Meanwhile, xanthones were predominant in G. acuta. From the perspective of chemical compound compositions, the leaf is recommended as the most valuable medicinal section for each of these three species.

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.

Polyphenol profile and antioxidant activity of the fruit and leaf of Vaccinium glaucoalbum from the Tibetan Himalayas.

Vaccinium glaucoalbum, a perennial evergreen shrub, is naturally distributed in high-altitude areas. In this study, the composition and content of polyphenolic compounds in the fruit and leaf of V. glaucoalbum were characterized. In total, 24 chemical compounds were detected and identified by HPLC-DAD and HPLC-ESI-MS2. Among all the compounds determined, 15 were anthocyanins and detected in fruit, 5 were flavonols and monitored in leaf, and 4 were chlorogenic acids and found in both fruit and leaf. The total anthocyanin content (TAC) of fruit (682mg/100gFW) was the highest among wild Vaccinium berries in China which have been investigated for now, and the total flavonol content of leaf was 2764mg/100gFW. The antioxidant activity of both fruit and leaf was assessed by DPPH and FRAP assays. Given its high TAC and strong antioxidant activity, the fruit of V. glaucoalbum has great potential in functional food.

Flavone synthases from Lonicera japonica and L. macranthoides reveal differential flavone accumulation.

Flavones are important secondary metabolites found in many plants. In Lonicera species, flavones contribute both physiological and pharmaceutical properties. However, flavone synthase (FNS), the key enzyme responsible for flavone biosynthesis, has not yet been characterized in Lonicera species. In this study, FNSII genes were identified from Lonicera japonica Thunb. and L. macranthoides Hand.-Mazz. In the presence of NADPH, the recombinant cytochrome P450 proteins encoded by LjFNSII-1.1, LjFNSII-2.1, and LmFNSII-1.1 converted eriodictyol, naringenin, and liquiritigenin to the corresponding flavones directly. The different catalytic properties between LjFNSII-2.1 and LjFNSII-1.1 were caused by a single amino acid substitution at position 242 (glutamic acid to lysine). A methionine at position 206 and a leucine at position 381 contributed considerably to the high catalytic activity of LjFNSII-1.1. In addition, LjFNSII-1.1&2.1 and LmFNSII-1.1 also biosynthesize flavones that were further modified by O-glycosylation in transgenic tobacco. The expression levels of the FNSII genes were consistent with flavone accumulation patterns in flower buds. Our findings suggested that the weak catalytic activity of LmFNSII-1.1 and the relatively low expression of LmFNSII-1.1 in flowers might be responsible for the low levels of flavone accumulation in flower buds of L. macranthoides.