Integrated metabolome and transcriptome revealed the flavonoid biosynthetic pathway in developing Vernonia amygdalina leaves.

BACKGROUND: Vernonia amygdalina as a tropical horticultural crop has been widely used for medicinal herb, feed, and vegetable. Recently, increasing studies revealed that this species possesses multiple pharmacological properties. Notably, V. amygdalina leaves possess an abundance of flavonoids, but the specific profiles of flavonoids and the mechanisms of fl avonoid bi osynthesis in developing leaves are largely unknown. METHODS: The total flavonoids of V. amygdalina leaves were detected using ultraviolet spectrophotometer. The temporal flavonoid profiles of V. amygdalina leaves were analyzed by LC-MS. The transcriptome analysis of V. amygdalina leaves was performed by Illumina sequencing. Functional annotation and differential expression analysis of V. amygdalina genes were performed by Blast2GO v2.3.5 and RSEM v1.2.31, respectively. qRT-PCR analysis was used to verify the gene expressions in developing V. amygdalina leaves. RESULTS: By LC-MS analysis, three substrates (p-coumaric acid, trans-cinnamic acid, and phenylalanine) for flavonoid biosynthesis were identified in V. amygdalina leaves. Additionally, 42 flavonoids were identified from V. amygdalina leaves, including six dihydroflavones, 14 flavones, eight isoflavones, nine flavonols, two xanthones, one chalcone, one cyanidin, and one dihydroflavonol. Glycosylation and methylation were common at the hydroxy group of C3, C7, and C4' positions. Moreover, dynamic patterns of different flavonoids showed diversity. By Illumina sequencing, the obtained over 200 million valid reads were assembled into 60,422 genes. Blast analysis indicated that 31,872 genes were annotated at least in one of public databases. Greatly increasing molecular resources makes up for the lack of gene information in V. amygdalina. By digital expression profiling and qRT-PCR, we specifically characterized some key enzymes, such as Va-PAL1, Va-PAL4, Va-C4H1, Va-4CL3, Va-ACC1, Va-CHS1, Va-CHI, Va-FNSII, and Va-IFS3, involved in flavonoid biosynthesis. Importantly, integrated metabolome and transcriptome data of V. amygdalina leaves, we systematically constructed a flavonoid biosynthetic pathway with regards to material supplying, flavonoid scaffold biosynthesis, and flavonoid modifications. Our findings contribute significantly to understand the underlying mechanisms of flavonoid biosynthesis in V. amygdalina leaves, and also provide valuable information for potential metabolic engineering.

Effects of exogenous abscisic acid on oil content, fatty acid composition, biodiesel properties and lipid components in developing Siberian apricot (Prunus sibirica) seeds.

Previous studies in Siberian apricot (Prunus sibirica) seed kernel (SASK) have suggested the involvement of abscisic acid (ABA) signaling pathway in oil accumulation. However, there are few reports on the effects of ABA on the metabolism of fatty acids (FA) in seed development. Here, we first evaluated the response of developing SASK to ABA treatment, with a focus on oil content, FA composition, biodiesel properties, lipid compounds and gene expressions. Compared with control samples, the application of exogenous ABA increased the total oil content by 6.55% in mature SASK. The C18:1 content markedly increased in ABA treatment, and conversely C16:0 decreased. Exogenous ABA also improved the biodiesel properties of SASK oil, making it better suited to the specifications of biodiesel standards. Furthermore, the molecular species of phosphatidylcholine (PC), phosphatidic acid (PA), diacylglycerol (DAG) and triacylglycerol (TAG) were detected using lipidomics analysis. The 18:1/18:1 was the main component in PA, PC and DAG, while the main components of 18:1/18:1/18:2, 18:1/18:1/18:3, 18:2/18:2/18:2 and 18:1/18:1/18:1 in TAG. Most lipid species gradually increased with SASK maturity. In addition, the relative contents of TAG-18:1/18:1/18:2 and TAG-18:1/18:1/18:1 in developing SASK increased with the application of exogenous ABA. We also detected elevated gene expression of key genes involved in ABA chemical pathway, which likely affected FA biosynthesis and accumulation. Our results provide insight into the effects of ABA on the oil accumulation in developing SASK, which has direct applications to improving the quality of SASK-derived biodiesel.

Genome-wide characterization of the NUCLEAR FACTOR-Y (NF-Y) family in Citrus grandis identified CgNF-YB9 involved in the fructose and glucose accumulation.

BACKGROUND: Nuclear factor Y (NF-Y) is increasingly known to be involved in many aspects of plant growth and development. To date, the systematic characterization of NF-Y family has never been reported in Citrus grandis. OBJECTIVE: Genome-wide characterization of C. grandis NF-Y (CgNF-Y) family and analysis of their role in sucrose metabolism. METHODS: NF-Y conserved models were employed to identify CgNF-Y genes from genomic data. Phylogenetic tree was generated by the neighbor-joining method using program MEGA 7.0. Based on our previous transcriptomic data, the transcription levels were calculated by RSEM software and were clustered by ShortTime-series Expression Miner. The plant expression vector of CgNF-YB9 was constructed using In-Fusion Cloning and transferred into tobacco by leaf disc transformation method. Soluble sugars and gene expressions were analysis by HPLC and qRT-PCR, respectively. RESULTS: A total of 24 CgNF-Y genes (6 CgNF-YAs, 13 CgNF-YBs and 5 CgNF-YCs) were identified with conserved domains. Phylogenetic analysis of the NF-Y proteins indicated that NF-YA, NF-YB and NF-YC could be categorized into four, five and three clades, respectively. Expression profiling analysis reflected spatio-temporally distinct expression patterns for CgNF-Y genes. Importantly, we observed a positive correlation between the expression level of CgNF-YB9 and the content of soluble sugar. Moreover, CgNF-YB9-corelated genes were enriched in carbohydrate metabolism. In CgNF-YB9 overexpression lines, sucrose content showed a decrease, whereas glucose and fructose contents displayed an increase. As expected, the transcription levels of sucrose-phosphate synthase and vacuolar invertase in transgenic Line 3 were observed with significantly down- and up-regulated, respectively. CONCLUSIONS: The structure, phylogenetic relationship and expression pattern of 24 CgNF-Y genes were identified, and CgNF-YB9 was involved in sucrose metabolism.

WRINKLED1 transcription factor orchestrates the regulation of carbon partitioning for C18:1 (oleic acid) accumulation in Siberian apricot kernel.

WRINKLED1 (WRI1), an APETALA2 (AP2)-type transcription factor, has been shown to be required for the regulation of carbon partitioning into fatty acid (FA) synthesis in plant seeds. To our knowledge, the regulatory network of WRI1 remains unknown in Prunus sibirica kernel (PSK), a novel woody biodiesel feedstock in China. In this study, based on the transcriptional data from developing oilseeds of multiple plant species, we identified 161 WRI1-coexpressed genes using weighted gene co-expression network analysis (WGCNA). The major portion of WRI1-coexpressed genes was characterized to be involved in carbon partitioning and FA biosynthesis. Additionally, we detected the temporal patterns for oil content and FA compositions in developing PSK from two different germplasms (AS-85 and AS-86). The major differences between the two germplasms are higher contents of oil and C18:1 in AS-85 than in AS-86 at a mature stage. Thus, AS-85 and AS-86 are desirable materials to explore the molecular and metabolic mechanisms of oil accumulation in Siberian apricot. Expression analysis in developing PSK of AS-85 and AS-86 indicated that the expression level of P. sibirica WRI1 (PsWRI1) was closely correlated to accumulative rate of oil. Also, the comparison of expression profiles in developing PSK of AS-85 and AS-86 displayed that the pPK, E1-α, E2, TAL, BC, MCMT, BS, SAD and FAD2 have a high correlation with PsWRI1. Transient expression showed that ProSAD- and ProBS-driving GUS expression showed no substantial difference between AS-85 and AS-86, while the expression level of ProPEPCK-AS-85 driving GUS was significantly higher than that of ProPEPCK-AS-86 driving GUS. Additionally, transient co-transformation with PsWRI1 revealed that ProSAD, ProPEPCK and ProBS activity could be specifically up-regulated by PsWRI1. This regulatory mechanism of PsWRI1 may create a steep concentration difference, thereby facilitating carbon flux into C18:1 accumulation in developing PSK. Overall, all our findings imply a versatile mechanism of WRI1 to optimize carbon allocation for oil accumulation, which can provide reference for researching the woody biodiesel plants.