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Research Highlights: The present study firstly reported the metabolic dynamics of astringent seed, a special type of abortion in Chinese fir, during the astringent material stages. The results provide a reference for further study on its occurrence mechanism and enrich the understanding of the plant seed developmental physiology. Background and Objectives: Astringent seed is a type of abortive phenomenon in Chinese fir, which significantly reduces the yield and quality of elite seeds for its high-incidence and indistinguishableness in seed orchard. Embryo defects can be observed in the astringent seed, accompanied with rapid accumulation of secondary metabolites. However, types of those metabolites in astringent seed, dynamic changes during seed growth process, and different accumulative characteristics compared to germinable seed have not been explained. Materials and Methods: Astringent and germinable seed samples were collected at four stages aim to determine the differences in their metabolic patterns. A liquid chromatography-mass spectrometry (LC-MS) detection was used to generate the raw metabolic peaks. Bioinformatics statistical strategies were used to further investigation. Results: A total of 421 metabolites were screened and 112 metabolites were identified as the different expressive metabolites including 68 up-regulated and 44 down-regulated metabolites. Those different expressive metabolites were grouped into 26 classes. Flavone, flavonol, and amino acid derivatives compounds were the most varied metabolites. Four subcategories which could represent the diverse basic expressive patterns or accumulative activity in different sample groups were further clustered. Moreover, pathways related to biosynthesis/degradation/metabolism of flavonoid-like compounds, amino acid/nucleotides derivatives, zeatin, and IAA were clearly enriched. Conclusions: Significant metabolic differences were observed across and between astringent and germinable seeds 105 d after pollination. Massive accumulation of flavonoids-like compounds, significant reduction of amino acids/nucleotides and their derivatives, and the abnormal expression of phytohormones, lipids and other secondary metabolites are the main metabolic characteristics in astringent seeds. [ABSTRACT FROM AUTHOR]

Pollen grains produce certain metabolites, which can improve or inhibit germination and tube growth. Metabolomic analysis of germinating and growing Chinese fir pollen has not been reported. Therefore, this study aimed to analyse metabolites changes, content and expression in the germinating pollen of Chinese fir. To understand the metabolic differences, two clones from Chinese fir were selected. Metabolomics analyses were performed on three stages (1-, 24- and 48-h) during in vitro pollen germination. The metabolites profiles at different time points were analyzed by using liquid chromatography-mass spectrometry. The results showed that 171 peaks were screened; the corresponding differential metabolites of 121 peaks were classified into nine types of substances. The expression of metabolites showed significant differences across and between clones, and the variation was evident at all germination stages. The expression was obvious at the early stage of germination, which differed clearly from that of the late stage after pollen tube growth. Moreover, the metabolites were mainly enriched in 14 metabolic pathways. Pollen germination and tube growth and metabolites expressions changed per incubation time. Since this work is preliminary, we suggest further investigations to understand the relationship between the differential metabolites and pollen development, and factors affecting pollen germination process. [ABSTRACT FROM AUTHOR]

• The difference between Ar and Af leaf samples was first characterized applying transcriptome and metabolome analysis. • Correlation analysis between metabolites and metabolic enzyme gene expression revealed flavonoid metabolic differences between Ar and Af. • This study screened and obtained key genes potentially involved in the differentially accumulation of flavonoids between Ar leaves and Af leaves. • This study provides an in-depth understanding of differentially flavonoid accumulation between Ar leaves and Af leaves. Anoectochilus roxburghii (Wall.) Lindl (Ar) and Anoectochilus formosanus Hayata (Af) are two species of valuable Chinese herbal medicines that belong to the genus Anoectochilus in Orchidaceae and have often been applied as the Chinese herbal medicine "Jin Xian Lian". However, these species exhibit obvious differences in leaf color, and the difference in their medicinal value remains unclear. In this study, the metabolomes of leaf samples of Ar and Af were analyzed by high-performance liquid chromatography-mass spectrometry (LC–MS) in the positive and negative ion modes. The analysis of the leaves of both species in the positive ion mode identified 4013 metabolites enriched in 106 metabolic pathways, and the analysis in the negative ion mode found 1530 metabolites enriched in 98 metabolic pathways. A differential metabolic profiling analysis of the results obtained in positive ion mode found that 816 differentially accumulated metabolites were enriched in 80 metabolic pathways, whereas 297 differentially accumulated metabolites enriched in 55 metabolic pathways were obtained from the analysis in the negative ion mode. The number of flavonoids was higher than those of alkaloids and terpenoids. A quantitative analysis of the differentially accumulated metabolites showed that the most highly upregulated metabolites (Af/Ar) among the top 20 differentially accumulated metabolites in Af were mainly related to amino acid-related metabolic pathways, whereas the most highly downregulated metabolites (Af/Ar) in Af were mainly associated with flavonoid-related metabolic pathways. In addition, the transcriptome sequencing of Ar and Af leaf samples yielded 40.12 Gb of transcriptomic data. The analysis of differentially expressed genes (DEGs) classified 555 metabolism-related DEGs into 23 metabolism-related KEGG pathways, and the top DEGs were involved in the phenylpropanoid biosynthetic pathways, which included up to 138 DEGs associated with the flavonoid metabolism pathway. In addition, significant differences in the flavonoid-related KEGG pathways were found. A comprehensive analysis of the metabolomes and transcriptomes showed that the genes annotated as chalcone synthase (CHS), chalcone isomerase (CHI), flavonol synthase (FLS), caffeoyl-CoA O -methyltransferase (CCOAOMT) and anthocyanidin synthase (ANS) were downregulated in Af leaves compared with Ar leaves, which might account for the downregulation of flavonoid-related metabolites in Af. In summary, both the metabolomic and transcriptomic analyses showed significant differences in flavonoid metabolism between the Ar and Af leaves, and the mechanisms underlying the molecular regulation of these differences were preliminarily analyzed. [ABSTRACT FROM AUTHOR]

• A. roxburghii was first characterized using high-throughput sequencing technologies and metabolomics. • Correlation analysis between metabolites and regulatory genes revealed flavonoid accumulation-related regulatory genes. • This study screened and obtained key genes potentially involved in the accumulation of flavonoids in A. roxburghii. • This study provides an in-depth understanding of the molecular mechanisms underlying flavonoid accumulation in A. roxburghii. Anoectochilus roxburghii , a species used in Chinese herbal medicine, has unique characteristics. This plant has important medicinal and ornamental value and is distributed primarily in China, including Fujian, Zhejiang, Jiangxi, and Guizhou provinces, and in Taiwan. Flavonoids are involved in leaf pigment formation and are major pharmacodynamic substances. However, the molecular mechanisms that regulate accumulation of flavonoids remain unclear, which has significantly limited their application. To elucidate the molecular mechanisms underlying the regulation of flavonoid accumulation in A. roxburghii , root, stem and leaf samples were collected for constructing transcriptomic and metabolomic datasets using RNA sequencing and liquid chromatography-mass spectrometry (LC–MS) techniques. Sequencing of the transcriptomes of the different organs generated 60.2 Gb of data, which were assembled into 186,865 unigenes. Metabolomic analysis resulted in the identification of 10,690 metabolites. Based on the ESI+ mode, 4,010, 4,008 and 4,013 metabolites were annotated in roots, stems, and leaves, respectively; based on the ESI– mode, 1,530, 1,530, and 1,531 metabolites were annotated, respectively. Differential analyses of the transcriptome and flavonoid metabolism of the different organs revealed the greatest significant differences between roots and leaves, followed by differences between stems and leaves; differences between roots and stems were the smallest. According to analysis of differentially expressed genes (DEGs), the secondary metabolism-related DEGs between roots and stems, between roots and leaves, and between leaves and stems were classified into 16, 21, and 19 secondary metabolic Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, respectively. Among these pathways, flavonoid biosynthesis, flavone and flavonol biosynthesis, and isoflavonoid biosynthesis significantly differed in terms of both gene expression and secondary metabolism. Moreover, the key genes involved in flavonoid accumulation were comprehensively analyzed using metabolic and transcriptomic data, and ten transcription factor-encoding genes and fourteen flavonoid biosynthesis genes were identified. Specifically, four of the ten transcription factor-encoding genes appear to activate CHS, CHI, CYP75A and ANR gene expression. The combined approach used in this study provides an in-depth understanding of the molecular regulatory mechanisms underlying flavonoid accumulation in A. roxburghii and is a powerful tool that can uncover valuable information for plant breeders. [ABSTRACT FROM AUTHOR]