Metabolomics technology has enabled an important method for the identification and quality control of Traditional Chinese Medical materials. In this study, we isolated metabolites from cultivated Dendrobium officinale and Dendrobium huoshanense stems of different growth years in the methanol/water phase and identified them using gas chromatography coupled with mass spectrometry (GC-MS). First, a metabolomics technology platform for Dendrobium was constructed. The metabolites in the Dendrobium methanol/water phase were mainly sugars and glycosides, amino acids, organic acids, alcohols. D. officinale and D. huoshanense and their growth years were distinguished by cluster analysis in combination with multivariate statistical analysis, including principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). Eleven metabolites that contributed significantly to this differentiation were subjected to t-tests (P<0.05) to identify biomarkers that discriminate between D. officinale and D. huoshanense, including sucrose, glucose, galactose, succinate, fructose, hexadecanoate, oleanitrile, myo-inositol, and glycerol. Metabolic profiling of the chemical compositions of Dendrobium species revealed that the polysaccharide content of D. huoshanense was higher than that of D. officinale, indicating that the D. huoshanense was of higher quality. Based on the accumulation of Dendrobium metabolites, the optimal harvest time for Dendrobium was in the third year. This initial metabolic profiling platform for Dendrobium provides an important foundation for the further study of secondary metabolites (pharmaceutical active ingredients) and metabolic pathways.

The content and size of stone cell clusters affects the quality of pear fruit, and monolignol polymerization and deposition in the cell walls constitute a required step for stone cell formation. Laccase (LAC) is the key enzyme responsible for the polymerization of monolignols. However, there are no reports on the LAC family in pear (Pyrus bretschneideri), and the identity of the members responsible for lignin synthesis has not been clarified. Here, 41 LACs were identified in the whole genome of pear. All Pyrus bretschneideri LACs (PbLACs) were distributed on 13 chromosomes and divided into four phylogenetic groups (I-IV). In addition, 16 segmental duplication events were found, implying that segmental duplication was a primary reason for the expansion of the PbLAC family. LACs from the genomes of three Rosaceae species (Prunus mummer, Prunus persica, and Fragaria vesca) were also identified, and an interspecies collinearity analysis was performed. The phylogenetic analysis, sequence alignments and spatiotemporal expression pattern analysis suggested that PbLAC1, 5, 6, 29, 36 and 38 were likely associated with lignin synthesis and stone cell formation in fruit. The two target genes of Pyr-miR1890 (a microRNA identified from pear fruit that is associated with lignin and stone cell accumulation), PbLAC1 and PbLAC14, were selected for genetic transformation. Interfamily transfer of PbLAC1 into Arabidopsis resulted in a significant increase (approximately 17%) in the lignin content and thicker cell walls in interfascicular fibre and xylem cells, which demonstrated that PbLAC1 is involved in lignin biosynthesis and cell wall development. However, the lignin content and cell wall thickness were not changed significantly in the PbLAC14-overexpressing transgenic Arabidopsis plants. This study revealed the function of PbLAC1 in lignin synthesis and provides important insights into the characteristics and evolution of the PbLAC family.

Stone cells content and size are the key factors determining the internal quality of the pear fruit. Synthesis of lignin and thickening of secondary cell wall are the keys to the development of stone cells. The polymerization of monolignols and secondary cell wall formation requires the participation of dirigent proteins (DIRs). In recent years, DIR family have been studied in higher plants, but lack of comprehensive study in the pear DIR (PbDIR) family. This study focuses on the identification and analysis of PbDIR family for the first time. We identified 35 PbDIRs from the pear genome, 89% of which are intronless genes. Phylogenetic tree and chromosome localization analysis showed that 35 PbDIRs were divided into four subfamilies (DIR-a, -b/d, -e, and -g) and irregularly distributed among 10 chromosomes. In addition, we identified 29, 26, and 14 DIRs from the other three Rosids (peach, Mei, and grape), respectively. Interspecies microsynteny analysis revealed the collinear gene pairs between pear and peach are the most. Temporal expression analysis showed that the expression changes of seven PbDIRs (DIR-a subfamily: PbDIR4 and PbDIR5; DIR-b/d subfamily: PbDIR11; DIR-g subfamily: PbDIR19; DIR-e subfamily: PbDIR23, 25 and 26) in fruits were consistent with the changes of fruit lignin and stone cells contents. In addition, the subfamily of PbDIRs in fruits showed significant responses after treatment with ABA, SA, and MeJA. According to the protein tertiary structure, key amino acid residues and expression patterns analysis found that PbDIR4 might be involved in the metabolism of lignin and related to stone cells contents in pear fruits. In this study, we systematically analyzed the structure, evolution, function and expression of PbDIR family, which not only confirmed the characteristics of PbDIR family, but also laid the foundation for revealing the role of DIR in pear stone cell development and lignin polymerization.

Fungal laccases play important roles in the degradation of lignocellulose. Although some PoLacs have been reported in several studies, still no comprehensive bioinformatics study of the LAC family in Pleurotus ostreatus has been reported. In this study, we identified 12 laccase genes in the whole genome sequence of P. ostreatus and their physical characteristics, gene distribution, phylogenic relationships, gene structure, conserved motifs, and cis-elements were also analyzed. The expression patterns of 12 PoLac genes at different developmental stages and under different culture substrates were also analyzed. The results revealed that PoLac2 and PoLac12 may be involved in the degradation of lignin and the formation of the fruiting body, respectively. Subsequently, we overexpressed PoLac2 in P. ostreatus by the Agrobacterium tumefaciens-mediated transformation (ATMT) method. The transformants’ laccase activity increased in varying degrees, and the gene expression level of PoLac2 in transformants was 2–8 times higher than that of the wild-type strain. Furthermore, the lignin degradation rate by transgenic fungus over 30 days was 2.36–6.3% higher than that of wild-type. Our data show that overexpression of PoLac2 significantly enhanced the lignin degradation of cotton-straw. To our knowledge, this study is the first report to demonstrate the functions of PoLac2 in P. ostreatus.