Genome-Wide Identification of Differentially Expressed Genes Associated with the High Yielding of Oleoresin in Secondary Xylem of Masson Pine (Pinus massoniana Lamb) by Transcriptomic Analysis

Abstract

Masson pine is an important timber and resource for oleoresin in South China. Increasing yield of oleoresin in stems can raise economic benefits and enhance the resistance to bark beetles. However, the genetic mechanisms for regulating the yield of oleoresin were still unknown. Here, high-throughput sequencing technology was used to investigate the transcriptome and compare the gene expression profiles of high and low oleoresin-yielding genotypes. A total of 40,690,540 reads were obtained and assembled into 137,499 transcripts from the secondary xylem tissues. We identified 84,842 candidate unigenes based on sequence annotation using various databases and 96 unigenes were candidates for terpenoid backbone biosynthesis in pine. By comparing the expression profiles of high and low oleoresin-yielding genotypes, 649 differentially expressed genes (DEGs) were identified. GO enrichment analysis of DEGs revealed that multiple pathways were related to high yield of oleoresin. Nine candidate genes were validated by QPCR analysis. Among them, the candidate genes encoding geranylgeranyl diphosphate synthase (GGPS) and (-)-alpha/beta-pinene synthase were up-regulated in the high oleoresin-yielding genotype, while tricyclene synthase revealed lower expression level, which was in good agreement with the GC/MS result. In addition, DEG encoding ABC transporters, pathogenesis-related proteins (PR5 and PR9), phosphomethylpyrimidine synthase, non-specific lipid-transfer protein-like protein and ethylene responsive transcription factors (ERFs) were also confirmed to be critical for the biosynthesis of oleoresin. The next-generation sequencing strategy used in this study has proven to be a powerful means for analyzing transcriptome variation related to the yield of oleoresin in masson pine. The candidate genes encoding GGPS, (-)-alpha/beta-pinene, tricyclene synthase, ABC transporters, non-specific lipid-transfer protein-like protein, phosphomethylpyrimidine synthase, ERFs and pathogen responses may play important roles in regulating the yield of oleoresin. These DEGs are worthy of special attention in future studies.

Fig 1. GO enrichment analysis of DEGs between high and low oleoresin-yielding masson pines using REVIGO.

(A) The interactive graph of the enriched GO terms for DEGs. Bubble color indicates the p-value; bubble size indicates the frequency of the GO term. Highly similar GO terms are linked by edges in the graph, where the line width indicates the degree of similarity. (B) The scatter plot showing the significance of the GO term for DEGs (terms remaining after reducing redundancy) in a two-dimensional space derived by applying multi-dimensional scaling to a matrix of GO term semantic similarities. Bubble color indicates the p-value for the false discovery rates. The circle size represents the frequency of the GO term (more general terms are represented by larger size bubbles).

Fig 2. Biosynthetic pathway of terpenoids in P. massoniana (adapted from Zulak et al [22]).

The number of unigenes homologous to gene families encoding these enzymes was provided in parentheses. The enzymes highlighted with red fonts showed that DEGs were detected in gene families encoding these enzymes. DXS: 1-deoxy-dxylulose-5-phosphate synthase; DXR: 1-deoxy-D-xylulose-5-phosphate reductoisomerase; MCT: 4-diphosphocytidyl-2Cmethyl-D-erythritol synthase; CMK: 4-diphosphocytidyl-2C–methyl-D-erythritol kinase; MECPS: 2C-methyl-D-erythritol 4-phosphate cytidylyltransferase; HDS: 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase; HDR: 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase; AACT: acetoacetyl-CoA thiolase; HMGS: 3-hydroxy-3-methylglutaryl-CoA synthase; HMGR: 3-hydroxy-3-methylglutaryl-CoA reductase; MVK: mevalonate kinase; PMVK: phosphomevalonate kinase; MVD: mevalonate diphosphate decarboxylase; IPPI: isopentenyl-diphosphate isomerase; FPPS: farnesyl diphosphate synthase; GPPS: geranyl diphosphate synthase; SesquiTPS: Sesquiterpene synthase; MonoTPS: Monoterpene synthase; DiTPS: Diterpene synthase; CYP720B: Abietadienol/abietadienal oxidase PtAO.

Fig 3. Clustering and heatmap of key DEG related to the yield of oleoresin based on their expression levels.

Three biological replicates were used for high-yielding genotype (H1, H2, H3) and low-yielding genotype (L1, L2, L3). Colored bars on the left of the heatmap mark the major distinct branches in the clustering tree grouping genes with similar expression pattern.

Fig 4. Quantitative RT-PCR validation of tag-mapped candidate genes associated with the yield of oleoresin.

Relative expression levels of qRT-PCR calculated using Elongation factor 1-alpha as the internal control were shown in the right y-axis. Relative expression levels of DGE were shown in the left y-axis.