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.

Reference Genes for qPCR Analysis in Resin-Tapped Adult Slash Pine As a Tool to Address the Molecular Basis of Commercial Resinosis

“…Increased monoterpene synthase gene expression was found in Picea sitchensis with a peak of expression between 7 and 16 days post-wounding (Byun-McKay et al, 2006 ). Transcriptomic studies detected that high level of expression of a monoterpene synthase was associated with increased resin production in 27 year-old bark streaked trees of Pinus massoniana not treated with stimulant paste (Liu et al, 2015 ). Although these various patterns of gene expression and terpene production are not from the same species and experimental conditions, they may reflect a highly conserved mechanism of response to damage in conifers.…”

Transcriptome and gene expression analysis revealed mechanisms for producing high oleoresin yields from Simao pine (Pinus kesiya var. langbianensis)

“…Many metabolic pathways, physiological processes, and growth and development mechanisms of non-model plant have been revealed by de novo transcriptome sequencing (Wu et al, 2014;Lang et al, 2015). The analysis of differentially expressed genes based on transcriptomic data from several samples is a powerful tool for revealing the biological mechanisms underlying differences among individuals (Lou et al, 2014;Liu et al, 2015;Shi et al, 2015). In particular, differential gene expression analyses showed that GGPS and (-)-alpha/ beta-pinene synthase were up-regulated in the high oleoresin-yielding genotype of Pinus massoniana .…”

“…Oleoresin including turpentine (monoterpenes and sesquiterpenes) and rosin (diterpenes) is mainly synthesized by MEP pathway. The level of gene expression of DXS and HDR in high and low Liu et al, (2015). Red genes are up-regulated that gene expression of high oleoresin yield genotype is larger than low oleoresin yield genotype.…”

“…The isopentenyl diphosphate from the MEP pathway was transformed into different terpenes by a series of post-modification enzymes, such as isopentenyl diphosphate isomerase (IDI), geranylgeranyl diphosphate synthase (GGPPS), geranyl diphosphate synthase (GPPS), terpenoid synthase (TPS), and cytochrome P450-dependent monooxygenase. Although several papers have reported the roles of DXS, DXR, HDR, and TPS in oleoresin biosynthesis (Phillips et al, 2003;Byun-Mckay et al, 2006;Kim et al, 2009;Liu et al, 2015), there have been no reports about the influence of the MEP pathway and expression the MEP enzyme on high and low oleoresin yields in Simao pine. In this study, the transcriptomes of high and low oleoresin-yielding individuals of Simao pine were analyzed, and the genes involved in the MEP pathway were identified and their functions were characterized.…”

Identification, expression, and phylogenetic analyses of terpenoid biosynthesis-related genes in secondary xylem of loblolly pine (Pinus taeda L.) based on transcriptome analyses

“…The geranyl diphosphate synthase (GPS), geranylgeranyl diphosphate synthase (GGPS) and farnesyl diphosphate synthase (FPS) are the precursors to monoterpenes, diterpenes and sesquiterpenes of conifer oleoresin, respectively (Zulak & Bohlmanu, 2010). Furthermore, previous studies have confirmed that ABC transporters (Jasinski et al, 2001), several cytochrome P450 family proteins (Banerjee & Hamberger, 2018), aldehyde/alcohol dehydrogenases (ALDH/ADH) (Teoh, Polichuk & Reed, 2009; Polichuk et al, 2010), pathogenesis-related proteins (Dafoe et al, 2009), non-specific lipid-transfer proteins (Liu et al, 2015), and ethylene responsive transcriptionfactors ( ERFs ) (Xu et al, 2007) are also crucial for terpenoid biosynthesis.…”