RNA Sequencing Reveals Phenylpropanoid Biosynthesis Genes and Transcription Factors for Hevea brasiliensis Reaction Wood Formation

Abstract

Given the importance of wood in many industrial applications, much research has focused on wood formation, especially lignin biosynthesis. However, the mechanisms governing the regulation of lignin biosynthesis in the rubber tree (Hevea brasiliensis) remain to be elucidated. Here, we gained insight into the mechanisms of rubber tree lignin biosynthesis using reaction wood (wood with abnormal tissue structure induced by gravity or artificial mechanical treatment) as an experimental model. We performed transcriptome analysis of rubber tree mature xylem from tension wood (TW), opposite wood (OW), and normal wood (NW) using RNA sequencing (RNA-seq). A total of 214, 1,280, and 32 differentially expressed genes (DEGs) were identified in TW vs. NW, OW vs. NW, and TW vs. OW, respectively. GO and KEGG enrichment analysis of DEGs from different comparison groups showed that zeatin biosynthesis, plant hormone signal transduction, phenylpropanoid biosynthesis, and plant-pathogen interaction pathways may play important roles in reaction wood formation. Sixteen transcripts involved in phenylpropanoid biosynthesis and 129 transcripts encoding transcription factors (TFs) were used to construct a TF-gene regulatory network for rubber tree lignin biosynthesis. Among them, MYB, C2H2, and NAC TFs could regulate all the DEGs involved in phenylpropanoid biosynthesis. Overall, this study identified candidate genes and TFs likely involved in phenylpropanoid biosynthesis and provides novel insights into the mechanisms regulating rubber tree lignin biosynthesis.

Keywords: lignin biosynthesis; phenylpropanoid biosynthesis pathway; reaction wood; rubber tree; rubber wood.

FIGURE 1

Global analysis of gene expression in different xylem tissues of rubber tree reaction wood. Volcano plots illustrate differentially expressed genes identified in TW vs. NW (A), OW vs. NW (B), and TW vs. OW (C) as individual dots. Red represents up-regulation and green represents down-regulation. (D) Venn diagram showing the total number of differentially expressed genes identified in TW vs. NW, OW vs. NW, and TW vs. OW, and overlap among these comparison groups.

FIGURE 2

GO and KEGG enrichment analysis of differentially expressed genes (DEGs) from the RNA-seq dataset. GO analysis of DEGs identified in TW vs. NW (A), OW vs. NW (C), and TW vs. OW (E). KEGG analysis of DEGs identified in TW vs NW (B), OW vs NW (D), and TW vs OW (F), respectively.

FIGURE 3

Differentially expressed genes (DEGs) and transcription factors in OW vs. NW. Heatmap showing the expression of the DEGs (A) and transcription factors (B) identified in OW vs. NW. Columns represent three different samples per tissue type and rows represent different transcripts. Each square represents a transcript and the color indicates the level of expression; red represents up-regulation and blue represents down-regulation.

FIGURE 4

Expression of genes in phenylpropanoid biosynthesis pathway in OW vs. NW. Red represents up-regulation and blue represents down-regulation. PAL, Phenylalanine ammonia-lyase; C4H, Cinnamate 4-hydroxylase; 4CL, 4-coumarate-CoA ligase; F5H, Ferulate 5-hydroxylase; CCoAOMT, Caffeoyl-CoA O-methyltransferase; CCR, Cinnamoyl-CoA reductase; CAD, Cinnamyl alcohol dehydrogenase; COMT, Caffeate O-methyltransferase; HCT, hydroxycinnamoyltransferase; POD, peroxidase.

FIGURE 5

Transcription factor-gene network in rubber tree lignin biosynthesis. Red circles represent genes and green triangles represent transcription factors.

FIGURE 6

Relative expression of five genes in TW vs. NW and OW vs. NW. Expression levels were determined by RT-qPCR using the 40S gene (A and C) or the Ubiquitin gene (B and D) as the internal control for normalization. Error bars indicate standard deviation of three biological replicates.