Transcriptome analysis of genes involved in secondary cell wall biosynthesis in developing internodes of Miscanthus lutarioriparius

Abstract

Miscanthus is a promising lignocellulosic bioenergy crop for bioethanol production. To identify candidate genes and regulation networks involved in secondary cell wall (SCW) development in Miscanthus, we performed de novo transcriptome analysis of a developing internode. According to the histological and in-situ histochemical analysis, an elongating internode of M. lutarioriparius can be divided into three distinct segments, the upper internode (UI), middle internode (MI) and basal internode (BI), each representing a different stage of SCW development. The transcriptome analysis generated approximately 300 million clean reads, which were de novo assembled into 79,705 unigenes. Nearly 65% of unigenes was annotated in seven public databases. Comparative profiling among the UI, MI and BI revealed four distinct clusters. Moreover, detailed expression profiling was analyzed for gene families and transcription factors (TFs) involved in SCW biosynthesis, assembly and modification. Based on the co-expression patterns, putative regulatory networks between TFs and SCW-associated genes were constructed. The work provided the first transcriptome analysis of SCW development in M. lutarioriparius. The results obtained provide novel insights into the biosynthesis and regulation of SCW in Miscanthus. In addition, the genes identified represent good candidates for further functional studies to unravel their roles in SCW biosynthesis and modification.

Figure 1

Anatomy of an elongating internode of M. lutariorioparius. Sections from three segments of the 2^nd internode from top were stained with Toluidine Blue O and observed under bright field. (A) upper internode (UI), (B) middle internode (MI), (C) basal internode (BI), (D,E and F) represent the magnified view of a vascular bundle in A, B, and C, respectively. Arrow head indicates the thickened fiber cells. Cc, companion cells, Pa, parenchyma, Sf, sclerenchyma fiber, St, sieve tube, V, vessel. Scale bar 100 μm in (A–C), and 50 μm in (D–F).

Figure 2

Lignification in an elongating internode of M. lutariorioparius. Lignification in three segments of an elongating internode (2^nd from top) was shown by Phloroglucinol-HCl staining (red color). (A) upper internode (UI), (B) middle internode (MI), (C) basal internode (BI), (D,E and F) are enlarged view of a specified vascular bundle in (A,B and C) respectively. Bar = 100 μm.

Figure 3

Immunolabeling of crystalline cellulose in an elongating M. lutarioriparius internode. The crystalline cellulose epitope in three sections of M. lutarioriparius internode was immunolabeled with CBM3a. The sections were counterstained with Calcofluor. (A) upper internode (UI), (B) middle internode (MI), (C) basal internode (BI). Bar = 100 μm.

Figure 4

Transcript profiling of genes across three internode segments. (A) hierarchical clustering of all differentially expressed genes (DEGs) based on Z-score normalized FPKM values in three internode segments. Blue indicates lower expression, and red indicates higher expression. (B) Venn diagram showing the up-regulated or down-regulated DEGs among three internode segments. (C) expression patterns of four significantly clustered profiles. UI, upper internode, MI, middle internode, BI, basal internode.

Figure 5

Enrichment analysis of KEGG pathways for differentially expressed genes. Significantly enriched KEGG pathways were plotted for DEGs among the three segments of internode. UI, upper internode, MI, middle internode, BI, basal internode.

Figure 6

A schematic overview of changes in transcript abundance in the middle internode compared to the upper internode. The log2 transformed FPKM values were imported into MapMan software to generate the overview of general metabolism in the middle internode (MI) compared to the upper internode (UI). DEGs are represented by colored squares and grouped according to functional annotation based on MapMan ontology. The fold change of DEGs is indicated by the scale bar. Red indicates up-regulation, whilst blue indicates down-regulation.

Figure 7

Expression profiles of cell wall biosynthesis genes and transcription factors during internode development. The heatmap was generated with Z-score normalized FPKM values. Up-regulated genes are shown in red, whilst down-regulated genes are in blue. (A) expression profiles of cellulose synthase genes. (B) expression patterns of cellulose synthase-like genes. (C–F) expression analysis of genes encoding cell wall structural proteins. (G) expression profiles of monolignol biosynthesis genes. (H) expression patterns of transcription factor genes. UI, upper internode, MI, middle internode, BI, basal internode.

Figure 8

Co-expression network of transcription factors with secondary cell wall biosynthesis genes. Regulatory networks showing transcription factors co-expressed with 6 cellulose synthase genes (A), 14 hemicellulose biosynthesis genes (B), 13 genes encoding cell wall structural proteins (C), and 18monolignol biosynthesis genes (D). Transcription factor genes are in blue color and cell wall component biosynthesis genes are in green color.

Figure 9

Validation of gene expression by quantitative real time RT-PCR. The expression of the UI was set as 1.0, and the relative expression level was calculated. UI, upper internode, MI, middle internode, BI, basal internode.