A systems biology approach uncovers a gene co-expression network associated with cell wall degradability in maize

Abstract

Understanding the mechanisms triggering variation of cell wall degradability is a prerequisite to improving the energy value of lignocellulosic biomass for animal feed or biorefinery. Here, we implemented a multiscale systems approach to shed light on the genetic basis of cell wall degradability in maize. We demonstrated that allele replacement in two pairs of near-isogenic lines at a region encompassing a major quantitative trait locus (QTL) for cell wall degradability led to phenotypic variation of a similar magnitude and sign to that expected from a QTL analysis of cell wall degradability in the F271 × F288 recombinant inbred line progeny. Using DNA sequences within the QTL interval of both F271 and F288 inbred lines and Illumina RNA sequencing datasets from internodes of the selected near-isogenic lines, we annotated the genes present in the QTL interval and provided evidence that allelic variation at the introgressed QTL region gives rise to coordinated changes in gene expression. The identification of a gene co-expression network associated with cell wall-related trait variation revealed that the favorable F288 alleles exploit biological processes related to oxidation-reduction, regulation of hydrogen peroxide metabolism, protein folding and hormone responses. Nested in modules of co-expressed genes, potential new cell-wall regulators were identified, including two transcription factors of the group VII ethylene response factor family, that could be exploited to fine-tune cell wall degradability. Overall, these findings provide new insights into the regulatory mechanisms by which a major locus influences cell wall degradability, paving the way for its map-based cloning in maize.

Fig 1. Selection and characterization of NILs with an introgressed genomic region at QTL positions for cell wall degradability.

(A) Experimental workflow used for introgression of F288 alleles at an 18–20 Mb region encompassing the QTL6.05 (referred to as the introgressed QTL6.05 region or QTL6.05ⁱ) into the background of F271. SNP: single nucleotide polymorphism. (B) The selected BC2S2 NILs 1 (referred to as 1^F271 and 1^F288) and NILs 2 (referred to as 2^F271 and 2^F288) were grown in the field under well-watered conditions for DNA and mRNA sampling and phenotyping. (C) Comparison of the cell wall degradability IVNDFD performance of NILs introgressed with F288 alleles at QTL6.05ⁱ (blue) and their respective recipient lines with F271 alleles (red). Open circles represent the means of two biological replicates.

Fig 2. Structure of the introgressed genomic QTL6.05ⁱ region.

(A) Large structural variation between B73, F271 and F288 in the introgressed genomic QTL6.05ⁱ region. Dark and light purple boxes represent clusters of QTL in bins 6.05 and 6.07, respectively, as previously defined [22]. The grey dotted line insertion represents a zoom of the genomic region that included the 1.5 Mb-genomic sequences from F271 and F288. Orange and green lines indicate sense and antisense DNA strands, respectively, of at least 910 bp and sharing at least 98% of identity; arrowed boxes represent genes and pseudogenes. (B) Number of annotated genes supported by RNA-seq predicted genes in the targeted QTL6.05 locus. (C) Gene content distribution in B73, F288 and F271 in the targeted QTL6.05 locus.

Fig 3. Transcriptomic adjustments in internodes of NILs introgressed at QTL6.05ⁱ.

(A) Venn diagram of upregulated or downregulated genes in the NILs^F288 relative to the NILs^F271. (B) Hierarchical clustering of DE genes in the NILs^F288 relative to the NILs^F271. The indicated scale is the natural log value of the normalized level of gene expression. (C) GO Biological Process analysis of DE genes in the NILs^F288 relative to the NILs^F271. Only Biological Processes with a Benjamini and Hochberg adjusted P-value below 0.05 are represented. Down_List: analyzed downregulated gene list; Up_List: analyzed upregulated gene list; Ref_List: reference list; ACC biosynthetic process: 1-aminocyclopropane-1-carboxylate biosynthetic process; Cell wall macrom. metabolic process: Cell wall macromolecule metabolic process; JA metabolic process: jasmonic acid metabolic process; Reg. hydrogen peroxide met. Process: Regulation of hydrogen peroxide metabolic process; SA-mediated signaling pathway: salicylic acid-mediated signaling pathway.

Fig 4. Identification of DE genes associated with cell wall-related traits.

(A) Number of common QTL6.05ⁱ DE genes correlating with at least one of the cell wall-related traits. (B) Gene expression levels of the 79 correlating DE genes in NILs introgressed into QTL6.05ⁱ. Columns represent gene expression profiles, and rows represent cell wall-related traits as follows: cell wall content (black), degradability (orange), sugar content (pink), lignin content (green) and p-hydroxycinnamic acids content (purple). IVNDFD: in vitro neutral detergent fiber digestibility; Hcell/NDF: hemicellulose content in % NDF; IVDMD: in vitro dry matter digestibility; EsterFA: esterified ferulic acid content in mg g^-1 NDF; LK/NDF: lignin klason in % NDF; NDF: neutral detergent fiber; ADL/NDF: acid detergent lignin in % NDF; Cell/NDF: cellulose content in % NDF; pCA: p-coumaric acid content mg g^-1 NDF; EtherFA: etherified ferulic acid content in mg g^-1 NDF.

Fig 5. Co-expression network of common QTL6.05ⁱ DE genes.

Downregulated genes in the NILs^F288 relative to the NILs^F271 are represented by diamonds, and upregulated genes by circles. Colored diamonds indicate genes with expression levels negatively correlating with cell wall degradability (IVNDFD) and/or hemicellulose content (Hcell/NDF), and/or positively correlating with cellulose content (Cell/NDF). Colored circles indicate genes with expression levels positively correlating with IVNDFD and/or Hcell/NDF, and/or negatively correlating with Cell/NDF. Node colors represent correlation with IVNDFD (yellow), IVNDFD and Hcell/NDF (blue); IVNDFD, Hcell/NDF and Cell/NDF (red); Hcell/NDF and Cell/NDF (orange). The “i” present in the nodes indicates DE genes located in the QTL6.05ⁱ region; numbered and larger nodes indicate genes located in the QTL6.05 locus (gene 03: ZmP5CS1; gene 06: ZmAKR4C9; gene 10: ZmFKB20-1; gene 14a: ZmESMD1; gene 36: ZmRPS23). The letters “A”, “D”, “E”, “H”, “N” and “U” present in the nodes indicate genes encoding ACC synthase, DREB TF, ERF-VII TF, HSP, NAC TF and universal stress protein, respectively. Blue stars indicate DE genes previously identified [38]; yellow stars indicate DE genes with SPE patterns co-expressed with ZmRPS23 (gene 36). Thick borders indicate gene with the GCCGCC motif in their promoter. Edges colored blue connect co-expressed genes with a PCC less than -0.995, red edges represent a PCC bigger than 0.995. Modules as determined by NetworkAnalyzer are designated M1-M7.