Genome-Wide Analysis of the Lysine Biosynthesis Pathway Network during Maize Seed Development

Abstract

Lysine is one of the most limiting essential amino acids for humans and livestock. The nutritional value of maize (Zea mays L.) is reduced by its poor lysine content. To better understand the lysine biosynthesis pathway in maize seed, we conducted a genome-wide analysis of the genes involved in lysine biosynthesis. We identified lysine biosynthesis pathway genes (LBPGs) and investigated whether a diaminopimelate pathway variant exists in maize. We analyzed two genes encoding the key enzyme dihydrodipicolinate synthase, and determined that they contribute differently to lysine synthesis during maize seed development. A coexpression network of LBPGs was constructed using RNA-sequencing data from 21 developmental stages of B73 maize seed. We found a large set of genes encoding ribosomal proteins, elongation factors and zein proteins that were coexpressed with LBPGs. The coexpressed genes were enriched in cellular metabolism terms and protein related terms. A phylogenetic analysis of the LBPGs from different plant species revealed different relationships. Additionally, six transcription factor (TF) families containing 13 TFs were identified as the Hub TFs of the LBPGs modules. Several expression quantitative trait loci of LBPGs were also identified. Our results should help to elucidate the lysine biosynthesis pathway network in maize seed.

Fig 1. Phylogenetic trees of lysine biosynthesis pathway gene orthologs.

Phylogenetic trees based on AK (aspartate kinase), ASD (aspartate-semialdehyde dehydrogenase), DHDPS (dihydrodipicolinate synthase), DapB (dihydrodipicolinate reductase), LL-DAP-AT (LL-diaminopimelate aminotransferase), DapF (diaminopimelate epimerase), and LysA (diaminopimelate decarboxylase) are shown. The AK tree contains two enzymes: the monofunctional enzyme AK (EC: 2.7.2.4; red branch) and the bifunctional enzyme HSDH (EC: 1.1.1.3; black branch)

Fig 2. Expression pattern of LBPGs coexpressed genes.

The module eigengenes are used to represent the genes expression pattern within each module. Each bar represents one sample (days after pollination) and the color represents the module color, the LBPGs was list below the module name. The value of the module eigengenes for each sample is displayed on the y axis.

Fig 3. The motifs enriched in the LBPGs modules.

The motifs enriched in 1-kb upstream sequences of the transcription start site of the LBPGs coexpressed module genes. (P-value was calculated by Ranksum test).

Fig 4. Expression patterns of dihydrodipicolinate synthase (DHDPS) genes.

(A) Expression levels reads per kilobase per million reads (RPKM) of DHDPS genes in 21 maize developmental stages. (B) Quantitative real-time PCR (qRT-PCR) validation of RNA-Seq-based DHDPS expression levels. qRT-PCR expression profiles (blue bars) of DHDPS genes closely match the RNA-Seq data (red lines). The correlation value (cor) was calculated using Pearson’s correlation coefficient. The error bars in the figure represent the standard deviation. (C) Heat map of expression profiles of DHDPS-coexpressed genes (Y axes). Numbers next to the heat map represent the number of days after pollination. (The error bars represent standard deviation).

Fig 5. Gene Ontology (GO) functional enrichment analysis of DHDPS coexpressed genes.

All represent all genes set (RefGen_v2 5b). DHDPS1 and DHDPS2 represent DHDPS1 and DHDPS2 coexpressed genes. GO categories among cell component, molecular function and biological process that show significant (P < 0.05) enrichment than all genes set.

Fig 6. Comparison of lysine content.

(A) Comparison of lysine biosynthesis pathway genes coexpressed genes (LBPGs) with the same number of randomly selected all genes set (Test) and with all genes set (All). (B) Comparison of DHDPS1-coexpressed genes (DHDPS1) and DHDPS2-coexpressed genes (DHDPS2) with all genes set (All). (** P < 0.01)