Transcriptome Dynamics during Maize Endosperm Development

<div><p>The endosperm is a major organ of the seed that plays vital roles in determining seed weight and quality. However, genome-wide transcriptome patterns throughout maize endosperm development have not been comprehensively investigated to date. Accordingly, we performed a high-throughput RNA sequencing (RNA-seq) analysis of the maize endosperm transcriptome at 5, 10, 15 and 20 days after pollination (DAP). We found that more than 11,000 protein-coding genes underwent alternative splicing (AS) events during the four developmental stages studied. These genes were mainly involved in intracellular protein transport, signal transmission, cellular carbohydrate metabolism, cellular lipid metabolism, lipid biosynthesis, protein modification, histone modification, cellular amino acid metabolism, and DNA repair. Additionally, 7,633 genes, including 473 transcription factors (TFs), were differentially expressed among the four developmental stages. The differentially expressed TFs were from 50 families, including the bZIP, WRKY, GeBP and ARF families. Further analysis of the stage-specific TFs showed that binding, nucleus and ligand-dependent nuclear receptor activities might be important at 5 DAP, that immune responses, signalling, binding and lumen development are involved at 10 DAP, that protein metabolic processes and the cytoplasm might be important at 15 DAP, and that the responses to various stimuli are different at 20 DAP compared with the other developmental stages. This RNA-seq analysis provides novel, comprehensive insights into the transcriptome dynamics during early endosperm development in maize.</p></div

Expression pattern analysis of 13 TF and 2 zein genes by qRT-PCR and RNA-seq.

<p>The <i>y-</i> axis shows the mRNA levels. The scale on the right indicates gene expression level based on RNA-Seq. The scale on the left shows relative gene expression levels based on qRT-PCR. The <i>x</i>-axis indicates the day of endosperm sampling after pollination. The letters correspond to the genes. The blue lines correspond with RNA-seq; and the gold lines correspond with qRT-PCR.</p

Distributions of the numbers of DEGs and DETFs.

<p>(A) Identification of the dynamic distribution of DEGs (fold change (FC) ≥ 2 (for up-regulation) or FC ≤ 0.5 (for down-regulation)) in developing endosperm via comparison of two stages; the red colour represents up-regulation, and green represents down-regulation. The scale bar depicts the percentages of DEGs in both directions (up and down). (B) A semi-matrix showing the dynamic changes in the DETFs, as determined by pair-wise comparisons of their expression at different developmental stages.</p

Discovery of a single AS event or multiple AS events in maize endosperm genes.

<p>(A) The heat map shows the dynamic changes in 5,905 genes with a single AS event during endosperm development, and the scale bar indicates whether an event occurred (red: event occurrence; yellow: no event occurrence). (B) An example of a single AS event occurring in a newly identified transcribed region (chromosome 7: 129960922–129965733); this developmentally regulated splicing event was only observed at 15 DAP. (C) Dynamic variation in multiple AS events in 5,116 genes is indicated at several developmental stages (the scale bar is defined in A). (D) An example of multiple AS events in <i>GRMZM2G051866</i>, indicating the diversity of newly identified transcribed regions (chromosome 10: 130393296–130404103) and the various developmentally regulated splicing events that occur at this locus during different developmental stages.</p

Statistical results for sequenced and mapped reads to the maize B73 genome.

<p>Statistical results for sequenced and mapped reads to the maize B73 genome.</p

Expression patterns of DEGs in developing maize endosperm.

<p>Fifteen expression patterns were characterized by the fluctuating expression of gene sets at 5, 10, 15 and 20 DAP. The DEG sets showed different tends of transition from down- to up-regulated states or from up- to down-regulated states over the developmental stages (TL-14 and 15). The up- and down-regulated gene sets are staggered or are depicted consecutively over the developmental stages of maize endosperm. The scaled expression levels of the DEGs are provided on the <i>y-</i>axis, the developmental stages are shown on the <i>x</i>-axis, the coloured lines represent the individual gene expression clusters, and the trend in expression of each gene set is depicted as a black line. ‘n’ represents the number of DEGs.</p

Differentially expressed genes among four endosperm developmental stages in maize.

<p>Differentially expressed genes among four endosperm developmental stages in maize.</p

Functional category enrichment analysis of genes in fifteen major clusters.

<p>Functional category enrichment analysis of genes in fifteen major clusters.</p

Enrichment and expression analysis of DETFs during maize endosperm development.

<p>(A) Pair-wise comparisons identified the dynamic distribution of the numbers of DETFs from 50 TF families. The scale bar represents the number of DETFs. (B) Heat map of DETFs identified at the time point of their peak expression based on normalised FPKM values. Twelve modules are classified according to normalised FPKM values of more than 0.5. The scale bar shows the normalised FPKM values.</p

Analysis of global gene expression based on FPKM values among different endosperm developmental stages.

<p>(A) Distribution of the transcripts at five expression levels based on the FPKM values are shown, and the expression values from 10 to 100 correspond with the most highly represented transcripts at the four developmental stages. The vertical axis represents the number of transcripts; and ‘N’ is the percentage of transcripts expressed at the corresponding level. (B) A total of 25,186 genes (FPKM value ≥ 1) were expressed during the four stages. Genes with shared and specific expression were detected during these stages.</p