Temporal patterns of gene expression in developing maize endosperm identified through transcriptome sequencing

Abstract

Endosperm is a filial structure resulting from a second fertilization event in angiosperms. As an absorptive storage organ, endosperm plays an essential role in support of embryo development and seedling germination. The accumulation of carbohydrate and protein storage products in cereal endosperm provides humanity with a major portion of its food, feed, and renewable resources. Little is known regarding the regulatory gene networks controlling endosperm proliferation and differentiation. As a first step toward understanding these networks, we profiled all mRNAs in the maize kernel and endosperm at eight successive stages during the first 12 d after pollination. Analysis of these gene sets identified temporal programs of gene expression, including hundreds of transcription-factor genes. We found a close correlation of the sequentially expressed gene sets with distinct cellular and metabolic programs in distinct compartments of the developing endosperm. The results constitute a preliminary atlas of spatiotemporal patterns of endosperm gene expression in support of future efforts for understanding the underlying mechanisms that control seed yield and quality.

Keywords: mRNA localization; time series.

Fig. 1.

Numbers of genes expressed during early maize kernel and endosperm development. (A) Frequency of the genes from the WGS and the FGS based on mRNA levels. (B) Numbers of expressed WGS genes, FGS genes, pseudogenes, and TE-related genes in the eight developmental stages. (C) Venn diagram of the numbers of expressed genes in unpollinated kernels, early developing kernels (2–6 DAP), and endosperm stages (8–12 DAP).

Fig. 2.

Identification of up- and down-regulated gene sets and the coexpressed TFs. (A) Numbers of mRNAs for all genes and the coexpressed TFs showing transitions from low to high (one-step-up) or high to low (one-step-down) in two consecutive developmental stages and two-step-up-down (up-down) or two-step-down-up (down-up) in our series of developmental stages. (B) Identification of the one-step-up (Left) and one-step-down (Right) transitions in mRNA levels for all genes and the coexpressed TFs during early kernel development. Seven expression patterns were identified to represent genes up- or down-regulated at 2, 3, 4, 6, 8, 10, and 12 DAP (Top). Gene expression profiles of individual genes are depicted in gray lines, and average expression profiles for each pattern are depicted in pink (one-step-up) or blue (one-step-down) lines. The total number of all genes (Left) and the coexpressed transcription-factor genes (Right) are indicated in parentheses for each expression pattern. End., endosperm; Ker., kernel.

Fig. 3.

Quantitive RT-PCR validation of TFs preferentially expressed in endosperm. (A) The mRNA levels of three TFs based on RNA-Seq (Left) and qRT-PCR (Right) data. Kernel (−E) denotes the kernel at 6 DAP with the endosperm removed. (B) Heatmap of qRT-PCR data for 13 TF genes (including 11 randomly chosen showing an up-at-6/8-DAP pattern and for two known endosperm-expressed TF genes, MRP-1 and Opaque-2) showing preferential expression in the endosperm clustered based on the Ct values of qRT-PCR of three bio-reps of 0, 2, 3, 4, and 6 DAP kernels; 4, 6, 8, 10, and 12 DAP hand-dissected endosperm; and 6-DAP kernel with the endosperm removed (−E, denoted by 6*).

Fig. 4.

Cell-specific expression of endosperm genes as determined by in situ hybridization. (A) Expression in the AL only. Section of an 8-DAP kernel hybridized with a probe for GRMZM2G091054 (AL9). (B) Expression in the BETL only. Section of a 6-DAP kernel hybridized with a probe for AC199820.4_FG006 (ZmMYBR29). (C) Expression in the BIZ only. Section of an 8-DAP kernel hybridized with a probe for GRMZM2G406170 (BURP domain-containing). (D) Expression in the BETL and BIZ. Section of an 8-DAP kernel hybridized with a probe for GRMZM2G088896 (uncharacterized). (E) Close-up of the image in A showing expression localized to the AL. (F) Close-up of the image in B showing expression localized to the BETL. (G) Close-up of the image in C showing expression localized to the BIZ. (H) Close-up of the image in D showing expression localized in the BETL and BIZ. (I) Expression in the CZ only. Section of a 7-DAP kernel hybridized with a probe for GRMZM2G006585 (ZmPLATZ12). (J) Expression in the ESR only. Section of a 7-DAP kernel hybridized with a probe for GRMZM2G120008 (uncharacterized). (K) Expression in the ESR, BETL, and BIZ. Section of a 7-DAP kernel hybridized with a probe for GRMZM2G128832 (uncharacterized). (L) Expression in the ESR, BETL, BIZ, and CZ. Section of a 7-DAP kernel hybridized with a probe for GRMZM2G046532 (uncharacterized). (M) Expression in the SE and CZ. Section of a 10-DAP kernel hybridized with a probe for GRMZM2G127160 (tryptophan aminotransferase). (N) Expression in the SE only. Section of a 10-DAP kernel hybridized with a probe for GRMZM2G023872 (ZmGRAS20). (O) Close-up of the image in N. (P) Negative control. Section of a 6-DAP kernel hybridized with a probe for GRMZM2G453555 (transposable element), which does not produce a signal. (Scale bars: 2 mm for A, B, C, D, G, I, J, K, L, M, N, and P; 500 µm for E, F, H, O). al, aleurone; betl, basal endosperm transfer layer; biz, basal intermediate zone; cz, conducting zone; emb, embryo; esr, embryo surrounding region; se, starchy endosperm.

Fig. 5.

Correlation of the temporal and spatial patterns of individual genes expressed during early kernel and endosperm development. Spatial patterns of mRNA accumulation were identified based on in situ hybridizations in this study (graph on Left) compared with the temporal accumulation of the same mRNAs based on our RNA-Seq data (heat map on Right; 0–6 DAP from kernel and 8–12 DAP from hand-dissected endosperm). Individual gene IDs are shown on the left.