Chromatin modification contributes to the expression divergence of three TaGS2 homoeologs in hexaploid wheat

Abstract

Plastic glutamine synthetase (GS2) is responsible for ammonium assimilation. The reason that TaGS2 homoeologs in hexaploid wheat experience different selection pressures in the breeding process remains unclear. TaGS2 were minimally expressed in roots but predominantly expressed in leaves, and TaGS2-B had higher expression than TaGS2-A and TaGS2-D. ChIP assays revealed that the activation of TaGS2-B expression in leaves was correlated with increased H3K4 trimethylation. The transcriptional silencing of TaGS2 in roots was correlated with greater cytosine methylation and less H3K4 trimethylation. Micrococcal nuclease and DNase I accessibility experiments indicated that the promoter region was more resistant to digestion in roots than leaves, which indicated that the closed nucleosome conformation of the promoter region was important to the transcription initiation for the spatial-temporal expression of TaGS2. In contrast, the transcribed regions possess different nuclease accessibilities of three TaGS2 homoeologs in the same tissue, suggesting that nucleosome conformation of the transcribed region was part of the fine adjustment of TaGS2 homoeologs. This study provides evidence that histone modification, DNA methylation and nuclease accessibility coordinated the control of the transcription of TaGS2 homoeologs. Our results provided important evidence that TaGS2-B experienced the strongest selection pressures during the breeding process.

Figure 1. TaGS2 function is essential for a normal yield in wheat.

(A) Phenotype of TaGS2 gene deletion mutants. (B) Identification of TaGS2 gene deletion mutants. (C) Plant height. (D) Spike number. Error bars indicate ± SE (n = 10). Asterisks indicate the significance of differences between wild-type and gs2 mutants as determined by the Student’s t-test: **P < 0.01, *P < 0.05.

Figure 2. The expression patterns of the TaGS2 homoeologs.

(A) Divergent expression of the TaGS2 homoeologs in the leaves of 7-d-old seedlings of the progenitor species. (B) Percentage of TaGS2-A and TaGS2-B occurring in the leaves of 7-d-old seedlings of tetraploid wheat. (C) Tissue-specific expression of TaGS2 at the booting stage. (D) Divergent expression of the three TaGS2 homoeologs in the leaves of 10-d-old seedlings. (E) The expression of TaGS2 at two developmental stages. For (A,D and E), the data are means ± SD of three independent biological replicates. Asterisks indicate a statistically significant difference (Student’s t-test: **P < 0.01).

Figure 3. Chromatin immunoprecipitation (ChIP) analysis of the three TaGS2 homoeologs.

(A) A diagram showing the three homoeologous TaGS2 genomic regions with ATG indicating the transcription start site. The black lines denote introns or intergenic regions, and the black rectangles denote coding regions. The regions analyzed for H3K4me3 enrichment in this study are marked by black bars below the diagram. (B) Leaves of 10-d-old seedlings. (C) Booting-stage leaves. (D) Roots of 10-d-old seedlings. (B) to (D) ChIP to examine the enrichment of H3K4me3 in WT. The data are means ± SD of three independent biological replicates.

Figure 4. Distribution of methylated cytosine in the 5′ region and exon 1 of the three TaGS2 homoeologs.

(A) to (C) Leaves of 10-d-old seedlings. (D) to (F) Booting-stage leaves. (G) to (I) Roots of 10-d-old seedlings. (A,D) and (G) TaGS2-A. (B,E) and (H) TaGS2-B. (C,F) and (I) TaGS2-D. The methylation status of CG/CHG/CHH sites in the 5′ regions and exon 1 of TaGS2 homoeologous genes in different tissues and developmental stages. Genomic DNA was treated with sodium bisulfite and used for PCR with primers that amplified fragments in the 5′ regions and exon 1 of the three TaGS2 homoeologous genes. The sequences were determined in 16 clones of each gene. Sequencing data were analyzed using Kismeth software (http://katahdin.mssm.edu/kismeth/revpage.pl). The colored lines above the x-axis show the percentage of methylation at individual cytosine sites. The short bars at the bottom of the graph show the positions of the cytosines.

Figure 5. Accessibility changes of three TaGS2 homoeologs to endonuclease in the roots and leaves.

The nuclei were digested with micrococcal nuclease and DNase I for an increasing time period as indicated. The amount of DNA was detected with quantitative PCR using primers specific for three TaGS2 homoeologs at the promoter and transcribed regions, respectively. Roots and leaves of 10-d-old seedlings and booting-stage leaves were measured. The data are the means ± SD of three independent biological replicates. (A) and (B) Degradation rates of three TaGS2 homoeologs with DNase I treatment. (C) and (D) Degradation rates of three TaGS2 homoeologs with micrococcal nuclease treatment.