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The biological functions of the circadian clock on growth and development have been well elucidated in model plants, while its regulatory roles in crop species, especially the roles on yield-related traits, are poorly understood. In this study, we characterized the core clock gene CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) homoeologs in wheat and studied their biological functions in seedling growth and spike development. TaCCA1 homoeologs exhibit typical diurnal expression patterns, which are positively regulated by rhythmic histone modifications including histone H3 lysine 4 trimethylation (H3K4me3), histone H3 lysine 9 acetylation (H3K9Ac), and histone H3 lysine 36 trimethylation (H3K36me3). TaCCA1s are preferentially located in the nucleus and tend to form both homo- and heterodimers. TaCCA1 overexpression (TaCCA1-OE) transgenic wheat plants show disrupted circadian rhythmicity coupling with reduced chlorophyll and starch content, as well as biomass at seedling stage, also decreased spike length, grain number per spike, and grain size at the ripening stage. Further studies using DNA affinity purification followed by deep sequencing [DNA affinity purification and sequencing (DAP-seq)] indicated that TaCCA1 preferentially binds to sequences similarly to “evening elements” (EE) motif in the wheat genome, particularly genes associated with photosynthesis, carbon utilization, and auxin homeostasis, and decreased transcriptional levels of these target genes are observed in TaCCA1-OE transgenic wheat plants. Collectively, our study provides novel insights into a circadian-mediated mechanism of gene regulation to coordinate photosynthetic and metabolic activities in wheat, which is important for optimal plant growth and crop yield formation.

Additional file 1: Figure S1. (A) and (B): Distribution of two sub-gene families on wheat chromosome. Distribution of two gene families on wheat chromosome. All wheat chromosomes have HKMT gene distribution, which are relatively uniform. However, chromosome 2 and chromosome 3 also tend to cluster. Compared with the HKMT genes, the number of PRMT genes is relatively small, and the distribution of PRMT genes in the whole wheat genome is uneven. In addition, compared with previous research results, most HMT genes have been located in a clear range, and there is only one HKMT gene on the U chromosome, may also indicate that the genome quality of wheat has been greatly improved. Figure S2. Analysis of cis-elements of promoters of TaHMT genes. The 2kb sequence before the CDS of each HMT gene was extracted as the promoter region and submitted to PlantCARE database for cis-regulatory response elements prediction, and the results were visualized by TBtools. Figure S3. Expression analysis all selected TaHMT genes. (A) Expression analysis of 19 genes in spike. (B) Expression analysis of 12 genes in tiller. The relative expression of the female parent of each combination was relative 1, and the expression values for each parent are placed in front of each combination. The horizontal column is the gene name and the vertical column is the name of the samples. Figure S4. The phenotype as well as sampling times at different periods of wheat development. (A) Different periods of wheat spike development and sampling were conducted on this basis at anther separation stage. The length of the red ruler represents 1mm. (B) Tillers from root base to stem about 0.5 cm in length were sampled which contains the apical meristem at about four-leaf stage; P: male parent, M: maternal parent. (C) Phenotypic trait combinations for different tillers at harvest time, from left to right, they were: high parent (HP), middle parent (MP) and low parent (LP). In the picture, the three plants were photographed as a whole. Figure S5. The phylogenetic trees of HKMT (A) and PRMT (B) proteins from wheat, rice, Arabidopsis, Sorghum bicolor, Hordeum vulgare, and Brachypodium distachyon were constructed by MEGA-X using Maximum likelihood method. The HKMT proteins were divided into seven subfamilies and PRMT were divided into four subfamilies based on the previous methods. The members of wheat were marked with red circles in Fig. S5B. Figure S6. Interaction networks of miRNAs and HMT targets. miRNAs were marked as a blue node and HMT targets were marked as a yellow node. The size of the node represents the number of action sites. The black solid line represents that the mismatch expectation is 3.0, the red thick solid line represents that the mismatch expectation is 1.5, and the green thick solid line represents that the mismatch expectation is 2.5.

Background: Known as the prerequisite component for the heterosis breeding system, the male sterile line determined the hybrid yield and seed purity. Therefore, a deep understanding of the mechanism and gene network that leads to male sterility is crucial. BS366, a temperature-sensitive genic male sterile (TGMS) line, were male sterile under cold condition (12℃ with 12 h daylight) but fertile under normal temperature (20℃ with 12 h daylight). Results: During the meiosis, BS366 was defective in forming tetrad and dyad due to the abnormal cell plate. During the pollen development, unusual vacuolated pollen that cannot accumulate starch grains at the binucleate stage was also observed. Transcriptome analysis revealed that genes involved in the meiotic process like sister chromatid segregation and microtubule-based movement were repressed, while genes involved in DNA and histone methylation were induced in BS366 under cold condition. MethylRAD was used for a reduced DNA methylation sequencing for BS366 under both cold and control conditions. The differentially methylated sites (DMSs) located in the gene region were mainly involved in carbohydrate and fatty acid metabolism, lipid metabolism, and transport. Genes differentially and methylated were mainly involved in cell division. Conclusions: These results indicated that methylation of genes involved in carbon metabolism or fatty acid metabolism would contribute to the male sterility in BS366, which will provide a novel insight into the molecular mechanism of wheat male sterility.