Deep sequencing of DNase-I treated chromatin (DNase-seq) can be used to identify DNase I-hypersensitive sites (DHSs) and facilitates genome-scale mining of de novo cis-regulatory DNA elements. Here, we adapted DNase-seq to generate genome-wide maps of DHSs using control and cold-treated leaf, stem, and root tissues of three widely studied grass species: Brachypodium distachyon, foxtail millet (Setaria italica), and sorghum (Sorghum bicolor). Functional validation demonstrated that 12 of 15 DHSs drove reporter gene expression in transiently transgenic B. distachyon protoplasts. DHSs under both normal and cold treatment substantially differed among tissues and species. Intriguingly, the putative DHS-derived transcription factors (TFs) are largely colocated among tissues and species and include 17 ubiquitous motifs covering all grass taxa and all tissues examined in this study. This feature allowed us to reconstruct a regulatory network that responds to cold stress. Ethylene-responsive TFs SHINE3, ERF2, and ERF9 occurred frequently in cold feedback loops in the tissues examined, pointing to their possible roles in the regulatory network. Overall, we provide experimental annotation of 322,713 DHSs and 93 derived cold-response TF binding motifs in multiple grasses, which could serve as a valuable resource for elucidating the transcriptional networks that function in the cold-stress response and other physiological processes.

Allopolyploidization, resulting in divergent genomes in the same cell, is believed to trigger a “genome shock”, leading to broad genetic and epigenetic changes. However, little is understood about chromatin and gene-expression dynamics as underlying driving forces during allopolyploidization. Here, we examined the genome-wide DNase I-hypersensitive site (DHS) and its variations in domesticated allotetraploid cotton ( Gossypium hirsutum and Gossypium barbadense , AADD) and its extant AA ( Gossypium arboreum ) and DD ( Gossypium raimondii ) progenitors. We observed distinct DHS distributions between G. arboreum and G. raimondii . In contrast, the DHSs of the two subgenomes of G. hirsutum and G. barbadense showed a convergent distribution. This convergent distribution of DHS was also present in the wild allotetraploids Gossypium darwinii and G. hirsutum var. yucatanense , but absent from a resynthesized hybrid of G. arboreum and G. raimondii , suggesting that it may be a common feature in polyploids, and not a consequence of domestication after polyploidization. We revealed that putative cis -regulatory elements (CREs) derived from polyploidization-related DHSs were dominated by several families, including Dof, ERF48, and BPC1. Strikingly, 56.6% of polyploidization-related DHSs were derived from transposable elements (TEs). Moreover, we observed positive correlations between DHS accessibility and the histone marks H3K4me3, H3K27me3, H3K36me3, H3K27ac, and H3K9ac, indicating that coordinated interplay among histone modifications, TEs, and CREs drives the DHS landscape dynamics under polyploidization. Collectively, these findings advance our understanding of the regulatory architecture in plants and underscore the complexity of regulome evolution during polyploidization.

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Agrobacterium-mediated genetic transformation is well established in the model grass Brachypodium distachyon. However, most protocols employ immature embryos because of their better regenerative capacity. A major problem associated with the immature embryo system is that they are available only during a limited time window of growing plants. In this study, we have developed an optimized Agrobacterium-mediated genetic transformation protocol that utilizes mature embryos. We have adopted seed shearing and photoautotrophic rooting (PR) in callus induction and root regeneration, respectively, with evident significant improvement in these aspects. We have also revealed that the newly developed chemical inducer Fipexide (FPX) had the ability to induce callus, shoots, and roots. By comparison, we have demonstrated that FPX shows higher efficiency in shoot generation than other frequently used chemicals in our mature embryo-based system. In addition, we demonstrated that the age of embryogenetic callus severely affects the transformation efficiency (TE), with the seven-week-old embryogenetic callus having the highest TE reaching 52.6%, which is comparable with that in immature embryo transformation. The new methodologies reported here will advance the development and utilization of Brachypodium as a new model system for grass genomics.