Rice transformation treatments leave specific epigenome changes beyond tissue culture.

During transgenic plant production, tissue culture often carries epigenetic, and genetic changes that underlie somaclonal variations, leading to unpredictable phenotypes. Additionally, specific treatments for rice (Oryza sativa) transformation processes may individually or jointly contribute to somaclonal variations, but their specific impacts on rice epigenomes toward transcriptional variations remain unknown. Here, the impact of individual transformation treatments on genome-wide DNA methylation and the transcriptome were examined. In addition to activating stress-responsive genes, individual transformation components targeted different gene expression modules that were enriched in specific functional categories. The transformation treatments strongly impacted DNA methylation and expression; 75% were independent of tissue culture. Furthermore, our genome-wide analysis showed that the transformation treatments consistently resulted in global hypo-CHH methylation enriched at promoters highly associated with downregulation, particularly when the promoters were colocalized with miniature inverted-repeat transposable elements. Our results clearly highlight the specificity of impacts triggered by individual transformation treatments during rice transformation with the potential association between DNA methylation and gene expression. These changes in gene expression and DNA methylation resulting from rice transformation treatments explain a significant portion of somaclonal variations, that is, way beyond the tissue culture effect.

HISTONE DEACETYLASE 15 and MOS4-associated complex subunits 3A/3B coregulate intron retention of ABA-responsive genes.

Histone deacetylases (HDAs) play an important role in transcriptional regulation of multiple biological processes. In this study, we investigated the function of HDA15 in abscisic acid (ABA) responses. We used immunopurification coupled with mass spectrometry-based proteomics to identify proteins interacting with HDA15 in Arabidopsis (Arabidopsis thaliana). HDA15 interacted with the core subunits of the MOS4-associated complex (MAC), MAC3A and MAC3B, with interaction between HDA15 and MAC3B enhanced by ABA. hda15 and mac3a/mac3b mutants were ABA-insensitive during seed germination and hyposensitive to salinity. RNA sequencing analysis demonstrated that HDA15 and MAC3A/MAC3B co-regulate ABA-responsive intron retention (IR). Furthermore, HDA15 reduced the histone acetylation level of genomic regions near ABA-responsive IR sites and the association of MAC3B with ABA-responsive pre-mRNA was dependent on HDA15. Our results indicate that HDA15 is involved in ABA responses by interacting with MAC3A/MAC3B to mediate splicing of introns.

Mycena genomes resolve the evolution of fungal bioluminescence.

Mushroom-forming fungi in the order Agaricales represent an independent origin of bioluminescence in the tree of life; yet the diversity, evolutionary history, and timing of the origin of fungal luciferases remain elusive. We sequenced the genomes and transcriptomes of five bonnet mushroom species (Mycena spp.), a diverse lineage comprising the majority of bioluminescent fungi. Two species with haploid genome assemblies ∼150 Mb are among the largest in Agaricales, and we found that a variety of repeats between Mycena species were differentially mediated by DNA methylation. We show that bioluminescence evolved in the last common ancestor of mycenoid and the marasmioid clade of Agaricales and was maintained through at least 160 million years of evolution. Analyses of synteny across genomes of bioluminescent species resolved how the luciferase cluster was derived by duplication and translocation, frequently rearranged and lost in most Mycena species, but conserved in the Armillaria lineage. Luciferase cluster members were coexpressed across developmental stages, with the highest expression in fruiting body caps and stipes, suggesting fruiting-related adaptive functions. Our results contribute to understanding a de novo origin of bioluminescence and the corresponding gene cluster in a diverse group of enigmatic fungal species.

Epigenomic regulation of OTU5 in Arabidopsis thaliana.

Epigenetic regulation by DNA methylation and histone marks is crucial to plant development. In Arabidopsis, the otu5 mutant exhibited altered root phenotypes resembling those of phosphate-deficient plants. In low phosphate (Pi) conditions, altered H3K4 and H3K27 trimethylation were associated with the expression of Pi homeostasis-related genes. However, the genetic effect of OTU5 on the epigenomes was left unexplored. We assessed genome-wide DNA methylation, gene expression and histone modifications of roots from both Col-0 and otu5 mutants. We found that OTU5 altered DNA methylation profile with a context-specific effect through targeting local genomic regions. Our analysis showed that in otu5 the abundance of H3K4me3 was clearly associated with the changes of DNA methylation, leading to the transcriptional difference from wildtype. We concluded that OTU5 induced cross-talks among epigenomes that altogether impacted the regulation of approximately 7060 genes. Of which 186 genes associated with root development were likely to be epigenetically regulated.

Establishing an optimized ATAC-seq protocol for the maize.

The advent of next-generation sequencing in crop improvement offers unprecedented insights into the chromatin landscape closely linked to gene activity governing key traits in plant development and adaptation. Particularly in maize, its dynamic chromatin structure is found to collaborate with massive transcriptional variations across tissues and developmental stages, implying intricate regulatory mechanisms, which highlights the importance of integrating chromatin information into breeding strategies for precise gene controls. The depiction of maize chromatin architecture using Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq) provides great opportunities to investigate cis-regulatory elements, which is crucial for crop improvement. In this context, we developed an easy-to-implement ATAC-seq protocol for maize with fewer nuclei and simple equipment. We demonstrate a streamlined ATAC-seq protocol with four key steps for maize in which nuclei purification can be achieved without cell sorting and using only a standard bench-top centrifuge. Our protocol, coupled with the bioinformatic analysis, including validation by read length periodicity, key metrics, and correlation with transcript abundance, provides a precise and efficient assessment of the maize chromatin landscape. Beyond its application to maize, our testing design holds the potential to be applied to other crops or other tissues, especially for those with limited size and amount, establishing a robust foundation for chromatin structure studies in diverse crop species.

Merit of integrating in situ transcriptomics and anatomical information for cell annotation and lineage construction in single-cell analyses of Populus.

Cell type annotation and lineage construction are two of the most critical tasks conducted in the analyses of single-cell RNA sequencing (scRNA-seq). Four recent scRNA-seq studies of differentiating xylem propose four models on differentiating xylem development in Populus. The differences are mostly caused by the use of different strategies for cell type annotation and subsequent lineage interpretation. Here, we emphasize the necessity of using in situ transcriptomes and anatomical information to construct the most plausible xylem development model.

The expression of long non-coding RNAs is associated with H3Ac and H3K4me2 changes regulated by the HDA6-LDL1/2 histone modification complex in Arabidopsis.

In recent years, eukaryotic long non-coding RNAs (lncRNAs) have been identified as important factors involved in a wide variety of biological processes, including histone modification, alternative splicing and transcription enhancement. The expression of lncRNAs is highly tissue-specific and is regulated by environmental stresses. Recently, a large number of plant lncRNAs have been identified, but very few of them have been studied in detail. Furthermore, the mechanism of lncRNA expression regulation remains largely unknown. Arabidopsis HISTONE DEACETYLASE 6 (HDA6) and LSD1-LIKE 1/2 (LDL1/2) can repress gene expression synergistically by regulating H3Ac/H3K4me. In this research, we performed RNA-seq and ChIP-seq analyses to further clarify the function of HDA6-LDL1/2. Our results indicated that the global expression of lncRNAs is increased in hda6/ldl1/2 and that this increased lncRNA expression is particularly associated with H3Ac/H3K4me2 changes. In addition, we found that HDA6-LDL1/2 is important for repressing lncRNAs that are non-expressed or show low-expression, which may be strongly associated with plant development. GO-enrichment analysis also revealed that the neighboring genes of the lncRNAs that are upregulated in hda6/ldl1/2 are associated with various developmental processes. Collectively, our results revealed that the expression of lncRNAs is associated with H3Ac/H3K4me2 changes regulated by the HDA6-LDL1/2 histone modification complex.