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Abstract The 3’ end of a gene, often called a terminator, modulates mRNA stability, localization, translation, and polyadenylation. Here, we adapted Plant STARR-seq, a massively parallel reporter assay, to measure the activity of over 50,000 terminators from the plantsArabidopsis thalianaandZea mays. We characterize thousands of plant terminators, including many that outperform bacterial terminators commonly used in plants. Terminator activity is species-specific, differing in tobacco leaf and maize protoplast assays. While recapitulating known biology, our results reveal the relative contributions of polyadenylation motifs to terminator strength. We built a computational model to predict terminator strength and used it to conduct in silico evolution that generated optimized synthetic terminators. Additionally, we discover alternative polyadenylation sites across tens of thousands of terminators; however, the strongest terminators tend to have a dominant cleavage site. Our results establish features of plant terminator function and identify strong naturally occurring and synthetic terminators. 

Abstract The genomes of flowering plants consist largely of transposable elements (TEs), some of which modulate gene regulation and function. However, the repetitive nature of TEs and difficulty of mapping individual TEs by short-read-sequencing have hindered our understanding of their regulatory potential. We demonstrate that long-read chromatin fiber sequencing (Fiber-seq) comprehensively identifies accessible chromatin regions (ACRs) and CpG methylation across the maize genome. We uncover stereotypical ACR patterns at young TEs that degenerate with evolutionary age, resulting in TE-enhancers preferentially marked by a novel plant-specific epigenetic feature: simultaneous hyper-CpG methylation and chromatin accessibility. We show that TE ACRs are co-opted as gene promoters and that ACR-containing TEs can facilitate gene amplification. Lastly, we uncover a pervasive epigenetic signature – hypo-5mCpG methylation and diffuse chromatin accessibility – directing TEs to specific loci, including the loci that sparked McClintock’s discovery of TEs. 

Abstract The precise onset of flowering is crucial to ensure successful plant reproduction. The geneFLOWERING LOCUS T(FT) encodes florigen, a mobile signal produced in leaves that initiates flowering at the shoot apical meristem. In response to seasonal changes,FTis induced in phloem companion cells located in distal leaf regions. Thus far, a detailed molecular characterization of theFT-expressing cells has been lacking. Here, we used bulk nuclei RNA-seq and single nuclei RNA (snRNA)-seq to investigate gene expression inFT-expressing cells and other phloem companion cells. Our bulk nuclei RNA-seq demonstrated thatFT-expressing cells in cotyledons and in true leaves differed transcriptionally. Within the true leaves, our snRNA-seq analysis revealed that companion cells with highFTexpression form a unique cluster in which many genes involved in ATP biosynthesis are highly upregulated. The cluster also expresses other genes encoding small proteins, including the flowering and stem growth inducer FPF1-LIKE PROTEIN 1 (FLP1) and the anti-florigen BROTHER OF FT AND TFL1 (BFT). In addition, we found that the promoters ofFTand the genes co-expressed withFTin the cluster were enriched for the consensus binding motifs of NITRATE-INDUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRESSOR 1 (NIGT1). Overexpression of the paralogousNIGT1.2andNIGT1.4repressedFTexpression and significantly delayed flowering under nitrogen-rich conditions, consistent with NIGT1s acting as nitrogen-dependentFTrepressors. Taken together, our results demonstrate that majorFT-expressing cells show a distinct expression profile that suggests that these cells may produce multiple systemic signals to regulate plant growth and development. 

Abstract BackgroundThe genetic information contained in the genome of an organism is organized in genes and regulatory elements that control gene expression. The genomes of multiple plants species have already been sequenced and the gene repertory have been annotated, however,cis-regulatory elements remain less characterized, limiting our understanding of genome functionality. These elements act as open platforms for recruiting both positive- and negative-acting transcription factors, and as such, chromatin accessibility is an important signature for their identification. ResultsIn this work we developed a transgenic INTACT [isolation of nuclei tagged in specific cell types] system in tetraploid wheat for nuclei purifications. Then, we combined the INTACT system together with the assay for transposase-accessible chromatin with sequencing [ATAC-seq] to identify open chromatin regions in wheat root tip samples. Our ATAC-seq results showed a large enrichment of open chromatin regions in intergenic and promoter regions, which is expected for regulatory elements and that is similar to ATAC-seq results obtained in other plant species. In addition, root ATAC-seq peaks showed a significant overlap with a previously published ATAC-seq data from wheat leaf protoplast, indicating a high reproducibility between the two experiments and a large overlap between open chromatin regions in root and leaf tissues. Importantly, we observed overlap between ATAC-seq peaks andcis-regulatory elements that have been functionally validated in wheat, and a good correlation between normalized accessibility and gene expression levels. ConclusionsWe have developed and validated an INTACT system in tetraploid wheat that allows rapid and high-quality nuclei purification from root tips. Those nuclei were successfully used to performed ATAC-seq experiments that revealed open chromatin regions in the wheat genome that will be useful to identify cis-regulatory elements. The INTACT system presented here will facilitate the development of ATAC-seq datasets in other tissues, growth stages, and under different growing conditions to generate a more complete landscape of the accessible DNA regions in the wheat genome. 

Abstract Argonaute 1 (AGO1), the principal protein component of microRNA-mediated regulation, plays a key role in plant growth and development. AGO1 physically interacts with the chaperone HSP90, which buffers cryptic genetic variation in plants and animals. We sought to determine whether genetic perturbation of AGO1 in Arabidopsis thaliana would also reveal cryptic genetic variation, and if so, whether AGO1-dependent loci overlap with those dependent on HSP90. To address these questions, we introgressed a hypomorphic mutant allele of AGO1 into a set of mapping lines derived from the commonly used Arabidopsis strains Col-0 and Ler. Although we identified several cases in which AGO1 buffered genetic variation, none of the AGO1-dependent loci overlapped with those buffered by HSP90 for the traits assayed. We focused on 1 buffered locus where AGO1 perturbation uncoupled the traits days to flowering and rosette leaf number, which are otherwise closely correlated. Using a bulk segregant approach, we identified a nonfunctional Ler hua2 mutant allele as the causal AGO1-buffered polymorphism. Introduction of a nonfunctional hua2 allele into a Col-0 ago1 mutant background recapitulated the Ler-dependent ago1 phenotype, implying that coupling of these traits involves different molecular players in these closely related strains. Taken together, our findings demonstrate that even though AGO1 and HSP90 buffer genetic variation in the same traits, these robustness regulators interact epistatically with different genetic loci, suggesting that higher-order epistasis is uncommon. Plain Language Summary Argonaute 1 (AGO1), a key player in plant development, interacts with the chaperone HSP90, which buffers environmental and genetic variation. We found that AGO1 buffers environmental and genetic variation in the same traits; however, AGO1-dependent and HSP90-dependent loci do not overlap. Detailed analysis of a buffered locus found that a nonfunctional HUA2 allele decouples days to flowering and rosette leaf number in an AGO1-dependent manner, suggesting that the AGO1-dependent buffering acts at the network level.