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Abstract Enhancers are cis-regulatory elements that shape gene expression in response to numerous developmental and environmental cues. In animals, several models have been proposed to explain how enhancers integrate the activity of multiple transcription factors. However, it remains largely unclear how plant enhancers integrate transcription factor activity. Here, we use Plant STARR-seq to characterize 3 light-responsive plant enhancers—AB80, Cab-1, and rbcS-E9—derived from genes associated with photosynthesis. Saturation mutagenesis revealed mutations, many of which clustered in short regions, that strongly reduced enhancer activity in the light, in the dark, or in both conditions. When tested in the light, these mutation-sensitive regions did not function on their own; rather, cooperative interactions with other such regions were required for full activity. Epistatic interactions occurred between mutations in adjacent mutation-sensitive regions, and the spacing and order of mutation-sensitive regions in synthetic enhancers affected enhancer activity. In contrast, when tested in the dark, mutation-sensitive regions acted independently and additively in conferring enhancer activity. Taken together, this work demonstrates that plant enhancers show evidence for both cooperative and additive interactions among their functional elements. This knowledge can be harnessed to design strong, condition-specific synthetic enhancers. 

Abstract Insulators arecis-regulatory elements that separate transcriptional units, whereas silencers are elements that repress transcription regardless of their position. In plants, these elements remain largely uncharacterized. Here, we use the massively parallel reporter assay Plant STARR-seq with short fragments of eight large insulators to identify more than 100 fragments that block enhancer activity. The short fragments can be combined to generate more powerful insulators that abolish the capacity of the strong viral 35S enhancer to activate the 35S minimal promoter. Unexpectedly, when tested upstream of weak enhancers, these fragments act as silencers and repress transcription. Thus, these elements are capable of both insulating or repressing transcription dependent upon regulatory context. We validate our findings in stable transgenicArabidopsis, maize, and rice plants. The short elements identified here should be useful building blocks for plant biotechnology efforts. 

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. 

Summary Seasonal changes in spring induce flowering by expressing the florigen, FLOWERING LOCUS T (FT), inArabidopsis.FTis expressed in unique phloem companion cells with unknown characteristics. The question of which genes are co-expressed withFTand whether they have roles in flowering remains elusive. Through tissue-specific translatome analysis, we discovered that under long-day conditions with the natural sunlight red/far-red ratio, theFT-producing cells express a gene encoding FPF1-LIKE PROTEIN 1 (FLP1). The masterFTregulator, CONSTANS (CO), controlsFLP1expression, suggestingFLP1’s involvement in the photoperiod pathway. FLP1 promotes early flowering independently ofFT,is active in the shoot apical meristem, and induces the expression ofSEPALLATA 3(SEP3), a key E-class homeotic gene. Unlike FT, FLP1 facilitates inflorescence stem elongation. Our cumulative evidence indicates that FLP1 may act as a mobile signal. Thus, FLP1 orchestrates floral initiation together with FT and promotes inflorescence stem elongation during reproductive transitions. 

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. 

Abstract The scarcity of accessible sites that are dynamic or cell type-specific in plants may be due in part to tissue heterogeneity in bulk studies. To assess the effects of tissue heterogeneity, we apply single-cell ATAC-seq toArabidopsis thalianaroots and identify thousands of differentially accessible sites, sufficient to resolve all major cell types of the root. We find that the entirety of a cell’s regulatory landscape and its transcriptome independently capture cell type identity. We leverage this shared information on cell identity to integrate accessibility and transcriptome data to characterize developmental progression, endoreduplication and cell division. We further use the combined data to characterize cell type-specific motif enrichments of transcription factor families and link the expression of family members to changing accessibility at specific loci, resolving direct and indirect effects that shape expression. Our approach provides an analytical framework to infer the gene regulatory networks that execute plant development.