Plant enhancers exhibit both cooperative and additive interactions among their functional elements.

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.

Impact on splicing in Saccharomyces cerevisiae of random 50-base sequences inserted into an intron.

Intron splicing is a key regulatory step in gene expression in eukaryotes. Three sequence elements required for splicing-5' and 3' splice sites and a branchpoint-are especially well-characterized in Saccharomyces cerevisiae, but our understanding of additional intron features that impact splicing in this organism is incomplete, due largely to its small number of introns. To overcome this limitation, we constructed a library in S. cerevisiae of random 50-nt (N50) elements individually inserted into the intron of a reporter gene and quantified canonical splicing and the use of cryptic splice sites by sequencing analysis. More than 70% of approximately 140,000 N50 elements reduced splicing by at least 20%. N50 features, including higher GC content, presence of GU repeats, and stronger predicted secondary structure of its pre-mRNA, correlated with reduced splicing efficiency. A likely basis for the reduced splicing of such a large proportion of variants is the formation of RNA structures that pair N50 bases-such as the GU repeats-with other bases specifically within the reporter pre-mRNA analyzed. However, multiple models were unable to explain more than a small fraction of the variance in splicing efficiency across the library, suggesting that complex nonlinear interactions in RNA structures are not accurately captured by RNA structure prediction methods. Our results imply that the specific context of a pre-mRNA may determine the bases allowable in an intron to prevent secondary structures that reduce splicing. This large data set can serve as a resource for further exploration of splicing mechanisms.

LTP2 hypomorphs show genotype-by-environment interaction in early seedling traits in Arabidopsis thaliana.

Isogenic individuals can display seemingly stochastic phenotypic differences, limiting the accuracy of genotype-to-phenotype predictions. The extent of this phenotypic variation depends in part on genetic background, raising questions about the genes involved in controlling stochastic phenotypic variation. Focusing on early seedling traits in Arabidopsis thaliana, we found that hypomorphs of the cuticle-related gene LIPID TRANSFER PROTEIN 2 (LTP2) greatly increased variation in seedling phenotypes, including hypocotyl length, gravitropism and cuticle permeability. Many ltp2 hypocotyls were significantly shorter than wild-type hypocotyls while others resembled the wild-type. Differences in epidermal properties and gene expression between ltp2 seedlings with long and short hypocotyls suggest a loss of cuticle integrity as the primary determinant of the observed phenotypic variation. We identified environmental conditions that reveal or mask the increased variation in ltp2 hypomorphs and found that increased expression of its closest paralog LTP1 is necessary for ltp2 phenotypes. Our results illustrate how decreased expression of a single gene can generate starkly increased phenotypic variation in isogenic individuals in response to an environmental challenge.

A single-cell view of the transcriptome during lateral root initiation in Arabidopsis thaliana.

Root architecture is a major determinant of plant fitness and is under constant modification in response to favorable and unfavorable environmental stimuli. Beyond impacts on the primary root, the environment can alter the position, spacing, density, and length of secondary or lateral roots. Lateral root development is among the best-studied examples of plant organogenesis, yet there are still many unanswered questions about its earliest steps. Among the challenges faced in capturing these first molecular events is the fact that this process occurs in a small number of cells with unpredictable timing. Single-cell sequencing methods afford the opportunity to isolate the specific transcriptional changes occurring in cells undergoing this fate transition. Using this approach, we successfully captured the transcriptomes of initiating lateral root primordia in Arabidopsis thaliana and discovered many upregulated genes associated with this process. We developed a method to selectively repress target gene transcription in the xylem pole pericycle cells where lateral roots originate and demonstrated that the expression of several of these targets is required for normal root development. We also discovered subpopulations of cells in the pericycle and endodermal cell files that respond to lateral root initiation, highlighting the coordination across cell files required for this fate transition.

Single-cell genomics in plants: current state, future directions, and hurdles to overcome.

Single-cell genomics has the potential to revolutionize the study of plant development and tissue-specific responses to environmental stimuli by revealing heretofore unknown players and gene regulatory processes. Here, I focus on the current state of single-cell genomics in plants, emerging technologies and applications, in addition to outlining possible future directions for experiments. I describe approaches to enable cheaper and larger experiments and technologies to measure multiple types of molecules to better model and understand cell types and their different states and trajectories throughout development. Lastly, I discuss the inherent limitations of single-cell studies and the technological hurdles that need to be overcome to widely apply single-cell genomics in crops to generate the greatest possible knowledge gain.

Identification of Plant Enhancers and Their Constituent Elements by STARR-seq in Tobacco Leaves.

Genetic engineering of cis-regulatory elements in crop plants is a promising strategy to ensure food security. However, such engineering is currently hindered by our limited knowledge of plant cis-regulatory elements. Here, we adapted self-transcribing active regulatory region sequencing (STARR-seq)-a technology for the high-throughput identification of enhancers-for its use in transiently transformed tobacco (Nicotiana benthamiana) leaves. We demonstrate that the optimal placement in the reporter construct of enhancer sequences from a plant virus, pea (Pisum sativum) and wheat (Triticum aestivum), was just upstream of a minimal promoter and that none of these four known enhancers was active in the 3' untranslated region of the reporter gene. The optimized assay sensitively identified small DNA regions containing each of the four enhancers, including two whose activity was stimulated by light. Furthermore, we coupled the assay to saturation mutagenesis to pinpoint functional regions within an enhancer, which we recombined to create synthetic enhancers. Our results describe an approach to define enhancer properties that can be performed in potentially any plant species or tissue transformable by Agrobacterium and that can use regulatory DNA derived from any plant genome.

Specificity of ARGONAUTE7-miR390 interaction and dual functionality in TAS3 trans-acting siRNA formation.

Trans-acting siRNA form through a refined RNAi mechanism in plants. miRNA-guided cleavage triggers entry of precursor transcripts into an RNA-DEPENDENT RNA POLYMERASE6 pathway, and sets the register for phased tasiRNA formation by DICER-LIKE4. Here, we show that miR390-ARGONAUTE7 complexes function in distinct cleavage or noncleavage modes at two target sites in TAS3a transcripts. The AGO7 cleavage, but not the noncleavage, function could be provided by AGO1, the dominant miRNA-associated AGO, but only when AGO1 was guided to a modified target site through an alternate miRNA. AGO7 was highly selective for interaction with miR390, and miR390 in turn was excluded from association with AGO1 due entirely to an incompatible 5' adenosine. Analysis of AGO1, AGO2, and AGO7 revealed a potent 5' nucleotide discrimination function for some, although not all, ARGONAUTEs. miR390 and AGO7, therefore, evolved as a highly specific miRNA guide/effector protein pair to function at two distinct tasiRNA biogenesis steps.

Dynamics of Gene Expression in Single Root Cells of Arabidopsis thaliana.

Single cell RNA sequencing can yield high-resolution cell-type-specific expression signatures that reveal new cell types and the developmental trajectories of cell lineages. Here, we apply this approach to Arabidopsis (Arabidopsis thaliana) root cells to capture gene expression in 3,121 root cells. We analyze these data with Monocle 3, which orders single cell transcriptomes in an unsupervised manner and uses machine learning to reconstruct single cell developmental trajectories along pseudotime. We identify hundreds of genes with cell-type-specific expression, with pseudotime analysis of several cell lineages revealing both known and novel genes that are expressed along a developmental trajectory. We identify transcription factor motifs that are enriched in early and late cells, together with the corresponding candidate transcription factors that likely drive the observed expression patterns. We assess and interpret changes in total RNA expression along developmental trajectories and show that trajectory branch points mark developmental decisions. Finally, by applying heat stress to whole seedlings, we address the longstanding question of possible heterogeneity among cell types in the response to an abiotic stress. Although the response of canonical heat-shock genes dominates expression across cell types, subtle but significant differences in other genes can be detected among cell types. Taken together, our results demonstrate that single cell transcriptomics holds promise for studying plant development and plant physiology with unprecedented resolution.

Preferences in a trait decision determined by transcription factor variants.

Few mechanisms are known that explain how transcription factors can adjust phenotypic outputs to accommodate differing environments. In Saccharomyces cerevisiae, the decision to mate or invade relies on environmental cues that converge on a shared transcription factor, Ste12. Specificity toward invasion occurs via Ste12 binding cooperatively with the cofactor Tec1. Here, we determine the range of phenotypic outputs (mating vs. invasion) of thousands of DNA-binding domain variants in Ste12 to understand how preference for invasion may arise. We find that single amino acid changes in the DNA-binding domain can shift the preference of yeast toward either mating or invasion. These mutations define two distinct regions of this domain, suggesting alternative modes of DNA binding for each trait. We characterize the DNA-binding specificity of wild-type Ste12 to identify a strong preference for spacing and orientation of both homodimeric and heterodimeric sites. Ste12 mutants that promote hyperinvasion in a Tec1-independent manner fail to bind cooperative sites with Tec1 and bind to unusual dimeric Ste12 sites composed of one near-perfect and one highly degenerate site. We propose a model in which Ste12 alone may have evolved to activate invasion genes, which could explain how preference for invasion arose in the many fungal pathogens that lack Tec1.

Deep learning of the regulatory grammar of yeast 5' untranslated regions from 500,000 random sequences.

Our ability to predict protein expression from DNA sequence alone remains poor, reflecting our limited understanding of cis-regulatory grammar and hampering the design of engineered genes for synthetic biology applications. Here, we generate a model that predicts the protein expression of the 5' untranslated region (UTR) of mRNAs in the yeast Saccharomyces cerevisiae. We constructed a library of half a million 50-nucleotide-long random 5' UTRs and assayed their activity in a massively parallel growth selection experiment. The resulting data allow us to quantify the impact on protein expression of Kozak sequence composition, upstream open reading frames (uORFs), and secondary structure. We trained a convolutional neural network (CNN) on the random library and showed that it performs well at predicting the protein expression of both a held-out set of the random 5' UTRs as well as native S. cerevisiae 5' UTRs. The model additionally was used to computationally evolve highly active 5' UTRs. We confirmed experimentally that the great majority of the evolved sequences led to higher protein expression rates than the starting sequences, demonstrating the predictive power of this model.

Complex Relationships between Chromatin Accessibility, Sequence Divergence, and Gene Expression in Arabidopsis thaliana.

Variation in regulatory DNA is thought to drive phenotypic variation, evolution, and disease. Prior studies of regulatory DNA and transcription factors across animal species highlighted a fundamental conundrum: Transcription factor binding domains and cognate binding sites are conserved, while regulatory DNA sequences are not. It remains unclear how conserved transcription factors and dynamic regulatory sites produce conserved expression patterns across species. Here, we explore regulatory DNA variation and its functional consequences within Arabidopsis thaliana, using chromatin accessibility to delineate regulatory DNA genome-wide. Unlike in previous cross-species comparisons, the positional homology of regulatory DNA is maintained among A. thaliana ecotypes and less nucleotide divergence has occurred. Of the ∼50,000 regulatory sites in A. thaliana, we found that 15% varied in accessibility among ecotypes. Some of these accessibility differences were associated with extensive, previously unannotated sequence variation, encompassing many deletions and ancient hypervariable alleles. Unexpectedly, for the majority of such regulatory sites, nearby gene expression was unaffected. Nevertheless, regulatory sites with high levels of sequence variation and differential chromatin accessibility were the most likely to be associated with differential gene expression. Finally, and most surprising, we found that the vast majority of differentially accessible sites show no underlying sequence variation. We argue that these surprising results highlight the necessity to consider higher-order regulatory context in evaluating regulatory variation and predicting its phenotypic consequences.

New generation of artificial MicroRNA and synthetic trans-acting small interfering RNA vectors for efficient gene silencing in Arabidopsis.

Artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs) are used for small RNA-based, specific gene silencing or knockdown in plants. Current methods to generate amiRNA or syn-tasiRNA constructs are not well adapted for cost-effective, large-scale production or for multiplexing to specifically suppress multiple targets. Here, we describe simple, fast, and cost-effective methods with high-throughput capability to generate amiRNA and multiplexed syn-tasiRNA constructs for efficient gene silencing in Arabidopsis (Arabidopsis thaliana) and other plant species. amiRNA or syn-tasiRNA inserts resulting from the annealing of two overlapping and partially complementary oligonucleotides are ligated directionally into a zero background BsaI/ccdB-based expression vector. BsaI/ccdB vectors for amiRNA or syn-tasiRNA cloning and expression contain a modified version of Arabidopsis MIR390a or TAS1c precursors, respectively, in which a fragment of the endogenous sequence was substituted by a ccdB cassette flanked by two BsaI sites. Several amiRNA and syn-tasiRNA sequences designed to target one or more endogenous genes were validated in transgenic plants that (1) exhibited the expected phenotypes predicted by loss of target gene function, (2) accumulated high levels of accurately processed amiRNAs or syn-tasiRNAs, and (3) had reduced levels of the corresponding target RNAs.

Functional analysis of three Arabidopsis ARGONAUTES using slicer-defective mutants.

In RNA-directed silencing pathways, ternary complexes result from small RNA-guided ARGONAUTE (AGO) associating with target transcripts. Target transcripts are often silenced through direct cleavage (slicing), destabilization through slicer-independent turnover mechanisms, and translational repression. Here, wild-type and active-site defective forms of several Arabidopsis thaliana AGO proteins involved in posttranscriptional silencing were used to examine several AGO functions, including small RNA binding, interaction with target RNA, slicing or destabilization of target RNA, secondary small interfering RNA formation, and antiviral activity. Complementation analyses in ago mutant plants revealed that the catalytic residues of AGO1, AGO2, and AGO7 are required to restore the defects of Arabidopsis ago1-25, ago2-1, and zip-1 (AGO7-defective) mutants, respectively. AGO2 had slicer activity in transient assays but could not trigger secondary small interfering RNA biogenesis, and catalytically active AGO2 was necessary for local and systemic antiviral activity against Turnip mosaic virus. Slicer-defective AGOs associated with miRNAs and stabilized AGO-miRNA-target RNA ternary complexes in individual target coimmunoprecipitation assays. In genome-wide AGO-miRNA-target RNA coimmunoprecipitation experiments, slicer-defective AGO1-miRNA associated with target RNA more effectively than did wild-type AGO1-miRNA. These data not only reveal functional roles for AGO1, AGO2, and AGO7 slicer activity, but also indicate an approach to capture ternary complexes more efficiently for genome-wide analyses.

Evolution and functional diversification of MIRNA genes.

MicroRNAs (miRNAs) are small regulatory RNAs found in diverse eukaryotic lineages. In plants, a minority of annotated MIRNA gene families are conserved between plant families, while the majority are family- or species-specific, suggesting that most known MIRNA genes arose relatively recently in evolutionary time. Given the high proportion of young MIRNA genes in plant species, new MIRNA families are likely spawned and then lost frequently. Unlike highly conserved, ancient miRNAs, young miRNAs are often weakly expressed, processed imprecisely, lack targets, and display patterns of neutral variation, suggesting that young MIRNA loci tend to evolve neutrally. Genome-wide analyses from several plant species have revealed that variation in miRNA foldback expression, structure, processing efficiency, and miRNA size have resulted in the unique functionality of MIRNA loci and resulting miRNAs. Additionally, some miRNAs have evolved specific properties and functions that regulate other transcriptional or posttranscriptional silencing pathways. The evolution of miRNA processing and functional diversity underscores the dynamic nature of miRNA-based regulation in complex regulatory networks.

Arabidopsis and Maize Terminator Strength is Determined by GC Content, Polyadenylation Motifs and Cleavage Probability.

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 plants Arabidopsis thaliana and Zea 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.

Arabidopsis and maize terminator strength is determined by GC content, polyadenylation motifs and cleavage probability.

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 plants Arabidopsis thaliana and Zea 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.

The regulatory potential of transposable elements in maize.

Since their initial discovery in maize, transposable elements (TEs) have emerged as being integral to the evolution of maize, accounting for 80% of its genome. However, the repetitive nature of TEs has hindered our understanding of their regulatory potential. Here, we demonstrate that long-read chromatin fiber sequencing (Fiber-seq) permits the comprehensive annotation of the regulatory potential of maize TEs. We uncover that only 94 LTR retrotransposons contain the functional epigenetic architecture required for mobilization within maize leaves. This epigenetic architecture degenerates with evolutionary age, resulting in solo TE enhancers being preferentially marked by simultaneous hyper-CpG methylation and chromatin accessibility, an architecture markedly divergent from canonical enhancers. We find that TEs shape maize gene regulation by creating novel promoters within the TE itself as well as through TE-mediated gene amplification. Lastly, we uncover a pervasive epigenetic code directing TEs to specific loci, including that locus that sparked McClintock's discovery of TEs.

A florigen-expressing subpopulation of companion cells expresses other small proteins and reveals a nitrogen-sensitive FT repressor.

The precise onset of flowering is crucial to ensure successful plant reproduction. The gene FLOWERING LOCUS T (FT) encodes florigen, a mobile signal produced in leaves that initiates flowering at the shoot apical meristem. In response to seasonal changes, FT is induced in phloem companion cells located in distal leaf regions. Thus far, a detailed molecular characterization of the FT-expressing cells has been lacking. Here, we used bulk nuclei RNA-seq and single nuclei RNA (snRNA)-seq to investigate gene expression in FT-expressing cells and other phloem companion cells. Our bulk nuclei RNA-seq demonstrated that FT-expressing cells in cotyledons and in true leaves differed transcriptionally. Within the true leaves, our snRNA-seq analysis revealed that companion cells with high FT expression 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 of FT and the genes co-expressed with FT in the cluster were enriched for the consensus binding motifs of NITRATE-INDUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRESSOR 1 (NIGT1). Overexpression of the paralogous NIGT1.2 and NIGT1.4 repressed FT expression and significantly delayed flowering under nitrogen-rich conditions, consistent with NIGT1s acting as nitrogen-dependent FT repressors. Taken together, our results demonstrate that major FT-expressing cells show a distinct expression profile that suggests that these cells may produce multiple systemic signals to regulate plant growth and development.

Florigen-producing cells express FPF1-LIKE PROTEIN 1 that accelerates flowering and stem growth in long days with sunlight red/far-red ratio in Arabidopsis.

Seasonal changes in spring induce flowering by expressing the florigen, FLOWERING LOCUS T (FT), in Arabidopsis. FT is expressed in unique phloem companion cells with unknown characteristics. The question of which genes are co-expressed with FT and 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, the FT-producing cells express a gene encoding FPF1-LIKE PROTEIN 1 (FLP1). The master FT regulator, CONSTANS (CO), controls FLP1 expression, suggesting FLP1's involvement in the photoperiod pathway. FLP1 promotes early flowering independently of FT, is active in the shoot apical meristem, and induces the expression of SEPALLATA 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.

Identification of MIR390a precursor processing-defective mutants in Arabidopsis by direct genome sequencing.

Transacting siRNA (tasiRNA) biogenesis in Arabidopsis is initiated by microRNA (miRNA) -guided cleavage of primary transcripts. In the case of TAS3 tasiRNA formation, ARGONAUTE7 (AGO7)-miR390 complexes interact with primary transcripts at two sites, resulting in recruitment of RNA-DEPENDENT RNA POLYMERASE6 for dsRNA biosynthesis. An extensive screen for Arabidopsis mutants with specific defects in TAS3 tasiRNA biogenesis or function was done. This yielded numerous ago7 mutants, one dcl4 mutant, and two mutants that accumulated low levels of miR390. A direct genome sequencing-based approach to both map and rapidly identify one of the latter mutant alleles was developed. This revealed a G-to-A point mutation (mir390a-1) that was calculated to stabilize a relatively nonpaired region near the base of the MIR390a foldback, resulting in misprocessing of the miR390/miR390* duplex and subsequent reduced TAS3 tasiRNA levels. Directed substitutions, as well as analysis of variation at paralogous miR390-generating loci (MIR390a and MIR390b), indicated that base pair properties and nucleotide identity within a region 4-6 bases below the miR390/miR390* duplex region contributed to the efficiency and accuracy of precursor processing.