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The genes for ribosomal RNA (rRNA) are encoded by ribosomal DNA (rDNA), whose structure is notable for being present in arrays of tens to thousands of tandemly repeated copies in eukaryotic genomes. The exact number of rDNA copies per genome is highly variable within a species, with differences between individuals measuring in potentially hundreds of copies and megabases of DNA. The extent to which natural variation in rDNA copy number impacts whole-organism phenotypes such as fitness and lifespan is poorly understood, in part due to difficulties in manipulating such large and repetitive tracts of DNA even in model organisms. Here, we used the natural resource of copy number variation in C. elegans wild isolates to generate new tools and investigated the phenotypic consequences of this variation. Specifically, we generated a panel of recombinant inbred lines (RILs) using a laboratory strain derivative with ~130 haploid rDNA copies and a wild isolate with ~417 haploid rDNA copies, one of the highest validated C. elegans rDNA copy number arrays. We find that rDNA copy number is stable in the RILs, rejecting prior hypotheses that predicted copy number instability and copy number reversion. To isolate effects of rDNA copy number on phenotype, we produced a series of near isogenic lines (NILs) with rDNA copy numbers representing the high and low end of the rDNA copy number spectrum in C. elegans wild isolates. We find no correlation between rDNA copy number and phenotypes of rRNA abundance, competitive fitness, early life fertility, lifespan, or global transcriptome under standard laboratory conditions. These findings demonstrate a remarkable ability of C. elegans to tolerate substantial variation in a locus critical to fundamental cell function. Our study provides strain resources for future investigations into the boundaries of this tolerance. 

Summary Day neutrality, or insensitivity to photoperiod (day length), is an important domestication trait in many crop species. Although the oilseed cropCamelina sativahas been cultivated since the Neolithic era, day-neutral accessions have yet to be described. We sought to leverage genetic diversity in existing germplasms to identifyC. sativaaccessions with low photoperiod sensitivity for future engineering of this trait. We quantified variation in the photoperiod response across 161 accessions ofC. sativaby measuring hypocotyl length of four-day-old seedlings grown in long-day and short-day conditions, finding wide variation in photoperiod response. Similarly, soil-grown adult plants from selected accessions showed variation in photoperiod response in several traits; however, photoperiod responses in seedling and adult traits were not correlated, suggesting complex mechanistic underpinnings. Although RNA-seq experiments of the reference accession Licalla identified several differentially regulatedArabidopsissyntelogs involved in photoperiod response, includingCOL2, FT, LHYandWOX4, expression of these genes in the accessions did not correlate with differences in their photoperiod sensitivity. Taken together, we show that all tested accessions show some degree of photoperiod response, and that this trait is likely complex, involving several and separable seedling and adult traits. Significance StatementDay neutrality (photoperiod insensitivity) is a common trait in domesticated crops; however, the ancient oilseed cropCamelina sativahas remained photoperiod-sensitive, which likely limits seed yields. Here, we show that photoperiod sensitivity is conserved across manyC. sativacultivars, albeit to different degrees, and we establish that photoperiod sensitivity is a complex trait, which will require genetic engineering to achieve day neutrality. 

Many of the most highly conserved elements in the human genome are “poison exons,” alternatively spliced exons that contain premature termination codons and permit post-transcriptional regulation of mRNA abundance through induction of nonsense-mediated mRNA decay (NMD). Poison exons are widely assumed to be highly conserved due to their presumed importance for organismal fitness, but this functional importance has never been tested in the context of a whole organism. Here, we report that a poison exon inSmndc1is conserved across mammals and plants and plays a molecular autoregulatory function in both kingdoms. We generated mouse andA.thalianamodels lacking this poison exon to find its loss leads to deregulation of SMNDC1 protein levels, pervasive alterations in mRNA processing, and organismal size restriction. Together, these models demonstrate the importance of poison exons for both molecular and organismal phenotypes that likely explain their extraordinary conservation. 

Abstract The rich diversity of angiosperms, both the planet's dominant flora and the cornerstone of agriculture, is integrally intertwined with a distinctive evolutionary history. Here, we explore the interplay between angiosperm genome organization and botanical diversity, empowered by genomic approaches ranging from genetic linkage mapping to analysis of gene regulation. Commonality in the genetic hardware of plants has enabled robust comparative genomics that has provided a broad picture of angiosperm evolution and implicated both general processes and specific elements in contributing to botanical diversity. We argue that the hardware of plant genomes—both in content and in dynamics—has been shaped by selection for rather substantial differences in gene regulation between plants and animals such as maize and human, organisms of comparable genome size and gene number. Their distinctive genome content and dynamics may reflect in part the indeterminate development of plants that puts strikingly different demands on gene regulation than in animals. Repeated polyploidization of plant genomes and multiplication of individual genes together with extensive rearrangement and differential retention provide rich raw material for selection of morphological and/or physiological variations conferring fitness in specific niches, whether natural or artificial. These findings exemplify the burgeoning information available to employ in increasing knowledge of plant biology and in modifying selected plants to better meet human needs. 

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 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.