Differential Regulation of Strand-Specific Transcripts from Arabidopsis Centromeric Satellite Repeats

Centromeres interact with the spindle apparatus to enable chromosome disjunction and typically contain thousands of tandemly arranged satellite repeats interspersed with retrotransposons. While their role has been obscure, centromeric repeats are epigenetically modified and centromere specification has a strong epigenetic component. In the yeast Schizosaccharomyces pombe, long heterochromatic repeats are transcribed and contribute to centromere function via RNA interference (RNAi). In the higher plant Arabidopsis thaliana, as in mammalian cells, centromeric satellite repeats are short (180 base pairs), are found in thousands of tandem copies, and are methylated. We have found transcripts from both strands of canonical, bulk Arabidopsis repeats. At least one subfamily of 180–base pair repeats is transcribed from only one strand and regulated by RNAi and histone modification. A second subfamily of repeats is also silenced, but silencing is lost on both strands in mutants in the CpG DNA methyltransferase MET1, the histone deacetylase HDA6/SIL1, or the chromatin remodeling ATPase DDM1. This regulation is due to transcription from Athila2 retrotransposons, which integrate in both orientations relative to the repeats, and differs between strains of Arabidopsis. Silencing lost in met1 or hda6 is reestablished in backcrosses to wild-type, but silencing lost in RNAi mutants and ddm1 is not. Twenty-four–nucleotide small interfering RNAs from centromeric repeats are retained in met1 and hda6, but not in ddm1, and may have a role in this epigenetic inheritance. Histone H3 lysine-9 dimethylation is associated with both classes of repeats. We propose roles for transcribed repeats in the epigenetic inheritance and evolution of centromeres

Automated assembly scaffolding using RagTag elevates a new tomato system for high-throughput genome editing

Advancing crop genomics requires efficient genetic systems enabled by high-quality personalized genome assemblies. Here, we introduce RagTag, a toolset for automating assembly scaffolding and patching, and we establish chromosome-scale reference genomes for the widely used tomato genotype M82 along with Sweet-100, a new rapid-cycling genotype that we developed to accelerate functional genomics and genome editing in tomato. This work outlines strategies to rapidly expand genetic systems and genomic resources in other plant species

Establishing Physalis as a Solanaceae model system enables genetic reevaluation of the inflated calyx syndrome

The highly diverse Solanaceae family contains several widely studied model and crop species. Fully exploring, appreciating, and exploiting this diversity requires additional model systems. Particularly promising are orphan fruit crops in the genus Physalis, which occupy a key evolutionary position in the Solanaceae and capture understudied variation in traits such as inflorescence complexity, fruit ripening and metabolites, disease and insect resistance, self-compatibility, and most notable, the striking inflated calyx syndrome (ICS), an evolutionary novelty found across angiosperms where sepals grow exceptionally large to encapsulate fruits in a protective husk. We recently developed transformation and genome editing in Physalis grisea (groundcherry). However, to systematically explore and unlock the potential of this and related Physalis as genetic systems, high-quality genome assemblies are needed. Here, we present chromosome-scale references for P. grisea and its close relative P. pruinosa and use these resources to study natural and engineered variation in floral traits. We first rapidly identified a natural structural variant in a bHLH gene that causes petal color variation. Further, and against expectations, we found that CRISPR-Cas9 targeted mutagenesis of 11 MADS-box genes, including purported essential regulators of ICS, had no effect on inflation. In a forward genetics screen, we identified huskless, which lacks ICS due to mutation of an AP2-like gene that causes sepals and petals to merge into a single whorl of mixed identity. These resources and findings elevate Physalis to a new Solanaceae model system, and establish a paradigm in the search for factors driving ICS

The Making of a Compound Inflorescence in Tomato and Related Nightshades

Variation in the branching of plant inflorescences determines flower number and, consequently, reproductive success and crop yield. Nightshade (Solanaceae) species are models for a widespread, yet poorly understood, program of eudicot growth, where short side branches are initiated upon floral termination. This “sympodial” program produces the few-flowered tomato inflorescence, but the classical mutants compound inflorescence (s) and anantha (an) are highly branched, and s bears hundreds of flowers. Here we show that S and AN, which encode a homeobox transcription factor and an F-box protein, respectively, control inflorescence architecture by promoting successive stages in the progression of an inflorescence meristem to floral specification. S and AN are sequentially expressed during this gradual phase transition, and the loss of either gene delays flower formation, resulting in additional branching. Independently arisen alleles of s account for inflorescence variation among domesticated tomatoes, and an stimulates branching in pepper plants that normally have solitary flowers. Our results suggest that variation of Solanaceae inflorescences is modulated through temporal changes in the acquisition of floral fate, providing a flexible evolutionary mechanism to elaborate sympodial inflorescence shoots

Extreme restructuring of cis-regulatory regions controlling a deeply conserved plant stem cell regulator.

A striking paradox is that genes with conserved protein sequence, function and expression pattern over deep time often exhibit extremely divergent cis-regulatory sequences. It remains unclear how such drastic cis-regulatory evolution across species allows preservation of gene function, and to what extent these differences influence how cis-regulatory variation arising within species impacts phenotypic change. Here, we investigated these questions using a plant stem cell regulator conserved in expression pattern and function over ~125 million years. Using in-vivo genome editing in two distantly related models, Arabidopsis thaliana (Arabidopsis) and Solanum lycopersicum (tomato), we generated over 70 deletion alleles in the upstream and downstream regions of the stem cell repressor gene CLAVATA3 (CLV3) and compared their individual and combined effects on a shared phenotype, the number of carpels that make fruits. We found that sequences upstream of tomato CLV3 are highly sensitive to even small perturbations compared to its downstream region. In contrast, Arabidopsis CLV3 function is tolerant to severe disruptions both upstream and downstream of the coding sequence. Combining upstream and downstream deletions also revealed a different regulatory outcome. Whereas phenotypic enhancement from adding downstream mutations was predominantly weak and additive in tomato, mutating both regions of Arabidopsis CLV3 caused substantial and synergistic effects, demonstrating distinct distribution and redundancy of functional cis-regulatory sequences. Our results demonstrate remarkable malleability in cis-regulatory structural organization of a deeply conserved plant stem cell regulator and suggest that major reconfiguration of cis-regulatory sequence space is a common yet cryptic evolutionary force altering genotype-to-phenotype relationships from regulatory variation in conserved genes. Finally, our findings underscore the need for lineage-specific dissection of the spatial architecture of cis-regulation to effectively engineer trait variation from conserved productivity genes in crops

Meristem maturation and inflorescence architecture-lessons from the Solanaceae

Plant apical meristems (AMs) grow continuously by delicately balancing cells leaving at the periphery to form lateral organs with slowly dividing central domain cells that replenish reservoirs of pluripotent cells. This balance can be modified by signals originating from within and outside the meristem, and their integration results in a gradual maturation process that often culminates with the meristem differentiating into a flower. Accompanying this ‘meristem maturation’ are changes in spacing and size of lateral organs and in rates at which lateral meristems are released from apical dominance. Modulation of distinct meristem maturation parameters through environmental and genetic changes underlies the remarkable diversity of shoot architectures. Here, we discuss recent studies relating the dynamics of meristem maturation with organization of floral branching systems — inflorescences — in the nightshades. From this context, we suggest general principles on how factors coordinating meristem maturation impact shoot organization more broadly

Engineering Quantitative Trait Variation for Crop Improvement by Genome Editing

Summary Major advances in crop yields are needed in the coming decades. However, plant breeding is currently limited by incremental improvements in quantitative traits that often rely on laborious selection of rare naturally occurring mutations in gene-regulatory regions. Here, we demonstrate that CRISPR/Cas9 genome editing of promoters generates diverse cis-regulatory alleles that provide beneficial quantitative variation for breeding. We devised a simple genetic scheme, which exploits trans-generational heritability of Cas9 activity in heterozygous loss-of-function mutant backgrounds, to rapidly evaluate the phenotypic impact of numerous promoter variants for genes regulating three major productivity traits in tomato: fruit size, inflorescence branching, and plant architecture. Our approach allows immediate selection and fixation of novel alleles in transgene-free plants and fine manipulation of yield components. Beyond a platform to enhance variation for diverse agricultural traits, our findings provide a foundation for dissecting complex relationships between gene-regulatory changes and control of quantitative traits

Tomato <i>CLV3</i> alleles used for interaction tests.

Heatmap representations of the SlCLV3 alleles used in interaction tests, with their locule number quantifications. Each interaction test consisted of a linear model generated from the relationship among four alleles: one 5’+3’ combinatorial allele, one 5’ allele, one 3’ allele, and WT. The R4 and R1 regions previously defined are highlighted by purple boxes on the SlCLV3 5’ non-coding sequence [34]. Purple arrowheads, gRNAs. (TIF)</p

The function of CLV3 in Arabidopsis and tomato is conserved despite extreme divergence in <i>cis</i>-regulatory sequences.

(A) A representative shoot apical meristem (SAM), demonstrating the conserved negative feedback loop between the signaling peptide CLV3 and the transcription factor WUS. CLV3 peptide indirectly inhibits WUS expression, while WUS promotes CLV3 expression. (B) Top-down view of Arabidopsis siliques from wild type (WT) and an Atclv3 null mutant. White arrows, individual locules. Scale bars, 1 mm. (C) Transverse sections of tomato fruits from WT and a Slclv3 null mutant. White arrows, individual locules. Scale bars, 1 cm. (D) AtCLV3 gene model and surrounding regulatory regions upstream (~3.8 kb) and downstream (~2.6 kb). mVISTA DNA sequence alignments of seven CLV3 orthologs from Brassicaceae species, using the AtCLV3 gene and its surrounding regulatory regions as the reference sequence. SlCLV3 could not be aligned to AtCLV3. Sequences conserved in all species are highlighted in dark green, and sequences conserved in at least half of the species are highlighted in light green. A representative diagram of the Arabidopsis SAM is shown to the left, indicating the location of CLV3 RNA expression relative to previously defined regions. L1, L2, and L3 layers are denoted by dotted black lines, the central zone (CZ) is outlined in yellow, and CLV3 transcripts are represented in red. (E) SlCLV3 gene model and surrounding regulatory regions upstream (~5.5 kb) and downstream (~0.7 kb). mVISTA DNA sequence alignments of seven CLV3 orthologs from Solanaceae species, using the SlCLV3 gene and its surrounding regulatory regions as the reference sequence. AtCLV3 could not be aligned to SlCLV3. Sequences conserved in all species are highlighted in dark red, and sequences conserved in at least half of species are highlighted in light red. Regions of the SlCLV3 promoter previously defined are highlighted in purple [34]. A representative diagram of the tomato SAM is shown to the left, indicating the location of CLV3 RNA expression relative to previously defined regions. (D)–(E) Conservation was calculated as sequences with 85% similarity in 20 bp windows. The gene models are annotated with the location of previously validated TFBSs (black and brown arrows), meristem open chromatin (yellow bars), and conserved non-coding sequences (CNSs) (blue arrows) defined by Conservatory [24,35,36]. Light blue, conserved UTRs. Dark blue, conserved exons. Pink, conserved regions.</p

Combined mutagenesis of regions upstream and downstream of <i>AtCLV3</i> uncovers functional redundancy between these regions.

(A) Schematic describing sequential CRISPR-Cas9 mutagenesis technique. Either a fixed 5’ allele was transformed with 3’-targeted gRNAs to induce new 3’ mutations, or a fixed 3’ allele was transformed with 5’-targeted gRNAs to induce new 5’ mutations. Transgenics were screened for new mutations, and alleles with both 5’ and 3’ mutations were selected. Genetic interaction tests were applied to explore the relationship between combined mutations in the 5’ and 3’ regions. (B) Heatmap representation of alleles generated from sequential CRISPR-Cas9 targeting with the AtCLV3 3’-gRNA array, in the background of the fixed 5’ mutant AtCLV3Reg5’-8, and their locule number quantifications. (C) Heatmap representation of alleles generated from sequential CRISPR-Cas9 targeting with the AtCLV3 3’-gRNA array, in the background of the fixed 5’ mutant AtCLV3Reg5’-7, and their locule number quantifications. (D) Heatmap representation of alleles generated from sequential CRISPR-Cas9 targeting with the AtCLV3 proximal 1.5 kb 5’-gRNA array, in the background of the fixed 3’ mutant AtCLV3Reg3’-3, and their locule number quantifications. (E) Representative silique images from WT and several alleles generated through sequential CRISPR-Cas9 editing. A top-down view is shown below. White arrows, individual locules. Scale bars, 1 mm. (F) Interaction tests performed between combined 5’+3’ alleles and similar individual 5’ and 3’ mutants (S1 Fig). p-values of the interaction effect were adjusted for multiple comparisons. (B)–(D) Locule number quantifications are represented by stacked bar plots and box plots. Box plots show the 25th, 50th (median) and 75th percentiles, with outliers as black points. Number of fruits sampled (n) is shown to the left, and mean and standard deviation (sd) are shown to the right. Grey boxes highlight identified regions of importance for regulation. R, redundant interaction type. S, synergistic interaction type. A, additive interaction type. Purple arrowheads, gRNAs. Black and brown arrows, validated WUS and STM TFBSs. Blue arrowheads, Conservatory CNSs. Green bars, mVISTA CNSs. Two-sided Dunnett’s compare with control tests were performed to compare WT and sequentially edited alleles to (B) AtCLV3Reg5’-8, (C) AtCLV3Reg5’-7, or (D) AtCLV3Reg3’-3.</p