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Abstract Regulatory networks coordinate metabolism to control how plants adapt to biotic and abiotic stresses. This coordination can align transcriptional shifts across metabolic pathways using cis-regulatory elements shared across the enzyme genes within these pathways. While the role of transcription factors (TFs) in controlling this process across pathways is well known, less is known regarding the role of shared cis-regulatory elements across the genes in a pathway. Sharing cis-regulatory elements across the genes in an enzyme complex or pathway, can create coordinated regulation of the pathway by a TF. However, it is unclear if all the genes in a pathway or enzyme complex need to be fully coordinated for maximal function. For example, if one gene in an enzyme complex loses a cis-regulatory element, it may not alter the function of the enzyme complexes function if post-transcriptional or compensatory transcriptional changes are sufficient to balance the complex. To test how cis-modular membership shapes the function of an enzyme complex, we used CRISPR/Cas9 to abolish a common cis-regulatory element across the promoters of nine genes required for the mitochondrial pyruvate dehydrogenase complex (mtPDC). This complex is composed of three apoenzymes and is a central hub coordinating carbon flow between glycolysis and the tricarboxylic acid (TCA) cycle. Different combinations of these cis-element mutations were tested across the genes in the complex inArabidopsis thalianaand the created genotypes were phenotyped for altered enzyme function using digital growth analysis, disease assays, metabolomics, and transcriptomics. This analysis revealed that mutating cis-element motifs of genes in this enzyme complex produced distinct phenotypes, displaying promoter-specific buffering and modularity. 

Abstract Arabidopsis thaliana (hereafter Arabidopsis) is a small plant with a fast generation time and a well-annotated genome, which makes it ideal for research labs. It is arguably the most used model species in basic plant sciences. Over the past half century, studies in Arabidopsis have generated enormous insight into fundamental principles of plant life, ranging from mechanistic molecular biology to the complexities of interacting ecosystems. Based on research in Arabidopsis, we now understand that while basic cellular metabolism is generally conserved across species, variation in specialized metabolite enzymes gives rise to complex bouquets of chemical weapons that are tightly interwoven with the environment. Understanding how these are produced, regulated, and—especially—how they are deployed remains a key research area for plant immunity. The breadth of work in Arabidopsis provides a unique window into this complicated aspect of life as a plant. We are happy to have an opportunity to share our common interest in these aspects in this review. Due to space constraints, we focus on compounds produced by Arabidopsis with demonstrated antimicrobial properties. We hope that this focus (despite our eagerness to write more) will inspire new avenues of research that will contribute to a more complete understanding of immunity. 

Abstract Diversity in plant specialized metabolites plays critical roles in plant–environment interactions. In longer evolutionary scales, e.g. between families or orders, this diversity arises from whole-genome and tandem duplication events. Less is known about the evolutionary patterns that shape chemical diversity at shorter scales, e.g. within a family. Utilizing the aliphatic glucosinolate pathway, we explored how the genes encoding the terminal structural modification enzyme GSL-OH evolved across the Brassicaceae and the genomic processes that control presence–absence variation of its products (R)-2-hydroxy-but-3-enyl and (S)-2-hydroxy-but-3-enyl glucosinolate. We implemented a phylo-functional approach to functionally validate GSL-OH orthologs across the Brassicaceae and used that information to map the genomic origin and trajectory of the locus. This uncovered a complex mechanism involving at least 3 ancestral loci with extensive gene loss across all species, creating unequal retention across the phylogenetic relationships. Convergent evolution in enantiomeric specificity was observed, where several independent species had tandem duplicates that diverged toward producing the R or S enantiomers. To explore potential biological differences between the enantiomers, we performed Trichoplusia ni larval choice assays and tested resistance against Botrytis cinerea in a detached leaf assay. We found that plants with the S-enantiomer were more susceptible to B. cinerea infection than to T. ni larval herbivory, while plants with the R-enantiomer seemed more susceptible to T. ni larval herbivory when compared to B. cinerea. Ultimately, we observed recurrent GSL-OH loss, uncovered a complex origin story for the gene, and measured the bioactivity of the enzyme's metabolic products. 

Abstract Data reduction methods are frequently employed in large genomics and phenomics studies to extract core patterns, reduce dimensionality, and alleviate multiple testing effects. Principal component analysis (PCA), in particular, identifies the components that capture the most variance within omics datasets. While data reduction can simplify complex datasets, it remains unclear how the use of PCA impacts downstream analyses such as quantitative trait loci (QTL) or genome-wide association (GWA) approaches and their biological interpretation. In QTL studies, an alternative to data reduction is the use of post-hoc data summarization approaches, such as hotspot analysis, which involves mapping individual traits and consolidating results based on shared genomic locations. To evaluate how different analytical approaches may alter the biological insights derived from multi-dimensional QTL datasets, we compared individual trait hotspots with PCA-based QTL mapping using transcriptomic and metabolomic data from a structured recombinant inbred line population. Interestingly, these two approaches identified different genomic regions and genetic architectures. These findings suggest that mapping PCA-reduced data does not merely streamline analyses but may generate a fundamentally different view of the underlying genetic architecture compared to individual trait mapping and hotspot analysis. Thus, the use of PCA and other data reduction techniques prior to QTL or GWAS mapping should be carefully considered to ensure alignment with the specific biological question being addressed. 

Summary Eukaryotic genomes harbor many forms of variation, including nucleotide diversity and structural polymorphisms, which experience natural selection and contribute to genome evolution and biodiversity. However, harnessing this variation for agriculture hinges on our ability to detect, quantify, catalog, and utilize genetic diversity.Here, we explore seven complete genomes of the emerging biofuel crop pennycress (Thlaspi arvense) drawn from across the species’s current genetic diversity to catalogue variation in genome structure and content.Across this new pangenome resource, we find contrasting evolutionary modes in different genomic regions. Gene-poor, repeat-rich pericentromeric regions experience frequent rearrangements, including repeated centromere repositioning. In contrast, conserved gene-dense chromosome arms maintain large-scale synteny across accessions, even in fast-evolving immune genes where microsynteny breaks down across species but the macrosynteny of gene cluster positioning is maintained.Our findings highlight that multiple elements of the genome experience dynamic evolution that conserves functional content on the chromosome scale but allows rearrangement and presence-absence variation on a local scale. This diversity is invisible to classical reference-based approaches and highlights the strength and utility of pangenomic resources. These results provide a valuable case study of rapid genomic structural evolution within a species and powerful resources for crop development in an emerging biofuel crop. 

Abstract Organisms regulate gene expression in response to environmental cues, a process known as plasticity, to adjust to changing environments. Research into natural variation and the evolution of plasticity frequently studies cis-regulatory elements with theory suggesting they are more important evolutionarily than trans-regulatory elements. Genome-wide association (GWA) studies have supported this idea, observing a predominance of cis-loci affecting plasticity. However, studies in structured populations provide a contrasting image, raising questions about the genetic architecture of natural variation in plasticity. To circumvent potential statistical difficulties present in GWA studies, we mapped loci underlying transcriptomic plasticity in response to salicylic acid (SA) using recombinant inbred lines generated from 2 random Arabidopsis thaliana accessions. We detected extensive transgressive segregation in the SA response, suggesting that plasticity to salicylate in Arabidopsis is polygenic. Most loci (>75%) underlying this variation act in trans, especially for loci influencing plasticity. Trans-acting loci were enriched in genome hotspots, with predominantly small-effect sizes distributed across many genes. This could potentially explain their under-discovery in GWA studies. This work reveals a potentially important role for trans-acting loci in plastic expression responses, with implications for understanding plant adaptation to different environments. 

Abstract Three cross-incompatibility loci each control a distinct reproductive barrier in both domesticated maize (Zea mays ssp. mays) and its wild teosinte relatives. These 3 loci, Teosinte crossing barrier1 (Tcb1), Gametophytic factor1 (Ga1), and Ga2, each play a key role in preventing hybridization between incompatible populations and are proposed to maintain the barrier between domesticated and wild subspecies. Each locus encodes both a silk-active and a matching pollen-active pectin methylesterase (PMEs). To investigate the diversity and molecular evolution of these gametophytic factor loci, we identified existing and improved models of the responsible genes in a new genome assembly of maize line P8860 that contains active versions of all 3 loci. We then examined 52 assembled genomes from 17 species to classify haplotype diversity and identify sites under diversifying selection during the evolution of these genes. We show that Ga2, the oldest of these 3 loci, was duplicated to form Ga1 at least 12 million years ago. Tcb1, the youngest locus, arose as a duplicate of Ga1 before or around the time of diversification of the Zea genus. We find evidence of positive selection during evolution of the functional genes at an active site in the pollen-expressed PME and predicted surface sites in both the silk- and pollen-expressed PMEs. The most common allele at the Ga1 locus is a conserved ga1 allele (ga1-Off), which is specific haplotype containing 3 full-length PME gene copies, all of which are noncoding due to conserved stop codons and are between 610 thousand and 1.5 million years old. We show that the ga1-Off allele is associated with and likely generates 24-nt siRNAs in developing pollen-producing tissue, and these siRNAs map to functional Ga1 alleles. In previously published crosses, the ga1-Off allele was associated with reduced function of the typically dominant functional alleles for the Ga1 and Tcb1 barriers. Taken together, this seems to be an example of an allele at a reproductive barrier locus being associated with an as yet undetermined mechanism capable of silencing the reproductive barrier. 

Abstract Transposable elements can be activated in response to environmental changes and lead to changes in DNA sequence. Their target sites of insertions have previously been thought to be random, but this theory has lately been contradicted. For instance, mobilization is favored towards genes involved in regulatory processes. This makes them interesting as potential players in rapid responses required under stressful environmental conditions. In this paper, we report the in-depth characterization of anArabidopsis thalianaCol-0-based line whose altered DNA methylation pattern made it vulnerable for transposable element movement. We identified a transposable element retrotransposition into a transporter of glucosinolate defense compounds. As a consequence of this transposable element movement, the plants showed tissue-specific changes in glucosinolate profiles and levels accompanied by rewiring of glucosinolate- and defense-related transcriptional changes. As this single transposable element had strong impact on the plants’ resistance to insect herbivory, our findings highlight the potential for transposable elements to play a role in plant adaptation. 

Abstract Recent technical and theoretical advances have generated an explosion in the identification of specialized metabolite pathways. In comparison, our understanding of how these pathways are regulated is relatively lagging. This and the relatively young age of specialized metabolite pathways has partly contributed to a default and common paradigm whereby specialized metabolite regulation is theorized as relatively simple with a few key transcription factors and the compounds are non-regulatory end-products. In contrast, studies into model specialized metabolites, such as glucosinolates, are beginning to identify a new understanding whereby specialized metabolites are highly integrated into the plants’ core metabolic, physiological, and developmental pathways. This model includes a greatly extended compendium of transcription factors controlling the pathway, key transcription factors that co-evolve with the pathway and simultaneously control core metabolic and developmental components, and finally the compounds themselves evolve regulatory connections to integrate into the plants signaling machinery. In this review, these concepts are illustrated using studies in the glucosinolate pathway within the Brassicales. This suggests that the broader community needs to reconsider how they do or do not integrate specialized metabolism into the regulatory network of their study species. 

SUMMARY Meristem function is underpinned by numerous genes that affect hormone levels, ultimately controlling phyllotaxy, the transition to flowering and general growth properties. Class I KNOX genes are major contributors to this process, promoting cytokinin biosynthesis but repressing gibberellin production to condition a replication competent state. We identified a suppressor mutant of theKNOX1mutantbrevipedicellus(bp) that we termedflasher(fsh), which promotes stem and pedicel elongation, suppresses early senescence, and negatively affects reproductive development. Map‐based cloning and complementation tests revealed thatfshis due to an E40K change in the flavin monooxygenaseGS‐OX5, a gene encoding a glucosinolate (GSL) modifying enzyme.In vitroenzymatic assays revealed thatfshpoorly converts substrate to product, yet the levels of several GSLs are higher in the suppressor line, implicatingFSHin feedback control of GSL flux.FSHis expressed predominantly in the vasculature in patterns that do not significantly overlap those ofBP, implying a non‐cell autonomous mode of meristem control via one or more GSL metabolites. Hormone analyses revealed that cytokinin levels are low inbp, butfshrestores cytokinin levels to near normal by activating cytokinin biosynthesis genes. In addition, jasmonate levels in thefshsuppressor are significantly lower than inbp, which is likely due to elevated expression of JA inactivating genes. These observations suggest the involvement of the GSL pathway in generating one or more negative effectors of growth that influence inflorescence architecture and fecundity by altering the balance of hormonal regulators.