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Abstract Cryptic genetic variants exert minimal phenotypic effects alone but are hypothesized to form a vast reservoir of genetic diversity driving trait evolvability through epistatic interactions^1–3. This classical theory has been reinvigorated by pan-genomics, which is revealing pervasive variation within gene families,cis-regulatory regions and regulatory networks^4–6. Testing the ability of cryptic variation to fuel phenotypic diversification has been hindered by intractable genetics, limited allelic diversity and inadequate phenotypic resolution. Here, guided by natural and engineeredcis-regulatory cryptic variants in a paralogous gene pair, we identified additional redundanttransregulators, establishing a regulatory network controlling tomato inflorescence architecture. By combining coding mutations withcis-regulatory alleles in populations segregating for all four network genes, we generated 216 genotypes spanning a wide spectrum of inflorescence complexity and quantified branching in over 35,000 inflorescences. Analysis of this high-resolution genotype–phenotype map using a hierarchical model of epistasis revealed a layer of dose-dependent interactions within paralogue pairs enhancing branching, culminating in strong, synergistic effects. However, we also identified a layer of antagonism between paralogue pairs, whereby accumulating mutations in one pair progressively diminished the effects of mutations in the other. Our results demonstrate how gene regulatory network architecture and complex dosage effects from paralogue diversification converge to shape phenotypic space, producing the potential for both strongly buffered phenotypes and sudden bursts of phenotypic change. 

Abstract Modern maize (Zea maysssp.mays) was domesticated fromTeosinte parviglumis(Zea maysssp.parviglumis), with subsequent introgressions fromTeosinte mexicana(Zea maysssp.mexicana), yielding increased kernel row number, loss of the hard fruit case and dissociation from the cob upon maturity, as well as fewer tillers. Molecular approaches have identified transcription factors controlling these traits, yet revealed that a complex regulatory network is at play. MaizeCODE deploys ENCODE strategies to catalog regulatory regions in the maize genome, generating histone modification and transcription factor ChIP-seq in parallel with transcriptomics datasets in 5 tissues of 3 inbred lines which span the phenotypic diversity of maize, as well as the teosinte inbred TIL11. Transcriptomic analysis reveals that pollen grains share features with endosperm, and express dozens of “proto-miRNAs” potential vestiges of gene drive and hybrid incompatibility. Integrated analysis with chromatin modifications results in the identification of a comprehensive set of regulatory regions in each tissue of each inbred, and notably of distal enhancers expressing non-coding enhancer RNAs bi-directionally, reminiscent of “super enhancers” in animal genomes. Furthermore, the morphological traits selected during domestication are recapitulated, both in gene expression and within regulatory regions containing enhancer RNAs, while highlighting the conflict between enhancer activity and silencing of the neighboring transposable elements. 

SUMMARY Carotenoids perform a broad range of important functions in humans; therefore, carotenoid biofortification of maize (Zea maysL.), one of the most highly produced cereal crops worldwide, would have a global impact on human health.PLASTID TERMINAL OXIDASE(PTOX) genes play an important role in carotenoid metabolism; however, the possible function ofPTOXin carotenoid biosynthesis in maize has not yet been explored. In this study, we characterized the maizePTOXlocus by forward‐ and reverse‐genetic analyses. While most higher plant species possess a single copy of thePTOXgene, maize carries two tandemly duplicated copies. Characterization of mutants revealed that disruption of either copy resulted in a carotenoid‐deficient phenotype. We identified mutations in thePTOXgenes as being causal of the classic maize mutant,albescent1. Remarkably, overexpression ofZmPTOX1significantly improved the content of carotenoids, especially β‐carotene (provitamin A), which was increased by ~threefold, in maize kernels. Overall, our study shows that maizePTOXlocus plays an important role in carotenoid biosynthesis in maize kernels and suggests that fine‐tuning the expression of this gene could improve the nutritional value of cereal grains. 

Summary Gene duplication is a powerful source of biological innovation giving rise to paralogous genes that undergo diverse fates. Redundancy between paralogous genes is an intriguing outcome of duplicate gene evolution, and its maintenance over evolutionary time has long been considered a paradox. Redundancy can also be dubbed ‘a geneticist's nightmare’: It hinders the predictability of genome editing outcomes and limits our ability to link genotypes to phenotypes. Genetic studies in yeast and plants have suggested that the ability of ancient redundant duplicates to compensate for dosage perturbations resulting from a loss of function depends on the reprogramming of gene expression, a phenomenon known as active compensation. Starting from considerations on the stoichiometric constraints that drive the evolutionary stability of redundancy, this review aims to provide insights into the mechanisms of active compensation between duplicates that could be targeted for breaking paralog dependencies – the next frontier in plant functional studies. 

Abstract Maize ear size and kernel number differ among lines, however, little is known about the molecular basis of ear length and its impact on kernel number. Here, we characterize a quantitative trait locus,qEL7, to identify a maize gene controlling ear length, flower number and fertility.qEL7encodes 1-aminocyclopropane-1- carboxylate oxidase2 (ACO2), a gene that functions in the final step of ethylene biosynthesis and is expressed in specific domains in developing inflorescences. Confirmation ofqEL7by gene editing ofZmACO2leads to a reduction in ethylene production in developing ears, and promotes meristem and flower development, resulting in a ~13.4% increase in grain yield per ear in hybrids lines. Our findings suggest that ethylene serves as a key signal in inflorescence development, affecting spikelet number, floral fertility, ear length and kernel number, and also provide a tool to improve grain productivity by optimizing ethylene levels in maize or in other cereals. 

Developmental transitions require precise temporal and spatial control of gene expression. In plants, such regulation is critical for flower formation, which involves the progressive maturation of stem cell populations within shoot meristems to floral meristems, followed by rapid sequential differentiation into floral organs. Across plant taxa, these transitions are orchestrated by the F-box transcriptional cofactor geneUNUSUAL FLORAL ORGANS(UFO). The conserved and pleiotropic functions ofUFOoffer a useful framework for investigating how evolutionary processes have shaped the intricatecis-regulation of key developmental genes. By pinpointing a conserved promoter sequence in an accessible chromatin region of the tomato ortholog ofUFO, we engineered in vivo a series ofcis-regulatory alleles that caused both loss- and gain-of-function floral defects. These mutant phenotypes were linked to disruptions in predicted transcription factor binding sites for known transcriptional activators and repressors. Allelic combinations revealed dosage-dependent interactions between opposing alleles, influencing the penetrance and expressivity of gain-of-function phenotypes. These phenotypic differences support that robustness in tomato flower development requires precise temporal control ofUFOexpression dosage. Bridging our analysis toArabidopsis, we found that although homologous sequences to the tomato regulatory region are dispersed within theUFOpromoter, they maintain similar control over floral development. However, phenotypes from disrupting these sequences differ due to the differing expression patterns ofUFO. Our study underscores the complexcis-regulatory control of dynamic developmental genes and demonstrates that critical short stretches of regulatory sequences that recruit both activating and repressing machinery are conserved to maintain developmental robustness. 

Heterozygous mutations in two genes encoding key regulators of development improve kernel row number in inbred and hybrid maize, revealing their potential for yield improvement. 

A striking paradox is that genes with conserved protein sequence, function and expression pattern over deep time often exhibit extremely divergentcis-regulatory sequences. It remains unclear how such drasticcis-regulatory evolution across species allows preservation of gene function, and to what extent these differences influence howcis-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. Usingin-vivogenome editing in two distantly related models,Arabidopsis thaliana(Arabidopsis) andSolanum lycopersicum(tomato), we generated over 70 deletion alleles in the upstream and downstream regions of the stem cell repressor geneCLAVATA3(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 tomatoCLV3are highly sensitive to even small perturbations compared to its downstream region. In contrast, ArabidopsisCLV3function 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 ArabidopsisCLV3caused substantial and synergistic effects, demonstrating distinct distribution and redundancy of functionalcis-regulatory sequences. Our results demonstrate remarkable malleability incis-regulatory structural organization of a deeply conserved plant stem cell regulator and suggest that major reconfiguration ofcis-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 ofcis-regulation to effectively engineer trait variation from conserved productivity genes in crops.