RESUMEN
Cis-regulatory mutations underlie important crop domestication and improvement traits1,2. However, limited allelic diversity has hindered functional dissection of the large number of cis-regulatory elements and their potential interactions, thereby precluding a deeper understanding of how cis-regulatory variation impacts traits quantitatively. Here, we engineered over 60 promoter alleles in two tomato fruit size genes3,4 to characterize cis-regulatory sequences and study their functional relationships. We found that targeted mutations in conserved promoter sequences of SlCLV3, a repressor of stem cell proliferation5,6, have a weak impact on fruit locule number. Pairwise combinations of these mutations mildly enhance this phenotype, revealing additive and synergistic relationships between conserved regions and further suggesting even higher-order cis-regulatory interactions within the SlCLV3 promoter. In contrast, SlWUS, a positive regulator of stem cell proliferation repressed by SlCLV3 (refs. 5,6), is more tolerant to promoter perturbations. Our results show that complex interplay among cis-regulatory variants can shape quantitative variation, and suggest that empirical dissections of this hidden complexity can guide promoter engineering to predictably modify crop traits.
Asunto(s)
Fenotipo , Células Vegetales/fisiología , Regiones Promotoras Genéticas/genética , Sitios de Carácter Cuantitativo , Secuencias Reguladoras de Ácidos Nucleicos , Solanum lycopersicum/genética , Células Madre/fisiología , Alelos , DomesticaciónRESUMEN
Divergence of gene function is a hallmark of evolution, but assessing functional divergence over deep time is not trivial. The few alleles available for cross-species studies often fail to expose the entire functional spectrum of genes, potentially obscuring deeply conserved pleiotropic roles. Here, we explore the functional divergence of WUSCHEL HOMEOBOX9 (WOX9), suggested to have species-specific roles in embryo and inflorescence development. Using a cis-regulatory editing drive system, we generate a comprehensive allelic series in tomato, which revealed hidden pleiotropic roles for WOX9. Analysis of accessible chromatin and conserved cis-regulatory sequences identifies the regions responsible for this pleiotropic activity, the functions of which are conserved in groundcherry, a tomato relative. Mimicking these alleles in Arabidopsis, distantly related to tomato and groundcherry, reveals new inflorescence phenotypes, exposing a deeply conserved pleiotropy. We suggest that targeted cis-regulatory mutations can uncover conserved gene functions and reduce undesirable effects in crop improvement.
Asunto(s)
Genes de Plantas , Pleiotropía Genética/genética , Proteínas de Homeodominio/genética , Proteínas de Plantas/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Alelos , Arabidopsis/genética , Sistemas CRISPR-Cas/genética , Cromatina/metabolismo , Regulación de la Expresión Génica de las Plantas , Inflorescencia/genética , Solanum lycopersicum/genética , Mutagénesis , Desarrollo de la Planta/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/metabolismo , Regiones Promotoras Genéticas , Solanaceae/genética , Solanaceae/crecimiento & desarrolloRESUMEN
Here we present an optimized protocol for immunolocalization of meiotic proteins during female meiosis in whole mount tissues. It ensures ovule morphology integrity and homogeneous reagent penetration. The method relies on paraformaldehyde tissue fixation, polyacrylamide embedding, tissue permeabilization, antibody incubation, counterstaining, and confocal microscopy analysis. This protocol has been used in diverse Arabidopsis ecotypes and in the legume Vigna unguiculata.
Asunto(s)
Inmunohistoquímica , Meiosis , Células Vegetales/fisiología , Arabidopsis/citología , Arabidopsis/metabolismo , Inmunohistoquímica/métodos , Microscopía ConfocalRESUMEN
Precise control of plant stem cell proliferation is necessary for the continuous and reproducible development of plant organs1,2. The peptide ligand CLAVATA3 (CLV3) and its receptor protein kinase CLAVATA1 (CLV1) maintain stem cell homeostasis within a deeply conserved negative feedback circuit1,2. In Arabidopsis, CLV1 paralogs also contribute to homeostasis, by compensating for the loss of CLV1 through transcriptional upregulation3. Here, we show that compensation4,5 operates in diverse lineages for both ligands and receptors, but while the core CLV signaling module is conserved, compensation mechanisms have diversified. Transcriptional compensation between ligand paralogs operates in tomato, facilitated by an ancient gene duplication that impacted the domestication of fruit size. In contrast, we found little evidence for transcriptional compensation between ligands in Arabidopsis and maize, and receptor compensation differs between tomato and Arabidopsis. Our findings show that compensation among ligand and receptor paralogs is critical for stem cell homeostasis, but that diverse genetic mechanisms buffer conserved developmental programs.
Asunto(s)
Meristema/citología , Meristema/genética , Desarrollo de la Planta/genética , Arabidopsis/citología , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Proliferación Celular/genética , Evolución Molecular , Regulación de la Expresión Génica de las Plantas , Redes Reguladoras de Genes , Genes de Plantas , Péptidos y Proteínas de Señalización Intercelular/genética , Ligandos , Solanum lycopersicum/citología , Solanum lycopersicum/genética , Solanum lycopersicum/crecimiento & desarrollo , Modelos Genéticos , Mutación , Plantas Modificadas Genéticamente , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal/genética , Células Madre/citología , Zea mays/citología , Zea mays/genética , Zea mays/crecimiento & desarrolloRESUMEN
Genome editing holds great promise for increasing crop productivity, and there is particular interest in advancing breeding in orphan crops, which are often burdened by undesirable characteristics resembling wild relatives. We developed genomic resources and efficient transformation in the orphan Solanaceae crop 'groundcherry' (Physalis pruinosa) and used clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease (Cas9) (CRISPR-Cas9) to mutate orthologues of tomato domestication and improvement genes that control plant architecture, flower production and fruit size, thereby improving these major productivity traits. Thus, translating knowledge from model crops enables rapid creation of targeted allelic diversity and novel breeding germplasm in distantly related orphan crops.
Asunto(s)
Producción de Cultivos/métodos , Domesticación , Edición Génica/métodos , Physalis/genética , Arabidopsis/genética , Proteína 9 Asociada a CRISPR , Sistemas CRISPR-Cas , Solanum lycopersicum/genética , Physalis/crecimiento & desarrollo , Plantas Modificadas GenéticamenteRESUMEN
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.
Asunto(s)
Productos Agrícolas/genética , Edición Génica , Genoma de Planta , Sistemas CRISPR-Cas , Regiones Promotoras Genéticas , Sitios de Carácter CuantitativoRESUMEN
Small RNA (sRNA)-mediated gene silencing represents a conserved regulatory mechanism controlling a wide diversity of developmental processes through interactions of sRNAs with proteins of the ARGONAUTE (AGO) family. On the basis of a large phylogenetic analysis that includes 206 AGO genes belonging to 23 plant species, AGO genes group into four clades corresponding to the phylogenetic distribution proposed for the ten family members of Arabidopsis thaliana. A primary analysis of the corresponding protein sequences resulted in 50 sequences of amino acids (blocks) conserved across their linear length. Protein members of the AGO4/6/8/9 and AGO1/10 clades are more conserved than members of the AGO5 and AGO2/3/7 clades. In addition to blocks containing components of the PIWI, PAZ, and DUF1785 domains, members of the AGO2/3/7 and AGO4/6/8/9 clades possess other consensus block sequences that are exclusive of members within these clades, suggesting unforeseen functional specialization revealed by their primary sequence. We also show that AGO proteins of animal and plant kingdoms share linear sequences of blocks that include motifs involved in posttranslational modifications such as those regulating AGO2 in humans and the PIWI protein AUBERGINE in Drosophila. Our results open possibilities for exploring new structural and functional aspects related to the evolution of AGO proteins within the plant kingdom, and their convergence with analogous proteins in mammals and invertebrates.
RESUMEN
The development of gametophytes relies on the establishment of a haploid gametophytic generation that initiates with the specification of gametophytic precursors. The majority of flowering plants differentiate a single gametophytic precursor in the ovule: the megaspore mother cell. Here we show that, in addition to argonaute9 (ago9), mutations in other ARGONAUTE (AGO) genes such as ago4, ago6, and ago8, also show abnormal configurations containing supernumerary gametophytic precursors in Arabidopsis thaliana Double homozygous ago4 ago9 individuals showed a suppressive effect on the frequency of ovules with multiple gametophytic precursors across three consecutive generations, indicating that genetic interactions result in compensatory mechanisms. Whereas overexpression of AGO6 in ago9 and ago4 ago9 confirms strong regulatory interactions among genes involved in RNA-directed DNA methylation, AGO8 is overexpressed in premeiotic ovules of ago4 ago9 individuals, suggesting that the regulation of this previously presumed pseudogene responds to the compensatory mechanism. The frequency of abnormal meiotic configurations found in ago4 ago9 individuals is dependent on their parental genotype, revealing a transgenerational effect. Our results indicate that members of the AGO4 clade cooperatively participate in preventing the abnormal specification of multiple premeiotic gametophytic precursors during early ovule development in A. thaliana.
Asunto(s)
Proteínas de Arabidopsis/genética , Proteínas Argonautas/genética , Redes Reguladoras de Genes , Óvulo Vegetal/genética , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/metabolismo , Proteínas Argonautas/metabolismo , Óvulo Vegetal/crecimiento & desarrolloRESUMEN
Here we describe a whole-mount immunolocalization protocol to follow the subcellular localization of proteins during female meiosis in Arabidopsis thaliana, a model species that is used to study sexual reproduction in flowering plants. By using confocal microscopy, the procedure allows one to follow megasporogenesis at all stages before differentiation of the functional megaspore. This in particular includes stages that occur during prophase I, such as the installation of the axial and central elements of the synaptonemal complex along the meiotic chromosomes. In contrast to procedures that require microtome sectioning or enzymatic isolation and smearing to separate female meiocytes from neighboring cells, this 3-day protocol preserves the constitution of the developing primordium and incorporates the architecture of the ovule to provide a temporal and spatial context to meiotic divisions. This opens up the possibility to systematically compare the dynamics of protein localization during female and male meiosis. Steps describe tissue collection and fixation, preparation of slides and polyacrylamide embedding, tissue permeabilization, antibody incubation, propidium iodide staining, and finally image acquisition by confocal microscopy. The procedure adds an essential technique to the toolkit of plant meiotic analysis, and it represents a framework for technical adaptations that could soon allow the analysis of plant reproductive alternatives to sexual reproduction.
Asunto(s)
Arabidopsis/genética , Inmunohistoquímica/métodos , Meiosis , Técnicas de Cultivo de Tejidos , Animales , Femenino , Flores/citología , Flores/genética , Masculino , Adhesión del TejidoRESUMEN
In angiosperms, the transition to the female gametophytic phase relies on the specification of premeiotic gamete precursors from sporophytic cells in the ovule. In Arabidopsis thaliana, a single diploid cell is specified as the premeiotic female gamete precursor. Here, we show that ecotypes of Arabidopsis exhibit differences in megasporogenesis leading to phenotypes reminiscent of defects in dominant mutations that epigenetically affect the specification of female gamete precursors. Intraspecific hybridization and polyploidy exacerbate these defects, which segregate quantitatively in F2 populations derived from ecotypic hybrids, suggesting that multiple loci control cell specification at the onset of female meiosis. This variation in cell differentiation is influenced by the activity of ARGONAUTE9 (AGO9) and RNA-DEPENDENT RNA POLYMERASE6 (RDR6), two genes involved in epigenetic silencing that control the specification of female gamete precursors. The pattern of transcriptional regulation and localization of AGO9 varies among ecotypes, and abnormal gamete precursors in ovules defective for RDR6 share identity with ectopic gamete precursors found in selected ecotypes. Our results indicate that differences in the epigenetic control of cell specification lead to natural phenotypic variation during megasporogenesis. We propose that this mechanism could be implicated in the emergence and evolution of the reproductive alternatives that prevail in flowering plants.
Asunto(s)
Arabidopsis/genética , Arabidopsis/fisiología , Epigénesis Genética/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Gametogénesis en la Planta/genética , Gametogénesis en la Planta/fisiología , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismoRESUMEN
Apomixis is a natural form of asexual reproduction through seeds that leads to viable offspring genetically identical to the mother plant. New evidence from sexual model species indicates that the regulation of female gametogenesis and seed formation is also directed by epigenetic mechanisms that are crucial to control events that distinguish sexuality from apomixis, with important implications for our understanding of the evolutionary forces that shape structural variation and diversity in plant reproduction.
Asunto(s)
Apomixis/genética , Epigénesis Genética , Plantas/genética , Desarrollo de la Planta , Fenómenos Fisiológicos de las Plantas , Semillas/genética , Semillas/crecimiento & desarrolloRESUMEN
The life cycle of flowering plants alternates between a predominant sporophytic (diploid) and an ephemeral gametophytic (haploid) generation that only occurs in reproductive organs. In Arabidopsis thaliana, the female gametophyte is deeply embedded within the ovule, complicating the study of the genetic and molecular interactions involved in the sporophytic to gametophytic transition. Massively parallel signature sequencing (MPSS) was used to conduct a quantitative large-scale transcriptional analysis of the fully differentiated Arabidopsis ovule prior to fertilization. The expression of 9775 genes was quantified in wild-type ovules, additionally detecting >2200 new transcripts mapping to antisense or intergenic regions. A quantitative comparison of global expression in wild-type and sporocyteless (spl) individuals resulted in 1301 genes showing 25-fold reduced or null activity in ovules lacking a female gametophyte, including those encoding 92 signalling proteins, 75 transcription factors, and 72 RNA-binding proteins not reported in previous studies based on microarray profiling. A combination of independent genetic and molecular strategies confirmed the differential expression of 28 of them, showing that they are either preferentially active in the female gametophyte, or dependent on the presence of a female gametophyte to be expressed in sporophytic cells of the ovule. Among 18 genes encoding pentatricopeptide-repeat proteins (PPRs) that show transcriptional activity in wild-type but not spl ovules, CIHUATEOTL (At4g38150) is specifically expressed in the female gametophyte and necessary for female gametogenesis. These results expand the nature of the transcriptional universe present in the ovule of Arabidopsis, and offer a large-scale quantitative reference of global expression for future genomic and developmental studies.