RESUMEN
Ascorbate (vitamin C) is an essential antioxidant in fresh fruits and vegetables. To gain insight into the regulation of ascorbate metabolism in plants, we studied mutant tomato plants (Solanum lycopersicum) that produce ascorbate-enriched fruits. The causal mutation, identified by a mapping-by-sequencing strategy, corresponded to a knock-out recessive mutation in a class of photoreceptor named PAS/LOV protein (PLP), which acts as a negative regulator of ascorbate biosynthesis. This trait was confirmed by CRISPR/Cas9 gene editing and further found in all plant organs, including fruit that accumulated 2 to 3 times more ascorbate than in the WT. The functional characterization revealed that PLP interacted with the 2 isoforms of GDP-L-galactose phosphorylase (GGP), known as the controlling step of the L-galactose pathway of ascorbate synthesis. The interaction with GGP occurred in the cytoplasm and the nucleus, but was abolished when PLP was truncated. These results were confirmed by a synthetic approach using an animal cell system, which additionally demonstrated that blue light modulated the PLP-GGP interaction. Assays performed in vitro with heterologously expressed GGP and PLP showed that PLP is a noncompetitive inhibitor of GGP that is inactivated after blue light exposure. This discovery provides a greater understanding of the light-dependent regulation of ascorbate metabolism in plants.
Asunto(s)
Antioxidantes , Galactosa , Galactosa/metabolismo , Antioxidantes/metabolismo , Ácido Ascórbico , Luz , Frutas/genética , Frutas/metabolismo , Fosforilasas/genética , Fosforilasas/metabolismo , Regulación de la Expresión Génica de las PlantasRESUMEN
Endoreduplication, during which cells increase their DNA content through successive rounds of full genome replication without cell division, is the major source of endopolyploidy in higher plants. Endoreduplication plays pivotal roles in plant growth and development and is associated with the activation of specific transcriptional programmes that are characteristic of each cell type, thereby defining their identity. In plants, endoreduplication is found in numerous organs and cell types, especially in agronomically valuable ones, such as the fleshy fruit (pericarp) of tomato presenting high ploidy levels. We used the tomato pericarp tissue as a model system to explore the transcriptomes associated with endoreduplication progression during fruit growth. We confirmed that expression globally scales with ploidy level and identified sets of differentially expressed genes presenting only developmental-specific, only ploidy-specific expression patterns or profiles resulting from an additive effect of ploidy and development. When comparing ploidy levels at a specific developmental stage, we found that non-endoreduplicated cells are defined by cell division state and cuticle synthesis while endoreduplicated cells are mainly defined by their metabolic activity changing rapidly over time. By combining this dataset with publicly available spatiotemporal pericarp expression data, we proposed a map describing the distribution of ploidy levels within the pericarp. These transcriptome-based predictions were validated by quantifying ploidy levels within the pericarp tissue. This in situ ploidy quantification revealed the dynamic progression of endoreduplication and its cell layer specificity during early fruit development. In summary, the study sheds light on the complex relationship between endoreduplication, cell differentiation and gene expression patterns in the tomato pericarp.
Asunto(s)
Endorreduplicación , Frutas , Regulación de la Expresión Génica de las Plantas , Ploidias , Solanum lycopersicum , Transcriptoma , Solanum lycopersicum/genética , Solanum lycopersicum/crecimiento & desarrollo , Solanum lycopersicum/metabolismo , Frutas/genética , Frutas/crecimiento & desarrollo , Frutas/metabolismo , Endorreduplicación/genética , Perfilación de la Expresión Génica , División Celular/genéticaRESUMEN
Plant organ size and shape are major agronomic traits that depend on cell division and expansion, which are both regulated by complex gene networks. In several eudicot species belonging to the rosid clade, organ growth is controlled by a repressor complex consisting of PEAPOD (PPD) and KINASE-INDUCIBLE DOMAIN INTERACTING (KIX) proteins. The role of these proteins in asterids, which together with the rosids constitute most of the core eudicot species, is unknown. We used Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated protein 9 genome editing to target SlKIX8 and SlKIX9 in the asterid model species tomato (Solanum lycopersicum) and analyzed loss-of-function phenotypes. Loss-of-function of SlKIX8 and SlKIX9 led to the production of enlarged, dome-shaped leaves and these leaves exhibited increased expression of putative Solanum lycopersicum PPD (SlPPD target genes. Unexpectedly, kix8 kix9 mutants carried enlarged fruits with increased pericarp thickness due to cell expansion. At the molecular level, protein interaction assays indicated that SlKIX8 and SlKIX9 act as adaptors between the SlPPD and SlTOPLESS co-repressor proteins. Our results show that KIX8 and KIX9 are regulators of organ growth in asterids and can be used in strategies to improve important traits in produce such as thickness of the fruit flesh.
Asunto(s)
Frutas/crecimiento & desarrollo , Frutas/genética , Reguladores del Crecimiento de las Plantas/genética , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/genética , Solanum lycopersicum/crecimiento & desarrollo , Solanum lycopersicum/genética , Productos Agrícolas/genética , Productos Agrícolas/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , FenotipoRESUMEN
Endoreduplication is the major source of somatic endopolyploidy in higher plants, and leads to variation in cell ploidy levels due to iterative rounds of DNA synthesis in the absence of mitosis. Despite its ubiquitous occurrence in many plant organs, tissues, and cells, the physiological meaning of endoreduplication is not fully understood, although several roles during plant development have been proposed, mostly related to cell growth, differentiation, and specialization via transcriptional and metabolic reprogramming. Here, we review recent advances in our knowledge of the molecular mechanisms and cellular characteristics of endoreduplicated cells, and provide an overview of the multi-scale effects of endoreduplication on supporting growth in plant development. In addition, the effects of endoreduplication in fruit development are discussed, since it is highly prominent during fruit organogenesis where it acts as a morphogenetic factor supporting rapid fruit growth, as illustrated by case of the model fleshy fruit, tomato (Solanum lycopersicum).
Asunto(s)
Endorreduplicación , Frutas , Organogénesis de las Plantas/genética , Ciclo Celular , MitosisRESUMEN
The pericarp is the predominant tissue determining the structural characteristics of most fruits. However, the molecular and genetic mechanisms controlling pericarp development remain only partially understood. Previous studies have identified that CLASS-II KNOX genes regulate fruit size, shape, and maturation in Arabidopsis thaliana and Solanum lycopersicum. Here we characterized the roles of the S. lycopersicum CLASS-II KNOX (TKN-II) genes in pericarp development via a detailed histological, anatomical, and karyotypical analysis of TKN-II gene clade mRNA-knockdown (35S:amiR-TKN-II) fruits. We identify that 35S:amiR-TKN-II pericarps contain more cells around their equatorial perimeter and fewer cell layers than the control. In addition, the cell sizes but not the ploidy levels of these pericarps were dramatically reduced. Further, we demonstrate that fruit shape and pericarp layer number phenotypes of the 35S:amiR-TKN-II fruits can be overridden by the procera mutant, known to induce a constitutive response to the plant hormone gibberellin. However, neither the procera mutation nor exogenous gibberellin application can fully rescue the reduced pericarp width and cell size phenotype of 35S:amiR-TKN-II pericarps. Our findings establish that TKN-II genes regulate tomato fruit anatomy, acting via gibberellin to control fruit shape but utilizing a gibberellin-independent pathway to control the size of pericarp cells.
Asunto(s)
Giberelinas , Solanum lycopersicum , Giberelinas/metabolismo , Frutas/metabolismo , Solanum lycopersicum/genética , Reguladores del Crecimiento de las Plantas/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
Cell fate maintenance is an integral part of plant cell differentiation and the production of functional cells, tissues, and organs. Fleshy fruit development is characterized by the accumulation of water and solutes in the enlarging cells of parenchymatous tissues. In tomato (Solanum lycopersicum), this process is associated with endoreduplication in mesocarp cells. The mechanisms that preserve this developmental program, once initiated, remain unknown. We show here that analysis of a previously identified tomato ethyl methanesulfonate-induced mutant that exhibits abnormal mesocarp cell differentiation could help elucidate determinants of fruit cell fate maintenance. We identified and validated the causal locus through mapping-by-sequencing and gene editing, respectively, and performed metabolic, cellular, and transcriptomic analyses of the mutant phenotype. The data indicate that disruption of the SlGBP1 gene, encoding GUANYLATE BINDING PROTEIN1, induces early termination of endoreduplication followed by late divisions of polyploid mesocarp cells, which consequently acquire the characteristics of young proliferative cells. This study reveals a crucial role of plant GBPs in the control of cell cycle genes, and thus, in cell fate maintenance. We propose that SlGBP1 acts as an inhibitor of cell division, a function conserved with the human hGBP-1 protein.
Asunto(s)
Frutas/citología , Frutas/crecimiento & desarrollo , Proteínas de Plantas/genética , Solanum lycopersicum/citología , Sistemas CRISPR-Cas , Ciclo Celular/genética , Diferenciación Celular , Tamaño de la Célula , Pared Celular/genética , Pared Celular/metabolismo , Endorreduplicación , Frutas/genética , Frutas/metabolismo , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Edición Génica , Regulación de la Expresión Génica de las Plantas , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Mutación , Pectinas/genética , Pectinas/metabolismo , Fenotipo , Células Vegetales , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , PloidiasRESUMEN
Ascorbate is a major antioxidant buffer in plants. Several approaches have been used to increase the ascorbate content of fruits and vegetables. Here, we combined forward genetics with mapping-by-sequencing approaches using an ethyl methanesulfonate (EMS)-mutagenized Micro-Tom population to identify putative regulators underlying a high-ascorbate phenotype in tomato fruits. Among the ascorbate-enriched mutants, the family with the highest fruit ascorbate level (P17C5, up to 5-fold wild-type level) had strongly impaired flower development and produced seedless fruit. Genetic characterization was performed by outcrossing P17C5 with cv. M82. We identified the mutation responsible for the ascorbate-enriched trait in a cis-acting upstream open reading frame (uORF) involved in the downstream regulation of GDP-l-galactose phosphorylase (GGP). Using a specific CRISPR strategy, we generated uORF-GGP1 mutants and confirmed the ascorbate-enriched phenotype. We further investigated the impact of the ascorbate-enriched trait in tomato plants by phenotyping the original P17C5 EMS mutant, the population of outcrossed P17C5 × M82 plants, and the CRISPR-mutated line. These studies revealed that high ascorbate content is linked to impaired floral organ architecture, particularly anther and pollen development, leading to male sterility. RNA-seq analysis suggested that uORF-GGP1 acts as a regulator of ascorbate synthesis that maintains redox homeostasis to allow appropriate plant development.
Asunto(s)
Solanum lycopersicum , Ácido Ascórbico , Fertilidad , Frutas/genética , Solanum lycopersicum/genética , Polen/genéticaRESUMEN
Cuticle function is closely related to the structure of the cutin polymer. However, the structure and formation of this hydrophobic polyester of glycerol and hydroxy/epoxy fatty acids has not been fully resolved. An apoplastic GDSL-lipase known as CUTIN SYNTHASE1 (CUS1) is required for cutin deposition in tomato (Solanum lycopersicum) fruit exocarp. In vitro, CUS1 catalyzes the self-transesterification of 2-monoacylglycerol of 9(10),16-dihydroxyhexadecanoic acid, the major tomato cutin monomer. This reaction releases glycerol and leads to the formation of oligomers with the secondary hydroxyl group remaining nonesterified. To check this mechanism in planta, a benzyl etherification of nonesterified hydroxyl groups of glycerol and hydroxy fatty acids was performed within cutin. Remarkably, in addition to a significant decrease in cutin deposition, mid-chain hydroxyl esterification of the dihydroxyhexadecanoic acid was affected in tomato RNA interference and ethyl methanesulfonate-cus1 mutants. Furthermore, in these mutants, the esterification of both sn-1,3 and sn-2 positions of glycerol was impacted, and their cutin contained a higher molar glycerol-to-dihydroxyhexadecanoic acid ratio. Therefore, in planta, CUS1 can catalyze the esterification of both primary and secondary alcohol groups of cutin monomers, and another enzymatic or nonenzymatic mechanism of polymerization may coexist with CUS1-catalyzed polymerization. This mechanism is poorly efficient with secondary alcohol groups and produces polyesters with lower molecular size. Confocal Raman imaging of benzyl etherified cutins showed that the polymerization is heterogenous at the fruit surface. Finally, by comparing tomato mutants either affected or not in cutin polymerization, we concluded that the level of cutin cross-linking had no significant impact on water permeance.
Asunto(s)
Lipasa/metabolismo , Lípidos de la Membrana/química , Solanum lycopersicum/enzimología , Esterificación , Ésteres/química , Metanosulfonato de Etilo/metabolismo , Ácidos Grasos/química , Frutas/enzimología , Frutas/genética , Glicerol/química , Lipasa/genética , Solanum lycopersicum/genética , Lípidos de la Membrana/metabolismo , Mutación , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Poliésteres/química , Polimerizacion , Polímeros/químicaRESUMEN
The thick cuticle covering and embedding the epidermal cells of tomato (Solanum lycopersicum) fruit acts not only as a protective barrier against pathogens and water loss but also influences quality traits such as brightness and postharvest shelf-life. In a recent study, we screened a mutant collection of the miniature tomato cultivar Micro-Tom and isolated several glossy fruit mutants in which the abundance of cutin, the polyester component of the cuticle, was strongly reduced. We employed a newly developed mapping-by-sequencing strategy to identify the causal mutation underlying the cutin deficiency in a mutant thereafter named gpat6-a (for glycerol-3-phosphate acyltransferase6). To this end, a backcross population (BC1F2) segregating for the glossy trait was phenotyped. Individuals displaying either a wild-type or a glossy fruit trait were then pooled into bulked populations and submitted to whole-genome sequencing prior to mutation frequency analysis. This revealed that the causal point mutation in the gpat6-a mutant introduces a charged amino acid adjacent to the active site of a GPAT6 enzyme. We further showed that this mutation completely abolished the GPAT activity of the recombinant protein. The gpat6-a mutant showed perturbed pollen formation but, unlike a gpat6 mutant of Arabidopsis (Arabidopsis thaliana), was not male sterile. The most striking phenotype was observed in the mutant fruit, where cuticle thickness, composition, and properties were altered. RNA sequencing analysis highlighted the main processes and pathways that were affected by the mutation at the transcriptional level, which included those associated with lipid, secondary metabolite, and cell wall biosynthesis.
Asunto(s)
Glicerol-3-Fosfato O-Aciltransferasa/metabolismo , Lípidos de la Membrana/metabolismo , Solanum lycopersicum/enzimología , Secuencia de Aminoácidos , Mapeo Cromosómico , Frutas/anatomía & histología , Frutas/enzimología , Frutas/genética , Frutas/crecimiento & desarrollo , Glicerol-3-Fosfato O-Aciltransferasa/genética , Solanum lycopersicum/anatomía & histología , Solanum lycopersicum/genética , Solanum lycopersicum/crecimiento & desarrollo , Modelos Moleculares , Mutación , Fenotipo , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Polen/anatomía & histología , Polen/enzimología , Polen/genética , Polen/crecimiento & desarrollo , Proteínas Recombinantes , Alineación de Secuencia , Análisis de Secuencia de ARNRESUMEN
Improving crop productivity and quality while promoting sustainable agriculture have become major goals in plant breeding. The cuticle is a natural film covering the aerial organs of plants and consists of lipid polyesters covered and embedded with wax. The cuticle protects plants against water loss and pathogens and affects traits with strong impacts on crop quality such as, for horticultural crops, fruit brightness, cracking, russeting, netting, and shelf life. Here we provide an overview of the most important cuticle-associated traits that can be targeted for crop improvement. To date, most studies on cuticle-associated traits aimed at crop breeding have been done on fleshy fruits. Less information is available for staple crops such as rice, wheat or maize. Here we present new insights into cuticle formation and properties resulting from the study of genetic resources available for the various crop species. Our review also covers the current strategies and tools aimed at exploiting available natural and artificially induced genetic diversity and the technologies used to transfer the beneficial alleles affecting cuticle-associated traits to commercial varieties.
Asunto(s)
Productos Agrícolas/fisiología , Frutas/fisiología , Variación Genética , Fitomejoramiento , Epidermis de la Planta/fisiología , Biotecnología , Productos Agrícolas/genética , Frutas/genética , Fenotipo , Fitomejoramiento/métodosRESUMEN
The cuticle is a protective layer synthesized by epidermal cells of the plants and consisting of cutin covered and filled by waxes. In tomato (Solanum lycopersicum) fruit, the thick cuticle embedding epidermal cells has crucial roles in the control of pathogens, water loss, cracking, postharvest shelf-life, and brightness. To identify tomato mutants with modified cuticle composition and architecture and to further decipher the relationships between fruit brightness and cuticle in tomato, we screened an ethyl methanesulfonate mutant collection in the miniature tomato cultivar Micro-Tom for mutants with altered fruit brightness. Our screen resulted in the isolation of 16 glossy and 8 dull mutants displaying changes in the amount and/or composition of wax and cutin, cuticle thickness, and surface aspect of the fruit as characterized by optical and environmental scanning electron microscopy. The main conclusions on the relationships between fruit brightness and cuticle features were as follows: (1) screening for fruit brightness is an effective way to identify tomato cuticle mutants; (2) fruit brightness is independent from wax load variations; (3) glossy mutants show either reduced or increased cutin load; and (4) dull mutants display alterations in epidermal cell number and shape. Cuticle composition analyses further allowed the identification of groups of mutants displaying remarkable cuticle changes, such as mutants with increased dicarboxylic acids in cutin. Using genetic mapping of a strong cutin-deficient mutation, we discovered a novel hypomorphic allele of GDSL lipase carrying a splice junction mutation, thus highlighting the potential of tomato brightness mutants for advancing our understanding of cuticle formation in plants.
Asunto(s)
Alelos , Frutas/fisiología , Lipasa/genética , Lípidos de la Membrana/deficiencia , Mutación/genética , Solanum lycopersicum/enzimología , Solanum lycopersicum/fisiología , Secuencia de Aminoácidos , Mapeo Cromosómico , Análisis por Conglomerados , Metanosulfonato de Etilo , Frutas/enzimología , Frutas/genética , Estudios de Asociación Genética , Sitios Genéticos , Lipasa/química , Lípidos/biosíntesis , Solanum lycopersicum/genética , Modelos Biológicos , Datos de Secuencia Molecular , Epidermis de la Planta/metabolismo , Ceras/metabolismoRESUMEN
Tomato (Solanum lycopersicum) is regarded as a model plant of the Solanaceae family. The genome sequencing of the tomato cultivar 'Heinz 1706' was recently completed. To accelerate the progress of tomato genomics studies, systematic bioresources, such as mutagenized lines and full-length cDNA libraries, have been established for the cultivar 'Micro-Tom'. However, these resources cannot be utilized to their full potential without the completion of the genome sequencing of 'Micro-Tom'. We undertook the genome sequencing of 'Micro-Tom' and here report the identification of single nucleotide polymorphisms (SNPs) and insertion/deletions (indels) between 'Micro-Tom' and 'Heinz 1706'. The analysis demonstrated the presence of 1.23 million SNPs and 0.19 million indels between the two cultivars. The density of SNPs and indels was high in chromosomes 2, 5 and 11, but was low in chromosomes 6, 8 and 10. Three known mutations of 'Micro-Tom' were localized on chromosomal regions where the density of SNPs and indels was low, which was consistent with the fact that these mutations were relatively new and introgressed into 'Micro-Tom' during the breeding of this cultivar. We also report SNP analysis for two 'Micro-Tom' varieties that have been maintained independently in Japan and France, both of which have served as standard lines for 'Micro-Tom' mutant collections. Approximately 28,000 SNPs were identified between these two 'Micro-Tom' lines. These results provide high-resolution DNA polymorphic information on 'Micro-Tom' and represent a valuable contribution to the 'Micro-Tom'-based genomics resources.
Asunto(s)
Genoma de Planta/genética , Polimorfismo de Nucleótido Simple , Solanum lycopersicum/genética , Cruzamiento , Mapeo Cromosómico , ADN Intergénico , ADN de Plantas/química , ADN de Plantas/genética , Biblioteca de Genes , Genómica , Mutación INDEL , Anotación de Secuencia Molecular , Mutación , Fenotipo , Análisis de Secuencia de ADN , Especificidad de la EspecieRESUMEN
The understanding of plant organ-size determination represents an important challenge, especially because of the significant role of plants as food and renewable energy sources and the increasing need for plant-derived products. Most of the knowledge on the regulation of organ growth and the molecular network controlling cell division and cell expansion, the main drivers of growth, is derived from arabidopsis. The increasing use of crops such as tomato for research is now bringing essential information on the mechanisms underlying size control in agronomically important organs. This review describes our current knowledge, still very scarce, of the cellular and molecular mechanisms governing tomato fruit size and proposes future research to better understand the regulation of growth in this important crop.
Asunto(s)
Arabidopsis , Solanum lycopersicum , División Celular , Frutas/genética , Regulación de la Expresión Génica de las Plantas , Solanum lycopersicum/genéticaRESUMEN
Improved plant varieties are important in our attempts to face the challenges of a growing human population and limited planet resources. Plant breeding relies on meiotic crossovers to combine favourable alleles into elite varieties1. However, meiotic crossovers are relatively rare, typically one to three per chromosome2, limiting the efficiency of the breeding process and related activities such as genetic mapping. Several genes that limit meiotic recombination were identified in the model species Arabidopsis thaliana2. Mutation of these genes in Arabidopsis induces a large increase in crossover frequency. However, it remained to be demonstrated whether crossovers could also be increased in crop species hybrids. We explored the effects of mutating the orthologues of FANCM3, RECQ44 or FIGL15 on recombination in three distant crop species, rice (Oryza sativa), pea (Pisum sativum) and tomato (Solanum lycopersicum). We found that the single recq4 mutation increases crossovers about three-fold in these crops, suggesting that manipulating RECQ4 may be a universal tool for increasing recombination in plants. Enhanced recombination could be used with other state-of-the-art technologies such as genomic selection, genome editing or speed breeding6 to enhance the pace and efficiency of plant improvement.
Asunto(s)
Cromosomas de las Plantas/genética , Productos Agrícolas/genética , Intercambio Genético , Proteínas de Plantas/genética , RecQ Helicasas/genética , ATPasas Asociadas con Actividades Celulares Diversas/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , ADN Helicasas/genética , Dosificación de Gen , Solanum lycopersicum/genética , Proteínas Asociadas a Microtúbulos/genética , Mutación , Oryza/genética , Pisum sativum/genéticaRESUMEN
The tomato is the model species of choice for fleshy fruit development and for the Solanaceae family. Ethyl methanesulfonate (EMS) mutants of tomato have already proven their utility for analysis of gene function in plants, leading to improved breeding stocks and superior tomato varieties. However, until recently, the identification of causal mutations that underlie particular phenotypes has been a very lengthy task that many laboratories could not afford because of spatial and technical limitations. Here, we describe a simple protocol for identifying causal mutations in tomato using a mapping-by-sequencing strategy. Plants displaying phenotypes of interest are first isolated by screening an EMS mutant collection generated in the miniature cultivar Micro-Tom. A recombinant F2 population is then produced by crossing the mutant with a wild-type (WT; non-mutagenized) genotype, and F2 segregants displaying the same phenotype are subsequently pooled. Finally, whole-genome sequencing and analysis of allele distributions in the pools allow for the identification of the causal mutation. The whole process, from the isolation of the tomato mutant to the identification of the causal mutation, takes 6-12 months. This strategy overcomes many previous limitations, is simple to use and can be applied in most laboratories with limited facilities for plant culture and genotyping.