RESUMEN
The F-box protein Coronatine Insensitive (COI) is a receptor for the jasmonic acid signaling pathway in plants. To investigate the functions of the six maize (Zea mays) COI proteins (COI1a, COI1b, COI1c, COI1d, COI2a, and COI2b), we generated single, double, and quadruple loss-of-function mutants. The pollen of the coi2a coi2b double mutant was inviable. The coi1 quadruple mutant (coi1-4x) exhibited shorter internodes, decreased photosynthesis, leaf discoloration, microelement deficiencies, and accumulation of DWARF8 and/or DWARF9, two DELLA family proteins that repress the gibberellic acid signaling pathway. Co-expression of COI and DELLA in Nicotiana benthamiana showed that the COI proteins trigger proteasome-dependent DELLA degradation. Many genes that are downregulated in the coi1-4x mutant are gibberellic acid-inducible. In addition, most of the proteins encoded by the downregulated genes are predicted to be bundle sheath- or mesophyll-enriched, including those encoding C4-specific photosynthetic enzymes. Heterologous expression of maize Coi genes in N. benthamiana showed that COI2a is nucleus-localized and interacts with maize jasmonate ZIM (zinc-finger inflorescence meristem) domain (JAZ) proteins, the canonical COI repressor partners. However, maize COI1a and COI1c showed only partial nuclear localization and reduced binding efficiency to the tested JAZ proteins. Together, these results show the divergent functions of the six COI proteins in regulating maize growth and defense pathways.
RESUMEN
Mediator is a conserved transcriptional co-activator that links transcription factors bound at enhancer elements to RNA Polymerase II. Mediator-RNA Polymerase II interactions can be sterically hindered by the Cyclin Dependent Kinase 8 (CDK8) module, a submodule of Mediator that acts to repress transcription in response to discrete cellular and environmental cues. The CDK8 module is conserved in all eukaryotes and consists of 4 proteins: CDK8, CYCLIN C (CYCC), MED12, and MED13. In this study, we have characterized the CDK8 module of Mediator in maize using genomic, molecular and functional resources. The maize genome contains single copy genes for Cdk8, CycC, and Med13, and two genes for Med12. Analysis of expression data for the CDK8 module demonstrated that all five genes are broadly expressed in maize tissues, and change their expression in response to phosphate and nitrogen limitation. We performed Dissociation (Ds) insertional mutagenesis, recovering two independent insertions in the ZmMed12a gene, one of which produces a truncated transcript. Our molecular identification of the maize CDK8 module, assays of CDK8 module expression under nutrient limitation, and characterization of transposon insertions in ZmMed12a establish the basis for molecular and functional studies of the role of these important transcriptional regulators in development and nutrient homeostasis in Zea mays.
Asunto(s)
Quinasa 8 Dependiente de Ciclina , Genes de Plantas , Zea mays , Quinasa 8 Dependiente de Ciclina/genética , Quinasa 8 Dependiente de Ciclina/metabolismo , Elementos Transponibles de ADN , Mutagénesis , ARN Polimerasa II/metabolismo , Factores de Transcripción/metabolismo , Zea mays/genéticaRESUMEN
Cultivated maize (Zea mays) has retained much of the genetic diversity of its wild ancestors. Here, we performed nontargeted liquid chromatography-mass spectrometry metabolomics to analyze the metabolomes of the 282 maize inbred lines in the Goodman Diversity Panel. This analysis identified a bimodal distribution of foliar metabolites. Although 15% of the detected mass features were present in >90% of the inbred lines, the majority were found in <50% of the samples. Whereas leaf bases and tips were differentiated by flavonoid abundance, maize varieties (stiff-stalk, nonstiff-stalk, tropical, sweet maize, and popcorn) showed differential accumulation of benzoxazinoid metabolites. Genome-wide association studies (GWAS), performed for 3,991 mass features from the leaf tips and leaf bases, showed that 90% have multiple significantly associated loci scattered across the genome. Several quantitative trait locus hotspots in the maize genome regulate the abundance of multiple, often structurally related mass features. The utility of maize metabolite GWAS was demonstrated by confirming known benzoxazinoid biosynthesis genes, as well as by mapping isomeric variation in the accumulation of phenylpropanoid hydroxycitric acid esters to a single linkage block in a citrate synthase-like gene. Similar to gene expression databases, this metabolomic GWAS data set constitutes an important public resource for linking maize metabolites with biosynthetic and regulatory genes.
Asunto(s)
Regulación de la Expresión Génica de las Plantas/genética , Variación Genética , Estudio de Asociación del Genoma Completo , Metaboloma , Zea mays/genética , Metabolómica , Fenotipo , Sitios de Carácter Cuantitativo/genética , Zea mays/química , Zea mays/metabolismoRESUMEN
The maize W22 inbred has served as a platform for maize genetics since the mid twentieth century. To streamline maize genome analyses, we have sequenced and de novo assembled a W22 reference genome using short-read sequencing technologies. We show that significant structural heterogeneity exists in comparison to the B73 reference genome at multiple scales, from transposon composition and copy number variation to single-nucleotide polymorphisms. The generation of this reference genome enables accurate placement of thousands of Mutator (Mu) and Dissociation (Ds) transposable element insertions for reverse and forward genetics studies. Annotation of the genome has been achieved using RNA-seq analysis, differential nuclease sensitivity profiling and bisulfite sequencing to map open reading frames, open chromatin sites and DNA methylation profiles, respectively. Collectively, the resources developed here integrate W22 as a community reference genome for functional genomics and provide a foundation for the maize pan-genome.
Asunto(s)
Elementos Transponibles de ADN/genética , Genes de Plantas/genética , Genoma de Planta/genética , Zea mays/genética , Cromatina/genética , Cromosomas de las Plantas/genética , Variaciones en el Número de Copia de ADN/genética , Metilación de ADN/genética , ADN de Plantas/genética , Genómica/métodos , Sistemas de Lectura Abierta/genética , Análisis de Secuencia de ADN/métodosRESUMEN
Insects such as the beet armyworm (Spodoptera exigua) cause extensive damage to maize (Zea mays). Maize plants respond by triggering defense signaling, changes in gene expression, and biosynthesis of specialized metabolites. Leaves of maize inbred line B73, which has an available genome sequence, were infested with S. exigua for 1 to 24 h, followed by comparisons of the transcript and metabolite profiles with those of uninfested controls. The most extensive gene expression responses occurred rapidly, within 4-6 h after caterpillar infestation. However, both gene expression and metabolite profiles were altered within 1 h and continued to change during the entire 24 h experiment. The defensive functions of three caterpillar-induced genes were examined using available Dissociation transposon insertions in maize inbred line W22. Whereas mutations in the benzoxazinoid biosynthesis pathway (Bx1 and Bx2) significantly improved caterpillar growth, the knockout of a 13-lipoxygenase (Lox8) involved in jasmonic acid biosynthesis did not. Interestingly, 9-lipoxygenases, which lead to the production of maize death acids, were more strongly induced by caterpillar feeding than 13-lipoxygenases, suggesting an as yet unknown function in maize defense against herbivory. Together, these results provide a comprehensive view of the dynamic transcriptomic and metabolomic responses of maize leaves to caterpillar feeding.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Spodoptera/fisiología , Zea mays/fisiología , Animales , Benzoxazinas/metabolismo , Ciclopentanos/metabolismo , Perfilación de la Expresión Génica , Técnicas de Inactivación de Genes , Herbivoria , Mutación , Oxilipinas/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Hojas de la Planta/genética , Zea mays/genéticaRESUMEN
Most terrestrial plants, including crops, engage in beneficial interactions with arbuscular mycorrhizal fungi. Vital to the association is mutual recognition involving the release of diffusible signals into the rhizosphere. Previously, we identified the maize no perception 1 (nope1) mutant to be defective in early signalling. Here, we report cloning of ZmNope1 on the basis of synteny with rice. NOPE1 encodes a functional homologue of the Candida albicans N-acetylglucosamine (GlcNAc) transporter NGT1, and represents the first plasma membrane GlcNAc transporter identified from plants. In C. albicans, exposure to GlcNAc activates cell signalling and virulence. Similarly, in Rhizophagus irregularis treatment with rice wild-type but not nope1 root exudates induced transcriptome changes associated with signalling function, suggesting a requirement of NOPE1 function for presymbiotic fungal reprogramming.
Asunto(s)
Micorrizas/metabolismo , Oryza/metabolismo , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/metabolismo , Zea mays/metabolismo , Clonación Molecular , Genes de Plantas , Mutación , Oryza/genética , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/genética , Raíces de Plantas/metabolismo , Raíces de Plantas/microbiología , Transducción de Señal , Simbiosis , Zea mays/genéticaRESUMEN
Phosphorus is an essential nutrient for all plants, but also one of the least mobile, and consequently least available, in the soil. Plants have evolved a series of molecular, metabolic and developmental adaptations to increase the acquisition of phosphorus and to maximize the efficiency of use within the plant. In Arabidopsis (Arabidopsis thaliana), the AtPHO1 protein regulates and facilitates the distribution of phosphorus. To investigate the role of PHO1 proteins in maize (Zea mays), the B73 reference genome was searched for homologous sequences, and four genes identified that were designated ZmPho1;1, ZmPho1;2a, ZmPho1;2b and ZmPho1;3. ZmPho1;2a and ZmPho1;2b are the most similar to AtPHO1, and represent candidate co-orthologs that we hypothesize to have been retained following whole genome duplication. Evidence was obtained for the production of natural anti-sense transcripts associated with both ZmPho1;2a and ZmPho1;2b, suggesting the possibility of regulatory crosstalk between paralogs. To characterize functional divergence between ZmPho1;2a and ZmPho1;2b, a program of transposon mutagenesis was initiated using the Ac/Ds system, and, here, we report the generation of novel alleles of ZmPho1;2a and ZmPho1;2b.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Mutagénesis , Proteínas de Plantas/genética , Zea mays/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Elementos Transponibles de ADN , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Genoma de Planta , Familia de Multigenes , Fosfatos/metabolismo , Proteínas de Plantas/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Zea mays/metabolismoRESUMEN
Benzoxazinoids are important defense compounds in grasses. Here, we investigated the biosynthesis and biological roles of the 8-O-methylated benzoxazinoids, DIM2BOA-Glc and HDM2BOA-Glc. Using quantitative trait locus mapping and heterologous expression, we identified a 2-oxoglutarate-dependent dioxygenase (BX13) that catalyzes the conversion of DIMBOA-Glc into a new benzoxazinoid intermediate (TRIMBOA-Glc) by an uncommon reaction involving a hydroxylation and a likely ortho-rearrangement of a methoxy group. TRIMBOA-Glc is then converted to DIM2BOA-Glc by a previously described O-methyltransferase BX7. Furthermore, we identified an O-methyltransferase (BX14) that converts DIM2BOA-Glc to HDM2BOA-Glc. The role of these enzymes in vivo was demonstrated by characterizing recombinant inbred lines, including Oh43, which has a point mutation in the start codon of Bx13 and lacks both DIM2BOA-Glc and HDM2BOA-Glc, and Il14H, which has an inactive Bx14 allele and lacks HDM2BOA-Glc in leaves. Experiments with near-isogenic maize lines derived from crosses between B73 and Oh43 revealed that the absence of DIM2BOA-Glc and HDM2BOA-Glc does not alter the constitutive accumulation or deglucosylation of other benzoxazinoids. The growth of various chewing herbivores was not significantly affected by the absence of BX13-dependent metabolites, while aphid performance increased, suggesting that DIM2BOA-Glc and/or HDM2BOA-Glc provide specific protection against phloem feeding insects.
Asunto(s)
Benzoxazinas/metabolismo , Zea mays/metabolismo , Mutación/genética , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sitios de Carácter Cuantitativo , Zea mays/genéticaRESUMEN
As a response to insect attack, maize (Zea mays) has inducible defenses that involve large changes in gene expression and metabolism. Piercing/sucking insects such as corn leaf aphid (Rhopalosiphum maidis) cause direct damage by acquiring phloem nutrients as well as indirect damage through the transmission of plant viruses. To elucidate the metabolic processes and gene expression changes involved in maize responses to aphid attack, leaves of inbred line B73 were infested with corn leaf aphids for 2 to 96 h. Analysis of infested maize leaves showed two distinct response phases, with the most significant transcriptional and metabolic changes occurring in the first few hours after the initiation of aphid feeding. After 4 d, both gene expression and metabolite profiles of aphid-infested maize reverted to being more similar to those of control plants. Although there was a predominant effect of salicylic acid regulation, gene expression changes also indicated prolonged induction of oxylipins, although not necessarily jasmonic acid, in aphid-infested maize. The role of specific metabolic pathways was confirmed using Dissociator transposon insertions in maize inbred line W22. Mutations in three benzoxazinoid biosynthesis genes, Bx1, Bx2, and Bx6, increased aphid reproduction. In contrast, progeny production was greatly decreased by a transposon insertion in the single W22 homolog of the previously uncharacterized B73 terpene synthases TPS2 and TPS3. Together, these results show that maize leaves shift to implementation of physical and chemical defenses within hours after the initiation of aphid feeding and that the production of specific metabolites can have major effects in maize-aphid interactions.
Asunto(s)
Áfidos/fisiología , Perfilación de la Expresión Génica , Metabolómica , Enfermedades de las Plantas/inmunología , Reguladores del Crecimiento de las Plantas/metabolismo , Zea mays/genética , Animales , Benzoxazinas/metabolismo , Ciclopentanos/metabolismo , Elementos Transponibles de ADN , Regulación de la Expresión Génica de las Plantas , Interacciones Huésped-Parásitos , Mutación , Oxilipinas/metabolismo , Floema/genética , Floema/inmunología , Floema/metabolismo , Hojas de la Planta/genética , Hojas de la Planta/inmunología , Hojas de la Planta/metabolismo , Ácido Salicílico/metabolismo , Factores de Tiempo , Zea mays/inmunología , Zea mays/metabolismoRESUMEN
Plants show considerable within-species variation in their resistance to insect herbivores. In the case of Zea mays (cultivated maize), Rhopalosiphum maidis (corn leaf aphids) produce approximately twenty times more progeny on inbred line B73 than on inbred line Mo17. Genetic mapping of this difference in maize aphid resistance identified quantitative trait loci (QTL) on chromosomes 4 and 6, with the Mo17 allele reducing aphid reproduction in each case. The chromosome 4 QTL mapping interval includes several genes involved in the biosynthesis of DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one), a maize defensive metabolite that also is required for callose accumulation in response to aphid feeding. Consistent with the known association of callose with plant defence against aphids, R. maidis reproduction on B73×Mo17 recombinant inbred lines was negatively correlated with both DIMBOA content and callose formation. Further genetic mapping, as well as experiments with near-isogenic lines, confirmed that the Mo17 allele causes increased DIMBOA accumulation relative to the B73 allele. The chromosome 6 aphid resistance QTL functions independently of DIMBOA accumulation and has an effect that is additive to that of the chromosome 4 QTL. Thus, at least two separate defence mechanisms account for the higher level of R. maidis resistance in Mo17 compared with B73.
Asunto(s)
Áfidos/fisiología , Resistencia a la Enfermedad/genética , Endogamia , Enfermedades de las Plantas/parasitología , Sitios de Carácter Cuantitativo/genética , Zea mays/genética , Zea mays/parasitología , Animales , Benzoxazinas/química , Benzoxazinas/metabolismo , Mapeo Cromosómico , Glucanos/metabolismo , Modelos Biológicos , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/inmunología , Reproducción , Zea mays/inmunologíaRESUMEN
In maize, mutations in the pr1 locus lead to the accumulation of pelargonidin (red) rather than cyanidin (purple) pigments in aleurone cells where the anthocyanin biosynthetic pathway is active. We characterized pr1 mutation and isolated a putative F3'H encoding gene (Zmf3'h1) and showed by segregation analysis that the red kernel phenotype is linked to this gene. Genetic mapping using SNP markers confirms its position on chromosome 5L. Furthermore, genetic complementation experiments using a CaMV 35S::ZmF3'H1 promoter-gene construct established that the encoded protein product was sufficient to perform a 3'-hydroxylation reaction. The Zmf3'h1-specific transcripts were detected in floral and vegetative tissues of Pr1 plants and were absent in pr1. Four pr1 alleles were characterized: two carry a 24 TA dinucleotide repeat insertion in the 5'-upstream promoter region, a third has a 17-bp deletion near the TATA box, and a fourth contains a Ds insertion in exon1. Genetic and transcription assays demonstrated that the pr1 gene is under the regulatory control of anthocyanin transcription factors red1 and colorless1. The cloning and characterization of pr1 completes the molecular identification of all genes encoding structural enzymes of the anthocyanin pathway of maize.
Asunto(s)
Antocianinas/biosíntesis , Vías Biosintéticas/genética , Genes de Plantas/genética , Proteínas de Plantas/genética , Zea mays/genética , Alelos , Secuencia de Aminoácidos , Antocianinas/metabolismo , Arabidopsis/genética , Secuencia de Bases , Mapeo Cromosómico , Cromosomas de las Plantas/genética , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Regulación de la Expresión Génica de las Plantas , Prueba de Complementación Genética , Datos de Secuencia Molecular , Mutagénesis Insercional/genética , Mutación/genética , Fenotipo , Pigmentación/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Alineación de Secuencia , Zea mays/enzimologíaRESUMEN
The maize (Zea mays) transposable element Dissociation (Ds) was mobilized for large-scale genome mutagenesis and to study its endogenous biology. Starting from a single donor locus on chromosome 10, over 1500 elements were distributed throughout the genome and positioned on the maize physical map. Genetic strategies to enrich for both local and unlinked insertions were used to distribute Ds insertions. Global, regional, and local insertion site trends were examined. We show that Ds transposed to both linked and unlinked sites and displayed a nonuniform distribution on the genetic map around the donor r1-sc:m3 locus. Comparison of Ds and Mutator insertions reveals distinct target preferences, which provide functional complementarity of the two elements for gene tagging in maize. In particular, Ds displays a stronger preference for insertions within exons and introns, whereas Mutator insertions are more enriched in promoters and 5'-untranslated regions. Ds has no strong target site consensus sequence, but we identified properties of the DNA molecule inherent to its local structure that may influence Ds target site selection. We discuss the utility of Ds for forward and reverse genetics in maize and provide evidence that genes within a 2- to 3-centimorgan region flanking Ds insertions will serve as optimal targets for regional mutagenesis.
Asunto(s)
Elementos Transponibles de ADN , Genoma de Planta , Zea mays/genética , Mapeo Cromosómico , Cromosomas de las Plantas , ADN de Plantas/genética , Mutagénesis Insercional , Análisis de Secuencia de ADNRESUMEN
We describe genetic screens, molecular methods and web resources newly available to utilize Dissociation (Ds) as an insertional mutagen in maize. Over 1700 Ds elements have been distributed throughout the maize genome to serve as donor elements for local or regional mutagenesis. Two genetic screens are described to identify Ds insertions in genes-of-interest (goi). In scheme I, Ds is used to generate insertion alleles when a recessive reference allele is available. A Ds insertion will enable the cloning of the target gene and can be used to create an allelic series. In scheme II, Ds insertions in a goi are identified using a PCR-based screen to identify the rare insertion alleles among a population of testcross progeny. We detail an inverse PCR protocol to rapidly amplify sequences flanking Ds insertion alleles and describe a high-throughput 96-well plate-based DNA extraction method for the recovery of high-quality genomic DNA from seedling tissues. We also describe several web-based tools for browsing, searching and accessing the genetic materials described. The development of these Ds insertion lines promises to greatly accelerate functional genomics studies in maize.
Asunto(s)
Elementos Transponibles de ADN/genética , Mutagénesis Insercional/métodos , Zea mays/genética , Biología Computacional , Internet , Programas InformáticosRESUMEN
A collection of Activator (Ac)-containing, near-isogenic W22 inbred lines has been generated for use in regional mutagenesis experiments. Each line is homozygous for a single, precisely positioned Ac element and the Ds reporter, r1-sc:m3. Through classical and molecular genetic techniques, 158 transposed Ac elements (tr-Acs) were distributed throughout the maize genome and 41 were precisely placed on the linkage map utilizing multiple recombinant inbred populations. Several PCR techniques were utilized to amplify DNA fragments flanking tr-Ac insertions up to 8 kb in length. Sequencing and database searches of flanking DNA revealed that the majority of insertions are in hypomethylated, low- or single-copy sequences, indicating an insertion site preference for genic sequences in the genome. However, a number of Ac transposition events were to highly repetitive sequences in the genome. We present evidence that suggests Ac expression is regulated by genomic context resulting in subtle variations in Ac-mediated excision patterns. These tr-Ac lines can be utilized to isolate genes with unknown function, to conduct fine-scale genetic mapping experiments, and to generate novel allelic diversity in applied breeding programs.