RESUMEN
Efficient splicing requires a balance between high-fidelity splice-site (SS) selection and speed. In Saccharomyces cerevisiae, Pre-mRNA processing factor 8 (Prp8) helps to balance precise SS selection and rapid, efficient intron excision and exon joining. argonaute1-52 (ago1-52) and incurvata13 (icu13) are hypomorphic alleles of the Arabidopsis thaliana genes ARGONAUTE1 (AGO1) and AUXIN RESISTANT6 (AXR6) that harbor point mutations creating a novel 3'SS and 5'SS, respectively. The spliceosome recognizes these novel SSs, as well as the intact genuine SSs, producing a mixture of wild-type and aberrant mature mRNAs. Here, we characterized five novel mutant alleles of PRP8 (one of the two Arabidopsis co-orthologs of yeast Prp8), naming these alleles morphology of ago1-52 suppressed5 (mas5). In the mas5-1 background, the spliceosome preferentially recognizes the intact genuine 3'SS of ago1-52 and 5'SS of icu13. Since point mutations that damage genuine SSs make the spliceosome prone to recognizing cryptic SSs, we also tested alleles of four genes carrying damaged genuine SSs, finding that mas5-1 did not suppress their missplicing. The mas5-1 and mas5-3 mutations represent a novel class of missplicing suppressors that increase splicing fidelity by hampering the use of novel SSs, but do not alter general pre-mRNA splicing.
Asunto(s)
Arabidopsis , Alelos , Arabidopsis/genética , Factor VIII/genética , Intrones/genética , Mutación , Precursores del ARN/genética , Sitios de Empalme de ARN/genética , Empalme del ARN/genética , Saccharomyces cerevisiae/genéticaRESUMEN
Cytokinesis in plant cells begins with the fusion of vesicles that transport cell wall materials to the center of the cell division plane, where the cell plate forms and expands radially until it fuses with the parental cell wall. Vesicle fusion is facilitated by trans-SNARE complexes, with assistance from Sec1/Munc18 (SM) proteins. The SNARE protein KNOLLE and the SM protein KEULE are required for membrane fusion at the cell plate. Due to the crucial function of KEULE, all Arabidopsis (Arabidopsis thaliana) keule mutants identified to date are seedling lethal. Here, we identified the Arabidopsis serrata4-1 (sea4-1) and sea4-2 mutants, which carry recessive, hypomorphic alleles of KEULE. Homozygous sea4-1 and sea4-2 plants are viable and fertile but have smaller rosettes and fewer leaves at bolting than the wild type. Their leaves are serrated, small, and wavy, with a complex venation pattern. The mutant leaves also develop necrotic patches and undergo premature senescence. RNA-seq revealed transcriptome changes likely leading to reduced cell wall integrity and an increase in the unfolded protein response. These findings shed light on the roles of KEULE in postembryonic development, particularly in the patterning of rosette leaves and leaf margins.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Regulación de la Expresión Génica de las Plantas , Mutación , Hojas de la Planta , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Pared Celular/metabolismo , Pared Celular/genética , Fenotipo , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/genética , Hojas de la Planta/metabolismoRESUMEN
Ribosome biogenesis is crucial for cellular metabolism and has important implications for disease and aging. Human (Homo sapiens) glioma tumor-suppressor candidate region gene2 (GLTSCR2) and yeast (Saccharomyces cerevisiae) Nucleolar protein53 (Nop53) are orthologous proteins with demonstrated roles as ribosome biogenesis factors; knockdown of GLTSCR2 impairs maturation of 18S and 5.8S ribosomal RNAs (rRNAs), and Nop53 is required for maturation of 5.8S and 25S rRNAs. Here, we characterized SMALL ORGAN4 (SMO4), the most likely ortholog of human GLTSCR2 and yeast Nop53 in Arabidopsis (Arabidopsis thaliana). Loss of function of SMO4 results in a mild morphological phenotype; however, we found that smo4 mutants exhibit strong cytological and molecular phenotypes: nucleolar hypertrophy and disorganization, overaccumulation of 5.8S and 18S rRNA precursors, and an imbalanced 40S:60S ribosome subunit ratio. Like yeast Nop53 and human GLTSCR2, Arabidopsis SMO4 participates in 5.8S rRNA maturation. In yeast, Nop53 cooperates with mRNA transport4 (Mtr4) for 5.8S rRNA maturation. In Arabidopsis, we found that SMO4 plays similar roles in the 5.8S rRNA maturation pathway than those described for MTR4. However, SMO4 seems not to participate in the degradation of by-products derived from the 5'-external transcribed spacer (ETS) of 45S pre-rRNA, as MTR4 does.
Asunto(s)
Arabidopsis/anatomía & histología , Arabidopsis/genética , Factor Promotor de Maduración/genética , ARN Ribosómico 5.8S/genética , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Variación Genética , Genotipo , Mutación , FenotipoRESUMEN
Ribosome biogenesis is fundamental to growth and development in eukaryotes and is linked to human diseases and cancer. Arabidopsis thaliana MORPHOLOGY OF ARGONAUTE1-52 SUPPRESSED 2 (MAS2) participates in splicing and 45S ribosomal DNA (rDNA) expression. In a screen for MAS2 interactors, we identified RIBOSOMAL RNA PROCESSING 7 (RRP7), an ortholog of yeast rRNA processing protein 7 (Rrp7), which is required for 18S ribosomal RNA (rRNA) maturation. Arabidopsis rrp7 mutants exhibit a pleiotropic phenotype including slow growth, altered shoot phyllotaxy, aberrant venation in lateral organs, partial infertility, and abscisic acid hypersensitivity in seedlings. In Arabidopsis, RRP7 localizes mainly to the nucleolus, the site of the 45S rDNA transcription that produces a 45S pre-rRNA primary transcript, precursor of the 25S, 18S and 5.8S rRNAs. Lack of RRP7 function perturbs 18S rRNA maturation, causes nucleolar hypertrophy, and results in an increased 25S/18S rRNA ratio. Arabidopsis contains hundreds of 45S rDNA genes whose expression is epigenetically regulated, and deregulated, in rrp7 mutants. Double mutant analysis revealed synergistic interactions between RRP7 alleles and alleles of MAS2, NUCLEOLIN1 (NUC1), and HISTONE DEACETYLASE 6 (HDA6), which encode epigenetic regulators of 45S rDNA transcription. Our results reveal the evolutionarily conserved but divergent roles of RRP7 as a ribosome biogenesis factor.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , ARN Ribosómico 18S/metabolismo , Proteínas de Unión al ARN/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , ADN Ribosómico/genética , ADN Ribosómico/metabolismo , Regulación de la Expresión Génica de las Plantas , ARN Ribosómico 18S/genética , Proteínas de Unión al ARN/genéticaRESUMEN
In Arabidopsis thaliana, ARGONAUTE1 (AGO1) plays a central role in microRNA (miRNA) and small interfering RNA (siRNA)-mediated silencing and is a key component in antiviral responses. The polerovirus F-box P0 protein triggers AGO1 degradation as a viral counterdefense. Here, we identified a motif in AGO1 that is required for its interaction with the S phase kinase-associated protein1-cullin 1-F-box protein (SCF) P0 (SCFP0) complex and subsequent degradation. The AGO1 P0 degron is conserved and confers P0-mediated degradation to other AGO proteins. Interestingly, the degron motif is localized in the DUF1785 domain of AGO1, in which a single point mutation (ago1-57, obtained by forward genetic screening) compromises recognition by SCFP0 Recapitulating formation of the RNA-induced silencing complex in a cell-free system revealed that this mutation impairs RNA unwinding, leading to stalled forms of AGO1 still bound to double-stranded RNAs. In vivo, the DUF1785 is required for unwinding perfectly matched siRNA duplexes, but is mostly dispensable for unwinding imperfectly matched miRNA duplexes. Consequently, its mutation nearly abolishes phased siRNA production and sense transgene posttranscriptional gene silencing. Overall, our work sheds new light on the mode of AGO1 recognition by P0 and the in vivo function of DUF1785 in RNA silencing.
Asunto(s)
Arabidopsis/metabolismo , ARN Bicatenario/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Mutación Puntual/genética , Interferencia de ARNRESUMEN
All critical developmental and physiological events in a plant's life cycle depend on the proper activation and repression of specific gene sets, and this often involves epigenetic mechanisms. Some Arabidopsis thaliana mutants with disorders of the epigenetic machinery exhibit pleiotropic defects, including incurved leaves and early flowering, due to the ectopic and heterochronic derepression of developmental regulators. Here, we studied one such mutant class, the incurvata11 (icu11) loss-of-function mutants. We have identified ICU11 as the founding member of a small gene family that we have named CUPULIFORMIS (CP). This family is part of the 2-oxoglutarate/Fe(II)-dependent dioxygenase superfamily. ICU11 and its closest paralog, CP2, have unequally redundant functions: although cp2 mutants are phenotypically wild type, icu11 cp2 double mutants skip vegetative development and flower upon germination. This phenotype is reminiscent of loss-of-function mutants of the Polycomb-group genes EMBRYONIC FLOWER1 (EMF1) and EMF2 Double mutants harboring icu11 alleles and loss-of-function alleles of genes encoding components of the epigenetic machinery exhibit synergistic, severe phenotypes, and some are similar to those of emf mutants. Hundreds of genes are misexpressed in icu11 plants, including SEPALLATA3 (SEP3), and derepression of SEP3 causes the leaf phenotype of icu11 ICU11 and CP2 are nucleoplasmic proteins that act as epigenetic repressors through an unknown mechanism involving histone modification, but not DNA methylation.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Epigénesis Genética/genética , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Mutación/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismoRESUMEN
The diverse eukaryotic proteins that contain zinc fingers participate in many aspects of nucleic acid metabolism, from DNA transcription to RNA degradation, post-transcriptional gene silencing, and small RNA biogenesis. These proteins can be classified into at least 30 types based on structure. In this review, we focus on the CCHC-type zinc fingers (ZCCHC), which contain an 18-residue domain with the CX2CX4HX4C sequence, where C is cysteine, H is histidine, and X is any amino acid. This motif, also named the "zinc knuckle", is characteristic of the retroviral Group Antigen protein and occurs alone or with other motifs. Many proteins containing zinc knuckles have been identified in eukaryotes, but only a few have been studied. Here, we review the available information on ZCCHC-containing factors from three evolutionarily distant eukaryotes-Saccharomyces cerevisiae, Arabidopsis thaliana, and Homo sapiens-representing fungi, plants, and metazoans, respectively. We performed systematic searches for proteins containing the CX2CX4HX4C sequence in organism-specific and generalist databases. Next, we analyzed the structural and functional information for all such proteins stored in UniProtKB. Excluding retrotransposon-encoded proteins and proteins harboring uncertain ZCCHC motifs, we found seven ZCCHC-containing proteins in yeast, 69 in Arabidopsis, and 34 in humans. ZCCHC-containing proteins mainly localize to the nucleus, but some are nuclear and cytoplasmic, or exclusively cytoplasmic, and one localizes to the chloroplast. Most of these factors participate in RNA metabolism, including transcriptional elongation, polyadenylation, translation, pre-messenger RNA splicing, RNA export, RNA degradation, microRNA and ribosomal RNA biogenesis, and post-transcriptional gene silencing. Several human ZCCHC-containing factors are derived from neofunctionalized retrotransposons and act as proto-oncogenes in diverse neoplastic processes. The conservation of ZCCHCs in orthologs of these three phylogenetically distant eukaryotes suggests that these domains have biologically relevant functions that are not well known at present.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas Fúngicas/genética , Saccharomyces cerevisiae/genética , Dedos de Zinc/genética , Animales , Proteínas de Unión al ADN/genética , Estudio de Asociación del Genoma Completo/métodos , HumanosRESUMEN
Forward genetic screens have successfully identified many genes and continue to be powerful tools for dissecting biological processes in Arabidopsis and other model species. Next-generation sequencing technologies have revolutionized the time-consuming process of identifying the mutations that cause a phenotype of interest. However, due to the cost of such mapping-by-sequencing experiments, special attention should be paid to experimental design and technical decisions so that the read data allows to map the desired mutation. Here, we simulated different mapping-by-sequencing scenarios. We first evaluated which short-read technology was best suited for analyzing gene-rich genomic regions in Arabidopsis and determined the minimum sequencing depth required to confidently call single nucleotide variants. We also designed ways to discriminate mutagenesis-induced mutations from background Single Nucleotide Polymorphisms in mutants isolated in Arabidopsis non-reference lines. In addition, we simulated bulked segregant mapping populations for identifying point mutations and monitored how the size of the mapping population and the sequencing depth affect mapping precision. Finally, we provide the computational basis of a protocol that we already used to map T-DNA insertions with paired-end Illumina-like reads, using very low sequencing depths and pooling several mutants together; this approach can also be used with single-end reads as well as to map any other insertional mutagen. All these simulations proved useful for designing experiments that allowed us to map several mutations in Arabidopsis.
Asunto(s)
Arabidopsis/genética , Mapeo Cromosómico/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Mutagénesis Insercional/métodos , Polimorfismo de Nucleótido Simple/genética , ADN Bacteriano/genética , Genoma de Planta/genética , Mutagénesis Sitio-Dirigida/métodosRESUMEN
When plants grow in close proximity basic resources such as light can become limiting. Under such conditions plants respond to anticipate and/or adapt to the light shortage, a process known as the shade avoidance syndrome (SAS). Following genetic screening using a shade-responsive luciferase reporter line (PHYB:LUC), we identified DRACULA2 (DRA2), which encodes an Arabidopsis homolog of mammalian nucleoporin 98, a component of the nuclear pore complex (NPC). DRA2, together with other nucleoporins, participates positively in the control of the hypocotyl elongation response to plant proximity, a role that can be considered dependent on the nucleocytoplasmic transport of macromolecules (i.e. is transport dependent). In addition, our results reveal a specific role for DRA2 in controlling shade-induced gene expression. We suggest that this novel regulatory role of DRA2 is transport independent and that it might rely on its dynamic localization within and outside of the NPC. These results provide mechanistic insights in to how SAS responses are rapidly established by light conditions. They also indicate that nucleoporins have an active role in plant signaling.
Asunto(s)
Transporte Activo de Núcleo Celular/fisiología , Proteínas de Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas , Hipocótilo/crecimiento & desarrollo , Proteínas de Complejo Poro Nuclear/genética , Transporte Activo de Núcleo Celular/genética , Arabidopsis/genética , Hipocótilo/genética , Luz , Poro Nuclear/genética , Poro Nuclear/metabolismo , Plantas Modificadas Genéticamente/genéticaRESUMEN
The shaping of organs in plants depends on the intercellular flow of the phytohormone auxin, of which the directional signaling is determined by the polar subcellular localization of PIN-FORMED (PIN) auxin transport proteins. Phosphorylation dynamics of PIN proteins are affected by the protein phosphatase 2A (PP2A) and the PINOID kinase, which act antagonistically to mediate their apical-basal polar delivery. Here, we identified the ROTUNDA3 (RON3) protein as a regulator of the PP2A phosphatase activity in Arabidopsis thaliana. The RON3 gene was map-based cloned starting from the ron3-1 leaf mutant and found to be a unique, plant-specific gene coding for a protein with high and dispersed proline content. The ron3-1 and ron3-2 mutant phenotypes [i.e., reduced apical dominance, primary root length, lateral root emergence, and growth; increased ectopic stages II, IV, and V lateral root primordia; decreased auxin maxima in indole-3-acetic acid (IAA)-treated root apical meristems; hypergravitropic root growth and response; increased IAA levels in shoot apices; and reduced auxin accumulation in root meristems] support a role for RON3 in auxin biology. The affinity-purified PP2A complex with RON3 as bait suggested that RON3 might act in PIN transporter trafficking. Indeed, pharmacological interference with vesicle trafficking processes revealed that single ron3-2 and double ron3-2 rcn1 mutants have altered PIN polarity and endocytosis in specific cells. Our data indicate that RON3 contributes to auxin-mediated development by playing a role in PIN recycling and polarity establishment through regulation of the PP2A complex activity.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Ácidos Indolacéticos/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteína Fosfatasa 2/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Hibridación in Situ , Proteínas de Transporte de Membrana/genética , Microscopía Confocal , Modelos Biológicos , Mutación , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The characterization of mutants with altered leaf shape and pigmentation has previously allowed the identification of nuclear genes that encode plastid-localized proteins that perform essential functions in leaf growth and development. A large-scale screen previously allowed us to isolate ethyl methanesulfonate-induced mutants with small rosettes and pale green leaves with prominent marginal teeth, which were assigned to a phenotypic class that we dubbed Angulata. The molecular characterization of the 12 genes assigned to this phenotypic class should help us to advance our understanding of the still poorly understood relationship between chloroplast biogenesis and leaf morphogenesis. In this article, we report the phenotypic and molecular characterization of the angulata7-1 (anu7-1) mutant of Arabidopsis thaliana, which we found to be a hypomorphic allele of the EMB2737 gene, which was previously known only for its embryonic-lethal mutations. ANU7 encodes a plant-specific protein that contains a domain similar to the central cysteine-rich domain of DnaJ proteins. The observed genetic interaction of anu7-1 with a loss-of-function allele of GENOMES UNCOUPLED1 suggests that the anu7-1 mutation triggers a retrograde signal that leads to changes in the expression of many genes that normally function in the chloroplasts. Many such genes are expressed at higher levels in anu7-1 rosettes, with a significant overrepresentation of those required for the expression of plastid genome genes. Like in other mutants with altered expression of plastid-encoded genes, we found that anu7-1 exhibits defects in the arrangement of thylakoidal membranes, which appear locally unappressed.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cloroplastos/metabolismo , Hojas de la Planta/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Proteínas del Choque Térmico HSP40/genética , Proteínas del Choque Térmico HSP40/metabolismo , Hojas de la Planta/genética , Tilacoides/metabolismoRESUMEN
Ribosome biogenesis requires stoichiometric amounts of ribosomal proteins and rRNAs. Synthesis of rRNAs consumes most of the transcriptional activity of eukaryotic cells, but its regulation remains largely unclear in plants. We conducted a screen for ethyl methanesulfonate-induced suppressors of Arabidopsis thaliana ago1-52, a hypomorphic allele of AGO1 (ARGONAUTE1), a key gene in microRNA pathways. We identified nine extragenic suppressors as alleles of MAS2 (MORPHOLOGY OF AGO1-52 SUPPRESSED2). Positional cloning showed that MAS2 encodes the putative ortholog of NKAP (NF-κ B activating protein), a conserved eukaryotic protein involved in transcriptional repression and splicing in animals. The mas2 point mutations behave as informational suppressors of ago1 alleles that cause missplicing. MAS2 is a single-copy gene whose insertional alleles are embryonic lethal. In yeast two-hybrid assays, MAS2 interacted with splicing and ribosome biogenesis proteins, and fluorescence in situ hybridization showed that MAS2 colocalizes with the 45S rDNA at the nucleolar organizer regions (NORs). The artificial microRNA amiR-MAS2 partially repressed MAS2 and caused hypomethylation of 45S rDNA promoters as well as partial NOR decondensation, indicating that MAS2 negatively regulates 45S rDNA expression. Our results thus reveal a key player in the regulation of rRNA synthesis in plants.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas Co-Represoras/genética , ADN Ribosómico/genética , Silenciador del Gen , Genes Esenciales , Homología de Secuencia de Aminoácido , Alelos , Animales , Arabidopsis/embriología , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Proteínas Co-Represoras/química , Proteínas Co-Represoras/metabolismo , Secuencia Conservada , Metilación de ADN/genética , ADN Intergénico , Epistasis Genética , Genes Supresores , Pleiotropía Genética , Humanos , Mutación/genética , Proteínas Nucleares/química , Región Organizadora del Nucléolo/metabolismo , Fenotipo , Unión Proteica , Transporte de Proteínas , Empalme del ARN/genética , Proteínas Represoras , Semillas/metabolismo , Fracciones Subcelulares/metabolismo , Supresión Genética , Transcripción Genética , Técnicas del Sistema de Dos HíbridosRESUMEN
Functional redundancy often hampers the analysis of gene families. To overcome this difficulty, we constructed Arabidopsis thaliana lines that expressed artificial microRNAs designed to simultaneously target two to six paralogous genes encoding members of transcription factor families. Of the 576 genes that we chose as targets, only 122 had already been functionally studied at some level. As a simple indicator of the inhibitory effects of our amiRNAs on their targets, we examined the amiRNA-expressing transgenic lines for morphological phenotypes at the rosette stage. Of 338 transgenes tested, 21 caused a visible morphological phenotype in leaves, a proportion that is much higher than that expected as a result of insertional mutagenesis. Also, our collection probably represents many other mutant phenotypes, not just those in leaves. This robust, versatile method enables functional examination of redundant transcription factor paralogs, and is particularly useful for genes that occur in tandem.
Asunto(s)
Arabidopsis/genética , MicroARNs/genética , Factores de Transcripción/genética , Proteínas de Arabidopsis/genética , Flores/genética , Silenciador del Gen , Genómica , Fenotipo , Hojas de la Planta/genética , Plantas Modificadas Genéticamente , Plantones/genética , Factores de Transcripción/metabolismo , TransgenesRESUMEN
The study and eventual manipulation of leaf development in plants requires a thorough understanding of the genetic basis of leaf organogenesis. Forward genetic screens have identified hundreds of Arabidopsis mutants with altered leaf development, but the genome has not yet been saturated. To identify genes required for leaf development we are screening the Arabidopsis Salk Unimutant collection. We have identified 608 lines that exhibit a leaf phenotype with full penetrance and almost constant expressivity and 98 additional lines with segregating mutant phenotypes. To allow indexing and integration with other mutants, the mutant phenotypes were described using a custom leaf phenotype ontology. We found that the indexed mutation is present in the annotated locus for 78% of the 553 mutants genotyped, and that in half of these the annotated T-DNA is responsible for the phenotype. To quickly map non-annotated T-DNA insertions, we developed a reliable, cost-effective and easy method based on whole-genome sequencing. To enable comprehensive access to our data, we implemented a public web application named PhenoLeaf (http://genetics.umh.es/phenoleaf) that allows researchers to query the results of our screen, including text and visual phenotype information. We demonstrated how this new resource can facilitate gene function discovery by identifying and characterizing At1g77600, which we found to be required for proximal-distal cell cycle-driven leaf growth, and At3g62870, which encodes a ribosomal protein needed for cell proliferation and chloroplast function. This collection provides a valuable tool for the study of leaf development, characterization of biomass feedstocks and examination of other traits in this fundamental photosynthetic organ.
Asunto(s)
Arabidopsis/genética , Bases de Datos Genéticas , Hojas de la Planta/genética , Genética Inversa/métodos , Arabidopsis/crecimiento & desarrollo , Arabidopsis/ultraestructura , Secuencia de Bases , Cloroplastos/metabolismo , Mapeo Cromosómico , ADN Bacteriano/genética , ADN de Plantas/química , ADN de Plantas/genética , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento , Datos de Secuencia Molecular , Mutagénesis Insercional , Fenotipo , Fotosíntesis , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/ultraestructura , Análisis de Secuencia de ADNRESUMEN
Epigenetic regulatory states can persist through mitosis and meiosis, but the connection between chromatin structure and DNA replication remains unclear. Arabidopsis INCURVATA2 (ICU2) encodes the catalytic subunit of DNA polymerase α, and null alleles of ICU2 have an embryo-lethal phenotype. Analysis of icu2-1, a hypomorphic allele of ICU2, demonstrated that ICU2 functions in chromatin-mediated cellular memory; icu2-1 strongly impairs ICU2 function in the maintenance of repressive epigenetic marks but does not seem to affect ICU2 polymerase activity. To better understand the global function of ICU2 in epigenetic regulation, here we performed a microarray analysis of icu2-1 mutant plants. We found that the genes up-regulated in the icu2-1 mutant included genes encoding transcription factors and targets of the Polycomb Repressive Complexes. The down-regulated genes included many known players in salicylic acid (SA) biosynthesis and accumulation, ABA signaling and ABA-mediated responses. In addition, we found that icu2-1 plants had reduced SA levels in normal conditions; infection by Fusarium oxysporum induced SA accumulation in the En-2 wild type but not in the icu2-1 mutant. The icu2-1 plants were also hypersensitive to salt stress and exogenous ABA in seedling establishment, post-germination growth and stomatal closure, and accumulated more ABA than the wild type in response to salt stress. The icu2-1 mutant also showed high tolerance to the oxidative stress produced by 3-amino-1,2,4-triazole (3-AT). Our results uncover a role for ICU2 in the regulation of genes involved in ABA signaling as well as in SA biosynthesis and accumulation.
Asunto(s)
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , ADN Polimerasa I/metabolismo , Ácido Salicílico/metabolismo , Transducción de Señal , Amitrol (Herbicida)/metabolismo , Fusarium/metabolismo , Estrés Oxidativo , Reguladores del Crecimiento de las Plantas/metabolismo , Estomas de Plantas/metabolismo , Factores de Transcripción/metabolismoAsunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Cloroplastos/genética , GTP Fosfohidrolasas/genética , Mutación , Fotosíntesis/genética , Fitocromo B/genética , Hojas de la Planta/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Clorofila/metabolismo , Cloroplastos/metabolismo , GTP Fosfohidrolasas/metabolismo , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Hipocótilo/genética , Hipocótilo/crecimiento & desarrollo , Hipocótilo/metabolismo , Fenotipo , Fitocromo B/metabolismo , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/metabolismo , Plantas Modificadas GenéticamenteRESUMEN
Auxin plays a pivotal role in plant development by modulating the activity of SCF ubiquitin ligase complexes. Here, we positionally cloned Arabidopsis (Arabidopsis thaliana) incurvata13 (icu13), a mutation that causes leaf hyponasty and reduces leaf venation pattern complexity and auxin responsiveness. We found that icu13 is a novel recessive allele of AUXIN RESISTANT6 (AXR6), which encodes CULLIN1, an invariable component of the SCF complex. Consistent with a role for auxin in vascular specification, the vascular defects in the icu13 mutant were accompanied by reduced expression of auxin transport and auxin perception markers in provascular cells. This observation is consistent with the expression pattern of AXR6, which we found to be restricted to vascular precursors and hydathodes in wild-type leaf primordia. AXR1, RELATED TO UBIQUITIN1-CONJUGATING ENZYME1, CONSTITUTIVE PHOTOMORPHOGENIC9 SIGNALOSOME5A, and CULLIN-ASSOCIATED NEDD8-DISSOCIATED1 participate in the covalent modification of CULLIN1 by RELATED TO UBIQUITIN. Hypomorphic alleles of these genes also display simple venation patterns, and their double mutant combinations with icu13 exhibited a synergistic, rootless phenotype reminiscent of that caused by loss of function of MONOPTEROS (MP), which forms an auxin-signaling module with BODENLOS (BDL). The phenotypes of double mutant combinations of icu13 with either a gain-of-function allele of BDL or a loss-of-function allele of MP were synergistic. In addition, a BDL:green fluorescent protein fusion protein accumulated in icu13, and BDL loss of function or MP overexpression suppressed the phenotype of icu13. Our results demonstrate that the MP-BDL module is required not only for root specification in embryogenesis and vascular postembryonic development but also for leaf flatness.
Asunto(s)
Alelos , Proteínas de Arabidopsis/metabolismo , Arabidopsis/embriología , Proteínas Cullin/metabolismo , Ácidos Indolacéticos/metabolismo , Complejos Multiproteicos/metabolismo , Hojas de la Planta/anatomía & histología , Haz Vascular de Plantas/embriología , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Transporte Biológico , Tipificación del Cuerpo/genética , Clonación Molecular , Proteínas Cullin/genética , Epistasis Genética , Regulación de la Expresión Génica de las Plantas , Genes de Plantas/genética , Proteínas Fluorescentes Verdes/metabolismo , Heterocigoto , Mutación/genética , Fenotipo , Hojas de la Planta/metabolismo , Haz Vascular de Plantas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Supresión GenéticaRESUMEN
A number of Arabidopsis (Arabidopsis thaliana) mutants exhibit leaf reticulation, having green veins that stand out against paler interveinal tissues, fewer cells in the interveinal mesophyll, and normal perivascular bundle sheath cells. Here, to examine the basis of leaf reticulation, we analyzed the Arabidopsis RETICULATA-RELATED (RER) gene family, several members of which cause leaf reticulation when mutated. Although transcripts of RE, RER1, and RER3 were mainly detected in the bundle sheath cells of expanded leaves, functional RER3:GREEN FLUORESCENT PROTEIN was visualized in the chloroplast membranes of all photosynthetic cells. Leaf reticulation in the re and rer3 loss-of-function mutants occurred, along with accumulation of reactive oxygen species, in a photoperiod-dependent manner. A comparison of re and rer3 leaf messenger RNA expression profiles showed more than 200 genes were similarly misexpressed in both mutants. In addition, metabolic profiles of mature leaves revealed that several biosynthetic pathways downstream of pyruvate are altered in re and rer3. Double mutant analysis showed that only re rer1 and rer5 rer6 exhibited synergistic phenotypes, indicating functional redundancy. The redundancy between RE and its closest paralog, RER1, was confirmed by overexpressing RER1 in re mutants, which partially suppressed leaf reticulation. Our results show that RER family members can be divided into four functional modules with divergent functions. Moreover, these results provide insights into the origin of the reticulated phenotype, suggesting that the RER proteins functionally interconnect photoperiodic growth, amino acid homeostasis, and reactive oxygen species metabolism during Arabidopsis leaf growth.
Asunto(s)
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Muerte Celular/genética , Regulación de la Expresión Génica de las Plantas , Luz , Metaboloma , Familia de Multigenes , Mutación , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa/metabolismo , Fotoperiodo , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Plantas Modificadas Genéticamente , Especies Reactivas de Oxígeno/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Zinc knuckle (ZCCHC) motif-containing proteins are present in unicellular and multicellular eukaryotes and most ZCCHC proteins with known functions participate in the metabolism of various classes of RNA, such as mRNAs, ribosomal RNAs, and microRNAs. The Arabidopsis (Arabidopsis thaliana) genome encodes 69 ZCCHC-containing proteins, but the functions of most remain unclear. One of these proteins is CAX-INTERACTING PROTEIN 4 (CXIP4), which has been classified as a PTHR31437 family member, along with human SREK1-interacting protein 1 (SREK1IP1), which is thought to function in pre-mRNA splicing and RNA methylation. Metazoan SREK1IP1-like and plant CXIP4-like proteins only share a ZCCHC motif, and their functions remain almost entirely unknown. We studied two loss-of-function alleles of Arabidopsis CXIP4, the first mutations in PTHR31437 family genes described to date: cxip4-1 is likely null and shows early lethality, and cxip4-2 is hypomorphic and viable, with pleiotropic morphological defects. The cxip4-2 mutant exhibited deregulation of defense genes and upregulation of transcription factor encoding genes, some of which might explain its developmental defects. This mutant also exhibited increased intron retention events, and the specific functions of misspliced genes, such as those involved in "gene silencing by DNA methylation" and "mRNA polyadenylation factor" suggest that CXIP4 has additional functions. The CXIP4 protein localizes to the nucleus in a pattern resembling nuclear speckles, which are rich in splicing factors. Therefore, CXIP4 is required for plant survival and proper development, and mRNA maturation.