RESUMO
IgA nephropathy (IgAN) is one of the most common primary glomerulonephritis. Previously identified genome-wide association study (GWAS) loci explain only a fraction of disease risk. To identify novel susceptibility loci in Han Chinese, we conduct a four-stage GWAS comprising 8,313 cases and 19,680 controls. Here, we show novel associations at ST6GAL1 on 3q27.3 (rs7634389, odds ratio (OR)=1.13, P=7.27 × 10(-10)), ACCS on 11p11.2 (rs2074038, OR=1.14, P=3.93 × 10(-9)) and ODF1-KLF10 on 8q22.3 (rs2033562, OR=1.13, P=1.41 × 10(-9)), validate a recently reported association at ITGAX-ITGAM on 16p11.2 (rs7190997, OR=1.22, P=2.26 × 10(-19)), and identify three independent signals within the DEFA locus (rs2738058, P=1.15 × 10(-19); rs12716641, P=9.53 × 10(-9); rs9314614, P=4.25 × 10(-9), multivariate association). The risk variants on 3q27.3 and 11p11.2 show strong association with mRNA expression levels in blood cells while allele frequencies of the risk variants within ST6GAL1, ACCS and DEFA correlate with geographical variation in IgAN prevalence. Our findings expand our understanding on IgAN genetic susceptibility and provide novel biological insights into molecular mechanisms underlying IgAN.
Assuntos
Povo Asiático/genética , Glomerulonefrite por IGA/genética , Adulto , Antígenos CD/genética , Antígeno CD11b/genética , Antígeno CD11c/genética , Estudos de Casos e Controles , China , Proteína DEFICIENS/genética , Fatores de Transcrição de Resposta de Crescimento Precoce/genética , Feminino , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Proteínas de Choque Térmico/genética , Humanos , Fatores de Transcrição Kruppel-Like/genética , Liases/genética , Masculino , Pessoa de Meia-Idade , Razão de Chances , Polimorfismo de Nucleotídeo Único , Sialiltransferases/genética , Adulto JovemRESUMO
IgA nephropathy (IgAN) is a complex syndrome with high genetic heterogeneity. More recently, a genome-wide association study (GWAS) from Southern Han population revealed that variants within 8p23.1, where the DEFA genes encoding a-defensins assembled, were associated with susceptibility to IgAN. To replicate the association and fine-map the genetic variants, a case-control genetic study from an independent Northern Han cohort was conducted. A total of 60 single-nucleotide polymorphisms in a region spanning 350 kb encompassing the DEFA genes cluster were analyzed in 2096 individuals. Copy number variations of DEFA1A3 within the loci were also checked for the independent association. Functional significance of the associated variants was further examined by the in silico method as well as by cis-acting expression quantitative trait loci analysis with mRNA. It showed that 17 out of 60 (28.3%) variants were associated with susceptibility to IgAN. Two independent signals with functional potentials were discovered (rs2738058, P=4.64 × 10(-5), odds ratio (OR)=0.76, 95% confidence interval (CI) 0.66-0.87 and rs9644778, P=4.78 × 10(-3), OR=1.21, 95% CI 1.06-1.39). Besides, marginally significant association of rs9644778 risk genotype with lower proportion of gross hematuria (CC+CA vs AA 35.2% vs 30.2%, P=0.073) was observed. In conclusion, DEFA gene polymorphisms have potentially pathogenic roles in IgAN, and the role of mucosal immunity in the pathogenesis of IgAN has to be emphasized.
Assuntos
Povo Asiático/genética , Proteína DEFICIENS/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Variação Genética , Glomerulonefrite por IGA/epidemiologia , Glomerulonefrite por IGA/genética , Alelos , Estudos de Casos e Controles , China/epidemiologia , Variações do Número de Cópias de DNA , Feminino , Expressão Gênica , Ligação Genética , Genótipo , Glomerulonefrite por IGA/diagnóstico , Humanos , Desequilíbrio de Ligação , Masculino , Família Multigênica , Razão de Chances , Peptídeos Cíclicos/genética , Fenótipo , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Índice de Gravidade de Doença , alfa-Defensinas/genéticaRESUMO
BACKGROUND AND AIMS: DEFICIENS (DEF)- and GLOBOSA (GLO)-like proteins constitute two sister clades of floral homeotic transcription factors that were already present in the most recent common ancestor (MRCA) of extant angiosperms. Together they specify the identity of petals and stamens in flowering plants. In core eudicots, DEF- and GLO-like proteins are functional in the cell only as heterodimers with each other. There is evidence that this obligate heterodimerization contributed to the canalization of the flower structure of core eudicots during evolution. It remains unknown as to whether this strict heterodimerization is an ancient feature that can be traced back to the MRCA of extant flowering plants or if it evolved later during the evolution of the crown group angiosperms. METHODS: The interactions of DEF- and GLO-like proteins of the early-diverging angiosperms Amborella trichopoda and Nuphar advena and of the magnoliid Liriodendron tulipifera were analysed by employing yeast two-hybrid analysis and electrophoretic mobility shift assay (EMSA). Character-state reconstruction, including data from other species as well, was used to infer the ancestral interaction patterns of DEF- and GLO-like proteins. KEY RESULTS: The yeast two-hybrid and EMSA data suggest that DEF- and GLO-like proteins from early-diverging angiosperms both homo- and heterodimerize. Character-state reconstruction suggests that the ability to form heterodimeric complexes already existed in the MRCA of extant angiosperms and that this property remained highly conserved throughout angiosperm evolution. Homodimerization of DEF- and GLO-like proteins also existed in the MRCA of all extant angiosperms. DEF-like protein homodimerization was probably lost very early in angiosperm evolution and was not present in the MRCA of eudicots and monocots. GLO-like protein homodimerization might have been lost later during evolution, but very probably was not present in the MRCA of eudicots. CONCLUSIONS: The flexibility of DEF- and GLO-like protein interactions in early-diverging angiosperms may be one reason for the highly diverse flower morphologies observed in these species. The results strengthen the hypothesis that a reduction in the number of interaction partners of DEF- and GLO-like proteins, with DEF-GLO heterodimers remaining the only DNA-binding dimers in core eudicots, contributed to developmental robustness, canalization of flower development and the diversification of angiosperms.
Assuntos
Evolução Biológica , Proteína DEFICIENS/genética , Proteínas de Homeodomínio/genética , Magnoliopsida/genética , Proteínas de Plantas/genética , Proteína DEFICIENS/classificação , Flores/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Homeodomínio/classificação , Proteínas de Domínio MADS/classificação , Proteínas de Domínio MADS/genética , Filogenia , Proteínas de Plantas/classificação , Multimerização Proteica , Fatores de Transcrição/classificação , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
The transcriptional network topology of B function in Antirrhinum, required for petal and stamen development, is thought to rely on initial activation of transcription of DEFICIENS (DEF) and GLOBOSA (GLO), followed by a positive autoregulatory loop maintaining gene expression levels. Here, we show that the mutant compacta (co), whose vegetative growth and petal size are affected, plays a role in B function. Late events in petal morphogenesis such as development of conical cell area and scent emissions were reduced in co and defâ(nicotianoides) (defâ(nic) ), and absent in co defâ(nic) double mutants, suggesting a role for CO in petal identity. Expression of DEF was down-regulated in co but surprisingly GLO was not affected. We investigated the levels of DEF and GLO at late stages of petal development in the co, defâ(nic) and glo-1 mutants, and established a reliable transformation protocol that yielded RNAi-DEF lines. We show that the threshold levels of DEF or GLO required to obtain petal tissue are approximately 11% of wild-type. The relationship between DEF and GLO transcripts is not equal or constant and changes during development. Furthermore, down-regulation of DEF or GLO does not cause parallel down-regulation of the partner. Our results demonstrate that, at late stages of petal development, the B function transcriptional network topology is not based on positive autoregulation, and has additional components of transcriptional maintenance. Our results suggest changes in network topology that may allow changes in protein complexes that would explain the fact that not all petal traits appear early in development.
Assuntos
Antirrhinum/genética , Flores/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Óleos Voláteis/metabolismo , Proteínas de Plantas/genética , Monoterpenos Acíclicos , Alcenos/análise , Alcenos/metabolismo , Antirrhinum/anatomia & histologia , Antirrhinum/crescimento & desenvolvimento , Antirrhinum/metabolismo , Benzoatos/análise , Benzoatos/metabolismo , Proteína DEFICIENS/genética , Proteína DEFICIENS/metabolismo , Flores/crescimento & desenvolvimento , Flores/metabolismo , Expressão Gênica , Regulação da Expressão Gênica de Plantas/genética , Redes Reguladoras de Genes , Genótipo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Modelos Biológicos , Monoterpenos/análise , Monoterpenos/metabolismo , Mutação , Óleos Voláteis/análise , Fenótipo , Folhas de Planta/anatomia & histologia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Interferência de RNARESUMO
An important objective in nowadays research is the discovery of new biomarkers that can detect colon tumours in early stages and indicate with accuracy the status of the disease. The aim of our study was to identify potential biomarkers for colon cancer onset and progression. We assessed gene expression profiles of a list of 10 candidate genes (MMP-1, MMP-3, MMP-7, DEFA 1, DEFA-5, DEFA-6, IL-8, CXCL-1, SPP-1, CTHRC-1) by quantitative real time PCR in triplets of colonic mucosa (normal, adenoma, tumoral tissue) collected from the same patient during surgery for a group of 20 patients. Additionally we performed immunohistochemistry for DEFA1-3 and SPP1. We remarked that DEFA5 and DEFA6 are key factors in adenoma formation (p<0.05). MMP7 is important in the transition from a benign to a malignant status (p <0.01) and further in metastasis being a prognostic indicator for tumor transformation and for the metastatic potential of cancer cells. IL8, irrespective of tumor stage, has a high mRNA level in adenocarcinoma (p< 0.05). The level of expression for SPP1 is correlated with tumor level. We suggest that high levels of DEFAS, DEFA6 (key elements in adenoma formation), MMP7 (marker of colon cancer onset and progression to metastasis), SPP1 (marker of progression) and IL8 could be used to diagnose an early stage colon cancer and to evaluate the prognostic of progression for colon tumors. Further, if DEFA5 and DEFA6 level of expression are low but MMP7, SPP1 and IL8 level are high we could point out that the transition from adenoma to adenocarcinoma had already occurred. Thus, DEFA5, DEFA6, MMP7, IL8 and SPP1 consist in a valuable panel of biomarkers, whose detection can be used in early detection and progressive disease and also in prognostic of colon cancer.
Assuntos
Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Neoplasias do Colo/química , Neoplasias do Colo/genética , Idoso , Transformação Celular Neoplásica/genética , Neoplasias do Colo/enzimologia , Neoplasias do Colo/patologia , Proteína DEFICIENS/análise , Proteína DEFICIENS/genética , Progressão da Doença , Detecção Precoce de Câncer , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Interleucina-8/análise , Interleucina-8/genética , Masculino , Metaloproteinase 7 da Matriz/análise , Metaloproteinase 7 da Matriz/genética , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Osteopontina/análise , Osteopontina/genética , Reação em Cadeia da Polimerase , Valor Preditivo dos Testes , Prognóstico , RNA Mensageiro/análise , Sensibilidade e EspecificidadeRESUMO
Homeotic class B genes GLOBOSA (GLO)/PISTILLATA (PI) and DEFICIENS (DEF)/APETALA3 (AP3) are involved in the development of petals and stamens in Arabidopsis. However, functions of these genes in the development of floral organs in torenia are less well known. Here, we demonstrate the unique floral phenotypes of transgenic torenia formed due to the modification of class B genes, TfGLO and TfDEF. TfGLO-overexpressing plants showed purple-stained sepals that accumulated anthocyanins in a manner similar to that of petals. TfGLO-suppressed plants showed serrated petals and TfDEF-suppressed plants showed partially decolorized petals. In TfGLO-overexpressing plants, cell shapes on the surfaces of sepals were altered to petal-like cell shapes. Furthermore, TfGLO- and TfDEF-suppressed plants partially had sepal-like cells on the surfaces of their petals. We isolated putative class B gene-regulated genes and examined their expression in transgenic plants. Three xyloglucan endo-1,4-beta-D: -glucanase genes were up-regulated in TfGLO- and TfDEF-overexpressing plants and down-regulated in TfGLO- and TfDEF-suppressed plants. In addition, 10 anthocyanin biosynthesis-related genes, including anthocyanin synthase and chalcone isomerase, were up-regulated in TfGLO-overexpressing plants and down-regulated in TfGLO-suppressed plants. The expression patterns of these 10 genes in TfDEF transgenic plants were diverse and classified into several groups. HPLC analysis indicated that sepals of TfGLO-overexpressing plants accumulate the same type of anthocyanins and flavones as wild-type plants. The difference in phenotypes and expression patterns of the 10 anthocyanin biosynthesis-related genes between TfGLO and TfDEF transgenic plants indicated that TfGLO and TfDEF have partial functional divergence, while they basically work synergistically in torenia.
Assuntos
Proteína DEFICIENS/genética , Gleiquênias/genética , Regulação da Expressão Gênica de Plantas , Variação Genética , Proteínas de Homeodomínio/genética , Proteínas de Plantas/genética , Antocianinas/biossíntese , Gleiquênias/metabolismo , Gleiquênias/ultraestrutura , Flavonas/biossíntese , Microscopia Eletrônica de Varredura , Filogenia , Plantas Geneticamente ModificadasRESUMO
BACKGROUND: Atopic dermatitis (AD) patients have been recognized to have an increased susceptibility to cutaneous colonization and infection by bacteria, fungi and viruses. OBJECTIVE: We wanted to evaluate the associations of single nucleotide polymorphism (SNP) and the haplotype in the defensin (DEFA) and defensin (DEFB) genes, and so we performed genotyping for the SNPs in these genes in both AD patients and normal controls. METHOD: We genotyped 27 SNPs from the DEFA 4, 5 and 6 genes and the DEFB1 gene for 1089 case-control samples (631 AD patients and 458 normal controls). We analyzed the SNPs and haplotypes in each gene. RESULT: We identified that two SNPs and the haplotype CT in the DEFB1 gene are associated with AD in Koreans. The rs5743399 (-2266T/C) SNP is associated with AD, and especially with the high IgE, extrinsic type, and the rs5743409 (-1241T/G) SNP is associated with AD. On the haplotype analysis of these two SNPs, the haplotype CT is associated with AD, and especially with the allergic, extrinsic type of AD. However, we could not find any significant associations between the SNPs in the three DEFA genes and AD. CONCLUSION: We found that the rs5743399 SNP, the rs5743409 SNP and the CT haplotype in the DEFB1 gene were significantly associated with the susceptibility to AD. We also found that rs5743399 polymorphism and the haplotype CT in this gene showed a strong association with the allergic, extrinsic type of AD. These results suggest that the DEFB1 gene has a main effect on the skin inflammation and/or skin responsiveness to any kind of allergic reaction.
Assuntos
Proteína DEFICIENS/genética , Dermatite Atópica/genética , Haplótipos/genética , beta-Defensinas/genética , Adolescente , Alelos , Estudos de Casos e Controles , Criança , Pré-Escolar , Proteína DEFICIENS/imunologia , Dermatite Atópica/epidemiologia , Dermatite Atópica/imunologia , Feminino , Frequência do Gene/genética , Predisposição Genética para Doença , Genótipo , Humanos , Lactente , Coreia (Geográfico)/epidemiologia , Masculino , Polimorfismo de Nucleotídeo Único , Adulto Jovem , beta-Defensinas/imunologiaRESUMO
Floral homeotic and flower development mutants of Primula, including double, Hose in Hose, Jack in the Green and Split Perianth, have been cultivated since the late 1500s as ornamental plants but until recently have attracted limited scientific attention. Here we describe the characterization of a new mutant phenotype, sepaloid, that produces flowers comprising only sepals and carpels. The sepaloid mutation is recessive, and is linked to the S locus that controls floral heteromorphy. The phenotype shows developmental variability, with flowers containing three whorls of sepals surrounding fertile carpels, two whorls of sepals with a diminished third whorl of sepals surrounding a fourth whorl of carpels, or three whorls of sepals surrounding abnormal carpels. In some respects, these phenotypes resemble the Arabidopsis and Antirrhinum homeotic B-function mutants apetala3/deficiens (ap3/def) and pistillata/globosa (pi/glo). We have isolated the Primula vulgaris B-function genes PvDEFICIENS (PvDEF) and PvGLOBOSA (PvGLO), expression of both of which is affected in the sepaloid mutant. PvGLO, like sepaloid, is linked to the S locus, whereas PvDEF is not. However, our analyses reveal that sepaloid and PvGLO represent different genes. We conclude that SEPALOID is an S-linked independent regulator of floral organ identity genes including PvDEF and PvGLO.
Assuntos
Proteína DEFICIENS/genética , Flores/anatomia & histologia , Proteínas de Homeodomínio/genética , Proteínas de Plantas/genética , Primula/genética , Alelos , Clonagem Molecular , DNA Complementar/genética , Flores/genética , Regulação da Expressão Gênica de Plantas , Biblioteca Gênica , Genes de Plantas , Padrões de Herança , Dados de Sequência Molecular , Mutação , Fenótipo , Filogenia , RNA de Plantas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de Proteína , Técnicas do Sistema de Duplo-HíbridoRESUMO
The development of the ovary into a fruit depends on pollination and fertilization. It has been proposed that the restriction of ovary growth before pollination is because of the stamens acting as negative regulators. Accordingly, the silencing of genes responsible for stamen identity has been correlated with parthenocarpy in different species. The tomato (Solanum lycopersicum L.) parthenocarpic fruit (pat) mutation associates autonomous ovary development with homeotic transformation of the anthers and aberrancy of ovules in the ovary. In this study, we tested the hypothesis that stamen aberrations and parthenocarpy in pat are driven by cues coming from the altered expression of class B MADS box genes. The data showed that the Pat locus is not allelic to either of the two tomato mutations putatively involved in the B function, stamenless (sl)-2 and pistillate (pi) or to genes encoding class B transcription factors. Whereas pat pi double mutants were not recovered because of tight linkage, pat sl-2 double mutants showed mainly epistatic effects. The developmental regulation of the Sl DEFICIENS (DEF) gene in the wild-type (WT) at anthesis as well as its differential transcription in the pat ovary suggest that it plays a role in the control of ovary growth. Accordingly, when compared with the WT, the gene was also differentially expressed in the parthenocarpic fruit-2 (pat-2) mutant, that is not allelic to pat and has normal ovule development. Altogether the results indicate that in tomato SlDEF plays a role in the control of ovary growth and that the pat mutation is located upstream of this regulatory cascade.
Assuntos
Proteína DEFICIENS/genética , Genes de Plantas , Mutação , Solanum lycopersicum/genética , Solanum lycopersicum/crescimento & desenvolvimentoRESUMO
APETALA3 (AP3)/DEFICIENS (DEF) is a MADS-box transcription factor that is involved in establishing the identity of petal and stamen floral organs. The AP3/DEF gene lineage has been extensively examined throughout the angiosperms in order to better understand its role in floral diversity and evolution. As a result, a large number of cloned AP3/DEF orthologues are available, which can be used for the design of taxon specific primers for phylogeny reconstruction of close relatives of the group of interest. Following this reasoning, we investigated the phylogenetic utility of the two AP3/DEF paralogues (ImpDEF1 and ImpDEF2) that were recently identified in the genus Impatiens (Balsaminaceae). K-domain introns 4 and 5 of both AP3/DEF duplicates were amplified and sequenced for 59 Impatiens species. Phylogenetic analyses of the separated and combined ImpDEF1 and ImpDEF2 data sets result in highly congruent topologies with the previously obtained chloroplast atpB-rbcL data set. Combination of chloroplast and nuclear matrices results in a well-supported evolutionary hypothesis of Impatiens. Our results show that introns 4 and 5 in AP3/DEF-like genes are a valuable source of characters for phylogenetic studies at the infrageneric level.
Assuntos
Proteína DEFICIENS/genética , Evolução Molecular , Impatiens/genética , Filogenia , Sequência de Aminoácidos , Sequência de Bases , Teorema de Bayes , Primers do DNA , DNA de Cloroplastos/genética , Dosagem de Genes/genética , Variação Genética , Íntrons/genética , Modelos Genéticos , Dados de Sequência Molecular , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
The class B genes, which belong to the MADS-box gene family, play important roles in regulating the development of petals and stamens in flowering plants. To understand the molecular mechanisms of floral development in Agapanthus praecox ssp. orientalis (Agapanthaceae), we isolated and characterized the homologs of the Antirrhinum majus genes GLOBOSA and DEFICIENS in this plant. These were designated as ApGLO and ApDEF, respectively. ApGLO and ApDEF contain open reading frames that encode deduced protein with 210 and 214 amino acid residues, respectively. Phylogenetic analysis indicated that ApGLO and ApDEF belong to the monocot class B gene family. In situ hybridization experiments revealed that hybridization signals of ApGLO and ApDEF were observed in whorl 1 as well as in whorls 2 and 3. Moreover, the flowers of transgenic Arabidopsis plants that ectopically expressed ApGLO formed petal-like organs in whorl 1. These observations indicate that the flower developmental mechanism of Agapanthus follows the modified ABC model.
Assuntos
Proteína DEFICIENS/genética , Flores/genética , Proteínas de Homeodomínio/genética , Magnoliopsida/genética , Proteínas de Plantas/genética , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Flores/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Genótipo , Hibridização In Situ , Proteínas de Domínio MADS/genética , Magnoliopsida/crescimento & desenvolvimento , Modelos Genéticos , Dados de Sequência Molecular , Fenótipo , Filogenia , Plantas Geneticamente Modificadas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
DEFICIENS (DEF) and GLOBOSA (GLO) function in petal and stamen organ identity in Antirrhinum and are orthologs of APETALA3 and PISTILLATA in Arabidopsis. These genes are known as B-function genes for their role in the ABC genetic model of floral organ identity. Phylogenetic analyses show that DEF and GLO are closely related paralogs, having originated from a gene duplication event after the separation of the lineages leading to the extant gymnosperms and the extant angiosperms. Several additional gene duplications followed, providing multiple potential opportunities for functional divergence. In most angiosperms studied to date, genes in the DEF/GLO MADS-box subfamily are expressed in the petals and stamens during flower development. However, in some angiosperms, the expression of DEF and GLO orthologs are occasionally observed in the first and fourth whorls of flowers or in nonfloral organs, where their function is unknown. In this article we review what is known about function, phylogeny, and expression in the DEF/GLO subfamily to examine their evolution in the angiosperms. Our analyses demonstrate that although the primary role of the DEF/GLO subfamily appears to be in specifying the stamens and inner perianth, several examples of potential sub- and neofunctionalization are observed.
Assuntos
Proteína DEFICIENS/genética , Evolução Molecular , Flores/genética , Proteínas de Homeodomínio/genética , Magnoliopsida/genética , Proteínas de Plantas/genética , Proteína DEFICIENS/fisiologia , Flores/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Proteínas de Homeodomínio/fisiologia , Magnoliopsida/classificação , Magnoliopsida/crescimento & desenvolvimento , Modelos Genéticos , Filogenia , Proteínas de Plantas/fisiologiaRESUMO
The class B MADS box transcription factors DEFICIENS (DEF) and GLOBOSA (GLO) of Antirrhinum majus together control the organogenesis of petals and stamens. Toward an understanding of how the downstream molecular mechanisms controlled by DEF contribute to petal organogenesis, we conducted expression profiling experiments using macroarrays comprising >11,600 annotated Antirrhinum unigenes. First, four late petal developmental stages were compared with sepals. More than 500 ESTs were identified that comprise a large number of stage-specifically regulated genes and reveal a highly dynamic transcriptional regulation. For identification of DEF target genes that might be directly controlled by DEF, we took advantage of the temperature-sensitive def-101 mutant. To enhance the sensitivity of the profiling experiments, one petal developmental stage was selected, characterized by increased transcriptome changes that reflect the onset of cell elongation processes replacing cell division processes. Upon reduction of the DEF function, 49 upregulated and 52 downregulated petal target genes were recovered. Eight target genes were further characterized in detail by RT-PCR and in situ studies. Expression of genes responding rapidly toward an altered DEF activity is confined to different petal tissues, demonstrating the complexity of the DEF function regulating diverse basic processes throughout petal morphogenesis.
Assuntos
Antirrhinum/crescimento & desenvolvimento , Antirrhinum/genética , Proteína DEFICIENS/genética , Flores/crescimento & desenvolvimento , Flores/genética , Regulação da Expressão Gênica de Plantas/genética , Divisão Celular/genética , Regulação para Baixo/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/genética , Marcação de Genes , Genes Reguladores/genética , Mutação/genética , Análise de Sequência com Séries de Oligonucleotídeos , Organogênese/genética , Proteínas de Plantas/genética , Transdução de Sinais/genética , Fatores de Transcrição/genética , Regulação para Cima/genéticaRESUMO
Traditionally, developmental studies in plant biology have suffered from the lack of a convenient means to study gene function in non-model plant species. Here we show that virus-induced gene silencing (VIGS) is an effective new tool to study the function of orthologs of floral homeotic genes such as DEFICIENS (DEF) in non-model systems. We used a tobacco rattle virus (TRV)-based VIGS approach to study the function of the Nicotiana benthamiana DEF ortholog (NbDEF). Silencing of NbDEF in N. benthamiana using TRV-VIGS was similar to that of Antirrhinum def and Arabidopsis ap3 mutants and caused transformation of petals into sepals and stamens into carpels. Molecular analysis of the NbDEF -silenced plants revealed a dramatic reduction of the levels of NbDEF mRNA and protein in flowers. NbDEF silencing was specific and has no effect on the mRNA levels of NbTM6, the closest paralog of NbDEF. A dramatic reduction of the levels of N. benthamiana GLOBOSA (NbGLO) mRNA and protein was also observed in flowers of NbDEF-silenced plants, suggesting that cross-regulation of this GLO-like gene by NbDEF. Taken together, our results suggest that NbDEF is a functional homolog of Antirrhinum DEF. Our results are significant in that they show that TRV efficiently induces gene silencing in young and differentiating flowers and that VIGS is a promising new tool for analyses of developmental gene function in non-model organisms.
Assuntos
Proteína DEFICIENS/genética , Inativação Gênica , Nicotiana/genética , Vírus de Plantas/genética , Proteína DEFICIENS/metabolismo , Flores/genética , Flores/crescimento & desenvolvimento , Flores/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Vetores Genéticos/genética , Imuno-Histoquímica , Dados de Sequência Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Nicotiana/metabolismo , Nicotiana/virologiaRESUMO
Frameshift mutations generally result in loss-of-function changes since they drastically alter the protein sequence downstream of the frameshift site, besides creating premature stop codons. Here we present data suggesting that frameshift mutations in the C-terminal domain of specific ancestral MADS-box genes may have contributed to the structural and functional divergence of the MADS-box gene family. We have identified putative frameshift mutations in the conserved C-terminal motifs of the B-function DEF/AP3 subfamily, the A-function SQUA/AP1 subfamily and the E-function AGL2 subfamily, which are all involved in the specification of organ identity during flower development. The newly evolved C-terminal motifs are highly conserved, suggesting a de novo generation of functionality. Interestingly, since the new C-terminal motifs in the A- and B-function subfamilies are only found in higher eudicotyledonous flowering plants, the emergence of these two C-terminal changes coincides with the origin of a highly standardized floral structure. We speculate that the frameshift mutations described here are examples of co-evolution of the different components of a single transcription factor complex. 3' terminal frameshift mutations might provide an important but so far unrecognized mechanism to generate novel functional C-terminal motifs instrumental to the functional diversification of transcription factor families.
Assuntos
Evolução Molecular , Mutação da Fase de Leitura , Genes de Plantas , Proteínas de Domínio MADS/química , Proteínas de Domínio MADS/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Sequência de Bases , Sequência Conservada , Proteína DEFICIENS/química , Proteína DEFICIENS/genética , Bases de Dados Genéticas , Flores/genética , Proteínas de Domínio MADS/classificação , Proteínas de Domínio MADS/fisiologia , Modelos Genéticos , Filogenia , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
The DEFICIENS (DEF) gene is required for establishing petal and stamen identity in Antirrhinum and is expressed in all three layers of the floral meristem in whorls 2 and 3. Expression of DEF in a subset of meristem layers gives rise to organs with characteristic shapes and cell types, reflecting altered patterns and levels of DEF gene activity. To determine how the contributions of layers and gene activity interact, we exploited a DEF allele which carries a transposon insertion in the MADS box region to generate periclinal chimeras expressing alleles with different activities. By comparing the phenotype, development and expression patterns of these chimeras we show that expression of DEF in L1 makes a major contribution to morphology in whorl 2, irrespective of the allele. By contrast L1 expression is largely unable to rescue whorl 3, possibly because of a non-autonomous inhibitor of DEF activity in this whorl.