RESUMEN
Leaf trichomes in Arabidopsis thaliana develop through several distinct cellular processes, such as patterning, differentiation, and growth. Although recent studies have identified several key transcription factors as regulating early patterning and differentiation steps, it is still largely unknown how these regulatory proteins mediate subsequent trichome development, which is accompanied by rapid cell growth and branching. Here, we report a novel trichome mutation in Arabidopsis, which in contrast with previously identified mutants, increases trichome cell size without altering its overall patterning or branching. We show that the corresponding gene encodes a GT-2-LIKE1 (GTL1) protein, a member of the trihelix transcription factor family. GTL1 is present within the nucleus during the postbranching stages of trichome development, and its loss of function leads to an increase in the nuclear DNA content only in trichomes that have completed branching. Our data further demonstrate that the gtl1 mutation modifies the expression of several cell cycle genes and partially rescues the ploidy defects in the cyclin-dependent kinase inhibitor mutant siamese. Taken together, this study provides the genetic evidence for the requirement of transcriptional regulation in the repression of ploidy-dependent plant cell growth as well as for an involvement of GTL trihelix proteins in this regulation.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/citología , Arabidopsis/metabolismo , Hojas de la Planta/citología , Hojas de la Planta/metabolismo , Ploidias , Factores de Transcripción/fisiología , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Caulimovirus/genética , Aumento de la Célula , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Microscopía Fluorescente , Datos de Secuencia Molecular , Plantas Modificadas Genéticamente/citología , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/metabolismo , Regiones Promotoras Genéticas/genética , Homología de Secuencia de Aminoácido , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
We have isolated two dominant mutants from screening approximately 50,000 RIKEN activation-tagging lines that have short inflorescence internodes. The activation T-DNAs were inserted near a putative basic helix-loop-helix (bHLH) gene and expression of this gene was increased in the mutant lines. Overexpression of this bHLH gene produced the original mutant phenotype, indicating it was responsible for the mutants. Specific expression was observed during seed development. The loss-of-function mutation of the RETARDED GROWTH OF EMBRYO1 (RGE1) gene caused small and shriveled seeds. The embryo of the loss-of-function mutant showed retarded growth after the heart stage although abnormal morphogenesis and pattern formation of the embryo and endosperm was not observed. We named this bHLH gene RGE1. RGE1 expression was determined in endosperm cells using the beta-glucuronidase reporter gene and reverse transcription-polymerase chain reaction. Microarray and real-time reverse transcription-polymerase chain reaction analysis showed specific down-regulation of putative GDSL motif lipase genes in the rge1-1 mutant, indicating possible involvement of these genes in seed morphology. These data suggest that RGE1 expression in the endosperm at the heart stage of embryo development plays an important role in controlling embryo growth.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/embriología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Semillas/crecimiento & desarrollo , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/química , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Perfilación de la Expresión Génica , Secuencias Hélice-Asa-Hélice , Datos de Secuencia Molecular , Mutagénesis Insercional , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Semillas/genética , Semillas/metabolismo , Alineación de SecuenciaRESUMEN
In a screen for morphological mutants from the T1 generation of approximately 50 000 activation-tagging lines, we isolated four dominant mutants that showed hyponastic leaves, downward-pointing flowers and decreased apical dominance. We designated them isoginchaku (iso). The iso-1D and iso-2D are allelic mutants caused by activation of the AS2 gene. The T-DNAs were inserted in the 3' downstream region of AS2. Iso-3D and iso-4D are the other allelic mutants caused by activation of the ASL1/LBD36 gene. These two genes belong to the AS2 family that is composed of 42 genes in Arabidopsis. The only recessive mutation isolated from this gene family was of AS2, which resulted in a leaf morphology mutant. Applying reverse genetics using a database of activation-tagged T-DNA flanking sequences, we found a dominant mutant that we designated peacock1-D (pck1-D) in which the ASL5/LBD12 gene was activated by a T-DNA. The pck1-D mutants have lost apical dominance, have epinastic leaves and are sterile. These results strongly suggest that activation tagging is a powerful mutant-mining tool especially for genes that make up a gene family.
Asunto(s)
Arabidopsis/crecimiento & desarrollo , Arabidopsis/genética , ADN Bacteriano/genética , Genes de Plantas/genética , Familia de Multigenes/genética , Mutagénesis Insercional/genética , Alelos , Arabidopsis/anatomía & histología , Flores/anatomía & histología , Flores/genética , Flores/crecimiento & desarrollo , Genes Dominantes/genética , Morfogénesis/genética , Fenotipo , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrolloRESUMEN
Plant genomic resources harbouring gain-of-function mutations remain rare, even though this type of mutation is believed to be one of the most useful for elucidating the function of unknown genes that have redundant partners in the genome. An activation-tagging T-DNA was introduced into the genome of Arabidopsis creating 55,431 independent transformed lines. Of these T1 lines, 1,262 showed phenotypes different from those of wild-type plants. We called these lines 'AT1Ps' (activation T1 putants). The phenotypes observed include abnormalities in morphology, growth rate, plant colour, flowering time and fertility. Similar phenotypes re-appeared either in dominant or semi-dominant fashion in 17% of 177 AT2P plants tested. Plasmid rescue or an adaptor-PCR method was used to identify 1172 independent genomic loci of T-DNA integration sites in the AT1P plants. Mapping of the integration sites revealed that the chromosomal distribution of these sites is similar to that observed in conventional T-DNA knock-out lines, except that the intragenic type of integration is slightly lower (27%) in the AT1P plants compared to that observed in other random knock-out populations (30-35%). Ten AT2P lines that showed dominant phenotypes were chosen to monitor expression levels of genes adjacent to the T-DNA integration sites by RT-PCR. Activation was observed in 7 out of 17 of the adjacent genes detected. Genes located up to 8.2 kb away from the enhancer sequence were activated. One of the seven activated genes was located close to the left-border sequence of the T-DNA, having an estimated distance of 5.7 kb from the enhancer. Surprisingly, one gene, the first ATG of which is located 12 kb away from the enhancer, showed reduced mRNA accumulation in the tagged line. Application of the database generated to Arabidopsis functional genomics research is discussed. The sequence database of the 1172 loci from the AT1P plants is available (http://pfgweb.gsc.riken.go.jp/index.html).