RESUMO
We have designed a novel tiling array, AtMap1, for genomic deletion mapping. AtMap1 is a 60-mer oligonucleotide microarray consisting of 42 497 data probes designed from the genomic sequence of Arabidopsis thaliana Col-0. The average probe interval is 2.8 kb. The performance of the AtMap1 array was assessed using the deletion mutants mag2-2, rot3-1 and zig-2. Eight of the probes showed threefold lower signals in mag2-2 than Col-0. Seven of these probes were located in one region on chromosome 3. We considered these adjacent probes to represent one deletion. This deletion was consistent with a reported deleted region. The other probe was located near the end of chromosome 4. A newly identified deletion around the probe was confirmed by PCR. We also detected the responsible deletions for rot3-1 and zig-2. Thus we concluded that the AtMap1 array was sufficiently sensitive to identify a deletion without any a priori knowledge of the deletion. An analysis of the result of hybridization of Ler and previously reported polymorphism data revealed that the signal decrease tended to depend on the overlap size of sequence polymorphisms. Mutation mapping is time-consuming, laborious and costly. The AtMap1 array removes these limitations.
Assuntos
Arabidopsis/genética , Deleção de Genes , Genoma de Planta , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Mapeamento Cromossômico , Sondas de DNA , DNA de Plantas/genéticaRESUMO
We characterized three Arabidopsis polyamine oxidase genes, AtPAO2, AtPAO3 and AtPAO4. Transient expression of these genes as monomeric red fluorescent protein fusion proteins in Arabidopsis root cells revealed that all are peroxisomal proteins. Quantitative analysis of their transcripts in various organs suggested that AtPAO4 is the major isoform in root peroxisomes. Analysis of recombinant AtPAO4 protein indicated that it is a flavoprotein that catalyzed the oxidative conversion of spermine to spermidine. AtPAO4-deficient mutants established by using T-DNA insertion and RNA interference techniques had markedly increased spermine and decreased spermidine levels in the roots. These results suggest that AtPAO4 is a root peroxisomal polyamine oxidase that participates in polyamine catabolism. Microarray analysis showed that AtPAO4 deficiency induced alterations in the expression of genes related to the drought stress response and flavonoid biosynthesis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Peroxissomos/metabolismo , Proteínas de Arabidopsis/genética , DNA Bacteriano/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Mutagênese Insercional , Análise de Sequência com Séries de Oligonucleotídeos , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Peroxissomos/genética , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Interferência de RNA , RNA de Plantas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Espermidina/metabolismo , Espermina/metabolismo , Especificidade por Substrato , Poliamina OxidaseRESUMO
Expression of miR398 is induced in response to copper deficiency and is involved in the degradation of mRNAs encoding copper/zinc superoxide dismutase in Arabidopsis thaliana. We found that SPL7 (for SQUAMOSA promoter binding protein-like7) is essential for this response of miR398. SPL7 is homologous to Copper response regulator1, the transcription factor that is required for switching between plastocyanin and cytochrome c(6) in response to copper deficiency in Chlamydomonas reinhardtii. SPL7 bound directly to GTAC motifs in the miR398 promoter in vitro, and these motifs were essential and sufficient for the response to copper deficiency in vivo. SPL7 is also required for the expression of multiple microRNAs, miR397, miR408, and miR857, involved in copper homeostasis and of genes encoding several copper transporters and a copper chaperone, indicating its central role in response to copper deficiency. Consistent with this idea, the growth of spl7 plants was severely impaired under low-copper conditions.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Cobre/metabolismo , Fatores de Transcrição/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , MicroRNAs/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , RNA Mensageiro/metabolismo , RNA de Plantas/genética , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
Plant survival requires the ability to acclimate to heat. When plants are subjected to heat shock, the expression of various genes is induced, and the plants become tolerant of higher temperatures. We found that transient treatment with geldanamycin and radicicol, two heat shock protein 90 (HSP90) inhibitors, induced heat-inducible genes and heat acclimation in Arabidopsis thaliana seedlings. Heat shock reduced the activity of exogenously expressed glucocorticoid receptor (GR). Since GR activity depends on HSP90, this suggests that heat shock reduces cytosolic HSP90 activity in vivo. Microarray analysis revealed that many of the genes that are up-regulated by both heat shock and HSP90 inhibitors are involved in protein folding and degradation, suggesting that the activation of a protein maintenance system is a crucial part of this response. Most of these genes have heat shock response element-like motifs in their promoters, which suggests that heat shock transcription factor (HSF) is involved in the response to HSP90 inhibition. Several HSF genes are expressed constitutively in A. thaliana, including AtHsfA1d. Recombinant AtHsfA1d protein recognizes the heat shock response element motif and interacts with A. thaliana cytosolic HSP90, HSP90.2. Overexpression of a dominant negative form of HSP90.2 induced the heat-inducible gene. Thus, it appears that in the absence of heat shock, cytosolic HSP90 negatively regulates heat-inducible genes by actively suppressing HSF function. Upon heat shock, cytosolic HSP90 is transiently inactivated, which may lead to HSF activation.