RESUMO
In Arabidopsis (Arabidopsis thaliana), small gene families encode multiple isoforms for many of the components of the mitochondrial protein import apparatus. There are three isoforms of the TRANSLOCASE OF THE INNER MEMBRANE17 (Tim17). Transcriptome analysis indicates that AtTim17-1 is only detectable in dry seed. In this study, two independent transfer DNA insertional mutant lines of tim17-1 exhibited a germination-specific phenotype, showing a significant increase in the rate of germination. Microarray analyses revealed that Attim17-1 displayed alterations in the temporal sequence of transcriptomic events during germination, peaking earlier compared with the wild type. Promoter analysis of AtTim17-1 further identified an abscisic acid (ABA)-responsive element, which binds ABA-responsive transcription factors, acting to repress the expression of AtTim17-1. Attim17-1 dry seeds contained significantly increased levels of ABA and gibberellin, 2- and 5-fold, respectively. These results support the model that mitochondrial biogenesis is regulated in a tight temporal sequence of events during germination and that altering mitochondrial biogenesis feeds back to alter the germination rate, as evidenced by the altered levels of the master regulatory hormones that define germination.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Germinação/fisiologia , Proteínas de Membrana Transportadoras/metabolismo , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacologia , Arabidopsis/efeitos dos fármacos , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Técnicas de Inativação de Genes , Germinação/efeitos dos fármacos , Germinação/genética , Giberelinas/metabolismo , Proteínas de Membrana Transportadoras/genética , Mitocôndrias/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Mutação , Regiões Promotoras Genéticas , Isoformas de Proteínas , Sementes/efeitos dos fármacos , Sementes/fisiologia , Fatores de TempoRESUMO
Mitochondrial components dynamically change in response to environmental and developmental cues. However, the regulatory pathways that underlie these changes are largely unknown. A global analysis of changes in mitochondrial components at the transcript, protein and metabolite levels was undertaken, to gain a greater insight into how mitochondrial functions are regulated and respond to various internal or external cues. At the transcript level, large-scale changes in groups of genes suggest the presence of co-regulatory mechanisms for these components. Furthermore, the pathways that regulate these changes appear to be integrated into regulatory pathways that alter a variety of functions in cells. However, the changes in transcripts are not always observed at the protein or the metabolite level. This is likely to be due to post-transcriptional levels of regulation and also the fact that in-depth profiles, which have been obtained for transcripts from a variety of studies, are currently not available for proteins and metabolites. Thus, while transcripts for genes give us a picture of what the cells are 'thinking' in relation to mitochondrial components, some of these responses may be lost in translation.
Assuntos
Mitocôndrias/metabolismo , Plantas/metabolismo , Regulação da Expressão Gênica de Plantas , Metabolômica , Mitocôndrias/genética , Oxirredução , Estresse Oxidativo , Plantas/genética , Proteômica , Estresse Fisiológico , TranscriptomaRESUMO
To obtain a global overview of how mitochondria respond to stress, we aimed to define the plant mitochondrial stress response (MSR). By combining a set of 1196 Arabidopsis thaliana genes that putatively encode mitochondrial proteins with 16 microarray experiments on stress-related conditions, 45 nuclear encoded genes were defined as widely stress-responsive. Using green fluorescent protein (GFP) fusion assays, the mitochondrial targeting of a large number of these proteins was tested, confirming in total 26 proteins as mitochondrially targeted. Several of these proteins were observed to be dual targeted to mitochondria and plastids, including the small heat shock proteins sHSP23.5 and sHSP23.6. In addition to the well defined stress components of mitochondria, such as alternative oxidases, nicotinamide adenine dinucleotide (NAD(P)H) dehydrogenases, and heat shock proteins, a variety of other proteins, many with unknown function, were identified. The mitochondrial carrier protein family was over-represented in the stress-responsive genes, suggesting that stress induces altered needs for metabolite transport across the mitochondrial inner membrane. Although the genes encoding many of these proteins contain common cis-acting regulatory elements, it was apparent that a number of distinct regulatory processes or signals likely triggered the MSR. Therefore, these genes provide new model systems to study mitochondrial retrograde regulation, in addition to the widely used alternative oxidase model. Additionally, as changes in proteins responsive to stress did not correlate well with changes at a transcript level, it suggests that post-transcriptional mechanisms also play an important role in defining the MSR.
Assuntos
Arabidopsis/genética , Mitocôndrias/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Fluorescência Verde/genética , Homeostase , Mitocôndrias/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , OxirreduçãoRESUMO
In this study we analyzed transcript abundance and promoters of genes encoding mitochondrial proteins to identify signaling pathways that regulate stress-induced gene expression. We used Arabidopsis (Arabidopsis thaliana) alternative oxidase AOX1a, external NADP H-dehydrogenase NDB2, and two additional highly stress-responsive genes, At2g21640 and BCS1. As a starting point, the promoter region of AOX1a was analyzed and functional analysis identified 10 cis-acting regulatory elements (CAREs), which played a role in response to treatment with H(2)O(2), rotenone, or both. Six of these elements were also functional in the NDB2 promoter. The promoter region of At2g21640, previously defined as a hallmark of oxidative stress, shared two functional CAREs with AOX1a and was responsive to treatment with H(2)O(2) but not rotenone. Microarray analysis further supported that signaling pathways induced by H(2)O(2) and rotenone are not identical. The promoter of BCS1 was not responsive to H(2)O(2) or rotenone, but highly responsive to salicylic acid (SA), whereas the promoters of AOX1a and NDB2 were unresponsive to SA. Analysis of transcript abundance of these genes in a variety of defense signaling mutants confirmed that BCS1 expression is regulated in a different manner compared to AOX1a, NDB2, and At2g21640. These mutants also revealed a pathway associated with programmed cell death that regulated AOX1a in a manner distinct from the other genes. Thus, at least three distinctive pathways regulate mitochondrial stress response at a transcriptional level, an SA-dependent pathway represented by BCS1, a second pathway that represents a convergence point for signals generated by H(2)O(2) and rotenone on multiple CAREs, some of which are shared between responsive genes, and a third pathway that acts via EDS1 and PAD4 regulating only AOX1a. Furthermore, posttranscriptional regulation accounts for changes in transcript abundance by SA treatment for some genes.