Specificity versus redundancy in the RAP2.4 transcription factor family of Arabidopsis thaliana: transcriptional regulation of genes for chloroplast peroxidases

Background The Arabidopsis ERFIb / RAP2.4 transcription factor family consists of eight members with highly conserved DNA binding domains. Selected members have been characterized individually, but a systematic comparison is pending. The redox-sensitive transcription factor RAP2.4a mediates chloroplast-to- nucleus redox signaling and controls induction of the three most prominent chloroplast peroxidases, namely 2-Cys peroxiredoxin A (2CPA) and thylakoid- and stromal ascorbate peroxidase (tAPx and sAPx). To test the specificity and redundancy of RAP2.4 transcription factors in the regulation of genes for chloroplast peroxidases, we compared the DNA-binding sites of the transcription factors in tertiary structure models, analyzed transcription factor and target gene regulation by qRT-PCR in RAP2.4, 2-Cys peroxiredoxin and ascorbate peroxidase T-DNA insertion lines and RAP2.4 overexpressing lines of Arabidopsis thaliana and performed promoter binding studies. Results All RAP2.4 proteins bound the tAPx promoter, but only the four RAP2.4 proteins with identical DNA contact sites, namely RAP2.4a, RAP2.4b, RAP2.4d and RAP2.4h, interacted stably with the redox-sensitive part of the 2CPA promoter. Gene expression analysis in RAP2.4 knockout lines revealed that RAP2.4a is the only one supporting 2CPA and chloroplast APx expression. Rap2.4h binds to the same promoter region as Rap2.4a and antagonizes 2CPA expression. Like the other six RAP2.4 proteins, Rap2.4 h promotes APx mRNA accumulation. Chloroplast ROS signals induced RAP2.4b and RAP2.4d expression, but these two transcription factor genes are (in contrast to RAP2.4a) insensitive to low 2CP availability, and their expression decreased in APx knockout lines. RAP2.4e and RAP2.4f gradually responded to chloroplast APx availability and activated specifically APx expression. These transcription factors bound, like RAP2.4c and RAP2.4g, the tAPx promoter, but hardly the 2CPA promoter. Conclusions The RAP2.4 transcription factors form an environmentally and developmentally regulated transcription factor network, in which the various members affect the expression intensity of the others. Within the transcription factor family, RAP2.4a has a unique function as a general transcriptional activator of chloroplast peroxidase activity. The other RAP2.4 proteins mediate the fine-control and adjust the relative availability of 2CPA, sAPx and tAPx

Identifizierung und Charakterisierung von ERF1b-Transkriptionsfaktor- Bindungsmotiven und ihren Zielgenen

The Ethylene Response Factor (ERF) gene family is a subset of AP2 (Apetala2)/ERF superfamily. It is characterized by exhibiting an AP2/ERF domain of 60 to 70 amino acids involved in DNA binding. In Arabidopsis thaliana ERF1b transcription factor family contains eight members (Nakano et al. 2006). All proteins in the group are characterized by a conserved AP2 domain, but non-conserved C- and N-termini. To investigate the functional redundancy or specificity of the transcription factors, the binding sites of Rap2.4a and Rap2.4d were defined. A new method, called stringent inverse yeast-one-hybrid, was developed to screen full genomes for transcription factor binding motifs and to identify transcription factor target genes. It allowed efficient, fast and easy screening of the A. thaliana genome, including the organellar genomes, for binding sites of Rap2.4a and Rap2.4d. Of the Rap2.4a and Rap2.4d binding sites detected on the nuclear genome, 88 % and 94.7 %, respectively, were found in the promoter regions of genes, demonstrating a high specificity of the screen for regulatory elements. The transcript dynamics of Rap2.4d and Rap2.4c were shown to follow similar patterns in response to cold other abiotic stresses, suggesting that the two genes are redundant in function. Expression analysis of cold regulated genes (COR6.6, COR47, COR15A, PLD1) in a Rap2.4c overexpression line showed that Rap2.4c is a general negative regulator of cold responsive genes. Transient reporter gene activity analysis demonstrated that Rap2.4c exerts its regulatory function on the promoter region of cold responsive genes. Yeast- one-hybrid assay and analysis of T-DNA insertion lines showed that Rap2.4a and Rap2.4h regulate the expression of 2CPA antagonistically, possibly by competing for the same binding site. However, Rap2.4h is a stronger competitor than Rap2.4a as demonstrated by vigorous binding of 2CPA promoter. Promoter analysis using the identified motifs, yeast-one-hybrid assay and expression analysis of T-DNA insertion lines indicated that Rap2.4a and Rap2.4d positively regulate expression sAPx and tAPx.Die Ethylene Response Faktoren (ERF) Gene gehören zur AP2 (Apetala2)/ERF Superfamilie. Diese ist durch eine AP2/ERF DNA Bindedomäne charakterisiert, die aus 60-70 Aminosäuren besteht. Die Familie der ERFIb Transkriptionsfaktoren beinhaltet 8 Mitglieder (Nakano et al. 2006) mit konservierter AP2 Domäne und nicht-konservierte C- und N-Termini. Um eine mögliche funktionelle Redundanz von der Spezifität der Transkriptionsfaktoren abzugrenzen, wurden die Bindemotive der Faktoren Rap2.4a und Rap2.4d identifiziert. Hierfür wurde eine neue Methode, das sogenannte „Stringente inverse Hefe-1-Hybrid-System“, entwickelt. Es ermöglicht eine genomweite Überprüfung von Transkriptionsfaktor-Bindestellen und zeichnet sich durch eine einfache und effiziente Handhabung aus. In der vorliegenden Arbeit wurde es zur Identifizierung von Rap2.4a-und Rap2.4d-Bindestellen in Arabidopsis thaliana, einschließlich der Organellgenome, eingesetzt. 88 % bzw. 94.7 % der in genomischer DNA identifizierten Rap2.4a-und Rap2.4d-Bindemotive liegen in Promotorabschnitten. Dies bestätigte die hohe Spezifität der Methode. Die Transkriptspiegel von Rap2.4d und Rap2.4c werden durch Kälte und anderen abiotischen Stressformen ähnlich reguliert, was für eine funktionelle Redundanz der Gene spricht. Expressionsanalysen zu den kälteregulierten Genen COR6.6, COR47, COR15A und PLD1 in Rap2.4c-Überexpressionlinien zeigten, dass der Transkriptionsfaktor einen generellen, reprimierenden Einfluss auf kälteregulierte Gene hat. Hefe-1-Hybrid Resultate sowie T-DNA Insertionsanalysen zeigten, dass Rap2.4a und Rap2.4h die Expression von 2CPA antagonistisch regulieren. Möglich wäre eine Konkurrenz um die gleiche Bindestelle auf dem 2CPA-Promotor, wobei Rap2.4h eine stärkere Bindeaffinität hat als Rap2.4a. Promotoranalysen, Hefe-1-Hybrid Experimente und Expressionsanalysen lassen darauf schließen, dass Rap2.4a und Rap2.4d die Expression von sAPx und tAPx positiv regulieren