Redox regulation of the MED28 and MED32 mediator subunits is important for development and senescence.

Mediator is a conserved multi-protein complex that acts as a bridge between promoter-bound transcriptional regulators and RNA polymerase II. While redox signaling is important in adjusting plant metabolism and development, the involvement of Mediator in redox homeostasis and regulation only recently started to emerge. Our previous results show that the MED10a, MED28, and MED32 Mediator subunits form various types of covalent oligomers linked by intermolecular disulfide bonds in vitro. To link that with biological significance we have characterized Arabidopsis med32 and med28 mutants and found that they are affected in root development and senescence, phenotypes possibly associated to redox changes.

Biochemical and redox characterization of the mediator complex and its associated transcription factor GeBPL, a GLABROUS1 enhancer binding protein.

The eukaryotic mediator integrates regulatory signals from promoter-bound transcription factors (TFs) and transmits them to RNA polymerase II (Pol II) machinery. Although redox signalling is important in adjusting plant metabolism and development, nothing is known about a possible redox regulation of mediator. In the present study, using pull-down and yeast two-hybrid assays, we demonstrate the association of mediator (MED) subunits MED10a, MED28 and MED32 with the GLABROUS1 (GL1) enhancer-binding protein-like (GeBPL), a plant-specific TF that binds a promoter containing cryptochrome 1 response element 2 (CryR2) element. All the corresponding recombinant proteins form various types of covalent oligomers linked by intermolecular disulfide bonds that are reduced in vitro by the thioredoxin (TRX) and/or glutathione/glutaredoxin (GRX) systems. The presence of recombinant MED10a, MED28 and MED32 subunits or changes of its redox state affect the DNA-binding capacity of GeBPL suggesting that redox-driven conformational changes might modulate its activity. Overall, these results advance our understanding of how redox signalling affects transcription and identify mediator as a novel actor in redox signalling pathways, relaying or integrating redox changes in combination with specific TFs as GeBPL.

GIP1 protein is a novel cofactor that regulates DNA-binding affinity of redox-regulated members of bZIP transcription factors involved in the early stages of Arabidopsis development.

In response to environmental light signals, gene expression adjustments play an important role in regulation of photomorphogenesis. LHCB2.4 is among the genes responsive to light signals, and its expression is regulated by redox-regulated members of G-group bZIP transcription factors. The biochemical interrelations of GBF1-interacting protein 1 (GIP1) and the G-group bZIP transcription factors have been investigated. GIP1, previously shown to enhance DNA-binding activities of maize GBF1 and Arabidopsis GBF3, is a plant specific protein that reduces DNA-binding activity of AtbZIP16, AtbZIP68, and AtGBF1 under non-reducing conditions through direct physical interaction shown by the yeast two-hybrid and pull-down assays. Fluorescence microscopy studies using cyan fluorescent protein (CFP)-fusion protein indicate that GIP1 is exclusively localized in the nucleus. Under non- reducing conditions, GIP1 exhibits predominantly high molecular weight forms, whereas it predominates in low molecular weight monomers under reducing conditions. While reduced GIP1 induced formation of DNA-protein complexes of G-group bZIPs, oxidized GIP1 decreased the amount of those complexes and instead induced its chaperone function suggesting functional switching from redox to chaperone activity. Finally analysis of transgenic plants overexpressing GIP1 revealed that GIP1 is a negative co-regulator in red and blue light mediated hypocotyl elongation. By regulating the repression effect by bZIP16 and the activation effect by bZIP68 and GBF1 on LHCB2.4 expression, GIP1 functions to promote hypocotyl elongation during the early stages of Arabidopsis seedling development.

The radical induced cell death protein 1 (RCD1) supports transcriptional activation of genes for chloroplast antioxidant enzymes.

The rimb1 (redox imbalanced 1) mutation was mapped to the RCD1 locus (radical-induced cell death 1; At1g32230) demonstrating that a major factor involved in redox-regulation genes for chloroplast antioxidant enzymes and protection against photooxidative stress, RIMB1, is identical to the regulator of disease response reactions and cell death, RCD1. Discovering this link let to our investigation of its regulatory mechanism. We show in yeast that RCD1 can physically interact with the transcription factor Rap2.4a which provides redox-sensitivity to nuclear expression of genes for chloroplast antioxidant enzymes. In the rimb1 (rcd1-6) mutant, a single nucleotide exchange results in a truncated RCD1 protein lacking the transcription factor binding site. Protein-protein interaction between full-length RCD1 and Rap2.4a is supported by H2O2, but not sensitive to the antioxidants dithiotreitol and ascorbate. In combination with transcript abundance analysis in Arabidopsis, it is concluded that RCD1 stabilizes the Rap2.4-dependent redox-regulation of the genes encoding chloroplast antioxidant enzymes in a widely redox-independent manner. Over the years, rcd1-mutant alleles have been described to develop symptoms like chlorosis, lesions along the leaf rims and in the mesophyll and (secondary) induction of extra- and intra-plastidic antioxidant defense mechanisms. All these rcd1 mutant characteristics were observed in rcd1-6 to succeed low activation of the chloroplast antioxidant system and glutathione biosynthesis. We conclude that RCD1 protects plant cells from running into reactive oxygen species (ROS)-triggered programs, such as cell death and activation of pathogen-responsive genes (PR genes) and extra-plastidic antioxidant enzymes, by supporting the induction of the chloroplast antioxidant system.

PAPP5 is involved in the tetrapyrrole mediated plastid signalling during chloroplast development.

The initiation of chloroplast development in the light is dependent on nuclear encoded components. The nuclear genes encoding key components in the photosynthetic machinery are regulated by signals originating in the plastids. These plastid signals play an essential role in the regulation of photosynthesis associated nuclear genes (PhANGs) when proplastids develop into chloroplasts. One of the plastid signals is linked to the tetrapyrrole biosynthesis and accumulation of the intermediates the Mg-ProtoIX and its methyl ester Mg-ProtoIX-ME. Phytochrome-Associated Protein Phosphatase 5 (PAPP5) was isolated in a previous study as a putative Mg-ProtoIX interacting protein. In order to elucidate if there is a biological link between PAPP5 and the tetrapyrrole mediated signal we generated double mutants between the Arabidopsis papp5 and the crd mutants. The crd mutant over-accumulates Mg-ProtoIX and Mg-ProtoIX-ME and the tetrapyrrole accumulation triggers retrograde signalling. The crd mutant exhibits repression of PhANG expression, altered chloroplast morphology and a pale phenotype. However, in the papp5crd double mutant, the crd phenotype is restored and papp5crd accumulated wild type levels of chlorophyll, developed proper chloroplasts and showed normal induction of PhANG expression in response to light. Tetrapyrrole feeding experiments showed that PAPP5 is required to respond correctly to accumulation of tetrapyrroles in the cell and that PAPP5 is most likely a component in the plastid signalling pathway down stream of the tetrapyrrole Mg-ProtoIX/Mg-ProtoIX-ME. Inhibition of phosphatase activity phenocopied the papp5crd phenotype in the crd single mutant demonstrating that PAPP5 phosphatase activity is essential to mediate the retrograde signal and to suppress PhANG expression in the crd mutant. Thus, our results suggest that PAPP5 receives an inbalance in the tetrapyrrole biosynthesis through the accumulation of Mg-ProtoIX and acts as a negative regulator of PhANG expression during chloroplast biogenesis and development.

The CRYPTOCHROME1-dependent response to excess light is mediated through the transcriptional activators ZINC FINGER PROTEIN EXPRESSED IN INFLORESCENCE MERISTEM LIKE1 and ZML2 in Arabidopsis.

Exposure of plants to light intensities that exceed the electron utilization capacity of the chloroplast has a dramatic impact on nuclear gene expression. The photoreceptor Cryptochrome 1 (cry1) is essential to the induction of genes encoding photoprotective components in Arabidopsis thaliana. Bioinformatic analysis of the cry1 regulon revealed the putative cis-element CryR1 (GnTCKAG), and here we demonstrate an interaction between CryR1 and the zinc finger GATA-type transcription factors ZINC FINGER PROTEIN EXPRESSED IN INFLORESCENCE MERISTEM LIKE1 (ZML1) and ZML2. The ZML proteins specifically bind to the CryR1 cis-element as demonstrated in vitro and in vivo, and TCTAG was shown to constitute the core sequence required for ZML2 binding. In addition, ZML2 activated transcription of the yellow fluorescent protein reporter gene driven by the CryR1 cis-element in Arabidopsis leaf protoplasts. T-DNA insertion lines for ZML2 and its homolog ZML1 demonstrated misregulation of several cry1-dependent genes in response to excess light. Furthermore, the zml1 and zml2 T-DNA insertion lines displayed a high irradiance-sensitive phenotype with significant photoinactivation of photosystem II (PSII), indicated by reduced maximum quantum efficiency of PSII, and severe photobleaching. Thus, we identified the ZML2 and ZML1 GATA transcription factors as two essential components of the cry1-mediated photoprotective response.

Redox-mediated mechanisms regulate DNA binding activity of the G-group of basic region leucine zipper (bZIP) transcription factors in Arabidopsis.

Plant genes that contain the G-box in their promoters are responsive to a variety of environmental stimuli. Bioinformatics analysis of transcriptome data revealed that the G-box element is significantly enriched in promoters of high light-responsive genes. From nuclear extracts of high light-treated Arabidopsis plants, we identified the AtbZIP16 transcription factor as a component binding to the G-box-containing promoter fragment of light-harvesting chlorophyll a/b-binding protein2.4 (LHCB2.4). AtbZIP16 belongs to the G-group of Arabidopsis basic region leucine zipper (bZIP) type transcription factors. Although AtbZIP16 and its close homologues AtbZIP68 and AtGBF1 bind the G-box, they do not bind the mutated half-sites of the G-box palindrome. In addition, AtbZIP16 interacts with AtbZIP68 and AtGBF1 in the yeast two-hybrid system. A conserved Cys residue was shown to be necessary for redox regulation and enhancement of DNA binding activity in all three proteins. Furthermore, transgenic Arabidopsis lines overexpressing the wild type version of bZIP16 and T-DNA insertion mutants for bZIP68 and GBF1 demonstrated impaired regulation of LHCB2.4 expression. Finally, overexpression lines for the mutated Cys variant of bZIP16 provided support for the biological significance of Cys(330) in redox regulation of gene expression. Thus, our results suggest that environmentally induced changes in the redox state regulate the activity of members of the G-group of bZIP transcription factors.

Interplay between Heat Shock Protein 90 and HY5 controls PhANG expression in response to the GUN5 plastid signal.

The presence of genes encoding organellar proteins in different cellular compartments necessitates a tight coordination of expression by the different genomes of the eukaryotic cell. This coordination of gene expression is achieved by organelle-to-nucleus or retrograde communication. Stress-induced perturbations of the tetrapyrrole pathway trigger large changes in nuclear gene expression in plants. Recently, we identified HSP90 proteins as ligands of the putative plastid signal Mg-ProtoIX. In order to investigate whether the interaction between HSP90 and Mg-ProtoIX is biologically relevant, we produced transgenic lines with reduced levels of cytosolic HSP90 in wild-type and gun5 backgrounds. Our work reveals that HSP90 proteins respond to the tetrapyrrole-mediated plastid signal to control expression of photosynthesis-associated nuclear genes (PhANG) during the response to oxidative stress. We also show that the hy5 mutant is insensitive to tetrapyrrole accumulation and that Mg-ProtoIX, cytosolic HSP90, and HY5 are all part of the same signaling pathway. These findings suggest that a regulatory complex controlling gene expression that includes HSP90 proteins and a transcription factor that is modified by tetrapyrroles in response to changes in the environment is evolutionarily conserved between yeast and plants.

Ascorbate regulation of 2-Cys peroxiredoxin-A promoter activity is light-dependent.

The 2-Cys peroxiredoxin-A (2CPA) promoter is a model promoter to study redox and ABA-dependent stress signaling. Here, an Arabidopsis reporter gene line expressing luciferase under control of the 2CPA promoter was used to study the impact of ascorbate on reporter gene transcription in a series of protoplast and leaf slice incubation experiments. It was shown that ascorbate has a dual function on gene expression regulation. First, a comparison of responses to ascorbate, dehydroascorbate and reduced and oxidized glutathione demonstrated that ascorbate feeding supports gene expression regulation by increasing the catalytic capacity in redox signaling, as defined by the concentration of low molecular weight antioxidants and their oxidized counterparts. Second, ascorbate had a specific and light-dependent effect on 2CPA transcription, which cannot be substituted by reduced glutathione. Based on the differences between ascorbate and glutathione in the subcellular redox-cycling capacities, it is concluded that ascorbate feeding modulates chloroplast-specific regulation of 2CPA expression.

The redox-sensitive transcription factor Rap2.4a controls nuclear expression of 2-Cys peroxiredoxin A and other chloroplast antioxidant enzymes.

BACKGROUND: The regulation of the chloroplast antioxidant capacity depends on nuclear gene expression. For the 2-Cys peroxiredoxin-A gene (2CPA) a cis-regulatory element was recently characterized, which responds to photosynthetic redox signals. RESULTS: In a yeast-one-hybrid screen for cis-regulatory binding proteins, the transcription factor Rap2.4a was isolated. Rap2.4a controls the transcript abundance of the prominent chloroplast antioxidant enzyme through binding to the CGCG core of a CE3-like element. Rap2.4a activity is regulated by dithiol/disulfide transition of regulatory cysteinyl residues and subsequent changes in the quaternary structure. The mid-point redox potential of Rap2.4a activation is -269 mV (pH 7.0). CONCLUSION: The redox sensitivity of Rap2.4a establishes an efficient switch mechanism for redox control of nuclear gene activity of chloroplast antioxidants, in which Rap2.4 is a redox-sensor and a transducer of redox information.