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

Shaikhali J, Heiber I, Seidel T, et al. The redox-sensitive transcription factor Rap2.4a controls nuclear expression of 2-Cys peroxiredoxin A and other chloroplast antioxidant enzymes. BMC Plant Biology. 2008;8(1):48

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

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

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

<i>PhANG</i> expression during chloroplast development.

<p>Relative expression levels of A) <i>LHCB2.4</i> (At3g27690) complemented with Western blot analysis of LHCBII protein and β-Tubulin as protein loading control, B) <i>GLK1</i> (At2g20570) and C) <i>GLK2</i> (At5g44190) in seedlings grown for three days in dark and exposed to 12 h and 24 h of illumination. Expression levels were compared to the respective dark control for each genotype and relative expression was calculated using Ubiquitin-protein ligase (At4g36800) as a reference gene. Data represents the mean (± SD) from three independent biological replicates. Significant differences relative to Col0 (<i>crd</i> and <i>papp5</i>) and to <i>crd</i> (<i>papp5crd</i>) were calculated according to <i>t-test</i> (a, P≤0.001; b, P≤0.005; c, P≤0.01). The bands were quantified using ImageJ software and the relative band intensities were obtained and related to Col0 12 h samples.</p

Chloroplast development during de-etiolation.

<p>Sequential electron microscopy images from Col0 (A, E, I and M), <i>papp5</i> (B, F, J and N), <i>crd</i> (C, G, K and O) and <i>papp5crd</i> (D, H, L and P) during the first 24 hours of illumination (100 µmol photons m⁻² sec⁻¹). Samples were collected 4 h, 8 h, 12 h and 24 h following transfer to light and compared to the dark sample. Arrows indicate examples of grana structures. Scale bar = 1 µm.</p

Tetrapyrrole accumulation maintained in <i>papp5crd</i>.

<p>A) Relative fluorescence corresponding to Mg-ProtoIX and Mg-ProtoIX-ME in seedlings from Col0, <i>papp5</i>, <i>crd</i>, and <i>papp5crd</i> during the dark to light transition. 3-d-old seedlings were transferred to constant light (100 µmol photons light cm⁻² sec⁻¹) and samples were collected following 12 h and 24 h exposure. Each data point represents the mean (± SD) of four independent biological replicates. Fluorescence data is complemented with Western blot analysis of the CRD protein levels in the different genotypes. β-Tubulin was used as protein loading control. B) Relative expression of <i>APX2</i> (At3g09640) in Col0, <i>papp5</i>, <i>crd</i>, and <i>papp5crd</i>. Expression levels were compared to the respective dark control for each genotype and relative expression was calculated using Ubiquitin-protein ligase (At4g36800) as a reference gene. Each bar represents the mean (± SD) of at least three independent biological samples. Significant differences relative to Col0 (<i>crd</i> and <i>papp5</i>) and to <i>crd</i> (<i>papp5crd</i>) were calculated according to <i>t-test</i> (a, P<0.001; b, c, P<0.01)</p

Chlorophyll determination during de-etiolation.

<p>Chlorophyll content in Col0, <i>papp5</i>, <i>crd</i> and <i>papp5crd</i> seedlings following A) 12 h and B) 24 h of illumination. C) Chlorophyll a/b ratio in the different lines during 12 h and 24 h of illumination. Significant differences relative to Col0 (<i>crd</i>) and to <i>crd</i> (<i>papp5crd</i>) according to <i>t-test</i> (a, P<0.001; b, P<0.005; c, P<0.01; d, P<0.05) are shown.</p

Okadaic acid treatment phenocopies <i>papp5crd</i> in <i>crd</i> single mutant.

<p>Relative expression of <i>LHCB2.4</i> (At3g27690), <i>GLK1</i> (At2g20570) and <i>GLK2</i> (At5g44190) in Col0 and <i>crd</i> plants following treatment with 10 nM okadaic acid during de-etiolation. Samples were collected following 12 h exposure to light. Each bar represents the mean (± SD) of three independent biological replicates. Significant differences relative to Col0 were calculated according to <i>t-test</i> (b, P<0.005; c, P<0.01; d, P<0.05).</p

The <i>papp5</i> mutant is insensitive to tetrapyrrole feeding.

<p>Feeding experiments were performed to increase the levels of tetrapyrroles in the plants. During the dark period, two-week-old plants from Col0 and <i>papp5</i> grown under LD conditions (15 hours light/9 hours dark) were treated with A) 50 µM Mg-ProtoIX or B) 10 mM ALA. Relative fluorescence corresponding to Mg-ProtoIX and Mg-ProtoIX-ME is shown. Samples were collected 4 h and 1 h into the light period following the Mg-ProtoIX and ALA treatment, respectively. Each bar represents the mean (± SD) of three independent biological replicates. Relative expression of <i>LHCB2.4</i> (At3g27690), <i>GLK1</i> (At2g20570) and <i>GLK2</i> (At5g44190) in Col0 and <i>papp5</i> plants following C) Mg-ProtoIX or D) ALA feeding is shown. Expression levels were compared to the respective mock control for each genotype and relative expression was calculated using Ubiquitin-like protein (At4g36800) as internal standard. Each bar represents the mean (± SD) of at least three independent biological replicates. Significant differences relative to untreated control or Col0 were calculated according to <i>t-test</i> (a, P<0.001; b, P<0.005; d, P<0.05).</p