Redox regulation of PEP activity during seedling establishment in Arabidopsis thaliana

Activation of the plastid-encoded RNA polymerase is tightly controlled and involves a network of phosphorylation and, as yet unidentified, thiol-mediated events. Here, we characterize PLASTID REDOX INSENSITIVE2, a redox-regulated protein required for full PEP-driven transcription. PRIN2 dimers can be reduced into the active monomeric form by thioredoxins through reduction of a disulfide bond. Exposure to light increases the ratio between the monomeric and dimeric forms of PRIN2. Complementation of prin2-2 with different PRIN2 protein variants demonstrates that the monomer is required for light-activated PEP-dependent transcription and that expression of the nuclear-encoded photosynthesis genes is linked to the activity of PEP. Activation of PEP during chloroplast development likely is the source of a retrograde signal that promotes nuclear LHCB expression. Thus, regulation of PRIN2 is the thiol-mediated mechanism required for full PEP activity, with PRIN2 monomerization via reduction by TRXs providing a mechanistic link between photosynthetic electron transport and activation of photosynthetic gene expression.</p

I samklang : harmoni mellan cellens kloroplaster och kärna

Photosynthetic eukaryots emerged as a result of several billion years of evolution between proeukaryotic cell and ancestral cyanobacteria that formed modern chloroplasts. The symbiotic relationship led to significant rearrangements in the genomes of the plastid and the nucleus: as many as 90 % of all the plastid genes were transferred to the nucleus. The gene transfer has been accompanied by the development of sophisticated regulatory signaling networks originating in the organelle (retrograde) and in the nucleus (anterograde) that coordinate development of the plastid and ensure adequate cell responses to stress signals. In this thesis I have demonstrated that transcriptional activity of PEP in the chloroplast is essential for proper embryo and seedling development in Arabidopsis thaliana. The function of PEP is dependent on the nuclear encoded PEPassociated factor PRIN2 that is able to sense the redox status of the plastid during seedling development and different stress. In response to the plastid status PRIN2 modulates the transcription activity of the PEP enzyme complex. We further established that PRIN2, as an essential component for full PEP activity, is also required to emit the Plastid Gene Expression (PGE) retrograde signal to regulate the Photosynthesis-Associated Nuclear Genes (PhANG) in the nucleus during early seedling growth via GUN1. On the other hand, regulation of PhANG expression during the High Light (HL) conditions requires functional PRIN2 and PEP activity but is GUN1-independent. Another retrograde signal produced by the developing chloroplast is associated with the tetrapyrrole biosynthesis pathway. We have established that accumulation of the chlorophyll intermediate MgProtoIX-ME in the crd mutant triggers repression of the PhANG expression, and this negative signal is mediated by a cytoplasmic protein complex containing the PAPP5 phosphatase. The nuclear targets that receive the tetrapyrrole mediated signal are GLK1 and GLK2 transcription factors that control the PhANG expression and the expression of the enzymes involved in the biosynthesis of chlorophyll.Fotosyntetiserande eukaryoter uppstod från en endosymbiotisk interaktion under några miljarder år mellan en ur-eukaryot och kloroplastens förfader, den prokaryota cyanobakterien. Den symbiotiska händelsen ledde till att kloroplastens och kärnans genom blev väsentligt förändrade. Så småningom överförde kloroplasten så många som 90 % av dess gener till cellkärnan. För att koordinera genutrycket från de två genomen utvecklade växtcellen ett sofistikerat signalsystemen som inkluderar: plastid-kärn (retrograd) och kärn-plastid (anterograd) signalering som styr kloroplastens utveckling och förmåga att anpassa sig till stressförhållanden. Den här avhandlingen beskriver kloroplastens maskineri för genuttryck (PEP) som en nödvändig komponent för embryo- och växtutvecklingen hos Arabidopsis thaliana. PEP funktionen är beroende av det kärnkodade kloroplastproteinet PRIN2 som är associerat med PEP. PRIN2 mottar redox signaler från plastiden och förändrar genuttrycksaktivitet under kloroplastens utvecklingen eller under olika stressförhållanden. Jag visar dessutom att PRIN2 spelar en viktig roll i överföring av kloroplastens signal som kommunicerar genuttrycksaktivitet (PGE) via GUN1 till kärnan där den styr uttryck av de kärnkodade fotosyntetesgenerna (PhANG). Under högljus stressförhållanden styrs dock PhANG-uttrycket av signaler som uppstår från PEP-aktivitet och PRIN2 men som är oberoende av GUN1. Vidare finns det en annan retrograd signal som har sitt ursprung i biosyntesen av tetrapyrroler. Jag har visat att ackumuleringen av tetrapyrrolen MgProtoIX-ME i crd-mutanten framkallar nedreglering av PhANG-uttryck genom interaktion med ett fosfatas (PAPP5) i cytosolen. GLK1 and GLK2 är två transkriptionsfaktorer som tar emot den tetrapyrrole-medierade signalen i sin tur styr biosyntes av chlorofyll och PhANG uttryck

Plastid encoded RNA polymerase activity and expression of photosynthesis genes required for embryo and seed development in Arabidopsis

Chloroplast biogenesis and function is essential for proper plant embryo and seed development but the molecular mechanisms underlying the role of plastids during embryogenesis are poorly understood. Expression of plastid encoded genes is dependent on two different transcription machineries; a plastid-encoded bacterial-type RNA polymerase (PEP) and a nuclear-encoded phage-type RNA polymerase (NEP), which recognize distinct types of promoters. However, the division of labor between PEP and NEP during plastid development and in mature chloroplasts is unclear. We show here that PLASTID REDOX INSENSITIVE 2 (PRIN2) and CHLOROPLAST STEM-LOOP BINDING PROTEIN 41 kDa (CSP41b), two proteins identified in plastid nucleoid preparations, are essential for proper plant embryo development. Using Co-IP assays and native PAGE we have shown a direct physical interaction between PRIN2 and CSP41b. Moreover, PRIN2 and CSP41b form a distinct protein complex in vitro that binds DNA. The prin2.2 and csp41b-2 single mutants displayed pale phenotypes, abnormal chloroplasts with reduced transcript levels of photosynthesis genes and defects in embryo development. The respective csp41b-2prin2.2 homo/heterozygote double mutants produced abnormal white colored ovules and shrunken seeds. Thus, the csp41b-2prin2.2 double mutant is embryo lethal. In silico analysis of available array data showed that a large number of genes traditionally classified as PEP dependent genes are transcribed during early embryo development from the pre-globular stage to the mature-green-stage. Taken together, our results suggest that PEP activity and consequently the switch from NEP to PEP activity, is essential during embryo development and that the PRIN2-CSP41b DNA binding protein complex possibly is important for full PEP activity during this process

Redox regulation of PEP activity during seedling establishment in Arabidopsis thaliana

Activation of the plastid-encoded RNA polymerase is tightly controlled and involves a network of phosphorylation and, as yet unidentified, thiol-mediated events. Here, we characterize PLASTID REDOX INSENSITIVE2, a redox-regulated protein required for full PEP-driven transcription. PRIN2 dimers can be reduced into the active monomeric form by thioredoxins through reduction of a disulfide bond. Exposure to light increases the ratio between the monomeric and dimeric forms of PRIN2. Complementation of prin2-2 with different PRIN2 protein variants demonstrates that the monomer is required for light-activated PEP-dependent transcription and that expression of the nuclear-encoded photosynthesis genes is linked to the activity of PEP. Activation of PEP during chloroplast development likely is the source of a retrograde signal that promotes nuclear LHCB expression. Thus, regulation of PRIN2 is the thiol-mediated mechanism required for full PEP activity, with PRIN2 monomerization via reduction by TRXs providing a mechanistic link between photosynthetic electron transport and activation of photosynthetic gene expression

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

Working model for the role of PAPP5 in tetrapyrrole mediated plastid signalling.

<p>PAPP5 pereceives an inbalance in the tetrapyrrole biosynthesis through the accumulation of Mg-ProtoIX/Mg-ProtoIXME and acts as a negative regulator of chloroplast biogenesis and development. The tetrapyrrole/PAPP5-mediated plastid signal blocks the induction of the genes encoding the GLK1/2 transcription factors. GLK1 and GLK2 are essential for the induction of <i>PhANG</i> expression and chloroplast development.</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

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

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