SUMMARYThe photosynthetic apparatus is composed of proteins encoded by genes from both the nuclear and the chloroplastic genomes. The activities of the nuclear and chloroplast genomes must therefore be closely coordinated through intracellular signalling. The plastids produce multiple retrograde signals at different times of their development, and in response to changes in the environment. These signals regulate the expression of nuclear-encoded photosynthesis genes to match the current status of the plastids. Using forward genetics we identified PLASTID REDOX INSENSITIVE 2 (PRIN2), a chloroplast component involved in redox-mediated retrograde signalling. The allelic mutants prin2-1 and prin2-2 demonstrated a misregulation of photosynthesis-associated nuclear gene expression in response to excess light, and an inhibition of photosynthetic electron transport. As a consequence of the misregulation of LHCB1.1 and LHCB2.4, the prin2 mutants displayed a high irradiance-sensitive phenotype with significant photoinactivation of photosystem II, indicated by a reduced variable to maximal fluorescence ratio (F v /F m ). PRIN2 is localized to the nucleoids, and plastid transcriptome analyses demonstrated that PRIN2 is required for full expression of genes transcribed by the plastid-encoded RNA polymerase (PEP). Similarly to the prin2 mutants, the ys1 mutant with impaired PEP activity also demonstrated a misregulation of LHCB1.1 and LHCB2.4 expression in response to excess light, suggesting a direct role for PEP activity in redox-mediated retrograde signalling. Taken together, our results indicate that PRIN2 is part of the PEP machinery, and that the PEP complex responds to photosynthetic electron transport and generates a retrograde signal, enabling the plant to synchronize the expression of photosynthetic genes from both the nuclear and plastidic genomes.

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 ciselement 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.

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.

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.

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.

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