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The plastid-encoded genes of higher plants are transcribed by at least two types of RNA polymerases, the nuclear-encoded RNA polymerase (NEP) and the plastid-encoded RNA polymerase (PEP). In mature photosynthesizing leaves, the vast majority of the genes are transcribed by PEP. However, the regulatory mechanisms controlling plastid transcription during early light response is unclear. Chloroplast development is suggested to be associated with a shift in the usage of the primary RNA polymerase from NEP to PEP as the expression of the plastid-encoded photosynthesis genes is induced upon light exposure. Assembly of the PEP complex has been suggested as a rate-limiting step for full activation of plastid-encoded photosynthesis gene expression. However, two sigma factor mutants, sig2 and sig6, with reduced PEP activity, showed significantly lower expression of the plastid-encoded photosynthesis genes already in the dark and during the first hours of light exposure indicating that PEP activity is required for basal expression of plastid-encoded photosynthesis genes in the dark and during early light response. Furthermore, in etioplasts and proplastids a fully assembled PEP complex was revealed on Blue Native PAGE. Our results indicate that a full assembly of the PEP complex is possible in the dark and that PEP drives basal transcriptional activity of plastid-encoded photosynthesis genes in the dark. Assembly of the complex is most likely not a rate-limiting step for full activation of plastid-encoded photosynthesis gene expression which is rather achieved either by the abundance of the PEP complex or by some posttranslational regulation of the individual PEP components.

Linear electron transport in the thylakoid membrane drives photosynthetic NADPH and ATP production, while cyclic electron flow (CEF) around photosystem I only promotes the translocation of protons from stroma to thylakoid lumen. The chloroplast NADH dehydrogenase‐like complex (NDH) participates in one CEF route transferring electrons from ferredoxin back to the plastoquinone pool with concomitant proton pumping to the lumen. CEF has been proposed to balance the ratio of ATP/NADPH production and to control the redox poise particularly in fluctuating light conditions, but the mechanisms regulating the NDH complex remain unknown. We have investigated potential regulation of the CEF pathways by the chloroplast NADPH‐thioredoxin reductase (NTRC) in vivo by using an Arabidopsis knockout line of NTRC as well as lines overexpressing NTRC. Here, we present biochemical and biophysical evidence showing that NTRC stimulates the activity of NDH‐dependent CEF and is involved in the regulation of generation of proton motive force, thylakoid conductivity to protons, and redox balance between the thylakoid electron transfer chain and the stroma during changes in light conditions. Furthermore, protein–protein interaction assays suggest a putative thioredoxin‐target site in close proximity to the ferredoxin‐binding domain of NDH, thus providing a plausible mechanism for redox regulation of the NDH ferredoxin:plastoquinone oxidoreductase activity.

Sucrose non-fermenting 1 (SNF1)-related protein kinase 1.1 (SnRK1.1; also known as KIN10 or SnRK1α) has been identified as the catalytic subunit of the complex SnRK1, the Arabidopsis thaliana homologue of a central integrator of energy and stress signalling in eukaryotes dubbed AMPK/Snf1/SnRK1. A nuclear localization of SnRK1.1 has been previously described and is in line with its function as an integrator of energy and stress signals. Here, using two biological models (Nicotiana benthamiana and Arabidopsis thaliana), native regulatory sequences, different microscopy techniques, and manipulations of cellular energy status, it was found that SnRK1.1 is localized dynamically between the nucleus and endoplasmic reticulum (ER). This distribution was confirmed at a spatial and temporal level by co-localization studies with two different fluorescent ER markers, one of them being the SnRK1.1 phosphorylation target HMGR. The ER and nuclear localization displayed a dynamic behaviour in response to perturbations of the plastidic electron transport chain. These results suggest that an ER-associated SnRK1.1 fraction might be sensing the cellular energy status, being a point of crosstalk with other ER stress regulatory pathways. Fil: Blanco, Nicolás Ernesto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosintéticos y Bioquímicos. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos; Argentina. Universidad de Umea; Suecia Fil: Liebsch, Daniela Elisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad de Umea; Suecia Fil: Guinea Díaz, Manuel. University of Turku. Department of Biochemistry. Molecular Plant Biology; Finlandia Fil: Strand, Asa. Universidad de Umea; Suecia Fil: Whelan, James. La Trobe University; Australia

Linear electron transport in the thylakoid membrane drives both photosynthetic NADPH and ATP production, while cyclic electron flow (CEF) around photosystem I only promotes the translocation of protons from stroma to thylakoid lumen. The chloroplast NADH-dehydrogenase-like complex (NDH) participates in one CEF route transferring electrons from ferredoxin back to the plastoquinone pool with concomitant proton pumping to the lumen. CEF has been proposed to balance the ratio of ATP/NADPH production and to control the redox poise particularly in fluctuating light conditions, but the mechanisms regulating the NDH complex remain unknown. We have investigated potential regulation of the CEF pathways by the chloroplast NADPH-thioredoxin reductase (NTRC)in vivoby using an Arabidopsis knockout line ofNTRCas well as lines overexpressing NTRC. Here we present biochemical and biophysical evidence showing that NTRC activates the NDH-dependent CEF and regulates the generation of proton motive force, thylakoid conductivity to protons and redox balance between the thylakoid electron transfer chain and the stroma during changes in light conditions. Further, protein–protein interaction assays suggest a putative thioredoxin-target site in close proximity to the ferredoxin binding domain of NDH, thus providing a plausible mechanism for regulation of the NDH ferredoxin:plastoquinone oxidoreductase activity by NTRC.One sentence summaryChloroplast thioredoxins regulate photosynthetic cyclic electron flow that balances the activities of light and carbon fixation reactions and improves plant fitness under fluctuating light conditions.

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., The plastid-encoded RNA polymerase PEP is regulated according to plastid redox state. Here, the authors show that the redox-regulated PRIN2 protein is reduced to monomeric form in a thiol-dependent manner in response to light and that PRIN2 monomers are required for PEP activity and retrograde signaling.

Highlight This work shows the dominant effect of NADPH thioredoxin reductase C (NTRC) over sulfiredoxin on 2-Cys peroxiredoxin (2-Cys Prx) overoxidation, and uncovers an NTRC-independent, light-dependent component contributing to 2-Cys Prx overoxidation in Arabidopsis thaliana chloroplasts., Hydrogen peroxide is a harmful by-product of photosynthesis, which also has important signalling activity. Therefore, the level of hydrogen peroxide needs to be tightly controlled. Chloroplasts harbour different antioxidant systems including enzymes such as the 2-Cys peroxiredoxins (2-Cys Prxs). Under oxidizing conditions, 2-Cys Prxs are susceptible to inactivation by overoxidation of their peroxidatic cysteine, which is enzymatically reverted by sulfiredoxin (Srx). In chloroplasts, the redox status of 2-Cys Prxs is highly dependent on NADPH-thioredoxin reductase C (NTRC) and Srx; however, the relationship of these activities in determining the level of 2-Cys Prx overoxidation is unknown. Here we have addressed this question by a combination of genetic and biochemical approaches. An Arabidopsis thaliana double knockout mutant lacking NTRC and Srx shows a phenotype similar to the ntrc mutant, while the srx mutant resembles wild-type plants. The deficiency of NTRC causes reduced overoxidation of 2-Cys Prxs, whereas the deficiency of Srx has the opposite effect. Moreover, in vitro analyses show that the disulfide bond linking the resolving and peroxidatic cysteines protects the latter from overoxidation, thus explaining the dominant role of NTRC on the level of 2-Cys Prx overoxidation in vivo. The overoxidation of chloroplast 2-Cys Prxs shows no circadian oscillation, in agreement with the fact that neither the NTRC nor the SRX genes show circadian regulation of expression. Additionally, the low level of 2-Cys Prx overoxidation in the ntrc mutant is light dependent, suggesting that the redox status of 2-Cys Prxs in chloroplasts depends on light rather than the circadian clock.

Despite being the primary source of energy in the biosphere, photosynthesis is a process that inevitably produces reactive oxygen species. Chloroplasts are a major source of hydrogen peroxide production in plant cells; therefore, different systems for peroxide reduction, such as ascorbate peroxidase and peroxiredoxins (Prxs), are found in this organelle. Most of the reducing power required for hydrogen peroxide reduction by these systems is provided by Fd reduced by the photosynthetic electron transport chain; hence, the function of these systems is highly dependent on light. Recently, it was described a novel plastidial enzyme, stated NTRC, formed by a thioredoxin reductase (NTR) domain at the N-terminus and a thioredoxin (Trx) domain at the C-terminus. NTRC is able to conjugate both NTR and Trx activities to efficiently reduce 2-Cys Prx using NADPH as a source of reducing power. Based on these results, it was proposed that NTRC is a new pathway to transfer reducing power to the chloroplast detoxification system, allowing the use of NADPH, besides reduced Fd, for such function. In this article, the most important features of NTRC are summarized and the implications of this novel activity in the context of chloroplast protection against oxidative damage are discussed.

Thioredoxins (TRXs) are protein oxidoreductases that control the structure and function of cellular proteins by cleavage of a disulphide bond between the side chains of two cysteine residues. Oxidized thioredoxins are reactivated by thioredoxin reductases (TR) and a TR-dependent reduction of TRXs is called a thioredoxin system. Thiol-based redox regulation is an especially important mechanism to control chloroplast proteins involved in biogenesis, in regulation of light harvesting and distribution of light energy between photosystems, in photosynthetic carbon fixation and other biosynthetic pathways, and in stress responses of plants. Of the two plant plastid thioredoxin systems, the ferredoxin-dependent system relays reducing equivalents from photosystem I via ferredoxin and ferredoxin-thioredoxin reductase (FTR) to chloroplast proteins, while NADPH-dependent thioredoxin reductase (NTRC) forms a complete thioredoxin system including both reductase and thioredoxin domains in a single polypeptide. Chloroplast thioredoxins transmit environmental light signals to biochemical reactions, which allows fine tuning of photosynthetic processes in response to changing environmental conditions. In this paper we focus on the recent reports on specificity and networking of chloroplast thioredoxin systems and evaluate the prospect of improving photosynthetic performance by modifying the activity of thiol regulators in plants. This article is part of the themed issue ‘Enhancing photosynthesis in crop plants: targets for improvement'.

Mitochondria and chloroplasts depend upon each other; photosynthesis provides substrates for mitochondrial respiration and mitochondrial metabolism is essential for sustaining photosynthetic carbon assimilation. In addition, mitochondrial respiration protects photosynthesis against photoinhibition by dissipating excess redox equivalents from the chloroplasts. Genetic defects in mitochondrial function result in an excessive reduction and energization of the chloroplast. Thus, it is clear that the activities of mitochondria and plastids need to be coordinated, but the manner by which the organelles communicate to coordinate their activities is unknown. The regulator of alternative oxidase ( rao1) mutant was isolated as a mutant unable to induce AOX1a expression in response to the inhibitor of the mitochondrial cytochrome c reductase (complex III), antimycin A. RAO1 encodes the nuclear localized cyclin-dependent kinase E1 (CDKE1). Interestingly, the rao1 mutant demonstrates a genome uncoupled phenotype also in response to redox changes in the photosynthetic electron transport chain. Thus, CDKE1 was shown to regulate both LIGHT HARVESTING COMPLEX B ( LHCB ) and ALTERNATIVE OXIDASE 1 ( AOX1a ) expression in response to retrograde signals. Our results suggest that CDKE1 is a central nuclear component integrating mitochondrial and plastid retrograde signals and plays a role in regulating energy metabolism during the response to stress.

Protein disulphide–dithiol interchange is a universal mechanism of redox regulation in which thioredoxins (Trxs) play an essential role. In heterotrophic organisms, and non-photosynthetic plant organs, NADPH provides the required reducing power in a reaction catalysed by NADPH-dependent thioredoxin reductase (NTR). It has been considered that chloroplasts constitute an exception because reducing equivalents for redox regulation in this organelle is provided by ferredoxin (Fd) reduced by the photosynthetic electron transport chain, not by NADPH. This view was modified by the discovery of a chloroplast-localised NTR, denoted NTRC, a bimodular enzyme formed by NTR and Trx domains with high affinity for NADPH. In this review, we will summarize the present knowledge of the biochemical properties of NTRC and discuss the implications of this enzyme on plastid redox regulation in plants. Ministerio de Ciencia e Innovación de España y Fondos FEDER de la Comisión Europea. BIO2010-15430 Junta de Andalucía. BIO-182 y CVI-5919

Photosynthesis is a process that inevitably produces reactive oxygen species, such as hydrogen peroxide, which is reduced by chloroplast-localized detoxification mechanisms one of which involves 2-Cys peroxiredoxins (2-Cys Prxs). Arabidopsis chloroplasts contain two very similar 2-Cys Prxs (denoted A and B). These enzymes are reduced by two pathways: NADPH thioredoxin reductase C (NTRC), which uses NADPH as source of reducing power; and plastidial thioredoxins (Trxs) coupled to photosynthetically reduced ferredoxin of which Trx x is the most efficient reductant in vitro. With the aim of establishing the functional relationship between NTRC, Trx x, and 2-Cys Prxs in vivo, an Arabidopsis Trx x knock-out mutant has been identified and a double mutant (denoted Δ2cp) with, This work was supported by grant BIO2007-60644 from Ministerio de Educacion y Ciencia, and grants P06-CVI01578 and BIO-182 from Junta de Andalucia (Spain). K-JD acknowledges support by the DFG (Di346). PP, KK, and MG were supported by pre-doctoral fellowships from Seville University, CSIC, and Junta de Andalucia, respectively.