The translocon protein FtsHi1 is an ATP-dependent DNA/RNA helicase that prevents R-loop accumulation in chloroplasts.

R-loops, three-stranded nucleic acid structures consisting of a DNA: RNA hybrid and displaced single-stranded DNA, play critical roles in gene expression and genome stability. How R-loop homeostasis is integrated into chloroplast gene expression remains largely unknown. We found an unexpected function of FtsHi1, an inner envelope membrane-bound AAA-ATPase in chloroplast R-loop homeostasis of Arabidopsis thaliana. Previously, this protein was shown to function as a component of the import motor complex for nuclear-encoded chloroplast proteins. However, this study provides evidence that FtsHi1 is an ATP-dependent helicase that efficiently unwinds both DNA-DNA and DNA-RNA duplexes, thereby preventing R-loop accumulation. Over-accumulation of R-loops could impair chloroplast transcription but not necessarily genome integrity. The dual function of FtsHi1 in both protein import and chloroplast gene expression may be important to coordinate the biogenesis of nuclear- and chloroplast-encoded subunits of multi-protein photosynthetic complexes. This study suggests a mechanical link between protein import and R-loop homeostasis in chloroplasts of higher plants.

Rational design of geranylgeranyl diphosphate synthase enhances carotenoid production and improves photosynthetic efficiency in Nicotiana tabacum.

Restricted genetic diversity can supply only a limited number of elite genes for modern plant cultivation and transgenesis. In this study, we demonstrate that rational design enables the engineering of geranylgeranyl diphosphate synthase (NtGGPPS), an enzyme of the methylerythritol phosphate pathway (MEP) in the model plant Nicotiana tabacum. As the crucial bottleneck in carotenoid biosynthesis, NtGGPPS1 interacts with phytoene synthase (NtPSY1) to channel GGPP into the production of carotenoids. Loss of this enzyme in the ntggpps1 mutant leads to decreased carotenoid accumulation. With the aim of enhancing NtGGPPS1 activity, we undertook structure-guided rational redesign of its substrate binding pocket in combination with sequence alignment. The activity of the designed NtGGPPS1 (a pentuple mutant of five sites V154A/I161L/F218Y/I209S/V233E, d-NtGGPPS1) was measured by a high-throughput colorimetric assay. d-NtGGPPS1 exhibited significantly higher conversion of IPP and each co-substrate (DMAPP ~1995.5-fold, GPP ~25.9-fold, and FPP ~16.7-fold) for GGPP synthesis compared with wild-type NtGGPPS1. Importantly, the transient and stable expression of d-NtGGPPS1 in the ntggpps1 mutant increased carotenoid levels in leaves, improved photosynthetic efficiency, and increased biomass relative to NtGGPPS1. These findings provide a firm basis for the engineering of GGPPS and will facilitate the development of quality and yield traits. Our results open the door for the structure-guided rational design of elite genes in higher plants.

The plastid-encoded protein Orf2971 is required for protein translocation and chloroplast quality control.

Photosynthesis and the biosynthesis of many important metabolites occur in chloroplasts. In these semi-autonomous organelles, the chloroplast genome encodes approximately 100 proteins. The remaining chloroplast proteins, close to 3,000, are encoded by nuclear genes whose products are translated in the cytosol and imported into chloroplasts. However, there is still no consensus on the composition of the protein import machinery including its motor proteins and on how newly imported chloroplast proteins are refolded. In this study, we have examined the function of orf2971, the largest chloroplast gene of Chlamydomonas reinhardtii. The depletion of Orf2971 causes the accumulation of protein precursors, partial proteolysis and aggregation of proteins, increased expression of chaperones and proteases, and autophagy. Orf2971 interacts with the TIC (translocon at the inner chloroplast envelope) complex, catalyzes ATP (adenosine triphosphate) hydrolysis, and associates with chaperones and chaperonins. We propose that Orf2971 is intimately connected to the protein import machinery and plays an important role in chloroplast protein quality control.

The DnaJ proteins DJA6 and DJA5 are essential for chloroplast iron-sulfur cluster biogenesis.

Fe-S clusters are ancient, ubiquitous and highly essential prosthetic groups for numerous fundamental processes of life. The biogenesis of Fe-S clusters is a multistep process including iron acquisition, sulfur mobilization, and cluster formation. Extensive studies have provided deep insights into the mechanism of the latter two assembly steps. However, the mechanism of iron utilization during chloroplast Fe-S cluster biogenesis is still unknown. Here we identified two Arabidopsis DnaJ proteins, DJA6 and DJA5, that can bind iron through their conserved cysteine residues and facilitate iron incorporation into Fe-S clusters by interactions with the SUF (sulfur utilization factor) apparatus through their J domain. Loss of these two proteins causes severe defects in the accumulation of chloroplast Fe-S proteins, a dysfunction of photosynthesis, and a significant intracellular iron overload. Evolutionary analyses revealed that DJA6 and DJA5 are highly conserved in photosynthetic organisms ranging from cyanobacteria to higher plants and share a strong evolutionary relationship with SUFE1, SUFC, and SUFD throughout the green lineage. Thus, our work uncovers a conserved mechanism of iron utilization for chloroplast Fe-S cluster biogenesis.

Functional redox links between lumen thiol oxidoreductase1 and serine/threonine-protein kinase STN7.

In response to changing light quantity and quality, photosynthetic organisms perform state transitions, a process which optimizes photosynthetic yield and mitigates photo-damage. The serine/threonine-protein kinase STN7 phosphorylates the light-harvesting complex of photosystem II (PSII; light-harvesting complex II), which then migrates from PSII to photosystem I (PSI), thereby rebalancing the light excitation energy between the photosystems and restoring the redox poise of the photosynthetic electron transport chain. Two conserved cysteines forming intra- or intermolecular disulfide bonds in the lumenal domain (LD) of STN7 are essential for the kinase activity although it is still unknown how activation of the kinase is regulated. In this study, we show lumen thiol oxidoreductase 1 (LTO1) is co-expressed with STN7 in Arabidopsis (Arabidopsis thaliana) and interacts with the LD of STN7 in vitro and in vivo. LTO1 contains thioredoxin (TRX)-like and vitamin K epoxide reductase domains which are related to the disulfide-bond formation system in bacteria. We further show that the TRX-like domain of LTO1 is able to oxidize the conserved lumenal cysteines of STN7 in vitro. In addition, loss of LTO1 affects the kinase activity of STN7 in Arabidopsis. Based on these results, we propose that LTO1 helps to maintain STN7 in an oxidized active state in state 2 through redox interactions between the lumenal cysteines of STN7 and LTO1.

Stt7-dependent phosphorylation during state transitions in the green alga Chlamydomonas reinhardtii.

Photosynthetic organisms are able to adapt to changes in light conditions by balancing the light excitation energy between the light-harvesting systems of photosystem (PS) II and photosystem I to optimize the photosynthetic yield. A key component in this process, called state transitions, is the chloroplast protein kinase Stt7/STN7, which senses the redox state of the plastoquinone pool. Upon preferential excitation of photosystem II, this kinase is activated through the cytochrome b(6)f complex and required for the phosphorylation of the light-harvesting system of photosystem II, a portion of which migrates to photosystem I (state 2). Preferential excitation of photosystem I leads to the inactivation of the kinase and to dephosphorylation of light-harvesting complex (LHC) II and its return to photosystem II (state 1). Here we compared the thylakoid phosphoproteome of the wild-type strain and the stt7 mutant of Chlamydomonas under state 1 and state 2 conditions. This analysis revealed that under state 2 conditions several Stt7-dependent phosphorylations of specific Thr residues occur in Lhcbm1/Lhcbm10, Lhcbm4/Lhcbm6/Lhcbm8/Lhcbm9, Lhcbm3, Lhcbm5, and CP29 located at the interface between PSII and its light-harvesting system. Among the two phosphorylation sites detected specifically in CP29 under state 2, one is Stt7-dependent. This phosphorylation may play a crucial role in the dissociation of CP29 from PSII and/or in its association to PSI where it serves as a docking site for LHCII in state 2. Moreover, Stt7 was required for the phosphorylation of the thylakoid protein kinase Stl1 under state 2 conditions, suggesting the existence of a thylakoid protein kinase cascade. Stt7 itself is phosphorylated at Ser(533) in state 2, but analysis of mutants with a S533A/D change indicated that this phosphorylation is not required for state transitions. Moreover, we also identified phosphorylation sites that are redox (state 2)-dependent but independent of Stt7 and additional phosphorylation sites that are redox-independent.

Biochemical and structural studies of the large Ycf4-photosystem I assembly complex of the green alga Chlamydomonas reinhardtii.

Ycf4 is a thylakoid protein essential for the accumulation of photosystem I (PSI) in Chlamydomonas reinhardtii. Here, a tandem affinity purification tagged Ycf4 was used to purify a stable Ycf4-containing complex of >1500 kD. This complex also contained the opsin-related COP2 and the PSI subunits PsaA, PsaB, PsaC, PsaD, PsaE, and PsaF, as identified by mass spectrometry (liquid chromatography-tandem mass spectrometry) and immunoblotting. Almost all Ycf4 and COP2 in wild-type cells copurified by sucrose gradient ultracentrifugation and subsequent ion exchange column chromatography, indicating the intimate and exclusive association of Ycf4 and COP2. Electron microscopy revealed that the largest structures in the purified preparation measure 285 x 185 A; these particles may represent several large oligomeric states. Pulse-chase protein labeling revealed that the PSI polypeptides associated with the Ycf4-containing complex are newly synthesized and partially assembled as a pigment-containing subcomplex. These results indicate that the Ycf4 complex may act as a scaffold for PSI assembly. A decrease in COP2 to 10% of wild-type levels by RNA interference increased the salt sensitivity of the Ycf4 complex stability but did not affect the accumulation of PSI, suggesting that COP2 is not essential for PSI assembly.

Potential for hydrogen production with inducible chloroplast gene expression in Chlamydomonas.

An inducible chloroplast gene expression system was developed in Chlamydomonas reinhardtii by taking advantage of the properties of the copper-sensitive cytochrome c(6) promoter and of the nucleus-encoded Nac2 chloroplast protein. This protein is specifically required for the stable accumulation of the chloroplast psbD RNA and acts on its 5' UTR. A construct containing the Nac2 coding sequence fused to the cytochrome c(6) promoter was introduced into the nac2-26 mutant strain deficient in Nac2. In this transformant, psbD is expressed in copper-depleted but not in copper-replete medium. Because psbD encodes the D2 reaction center polypeptide of photosystem II (PSII), the repression of psbD leads to the loss of PSII. We have tested this system for hydrogen production. Upon addition of copper to cells pregrown in copper-deficient medium, PSII levels declined to a level at which oxygen consumption by respiration exceeded oxygen evolution by PSII. The resulting anaerobic conditions led to the induction of hydrogenase activity. Because the Cyc6 promoter is also induced under anaerobic conditions, this system opens possibilities for sustained cycling hydrogen production. Moreover, this inducible gene expression system is applicable to any chloroplast gene by replacing its 5' UTR with the psbD 5' UTR in the same genetic background. To make these strains phototrophic, the 5' UTR of the psbD gene was replaced by the petA 5' UTR. As an example, we show that the reporter gene aadA driven by the psbD 5' UTR confers resistance to spectinomycin in the absence of copper and sensitivity in its presence in the culture medium.

One of two alb3 proteins is essential for the assembly of the photosystems and for cell survival in Chlamydomonas.

Proteins of the YidC/Oxa1p/ALB3 family play an important role in inserting proteins into membranes of mitochondria, bacteria, and chloroplasts. In Chlamydomonas reinhardtii, one member of this family, Albino3.1 (Alb3.1), was previously shown to be mainly involved in the assembly of the light-harvesting complex. Here, we show that a second member, Alb3.2, is located in the thylakoid membrane, where it is associated with large molecular weight complexes. Coimmunoprecipitation experiments indicate that Alb3.2 interacts with Alb3.1 and the reaction center polypeptides of photosystem I and II as well as with VIPP1, which is involved in thylakoid formation. Moreover, depletion of Alb3.2 by RNA interference to 25 to 40% of wild-type levels leads to a reduction in photosystems I and II, indicating that the level of Alb3.2 is limiting for the assembly and/or maintenance of these complexes in the thylakoid membrane. Although the levels of several photosynthetic proteins are reduced under these conditions, other proteins are overproduced, such as VIPP1 and the chloroplast chaperone pair Hsp70/Cdj2. These changes are accompanied by a large increase in vacuolar size and, after a prolonged period, by cell death. We conclude that Alb3.2 is required directly or indirectly, through its impact on thylakoid protein biogenesis, for cell survival.

Tab2 is a novel conserved RNA binding protein required for translation of the chloroplast psaB mRNA.

The chloroplast psaB mRNA encodes one of the reaction centre polypeptides of photosystem I. Protein pulse-labelling profiles indicate that the mutant strain of Chlamydomonas reinhardtii, F14, affected at the nuclear locus TAB2, is deficient in the translation of psaB mRNA and thus deficient in photosystem I activity. Genetic studies reveal that the target site for Tab2 is situated within the psaB 5'UTR. We have used genomic complementation to isolate the nuclear Tab2 gene. The deduced amino acid sequence of Tab2 (358 residues) displays 31-46% sequence identity with several orthologues found only in eukaryotic and prokaryotic organisms performing oxygenic photosynthesis. Directed mutagenesis indicates the importance of a highly conserved C-terminal tripeptide in Tab2 for normal psaB translation. The Tab2 protein is localized in the chloroplast stroma where it is associated with a high molecular mass protein complex containing the psaB mRNA. Gel mobility shift assays reveal a direct and specific interaction between Tab2 and the psaB 5'UTR. We propose that Tab2 plays a key role in the initial steps of PsaB translation and photosystem I assembly.

Light activates binding of membrane proteins to chloroplast RNAs in Chlamydomonas reinhardtii.

Several membrane proteins were previously shown to bind to the 5' leader of the chloroplast psbC mRNA in the unicellular eukaryotic alga Chlamydomonas reinhardtii. This study showed that these proteins have affinity for AU-rich RNAs, as determined by competition experiments. In addition, their binding activities are enhanced 13-15-fold by light, and a 46 kDa protein is activated within 1-10 min. This activation could be mediated by the modulation of ADP pools by the light-dependent reactions of photosynthesis and ATP synthase because (1) two inhibitors that block ATP synthesis also prevent this activation and (2) ADP inhibits the RNA-binding activity of this protein in vitro. An inhibitor of Photosystem II diminishes this induction, suggesting that reducing potential generated by the photosynthetic electron transport chain modulates this RNA-binding activity. The RNA-binding activities of two proteins (of 46 and 47 kDa) are inhibited by Mg-protoporphyrin IX methyl ester in vitro suggesting they could be regulated by these intermediates in the chlorophyll biosynthetic pathway.

Chlamydomonas, a model system for studying the assembly and dynamics of photosynthetic complexes.

The green unicellular alga Chlamydomonas reinhardtii has emerged as a powerful model system for studying the biosynthesis of the photosynthetic apparatus and the acclimation of this system to changes in light conditions. The assembly of the photosynthetic complexes involves the coordinate interaction between the nuclear and chloroplast genetic systems. Many factors involved in specific chloroplast post-transcriptional steps have been identified and characterized. Chlamydomonas is able to adapt to changes in light quality and in cellular ATP content by performing state transition, a process that leads to a redistribution of light excitation energy between photosystem II and photosystem I and that involves the redox state of the plastoquinone pool, the cytochrome b(6)f complex and one or several kinases specific for the light-harvesting system. Genetic approaches have provided new insights into this process.