Département de biologie moléculaire
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Conditional repression of essential chloroplast genes: Evidence for new plastid signaling pathways.
Biochim Biophys Acta, ; 1847 (9): 986-992
The development of a repressible chloroplast gene expression system in Chlamydomonas reinhardtii has opened the door for studying the role of essential chloroplast genes. This approach has been used to analyze three chloroplast genes of this sort coding for the α subunit of RNA polymerase (rpoA), a ribosomal protein (rps12) and the catalytic subunit of the ATP-dependent ClpP protease (clpP1). Depletion of the three corresponding proteins leads to growth arrest and cell death. Shutdown of chloroplast transcription and translation increases the abundance of a set of plastid transcripts that includes mainly those involved in transcription, translation and proteolysis and reveals multiple regulatory feedback loops in the chloroplast gene circuitry. Depletion of ClpP profoundly affects plastid protein homeostasis and elicits an autophagy-like response with extensive cytoplasmic vacuolization of cells. It also triggers changes in chloroplast and nuclear gene expression resulting in increased abundance of chaperones, proteases, ubiquitin-related proteins and proteins involved in lipid trafficking and thylakoid biogenesis. These features are hallmarks of an unfolded protein response in the chloroplast and raise new questions on plastid protein homeostasis and plastid signaling. This article is part of a Special Issue entitled: Chloroplast Biogenesis.
Controlling expression of genes in the unicellular alga Chlamydomonas reinhardtii with a vitamin-repressible riboswitch.
Methods Enzymol, ; 550 : 267-281
Chloroplast genomes of land plants and algae contain generally between 100 and 150 genes. These genes are involved in plastid gene expression and photosynthesis and in various other tasks. The function of some chloroplast genes is still unknown and some of them appear to be essential for growth and survival. Repressible and reversible expression systems are highly desirable for functional and biochemical characterization of these genes. We have developed a genetic tool that allows one to regulate the expression of any coding sequence in the chloroplast genome of the unicellular alga Chlamydomonas reinhardtii. Our system is based on vitamin-regulated expression of the nucleus-encoded chloroplast Nac2 protein, which is specifically required for the expression of any plastid gene fused to the psbD 5'UTR. With this approach, expression of the Nac2 gene in the nucleus and, in turn, that of the chosen chloroplast gene artificially driven by the psbD 5'UTR, is controlled by the MetE promoter and Thi4 riboswitch, which can be inactivated in a reversible way by supplying vitamin B12 and thiamine to the growth medium, respectively. This system opens interesting possibilities for studying the assembly and turnover of chloroplast multiprotein complexes such as the photosystems, the ribosome, and the RNA polymerase. It also provides a way to overcome the toxicity often associated with the expression of proteins of biotechnological interest in the chloroplast.


Chloroplast unfolded protein response, a new plastid stress signaling pathway?
Plant Signal Behav, ; 9 (10): e972874
A unique feature of the ATP-dependent ClpP protease of eukaryotic photosynthetic organisms is that its catalytic subunit ClpP1 is encoded by the chloroplast genome. Attempts to inactivate this subunit through chloroplast transformation have failed because it is essential for cell survival. To study the function of ClpP we have developed a repressible chloroplast gene expression system in Chlamydomonas reinhardtii. This system is based on the use of a chimeric nuclear gene in which the vitamin-repressible MetE promoter and Thi4 riboswitch have been fused to the coding sequence of Nac2. Upon entry into the chloroplast the Nac2 protein specifically interacts with the psbD 5'UTR and is required for the proper processing/translation of the psbD mRNA. This property can be conveyed to any chloroplast mRNA by replacing its 5'UTR with that of psbD. In this study we have chosen clpP1 as plastid target gene and examined the cellular events induced upon depletion of ClpP through transcriptomic, proteomic, biochemical and electron microscope analysis. Among the most striking features, a massive increase in protein abundance occurs for plastid chaperones, proteases and proteins involved in membrane assembly/disassembly strongly suggesting the existence of a chloroplast unfolded protein response.


Comparative phosphoproteome profiling reveals a function of the STN8 kinase in fine-tuning of cyclic electron flow (CEF).
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Proc Natl Acad Sci U S A, ; 108 (31): 12955-12960
Important aspects of photosynthetic electron transport efficiency in chloroplasts are controlled by protein phosphorylation. Two thylakoid-associated kinases, STN7 and STN8, have distinct roles in short- and long-term photosynthetic acclimation to changes in light quality and quantity. Although some substrates of STN7 and STN8 are known, the complexity of this regulatory kinase system implies that currently unknown substrates connect photosynthetic performance with the regulation of metabolic and regulatory functions. We performed an unbiased phosphoproteome-wide screen with Arabidopsis WT and stn8 mutant plants to identify unique STN8 targets. The phosphorylation status of STN7 was not affected in stn8, indicating that kinases other than STN8 phosphorylate STN7 under standard growth conditions. Among several putative STN8 substrates, PGRL1-A is of particular importance because of its possible role in the modulation of cyclic electron transfer. The STN8 phosphorylation site on PGRL1-A is absent in both monocotyledonous plants and algae. In dicots, spectroscopic measurements with Arabidopsis WT, stn7, stn8, and stn7/stn8 double-mutant plants indicate a STN8-mediated slowing down of the transition from cyclic to linear electron flow at the onset of illumination. This finding suggests a possible link between protein phosphorylation by STN8 and fine-tuning of cyclic electron flow during this critical step of photosynthesis, when the carbon assimilation is not commensurate to the electron flow capacity of the chloroplast.
Reprint of: Regulation of photosynthetic electron transport.
Biochim Biophys Acta, ; 1807 (8): 878-886
The photosynthetic electron transport chain consists of photosystem II, the cytochrome b(6)f complex, photosystem I, and the free electron carriers plastoquinone and plastocyanin. Light-driven charge separation events occur at the level of photosystem II and photosystem I, which are associated at one end of the chain with the oxidation of water followed by electron flow along the electron transport chain and concomitant pumping of protons into the thylakoid lumen, which is used by the ATP synthase to generate ATP. At the other end of the chain reducing power is generated, which together with ATP is used for CO(2) assimilation. A remarkable feature of the photosynthetic apparatus is its ability to adapt to changes in environmental conditions by sensing light quality and quantity, CO(2) levels, temperature, and nutrient availability. These acclimation responses involve a complex signaling network in the chloroplasts comprising the thylakoid protein kinases Stt7/STN7 and Stl1/STN7 and the phosphatase PPH1/TAP38, which play important roles in state transitions and in the regulation of electron flow as well as in thylakoid membrane folding. The activity of some of these enzymes is closely connected to the redox state of the plastoquinone pool, and they appear to be involved both in short-term and long-term acclimation. This article is part of a Special Issue entitled: Regulation of Electron Transport in Chloroplasts.


State transitions at the crossroad of thylakoid signalling pathways.
Photosynth Res, ; 106 (1-2): 33-46
In order to maintain optimal photosynthetic activity under a changing light environment, plants and algae need to balance the absorbed light excitation energy between photosystem I and photosystem II through processes called state transitions. Variable light conditions lead to changes in the redox state of the plastoquinone pool which are sensed by a protein kinase closely associated with the cytochrome b(6)f complex. Preferential excitation of photosystem II leads to the activation of the kinase which phosphorylates the light-harvesting system (LHCII), a process which is subsequently followed by the release of LHCII from photosystem II and its migration to photosystem I. The process is reversible as dephosphorylation of LHCII on preferential excitation of photosystem I is followed by the return of LHCII to photosystem II. State transitions involve a considerable remodelling of the thylakoid membranes, and in the case of Chlamydomonas, they allow the cells to switch between linear and cyclic electron flow. In this alga, a major function of state transitions is to adjust the ATP level to cellular demands. Recent studies have identified the thylakoid protein kinase Stt7/STN7 as a key component of the signalling pathways of state transitions and long-term acclimation of the photosynthetic apparatus. In this article, we present a review on recent developments in the area of state transitions.
Stt7-dependent phosphorylation during state transitions in the green alga Chlamydomonas reinhardtii.
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Mol Cell Proteomics, ; 9 (6): 1281-1295
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.
The PPH1 phosphatase is specifically involved in LHCII dephosphorylation and state transitions in Arabidopsis.
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Proc Natl Acad Sci U S A, ; 107 (10): 4782-4787
The ability of plants to adapt to changing light conditions depends on a protein kinase network in the chloroplast that leads to the reversible phosphorylation of key proteins in the photosynthetic membrane. Phosphorylation regulates, in a process called state transition, a profound reorganization of the electron transfer chain and remodeling of the thylakoid membranes. Phosphorylation governs the association of the mobile part of the light-harvesting antenna LHCII with either photosystem I or photosystem II. Recent work has identified the redox-regulated protein kinase STN7 as a major actor in state transitions, but the nature of the corresponding phosphatases remained unknown. Here we identify a phosphatase of Arabidopsis thaliana, called PPH1, which is specifically required for the dephosphorylation of light-harvesting complex II (LHCII). We show that this single phosphatase is largely responsible for the dephosphorylation of Lhcb1 and Lhcb2 but not of the photosystem II core proteins. PPH1, which belongs to the family of monomeric PP2C type phosphatases, is a chloroplast protein and is mainly associated with the stroma lamellae of the thylakoid membranes. We demonstrate that loss of PPH1 leads to an increase in the antenna size of photosystem I and to a strong impairment of state transitions. Thus phosphorylation and dephosphorylation of LHCII appear to be specifically mediated by the kinase/phosphatase pair STN7 and PPH1. These two proteins emerge as key players in the adaptation of the photosynthetic apparatus to changes in light quality and quantity.


Phosphorylation of photosystem II controls functional macroscopic folding of photosynthetic membranes in Arabidopsis.
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Plant Cell, ; 21 (12): 3950-3964
Photosynthetic thylakoid membranes in plants contain highly folded membrane layers enriched in photosystem II, which uses light energy to oxidize water and produce oxygen. The sunlight also causes quantitative phosphorylation of major photosystem II proteins. Analysis of the Arabidopsis thaliana stn7xstn8 double mutant deficient in thylakoid protein kinases STN7 and STN8 revealed light-independent phosphorylation of PsbH protein and greatly reduced N-terminal phosphorylation of D2 protein. The stn7xstn8 and stn8 mutants deficient in light-induced phosphorylation of photosystem II had increased thylakoid membrane folding compared with wild-type and stn7 plants. Significant enhancement in the size of stacked thylakoid membranes in stn7xstn8 and stn8 accelerated gravity-driven sedimentation of isolated thylakoids and was observed directly in plant leaves by transmission electron microscopy. Increased membrane folding, caused by the loss of light-induced protein phosphorylation, obstructed lateral migration of the photosystem II reaction center protein D1 and of processing protease FtsH between the stacked and unstacked membrane domains, suppressing turnover of damaged D1 in the leaves exposed to high light. These findings show that the high level of photosystem II phosphorylation in plants is required for adjustment of macroscopic folding of large photosynthetic membranes modulating lateral mobility of membrane proteins and sustained photosynthetic activity.
Biochemical and structural studies of the large Ycf4-photosystem I assembly complex of the green alga Chlamydomonas reinhardtii.
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Plant Cell, ; 21 (8): 2424-2442
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.
Analysis of the chloroplast protein kinase Stt7 during state transitions.
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PLoS Biol, ; 7 (3): e45
State transitions allow for the balancing of the light excitation energy between photosystem I and photosystem II and for optimal photosynthetic activity when photosynthetic organisms are subjected to changing light conditions. This process is regulated by the redox state of the plastoquinone pool through the Stt7/STN7 protein kinase required for phosphorylation of the light-harvesting complex LHCII and for the reversible displacement of the mobile LHCII between the photosystems. We show that Stt7 is associated with photosynthetic complexes including LHCII, photosystem I, and the cytochrome b6f complex. Our data reveal that Stt7 acts in catalytic amounts. We also provide evidence that Stt7 contains a transmembrane region that separates its catalytic kinase domain on the stromal side from its N-terminal end in the thylakoid lumen with two conserved Cys that are critical for its activity and state transitions. On the basis of these data, we propose that the activity of Stt7 is regulated through its transmembrane domain and that a disulfide bond between the two lumen Cys is essential for its activity. The high-light-induced reduction of this bond may occur through a transthylakoid thiol-reducing pathway driven by the ferredoxin-thioredoxin system which is also required for cytochrome b6f assembly and heme biogenesis.


Redundant cis-acting determinants of 3' processing and RNA stability in the chloroplast rbcL mRNA of Chlamydomonas.
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Plant J, ; 53 (3): 566-577
We have designed a screen for mutants affected in 3' maturation of the chloroplast rbcL mRNA in Chlamydomonas reinhardtii. We inserted a spectinomycin resistance cassette, 5'atpA::aadA::3'rbcL, in a peripheral domain of tscA, the gene for a small non-coding RNA involved in trans-splicing of psaA. Depending on the orientation of the cassette, a polar effect was observed which was due to processing at the 3'rbcL element: the chimeric tscA RNA was truncated and splicing of psaA was blocked. We selected phenotypic revertants of this insertion mutant that restored psaA splicing, which correlated with the presence of chimeric transcripts that regained the 3' part of tscA. We analyzed two nuclear and six chloroplast suppressors. Five chloroplast mutations altered a short element in the center of the second inverted repeat in the 3'rbcL (IR2), and one deleted a larger region including this element. These mutations revealed a cis-acting element in IR2 which is required for 3' processing. When the same mutations were inserted in the 3' untranslated region (UTR) of the native rbcL gene, the rbcL mRNA accumulated to normal levels, but in strong alleles its 3' end was located upstream, near the end of the first inverted repeat (IR1). Deletion of either IR1 or IR2 allowed stable accumulation of rbcL mRNA, but deletion of both resulted in its complete absence. This indicated that the two inverted repeats function as redundant mRNA stability determinants in the 3' UTR of rbcL.


Potential for hydrogen production with inducible chloroplast gene expression in Chlamydomonas.
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Proc Natl Acad Sci U S A, ; 104 (44): 17548-17553
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.
Role of thylakoid protein kinases in photosynthetic acclimation.
FEBS Lett, ; 581 (15): 2768-2775
Photosynthetic organisms are able to adjust to changes in light quality through state transition, a process which leads to a balancing of the light excitation energy between the antennae systems of photosystem II and photosystem I. A genetic approach has been used in Chlamydomonas with the aim of elucidating the signaling chain involved in state transitions. This has led to the identification of a small family of Ser-Thr protein kinases associated with the thylakoid membrane and conserved in algae and land plants. These kinases appear to be involved both in short and long term adaptations to changes in the light environment.


ATAB2 is a novel factor in the signalling pathway of light-controlled synthesis of photosystem proteins.
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EMBO J, ; 25 (24): 5907-5918
Plastid translational control depends to a large extent on the light conditions, and is presumably mediated by nucleus-encoded proteins acting on organelle gene expression. However, the molecular mechanisms of light signalling involved in translation are still poorly understood. We investigated the role of the Arabidopsis ortholog of Tab2, a nuclear gene specifically required for translation of the PsaB photosystem I subunit in the unicellular alga Chlamydomonas. Inactivation of ATAB2 strongly affects Arabidopsis development and thylakoid membrane biogenesis and leads to an albino phenotype. Moreover the rate of synthesis of the photosystem reaction center subunits is decreased and the association of their mRNAs with polysomes is affected. ATAB2 is a chloroplast A/U-rich RNA-binding protein that presumably functions as an activator of translation with at least two targets, one for each photosystem. During early seedling development, ATAB2 blue-light induction is lowered in photoreceptor mutants, notably in those lacking cryptochromes. Considering its role in protein synthesis and its photoreceptor-mediated expression, ATAB2 represents a novel factor in the signalling pathway of light-controlled translation of photosystem proteins during early plant development.
One of two alb3 proteins is essential for the assembly of the photosystems and for cell survival in Chlamydomonas.
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Plant Cell, ; 18 (6): 1454-1466
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.


State transitions and light adaptation require chloroplast thylakoid protein kinase STN7.
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Nature, ; 433 (7028): 892-895
Photosynthetic organisms are able to adjust to changing light conditions through state transitions, a process that involves the redistribution of light excitation energy between photosystem II (PSII) and photosystem I (PSI). Balancing of the light absorption capacity of these two photosystems is achieved through the reversible association of the major antenna complex (LHCII) between PSII and PSI (ref. 3). Excess stimulation of PSII relative to PSI leads to the reduction of the plastoquinone pool and the activation of a kinase; the phosphorylation of LHCII; and the displacement of LHCII from PSII to PSI (state 2). Oxidation of the plastoquinone pool by excess stimulation of PSI reverses this process (state 1). The Chlamydomonas thylakoid-associated Ser-Thr kinase Stt7, which is required for state transitions, has an orthologue named STN7 in Arabidopsis. Here we show that loss of STN7 blocks state transitions and LHCII phosphorylation. In stn7 mutant plants the plastoquinone pool is more reduced and growth is impaired under changing light conditions, indicating that STN7, and probably state transitions, have an important role in response to environmental changes.
The FLP proteins act as regulators of chlorophyll synthesis in response to light and plastid signals in Chlamydomonas.
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Genes Dev, ; 19 (1): 176-187
In photosynthetic organisms the accumulation of harmful photodynamic chlorophyll precursors is prevented because of the tight regulation of the tetrapyrrole pathway. FLU is one of the regulatory factors involved in this process in land plants. We have examined the function of a Flu-like gene (FLP) from Chlamydomonas that gives rise to two FLP transcripts through alternative splicing. These transcripts are translated into a short and a long protein that differ by only 12 amino acids but that interact differently with glutamyl-tRNA reductase, an enzyme involved in an early step of the chlorophyll biosynthetic pathway. Expression of FLPs is light-regulated at the level of RNA accumulation and splicing and is altered by mutations affecting the pathway. The relative levels of the long and short forms of FLP can be correlated with the accumulation of specific porphyrin intermediates, some of which have been implicated in a signaling chain from the chloroplast to the nucleus. Reciprocally, reduction of the FLP proteins by RNA interference leads to the accumulation of several porphyrin intermediates and to photobleaching when cells are transferred from the dark to the light. Thus the FLP proteins act as regulators of chlorophyll synthesis, and their expression is controlled by light and plastid signals.