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The Metabolic Status Drives Acclimation of Iron Deficiency Responses in Chlamydomonas reinhardtii as Revealed by Proteomics Based Hierarchical Clustering and Reverse Genetics*

  • Ricarda Höhner
    Affiliations
    Institute of Plant Biology and Biotechnology, University of Münster, Schlossplatz 8, Münster 48143, Germany;
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  • Johannes Barth
    Affiliations
    Institute of Plant Biology and Biotechnology, University of Münster, Schlossplatz 8, Münster 48143, Germany;
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  • Leonardo Magneschi
    Affiliations
    Institute of Plant Biology and Biotechnology, University of Münster, Schlossplatz 8, Münster 48143, Germany;
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  • Daniel Jaeger
    Affiliations
    Institute of Plant Biology and Biotechnology, University of Münster, Schlossplatz 8, Münster 48143, Germany;
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  • Anna Niehues
    Affiliations
    Institute of Plant Biology and Biotechnology, University of Münster, Schlossplatz 8, Münster 48143, Germany;
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  • Till Bald
    Affiliations
    Institute of Plant Biology and Biotechnology, University of Münster, Schlossplatz 8, Münster 48143, Germany;
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  • Arthur Grossman
    Affiliations
    Department of Plant Biology, Carnegie Institution for Science, Stanford, California 94305
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  • Christian Fufezan
    Correspondence
    To whom correspondence should be addressed: Prof. Dr. M. Hippler & Dr. C. Fufezan, Institute of Biology and Biotechnology of Plants, University of Münster, Schlossplatz 8, 48143 Münster, Germany. Tel.: ++49-(0)251-8324790 or 24861
    Affiliations
    Institute of Plant Biology and Biotechnology, University of Münster, Schlossplatz 8, Münster 48143, Germany;
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  • Michael Hippler
    Correspondence
    To whom correspondence should be addressed: Prof. Dr. M. Hippler & Dr. C. Fufezan, Institute of Biology and Biotechnology of Plants, University of Münster, Schlossplatz 8, 48143 Münster, Germany. Tel.: ++49-(0)251-8324790 or 24861
    Affiliations
    Institute of Plant Biology and Biotechnology, University of Münster, Schlossplatz 8, Münster 48143, Germany;
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  • Author Footnotes
    * M.H. and C.F. acknowledge support from the Deutsche Forschungsgemeinschaft. M.H. also acknowledges support from the FP7-funded Sunbiopath Project (GA245070). L.M. acknowledges support from the Alexander von Humboldt Stiftung/Foundation during his stay at the Institute of Plant Biology and Biotechnology (University of Münster, Germany). A.R.G. acknowledges support from the Department of Energy, Grant No. DE-FG02-12ER16338.
    This article contains supplemental Figs. S1 to S4 and Tables S1 to S3.
Open AccessPublished:July 02, 2013DOI:https://doi.org/10.1074/mcp.M113.029991
      Iron is a crucial cofactor in numerous redox-active proteins operating in bioenergetic pathways including respiration and photosynthesis. Cellular iron management is essential to sustain sufficient energy production and minimize oxidative stress. To produce energy for cell growth, the green alga Chlamydomonas reinhardtii possesses the metabolic flexibility to use light and/or carbon sources such as acetate. To investigate the interplay between the iron-deficiency response and growth requirements under distinct trophic conditions, we took a quantitative proteomics approach coupled to innovative hierarchical clustering using different “distance-linkage combinations” and random noise injection. Protein co-expression analyses of the combined data sets revealed insights into cellular responses governing acclimation to iron deprivation and regulation associated with photosynthesis dependent growth. Photoautotrophic growth requirements as well as the iron deficiency induced specific metabolic enzymes and stress related proteins, and yet differences in the set of induced enzymes, proteases, and redox-related polypeptides were evident, implying the establishment of distinct response networks under the different conditions. Moreover, our data clearly support the notion that the iron deficiency response includes a hierarchy for iron allocation within organelles in C. reinhardtii. Importantly, deletion of a bifunctional alcohol and acetaldehyde dehydrogenase (ADH1), which is induced under low iron based on the proteomic data, attenuates the remodeling of the photosynthetic machinery in response to iron deficiency, and at the same time stimulates expression of stress-related proteins such as NDA2, LHCSR3, and PGRL1. This finding provides evidence that the coordinated regulation of bioenergetics pathways and iron deficiency response is sensitive to the cellular and chloroplast metabolic and/or redox status, consistent with systems approach data.
      The green alga Chlamydomonas reinhardtii has an enormous metabolic versatility (
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      Transition metals like copper, manganese, and iron possess the ability to donate and accept electrons, making these metals suitable cofactors in enzymes that catalyze redox reactions. In particular, iron is used as a cofactor in numerous biochemical pathways and is therefore an essential nutrient. Cells require relatively high levels of iron because it is present in heme-, iron-sulfur and other proteins that function in respiratory and photosynthetic energy transducing. Correspondingly, in eukaryotic cells, the mitochondrion is a major iron-utilizing compartment. It is well established that iron is transported into mitochondria for heme synthesis and iron-sulfur cluster assembly. This is required for the formation of a functional respiratory electron transport machinery (
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      Photosystem I (PSI) is a prime target of iron deficiency as it contains 12 atoms of iron per core complex. In algae, the degradation of PSI is also linked to remodeling of PSI-associated light-harvesting antenna (LHCI) (
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      ) and induction of the “iron-stress-induced” gene isiA. The ISIA protein, which has significant sequence similarity with CP43, a chlorophyll a-binding protein of photosystem II (PSII; (
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      The marine diatom Thalassiosira oceanica shows a remarkable retrenchment of cellular metabolism and remodeling of bioenergetic pathways in response to iron availability (
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      Comparative quantitative proteomics to investigate the remodeling of bioenergetic pathways under iron deficiency in Chlamydomonas reinhardtii.
      ), are important for minimizing photo-oxidative stress and optimizing photosynthetic function. As observed for T. oceanica, under conditions of low iron availability (in the presence of organic carbon) a hierarchy of iron allocation responses in C. reinhardtii result in the down-regulation of iron-rich photosynthetic complexes while iron-rich mitochondrial complexes remain stable (
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      Comparative quantitative proteomics to investigate the remodeling of bioenergetic pathways under iron deficiency in Chlamydomonas reinhardtii.
      ). Notably, under photoautotrophic and mixotrophic conditions C. reinhardtii displays distinct iron deprivation responses, suggesting that the cell's response to iron deficiency is also dependent on trophic conditions (
      • Busch A.
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      • Rensch S.
      • Hippler M.
      Ferritin is required for rapid remodeling of the photosynthetic apparatus and minimizes photo-oxidative stress in response to iron availability in Chlamydomonas reinhardtii.
      ,
      • Terauchi A.M.
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      • Kobayashi M.C.
      • Niyogi K.K.
      • Merchant S.S.
      Trophic status of Chlamydomonas reinhardtii influences the impact of iron deficiency on photosynthesis.
      ,
      • Urzica E.I.
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      • Pellegrini M.
      • Merchant S.S.
      Systems and trans-system level analysis identifies conserved iron deficiency responses in the plant lineage.
      ). Thus bioenergetics pathways are remodeled in response to iron availability as well as to the type of carbon source available. Moreover, recent data has indicated that the regulation of iron-induced remodeling of the photosynthetic apparatus is linked to energy metabolism. Depletion of Proton Gradient Regulation Like1 protein (PGRL1) in C. reinhardtii has revealed a decreased efficiency of cyclic electron transfer under low iron conditions resulting in higher vulnerability toward iron deprivation (
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      PGRL1 participates in iron-induced remodeling of the photosynthetic apparatus and in energy metabolism in Chlamydomonas reinhardtii.
      ).
      It was our aim to generate a more comprehensive picture of how the proteome of C. reinhardtii varies in response to low iron under distinct trophic conditions and how these changes compare with differences observed for cells grown under photoautotrophic and photoheterotrophic iron replete conditions. Quantitative proteomics in conjunction with a novel hierarchical clustering approach revealed information about the responses of C. reinhardtii to low iron conditions and the iron requirements of photoautotrophic growth. These analyses provide novel insights into the relationships between protein networks required for photosynthesis and iron deprivation-elicited stress responses; these studies are providing the knowledge required for modulating the level of available iron to improve the photosynthetic performance of plants (
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      ).

      RESULTS

      Iron deficiency responses in C. reinhardtii are influenced by the trophic status of the cells (
      • Busch A.
      • Rimbauld B.
      • Naumann B.
      • Rensch S.
      • Hippler M.
      Ferritin is required for rapid remodeling of the photosynthetic apparatus and minimizes photo-oxidative stress in response to iron availability in Chlamydomonas reinhardtii.
      ,
      • Terauchi A.M.
      • Peers G.
      • Kobayashi M.C.
      • Niyogi K.K.
      • Merchant S.S.
      Trophic status of Chlamydomonas reinhardtii influences the impact of iron deficiency on photosynthesis.
      ,
      • Urzica E.I.
      • Casero D.
      • Yamasaki H.
      • Hsieh S.I.
      • Adler L.N.
      • Karpowicz S.J.
      • Blaby-Haas C.E.
      • Clarke S.G.
      • Loo J.A.
      • Pellegrini M.
      • Merchant S.S.
      Systems and trans-system level analysis identifies conserved iron deficiency responses in the plant lineage.
      ,
      • Naumann B.
      • Busch A.
      • Allmer J.
      • Ostendorf E.
      • Zeller M.
      • Kirchhoff H.
      • Hippler M.
      Comparative quantitative proteomics to investigate the remodeling of bioenergetic pathways under iron deficiency in Chlamydomonas reinhardtii.
      ). Yet, although many aspects of the cellular iron deficiency response are driven by low iron availability, other properties appear to be governed by the cell's metabolic status. To dissect these responses and obtain more detailed insights into these regulatory processes we performed an in-depth quantitative proteomics approach using metabolic labeling. To this end iron sufficient and deficient cells were grown under photoheterotrophic (PH) or -autotrophic (PA) growth conditions in 15N or 14N labeled media, respectively. The iron deficiency status was verified using low temperature fluorescence emission spectroscopy and immunoblotting using antibodies directed against marker proteins, diminished in response to iron deprivation (see supplemental Fig. S1). Moreover PH grown iron sufficient cells were labeled with 15N and compared with 14N labeled cells grown under PA conditions. Here also a swapping experiment was performed to detect and avoid isotope effects. Quantitative analysis showed very good correlation among the label swap experiments (see supplemental Fig. S2). 15N labeled iron sufficient cells or isolated chloroplasts were mixed on equal protein basis with the respective complement from 14N labeled iron deficient or iron sufficient material (Fig. 1). In addition mitochondria were isolated from 15N labeled iron sufficient and 14N labeled iron deficient cells grown under PH conditions and also mixed on equal protein basis (Fig. 1). Mixed samples were fractionated by SDS-PAGE and protein bands were excised from the gels. On average about 56 bands were cut per gel. SDS-PAGE bands were in-gel digested with trypsin and peptides were analyzed by LC-MS/MS. We completed and processed two independent biological replicates. For the whole cell and chloroplast sample sets we investigated 337 and 523 excised and tryptically digested protein bands, respectively. Moreover, for isolated mitochondria in total 124 excised and proteolytically digested protein bands were examined by LC-MS/MS. The mass spectrometric analyses resulted in the identification of peptides and proteins and subsequently allowed the quantitation of protein ratios. The results stemming from the two independent biological replicates are summarized in Fig. 2 and are plotted as number of quantified proteins and their respective proteins ratios starting from the lowest to the highest observed ratios. As shown in Fig. 2, from the six independent experimental set-ups at minimum 1316 and at maximum 1944 proteins could be quantified. These high numbers can be attributed to the enhancement procedure using piqDB, (Fufezan and coworkers, unpublished), which is similar to SuperHirn (
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      • Muller M.
      SuperHirn - a novel tool for high resolution LC-MS-based peptide/protein profiling.
      ). Consequently, such large amount of protein ratios provided a good basis for further exploration. Thus coregulation analyses of the proteomics data was performed using a novel and advanced hierarchical clustering approach, that injects noise into the data set and uses a large number of iterations, using pyGCluster (Jaeger et al., manuscript in prep). Briefly, pyGCluster uses the uncertainty of the data at hand by including the standard deviation (std) into the agglomerative hierarchical clustering approach. For each iteration, a new data set is generated in which each data point is randomly sampled out of the Gauss distribution described by its mean and std. The generated data set is described by the original data yet it will cluster differently. pyGCluster then applies nine different distance-linkage combinations for each of those perturbed data sets to ultimately, after a high number of iterations, isolate the high frequency clusters and merge those into coregulated communities. The advantages lie in less user bias and most importantly in reducing the data set at hand to very few proteins that show coregulations although heavily perturbed based on the uncertainty of their data.
      Figure thumbnail gr2
      Fig. 2The ratio for all quantified proteins of either iron deficient (14N(−Fe)) to iron sufficient conditions (15N (+Fe)) from photoheterotrophic conditions (PH) or photoautotrophic conditions (PA) or the ratio of 14N PA to 15N PH is shown for whole cells (upper panel) and enriched chloroplasts (lower panel). Error bars indicate standard deviations among different combinations of peptide, band, and charge states and between two independent biological samples.
      Quantitative mass spectrometric data from whole cell extracts (WC) and isolated chloroplasts (C) have been clustered using pyGCluster (see Material and Methods). It is expected that chloroplast localized proteins harbor similar trends in the six settings compared, which is indeed observed for all chloroplast proteins found in the clusters. Notably among the 109 proteins clustered, 53 were reported to be part of a chloroplast proteome (
      • Terashima M.
      • Specht M.
      • Hippler M.
      The chloroplast proteome: a survey from the Chlamydomonas reinhardtii perspective with a focus on distinctive features.
      ) (supplemental Table S3). For nonchloroplast proteins, tendencies seen in whole cells should be representative, whereas changes observed in enriched chloroplast fractions might be biased by the organelle enrichment and purity, yet, jointed clustering might be reached because of these properties. The results are illustrated as cNode maps (Fig. 3) that highlight well-defined high frequency clusters of proteins, their relation to each other and how they are grouped into communities. These communities are colored coded and labeled with roman numerals in the cNode map. A total of 23 communities have been identified. The proteins within each community are illustrated as heat maps, a selection of heat maps is shown in Fig. 3. All heat maps can be found in the supplemental Fig. S3 (communities 0 - XXII). The heat maps also show the cFrequencies of each protein within a community. The cFrequencies are the sum of all cluster frequencies the protein is part of within a community. Because each cluster is found with different frequencies by the 9 hierarchical clustering assays performed here, the median frequency of those was used to represent each cluster. The colors in the heat maps between purple and yellow represent reduced and increased protein levels, respectively, comparing iron deficient (−Fe) and iron sufficient (+Fe) conditions or PA and PH settings. Ratios (i.e. -Fe/+Fe or PA/PH) are log2 transformed and the uncertainty, i.e. the standard deviation of a ratio is reflected in its box size. The higher the standard deviation and therefore the higher the uncertainty of a given ratio, the smaller is the box. This is illustrated in the legend.
      Figure thumbnail gr3
      Fig. 3Results obtained by multiple agglomerative hierarchical clustering approaches using pyGCluster of protein ratios comparing iron deficient (−Fe) with iron sufficient (+Fe) in photoautotrophic growth conditions (PA) or in photoheterotrophic growth conditions (PH) or comparing PA with PH in iron sufficient growth conditions in whole cells or isolated chloroplasts. Shown are the cNode map and five heat maps for selected communities. The cNode map highlights the most frequent clusters with their clusterID and their membership to one of the 23 communities identified. Communities are indicated in different colors and roman numerals. The cluster frequency is illustrated by the node size as shown in the legend (gray nodes). For detailed information on the clusters see Supplemental Table T2. The cFrequency of a protein is equal to the sum of all cluster frequencies the protein is part of, within its community. Heat maps are subdivided to illustrate the separation of subcommunities visible as branches in the cNode map.

      Subunits of the cyt b6f Complex and Photosystem I As Well As Proteins Involved in Chlorophyll Biosynthesis Exhibit a Uniform Down-regulation Under Iron Deprivation

      Community III (orange, Fig. 3 top right) contains 17 proteins, which are all down-regulated under low iron. This cluster is dominated by four subunits of the cytochrome b6f complex (cyt b6f complex, cytf, Rieske, cytb6 and subunit IV) and five enzymes involved in chlorophyll biosynthesis (the geranyl-geranylreductase (GGR), uroporphyrinogen-III decarboxylase (UROD1), light-dependent protochlorophyllide reductase (POR), copper target homolog 1 (CTH1), and porphobilinogen deaminase (PBGD1)). This cluster supports earlier notions that cyt b6f complex and the chlorophyll biosynthesis pathway are prime targets of iron-deficiency (
      • Moseley J.L.
      • Allinger T.
      • Herzog S.
      • Hoerth P.
      • Wehinger E.
      • Merchant S.
      • Hippler M.
      Adaptation to Fe-deficiency requires remodeling of the photosynthetic apparatus.
      ,
      • Spiller S.C.
      • Castelfranco A.M.
      • Castelfranco P.A.
      Effects of Iron and Oxygen on Chlorophyll Biosynthesis : I. In Vivo observations on iron and oxygen-deficient plants.
      ,
      • Chereskin B.M.
      • Castelfranco P.A.
      Effects of Iron and Oxygen on Chlorophyll Biosynthesis : II. Observations on the biosynthetic pathway in isolated etiochloroplasts.
      ,
      • Pinta V.
      • Picaud M.
      • Reiss-Husson F.
      • Astier C.
      Rubrivivax gelatinosus acsF (previously orf358) codes for a conserved, putative binuclear-iron-cluster-containing protein involved in aerobic oxidative cyclization of Mg-protoporphyrin IX monomethylester.
      ,
      • Moseley J.
      • Quinn J.
      • Eriksson M.
      • Merchant S.
      The Crd1 gene encodes a putative di-iron enzyme required for photosystem I accumulation in copper deficiency and hypoxia in Chlamydomonas reinhardtii.
      ). A light-harvesting-like protein LIL (Lhl3) shows the same tendency as the chlorophyll biosynthesis proteins. Another photosynthetic protein complex that is preferentially down-regulated under iron-deprivation is photosystem I. In community IV (yellow, Fig. 3 left bottom) seven out of eight proteins belong to the photosystem I complex (PsaA, B, D, E, F, L, H (Cre07.g330250)). The only nonphotosystem I protein in this cluster is a protein of unknown function (Cre05.g244500). All photosystem I complex subunits remain unchanged or slightly increase when iron replete PA/PH conditions are compared.
      Core subunits of photosystem II (PSII) and the ATPase and many light-harvesting proteins are not among the clustered proteins. The polypeptides belonging to PSII and ATPase multiprotein complexes as well as to the light-harvesting complement show only moderate down-regulation under low iron availability (supplemental Fig. S4A). Moreover it appears that the impact on these complexes under low iron is generally more pronounced under PA rather than under PH conditions. In contrast, light-harvesting proteins that are associated to PSII, with the exception of Lhcb4 and Lhcb5, and listed in supplemental Fig. S4A are not reduced in amounts by iron deficiency. These data correlate very well with published results (
      • Naumann B.
      • Busch A.
      • Allmer J.
      • Ostendorf E.
      • Zeller M.
      • Kirchhoff H.
      • Hippler M.
      Comparative quantitative proteomics to investigate the remodeling of bioenergetic pathways under iron deficiency in Chlamydomonas reinhardtii.
      ) and underpin the differential regulation of photosynthetic complex proteins in response to iron deprivation.

      Clustering Reveals Response Networks of Acclimation to Iron Deficiency

      A complete different picture can be seen in community VI (green, Fig. 3 top right). Here proteins are increased in expression under iron deprivation and mainly decreased under PA/PH iron replete conditions. The protein with the highest cFrequency belongs to the reticulon protein family (Cre06.g308950.t1.1), such proteins are found to be associated with the endoplasmic reticulum (ER) (
      • van de Velde H.J.
      • Roebroek A.J.
      • Senden N.H.
      • Ramaekers F.C.
      • Van de Ven W.J.
      NSP-encoded reticulons, neuroendocrine proteins of a novel gene family associated with membranes of the endoplasmic reticulum.
      ). The Chlamydomonas protein possesses a characteristic dilysine motif at the C-terminal and may be associated with ER channel-like membrane complexes as found for the reticulon NSP-A that is connected with an ER Ca2+-ATPase (
      • van de Velde H.J.
      • Roebroek A.J.
      • Senden N.H.
      • Ramaekers F.C.
      • Van de Ven W.J.
      NSP-encoded reticulons, neuroendocrine proteins of a novel gene family associated with membranes of the endoplasmic reticulum.
      ). Other members of this community are a 6-phosphogluconate dehydrogenase (GND1a, Cre12.g526800.t1.1), a putative glutathione S-transferase (Cre12.g538100.t1.1) and a dynamin related GTPase (DRP1), followed by two proteases, ASP, an aspartic-type endopeptidase and CEP (a candidate serine carboxypeptidase) and a putative mitochondrial phosphate carrier 1 (MPC1). Lower cFrequency members are a septin (SEP1), a granule bound starch synthase I (STA2), GSH1, a gamma-glutamylcysteine synthetase, a flagellar associated protein (AGG3/AGG4; star behind the name tag in Fig. 3 indicates a merge tag with models Cre10.g456100.t1.1/Cre10.g456050.t1.1 because of non proteotypic peptides matching on both protein sequences), Cre12.g547300.t1.2 (a putative ABC transporter), as well as two proteins of unknown function (Cre08.g382950.t1.1 and Cre01.g010400.t1.1).
      Bifunctional alcohol and acetaldehyde dehydrogenase (ADH1), glyceraldehyde-3-phosphate dehydrogenase (plastidic), and monodehydroascorbate reductase (MDAR) are present in correlated subcommunities, having highest cFrequency in their communities. Another related subcommunity consists of four proteins, which differs by the fact that all these proteins are more increased under PA than PH iron-deficiency. These proteins are a DegP protease (DEG5A), a putative glutathione S-transferease (Cre01.g064400.t1.1), a protein of unknown function (Cre16.g683150.t1.1), and a protein possessing an acetyl-CoA-synthetase-like superfamily domain (Cre03.g182050.t1.1).
      Overall, members of community VI are involved in bioenergetic remodeling and stress responses as many metabolic enzymes, proteases, and redox related enzymes dominate in this assembly. Importantly the clustering reveals that certain responses are distinct among the different conditions analyzed, a scenario that is also observed in community XI.

      Clustering Reveals Responses that are Dependent on the Trophic and Iron Status of the Cells

      Proteins that are down-regulated under PA growth conditions (−Fe/+Fe), up or only slightly down-regulated under PH growth (−Fe/+Fe) or up-regulated under PA/PH (+Fe) assembled together and are represented in community XI (light blue, Fig. 3 top left). A candidate Peptidyl-prolyl cis-trans isomerase (CYN37) shows the highest cFrequency in this assembly, followed by a putative metallopeptidase (Cre01.g023350.t1.2), a translation factor for expression of the chloroplast-encoded psbA gene (TBA1) (
      • Somanchi A.
      • Barnes D.
      • Mayfield S.P.
      A nuclear gene of Chlamydomonas reinhardtii, Tba1, encodes a putative oxidoreductase required for translation of the chloroplast psbA mRNA.
      ), a cytochrome c peroxidase (CCPR1) and a thioredoxin o (TRXo). Proteins having a lower cFrequency are represented by a voltage-dependent anion-selective channel protein (VDAC), a low-CO2-inducible protein (LCIC), a putative peptidase (Cre10.g423300.t1.1), a putative plant-like transcription factor (Cre02.g091550.t1.1), and the AAA-metalloprotease FTSH1.
      Three proteins are represented in an adjacent subcommunity. These proteins exhibit a distinctive up-regulation under PA/PH (+Fe) and a diminishment under iron-deficiency. The proteins present in this cluster are a stomatin-like protein (Cre33.g782550.t1.1), a NADP malic enzyme (MME5) as well as a protein that contains a thioredoxin domain and four putative EF-hand motifs (Cre03.g202950.t1.2). According to their regulation, these proteins might be important for phototrophic growth.
      An additional related subcommunity shows a characteristic and specific up-regulation under PA/PH (+Fe) and a pronounced down-regulation under PA (−Fe/+Fe) growth conditions. It is represented by five proteins which are a hydroxypuruvate reductase (HPR1), a HVA22-like regulatory protein that was shown to be abscisic acid-induced in barley (Cre17.g696850.t1.1) (
      • Shen Q.
      • Uknes S.J.
      • Ho T.H.
      Hormone response complex in a novel abscisic acid and cycloheximide-inducible barley gene.
      ,
      • Shen Q.
      • Chen C.N.
      • Brands A.
      • Pan S.M.
      • Ho T.H.
      The stress- and abscisic acid-induced barley gene HVA22: developmental regulation and homologues in diverse organisms.
      ), another low-CO2-inducible protein (LCIB), a protein of unknown function (Cre11.g474400.t1.1), and a carbonic anhydrase 3 (CAH3).
      Proteins of another subcommunity show a uniform down-regulation under PA growth conditions (−Fe/+Fe) and exhibit a slight up-regulation in the other two growth settings. Out of seven proteins present in this community, three proteins belong to the glycine cleavage multienzyme complex (GCSH, GCST and GCSP). This complex has glycine decarboxylase activity and is required for efficient photorespiration as demonstrated by analysis of an Arabidopsis thaliana mutant devoid of glycine decarboxylase activity (
      • Somerville C.R.
      • Ogren W.L.
      Mutants of the cruciferous plant Arabidopsis thaliana lacking glycine decarboxylase activity.
      ). Another photorespiratory enzyme in this cluster is the serine glyoxylate aminotransferase (Cre01.g005150.t1.1) (
      • Bauwe H.
      • Hagemann M.
      • Fernie A.R.
      Photorespiration: players, partners and origin.
      ). Other members of this assembly are a N-acetyl-gamma-glutamyl-phosphate reductase (ARGC1), the large subunit of carbonyl phosphate synthase (Cre28.g776100.t1.1), and a putative 10-formyltetrahydrofolate synthetase (FTHFS) (Cre13.g566000.t1.1). The latter enzyme is involved in formate dependent synthesis of 10-formyltetrahydrofolate, a precursor for 5,10-methylene tetrahydrofolate that is required for synthesis of serine and thus also linked to the photorespiratory process (
      • Shingles R.
      • Woodrow L.
      • Grodzinski B.
      Effects of Glycolate Pathway Intermediates on Glycine Decarboxylation and Serine Synthesis in Pea (Pisum sativum L.).
      ). 5,10 methylene tetrahydrofolate is also produced by glycine decarboxylase activity which is then used as the main single carbon donor for synthesis of serine (
      • Rebeille F.
      • Neuburger M.
      • Douce R.
      Interaction between glycine decarboxylase, serine hydroxymethyltransferase and tetrahydrofolate polyglutamates in pea leaf mitochondria.
      ). Yet, the synthesis via the 10-formyltetrahydrofolate synthetase provides independent 5,10 methylene tetrahydrofolate for the synthesis of serine (
      • Prabhu V.
      • Chatson K.B.
      • Abrams G.D.
      • King J.
      13C nuclear magnetic resonance detection of interactions of serine hydroxymethyltransferase with C1-tetrahydrofolate synthase and glycine decarboxylase complex activities in Arabidopsis.
      ). Interestingly using 13C nuclear magnetic resonance, interactions among serine hydroxymethyltransferase, tetrahydrofolate synthase, and glycine decarboxylase complex activities were shown (
      • Prabhu V.
      • Chatson K.B.
      • Abrams G.D.
      • King J.
      13C nuclear magnetic resonance detection of interactions of serine hydroxymethyltransferase with C1-tetrahydrofolate synthase and glycine decarboxylase complex activities in Arabidopsis.
      ). Accordingly our finding of coclustering of the glycine cleavage multienzyme complex, the serine glyoxylate aminotransferase and 10-formyltetrahydrofolate synthetase supports the assumption of coordinated action in serine biosynthesis and reveal that the process of photorespiration is active under PH (−Fe/+Fe) and PA/PH (+Fe), whereas it appears to be not active under PA (−Fe/+Fe).

      Respiratory Complexes are More Severely Down-regulated Under Autotrophic Low Iron Conditions

      The community XIII (blue, Fig. 3 bottom middle) consists of 12 proteins. Among those, seven proteins are subunits of mitochondrial multiprotein respiratory protein complexes (three subunits of complex I, three subunits of complex III, and one subunit of complex IV). Considering the whole cell data, subunits of the respiratory complexes are slightly induced under PH (−Fe/+Fe) and PA/PH (+Fe), but down-regulated under PA (−Fe/+Fe). Looking at the entire list of mitochondrial respiratory proteins organized in complexes I to IV as well as in ATPase in whole cells or isolated mitochondria (supplemental Fig. S4B), it appears that, except for complex II proteins and subunits of the mitochondrial ATPase, protein expression has a stronger impact under PA (−Fe/+Fe) or PA/PH (+Fe) than under iron deficient PH growth condition. The rather minor down-regulation of respiratory complexes under PH low iron availability has been already described (
      • Naumann B.
      • Busch A.
      • Allmer J.
      • Ostendorf E.
      • Zeller M.
      • Kirchhoff H.
      • Hippler M.
      Comparative quantitative proteomics to investigate the remodeling of bioenergetic pathways under iron deficiency in Chlamydomonas reinhardtii.
      ) and is confirmed by the current study. However a new aspect is that autotrophic growth under low iron induces a remodeling of the respiratory machinery.

      Deletion of ADH1 Attenuates the Remodeling of the Photosynthetic Machinery in Response to Iron Deficiency and Stimulates Expression of Stress-related Proteins

      To substantiate our quantitative proteomic data, we investigated the up-regulation of ADH1 by immunochemical methods and analyzed the iron-deficiency response in an ADH1 deletion mutant that has been recently reported (
      • Magneschi L.
      • Catalanotti C.
      • Subramanian V.
      • Dubini A.
      • Yang W.
      • Mus F.
      • Posewitz M.C.
      • Seibert M.
      • Perata P.
      • Grossman A.R.
      A mutant in the ADH1 gene of Chlamydomonas reinhardtii elicits metabolic restructuring during anaerobiosis.
      ). To analyze the induction of ADH1 under low iron conditions and possible acclimation phenotype of the ADH1 deletion mutant in response to the iron-deficiency response we shifted wild type and mutants grown from iron-sufficient acetate-containing medium for 2 days into iron-free culture solution. Such a shift causes a pronounced remodeling of the photosynthetic complexes leading to functional uncoupling of PSI associated LHCI with PSI and diminishment of PSI (
      • Moseley J.L.
      • Allinger T.
      • Herzog S.
      • Hoerth P.
      • Wehinger E.
      • Merchant S.
      • Hippler M.
      Adaptation to Fe-deficiency requires remodeling of the photosynthetic apparatus.
      ). After shifting to iron-free medium, growth performance of wild type and ADH1-deficient cells was comparable (4.53 ± 0.46 × 106 cells/ml for WT versus 3.93 ± 0.25 × 106 cells/ml for adh1 after 2 days from the shift; Fig. 4A, upper panel). Interestingly, the decrease in chlorophyll per cell content associated with the iron-starvation response appeared to be less pronounced in the mutant devoid of ADH1 (Fig. 4A, lower panel). Low temperature fluorescence spectroscopy of wild type cells after 2 days of iron-deficient growth showed a maximum emission at 705 nm whereas iron sufficient cells peaked at 714 nm (Fig. 4B), revealing that PSI and LHCI are functionally uncoupled under low iron as described (
      • Moseley J.L.
      • Allinger T.
      • Herzog S.
      • Hoerth P.
      • Wehinger E.
      • Merchant S.
      • Hippler M.
      Adaptation to Fe-deficiency requires remodeling of the photosynthetic apparatus.
      ). Interestingly, this shift in maximal fluorescence emission is not observed in adh1 mutant cells grown in iron-free medium for 2 days. To obtain further insights into the acclimation to iron-deficiency, cells were harvested from the outlined conditions and fractionated by SDS-PAGE, immunoblotted, and analyzed by a set of different antibodies (Figs. 4C, 4D). Analysis of ATPb protein levels by anti-ATPb antibodies was used as a loading control. As expected the immunoblot data revealed for wild type a down-regulation of PSI and PSII as visualized by decreases of PSAD and PSBA in day 1 and 2 of iron deficiency, respectively. On contrary this down-regulation is less pronounced in the ADH1 deletion mutant, particularly, PsbA appears to be two-times more abundant after 2 days of iron-deficiency as compared with the wild type (Fig. 4D). Additionally down-regulation of LHCA3 is less pronounced in adh1 (Fig. 4C), which likely explains the minor blue shift of the low temperature fluorescence emission spectrum in comparison to wild type, as degradation of LHCA3 correlates with changes in low temperature fluorescence emission of PSI-LHCI (
      • Naumann B.
      • Stauber E.J.
      • Busch A.
      • Sommer F.
      • Hippler M.
      N-terminal processing of Lhca3 Is a key step in remodeling of the photosystem I-light-harvesting complex under iron deficiency in Chlamydomonas reinhardtii.
      ). Interestingly, degradation of FDX1 on the other hand is paralleled in wild type and adh1 mutant. In contrast, the iron-deficiency induced expression of NDA2, LHCSR3 and PGRL1 is significantly stimulated in adh1 (Fig. 4D). Notably, iron-deficiency stimulated expression of the three proteins is also observed in the quantitative proteomics data (supplemental Fig. S4C), thereby confirming the mass spectrometric data.

      DISCUSSION

      In this work we compared iron deficiency responses under different metabolic conditions with data obtained from iron sufficient cells grown under PA and PH settings. Taking advantage of quantitative mass spectrometric data stemming from these distinct conditions, protein co-expression and the resulting network analyses provided new insights into coordinated remodeling of bioenergetics pathways and iron deficiency responses in C. reinhardtii and revealed interdependencies with the cellular metabolic status. Analyses of an adh1 knockout mutant suggest that the low iron regulatory acclimation networks are particularly sensitive to the chloroplast metabolic and/or redox status.

      Iron Deficiency Uniformly Down-regulates Assemblies of Proteins Connected to PSI, the cyt b6f Complex and Chlorophyll Biosynthesis

      A hallmark of the iron deficiency response in cyanobacteria, algae and higher plants is the down-regulation of PSI (see above). This is also reflected in community IV shown in Fig. 3, where most of its members are PSI subunits. Moreover, the down-regulation of four subunits belonging to the cyt b6f complex can be observed in community III (Fig. 3). Down-regulation of the cyt b6f complex under iron deprivation is expected (
      • Moseley J.L.
      • Allinger T.
      • Herzog S.
      • Hoerth P.
      • Wehinger E.
      • Merchant S.
      • Hippler M.
      Adaptation to Fe-deficiency requires remodeling of the photosynthetic apparatus.
      ). Nevertheless, coclustering of subunits belonging to a common multiprotein complex as already seen for PSI subunits underscores the robustness of the quantitative data and the strength of the clustering algorithm, pyGCluster. A protein that clusters with the subunits of the cyt b6f complex is CTH1. CTH1 is a diiron protein that functions as the aerobic cyclase in chlorophyll biosynthesis and is another key chloroplast target under iron deficiency (
      • Moseley J.L.
      • Allinger T.
      • Herzog S.
      • Hoerth P.
      • Wehinger E.
      • Merchant S.
      • Hippler M.
      Adaptation to Fe-deficiency requires remodeling of the photosynthetic apparatus.
      ,
      • Chereskin B.M.
      • Castelfranco P.A.
      Effects of Iron and Oxygen on Chlorophyll Biosynthesis : II. Observations on the biosynthetic pathway in isolated etiochloroplasts.
      ,
      • Pinta V.
      • Picaud M.
      • Reiss-Husson F.
      • Astier C.
      Rubrivivax gelatinosus acsF (previously orf358) codes for a conserved, putative binuclear-iron-cluster-containing protein involved in aerobic oxidative cyclization of Mg-protoporphyrin IX monomethylester.
      ,
      • Tottey S.
      • Block M.A.
      • Allen M.
      • Westergren T.
      • Albrieux C.
      • Scheller H.V.
      • Merchant S.
      • Jensen P.E.
      Arabidopsis CHL27, located in both envelope and thylakoid membranes, is required for the synthesis of protochlorophyllide.
      ,
      • Moseley J.L.
      • Page M.D.
      • Pergam N.
      • Eriksson M.
      • Quinn J.
      • Soto J.
      • Theg S.
      • Hippler M.
      • Merchant S.
      Reciprocal expression of two di-iron enzymes affecting photosystem I and light-harvesting complex accumulation.
      ). The diminishment of CTH1 under iron deprivation is likely responsible for the accumulation of Mg-protoporphyrin IX and Mg-protoporphyrin IX monomethyl ester under such conditions (
      • Spiller S.C.
      • Castelfranco A.M.
      • Castelfranco P.A.
      Effects of Iron and Oxygen on Chlorophyll Biosynthesis : I. In Vivo observations on iron and oxygen-deficient plants.
      ,
      • Chereskin B.M.
      • Castelfranco P.A.
      Effects of Iron and Oxygen on Chlorophyll Biosynthesis : II. Observations on the biosynthetic pathway in isolated etiochloroplasts.
      ). The regulation of CTH1 goes in line with the down-regulation of GGR, POR, PBGD1, and UROD1 that are also related to the tetrapyrole biosynthesis process. The impact in chlorophyll biosynthesis, however, has a very distinct effect on chlorophyll binding proteins and protein complexes as discussed above. Remarkably, LHCSR3 is up-regulated under PH iron deficiency (Fig. 4D; supplemental Fig. S4C) as well as other LHCII polypeptides (supplemental Fig. S4A) despite a decrease in chlorophyll biosynthesis. Thus the alteration of the abundance of chlorophyll binding proteins is more likely to be a regulatory mechanism than an answer to chlorophyll deficiency under iron deprivation in agreement with previous conclusions (
      • Moseley J.L.
      • Allinger T.
      • Herzog S.
      • Hoerth P.
      • Wehinger E.
      • Merchant S.
      • Hippler M.
      Adaptation to Fe-deficiency requires remodeling of the photosynthetic apparatus.
      ,
      • Naumann B.
      • Busch A.
      • Allmer J.
      • Ostendorf E.
      • Zeller M.
      • Kirchhoff H.
      • Hippler M.
      Comparative quantitative proteomics to investigate the remodeling of bioenergetic pathways under iron deficiency in Chlamydomonas reinhardtii.
      ).

      Remodeling of Bioenergetics Pathways Under Iron-deficiency is Modulated by the Cellular Metabolic Status

      As found in an earlier study (
      • Naumann B.
      • Busch A.
      • Allmer J.
      • Ostendorf E.
      • Zeller M.
      • Kirchhoff H.
      • Hippler M.
      Comparative quantitative proteomics to investigate the remodeling of bioenergetic pathways under iron deficiency in Chlamydomonas reinhardtii.
      ) mitochondrial protein complexes are only marginally affected by iron deficiency in C. reinhardtii, although this impact is stronger under PA than under PH growth conditions, which is also reflected in respiratory activity (Table I, supplemental Fig. S4B). Respiratory complex I itself contains at least 26 atoms of iron (
      • Brandt U.
      Energy converting NADH:quinone oxidoreductase (complex I).
      ), which is more than the entire photosynthetic machinery possesses, yet this complex as well as the other iron rich respiratory electron transfer complexes remain unaffected by iron deficiency (supplemental Fig. S4B). Down-regulation of the photosynthetic electron transfer complexes PSI as well as cyt b6f is therefore a result of a coordinated regulation. Notably, a bifunctional alcohol and acetaldehyde dehydrogenase (ADH1) is induced under iron deprivation (Fig. 3, Fig. 4C). Interestingly expression of ADH1 follows a day/night cycle and was mainly insensitive to oxygen availability ((
      • Whitney L.A.
      • Loreti E.
      • Alpi A.
      • Perata P.
      Alcohol dehydrogenase and hydrogenase transcript fluctuations during a day-night cycle in Chlamydomonas reinhardtii: the role of anoxia.
      ); Barth and Fufezan, unpublished results). A proteomic study found the chloroplast localized ADH1 to be 1.5 fold up-regulated under anaerobic conditions (
      • Terashima M.
      • Specht M.
      • Naumann B.
      • Hippler M.
      Characterizing the anaerobic response of Chlamydomonas reinhardtii by quantitative proteomics.
      ). Moreover, ADH1 is also induced under copper and zinc deficiency (
      • Hsieh S.I.
      • Castruita M.
      • Malasarn D.
      • Urzica E.
      • Erde J.
      • Page M.D.
      • Yamasaki H.
      • Casero D.
      • Pellegrini M.
      • Merchant S.S.
      • Loo J.A.
      The proteome of copper, iron, zinc, and manganese micronutrient deficiency in Chlamydomonas reinhardtii.
      ). As discussed for anoxic growth in C. reinhardtii (
      • Magneschi L.
      • Catalanotti C.
      • Subramanian V.
      • Dubini A.
      • Yang W.
      • Mus F.
      • Posewitz M.C.
      • Seibert M.
      • Perata P.
      • Grossman A.R.
      A mutant in the ADH1 gene of Chlamydomonas reinhardtii elicits metabolic restructuring during anaerobiosis.
      ), ADH1 could oxidize two molecules of NAD(P)H per acetyl-CoA under low iron availability and thereby modulate the redox poise of the chloroplast by replenishing it with NAD(P)+. The induction of cyclic photosynthetic electron flow (CEF) (
      • Petroutsos D.
      • Terauchi A.M.
      • Busch A.
      • Hirschmann I.
      • Merchant S.S.
      • Finazzi G.
      • Hippler M.
      PGRL1 participates in iron-induced remodeling of the photosynthetic apparatus and in energy metabolism in Chlamydomonas reinhardtii.
      ) would likewise decrease the flow of electrons into the NADPH pool, acidify the thylakoid lumen and activate energy dependent nonphotochemical quenching, going along with the described induction of LHCSR3 (
      • Peers G.
      • Truong T.B.
      • Ostendorf E.
      • Busch A.
      • Elrad D.
      • Grossman A.R.
      • Hippler M.
      • Niyogi K.K.
      An ancient light-harvesting protein is critical for the regulation of algal photosynthesis.
      ), particularly under PH settings of low iron availability (
      • Terauchi A.M.
      • Peers G.
      • Kobayashi M.C.
      • Niyogi K.K.
      • Merchant S.S.
      Trophic status of Chlamydomonas reinhardtii influences the impact of iron deficiency on photosynthesis.
      ,
      • Naumann B.
      • Busch A.
      • Allmer J.
      • Ostendorf E.
      • Zeller M.
      • Kirchhoff H.
      • Hippler M.
      Comparative quantitative proteomics to investigate the remodeling of bioenergetic pathways under iron deficiency in Chlamydomonas reinhardtii.
      ); Fig. 4D). Most interestingly deletion of ADH1 leads to an increased expression of LHCSR3, PGRL1 and NDA2 after the onset of iron deprivation as compared with wild type (Fig. 4D), indicating that the redox poise of the chloroplast in adh1 knockout strain is more reducing than in the wild type. Beside increase of PGRL1 also induction of NDA2 can be seen as a response to diminish the chloroplast redox poise, as NDA2 can lower the stromal redox poise by oxidizing NADPH and reducing plastoquinone (
      • Jans F.
      • Mignolet E.
      • Houyoux P.A.
      • Cardol P.
      • Ghysels B.
      • Cuine S.
      • Cournac L.
      • Peltier G.
      • Remacle C.
      • Franck F.
      A type II NAD(P)H dehydrogenase mediates light-independent plastoquinone reduction in the chloroplast of Chlamydomonas.
      ). The attenuated degradation of PSI and PSII and the absence of the low iron induced low temperature fluorescence emission shift of PSI-LHCI in the ADH1 deletion mutant could be explained by the fact that stress protection in form of LHCSR3, PGRL1 and NDA2 is faster induced and therefore more protective. Alternatively, iron deficiency induced proteases could be less active and/or less induced in the adh1 mutant. On the other hand, iron deficiency induced degradation of FDX1 is unaltered when wild type and adh1 are compared (Fig. 4D), indicating that the iron deficiency response is active in the mutant. Summarizing these considerations, we can at least conclude that the deletion of ADH1 has a profound impact on the remodeling of the photosynthetic apparatus as well as in the iron dependent regulation of LHCSR3, PGRL1 and NDA2 expression. Similarly, the activation of a metabolic by-pass resulting in glycerol synthesis had been shown for the adh1 mutant placed under dark anoxic conditions. This new metabolic pathway has been suggested to compensate for the inability of adh1 to synthesize ethanol and concomitantly reoxidize NAD(P)H (
      • Magneschi L.
      • Catalanotti C.
      • Subramanian V.
      • Dubini A.
      • Yang W.
      • Mus F.
      • Posewitz M.C.
      • Seibert M.
      • Perata P.
      • Grossman A.R.
      A mutant in the ADH1 gene of Chlamydomonas reinhardtii elicits metabolic restructuring during anaerobiosis.
      ).
      Table IPhotosynthetic and respiratory rates of PH and PA grown cells in two different iron concentrations. The photosynthetic rate was measured at 1000 μE/m2/s2. Standard deviation is based on biological triplicates
      Fe [μm]PHPA
      Photosynthetic rate [pm O2/cell]Respiration rate [pm O2/cell]Photosynthetic rate [pm O2/cell]Respiration rate [pm O2/cell]
      0.10.180 ± 0.060−0.191 ± 0.0630.119 ± 0.036−0.048 ± 0.007
      180.601 ± 0.076−0.305 ± 0.0371.000 ± 0.260−0.207 ± 0.077
      ADH1 clustered with other metabolic enzymes namely 6-phosphogluconate dehydrogenase (GND1a) and glyceraldehyde-3-phosphate dehydrogenase (GAP1, plastidic) and shared up-regulation under low iron and down-regulation by PA/PH under iron sufficiency (Fig. 3). GND1a catalyzes the second step of the oxidative Pentose-Phosphate-Pathway (OPPP). The glycolytic enzyme GAP1 catalyzes the first step of the second phase of glycolysis and was found in vascular plants to be induced in expression under anaerobiosis and heat stress (
      • Russell D.A.
      • Sachs M.M.
      Differential Expression and Sequence-Analysis of the Maize Glyceraldehyde-3-Phosphate Dehydrogenase Gene Family.
      ) as well as under iron-deficiency (
      • Herbik A.
      • Giritch A.
      • Horstmann C.
      • Becker R.
      • Balzer H.J.
      • Baumlein H.
      • Stephan U.W.
      Iron and copper nutrition-dependent changes in protein expression in a tomato wild type and the nicotianamine-free mutant chloronerva.
      ). Impaired iron availability clearly decreases photosynthetic activity, so that plants as well as algae probably enter into an anaerobic growth mode. The increased expression of GAP1 probably mirrors increased request to synthesize ATP under conditions where ATP synthesis is diminished because of decreased photosynthetic and respiratory activities. In line, it was found that specific activities of GAP and GND1 were greater in leaf tissues unable to generate reducing equivalents and ATP by photosynthesis (
      • Wurtele E.S.
      • Nikolau B.J.
      Enzymes of Glucose Oxidation in Leaf Tissues : The Distribution of the Enzymes of Glycolysis and the Oxidative Pentose Phosphate Pathway between Epidermal and Mesophyll Tissues of C(3)-Plants and Epidermal, Mesophyll, and Bundle Sheath Tissues of C(4)-Plants.
      ). Thus induction of GAP1 and GND1 in C. reinhardtii under low iron likely reflects the necessity to generate NADH, NADPH, and ATP from the gluconeogenesis and successive oxidation of glucose. Under such circumstances ADH1 is required to oxidize NAD(P)H to allow advancement of glucose oxidizing by replenishing NAD(P)+. Its deletion, under conditions when GAP1 and GND1 are induced, on the other hand will therefore certainly increase the cellular redox poise, supporting the conclusions made above. As ADH1, GAP1, and GND1a are chloroplast localized (
      • Terashima M.
      • Specht M.
      • Naumann B.
      • Hippler M.
      Characterizing the anaerobic response of Chlamydomonas reinhardtii by quantitative proteomics.
      ), it is likely that the redox poise of the chloroplast is involved in communication with the nucleus to modulate gene expression. Interestingly, transcripts of genes encoding ADH1, GAP1, and GND1a are down-regulated by iron-deficiency (
      • Urzica E.I.
      • Casero D.
      • Yamasaki H.
      • Hsieh S.I.
      • Adler L.N.
      • Karpowicz S.J.
      • Blaby-Haas C.E.
      • Clarke S.G.
      • Loo J.A.
      • Pellegrini M.
      • Merchant S.S.
      Systems and trans-system level analysis identifies conserved iron deficiency responses in the plant lineage.
      ), whereas the protein amounts are up-regulated (herein; (
      • Urzica E.I.
      • Casero D.
      • Yamasaki H.
      • Hsieh S.I.
      • Adler L.N.
      • Karpowicz S.J.
      • Blaby-Haas C.E.
      • Clarke S.G.
      • Loo J.A.
      • Pellegrini M.
      • Merchant S.S.
      Systems and trans-system level analysis identifies conserved iron deficiency responses in the plant lineage.
      )), thus strengthening our interpretation that these proteins are in the same regulatory circuit. Moreover, the divergence of transcript and protein expression may implicate post-transcriptional regulation for expression control. It is tempting to speculate that this may occur via the cytosolic translational machinery and the redox poise of the chloroplast. Besides remodeling of bioenergetics pathways, the low iron response also induced proteases, redox- and oxidative stress related enzymes.

      The Iron Deficiency Response Induces Proteases, Redox- and Oxidative Stress Related Enzymes - Nonetheless Differences Between PH and PA Conditions are Evident

      In the iron deficiency response of C. reinhardtii several proteases are up-regulated. This up-regulation is especially pronounced under PH conditions (Fig. 3), as seen for a putative metallopeptidase (Cre01.g023350.t1.2) and a serine-type protease (Cre10.g423300.t1.1) as well as a peptidase M (Cre20.g758550.t1.2, supplemental Fig. S3 community XVIII). Two other proteases, a cysteine protease, CEP, and an aspartic acid protease, ASP, clustered together and are also induced under PA iron-deficiency but down-regulated under PA/PH iron sufficient conditions. Notably, CEP and ASP proteases are chloroplast-localized (
      • Terashima M.
      • Specht M.
      • Naumann B.
      • Hippler M.
      Characterizing the anaerobic response of Chlamydomonas reinhardtii by quantitative proteomics.
      ). Generally plant proteases are involved in many cellular functions, including chloroplast related properties (
      • van der Hoorn R.A.
      Plant proteases: from phenotypes to molecular mechanisms.
      ). However, the precise functions of the four proteases found in our protein assemblies are unknown. For strain C. reinhardtii W80, a cysteine protease was isolated that was also induced under photo-oxidative stress conditions (
      • Usui M.
      • Tanaka S.
      • Miyasaka H.
      • Suzuki Y.
      • Shioi Y.
      Characterization of cysteine protease induced by oxidative stress in cells of Chlamydomonas sp. strain W80.
      ). Photo-oxidative stress in the chloroplast of iron-deficient photoheterotrophic cells can be conceived, as PSI and the cytochrome b6f complex are down-regulated, whereas the PSII related light-harvesting protein complement remains stable or either increases or decreases in amount as in the case of LHCBM1 and LHCBM6/3/7 or LHCBM4, respectively (supplemental Fig. S3 community XVII). It was reported that under PH growth and iron deprivation the light-harvesting antenna was still functionally coupled to PSII, which caused significant photoinhibition (
      • Naumann B.
      • Busch A.
      • Allmer J.
      • Ostendorf E.
      • Zeller M.
      • Kirchhoff H.
      • Hippler M.
      Comparative quantitative proteomics to investigate the remodeling of bioenergetic pathways under iron deficiency in Chlamydomonas reinhardtii.
      ). Thus induction of the proteases could be related to the light-induced photo-oxidative stress that is particular for iron deficient PH grown cells and potentially involved in the degradation of Fe-containing proteins (e.g. FDX1, FAB2, cytf etc.) At the same time respiratory activity of these cells, as measured by oxygen uptake activity in the dark, is only 1.5 times lower as compared with iron sufficient PH grown cells (Table I). In contrast, respiratory activity of iron deficient PA grown cells is four times lower in regard to iron sufficient cells. Importantly the oxygen evolution activity of iron-sufficient PA grown cells is increased almost twofold compared with the activity of PH grown cells, whereas iron deficient PA grown cells produce less oxygen as compared with iron deficient PH cells (Table I). Thus iron deficient PH grown cells possess a higher photosynthetic activity as well as a higher respiratory rate than iron deficient PA grown cells. Apparently, PH grown cells suffer a higher degree of photo-oxidative stress for their pronounced bioenergetic capacity. Notably, we found proteins with a flavodoxin domain (AGG3 (Cre10.g456000.t1.1) and particularly AGG4 (Cre10.g456050.t1.1)) that are highly up-regulated under PH low iron conditions (Fig. 3, supplemental Fig. S3 community XVIII). AGG3 and AGG4 were found to be enriched in the flagella proteome and the membrane-matrix fraction of C. reinhardtii (
      • Pazour G.J.
      • Agrin N.
      • Leszyk J.
      • Witman G.B.
      Proteomic analysis of a eukaryotic cilium.
      ). AGG3 was shown to be involved in mediating the orientation of the alga toward a directional light source (
      • Iomini C.
      • Li L.
      • Mo W.
      • Dutcher S.K.
      • Piperno G.
      Two flagellar genes, AGG2 and AGG3, mediate orientation to light in Chlamydomonas.
      ). It was concluded that down-regulation of AGG3 blocks positive phototaxis (
      • Iomini C.
      • Li L.
      • Mo W.
      • Dutcher S.K.
      • Piperno G.
      Two flagellar genes, AGG2 and AGG3, mediate orientation to light in Chlamydomonas.
      ). It is unclear what impact overexpression of AGG3 would have, also the concrete function of AGG4, a close paralog of AGG3, is currently unknown but it is tempting to speculate that increase of AGG3 and AGG4 under iron deficiency impacts the phototaxis of the alga, thereby contributing to the overall strategy to minimize photooxidative stress and optimize photosynthetic function. Interestingly, under iron deficiency, flavodoxin substitutes for ferredoxin as electron acceptor of PSI in diatoms and cyanobacteria (
      • LaRoche J.
      • Boyd P.W.
      • McKay R.M.L.
      • Geider R.J.
      Flavodoxin as an in situ marker for iron stress in phytoplankton.
      ,
      • Leonhardt K.
      • Straus N.A.
      An iron stress operon involved in photosynthetic electron transport in the marine cyanobacterium Synechococcus sp. PCC 7002.
      ,
      • Straus N.A.
      Iron deprivation: physiology and gene regulation.
      ,
      • Ghassemian M.
      • Straus N.A.
      Fur regulates the expression of iron-stress genes in the cyanobacterium Synechococcus sp. strain PCC 7942.
      ). However, because of their localization and low sequence similarity to photosynthetic flavodoxins, AGG3 and AGG4 are rather involved in regulation of phototaxis than in photosynthesis.
      Having acetate as a fueling source, the iron deprived PH grown cells produce energy via respiration and apparently invest in a costly stress response to maintain photosynthetic activity. In line with this argumentation, a Peptidyl-prolyl cis-trans isomerases (CYN37), a translation factor for expression of the chloroplast-encoded psbA gene (TBA1) (
      • Somanchi A.
      • Barnes D.
      • Mayfield S.P.
      A nuclear gene of Chlamydomonas reinhardtii, Tba1, encodes a putative oxidoreductase required for translation of the chloroplast psbA mRNA.
      ), a cytochrome c peroxidase (CCPR1) and a thioredoxin o (TRXo) were induced solely under PH iron-deficiency and clustered together (Fig. 3, XI). Notably these proteins are also induced under PA iron plus conditions. CYN37 shares highest similarity with Arabidopsis TLP38, a luminal chloroplast protein (
      • Schubert M.
      • Petersson U.A.
      • Haas B.J.
      • Funk C.
      • Schroder W.P.
      • Kieselbach T.
      Proteome map of the chloroplast lumen of Arabidopsis thaliana.
      ). PPIases catalyze the cis-trans isomerization of the peptidyl-prolyl bond during protein folding, a rate-limiting step in this process (
      • Gothel S.F.
      • Marahiel M.A.
      Peptidyl-prolyl cis-trans isomerases, a superfamily of ubiquitous folding catalysts.
      ). Interestingly, Vener and colleagues (
      • Shapiguzov A.
      • Edvardsson A.
      • Vener A.V.
      Profound redox sensitivity of peptidyl-prolyl isomerase activity in Arabidopsis thylakoid lumen.
      ) found redox-dependent activity of chloroplast PPIase implying a link to the redox poise of the stroma and suggesting that protein folding catalysis under conditions of active photosynthesis is important. The finding of a constant up-regulation of CYN37 under iron deficiency stress or PA conditions underpins this notion. TBA1 encodes a putative oxidoreductase required for translation of the chloroplast psbA mRNA (
      • Somanchi A.
      • Barnes D.
      • Mayfield S.P.
      A nuclear gene of Chlamydomonas reinhardtii, Tba1, encodes a putative oxidoreductase required for translation of the chloroplast psbA mRNA.
      ). Up-regulation of chloroplast localized TBA1 may reflect high turnover of the D1 protein, thereby requiring increased translation of psbA RNA. The oxidoreductase domain may connect TBA1 activity to the redox poise of the stroma and implicate a regulation similar to CYN37. The mitochondrial cytochrome c peroxidase (CCPR1) is located at the intermembrane space, reduces hydrogen peroxide to water using the electrons provided by cytochrome c and is thereby involved in scavenging of reactive oxygen species in the mitochondria (
      • Volkov A.N.
      • Nicholls P.
      • Worrall J.A.
      The complex of cytochrome c and cytochrome c peroxidase: the end of the road?.
      ). The mitochondrial thioredoxin o (TRXo) that is found in the same cluster is another player in this scavenging system. It acts as an electron donor to mitochondrial type II peroxiredoxin F, which has been shown to be essential for redox homeostasis and root growth of A. thaliana under stress (
      • Finkemeier I.
      • Goodman M.
      • Lamkemeyer P.
      • Kandlbinder A.
      • Sweetlove L.J.
      • Dietz K.J.
      The mitochondrial type II peroxiredoxin F is essential for redox homeostasis and root growth of Arabidopsis thaliana under stress.
      ). Correspondingly, the mitochondrial manganese containing superoxide dismutases (MSD1 and MSD2) are strongly induced under PH low iron, likely combating superoxide formed by the respiratory electron transport (supplemental Fig. S3 community XVIII, supplemental Fig. S4D). Beside these responses, Fig. 3 reveals other redox and oxidative stress related proteins that are up-regulated under PH and PA iron-deficiency, though up-regulation of these proteins is stronger under PH low iron. These proteins are represented by a putative glutathione S-transferase (Cre12.g526800.t1.1) and GSH1, the gamma-glutamylcysteine synthetase. This enzyme catalyzes the second step of glutathione (GSH) biosynthesis by adding glycine to the C-terminal site of γ-glutamylcysteine to form GSH (
      • May M.J.
      • Leaver C.J.
      Arabidopsis thaliana gamma-glutamylcysteine synthetase is structurally unrelated to mammalian, yeast, and Escherichia coli homologs.
      ). GSH operates as a redox buffer but contributes also to other cellular functions; in particular it participates in heavy metal detoxification and in ROS scavenging (
      • Rouhier N.
      • Lemaire S.D.
      • Jacquot J.P.
      The role of glutathione in photosynthetic organisms: emerging functions for glutaredoxins and glutathionylation.
      ). GSH is involved in the generation of reduced ascorbate, another cellular redox buffer (
      • Pignocchi C.
      • Foyer C.H.
      Apoplastic ascorbate metabolism and its role in the regulation of cell signalling.
      ) and antioxidant (
      • Gutteridge J.M.
      • Halliwell B.
      Free radicals and antioxidants in the year 2000. A historical look to the future.
      ), which is required e.g. for the detoxification of ROS produced by PSI (
      • Ort D.R.
      • Baker N.R.
      A photoprotective role for O(2) as an alternative electron sink in photosynthesis?.
      ,
      • Asada K.
      The water-water cycle as alternative photon and electron sinks.
      ). Likewise monodehydroascorbate reductase (MDAR), which also participates in regeneration of ascorbate (
      • Hossain M.A.
      • Nakano Y.
      • Asada K.
      Monodehydroascorbate reductase in spinach-chloroplasts and its participation in regeneration of ascorbate for scavenging hydrogen-peroxide.
      ) by reducing monodehydroascorbate using NADPH, is up-regulated under PH low iron growth condition (Fig. 3). The putative glutathione S-transferase (Cre12.g526800.t1.1) might be functionally related to the glutathione S-transferase, GST1. GST1 expression is induced as an acclimation response to singlet oxygen treatment and its overexpression was sufficient to enforce resistance against singlet oxygen stress (
      • Ledford H.K.
      • Chin B.L.
      • Niyogi K.K.
      Acclimation to singlet oxygen stress in Chlamydomonas reinhardtii.
      ). Ledford and colleagues (
      • Ledford H.K.
      • Chin B.L.
      • Niyogi K.K.
      Acclimation to singlet oxygen stress in Chlamydomonas reinhardtii.
      ) discuss that GST1 could function as lipid peroxidase, thereby increasing lipid peroxidase activity of the cells, which in turn would protect against reactive oxygen species (ROS) and/or participate in signaling activity.
      Interestingly another glutathione S-transferase (Cre01.g064400.t1.1) and a DegP protease (DEG5A) are induced under PA iron-deprivation (Fig. 3), indicating functional relation between these two but on the contrary distinct tasks for the one induced under PH iron deficiency.

      Carbon-concentrating Mechanism, Photorespiration, and Arginine Biosynthesis are Linked Under Photoautotrophic Growth Conditions

      A common motif of regulation for growth under photoautotrophic condition is the induction of carbon-concentrating mechanism (CCM) related proteins. In C. reinhardtii and other aquatic unicellular organism, CCM is induced under low CO2 availability to improve photosynthesis by increasing the concentration of intracellular inorganic carbon to elevate the CO2 concentration at the site of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; for reviews see (
      • Wang Y.
      • Duanmu D.
      • Spalding M.H.
      Carbon dioxide concentrating mechanism in Chlamydomonas reinhardtii: inorganic carbon transport and CO2 recapture.
      ,
      • Yamano T.
      • Fukuzawa H.
      Carbon-concentrating mechanism in a green alga, Chlamydomonas reinhardtii, revealed by transcriptome analyses.
      )). Rubisco catalyzes the first step of photosynthetic CO2 fixation and beside the carboxylation of its substrate ribulose-1,5-bisphosphate (RuBP), it may also oxygenate it. Oxygenation produces the toxic component 2-phosphoglycolate (2-PG), which is recycled to 3-phosphoglycerate (3-PGA) during the process of photorespiration. Consequently an increase in the CO2 to O2 ratio will decrease photorespiration. Among the proteins that are found to be up-regulated under PA conditions compared with PH (communities VI and XI, Fig. 3) and found in a shared cluster are LCIC and LCIB. Studies of mRNA expression in response to low CO2 revealed that transcripts of lcib and lcic are among the most induced mRNA molecules recorded (
      • Wang Y.
      • Spalding M.H.
      An inorganic carbon transport system responsible for acclimation specific to air levels of CO2 in Chlamydomonas reinhardtii.
      ,
      • Yamano T.
      • Miura K.
      • Fukuzawa H.
      Expression analysis of genes associated with the induction of the carbon-concentrating mechanism in Chlamydomonas reinhardtii.
      ). Genetic analyses indicated that LCIB is involved in the accumulation of inorganic carbon in the chloroplast (
      • Wang Y.
      • Spalding M.H.
      An inorganic carbon transport system responsible for acclimation specific to air levels of CO2 in Chlamydomonas reinhardtii.
      ) and that correspondingly to their co-expression, LCIB and LCIC form a 350 kDa hexameric complex in the chloroplast (
      • Yamano T.
      • Tsujikawa T.
      • Hatano K.
      • Ozawa S.
      • Takahashi Y.
      • Fukuzawa H.
      Light and low-CO2-dependent LCIB-LCIC complex localization in the chloroplast supports the carbon-concentrating mechanism in Chlamydomonas reinhardtii.
      ). Induction of CCM proteins is also strongly supported by the fact that CAH3 (for review see (
      • Wang Y.
      • Duanmu D.
      • Spalding M.H.
      Carbon dioxide concentrating mechanism in Chlamydomonas reinhardtii: inorganic carbon transport and CO2 recapture.
      ,
      • Yamano T.
      • Fukuzawa H.
      Carbon-concentrating mechanism in a green alga, Chlamydomonas reinhardtii, revealed by transcriptome analyses.
      ) is significantly increased under iron sufficient PA settings. In accordance, LCIB/LCIC and CAH3 were found in CCM clusters when transcriptome-wide changes in relation to CO2 and the CO2-CCM regulators CIA5/CCM1 were analyzed (
      • Fang W.
      • Si Y.Q.
      • Douglass S.
      • Casero D.
      • Merchant S.S.
      • Pellegrini M.
      • Ladunga I.
      • Liu P.
      • Spalding M.H.
      Transcriptome-wide changes in Chlamydomonas reinhardtii gene expression regulated by carbon dioxide and the CO2-concentrating mechanism regulator CIA5/CCM1.
      ). Despite the fact that C. reinhardtii possesses a carbon-concentrating mechanism, photorespiratory products have been detected after shifting cells to low CO2 by using gas chromatography-coupled time of flight mass spectrometry (
      • Renberg L.
      • Johansson A.I.
      • Shutova T.
      • Stenlund H.
      • Aksmann A.
      • Raven J.A.
      • Gardestrom P.
      • Moritz T.
      • Samuelsson G.
      A metabolomic approach to study major metabolite changes during acclimation to limiting CO2 in Chlamydomonas reinhardtii.
      ). Accordingly, transcript levels for the photorespiratory enzymes, including HRP1, were increased in low CO2 grown cells (
      • Yamano T.
      • Miura K.
      • Fukuzawa H.
      Expression analysis of genes associated with the induction of the carbon-concentrating mechanism in Chlamydomonas reinhardtii.
      ,
      • Fang W.
      • Si Y.Q.
      • Douglass S.
      • Casero D.
      • Merchant S.S.
      • Pellegrini M.
      • Ladunga I.
      • Liu P.
      • Spalding M.H.
      Transcriptome-wide changes in Chlamydomonas reinhardtii gene expression regulated by carbon dioxide and the CO2-concentrating mechanism regulator CIA5/CCM1.
      ). HPR1 is involved in photorespiration as explained above, and is found in a coregulated community with LCIB and LCIC (Fig. 3, community XI). Thus a close connection between photorespiration and the CCM can be interfered as already suggested (
      • Yamano T.
      • Miura K.
      • Fukuzawa H.
      Expression analysis of genes associated with the induction of the carbon-concentrating mechanism in Chlamydomonas reinhardtii.
      ,
      • Fang W.
      • Si Y.Q.
      • Douglass S.
      • Casero D.
      • Merchant S.S.
      • Pellegrini M.
      • Ladunga I.
      • Liu P.
      • Spalding M.H.
      Transcriptome-wide changes in Chlamydomonas reinhardtii gene expression regulated by carbon dioxide and the CO2-concentrating mechanism regulator CIA5/CCM1.
      ). Coclustering of the glycine cleavage multienzyme complex, the serine glyoxylate aminotransferase and 10-formyltetrahydrofolate synthetase, as suggested above, indicates coordinated action in serine biosynthesis and supports that photorespiration is more active under PH low iron and PA/PH as compared with PH low iron. Other members of this assembly are a N-acetyl-gamma-glutamyl-phosphate reductase (ARGC1) and the large subunit of carbonyl phosphate synthase (Cre28.g776100.t1.1) (CPS), two enzymes that are involved in arginine biosynthesis. It is of note that these enzyme are all found in the chloroplast proteome of C. reinhardtii (
      • Terashima M.
      • Specht M.
      • Naumann B.
      • Hippler M.
      Characterizing the anaerobic response of Chlamydomonas reinhardtii by quantitative proteomics.
      ) and Arabidopsis thaliana (
      • Kleffmann T.
      • Russenberger D.
      • von Zychlinski A.
      • Christopher W.
      • Sjolander K.
      • Gruissem W.
      • Baginsky S.
      The Arabidopsis thaliana chloroplast proteome reveals pathway abundance and novel protein functions.
      ,
      • Peltier J.B.
      • Cai Y.
      • Sun Q.
      • Zabrouskov V.
      • Giacomelli L.
      • Rudella A.
      • Ytterberg A.J.
      • Rutschow H.
      • van Wijk K.J.
      The oligomeric stromal proteome of Arabidopsis thaliana chloroplasts.
      ). Knock-out of the small or large subunit of CPS resulted in a reticulate leaf phenotype that was correlated with a defect in mesophyll development (
      • Molla-Morales A.
      • Sarmiento-Manus R.
      • Robles P.
      • Quesada V.
      • Perez-Perez J.M.
      • Gonzalez-Bayon R.
      • Hannah M.A.
      • Willmitzer L.
      • Ponce M.R.
      • Micol J.L.
      Analysis of ven3 and ven6 reticulate mutants reveals the importance of arginine biosynthesis in Arabidopsis leaf development.
      ), implicating that CPS function and arginine may play a role in these developmental processes. CPS is involved in the conversion of glutamine and bicarbonate into carbamoyl phosphate (CP) and glutamate (
      • Holden H.M.
      • Thoden J.B.
      • Raushel F.M.
      Carbamoyl phosphate synthetase: an amazing biochemical odyssey from substrate to product.
      ). CP formation is the first committed step in the biosynthesis of arginine and in de novo biosynthesis of pyrimidines (
      • Zrenner R.
      • Stitt M.
      • Sonnewald U.
      • Boldt R.
      Pyrimidine and purine biosynthesis and degradation in plants.
      ). Because CPS, some CCM proteins, HPR1, three subunits of the glycine cleavage multienzyme complex, the serine glyoxylate aminotransferase and 10-formyltetrahydrofolate synthetase are coregulated, the question arises about the connection between these biological processes and the arginine and pyrimidine biosynthesis. During photorespiration, the formation of glycine from glyoxylate via the glutamate:glyoxylate aminotransferase (GGT) requires glutamate as a cosubstrate (
      • Bauwe H.
      • Hagemann M.
      • Fernie A.R.
      Photorespiration: players, partners and origin.
      ). Thus the creation of CP as a committed step in the arginine biosynthetic pathway could participate in glutamate production as substrate for the photorespiratory process, linking thereby arginine biosynthesis and photorespiration. This goes hand in hand with the observed increase in 2-oxogluterate, which is produced via GGT action induced after shifting C. reinhardtii cells into low CO2 conditions (
      • Renberg L.
      • Johansson A.I.
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      ), whereas glutamate is decreased because it is steadily consumed. Notably arginine accumulates under stress in A. thaliana (
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      • Fotopoulos V.
      • Pateraki I.
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      ,
      • Kalamaki M.S.
      • Merkouropoulos G.
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      ) and is considered an important intermediate for nitrogen storage (
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      • Rubio V.
      Arginine and nitrogen storage.
      ). In line our data suggest that arginine biosynthesis, as well as photorespiration, is characteristic for cells actively performing photosynthesis.

      Photoautotrophic Growth Regulation May Require Calcium and Redox Signaling

      Beside proteins involved in CCM, photorespiration or arginine biosynthesis as presented in community XI, other proteins were particularly up-regulated under PA iron sufficient conditions (Fig. 3). These proteins are a stomatin-like protein (Cre33.g782550.t1.1), a protein that contains a thioredoxin domain and four putative EF-hand motifs (Cre03.g202950.t1.2) as well as a NADP malic enzyme (MME5). MME5 is a chloroplast localized NADP-malic enzyme (
      • Terashima M.
      • Specht M.
      • Hippler M.
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      ) that either operates in decarboxylating malate to pyruvate or in catalyzing the carboxylation of pyruvate to malate thereby generating NADPH or oxidizing it to NADP+. As the direction is dependent on the chloroplast redox poise, the enzyme could thereby be important for the fine-tuning of the chloroplast redox status.
      In mammalians, stomatin and its paralogs are described, as homo-oligomeric, lipid raft-associated, integral membrane proteins that may interact with and modulate various ion channels and transporters (
      • Lapatsina L.
      • Brand J.
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      ). More particularly, stomatin was found to interact with a Ca2+ ATPase (
      • Rungaldier S.
      • Oberwagner W.
      • Salzer U.
      • Csaszar E.
      • Prohaska R.
      Stomatin interacts with GLUT1/SLC2A1, band 3/SLC4A1, and aquaporin-1 in human erythrocyte membrane domains.
      ). However, whether a link between C. reinhardtii stomatin and calcium exists remains to be shown.
      Four EF hands domains were found in Cre03.g202950.t1.2. EF hands present a common motif for proteins involved in the binding of Ca2+. The EF is a helix-loop-helix motif, which mostly occurs in pairs (not identical; up to 6 pairs in one protein), allowing high affinity binding to Ca2+. The pairing is important for the Ca2+-binding mechanism and Ca2+-induced conformational change, translating the Ca2+concentration into an output response (
      • Kretsinger R.H.
      Calcium-binding proteins.
      ). EF-hand containing proteins operate in all aspects of cell function and more than 3000 EF-hand related entries can be found in the NCBI RefSeq database. Likewise thioredoxin domains are contained in many proteins and enzymatically thioredoxin acts as a protein disulphideoxidoreductase that catalyzes the reversible oxidation of two cysteine thiol groups to a disulfide, thereby transferring two electrons and two protons (
      • Holmgren A.
      Thioredoxin.
      ). Thus combining EF-hand motifs with a thioredoxin domain would link calcium and redox signaling. From network analyses it distinctly appears that thioredoxins are central to chloroplast redox signaling (
      • Dietz K.J.
      • Jacquot J.P.
      • Harris G.
      Hubs and bottlenecks in plant molecular signalling networks.
      ). Recent data revealed that chloroplast calcium signaling may also contribute to retrograde signaling between chloroplast and the nucleus. In C. reinhardtii, the expression of the nuclear encoded LHCSR3 is regulated via the thylakoid membrane protein CAS (Ca2+-sensing receptor) and Ca2+ (
      • Petroutsos D.
      • Busch A.
      • Janssen I.
      • Trompelt K.
      • Bergner S.V.
      • Weinl S.
      • Holtkamp M.
      • Karst U.
      • Kudla J.
      • Hippler M.
      The Chloroplast Calcium Sensor CAS Is Required for Photoacclimation in Chlamydomonas reinhardtii.
      ). In A. thaliana, CAS is involved in chloroplast mediated activation of plant immune signaling (
      • Nomura H.
      • Komori T.
      • Uemura S.
      • Kanda Y.
      • Shimotani K.
      • Nakai K.
      • Furuichi T.
      • Takebayashi K.
      • Sugimoto T.
      • Sano S.
      • Suwastika I.N.
      • Fukusaki E.
      • Yoshioka H.
      • Nakahira Y.
      • Shiina T.
      Chloroplast-mediated activation of plant immune signalling in Arabidopsis.
      ) and in stomatal regulation in response to elevations of external Ca2+ through the modulation of cytoplasmic Ca2+ dynamics (
      • Weinl S.
      • Held K.
      • Schlucking K.
      • Steinhorst L.
      • Kuhlgert S.
      • Hippler M.
      • Kudla J.
      A plastid protein crucial for Ca2+-regulated stomatal responses.
      ,
      • Nomura H.
      • Komori T.
      • Kobori M.
      • Nakahira Y.
      • Shiina T.
      Evidence for chloroplast control of external Ca2+-induced cytosolic Ca2+ transients and stomatal closure.
      ), thereby regulating the availability of carbon dioxide and thus indirectly also the redox poise of the chloroplast stroma. Importantly, CAS has been also implicated in regulation of cyclic electron transfer (
      • Terashima M.
      • Petroutsos D.
      • Hudig M.
      • Tolstygina I.
      • Trompelt K.
      • Gabelein P.
      • Fufezan C.
      • Kudla J.
      • Weinl S.
      • Finazzi G.
      • Hippler M.
      Calcium-dependent regulation of cyclic photosynthetic electron transfer by a CAS, ANR1, and PGRL1 complex.
      ). Here consistently a link between redox poise and calcium can be anticipated.
      Notably the induction of a Peptidyl-prolyl cis-trans isomerase (CYN37) and a redox-related translation factor for expression of the chloroplast-encoded psbA gene (TBA1) (
      • Somanchi A.
      • Barnes D.
      • Mayfield S.P.
      A nuclear gene of Chlamydomonas reinhardtii, Tba1, encodes a putative oxidoreductase required for translation of the chloroplast psbA mRNA.
      ), under PA/PH conditions, as already discussed for PH iron deficiency, support the conclusion that photoautotrophic growth regulation is related to the chloroplast redox poise.

      Acknowledgments

      We thank Dr. Y. Takahashi and Dr. Eugen Urzica for fruitful comments and lively discussions.

      Supplementary Material

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