TY - JOUR
AU - Gontero, Brigitte
AB - Abstract Aquatic photosynthesis is responsible for about half of the global production and is undertaken by a huge phylogenetic diversity of algae that are poorly studied. The diversity of redox-regulation of phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was investigated in a wide range of algal groups under standard conditions. Redox-regulation of PRK was greatest in chlorophytes, low or absent in a red alga and most chromalveolates, and linked to the number of amino acids between two regulatory cysteine residues. GAPDH regulation was not strongly-related to the different forms of this enzyme and was less variable than for PRK. Addition of recombinant CP12, a protein that forms a complex with PRK and GAPDH, to crude extracts inhibited GAPDH and PRK inversely in the Plantae, but in most chromalveolates had little effect on GAPDH and inhibited or stimulated PRK depending on the species. Patterns of enzyme regulation were used to produce a phylogenetic tree in which cryptophytes and haptophytes, at the base of the chromalveolates, formed a distinct clade. A second clade comprised only chromalveolates. A third clade comprised a mixture of Plantae, an excavate and three chromalveolates: a marine diatom and two others (a xanthophyte and eustigmatophyte) that are distinguished by a low content of chlorophyll c and a lack of fucoxanthin. Regulation of both enzymes was greater in freshwater than in marine taxa, possibly because most freshwaters are more dynamic than oceans. This work highlights the importance of understanding enzyme regulation in diverse algae if their ecology and productivity is to be understood. Algae, CP12, glyceraldehyde-3-phosphate dehydrogenase, phosphoribulokinase, phylogeny Introduction Photosynthesis on land is largely the result of one phylogenetic group, the land plants or Embryophytes, but in the oceans and freshwaters it is performed by a huge phylogenetic diversity of algae (Falkowski et al., 2004). Although the biochemical properties and patterns of metabolic regulation are well known in land plants, relatively little is known about this in most groups of algae, even though they contribute about half of all the global primary production (Falkowski et al., 2004). In photosynthetic eukaryotes, carbon fixation is performed by the Calvin cycle within plastids that had a different evolutionary origin depending on the taxonomic group. A single endosymbiotic event 1600 million years ago produced the plastids of the supergroup Plantae (green algae, red algae, and glaucophytes) (Nozaki et al., 2007; Yoon et al., 2004). Land plants (Embryophyta) evolved from a green algal ancestor (Yoon et al., 2004) but the green algae were also involved in two subsequent secondary symbiotic events that gave rise to the Euglenophyceae (supergroup Excavata) and the Chlorarachniophyceae (supergroup Rhizaria). Other secondary symbiotic events involving red algae led to the Chromalveolata (Yoon et al., 2004). The major autotrophic chromalveolate groups, the heterokonts (diatoms, chrysophytes, eustigmatophytes, raphidophytes, and xanthophytes) and alveolates (dinoflagellates) have recently been proposed to have evolved from haptophytes, or the haptophyte–cryptophyte ancestor at the base of the chromalveolates, by tertiary endosymbiosis (Sanchez-Puerta and Delwiche, 2008). In addition to the Calvin cycle, plastids of land plants and green algae possess an oxidative pentose phosphate pathway. As both pathways are interconnected, operating them simultaneously would result in a futile cycle, using up energy in the form of ATP without net CO2 fixation. As a result, Calvin cycle enzymes such as phosphoribulokinase (PRK, EC 2.7.1.19), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.13), fructose-1,6-bisphosphatase (EC 3.1.3.11), and sedoheptulose-1,7-bisphosphatase (EC 3.1.3.37) are activated in the light and inactivated in the dark, while the key enzyme of the oxidative pentose phosphate, glucose-6-phosphate dehydrogenase (EC 1.1.1.49), is active in the dark but inhibited in the light. In land plants, this regulation has been well-documented and shown to involve various mechanisms including, but not exclusively, protein–protein interactions (Gontero et al., 2006). An in vivo study of pea leaves has shown that the formation and dissociation of PRK/GAPDH/CP12, where CP12 is a small intrinsically unstructured protein (Wedel and Soll, 1998; Graciet et al., 2003a; Marri et al., 2005; Tamoi et al., 2005; Oesterhelt et al., 2007), is controlled by light quantity, via thioredoxin, and regulates the enzyme activity (Howard et al., 2008). Although there is apparently no complete oxidative pentose phosphate pathway in the plastids of marine diatoms (Kroth et al., 2008), plastidic enzymes still need to be regulated. For example, a single isoform of GAPDH exists within the diatom plastid (Gruber et al., 2009) that participates in two opposite reactions: in the Calvin cycle using 1,3-bisphosphoglycerate to produce glyceraldehyde 3-phosphate and in the glycolytic pathway, that is present in the plastid (Kroth et al., 2008), catalysing the reverse reaction. The regulation of Calvin cycle enzymes by light is different in diatoms than in green algae and land plants (Michels et al., 2005; Wilhelm et al., 2006; Boggetto et al., 2007; Erales et al., 2008). For example, unlike green algae, PRK was not redox-regulated in the marine diatom, Odontella sinensis (Greville) Grunow (Michels et al., 2005) or the freshwater diatom, Asterionella formosa Hassall (Boggetto et al., 2007) using dithiothreitol (DTT), a reducing agent that, in vitro, mimics the action of thioredoxins in vivo, and therefore the effects of light on enzyme activities. For NADPH-dependent GAPDH, no redox-activation was observed in Odontella sinensis (Michels et al., 2005) whereas a strong activation upon reduction was observed in Asterionella formosa (Boggetto et al., 2007). The aim of the work reported here was to characterize the regulation of two key enzymes, PRK and GAPDH, in a wide phylogenetic range of marine and freshwater algae. This broad survey of 16 species was not intended to characterize the specific properties of each enzyme but to determine if the different algal groups that constitute the phytoplankton (Falkowski et al., 2004) have different patterns of regulation under uniform conditions. Materials and methods Phytoplankton cultivation and harvesting The 16 phytoplankton species used are shown in Supplementary Table S1 at JXB online. Cultures were typically grown in 1.5 l batch cultures at 20 °C under a 16/8 h (light:dark) regime at about 65 μmol m−2 s−1 (photosynthetically active radiation; 400–700 nm). All media recipes are available at the websites of CCAP (Culture Collection of Algae and Protozoa: http://www.ccap.ac.uk/index.htm) or CCMP (Provasoli-Guillard National Center for Culture of Marine Phytoplankton: https://ccmp.bigelow.org/node/58) apart from TAP which is given in Harris (1988). K-medium was prepared with artificial seawater at a concentration of 32‰ sea salts (Sigma-Aldrich, UK) and, in the case of the Thalassiosira pseudonana Hasle & Heimdal culture, supplemented with 200 μM NaSiO3.9H2O. All species were grown photoautotrophically apart from Chlamydomonas reinhardtii PA Dangeard and Euglena gracilis Klebs which were grown mixotrophically. Dense cultures were harvested by centrifugation (12 000 g for 15 min at 10 °C) or slow filtration onto polycarbonate filters [Emiliana huxleyi (Lohmann) WH Hay & H Mohler: 2 μm pore size; Alexandrium minutum Halim: 10 μm pore size]. Cells were resuspended in growth medium, transferred to Eppendorf tubes and centrifuged briefly. After removal of the supernatant, cell pellets were stored frozen at –20 °C until further analysis. Cultures of Dinobryon sociale Ehrenberg and Hemiselmis rufescens Parke were grown and harvested by CCMP. Between 3.0 l and 50 l of culture per species was used to produce the material analysed in this work. Extraction of proteins Cells were harvested and the pellets (about 2 g cells) were resuspended in a minimum volume of 15 mM TRIS, 4 mM EDTA, 5 mM cysteine, 0.1 mM NAD in the presence of a cocktail of protease inhibitors (0.5 μg ml−1, Sigma Inc., St Louis, MO, USA) at pH 7.9 on ice. Cysteine was added to all samples in order to stabilize enzyme activity, but all samples were kept on ice in order to minimize possible reduction by this agent. The resulting suspension was broken using a One Shot apparatus (Constant Systems, Northants, UK) at 100–130 MPa. The broken cells were subsequently centrifuged at 27 000 g for 25 min at 4 °C. The supernatant, containing the crude extract, was kept on ice after adding 10% glycerol (final concentration). Protein concentration in all crude extracts was assayed with the Bio-Rad (Hercules, CA, USA) protein dye reagent, using bovine serum albumin as a standard (Bradford, 1976). Measurement of enzyme activity Crude extracts were incubated at room temperature for 30 min before enzyme activity was measured, as tests on a range of species showed that full activation was reached within 5 min. Extracts for PRK and GAPDH assays were incubated without additions or with (i) 20 mM DTT, (ii) 1 mM NADPH, and (iii) 20 mM DTT plus 1 mM NADPH because previous work has shown that these conditions affect the activity of both enzymes via an effect on complex formation (Boggetto et al., 2007). For some species, 20 mM oxidized glutathione was added to check the redox state of the samples. Aliquots from the different incubation mixtures were withdrawn after 30 min, and the activities of PRK and GAPDH were measured. GAPDH activity was monitored with 0.2 mM NADPH as cofactor plus 1 mM BPGA that was synthesized as previously described (Graciet et al., 2003b). For samples incubated with NADPH, the final concentration of this cofactor in the assay reached 0.24 mM because of the carryover from NADPH incubation. PRK activity was measured as previously described (Gontero et al., 1988). Both activities were followed at 340 nm, using a Pye Unicam UV2 spectrophotometer (Cambridge, UK) without the addition of DTT in the assay cuvette. Activity was measured at least four times and was normalized to a protein basis (Bradford, 1976). All reagents for enzyme activity measurements were from Sigma Inc. Standard errors were calculated using a formula as previously described (Armitage, 1971) and significant differences among treatments were determined using t tests. Measurement in the presence of recombinant CP12 PRK and GAPDH activities were measured as described above in the absence of DTT and in the presence of 200 μM oxidized recombinant CP12 from Chlamydomonas reinhardtii, purified as described by Graciet et al. (2003a). CP12 protein was dialysed against 30 mM TRIS-HCl, 0.1 M NaCl, pH 7.9 and stored at –20 °C. Construction of a UPGMA tree based on enzyme regulation In order to group similar patterns of regulation, activity of PRK or of NADPH-GAPDH under four different conditions (crude extract alone, added DTT, added NADPH, and added DTT and NADPH) was correlated across the different algal cultures. In some species, the slope of response for PRK and GAPDH was different, but in others it was similar so a third correlation was calculated that combined both enzymes in order to take this into account. Each correlation coefficient (r) was converted to a measure of dissimilarity (d) where d=1–((r + 1)/2). The three dissimilarity values were averaged and used to build a tree using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) (Cornish-Bowden, 1983) that first identifies the least dissimilar pair and then recalculates the dissimilarity coefficient matrix to find the next least dissimilar correlation using the weighted average value for a group. Results Enzyme activity As shown in Table 1, the two cultures of Chlamydomonas reinhardtii, had the highest activities of PRK on a protein basis. In general, the activities of PRK were highest in the Plantae, although PRK activity was also high in Alexandrium minutum and Navicula pelliculosa (Brébisson ex Kützing) Hilse and Euglena gracilis. The activity of NADPH-GAPDH was also high in the Plantae, apart from Staurastrum cingulum (W West & GS West) GM Smith, and also high in Alexandrium minutum and Asterionella formosa strain CCAP 1005/19. The ratio of PRK to GAPDH activity in the crude extract tended to be highest in the Plantae but there was not a very strong phylogenetic pattern because other strains such as Euglena gracilis, Thalassiosira pseudonana, and Bumilleriopsis peterseniana Vischer & Pascher also had high ratios. The very high ratio in Euglena gracilis may be linked to the mixotrophic growth of these cells, even though the photosynthetic form of GAPDH (NADPH-GAPDH) was assayed. The ratio of PRK to GAPDH activity in the ‘fully-activated’ extract was close to, or less than one, in many chromalveolates (11 species) apart from Thalassiosira pseudonana, Pseudocharaciopsis ovalis (Chodat) Hibberd, and Bumilleriopsis peterseniana. The activity of the ‘fully activated’ extract corresponds to the maximal (highest) activity found under the different incubation conditions (Table 1). Table 1. Activities and ratios of activity for PRK and NADPH-GAPDH in different strains of algae Species  Crude extract (nmol product mg−1 protein min−1)   Ratio of PRK to GAPDH   PRK activity  GAPDH activity  Crude extract  Fully-activated  Plantae          Staurastrum cingulum  198 (5)  8 (4)  24.8 (12.4)  4.5 (0.3)  Chlamydomonas reinhardtii-Aa  1831 (243)  418 (100)  4.4 (1.2)  1.7 (0.5)  Chlamydomonas reinhardtii-Ba  3895 (257)  948 (22)  4.1 (0.3)  3.0 (0.2)  Porphyridium purpureum  563 (66)  174 (13)  3.2 (0.5)  1.8 (0.4)  Cyanophora paradoxa  620 (78)  69 (3)  9.0 (1.2)  6.5 (1.1)  Chromalveolata          Alexandrium minutum  331 (54)  476 (32)  0.7 (0.1)  0.5 (0.1)  Asterionella formosa strain 18  31 (7)  56 (8)  0.6 (0.2)  0.1 (0.0)  Asterionella formosa strain 19  18 (5)  191 (59)  0.1 (0.0)  0.03 (0.5)  Aulacoseira granulata  69 (9)  25 (7)  2.8 (0.9)  0.5 (0.0)  Navicula pelliculosa  176 (12)  9 (3)  19.6 (6.7)  0.4 (0.0)  Thalassiosira pseudonana  81 (4)  9 (1)  9.0 (1.1)  5.5 (0.4)  Pseudocharaciopsis ovalis  97 (9)  21 (4)  4.4 (0.9)  1.7 (0.2)  Bumilleriopsis peterseniana  74 (14)  8 (1)  9.4 (2.0)  1.4 (0.3)  Mallomonas caudata  40 (14)  40 (8)  1.0 (0.4)  0.2 (0.1)  Dinobryon sociale  75 (11)  25 (5)  3.0 (0.7)  0.7 (0.1)  Emiliania huxleyi  29 (1)  27 (3)  1.1 (0.1)  1.1 (0.1)  Hemiselmis rufescens  68 (2)  10 (1)  6.8 (0.7)  0.9 (0.2)  Excavata          Euglena gracilis  306 (202)  4 (1)  76.5 (20.0)  12.3 (1.5)  Species  Crude extract (nmol product mg−1 protein min−1)   Ratio of PRK to GAPDH   PRK activity  GAPDH activity  Crude extract  Fully-activated  Plantae          Staurastrum cingulum  198 (5)  8 (4)  24.8 (12.4)  4.5 (0.3)  Chlamydomonas reinhardtii-Aa  1831 (243)  418 (100)  4.4 (1.2)  1.7 (0.5)  Chlamydomonas reinhardtii-Ba  3895 (257)  948 (22)  4.1 (0.3)  3.0 (0.2)  Porphyridium purpureum  563 (66)  174 (13)  3.2 (0.5)  1.8 (0.4)  Cyanophora paradoxa  620 (78)  69 (3)  9.0 (1.2)  6.5 (1.1)  Chromalveolata          Alexandrium minutum  331 (54)  476 (32)  0.7 (0.1)  0.5 (0.1)  Asterionella formosa strain 18  31 (7)  56 (8)  0.6 (0.2)  0.1 (0.0)  Asterionella formosa strain 19  18 (5)  191 (59)  0.1 (0.0)  0.03 (0.5)  Aulacoseira granulata  69 (9)  25 (7)  2.8 (0.9)  0.5 (0.0)  Navicula pelliculosa  176 (12)  9 (3)  19.6 (6.7)  0.4 (0.0)  Thalassiosira pseudonana  81 (4)  9 (1)  9.0 (1.1)  5.5 (0.4)  Pseudocharaciopsis ovalis  97 (9)  21 (4)  4.4 (0.9)  1.7 (0.2)  Bumilleriopsis peterseniana  74 (14)  8 (1)  9.4 (2.0)  1.4 (0.3)  Mallomonas caudata  40 (14)  40 (8)  1.0 (0.4)  0.2 (0.1)  Dinobryon sociale  75 (11)  25 (5)  3.0 (0.7)  0.7 (0.1)  Emiliania huxleyi  29 (1)  27 (3)  1.1 (0.1)  1.1 (0.1)  Hemiselmis rufescens  68 (2)  10 (1)  6.8 (0.7)  0.9 (0.2)  Excavata          Euglena gracilis  306 (202)  4 (1)  76.5 (20.0)  12.3 (1.5)  Fully-activated activity refers to the maximal activity under the different incubation conditions. The mean is given with standard deviation in parenthesis. a Chlamydomonas reinhardtii-A was grown in TAP and Chlamydomonas reinhardtii-B was grown in JM with 6.25% EG. View Large In all species, control experiments confirmed that the activity of PRK and GAPDH in the crude extracts did not change during incubation in the presence of 5 mM cysteine (data not shown). This is in agreement with the mid-redox potential of cysteine that is around –140 mV and the known mid-redox potentials of PRK and GAPDH (Marri et al., 2005, 2009; Michels et al., 2005). Regulation of PRK PRK showed a variety of regulation patterns for algae derived from different endosymbioses (Fig. 1). In the Plantae, PRK from the Chlorophyceae was strongly up-regulated by DTT, compared to that from Cyanophora paradoxa Korshikov (Glaucophyceae) which had a slight activation and that from Porphyridium purpureum (Bory de Saint-Vincent) KM Drew et R Ross (Rhodophyta) where there was no significant effect of DTT. To check if the non-activation of the red algal enzyme was not observed because the enzyme was already reduced, an oxidant (oxidized glutathione) was added and no change in the activity was observed confirming the redox insensitivity of this enzyme (data not shown). In the case of the PRK from Chlamydomonas reinhardtii and Cyanophora paradoxa reducing the samples with DTT first and then adding the oxidant led to a decrease in activity confirming their redox sensitivity. Fig. 1. View largeDownload slide Comparison of regulation of the activity of PRK, by DTT, NADPH, and DTT plus NADPH in different algal species. The y-axis shows the ratio of treated to untreated crude extract. The horizontal line shows a ratio against the crude extract of 1. Means and one standard deviation are presented. Statistical differences from the crude extracts are designated as: NS, not significant; *P <0.05; **P <0.01; ***P <0.001. Note that ratios for Chlamydomonas reinhardtii grown on TAP medium are presented on a different scale. The top row includes the Plantae, apart from E. gracilis top right (Excavata) and the two bottom rows are all Chromalveolata. Fig. 1. View largeDownload slide Comparison of regulation of the activity of PRK, by DTT, NADPH, and DTT plus NADPH in different algal species. The y-axis shows the ratio of treated to untreated crude extract. The horizontal line shows a ratio against the crude extract of 1. Means and one standard deviation are presented. Statistical differences from the crude extracts are designated as: NS, not significant; *P <0.05; **P <0.01; ***P <0.001. Note that ratios for Chlamydomonas reinhardtii grown on TAP medium are presented on a different scale. The top row includes the Plantae, apart from E. gracilis top right (Excavata) and the two bottom rows are all Chromalveolata. In general, the PRK from chromalveolates was also not activated by DTT alone, apart from in Pseudocharaciopsis ovalis (Eustigmatophyceae), although weak (i.e. 1.1–1.3-fold stimulation over the crude extract) but statistically-significant activation was also observed in Dinobryon sociale and Emiliania huxleyi. In diatoms, the lack of regulation of PRK by DTT observed for Asterionella formosa confirmed previous results (Boggetto et al., 2007), and redox-insensitivity was confirmed by the lack of effect on activity of adding oxidized glutathione (data not shown). This lack of regulation was also observed in a different strain of Asterionella formosa (CCAP 1005/19) and in other diatoms, Aulacoseira granulata (Ehrenberg) Simonsen, Navicula pelliculosa, and Thalassiosira pseudonana. For most species, the response to the addition of NADPH and DTT was similar to the addition of DTT alone. However, a greater activation was observed with DTT plus NADPH than with DTT alone, in Chlamydomonas reinhardtii-B (P <0.001), Euglena gracilis (P <0.001), both strains of Asterionella formosa (P <0.05), and Dinobryon sociale (P <0.001). The effect of NADPH was therefore checked alone, but this either had no effect, or caused only a slight inhibition of PRK, apart from in Emiliania huxleyi where it caused a marked stimulation compared to the crude extract (44%, P <0.001). Relationship between structure of PRK and regulatory patterns Analysis of published sequences of PRK suggests that the number of amino acid residues between the two regulatory cysteine residues is critical for redox-regulation of this enzyme. Thus, sequences from the Chlorophyta all have 38 residues between the two cysteine residues at positions 16 and 55 (numbering from the spinach and green algal enzymes; Fig. 2) and algae from this group show redox-regulation (Fig. 1). By contrast, the three available sequences from Rhodophyta have seven amino acids fewer between the cysteine residues and PRK from Porphyridium purpureum is not redox-regulated (Figs 1, 2). The five available sequences from the Cryptophyta/Haptophyta have one less amino acid residue than the green algae between the regulatory cysteine residues (Fig. 2) and it was found that PRK from the two representatives that were tested from this group lacked or had very weak redox-regulation (Fig. 1). Apart from Pseudocharaciopsis ovalis, the other chromalveolates tested also showed no, or weak, redox-regulation and the heterokonts (diatoms, chrysophytes, raphidophytes) had five more amino acid residues between the regulatory cysteine residues than the green algae and land plants (Fig. 2). Interestingly, the Excavate Euglena gracilis also had this pattern of amino acids (Fig. 2) and PRK was also not redox-regulated. Two sequences from the dinoflagellates Lingulodinium polyedra (Stein) Dodge and Heterocapsa triquetra (Ehrenberg) F Stein and one from the Chlorarachniophyta Bigelowiella natans Moestrup had seven fewer amino acid residues between the regulatory cysteine residues than that found in green algae and land plants (Fig. 2). The only enzyme regulation result we have for this amino acid pattern is for the dinoflagellate Alexandrium minutum which lacked redox-regulation of PRK (Fig. 1). Fig. 2. View largeDownload slide Sequence alignment of the regulatory part of PRK proteins from a range of eukaryotic algae produced using ClustalW. Amino acids labels show identical residues in all sequences (*), conserved substitutions (:) and semi-conserved substitutions (.). The two cysteine residues (positions 16 and 55 in the Chlamydomonas enzyme), where present, are shaded in grey. #Lingulodinium polyedra was incorrectly named L. polyedrum on the sequence accession. Fig. 2. View largeDownload slide Sequence alignment of the regulatory part of PRK proteins from a range of eukaryotic algae produced using ClustalW. Amino acids labels show identical residues in all sequences (*), conserved substitutions (:) and semi-conserved substitutions (.). The two cysteine residues (positions 16 and 55 in the Chlamydomonas enzyme), where present, are shaded in grey. #Lingulodinium polyedra was incorrectly named L. polyedrum on the sequence accession. Regulation of NADPH-GAPDH The pattern of regulation of NADPH-GAPDH was less variable than for PRK, but the extent of activation was generally greater than for PRK, ranging from about 1.4–40-fold (Fig. 3). GAPDH was activated by DTT in all species, apart from in Cyanophora paradoxa, Thalassiosira pseudonana, and Alexandrium minutum. GAPDH from Hemiselmis rufescens showed a small, but statistically significant, activation by DTT. For Cyanophora, Thalassiosira, and Hemiselmis, NADPH and DTT together stimulated GAPDH. In all the diatoms, and commonly in the chromalveolates, NADPH alone caused an inhibition of GAPDH. One exception was the cryptophyte Hemiselmis rufescens that showed a modest activation (Fig. 3). In the Plantae, addition of NADPH alone had little effect on GAPDH activity, apart from in Staurastrum cingulum where there was a strong inhibition, similar to that of chromalveolates. Fig. 3. View largeDownload slide Comparison of regulation of the activity of NADPH-GAPDH, by DTT, NADPH, and DTT plus NADPH in different algal species. The y-axis shows the ratio of treated to untreated crude extract. The horizontal line shows a ratio against the crude extract of 1. Means and one standard deviation are presented. Statistical differences from the crude extracts are designated as: NS, not significant; *P <0.05; **P <0.01; ***P <0.001. Note that ratios for Navicula pelliculosa are presented on a different scale. The top row includes the Plantae, apart from E. gracilis top right (Excavata) and the two bottom rows are all Chromalveolata. Fig. 3. View largeDownload slide Comparison of regulation of the activity of NADPH-GAPDH, by DTT, NADPH, and DTT plus NADPH in different algal species. The y-axis shows the ratio of treated to untreated crude extract. The horizontal line shows a ratio against the crude extract of 1. Means and one standard deviation are presented. Statistical differences from the crude extracts are designated as: NS, not significant; *P <0.05; **P <0.01; ***P <0.001. Note that ratios for Navicula pelliculosa are presented on a different scale. The top row includes the Plantae, apart from E. gracilis top right (Excavata) and the two bottom rows are all Chromalveolata. Effect of Chlamydomonas reinhardtii CP12 on activity of PRK and GAPDH To test if the differences in PRK and GAPDH regulation described above were linked to the properties of CP12, excess, recombinant CP12 from Chlamydomonas reinhardtii was added to a range of algal strains. The pattern of regulation was still variable for PRK and GAPDH (Fig. 4). This was caused by binding of CP12 because no effects were observed in the presence of DTT which weakens the interaction between CP12 and both enzymes (data not shown). Recombinant CP12 inhibited both enzymes in Cyanophora paradoxa, Porphyridium purpureum, Pseudocharaciopsis ovalis, and Asterionella formosa. These species and Chlamydomonas reinhardtii, showed an apparent inverse relationship between the magnitude of the inhibition of GAPDH and PRK. In contrast to the Plantae, the chromalveolates generally showed almost no inhibition of GAPDH and either inhibition or stimulation of PRK and GAPDH. One exception was the Chromalveolate (Eustigmatophyte) Pseudocharaciopsis ovalis (Fig. 4) which showed a substantial inhibition of GAPDH by recombinant CP12. The absence of effect on PRK from Chlamydomonas reinhardti could be linked to the oxidized state of this enzyme in the sample. However, the effect of CP12 on PRK from Porphyridium purpureum cannot be linked to a possible reduced state of this enzyme because, as mentioned above, oxidized glutathione had no effect. Thus, in general, the effect of CP12 cannot be linked to the enzyme redox states. Fig. 4. View largeDownload slide Effect of adding exogenous CP12 from Chlamydomonas reinhardtii on the activity of PRK and GAPDH in Plantae (filled circles), (1), (2), (3), stand for Chlamydomonas reinhardtii, Cyanophora paradoxa, and Porphyridium purpureum; and chromalveolates (open triangles), (4), (5) (6), (7), (8), (9) stand for Pseudocharaciopsis ovalis, Alexandrium minutum, Dinobryon sociale, Hemiselmis rufescens, Bumilleriopsis peterseniana, and Asterionella formosa. The dashed lines show no-effect and error bars show one standard deviation. The two solid lines show a linear regression for each algal group. Fig. 4. View largeDownload slide Effect of adding exogenous CP12 from Chlamydomonas reinhardtii on the activity of PRK and GAPDH in Plantae (filled circles), (1), (2), (3), stand for Chlamydomonas reinhardtii, Cyanophora paradoxa, and Porphyridium purpureum; and chromalveolates (open triangles), (4), (5) (6), (7), (8), (9) stand for Pseudocharaciopsis ovalis, Alexandrium minutum, Dinobryon sociale, Hemiselmis rufescens, Bumilleriopsis peterseniana, and Asterionella formosa. The dashed lines show no-effect and error bars show one standard deviation. The two solid lines show a linear regression for each algal group. Relationship between enzyme regulation and algal phylogeny In order to assess whether or not PRK and GAPDH were regulated proportionally among species, activities measured under the four treatments were compared. For example, Dinobryon sociale and Emiliania huxleyi showed very different patterns of regulation: for GAPDH there was no obvious correlation between the two species, while for PRK there was a weak negative correlation (Fig. 5A). In the case of Aulacoseira granulata and Staurastrum cingulum (Fig. 5B), the responses of the individual enzymes were correlated; but the two enzymes were regulated to different extents. In the case of Chlamydomonas reinhardtii versus Staurastrum cingulum (Fig. 5C) the responses of the individual enzymes were correlated and there was a similar regulation pattern for both enzymes. Fig. 5. View largeDownload slide Example of correlations in regulation of enzyme (activity, nmol product mg−1 protein min−1) for PRK (open circles) and NADPH-GAPDH (filled circles) in (A) Emiliania huxleyi versus Dinobryon sociale; (B) Aulacoseira granulata versus Staurastrum cingulum; (C) Chlamydomonas reinhardtii-B versus Staurastrum cingulum. Fig. 5. View largeDownload slide Example of correlations in regulation of enzyme (activity, nmol product mg−1 protein min−1) for PRK (open circles) and NADPH-GAPDH (filled circles) in (A) Emiliania huxleyi versus Dinobryon sociale; (B) Aulacoseira granulata versus Staurastrum cingulum; (C) Chlamydomonas reinhardtii-B versus Staurastrum cingulum. A dissimilarity matrix calculated for the different algae, based on the types of correlations, is shown in Fig. 5. Three high-level groups were found with dissimilarity coefficients greater than 0.25. One group (Group III; Fig. 6) comprised Emiliania huxleyi and Hemiselmis rufescens, although these were distantly related to one another (dissimilarity coefficient of 0.371). Aulacoseira granulata and Dinobryon sociale were the most similar with a dissimilarity coefficient of 0.018 (Fig. 6). They formed part of a larger group that only comprised chromalveolates (Group II) including Alexandrium minutum and the two strains of Asterionella formosa which were closely related on the tree, as were Navicula pelliculosa and Mallomonas caudata Ivanov. The third group contained a mix of three chromalveolates, five Plantae, and an Excavate (Group I; Fig. 6). The most similar species pair was Pseudocharaciopsis ovalis and Chlamydomonas reinhardtii-A (see Table 1), with a dissimilarity coefficient of 0.019. This pair was closely related to Staurastrum cingulum, Bumilleriopsis peterseniana, and Chlamydomonas reinhardtii-B. Porphyridium purpureum was linked to this group. Thalassiosira pseudonana and Cyanophora paradoxa clustered together at a dissimilarity coefficient of 0.075 and linked to the previous group. Euglena gracilis, was the most dissimilar strain in this clade and linked to the rest at a dissimilarity coefficient of 0.195. Fig. 6. View largeDownload slide Tree based on the average dissimilarity between the different algal strains calculated with the Unweighted Pair Group Method with Arithmetic Mean. The three groups are designated by I, II, and III, respectively and d stands for the dissimilarity coefficient. Fig. 6. View largeDownload slide Tree based on the average dissimilarity between the different algal strains calculated with the Unweighted Pair Group Method with Arithmetic Mean. The three groups are designated by I, II, and III, respectively and d stands for the dissimilarity coefficient. Discussion Eukaryotic algae are polyphyletic and found in four of the five supergroups in the eukaryote ‘Tree of Life’ (Keeling et al., 2005), although their plastids are all ultimately derived from cyanobacteria. Although studies have been undertaken on algae from individual groups, the consequence of this evolutionary diversity on biochemical regulation of key Calvin cycle enzymes has not previously been fully considered. Regulation of PRK Data presented here show that although some weak redox-regulation of PRK was observed in several algal groups, strong regulation was mainly a feature of green algae and was also present in land plants. Cyanophora paradoxa had a similar pattern of regulation to the green algae but the extent of activation was smaller. Activation of PRK on reduction results from the rupture of a disulphide bridge between the two cysteine residues at positions 16 and 55 in land plants and green algae. In photosynthetic bacteria, where these two cysteine residues are missing, PRK is not redox-sensitive although it can be allosterically regulated by NADH (Miziorko, 2000). The work presented here confirms and expands that of Oesterhelt et al. (2007) who related the lack of redox-regulation of PRK in the red alga Galdieria sulphuraria (Galdieri) Merola to the seven fewer amino acids between the two cysteine residues. This pattern was also found in two other red algae, Chondrus crispus Stackhouse and Cyanidioschyzon merolae P De Luca, R Taddei & L Varano which also lacked the cysteine at position 16. These results suggest that this deletion is a feature of red algal PRK and therefore that this enzyme is unlikely to be redox-regulated in this algal group as confirmed both by the addition of reductant or oxidant in the samples. It was found that PRK from the glaucophyte Cyanophora paradoxa is redox-regulated so we hypothesize that its PRK sequence will be similar to that of land plants and green algae. The general lack of PRK redox-regulation within the chromalveolates appears to be related, in part, to the PRK sequence. In comparison to the green algae and land plants, the five sequences from cryptophytes and haptophytes, had one fewer amino acid between their cysteine resides, the six sequences from heterokonts had five more amino acids between the cysteine residues, and the two sequences from alveolata had seven fewer amino acids between the cysteine residues. The main exception to this overall pattern is the apparent redox-regulation of the eustigmatophyte Pseudocharaciopsis ovalis found here and the Raphidiophyceae, Heterosigma carterae Hulbert (Taylor), that seems to be redox-regulated (Hariharan et al., 1998) despite having an insertion in its sequence. The only available PRK sequence from a Chlorarachniophyta has a deletion similar to the alveolates (Fig. 2) so it is hypothesized that this PRK would also not be redox-regulated. Regulation of GAPDH The GapA form of GAPDH is found in cyanobacteria and in the Plantae: chlorophytes, rhodophytes, glaucophytes, and land plants (Petersen et al., 2006a). This form lacks a regulatory sequence so redox-regulation of GAPDH is only possible through the formation of a supramolecular complex with CP12. In streptophytes (charophytes and land plants) an additional form, GapB, with a C-terminal extension possessing two regulatory cysteine residues recruited from CP12, provides a mechanism for direct light–dark regulation. In the chromalveolates, GAPDH exists as GapC1 that derived from a cytosolic form and lacks regulatory cysteine residues (Harper and Keeling, 2003; Liaud et al., 2000). In the only species studied here with both GapA and GapB, Staurastrum cingulum, the response to the addition of DTT was similar to Chlamydomonas reinhardtii with the GapA form alone. Within the chromalveolates, GAPDH from Odontella sinensis and Coscinodiscus granii Gough was not redox-regulated (Michels et al., 2005) and a similar result was found with another marine diatom, Thalassiosira pseudonana. In contrast, GAPDH from the freshwater diatom, Asterionella formosa was redox-regulated (Boggetto et al., 2007; Erales et al., 2008) and a similar response was found in two other freshwater diatoms, Aulacoseira granulata and Navicula pelliculosa. In the other chromalveolates, GAPDH was redox-regulated in all species apart from in the marine dinoflagellate Alexandrium minutum. These variable responses may indicate that regulation of GAPDH is controlled more by the interaction with other proteins, such as PRK and above all, CP12, than by the particular type of GAPDH. CP12 CP12 exhibits a highly conserved C-terminal domain, including cysteine residues, but the N-terminal part is less conserved. Moreover, the cysteine residues of this part are absent from CP12 proteins in Rhodophyta, Cyanophora, and some cyanobacteria such as Synechococcus (Tamoi et al., 2005; Oesterhelt et al., 2007). In the latter species, the absence of the cysteine residues does not impair the formation of a supramolecular complex as a PRK/GAPDH/CP12 complex has been characterized in this species (Tamoi et al., 2005). The addition of exogenous CP12 from Chlamydomonas reinhardtii caused an almost complete inhibition of GAPDH activity in Chlamydomonas reinhardti implying a high affinity between GAPDH and CP12. By contrast, addition of exogenous CP12 from Chlamydomonas reinhardtii had little effect on the GapC1 enzymes from chromalveolates. In the Plantae, a decrease in GAPDH inhibition was associated with an increase in PRK inhibition and may thus reflect a preference for CP12 binding/affinity for PRK. In support of these data, little if any inhibition was observed with A4 GAPDH from Arabidopsis in the presence of oxidized CP12 (Marri et al., 2005) unlike the inhibition observed with the Chlamydomonas reinhardtii enzyme (Graciet et al., 2003b). These differences may result from a different affinity between these two A4 GAPDHs and CP12 since experiments performed in vitro with Arabidopsis using recombinant proteins showed that they interact with a dissociation constant in the submicromolar range (Kd of about 0.18 μM; Marri et al., 2008). By contrast, the dissociation constant from similar experiments with Chlamydomonas reinhardtii was 450-fold smaller (Kd of about 0.4 nM; Graciet et al., 2003a). In the chromalveolates, there was a large range of responses by PRK to the addition of CP12 ranging between about 41% stimulation in Alexandrium minutum and 62% inhibition in Asterionella formosa. One hypothesis is that CP12 from Chlamydomonas reinhardtii interacts more strongly with PRK from Asterionella formosa than does the native CP12. Indeed in Asterionella formosa, PRK readily dissociates from the PRK/GAPDH/CP12 complex during enzyme purification, while GAPDH co-purified with a CP12-like protein (Erales et al., 2008). The weak interaction between PRK and CP12 could be a consequence of the sequences of these proteins in Asterionella formosa: work to test this hypothesis is currently in progress. Regulation and phylogeny So far as we are aware, this is the first time that patterns of enzyme regulation have been used to produce a phylogenetic tree. The results show that different algal groups have patterns of redox-regulation linked, in part, to their evolutionary history. For example, the tree joined the haptophytes and cryptophytes (Group III) which is consistent with molecular data from the analysis of plastid genomes that links these two groups and frequently locates them at the base of the chromalveolate supergroup (Rice and Palmer, 2006; Sanchez-Puerta and Delwiche, 2008). A large grouping (Group II) was comprised entirely of chromalveolates (including a dinoflagellate, four diatom strains, and two chrysophytes), characterized by cells that possess chlorophyll c2 and, apart from the dinoflagellate Alexandrium minutum, the carotenoid fucoxanthin. This is consistent with the proposed single origin of chromalveolate plastids (Harper and Keeling, 2003). A second large grouping (Group I) was a mix of algal groups. It comprised all the Plantae and the green-lineage secondary endosymbiont Euglena gracilis. The close position of the green algae, red algae, and glaucophytes within this grouping suggests that pigment composition is not the most important factor determining how PRK and GAPDH are redox-regulated. Three chromalveolates were also found in this group, one was the marine diatom Thalassiosira pseudonana. We do not have an explanation for this anomalous placement at present, but it resulted from a lack of DTT regulation of PRK and GAPDH in our assay conditions. A similar lack of regulation has been found for another marine diatom Odontella sinensis (Michels et al., 2005). Two other chromalveolates, a eustigmatophyte Pseudocharaciopsis ovalis, and a xanthophyte Bumilleriopsis peterseniana were also in this grouping. In contrast to other chromalveolates, they do not contain chlorophyll c (or very low amounts in the case of the xanthophyte) or fucoxanthin. The different pigment composition in these chromalveolates may be linked in some way with their different pattern of enzyme regulation. Although the evolution of the chromalveolates is becoming better understood (Sanchez-Puerta and Delwiche, 2008), the precise position of the Eustigmatophyceae and Xanthophyceae is not well known, although a recent analysis of GapC1 sequences located both groups within the chromalveolates close to the Phaeophyceae (Xanthophyceae) and Bacillariophyceae (Eustigmatophyceae; Takishita et al., 2009). However, the evidence from the data presented here suggests that they may be distinct from the rest of the supergroup, at least regarding their enzyme regulation. This is particularly the case for Pseudocharaciopsis ovalis where the inhibition of its NADPH-GAPDH enzyme by exogenous recombinant CP12 was similar to that of the Plantae. Although chromalveolates and their putative plastid ancestors, the red algae, have a similar lack of PRK redox-regulation, this is caused by different numbers of residues between the regulatory cysteine pair: either an insertion in chromalveolates or a deletion in red algae (relative to green algae; Fig. 2). Similarly, even though Euglena gracilis is believed to have evolved from a secondary symbiotic event with a green alga (Rogers et al., 2007), it was found that PRK was redox-insensitive, which is in agreement with the five amino acid insertion that was found in its sequence. Interestingly, the insertion region in Euglena gracilis is very similar to that found in chromalveolates suggesting either horizontal gene transfer (Petersen et al., 2006b; Keeling, 2009) or a common origin. The plastidic GAPDH gene in Euglena gracilis is known to be related to that in some dinoflagellates, presumably by horizontal gene transfer (Fagan and Hastings, 2002), so it is possible that this mechanism could also have occurred with PRK. The chromalveolate PRK gene was also suggested to be derived by horizontal gene transfer, but from green algae (Petersen et al., 2006b). Recently, Moustafa et al. (2009) suggested that diatoms and other chromalveolates derived from endosymbiotic events with both red and green lineages so the patterns of PRK and GAPDH regulation found here may reflect that complicated ancestry and, in particular, may help to explain why chromalveolates and Plantae occurred together in one group of our phylogenetic tree and could also explain the results found here for the eustigmatophyte Pseudocharaciopsis ovalis. Environment also appears to influence regulation: GAPDH was not strongly redox-regulated in any of the marine species tested here (1–2-fold) compared to the freshwater species (1–45-fold). Similarly for PRK, none of the marine species showed strong redox-regulation (1–1.1-fold) compared to freshwater algae (0.7–4.2-fold). Further work is needed to confirm this and to determine why redox-regulation differs in marine and freshwater diatoms. One possibility is that the history of horizontal gene transfer differed in marine and freshwater algae. Another possibility is that the greater extent of redox-regulation in freshwater algae is linked to the more dynamic nature of productive freshwaters, compared to the open-oceans, with a more rapid light attenuation with depth and a more variable supply of carbon dioxide. Although it is possible that different growth and assay conditions will alter the regulation patterns of different species, this broad survey of algae grown under standardized conditions has shown clear links between the redox-regulation of key Calvin cycle enzymes, PRK and GAPDH, and algal phylogeny. While, in some cases, the responses can be linked to the amino acid sequence, in others it probably results from interactions with other proteins such as CP12. More work is needed to increase the number of species studied in many groups to distinguish clearly between species and phylogenetic-level effects. However, the results show that it is important to appreciate the large diversity in patterns of redox-regulation in order to understand current ecological distribution, and the likely impacts of environmental perturbation on species composition and productivity. Abbreviations Abbreviations BPGA 1,3-bisphosphoglycerate CP12 chloroplast protein DTT dithiothreitol EG Euglena gracilis medium JM Jaworski's medium GAPDH glyceraldehyde-3-phosphate dehydrogenase PRK phosphoribulokinase TAP TRIS acetate phosphate UPGMA Unweighted Pair Group Method with Arithmetic Mean We thank Annabelle Cortes-Maurel, Helene Lenoir, Marion Pavy, and Sebastian Meis for help with culturing the algae and Drs Athel Cornish-Bowden and Guillermo Mulliert-Carlin for advice on the construction of the phylogenetic tree. Travel between the laboratories in Lancaster and Marseille was funded by a British Council-Egide Alliance Programme and the project DIALOG funded by Programme Energie PIE2 from CNRS. The comments of two anonymous referees helped to improve the manuscript. 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TI - Phylogenetically-based variation in the regulation of the Calvin cycle enzymes, phosphoribulokinase and glyceraldehyde-3-phosphate dehydrogenase, in algae
JF - Journal of Experimental Botany
DO - 10.1093/jxb/erp337
DA - 2009-11-19
UR - https://www.deepdyve.com/lp/oxford-university-press/phylogenetically-based-variation-in-the-regulation-of-the-calvin-cycle-fm9fyzTyF3
SP - 735
EP - 745
VL - 61
IS - 3
DP - DeepDyve
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