ORIGINAL ARTICLE

Federico Valverde Æ Jose M. Ortega Æ Manuel Losada

Aurelio Serrano

Sugar-mediated transcriptional regulation of the Gap gene systemand concerted photosystem II functional modulationin the microalga Scenedesmus vacuolatus

Received: 1 September 2004 / Accepted: 2 February 2005 / Published online: 14 April 2005� Springer-Verlag 2005

Abstract Partial cDNAs corresponding to the GapA,GapC and GapN genes that encode the three differentglyceraldehyde-3-phosphate dehydrogenases (GAPDHs)of the green microalga Scenedesmus vacuolatus SAG211-8b have been cloned and characterized. Northernblot experiments, as well as immunoblots and activitymeasurements, demonstrate a differential regulation bysugars of the components of the algal Gap gene system.Addition of glucose or other metabolizable sugars tophotoautotrophic cultures promoted a drastic repressionof the GapA gene and depletion to negligible levels of thecorresponding GAPDHA, a chloroplastic protein in-volved in photosynthetic CO2 assimilation. By contrast,expression of the GapC and GapN genes encoding theircytosolic counterparts involved in glycolysis was en-hanced. However, no down-regulation of the GapA geneby glucose took place in the dark, indicating that theobserved effect is associated with sugar assimilation inthe light. Likewise, glucose promoted in illuminated al-gal cultures a severe decrease of photosystem II func-tionality, estimated by O2 evolution activity,thermoluminescence emission and D1 protein level,while again, no effect was observed in the dark. On thebasis of the correlation found between photosystem IIperformance and sugar transcriptional regulation of theGapA gene, a scenario of sugar-mediated regulation ofphotosynthetic metabolism in microalgae is proposedthat will help to explain the so-called glucose bleachingeffect in photosynthetic eukaryotes.

Keywords GAPDH Æ Photoinhibition Æ Scenedesmusvacuolatus Æ Sugar gene regulation

Abbreviations BPGA: 1,3-Bisphosphoglycerate Æ G3P:Glyceraldehyde-3-phosphate Æ GAPDH:Glyceraldehyde-3-phosphate dehydrogenase Æ PGA: 3-Phosphoglycerate Æ Pi: Inorganic orthophosphate Æ PSI,II: Photosystem I, II Æ TL: Thermoluminescence

Introduction

Plants and Chlorophycean (green) algae (Viridiplantae)possess three different glyceraldehyde-3-phosphate de-hydrogenases (GAPDHs) that show specific cell com-partmentalization and are encoded by distinct nuclearGap genes (Mateos and Serrano 1992) (see Fig. 1). In thechloroplast stroma, the phosphorylating NAD(P)+ -dependent GAPDHA (EC 1.2.1.13), encoded by theGapA gene (Martin and Cerff 1986), plays a crucial rolein the Calvin cycle, producing triose-phosphates that areeventually exported to the cytosol. Although the algalenzyme is a homotetramer (A4), plants posseseses asecond subunit called GAPDHB (encoded by a GapBgene absent in algae). GAPDHB has a regulatory C-terminal extension, and both the homotetramer and theheterotetramer (A2B2) are found in the plant chloro-plast stroma (Cerff and Chambers 1979). The cytosolicNAD+ -dependent phosphorylating GAPDHC (EC1.2.1.12), encoded by the GapC gene (Martin and Cerff1986), participates mainly in carbohydrate catabolismand is involved, in association with phosphoglyceratekinase, in the first substrate-level phosphorylation ofglycolysis (Fothergill-Gilmore and Michels 1993; Plax-ton 1996). It has also a fundamental role in the glu-coneogenic pathway that seems to be essential in thecarbon metabolism of photosynthetic organisms, bothbacteria and eukaryotes (Plaxton 1996; Valverde et al.1999). A NAD(P)+ -dependent, non-phosphorylatingglyceraldehyde-3-phosphate dehydrogenase (GAPDHN,EC 1.2.1.9) is also present in the cytosol of photosyn-thetic eukaryotes, in both microalgae and plants (Igle-

F. Valverde Æ J. M. Ortega Æ M. Losada Æ A. Serrano (&)Instituto de Bioquımica Vegetal y Fotosıntesis,Universidad de Sevilla-CSIC, 41092 Seville, SpainE-mail: aurelio@cica.esTel.: +34-95-4489524Fax: +34-95-4460065

Planta (2005) 221: 937–952DOI 10.1007/s00425-005-1501-0

sias et al. 1987; Mateos and Serrano 1992), and in someGram-positive bacteria (Habenicht 1997). This enzyme,encoded by the GapN gene, is a member of the aldehydedehydrogenases superfamily, a protein class structurallyquite different from the phosphorylating GAPDHs(Habenicht et al. 1994). GAPDHN catalyzes the directand irreversible oxidation of glyceraldehyde-3-phos-phate (G3P) to 3-phosphoglycerate coupled to NADP+

reduction, with water instead of inorganic orthophos-phate (Pi) as the substrate, thus impeding phosphory-lation and the subsequent ATP synthesis (Serrano et al.1993). This apparently wasted chemical energy cannonetheless be used, as elegantly proposed, to drive ex-port of reducing power and protons from the chloro-plast stroma to the cytosol by a multi-protein systeminvolving GAPDHA, GAPDHN and the triose-phos-phate/Pi translocator of the inner chloroplast membrane(Serrano et al. 1991, 1993; Flugge 1998) (Fig. 1). Theexclusive presence in photosynthetic eukaryotes of threeparalogous GAPDHs with specific metabolic roles anddistinct cellular localization makes it an interesting sys-tem to study regulatory mechanisms.

The addition of sugars to photoautotrophic algalcultures promotes drastic physiological changes in thecorresponding cells (Hilgarth et al. 1991; Villarejo et al.1995). This effect, also called ‘glucose bleaching’ due tothe frequently observed decrease in chlorophyll content,may result in complete loss of the photosynthetic capa-bility of microalgae (Bishop 1961). A similar effect isobserved in higher plants after addition of an assimilablehexose (Yang et al. 1993; Shin-Lon et al. 2001). Mixo-trophically growing algal cultures show decreasedactivity levels of some chloroplastic enzymes (Goldsch-midt-Clermont 1986; Kindle 1987), mostly those in-volved in inorganic carbon assimilation. Scenedesmus

vacuolatus (formerly, Chlorella fusca) strain SAG 211-8bis a green microalga with a very versatile metabolism,able to grow efficiently under different trophic condi-tions, from photoautotrophy in a minimal medium tostrict heterotrophy in the dark using a range of metab-olizable sugars (Serrano et al. 1991). This behaviourmakes it an ideal organism for the study of the GAPDHsystem, as a sensor able to report the physiological sta-tus of the algal cell in the analysis of sugar-mediatedregulatory mechanisms. In this work, we state that thethree algal Gap genes respond differently to environ-mental signals promoted by changes in trophic condi-tions. Moreover, since a good correlation withphotosystem II (PSII) functionality has also been found,the GAPDH enzyme system may be of additionalinterest to investigate common control mechanisms inphotosynthetic light reactions and carbohydratemetabolism of phototrophic eukaryotes.

Materials and Methods

Media and organisms

Scenedesmus vacuolatus strain SAG 211-8b (Chloro-phyta, Chlorophyceae) was cultured at 25�C in liquidSueoka minimal medium (Sueoka et al. 1967) bubbledwith air supplemented with 2% (v/v) CO2. Photoauto-trophic and mixotrophic cultures were illuminated with aconstant 100 lE/m2 s fluorescent white light. To attainmixo- and hetero-trophic conditions, glucose was addedat 17 mM final concentration to photoautotrophicallygrowing cell cultures (10 lg/ml of chlorophyll). Othersugars and sugar-analogues were used at the same finalconcentration (Sigma-Aldrich, St. Louis, MO, USA).

Fig. 1 Scheme of the GAPDHsystem in a model plant or algalcell. The three GAPDHenzymes are situated in thecontext of their place andfunction: 1 CytosolicGAPDHC involved inglycolysis; 2 ChloroplasticGAPDHA(B) involved inCalvin-Benson cycle; 3Cytosolic GAPDHNrepresented bypassing thesubstrate-level phosphorylationstep of glycolisis. G3Pglyceraldehyde-3-phosphate,BPGA 1,3-bisphosphoglycerate,PGA 3-phosphoglycerate. Theclosed circle in the chloroplastmembrane represents the Pi/triose-phosphate translocator

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Growth was monitored by measuring both the chloro-phyll content, determined after cell extraction withmethanol (MacKinney 1941), and total protein, estimatedby a modification of the Lowry protocol (Lowry et al.1951). Other microalgae were grown photoautotrophi-cally in minimal media as previously described (Mateosand Serrano 1992), except Ochromonas danica SAG 933-7, which was cultured in a rich medium under dim light asrecommended by SAG (Sammlung von Algenkulturen,University of Gottingen, Germany). Escherichia colistrains DH5a and XLI-Blue MRF’ were cultured in LBsuppliedwhen neededwith ampicillin (100 lg/ll) either insolid (1.5% w/v agar) or liquid media. All algal strainsused in this work were obtained from SAG.

DNA and RNA techniques

Genomic DNA from S. vacuolatus and other algalspecies was isolated as described in Doyle and Doyle(1990). cDNA was made from total RNA preparationsof both autotrophic and mixotrophic algal cellsemploying an AMV reverse transcriptase (Gibco BRL,Gaithersburg, MD, USA) and an oligo-dT primer asdescribed by the manufacturer (Amersham Biosciences,Uppsala, Sweden, ‘Time Saver cDNA synthesis kit’).An expression library was constructed from RNA ex-tracted from photoautotrophic cells in Lambda IIphagemid vector and a Ready-to-go lambda packagingkit (Amersham) following the instructions of the man-ufacturer. Specific RT-PCR was performed usingdegenerate primers constructed from two highly con-served amino acid regions, NGFGRIG and WYD-NEWG, located near the N- and C-termini of allGAPDH proteins (gap4, forward, 5¢-AAT(C)G-GA(CGT)TTC(T)GGA (CGT)A(C)GA(G)ATA(CT)GGA(CGT)A(C)G-3¢, and gap2, reverse, 5¢-ACCATG(A)CTG(A)TTG(A)CTC(T)ACCCC-3¢) (Valverde et al.1997) that amplified ca. 95% of the coding sequence ofphosphorylating GAPDH encoding GapA and C genes(0.9 kb approx.), and primers NPCO1 (forward, 5¢-T(C)TA (CGT)GCA(CGT)CCA(CGT)CCA(CGT)TTC(T)AAC(T)-3¢) and NPCO2 (reverse, 5¢-TGGGAA(G)GAA(G)CCA(CGT)TTC(T)GGA(CGT)CC-3¢) thatamplified a DNA fragment of 0.7 kb comprising ca.50% of the coding sequence of GapN genes encodingnon-phosphorylating enzymes (Valverde et al. 1999).The amplified DNA fragments were cloned in p-GEMTvector (Promega, MD, USA) and sequenced in bothstrands. Southern blots in high or low stringency con-ditions were performed as described in Sambrook et al.(1989). Detection of Gap genes expression was carriedout by slot blot screenings in a manifold blotting unitemploying 20 lg of total RNA extracted from the sameculture samples used for the immunochemical assays(see below) and the specific probes of S. vacuolatus Gapgenes. Hybridization and washes were performed athigh stringency conditions (Ausubel et al. 1992), andthe filters were eventually analyzed and quantified in a

Cyclone PhosphorImage system (Packard Corp., PaloAlto, CA USA).

Phylogenetic analyses

Multiple sequence analyses of GAPDH protein regionscorresponding to the cDNA fragments described aboveand those already deposited in databases were performedwith the CLUSTAL X version 1.8 program (Thompsonet al. 1997). These alignments were used to constructdistance phylogenetic trees (Neighbour-Joining method,Kimura distance calculations). Bootstrap values com-puted with 1,000 replicates are shown. Similar tree con-figurations were obtained employing a maximumparsimony method with the program PROTPARS(PHYLIP package version 3.5c). The following phos-phorylating GAPDH sequences from databases (namedfrom its corresponding genes but in standard style) wereused: Anabaena variabilis gap1 (L07497) and gap2(L07498); Synechocystis sp. PCC 6803 gap1 (X86375)and gap2 (P49433); Arabidopsis thalianaGapA (P25856),GapB (P25857), GapC (P25858) and GapCp(NP_178071); Nicotiana tabacum GapA (P09043), GapB(P09044) and GapC (P09094); Chlamydomonas rein-hardtii GapA (P50362) and GapC (P49644); Euglenagracilis GapA (P21904) and GapC (P21903); Cyanidiumcaldarium GapC (AJ313315); Cyanophora paradoxaGapC (AJ313316); Chondrus crispusGapA (P34919) andGapC (P34920);O. danicaGapCp (AJ512344) and GapC(AJ512345);Guillardia thetaGapCp (U40032) and GapC(U39873); Pyrenomonas salina GapCp (U40033) andGapC (U39897); Monocercomonas sp. GapC(AAC63603). The accession numbers for the GAPDHNsequences are: Streptococcus pneumoniae (AAK99832);Streptococcus mutans (Q59931); Clostridium acetobutyli-cum (NC003030); A. thaliana (AY037205); Nicotianaplumbaginifolia (P93338); Pisum sativum (P81406);Oryzasativa (TC53162); Zea mays (Q43272). The partial C.reinhardtii putative GapN sequence was obtained fromthe Joint Genomic Institute (JGI, DOE, USA), genomeproject release v2.0. The sequences of S. vacuolatus Gapgenes studied in this work were submitted to databasesand assigned the accession numbers AJ252208 (GapA),AJ252209 (GapC) and AJ252211 (GapN).

Enzyme activity measurements and immunochemicaltechniques

Samples of 50 ml recovered every 12 h from S. vacuol-atus cultures growing under different trophic conditionswere collected, centrifuged, washed in Tris–HCl 50 mM(pH 7.5) and kept frozen at �20�C. Cell pellets wereresuspended at a 3 ml/g ratio in a rupture buffer sup-plemented with a proteases inhibitors cocktail (Valverdeet al. 1997), disrupted by ultrasonic treatment as de-scribed in Serrano et al. (1991) and centrifuged 15 min at40,000 g, the supernatant being considered the protein

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cell-free extract. Phosphorylating GAPDH activity wasassayed in these extracts as previously described, eitherby the anabolic reaction - reduction of NAD(P)+, or thecatabolic one-oxidation of NAD(P)H (Mateos andSerrano 1992). Non-phosphorylating NADP+ reductionby GAPDHN was assayed as described in Valverde et al.(1999). Immunochemical experiments with GAPDHproteins blotted on nitrocellulose membranes were doneby the Western blot technique after SDS-PAGE elec-trophoresis in 12% polyacrylamide slab gels, using asemidry transfer unit (Biometra, Gottingen, Germany)(Sambrook et al. 1989). The highly specific polyclonalantibodies employed were raised in rabbit against thepurified cyanobacterial homologous proteins to the algalGAPDHC and GAPDHA—GAPDH1 and GAPDH2,respectively—and pea GAPDHN and were describedelsewhere (Valverde et al. 1997, 1999). Antibodiesagainst the structural D1 protein of photosystem II fromSynechocystis sp. PCC 6803 and against-FNR fromAnabaena sp. PCC 7119 were generous gifts from Prof.Rafael Picorell (E.E. Aula Dei, CSIC, Zaragoza, Spain)and Prof. Carlos Gomez-Moreno (Zaragoza University,Spain), respectively. Light-dependent oxygen evolutionactivity of whole algal cells was determined polaro-graphically using a Clark-type oxygen electrode (YellowSprings Instruments Co. Inc., Yellow Springs, OH,USA) and saturating white light.

Analytical column chromatofocusing

Cell-free extracts from autotrophic or mixotrophic cul-tures of S. vacuolatus were obtained as described above.Extracts were fractioned with ammonium sulphate from40% to 80% saturation and the final product dialyzedagainst buffer Tris–HCl pH 8.3 25 mM; 10% glycerol;1 mM DTT; 0.5 mM EDTA; 1 mM PMSF; 10 mM b-mercaptoethanol, which was also the equilibration bufferfor an FPLC-based mono P HR 5/20 chromatofocusingcolumn (Amersham). 20 mg of protein from the dialyzedextracts were loaded in the column and eluted with thesame buffer plus 1/10 of polybuffer 9-6 (Amersham),adjusted to pH 6.8 with HCl, at a 0.5 ml/min flow rate inan AKTA purifier FPLC apparatus (Amersham). Afterchecking that no GAPDH activity was out of the pHrange in study, 0.5 ml fractions were collected, tested forthe different GAPDH activities and pH was determined.Immunoblots with different anti-GAPDH antibodieswere performed in the peak activity fractions. Isoelectricpoints were ascribed to the pH of the fraction thatshowed maximal activity and protein amount for eachGAPDH type. Experiments were repeated at least threetimes with independent starting material, and no signif-icant change in the data was observed.

Thermoluminescence measurements

Thermoluminescence (TL) glow curves of intact cellswere measured using a home-built set-up designed by

Dr. J.M. Ducruet (CEA, Orsay, France) (Miranda andDucruet 1995). The sample cuvette consisted of a hori-zontal chamber (2 cm diameter) with a copper film onthe bottom. A double-stage Peltier plate (model DT1089-14; Marlow Industries, Dallas, TX, USA) wasmounted below the chamber for temperature regulation.The Peltier element was cooled by a temperature-con-trolled bath . S. vacuolatus cells were adsorbed on a pieceof filter paper (0.45 lm, Whatman) that was pressedagainst the copper film. Samples were dark-incubatedfor 2 min at 20�C, cooled to 0�C and illuminated with asaturating single-turnover flash of white light (HeinzWalz GmbH, Effeltrich, Germany) through an optic fi-ber. Light emission was recorded while warming samplesfrom 0�C to 70�C at a heating rate of 0.5�C/s by using aHamamatsu (Shizuoka, Japan) H5701-50 photomulti-plier. Signal recording, temperature regulation and sig-nal analysis were performed using a program designedby Dr. J.M. Ducruet (Miranda and Ducruet 1995).

Results

Occurrence of three Gap genes encoding differentGAPDH proteins in unicellular microalgae: cloning andcharacterization of S. vacuolatus GapA, GapC and GapNgenes

In order to identify the genes that encode the differentcomponents of the Gap system in the microalga S. vac-uolatus, a number of PCR reactions employing degen-erate primers were performed (Materials and methods).In this manner we amplified different sequences fromgenomic DNA and cDNA isolated from this and otherdiverse algae cultured in autotrophic conditions (datanot shown). Sequencing of the amplified cDNA frag-ments showed that they were mixtures of both GapA andGapC genes from the different algae analyzed, as de-duced from the alignment of their predicted amino acidsequences with that of known algal and plant Gap genes(Fig. 2a). This way, we have been able to identify GapAand GapC genes not only from S. vacuolatus (Chloro-phyceae) but also from other microalgae such as C.paradoxa (Glaucocystophyceae), C. caldarium (Rhodo-phyceae) and O. danica (Chromophyceae), representingdifferent algal phylogenetic groups (Van den Hoek et al.1995). RT-PCR experiments with cDNA from mixo-trophic S. vacuolatus cells amplified a single GapC genefragment corresponding to the same one identified inautotrophy-grown algae. Further on, PCR experimentsemploying recombinant lambda DNA from an auto-trophic S. vacuolatus cDNA gene library and the samedegenerate primers, identified several sequences belong-ing to the same GapA and GapC genes described above.In all these experiments no other Gap gene of thephosphorylating type was amplified.

A similar approach was employed to clone GapNgenes from diverse microalgae using degenerate primersthat amplified a fragment of around 0.7 kb from geno-

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mic DNA or cDNA extracted from autotrophic cul-tures. A Southern hybridization employing a specificprobe from the GapN gene from P. sativum (Habenicht

et al. 1994) demonstrated that these fragments belongedto the non-phosphorylating Gap gene family (data notshown). In the case of S. vacuolatus the genomic frag-ment was cloned, sequenced and identified as the firstalgal GapN gene characterized to date, inferred fromcomparison of its predicted amino acid sequence toknown plant GAPDHNs (Fig. 2a). An incomplete par-tial sequence retrieved from the genomic informationfrom C. reinhardtii (http://www.genome.jgi-psf.org/chlre1/chlre1.home.html) has been also identified as aputative GapN gene by sequence homology. In the caseof S. vacuolatus, a band was also amplified from cDNAof mixotrophic cells, its sequence exactly matching thatof the genomic band described above, except for aninternal 300 bp region with clear canonical intron fea-tures that was present in the genomic sequence.

The partial amino acid sequence alignment of theregion around the active site shown in Fig. 2a, identifiesand classifies the first genes cloned from S. vacuolatus asa GapC gene and a GapA gene, deduced from the uniqueresidues where the predicted NAD+/NADP+ depen-dence resides. Plants and algae GAPDHA show a Serineresidue at position 188 (Bacillus stearothermophilusGAPDH numbering, highlighted in Fig. 2a) that deter-mines the preference of the protein towards NADP+

(Corbier et al. 1990), and defines a family of GAPDHAenzymes involved in the chloroplastic Calvin–Bensoncycle. By contrast, the presence of a Proline residue in

Fig. 2 Identification of Gap genes and GAPDH proteins in S.vacuolatus. a Sequence alignment of the three different GAPDHsfrom S. vacuolatus compared with homologous plant and algaegenes. An asterisk marks identical residues. The active sites areshadowed in both GAPDHA, C and GAPDHN, with the mainCysteine marked by an arrowhead. The Proline/Serine residues thatdetermine the NAD+ /NADP+ specificity are highlighted. AtA,AtC, AtN: GAPDHA, GAPDHC, GAPDHN from A. thaliana;CrA, CrC: GAPDHA, GAPDHC from C. reinhardtii; SvA, SvC,SvN: GAPDHA, GAPDHC, GAPDHN from S. vacuolatus; PsN:GAPDHN from P. sativum. b Immunoblots with specific poly-clonal antibodies against different GAPDH proteins of cell-freeextracts (soluble protein fraction, 50 lg) or purified proteins(0.5 lg) from different photosynthetic organisms. Abbreviationsare: Cr, C. reinhardtii; Cc, C. caldarium; Cp, C. paradoxa; Eg, E.gracilis; Pp, Porphyridium purpureum; Od, O. danica; Sv, S.vacuolatus; G1 and G2, purified Synechocystis PCC6803 GAPDH1and GAPDH2, respectively; Am, Anthirrinum majus; So, Spinaceaoleracea; Mb, Monoraphydium braunii; GN, purified GAPDHNfrom P. sativum; Sy, Synechocystis PCC6803. c Preparativechromatofocusing from pH 8.3–6.8 of a semi-purified proteinextract from S. vacuolatus grown in autotrophic conditions. Closedsquares and circles represent protein quantity and pH respectively.Dotted line: Phosphorylating NAD+-dependent activity; brokenline: Phosphorylating NAD(P)+-dependent activity; line with dot:Non-phosphorylating NADP+-dependent activity. The isoelectricpoint of each enzyme is indicated above each activity peak, as wellas immunoblots showing the number of the fractions where thethree GAPDHs were detected with specific antibodies

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this position defines a well-described GAPDHC proteinfamily absolutely dependent on NAD+ that participatesin the cytosolic glycolytic/gluconeogenic pathways. Onthe other hand, the deduced amino acid sequence of theGapN gene isolated from S. vacuolatus presented a highidentity match with two other GADPDHN from plantsaround the putative active site that includes the catalyticcysteine residue.

To further demonstrate that the Gap genes we hadcloned belonged to S. vacuolatus genome, we performedSouthern blots in high stringency conditions employingthe cDNA fragments identified earlier as specific probes,and genomic DNA of the microalga digested with sev-eral restriction enzymes (data not shown). The patternof hybridization of the detected bands suggested that thethree Gap genes are present in the S. vacuolatus genomeas single copy genes.

Figure 2b shows immunodetection of the threeGAPDH proteins encoded by the diverse Gap genes incell-free extracts from different microalgae, using highlyspecific antibodies. We have observed that the antibod-ies raised against the GAPDHA cyanobacterial homo-log GAPDH2, detected a unique GAPDHA band(upper panel) in extracts from green and red microalgacorresponding to the unique chloroplastic GAPDHpolypeptide present in these organisms (Martin andSchnarrenberger 1997). This antibody showed no cross-reaction with purified GAPDH1, the cyanobacterialhomolog of GAPDHC (G1, upper panel). Plant leavesextracts exhibited two bands corresponding to paralo-gous proteins, the GAPDHA (38 kDa) and the GAP-DHB (41 kDa) (Cerff and Chambers 1979; Martin andCerff 1986). By contrast, the antibody raised againstGAPDH1 immunodetected a unique band of 38 kDa inextracts from both microalgae and plants correspondingto a single GAPDHC subunit (middle panel). In a sim-ilar way, this antibody showed no cross-reaction withpurified GAPDH2 (G2, middle panel). Antibodies raisedagainst the pea GAPDHN detected single 50-kDa bandsin extracts from both plants and phylogenetically diversemicroalgae, but was absent in cyanobacterial extracts inaccordance with previous reports (Mateos and Serrano1992; Valverde et al. 1997) (Fig. 2b, bottom panel). Thisband was surprisingly absent in O. danica extracts thatalso showed no GAPDHN activity in the growing con-ditions used. Overall, the immunochemical data clearlydemonstrate the presence of the three GAPDH proteinsin the photosynthetic eukaryotes tested, both microalgaeand vascular plants, and confirm previous reports basedon enzymatic activity measurements (Iglesias et al. 1987;Mateos and Serrano 1992; Habenicht et al. 1994).

We further performed a FPLC column chromatofo-cusing of partially purified protein extracts from S.vacuolatus grown under autotrophic conditions, wherewe were able to resolve three distinct GAPDH activitiesas well-defined non-overlapping peaks with differentisoelectric points (Fig. 2c). A strictly NAD+ -dependentGAPDH activity peak eluted at pH 7.9, and cross-re-acted specifically with an anti-GAPDHC antibody

showing a molecular mass (Mm) around 37 kDa. This isa moderately basic GAPDHC similar to the one de-scribed for the GAPDHC of the photosynthetic protistC. paradoxa (Serrano and Loffelhardt 1994). A secondenzyme eluted at a more acidic pH (7.3) showing apreferential NADP+ - and moderate NAD+ -dependentactivity, which could be attributed to a GAPDHA. Infact, this enzyme was specifically recognized by immu-noblot employing anti-GAPDHA antibodies showing aband around 38 kDa. Finally, at a slightly acidic pH of6.7 a strictly NADP+ -dependent activity was eluted,which was inhibited by phosphate and thus could beascribed to the non-phosphorylating GAPDHN. Thisprotein showed by immunoblot a Mm of 50 kDa whendetected with anti-GAPDHN antibodies.

A similar chromatofocusing experiment employingpartially purified extracts from S. vacuolatus grown inmixotrophic conditions showed the same GAPDH-activity peak elution pattern of autotrophic conditions,with no significant change in the isoelectric points abovedescribed (data not shown). Noteworthly, the GAP-DHA peak showed a drastic decrease both by activityand immunoblot assays, a phenomenon that is furtherdescribed below.

On the basis of the immunoblot data describedabove, and since no other GAPDH activity was found inthe extracts, we conclude that the three activities foundcorrespond to the enzymes described previously. Allthese experiments showed that the GAPDH system in S.vacuolatus consists of three different GAPDHs withparticular specificity for the nicotinamide cofactor andthat we had probably cloned the cDNAs that expressedthem.

Fig. 3 Distance phylogenetic trees of GAPDHA, GAPDHC (a)and GAPDHN (b) proteins from photosynthetic eukaryoticorganisms, both algae and higher plants. The trees were obtainedfrom two multiple alignments (CLUSTAL X version 1.8 program)of phosphorylating and non-phosphorylating partial GAPDHamino acid sequences inferred from the corresponding Gap genes.Bootstrap values in the most significant nodes indicate a highstatistical support of the main associated groups. Scale barsindicate estimated differences in protein sequences (substitutionsper site). In a the GAPDHC subtree is marked in light grey whilethe photosynthetic GAPDHA subtree (that also includes itsparalogous GAPDHB) is marked in dark grey. In b the bacterialGAPDHN node is light grey while a dark-grey circle highlights theplant and algae subtree. The partial phosphorylating GAPDHsequences from the following species were used: Anabaena variabilisgap1 and gap2; Synechocystis sp. PCC 6803 gap1 and gap2;Arabidopsis thaliana GapA, GapB, GapC and GapCp; Nicotianatabacum GapA, GapB and GapC; Chlamydomonas reinhardtiiGapA and GapC; Euglena gracilis GapA and GapC; Cyanidiumcaldarium GapC; Cyanophora paradoxa GapC; Chondrus crispusGapA and GapC; Ochromonas danica GapCp and GapC;Guillardia theta GapCp and GapC; Pyrenomonas salina GapCpand GapC; Monocercomonas sp. GapC; Scenedesmus vacuolatusGapA and GapC. The partial GAPDHN sequences from thefollowing species were used: Streptococcus pneumoniae; Streptococ-cus mutans; Clostridium acetobutylicum; Chlamydomonas rein-hardtii; Arabidopsis thaliana; Nicotiana plumbaginifolia; Pisumsativum; Oryza sativa; Zea mays and Scenedesmus vacuolatus

c

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On the other hand, the partial amino acid sequencededuced from the putative GapA cDNA clustered in adistance phylogenetic tree with those from other chlo-roplastic GAPDHs of plants and algae and their cy-anobacterial gap2-encoded homologous GAPDH2proteins (Fig. 3a, dark-grey circle), and could beundoubtedly identified as a GAPDHA. It showed thehighest similarity to the amino acid sequence of theGAPDHA from C. reinhardtii, deduced from the onlyother GapA gene from a green microalga sequenced to

date. The paralogous GapA and GapB genes from plantswere more closely related to these two green microalgalgenes than to GapA genes from thallophytic red algae(C. crispus) or the distantly related photosynthetic pro-tist E. gracilis. Similarly, the deduced amino acid se-quence of the putative GapC gene from S. vacuolatusclustered with other GAPDHC sequences of photosyn-thetic organisms (plant and algal GAPDHC proteinsand their cyanobacterial gap-1 encoded GAPDH1 ho-mologs), in a branch that also includes the C. reinhardtii

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protein (Fig. 3a, light-grey circle). Again, the S. vacu-olatus sequence appeared closer to sequences fromhomologous plant GapC genes than to those of E.gracilis and other photosynthetic protists.

The two algal GapC genes cloned from C. caldariumand C. paradoxa clustered together with the thallophyticRhodophycean C. crispus with a high bootstrap value,consistently with their postulated common evolutionaryorigin (Martin and Schnarrenberger 1997). A particularcase is the GapCp gene encoding the plastidic GAPDHof O. danica—a Chromophycean protist with a complexplastid generated by secondary endosymbiosis—whichprobably replaced a former chloroplastic GapA gene fora GapC-like gene of eukaryotic origin. Its product be-longs to a recently described plastidic GAPDHC class,the so-called GAPDHCp proteins proper of protistswith complex plastids, which acquired the Pro fi Seramino acid change specific for GAPDHA-type proteinsand became chloroplast NAD(P)+ -dependent enzymesinvolved in the Calvin–Benson cycle (Liaud et al. 1997).In the tree of Fig. 3a, the O. danica GAPDHCp bran-ches with other GAPDHCp proteins encoded by theGapCp genes from Cryptophycean protists (G. theta, P.salina). The other typical GapC gene found in O. danicaencoded a protein that grouped together with the GapCgene products of G. theta and P. salina—also membersof the Heterokonts clade—all of them encoding glyco-lytic GAPDHC proteins (Liaud et al. 1997).

This tree also includes a sequence from A. thalianathat belongs to the recently described plant GAPDHCpclass, strictly NAD+ -dependent enzymes present in theplastid stroma (Petersen et al. 2003). However, this novelglycolytic GAPDH was not found in S. vacuolatus,neither was any GapCp gene of this kind amplified inPCR reactions using genomic DNA or cDNA of thedifferent microalgae tested. Furthermore, there is noGapCp gene present in the almost complete genome se-quence of the green alga C. reinhardtii. In the phyloge-netic tree, plant GapCp shows a closer relation withplant GapC than with microalgal GapCp (Fig. 3a). Thesedata support the proposal that glycolytic plastidic Gap-Cp genes originated by GapC gene duplication late inevolution, in the land plants (Petersen et al. 2003).

Figure 3b shows a distance phylogenetic tree basedon an alignment of the S. vacuolatus GAPDHN se-quence with homologous protein sequences from plantsand Gram-positive bacteria. The S. vacuolatus GAP-DHN branches deeply in the green lineage subtree(dark-grey circle) with the rather compact clade ofhigher plant homologues, and is distinctly separatedfrom the bacterial subtree (light-grey circle). The partialamino acid sequence of a putative GAPDHN from theinformation available in the C. reinhardtii genome alsoclustered with plant GAPDHNs, supporting a commonevolutionary origin of the GapN genes from all photo-synthetic eukaryotes. The absence of this gene fromcyanobacterial genomes and the wide distributionof GAPDHN in algae, plants and Gram-positive bac-teria (Habenicht 1997; and this work) suggest possible

horizontal gene transfer and/or cryptic endosymbioticevents in algae similar to those earlier proposed for otherGap genes (Martin et al. 1993).

Transcriptional regulation by trophic conditionsof the Gap gene system in S. vacuolatus

Our interest was mainly centred on the long-term regu-lation of this system as affected by light and the presenceof a metabolizable sugar. Experimentally, it was fol-lowed by measuring the activity, protein and transcriptlevels of the three algal G3P dehydrogenases in batchcultures grown in minimal medium under continuouslight along 80 h periods. Growth curves estimated byboth cell chlorophyll and total protein contents showedparallel increases during the exponential phase and thebeginning of the stationary phase until growth stabil-ization at the stationary phase (Fig. 4a). Furthergrowth, up to 1 week, produced a gradual slow decreaseof both parameters, presumably due to the consumptionof nutrients (not shown). Specific activity of the threeGAPDHs showed distinctive changes with time(Fig. 4b). After increasing during the exponential phaseall the enzymes reached peak values at the end of thisgrowth phase, but, whereas the levels of the cytosolicGAPDHC and GAPDHN exhibited a further progres-sive increase during the stationary phase, the plastidic

Fig. 4 Evolution with time of specific activity of the threeGAPDHs in S. vacuolatus photoautotrophic batch cultures.a Protein and chlorophyll content along the course of theexperiment. Both parameters define an initial lag phase, anexponential phase of maximum growth rate and a final stationaryphase in which no net growth is observed. Data are from a typicalexperiment. b Time course of the three GAPDH specific activitiesdetermined in whole cell extracts from samples collected every 12 h.Data are means ± SE of at least three independent determinations

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GAPDHA remained stable or even decreased somewhat.The protein amount detected by immunoblot followed asimilar pattern (data not shown). It should be noted thatin all cases the specific activity levels of both GAPDHAand GAPDHC were three to five times higher than thoseof GAPDHN.

The above-described patterns drastically changedwhen glucose was added to exponentially growingautotrophic algal cultures, subsequently maintainedunder such mixotrophic conditions. As shown in theupper panels of Fig. 5a, under these conditions thechlorophyll levels ceased to increase ca. 24 h after sugaraddition and eventually decreased. This chlorophyll loss,caused by the addition of glucose (or other assimilablecarbon substrate) to cells or plants growing photoauto-trophically, has been described in many other photo-synthetic organisms. It has been generically named‘glucose bleaching¢ because often the gradual decrease inchlorophyll content causes a complete loss of the pig-ment, making the cells chlorotic (Bishop 1961; Villarejoet al. 1995). This outermost event does not seem tohappen in S. vacuolatus because we have observed thateven in the late exponential phase of mixotrophicgrowth, the algal cells still retain a substantial amount ofits original chlorophyll content. Although this effecttakes place concurrently with several other physiologicalchanges, such as cell enlargement, starch accumulationand loss of photosynthetic capability, the nature of theprocess is so far obscure (Jang and Sheen 1997; Shin-Lon et al. 2001). The three algal GAPDH enzymes re-acted differently to the presence of glucose in the culturemedium. Whereas the two cytosolic GAPDHs increasedtheir specific activities, the plastidic GAPDHA droppedto marginal levels at the latter stages of culture devel-opment (Fig. 5a). Immunoblots revealed that this de-crease in activity was parallel to a dramatic fall of theGAPDHA protein, while both GAPDHC and GAP-DHN showed significant increases.

To further characterize the sugar effect on theGAPDH system, S. vacuolatus photoautotrophic cul-tures in the exponential phase were shifted to darknessimmediately after glucose addition and then allowed togrow under heterotrophic conditions (Fig. 5b). Thisfigure compares growth rates, GAPDH activity andprotein levels of a heterotrophic culture with anothermaintained in the dark without sugar. The dark culturepromptly stopped growth in the absence of any assimi-lable substrate, as evidenced by the quick stabilization ofboth chlorophyll and total protein levels (Fig. 5b),ending eventually in culture death. Growth of hetero-trophic cultures took place in the dark, but both totalprotein and chlorophyll content were lower than in thephotoautotrophic cultures. On the other hand, afterseveral weeks under strict heterotrophic conditions, cellsremained viable for photoautotrophic growth. Theseresults suggest a better adaptation of S. vacuolatus tophotoautotrophic conditions, presumably prevalent inits natural oligotrophic environment.

The reaction of the GAPDH system to the presenceof glucose in S. vacuolatus heterotrophic (dark) cells(Fig. 5b) changes significantly from that in autotrophic(illuminated) ones. After cell transfer to the dark in theabsence of glucose the specific activity and protein levelsof all three GAPDHs decreased, although still retainingsignificant levels after 4 days starvation, which probablyreflects a relevant role in basal metabolism. Noteworthy,and differing from its cytosolic counterparts, chloro-plastic GAPDHA activity, protein and transcript levels(see below) remained comparatively high in heterotro-phic dark cultures even in the absence of sugar. Theseresults suggest a potential function for the algal GAP-DHA enzyme in a possible heterotrophic carbonmetabolism of the chloroplast—a sort of ‘‘chloroplasticglycolysis’’—something that had been previously pro-posed but not much investigated (Plaxton 1996). Incontrast, the levels of cytosolic GAPDHs increasedsteeply under heterotrophic conditions and reachedvalues similar to those of mixotrophic cultures and evenhigher than those of autotrophic ones (Fig. 5a), butdecreased steeply in the absence of sugar (Fig. 5b). Fi-nally, when comparing Fig. 5a and 5b, it can be ob-served that although retaining similar protein amounts,GAPDHA activity was lower in heterotrophy than inautotrophy. Although light-driven activation of keyenzymes of chloroplast carbon metabolism in microal-gae is somehow contradictory (Ocheretina et al. 2000;Tamoi et al. 2001) this effect may be explained by apossible posttranscriptional modulation by redox-med-iated protein–protein interaction, as is the case for the C.reinhardtii GAPDHA (Lebreton et al. 2003).

Total RNA was extracted from cells of S. vacuolatuscultures at different times before and after addition ofglucose, and a slot blot was carried out to check Gapgene expression using the GapA, GapC and GapN cDNAclones reported in this work as radiolabelled probes. Theblots were then quantified for the GapA, GapC andGapN signals and the resulting values normalized to atubulin control (Fig. 6a). This experiment showed thatin the long-term regulation, the transcript levels fol-lowed roughly the same temporal patterns of change asthe specific activity and protein levels. Because tran-script, protein and activity behaved similarly, the long-term regulation of S. vacuolatus GAPDHs seems to bepreferentially exerted at the transcriptional level.

To investigate whether the decrease of the GAPDHAprotein level after glucose addition was readily revers-ible, mixotrophic cells were shifted back to autotrophicconditions by removing the sugar (Fig. 6b). This allowedrecovery of GAPDHA to almost normal levels after24 h. Significantly, re-addition of glucose at this pointinduced a protein drop even faster and more severe(Fig. 6b, bottom). This may indicate an adaptative re-sponse of algal cells to the quickly changing environ-mental conditions, with periods of scarce nutrientavailability and others of lavish sugar abundance thatare common in their natural aquatic environments.

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Like many other Chlorophycean microalgae, S. vac-uolatus is able to use a wide range of carbohydrates asorganic carbon sources. In an attempt to better char-acterize the mechanism of sugar-mediated Gap generegulation in this microalga, the effect of differentassimilable sugars on the GAPDHA protein levels wastested by immunoblot experiments (Fig. 7, left-sidepanels). D-galactose and D-mannose produced a strongrepression of GAPDHA, similar to that observed withD-glucose, as was also the case with D-ribose, and su-crose (data not shown). Non-metabolizable sugar ana-logs, widely used as a tool to study sugar sensing inhigher plants (Jang and Sheen 1997), were also tested(Fig. 7, right-side panels). L-glucose (not transportedinto the cells) and 3-O-methyl-D-glucose (transportedbut not phosphorylated) did not trigger GAPDHArepression, whereas 2-deoxy-D-glucose (phosphorylatedbut not further metabolized), a toxic compound for thealga that severely reduced its growth rate in the light,

produced a partial repression. Since the common featureto all sugar effectors that efficiently promote GAPDHArepression is phosphorylation by the corresponding ki-nase (hexokinase, glucokinase or galactokinase), a‘‘sensing kinase’’ signal, similar to that reported forhigher plants (Jang and Sheen 1997; Smeekens 1998),might likewise participate in the mechanism that triggerssugar control in the S. vacuolatus GAPDH system.These results also suggest an early evolutionary originfor this signal transduction mechanism through thegreen evolutionary lineage.

Sugar-mediated changes in photosyntheticparameters of S. vacuolatus cells

Many genes encoding components of the photosyntheticapparatus are sugar-repressible, but the phenomenon isso far well documented only for higher plants and the

Fig. 5 Comparison of GAPDHactivity and protein levelsduring different growingconditions of S. vacuolatusbatch cultures. a Activity andprotein levels of GAPDHs inauxotrophy (control) andmixotrophy (+G). Glucose(17 mM) was added to one oftwo parallel cultures aftergrowing autotrophically for24 h (white background). Bothcontrol and mixotrophiccultures were further grown(grey background) for 4 daysuntil late stationary phase withsamples taken every 12 h. Dataare from a representativeexperiment. Above, growthcurves estimated by chlorophylland total protein contents.Below, levels of GAPDHsspecific activities with time.Immunoblots are presentedabove each graphic. Note themultiple but very close bandsobserved for GAPDHC.b Activity and protein levels ofGAPDHs in S. vacuolatus batchcultures in the dark with (+G)or without glucose (-G). Theexperiment is similar to thatshown in a but after glucoseaddition to one of the cultures(grey background) both werekept in the dark, as indicated bythe bars above each set ofgraphics. Above, chlorophylland protein content. Below,GAPDH activity measurements

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flagellated protist E. gracilis (Pego et al. 2000; Price et al.2004). Therefore, we considered it relevant to comple-ment the study of the regulation by trophic conditions ofthe S. vacuolatus Gap genes with the analysis of theconcomitant effect exerted on several photosyntheticparameters and protein components of the photosyn-thetic apparatus.

In order to test the functional state of the PSII invivo, we employed the TL technique, an outburst oflight emission occurring at characteristic temperatureswhen some materials preilluminated at low temperaturesare warmed gradually in the darkness. In plant materi-als, the outburst of light occurs at several temperaturesand it has been firmly established that most of these TLcomponents arise from the reversal of light-driven

charge separations in PSII reaction centres. TL emis-sions are due to the thermally activated recombinationbetween the positive charges accumulated in the inter-mediates of the water oxidation system on its donor side,and the negative charges stabilized on primary (QA) orsecondary (QB) quinone acceptors on its acceptor side.TL can be used (for methodological reviews see Mirandaand Ducruet 1995; Ducruet 2003) therefore, as a simplebut effective probe of both the donor and acceptorchemistry in PSII, since many of the emission bandshave been assigned to the specific charge pairs involved.Figure 8a shows the TL glow curves of S. vacuolatuscells cultured under different trophic conditions. Uponexcitation with a single saturating flash, the PSII ofautotrophic cells exhibited a TL band peaking at anemission temperature of around 30�C (L curve). Thisband has been assigned to the B-band originating fromthe processes of charge recombination between the sec-ondary quinone acceptor QB and the S2 (B2-band) andS3 (B1-band) states of the manganese cluster of the wateroxidation complex, and indicates a PSII reaction centerin optimal capability. In our system, a severe reductionof the TL B-band was observed in mixotrophic cells(48 h after glucose addition) when compared withautotrophic cells (cf. L and L+G curves). This changestrongly suggests a PSII inactivation after sugar additionin the light that leads to a drastic reduction of thephotosynthetic linear electron flow. Moreover, the TLcurve of heterotrophic algal cells (grown in the dark withglucose, D+G curve) showed a conspicuous B-band,indicative of a fully operational PSII. In dark-main-tained (starved) cells, however, the TL curve showed atwo-peak profile (D curve) with emission maximum at20�C (B1-band component of the B-band) and 43�C(afterglow emission band). The afterglow light emissionband seems to reflect a back-flow of electrons from re-ductants present in the stroma to the quinonic acceptorsof PSII, allowing their recombination with the S2 and S3states, and it has been proposed that it is related with

Fig. 6 a Regulation of Gap genes expression in S. vacuolatusautotrophic and mixotrophic batch cultures. An autotrophic algalculture in exponential phase was split in two, and glucose 17 mMwas added to one of them (+G). This point was considered thetime zero of the experiment. RNA samples were spotted on nylonmembranes and hybridized with specific probes for the differentGap genes and the C. reinhardtii b 2-tubulin gene as a loadingcontrol. Transcript levels of the Gap genes were quantified,normalized with the control and plotted in a bar graphic.b Recovery of the GAPDHA protein level after sugar removal fromS. vacuolatus culture. The panels show the detection of GAPDHAbyimmunoblot in protein extracts sampled every 12 h from a pair ofparallel autotrophic cultures where glucose was added at the timeindicated by the first arrow. After 36 h of mixotrophic growth, thesugar was removed as indicated by the asterisk and eventually re-added after a 24 h interval (second arrow) to only one of the cultures(below). The figure shows a representative experiment

Fig. 7 Effect of different sugars and sugar analogues on theGAPDHA levels in S. vacuolatus cells growing in the light.Immnunoblot analyses employing GAPDHA antibodies wereperformed on protein extracts from parallel cultures grownautotrophically (�) or 72 h after sugar/sugar analogue addition(+). Concentrations of the effectors (indicated above each panel)were in all cases equivalent to 0.5% (w/v)

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cyclic electron flow and/or chlororespiration (Ducruet2003).

Addition of glucose in the light promoted a sub-stantial decrease of the chlorophyll content of S. vacu-olatus cells, the so-called glucose-bleaching, previouslyreported for green algae (Bishop 1961; Serrano et al.1991). This effect was illustrated by a significant intensitydecrease of the absorbance band around 685 nm—theabsorbance peak in the red range of the main chlorophylla of PSII—in the UV-visible absorption spectrum ofmixotrophic cells (Fig. 8b). Chlorophyll levels, however,appeared unchanged in dark-maintained cells withoutglucose. The correlation between the TL curves and thechlorophyll content of mixotrophic cells would point to aspecific degradation of PSII upon the addition of glucosein the light. Both TL and chlorophyll content data

suggested that in the dark with or without glucose PSIIwas functional. Besides, when the light-dependent O2

evolution—a parameter indicative of the linear PSII-dependent photosynthetic electron flow—was tested inthe cell suspensions, a pattern clearly consistent with TLresults emerged (Fig. 8c): high rates were displayed byautotrophic, heterotrophic and dark-maintained cellswhereas mixotrophic cells exhibited a severe reduction ofthis activity. No significant changes were observed in theO2 uptake rate, which was low in all cell types. Since highrates of O2 evolution are indicative of efficient light-in-duced water splitting by PSII, it is obvious that bothautotrophic and heterotrophic cells possess a fully func-tional PSII that, on the other hand, is not negativelyaltered in dark-maintained cells. Overall, these photo-synthetic parameters point to a specific inactivation ofPSII in mixotrophic cells.

The levels of two relevant proteins directly involvedin photosynthetic processes were then tested by immu-noblot analyses in S. vacuolatus cell extracts and corre-lated with those of the GAPDHs (Fig. 9). Both the D1protein, one of the two core structural components ofPSII, and ferredoxin-NADP reductase (FNR), anextrinsic photosystem I (PSI)-associated electron-trans-fer flavoprotein, were immunodetected and quantifiedusing specific polyclonal antibodies during transition ofalgal cells from autotrophic to mixotrophic conditions(Fig. 9a). The D1 protein levels quickly decreased inmixotrophic cells, in agreement with reports for higherplants (Smeekens 1998), whereas the FNR protein levelsclearly increased. The sugar-mediated decrease of the D1protein would eventually reduce the number of func-

Fig. 8 Functional state analysis of photosystem II in S. vacuolatuscells grown under different trophic conditions. a Thermolumines-cence (TL) glow curves of S. vacuolatus cells after 72-h-growth indifferent conditions: D+G= dark with glucose; D= dark; L=light; L+G= light with glucose. b Absorption band in the redregion of the visible spectrum characteristic of the chlorophyll apigments associated with PSII in S. vacuolatus cells grown as in (a).The interval wavelength between 650 nm and 700 nm of the totalUV-visible absorption spectra (250–800 nm, normalized to zeroabsorbance at 720 nm) is shown. c Photosynthetic activity of PSIImeasured as light-dependent O2 evolution. Oxygen evolution wasmeasured polarographically in intact cells (50 lg of chlorophyllml�1) from 60-h-old cultures after 5 min adaptation to light. Cellswere then kept in darkness, and the rate of O2 consumptionmeasured after a brief lag time. Data is given in micromoles ofoxygen liberated/consumed per milligram of chlorophyll per hourand are the mean ± SE of four independent experiments

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tional PSII reaction centres, which are subjected to highturnover under illumination, while the up-regulation ofFNR could lead to an increase in PSI-associated elec-tron flow. In the same experiments the GAPDH systembehaved as described above (see Fig. 5a). In this sense,the GAPDHA suffered a marked decrease, in parallel tothe D1 protein reduction, reflecting a possible shutdownof the Calvin–Benson cycle and decrease in the linearphotosynthesis from PSII to PSI. On the other hand,GAPDHC and GAPDHN protein levels increased likethose of FNR, indicating a shift to optimize energyproduction from an abundant reduced compound (glu-cose) through glycolysis and cyclic photophosphoryla-tion around PSI. That is, anabolism is inhibited andcatabolism is enhanced. As shown in Fig. 9b, TL mea-surements in S. vacuolatus autotrophic and mixotrophiccells confirmed the above interpretation. While the TL

B-band signal intensity remained high in control auto-trophic cells, a dramatic decrease was observed verysoon after the addition of glucose, its complete disap-pearance being maintained in subsequent samples. Thisresult is once more indicative of a PSII inactivation byglucose that persisted as long as the sugar remained inthe culture.

Discussion

The enzymes of the GAPDH system perform key rolesin widely divergent and diametrically opposed pathwaysof carbon metabolism: while the chloroplastic GAP-DHA is involved in the Calvin–Benson cycle, the twocytosolic GAPDHC and GAPDHN participate in gly-colysis/gluconeogenesis (Plaxton 1996; Valverde et al.1999). Due to their key metabolic roles, studies on theregulation of the different GAPDH proteins at the genelevel or at the posttranslational modification or activa-tion in both algal and plant cells by different signals suchas the circadian-clock (Fagan et al. 1998), light-gener-ated reducing power (Li et al. 1997; Backhausen et al.1998), stress (Yang et al. 1993) and developmentalprocesses (Serrano et al. 1991; Valverde et al. 1997) arefrequent in the literature. Nevertheless, the regulation ofthe system as a whole, and concerted with the metaboliccondition of the cell, has only been reported before in acyanobacterium (Koksharova et al. 1998) and never inphotosynthetic eukaryotes. In this work, three Gap genesand the distinct GAPDHs that they encode have beenidentified and characterized in the microalga S. vacuol-atus. Moreover, the occurrence in this green alga of a

Fig. 9 Comparative evolution with time of several molecularindicators associated with photosynthesis in S. vacuolatus auto-trophic and mixotrophic cultures. a Samples were collected at theindicated times from two parallel cultures (glucose was added toone of them after 24 h of initial photoautotrophic growth, verticalline) and probed in immunoblots for FNR and D1 proteins.Samples were also probed for GAPDHA, GAPDHC and GAP-DHN (see same data Fig 5a). In each case the value of theimmunodetected band was quantified and normalized with tubulinas an internal control signal and bar diagrams are shown (relativeunits referred to the maximum value as 100% for each graphic). Arepresentative experiment is shown. b Thermoluminescence mea-surements of the PSII functional state in each sample as in (a). Thebar diagram represents values of thermoluminescence peak emis-sion referred to the maximum relative value (24 h) as 100%. Notethat 48 h after glucose addition the PSII signal completelydisappeared. Light bars, control autotrophic culture (�G); greybars, mixotrophic culture with glucose (+G)

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regulatory mechanism and sensor system for the coor-dinate function of these three enzymes has been un-veiled. Higher plants are able to adapt theirphotosynthetic metabolism to different nutritional con-ditions, a number of studies having reported that sugarsare key effectors that either repress the expression ofgenes encoding photosynthetic proteins or induce othersinvolved in carbohydrate catabolism and storage(Smeekens 1998; Rolland et al. 2002). In this work, wepresent the first coordinate study on sugar and light-mediated regulation of the GAPDH enzymes and someprotein markers of photosynthetic processes in a greenmicroalga. Based on the results obtained, an attempt hasbeen made to explain the molecular processes that trig-ger the changes in the GAPDH system in concert withthese photosynthetic markers under different trophicconditions.

During mixotrophic growth, a severe drop in somephotosynthetic activity parameters associated with PSII(oxygen evolution and thermoluminescence) and thedrastic decrease in the structural protein D1, wouldindicate a repression of PSII functionality triggered bysugar signalling. At the same time, the increase in FNRprotein may be related to the activation of PSI-associ-ated electron flow by the same signal (Heldt 1997). Inthis metabolic scenario, where anabolism is sloweddown, GAPDHA is dispensable and its transcription isdrastically cut, as part of a well-known general Calvincycle shutdown (Pego et al. 2000; Rolland et al. 2002). Incontrast, the two cytosolic GAPDHs are maintained andeven somewhat up-regulated to maximize catabolism ofthe excess sugar through glycolysis and related catabolicpathways.

We could explain some of our results as part of ashort-term response, which implies a light-dependentredox signal in the regulation of enzymes involved inchloroplast carbon metabolism similar to that occurringin plants (Shih and Goodman 1988), but in microalgaethis point is under debate (Ocheretina et al. 2000; Tamoiet al. 2001). Nevertheless, recent reports on the regula-tion of GAPDHA in C. reinhardtii by the small regula-tory peptide CP12, which is also present in plants, andits ability to link to phosphoribulokinase in a redox-dependent modulation (Lebreton et al. 2003) calls for aspecific regulation of GAPDHA in algae. Furthermore,the recent implication of a redox signal in the regulationof 14-3-3 proteins, and through them of GAPDH andcertain other enzymes involved in glucose metabolism,has been recently reported (Cotelle et al. 2000). On theother hand, chloroplastic GAPDH in algae might berepressed in the long term by a general mechanism,similar to that reported in plants to reduce levels of abroad spectrum of enzymes after addition of sucrose orglucose (Smeekens 1998; Rolland et al. 2002). Our re-sults on the repression of GAPDHA by sugar analogssuggest that the phosphorylation step by sugar kinases isnecessary to trigger the signal transduction thatrepresses photosynthesis-linked genes, but the fact thatrepression was not observed in pure heterotrophic

conditions points to a mechanism that depends on sugarmetabolization in the light. It is tempting to hypothesizethat a similar mechanism could be at least partly in-volved in the up-regulation of the cytosolic GAPDHs.Indeed, an increase in GapC mRNA levels in bothChlorella and Arabidopsis cells after glucose addition hasbeen reported (Hilgarth et al. 1991; Shin-Lon et al. 2001)and up-regulation of GapC by light was found inmicroarray and differential display experiments com-paring gene expression profiles in plants (Ma et al.2001). In this respect, we have analyzed the promoters ofthe components of the Gap gene system in A. thaliana(GapA, GapB, GapC and GapN) and C. reinhardtii(GapA, GapC and GapN) finding in both organisms clearand repeated cis-elements similar to those reported forother genes regulated by sugars (reviewed in Rollandet al. 2002), including elements analogous to E- and G-boxes, suspected to be activated both by sugars andlight.

Mechanisms involved in sugar regulation of geneexpression in photosynthetic organisms are complex andinclude both a signal dependent on the sugar and asecond one dependent on its metabolization (Mooreet al. 2003). Our study on the mechanisms that regulatethe GAPDH enzymes of a unicellular microalga hasshown that their regulation correlates with changes incertain parameters associated with photosynthetic effi-ciency. Moreover, it has been shown that this signaldepends on sugar metabolization in the presence of lightand therefore may reflect part of a more general mech-anism designed to achieve a coordinate gene expression.Such precise gene regulation could be needed to opti-mize energy resources in the changing environmentalconditions of the natural habitats proper to photosyn-thetic eukaryotes.

Acknowledgments The authors gratefully thank Dr. Carolyn Sil-flow (University of Minnesota, MN, USA), Prof. Rafael Picorell(Estacion Experimental Aula Dei, CSIC, Zaragoza, Spain) andProf. Carlos Gomez-Moreno (University of Zaragoza, Spain) forkindly providing the b 2-tubulin cDNA clone of C. reinhardtii andantibodies against D1 and FNR proteins, respectively. This workwas supported by Grants PB-97/1135, BMC2001-563 from MCYT(Spanish Government); Grupo CVI-261 of III-PAI (AndalusianRegional Government) and EU project contract MERG-CT-2004-505303.

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