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First published online June 28, 2007; 10.1104/pp.107.103341 Plant Physiology 144:1946-1959 (2007) © 2007 American Society of Plant Biologists Long-Term Response toward Inorganic Carbon Limitation in Wild Type and Glycolate Turnover Mutants of the Cyanobacterium Synechocystis sp. Strain PCC 68031,[W]Universität Rostock, Institut für Biowissenschaften, Abteilung Pflanzenphysiologie (M.E., H.B., M.H.) und Ökologie (H.S.), D–18059 Rostock, Germany; University of Amsterdam, Institute for Biodiversity and Ecosystem Dynamics, NL–1018WS Amsterdam, The Netherlands (E.A.v.W., H.C.P.M.); Universität Rostock, Institut für Pathologie, Elektronenmikroskopisches Zentrum, D–18055 Rostock, Germany (L.J.); Netherlands Institute of Ecology, Centre for Limnology, Department of Foodweb Studies, NL–3631AC Nieuwersluis, The Netherlands (B.W.I.); and Eawag, Swiss Federal Institute of Aquatic Sciences and Technology, Centre of Ecology, Evolution, and Biogeochemistry, CH–6047 Kastanienbaum, Switzerland (B.W.I.)
Concerted changes in the transcriptional pattern and physiological traits that result from long-term (here defined as up to 24 h) limitation of inorganic carbon (Ci) have been investigated for the cyanobacterium Synechocystis sp. strain PCC 6803. Results from reverse transcription-polymerase chain reaction and genome-wide DNA microarray analyses indicated stable up-regulation of genes for inducible CO2 and HCO3– uptake systems and of the rfb cluster that encodes enzymes involved in outer cell wall polysaccharide synthesis. Coordinated up-regulation of photosystem I genes was further found and supported by a higher photosystem I content and activity under low Ci (LC) conditions. Bacterial-type glycerate pathway genes were induced by LC conditions, in contrast to the genes for the plant-like photorespiratory C2 cycle. Down-regulation was observed for nitrate assimilation genes and surprisingly also for almost all carboxysomal proteins. However, for the latter the observed elongation of the half-life time of the large subunit of Rubisco protein may render compensation. Mutants defective in glycolate turnover (  glcD and gcvT) showed some transcriptional changes under high Ci conditions that are characteristic for LC conditions in wild-type cells, like a modest down-regulation of carboxysomal genes. Properties under LC conditions were comparable to LC wild type, including the strong response of genes encoding inducible high-affinity Ci uptake systems. Electron microscopy revealed a conspicuous increase in number of carboxysomes per cell in mutant glcD already under high Ci conditions. These data indicate that an increased level of photorespiratory intermediates may affect carboxysomal components but does not intervene with the expression of majority of LC inducible genes.
Growth of photoautotrophic organisms is often limited by the amount of available inorganic carbon (Ci). In aquatic systems, Ci is available as HCO3–, CO2, or both depending on the pH. Cyanobacteria face the challenge of a rather low affinity of Rubisco toward CO2 and constant fluctuations in Ci level by the development of a CO2 concentrating mechanism (CCM; for review, see Kaplan and Reinhold, 1999  ; Badger et al., 2006). The CCM consists mainly of two components: the carboxysome and high-affinity CO2 or HCO3– uptake systems. The carboxysome is an intracellular polyhedral inclusion body, which is surrounded by a unilamellar protein shell (Cannon et al., 2001; Kerfeld et al., 2005). Inside the carboxysome, the CO2-fixing enzyme Rubisco is concentrated together with carbonic anhydrase, which converts intracellularly accumulated HCO3– into CO2 as the substrate for Rubisco. The cytoplasmic HCO3– pool is fed by several constitutive as well as inducible CO2 and HCO3– uptake systems, which import Ci from the environment into the cytoplasm resulting in up to a 1,000-fold accumulation (Kaplan and Reinhold, 1999). For the cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis) five Ci uptake systems are known: (1) BCT1, an inducible high-affinity ATP-binding cassette-type HCO3– transporter, encoded by the cmpABCD operon; (2) SbtA, an inducible high-affinity Na+/HCO3– symporter; (3) BicA, a low-affinity Na+-dependent HCO3– transporter of the SulP family; (4) NDH-14, a constitutive low-affinity CO2 uptake system that is based on a modified NDH-1 complex and is located inside the thylakoid membrane; and (5) NDH-13 is a CO2 uptake system that applies another modified NDH-1 complex, but is inducible under Ci limiting conditions and has a very high affinity (Shibata et al., 2001; Zhang et al., 2004; for review, see Badger et al., 2006). Recently, a genome-wide DNA microarray has been employed to study short-term changes accompanying acclimation toward low concentrations of Ci (LC) in Synechocystis, where about 200 genes including those encoding BCT1, SbtA, and NDH-13 were found to be strongly up-regulated (Wang et al., 2004).
In higher plants, changes in Ci concentrations strongly influence the carbon-fixing rate of Rubisco, since under LC its second substrate, oxygen, is increasingly used by Rubisco. Oxygenase activity forms large equimolar amounts of 2-phosphoglycolate (2PG) and the Calvin cycle intermediate, 3-phosphoglycerate. The 2PG by-product inhibits the Calvin cycle enzyme triosephosphate isomerase (Husic et al., 1987
Despite great progress in understanding the dynamic reactions that relate to Ci availability, one interesting and central question is still open: What is the primary signal that induces the response toward Ci limitation? Different hypotheses have been put forward, some of which include a photorespiration-based mechanism (Kaplan and Reinhold, 1999
Using genome-wide DNA microarrays, it has been shown that shortly after a stress treatment numerous genes are transiently up-regulated, many of them encoding general stress proteins. However, after long-term acclimation to suboptimal conditions only a few genes remain transcribed at the elevated level, among those often functionally important proteins specific for a given stress condition were found to behave in this way (e.g. light acclimation by Hihara et al., 2001
Synechocystis wild-type or mutant cells were shifted after precultivation with buffered BG11 at pH 7 from HC (5% CO2) aeration to LC (0.035% CO2). At this pH, a large amount of Ci is available as CO2. Long-term alterations in transcript prevalence and physiological patterns following the transfer to LC conditions were investigated. Samples were taken at different time points during 24 h after the transfer to LC to follow the different stages of response as established by Wang et al. (2004) . In this study, we applied semiquantitative reverse transcription (RT)-PCR and a newly developed genome-wide DNA microarray for Synechocystis, which is composed of custom-selected 60-mer synthetic oligo probes on an Agilent technology platform (E. Aguirre von Wobeser, B.W. Ibelings, J. Huisman, and H.C.P. Matthijs, unpublished data). The array comprises up to four different, nonoverlapping probes for each single gene. Only changes, which were reproducibly detected at each gene-specific spot and in each biological replicate, were taken into consideration. In the following, we present and discuss only selected data (the complete microarray data set is given as Supplemental Table S1).
Different types of Ci transporters are crucial for CCM in cyanobacteria. The expression of their genes is known to be regulated by Ci. Therefore, their expression data may verify the stringency of our Ci limitation and the reliability of the microarray data obtained with the new platform, also with reference to the work of Wang et al. (2004)
To support the microarray data by a second independent method, we used semiquantitative RT-PCR. As representative examples, the transcriptional kinetics for sbtA and bicA are presented in Figure 1 . A slight induction of expression occurred already 1 h after transfer to LC; sbtA mRNA reached its highest level after 3 h and declined steadily afterward. Expression of bicA was already seen under HC conditions and increased slightly up to 3 h, while long-term cultivation at LC resulted in reduced bicA expression. The kinetics of the RT-PCR matched well with our microarray data set and that of Wang et al. (2004) .
Unexpectedly, we found significantly reduced transcript levels of the genes encoding most integral parts of the carboxysome and thus the second component of the CCM. Microarray data (Table I) indicated an expression level of only 26% to 69% for the genes of the ccmK-N operon (sll1028–sll1033), 29% to 49% for the genes of the ccmK3,4 operon (slr1838–slr1840), about 80% for the genes of the rbcLXS operon (slr0009–slr0012), and 57% for ccaA (slr1347) after 24 h at LC. Expression of ccmO, whose product is thought to be necessary for carboxysome assembly (Marco et al., 1994 ), continued unchanged. The alterations were again supported by RT-PCR analysis (Fig. 1). In former studies it was shown that the mentioned genes were insensitive toward LC conditions in Synechocystis (McGinn et al., 2003; Wang et al., 2004). In cells from Synechococcus PCC 7942, the transcription of ccmM, rbcL, and ccaA was even induced after transfer from HC to LC (Omata et al., 2001; Woodger et al., 2003). Admittedly, the increases in transcript abundances of the mentioned genes were much smaller compared to transcripts encoding inducible Ci uptake systems and only transient (Woodger et al., 2003).
Recently, metabolism of 2PG, generated by oxygenase activity of Rubisco, has been reinvestigated by our group in Synechocystis. Genes coding for enzymes of the plant-like photorespiratory C2 cycle and bacterial-type glycerate pathway were assigned (Eisenhut et al., 2006
Moreover, the microarray analysis revealed a third hypothetical route to degrade glycolate by its complete decarboxylation via formate with hydrogen as by-product. The existence of this pathway in cyanobacteria has been postulated, but was never proven (Norman and Colman, 1992 ). Based on data taken from the Kyoto Encyclopedia of Genes and Genomes database (http://www.genome.jp/kegg/pathway/map/map00630.html), most genes encoding the enzymes involved in this pathway could be found in the Synechocystis genome. Only for the conversion of glyoxylate into oxalate no clear candidate could be proposed. The decarboxylation of oxalate is catalyzed by oxalate decarboxylase, probably encoded by sll1358. According to our dataset this ORF is obviously cotranscribed with sll1359. The Sll1359 protein contains a cytochrome-C-like domain, making it a promising candidate for formate dehydrogenase, which may perform the final decarboxylation of formate to CO2. The expression of sll1358 and sll1359 was barely altered or reduced after short-term transfer to LC (Wang et al., 2004), while our data showed a concerted up-regulation (1.8- to 2.5-fold) 24 h after transfer to LC conditions (Table II). This can be taken as an indication for the operation of this putative glycolate breakdown route in Ci-limited cultures.
Though the results of microarray analyses for 12 (Wang et al., 2004
The overall picture that develops is that LC-acclimated cells are better protected against becoming overreduced and also that cyclic photophosphorylation is stimulated to support bioenergetic needs. In this regard it is interesting to note that long-term acclimation to LC conditions seems to include aspects of the high light response in Synechocystis. One example is the differential expression of ORFs sll1521 and sll0550 (Table III) encoding the flavoprotein1 (Flv1) and Flv3, respectively, which are involved in the Mehler reaction (Helman et al., 2003 ). The distinctive increase in transcript abundance of only sll0550 (5-fold) implies importance for Flv3 under Ci limiting conditions, which was verified by RT-PCR (Fig. 1). Probably, the increased Mehler reaction defends PSII from photodamage by regeneration of the electron acceptor NADP+, which is limiting under LC conditions (Wang et al., 2004). Flv3, but not Flv1 was also selectively increased after transfer of Synechocystis to high light conditions (Hihara et al., 2001).
As mentioned above, regulatory processes involved in Ci acclimation are mostly unknown. Therefore, in our data set we searched for genes encoding proteins with regulatory functions and that showed stable up-regulation under LC conditions. In addition to the known Ci depending transcriptional factors CmpR (Omata et al., 2001
Another group of newly identified Ci responsive genes forms the rfb cluster, which comprises 44 genes (slr0976–0985, slr1610–1619, slr1062–1087) encoding enzymes putatively involved in the biosynthesis of cell wall or outer membrane polysaccharides (Table III). The transcription of all genes in this cluster, with the exception of an inserted gene for a transposase (slr1075), was induced (2-fold on average, data not shown) 24 h after transfer to LC, while its expression was not significantly changed or even repressed until 12 h after downshift (Wang et al., 2004
One central regulatory point under LC conditions is the balancing of the carbon/nitrogen ratio. As expected, we could observe a striking transcriptional repression of genes encoding proteins crucial for nitrogen acquisition and assimilation. As found before (Wang et al., 2004
To reinvestigate the hypothesis that alterations in concentrations of photorespiratory metabolites could act as signal for sensing Ci limitation (Kaplan and Reinhold, 1999
However, several genes showing a changed behavior between mutant and wild-type cells were found. Particularly interesting are differences in gene expression, which overlap in the two mutant transcriptomes (Table IV
). A reduced transcript level of the rbcLXS and ccmK-N operon was detected for
Besides genes changed in common in the two mutants (Table IV), some additional genes were differentially regulated in either gcvT or glcD (Supplemental Table S3). These genes are mostly not directly linked to known Ci-acclimation processes and include also many genes for so-called hypothetical proteins. Possibly their expression may be indirectly affected by the mutations. In general, the mutant gcvT seems to be defective in nitrogen metabolism, since typical nitrogen-regulated genes such as gifA, gifB, glnN, and ntrX were found among the genes differentially expressed between wild type and this mutant. Interestingly, the salt-regulated genes ggpS and glpD coding for enzymes involved in the synthesis of the compatible solute glucosylglycerol (Marin et al., 2004) were also slightly up-regulated in mutant gcvT, however, we could not detect any GG accumulation (data not shown). In cells of the mutant glcD many genes coding for ATPase subunits as well as for plastocyanin (PetE) are coordinated down-regulated (Supplemental Table S3), which may be taken as an indication for lower energy demand. The whole microarray data set for the effects of knockout in gcvT and glcD, respectively, is given in Supplemental Table S2.
The observed down-regulation of rbcLXS and ccmK-N mRNA levels after long-term acclimation to LC conditions was contradictory to findings in short-term LC-acclimated cyanobacterial cells, which showed up-regulation of such genes (e.g. Woodger et al., 2003
The amount of the four carboxysomal shell proteins CcmK1 to 4 was analyzed too. Though transcription of these genes was also down-regulated, we observed no decrease in the amount of the four proteins (Fig. 4B) in LC cultures. Moreover, since the number of carboxysomes increases under LC conditions (see Fig. 6 ), a rise in CcmK1 to 4 was expected. These findings suppose a regulation at posttranscriptional level for carboxysomal shell proteins similar to RbcL. Determination of the crystal structures of CcmK2 and CcmK4 revealed a function in controlling metabolite flow by forming selective pores (Kerfeld et al., 2005 ). Perhaps the composition of the carboxysomal protein shell alters depending on the Ci availability to regulate its permeability.
Changes in Cell Morphology during Long-Term Acclimation toward LC Conditions
Acclimation to alterations in Ci availability includes global changes in metabolism and obviously also morphology. Carboxysomes represent one central compound of CCM. In these hexagonically shaped microcompartments CO2 is finally extracted from HCO3– and becomes fixed by Rubisco. As it can be seen in Figure 6, a significant rise in carboxysome number becomes evident for LC-grown cells. The average number for carboxysomes in a thin section of cells increased from about one per cell to 2.3 per cell in cultures shifted from HC to LC. Interestingly, cells from the mutant
The electron micrographs showed further that the C-storage product, glycogen, is degraded during acclimation toward LC conditions (Fig. 6). HC-grown cells contain much more glycogen granules in the thylakoid space compared to LC-grown cells. The first step in glycogen degradation is catalyzed by glycogen phosphorylase (GlgP). The genome of Synechocystis harbors two glgP genes: sll1356 and slr1367. Recently, the functional divergence of the two homologs was shown. When grown photoautotrophically, Sll1356 seemed to play the dominant role in glycogen consumption (Fu and Xu, 2006
In our DNA microarray analyses, genes of the rfb cluster, whose products are most probably involved in the synthesis of cell envelope components, were newly identified to be Ci responsive (see Table III). Our electron microscopy study revealed only slight differences in the cell wall composition such as more thickness of the outermost stained layer, similar to what was reported before for acclimation to LC conditions in Anabaena (Marcus et al., 1982
Our analysis of long-term response toward Ci limitation in Synechocystis showed a stable up-regulation of genes encoding inducible CO2 and HCO3– uptake systems, which are already induced after short-term transfer to LC conditions. However, genes encoding structural and enzymatic components of the carboxysome were down-regulated, while the number of carboxysomes per cell and protein levels of Rubisco increased. The amount of the carboxysomal shell proteins CcmK1 to 4 remained unaltered. These contradictory results indicate that different components of the cyanobacterial CCM are probably differently regulated: Ci transporters mainly by transcriptional changes and carboxysome components by posttranscriptional mechanisms as was indicated for the half-life in the case of Rubisco.
Besides the finding of already known genes involved in LC acclimation, our relatively long-term experiments revealed also the importance of additional processes. The stable induction of genes encoding proteins necessary for cell wall synthesis implies changes in Ci permeability. Up-regulation of PSI certainly helps to produce extra energy for Ci uptake, particularly CO2 conversion into HCO3– at NDH-1. Moreover, genes for proteins necessary to avoid overreduction and oxygen radical accumulation were found to be up-regulated in a later stage during LC acclimation, which supports the view of an overlapping response toward LC and high light (Hihara et al., 2001 Detailed investigation of the regulation of genes encoding enzymes involved in the metabolism of the Rubisco oxygenase activity product phosphoglycolate showed also a differential regulation. The plant-like C2 cycle enzymes are obviously not transcriptionally regulated after HC to LC transfer, while the bacterial-type glycerate pathway is activated. Moreover, the DNA microarray data indicated a third route for phosphoglycolate metabolism involving its complete breakdown to CO2 via oxalate and formate, since the genes for the necessary steps were induced in a coordinated way.
In addition to wild-type cells, the mutants
Strains and Culture Conditions The cyanobacterial strains used in this work are listed in Table V . The Glc-tolerant strain of Synechocystis sp. PCC 6803 was obtained from Professor Murata (National Institute for Basic Biology, Okazaki, Japan) and served as the wild type. Cultivation of mutants was performed at 50 µg mL–1 kanamycin (Km) or at 20 µg mL–1 spectinomycin. For the experiments, axenic cultures of the cyanobacteria (about 108 cells mL–1) were grown photoautotrophically in batch cultures (3 cm glass vessels with 5 mm glass tubes for aeration) at 29°C under continuous illumination of 130 µmol photons s–1 m–2 (warm light, Osram L58 W32/3) bubbling (flow rate about 5 mL min–1) with air enriched with CO2 (5% defined as HC) in the BG11 medium at pH 7.0. Ci limitation was set by transferring exponentially growing cultures (OD750 of 0.9, volume of 130 mL) from bubbling with CO2-enriched air to bubbling with pure air (about 0.035% CO2 defined as LC).
Absence of contaminating heterotrophic bacteria was routinely checked by spreading of 0.2 mL culture on LB plates.
Cells from 10 mL of culture were harvested by centrifugation at 4,000 rpm for 5 min at 4°C and were immediately frozen at –80°C. Total RNA was extracted after pretreatment with hot phenol and chloroform by the High Pure RNA isolation kit (Roche Diagnostics). The same RNA samples were used for labeling in DNA microarray experiments and RT-PCR.
For RT-PCR, 3 µg RNA was used for cDNA synthesis. RT was performed with random hexamers (Amersham Pharmacia), dNTP mix (MBI Fermentas), and RevertAid M-MuLV Reverse Transcriptase (MBI Fermentas). The generated cDNA was diluted 4-fold and used as template for RT-PCR. Amplification with gene-specific primers (see Table V) for 18 to 30 cycles depending on the abundance of the transcript was evaluated by electrophoresis in 1.5% agarose gels. The transcript abundance of the constitutively expressed rnpB encoding the RNA subunit of RNaseP served as a control (Wang et al., 2004
Direct cDNA labeling was performed as follows: 10 µg of total RNA and 0.5 µg random hexamers were used in a total volume of 15 µL. After incubation at 70°C for 10 min the samples were allowed to chill for 10 min at 4°C. Six microliters Reverse Transcriptase buffer (5x), 3 µL dithiothreitol (0.1 M), 0.6 µL dNTP mix (25 mM), and 1.4 µL milliQ water were added and mixed. As fluorescent dyes served either 2 µL Cy3 or Cy5 (1 mM, Amersham Pharmacia). Two microliters Superscript II reverse transcriptase (200 units µL–1, Invitrogen) were added, the mixtures incubated for 10 min at RT and to start synthesis of fluorescently labeled cDNA transferred for 110 min to 42°C. RNA was hydrolyzed by addition of 1.5 µL NaOH (1 M) at 70°C for 10 min. A total of 1.5 µL HCl (1 M) was added afterward to neutralize the alkali. The samples were purified using QIAquick PCR Purification kit (Quiagen) according to the protocol of the manufacturer. Finally, the concentration and fluorescent dye incorporation was checked photometrically on a Nano-drop instrument (Nano-drop Technologies). Labeled cDNA was hybridized to 60-mer oligonucleotide DNA microarrays (Agilent) designed from the complete Synechocystis genome sequence (Kaneko et al., 1996 The hybridization was performed according to the 60-mer oligionucleotide microarray protocol of Agilent (http://www.chem.agilent.com). Aliquots of Cy3- or Cy5-labeled cDNA were denatured at 98°C for 3 min and mixed with 125 µL hybridization buffer and 25 µL control targets (Agilent). This hybridization solution was exposed to the microarray surface in Agilent hybridization chambers for 17 h in a rotating oven at 60°C. The slides were then washed according to the manufacturer's protocol with one exception: acetonitrile was used instead of wash solution 3 to avoid the formation of precipitates on the slide. The scanning was performed with an Agilent Microarray Scanner (model G2505B) with default settings. Fluorescence intensity values were extracted using Feature Extraction software version 8.1.1.1 (Agilent). The mean signal of the pixels on each spot was used for all subsequent analysis. The values obtained for the different probes corresponding to the same gene were averaged to obtain one value per gene per sample. Given values are the means and SDs of at least two independent experiments using RNA isolated from separate cultures.
Low temperature emission spectra were measured at 77 K in the low temperature unit of a Hitachi F4010 spectrofluorometer (Hitachi) using dark-adapted cells frozen in glass capillaries. The red-sensitive photomultiplier was protected from scattered excitation light by a Schott RG 620 cutoff filter. Emission was recorded at 2 nm intervals from 620 to 780 nm. Redox changes of P700 were measured at 820 nm with a pulse-amplitude-modulated system equipped with an ED-800T emitter-detector unit (PAM101-103, Walz GmbH) as described in Schreiber et al. (1988)
Cells from 20 mL of culture were harvested by centrifugation at 4,000 rpm for 5 min at 4°C and were immediately frozen at –20°C. For protein isolation the pellets were resuspended in 500 µL 0.01 M HEPES buffer (pH 7.3) supplemented with 10 mM phenylmethylsulfonyl fluoride. Under ice cooling the suspensions were sonicated (2 x 1 min, 35 W) and the homogenates were centrifuged at 4,000 rpm for 20 min at 4°C. Protein contents were estimated by the method of Bradford (1976)
The half-life of the RbcL protein was analyzed by incubation of cells with lincomycin (final concentration 250 µg mL–1; Sigma) as a specific inhibitor of translation. Wild-type cells were precultivated for 24 h under HC and LC conditions in BG11, pH 7, respectively. After adjusting optical density (OD750 = 0.9), lincomycin (final concentration 250 µg mL–1) was applied. Cultures were kept on bubbling with 5% CO2 or with pure air for 24 h more. Soluble proteins were isolated from cells grown under HC conditions and from cells grown under LC conditions in the presence or absence of lincomycin. The specific RbcL protein content was estimated before and 3, 9, 6, 12, and 24 h after addition of lincomycin, respectively. The signal intensities were estimated by SigmaGel 1.0 and plotted versus time to calculate the half-life.
Synechocystis cells were harvested by centrifugation at 4,000 rpm for 5 min at 4°C and fixed with glutaraldehyde (4%, w/v) in phosphate-buffered saline (PBS). After washing with PBS, cells were postfixed with 1% OsO4 in PBS for 1 h, washed again, dehydrated in graded acetone concentrations (30%, 60%, 90%, 100%), and embedded in Araldite (Fluka). Ultrathin sections were prepared by an ultramicrotome (Ultracut E. Reichert, Optische Werke), placed on copper grids, stained by uranyl acetate and lead citrate, and studied with a transmission electron microscope (EM 902 A, Zeiss). Ultramicrographs were taken with a CCD camera (Proscan) and a Pentium computer, using the ITEM software (Soft Imaging Solutions, SIS).
The following materials are available in the online version of this article.
We would like to thank Ms. Klaudia Michl (Plant Physiology, University of Rostock, Rostock, Germany) for excellent technical assistance. The help of Gerhard Fulda (Electron Microscopic Center of Medical Faculty, Rostock, Germany) during the ultrastructural studies and Jasper Bok (Netherlands Institute of Ecology, Nieuwersluis, The Netherlands) in work with DNA microarrays is greatly acknowledged. Specific antibodies were kindly donated by Dr. E. Pistorius (RbcL, University of Bielefeld, Bielefeld, Germany) and Dr. G.C. Cannon (CsoS1, University of Southern Mississippi, Hattiesburg, Mississippi). Received June 4, 2007; accepted June 24, 2007; published June 28, 2007.
1 This work was supported by a scholarship from Consejo Nacional para la Ciencia y Tecnología (Mexico) and by a grant from the Earth and Life Sciences Foundation (to E.A.v.W.), which is subsidized by the Netherlands Organization for Scientific Research (grant to Dr. Jef Huisman, University of Amsterdam). Financial support for setting up the array facility and for purchase of the arrays used in this study was provided by the Bootsma fonds at the Royal Academy of Arts and Sciences (grant to B.W.I.). This work was also supported by a grant from the Deutsche Forschungsgemeinschaft (to M.H.).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Martin Hagemann (martin.hagemann{at}uni-rostock.de).
[W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.107.103341 * Corresponding author; e-mail martin.hagemann{at}uni-rostock.de; fax 49–0–3814986112.
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ABSTRACT
RESULTS AND DISCUSSION
I/I, white circles) were far stronger for LC-grown cells compared to HC-grown ones, indicating a higher P700 content. Activation of PSII by a strong white light pulse led to rereduction of oxidized P700, causing a drop in the absorbance signal measured for P700 (black circles). For HC-grown cells the amount of rereduction was about 65% of the value at saturating far-red light intensities, whereas for LC-grown cells just 45% of the P700+ got rereduced, the majority of the P700 was still locked in the oxidized state. The activation of PSI at transcriptional and activity level and unchanged PSII activity demonstrates an increase in cyclic electron flow, which helps cells to acquire the capacity for photophosphorylation that operates independently of the linear photosynthetic electron transfer pathway. In addition, cyclic electron flow plays a crucial role in the energy supply for CO2 to HCO3– interconversion at specialized NDH complexes such as NDH-13 as part of the CCM (see Zhang et al., 2004
0.05, n = 50).
-phosphate system in reaction buffer (0.1 M Tris/HCl, 0.1 M NaCl, 0.05 M MgCl2, pH 9.5). 
