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First published online March 24, 2006; 10.1104/pp.105.074765 Plant Physiology 141:508-521 (2006) © 2006 American Society of Plant Biologists
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ABSTRACT |
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 TOP ABSTRACT RESULTSDISCUSSIONMATERIALS AND METHODSLITERATURE CITED |
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-Tocopherol is synthesized exclusively in oxygenic phototrophs and is known to function as a lipid-soluble antioxidant. Here, we report that -tocopherol also has a novel function independent of its antioxidant properties in the cyanobacterium Synechocystis sp. PCC 6803. The photoautotrophic growth rates of wild type and mutants impaired in -tocopherol biosynthesis are identical, but the mutants exhibit elevated photosynthetic activities and glycogen levels. When grown photomixotrophically with glucose (Glc), however, these mutants cease growth within 24 h and exhibit a global macronutrient starvation response associated with nitrogen, sulfur, and carbon, as shown by decreased phycobiliprotein content (35% of the wild-type level) and accumulation of the nblA1-nblA2, sbpA, sigB, sigE, and sigH transcripts. Photosystem II activity and carboxysome synthesis are lost in the tocopherol mutants within 24 h of photomixotrophic growth, and the abundance of carboxysome gene (rbcL, ccmK1, ccmL) and ndhF4 transcripts decreases to undetectable levels. These results suggest that -tocopherol plays an important role in optimizing photosynthetic activity and macronutrient homeostasis in Synechocystis sp. PCC 6803. Several lines of evidence indicate that increased oxidative stress in the tocopherol mutants is unlikely to be the underlying cause of photosystem II inactivation and Glc-induced lethality. Interestingly, insertional inactivation of the pmgA gene, which encodes a putative serine-threonine kinase similar to RsbW and RsbT in Bacillus subtilis, results in a similar increase in glycogen and Glc-induced lethality. Based on these results, we propose that -tocopherol plays a nonantioxidant regulatory role in photosynthesis and macronutrient homeostasis through a signal transduction pathway that also involves PmgA.
-Tocopherol (vitamin E) is a lipid-soluble, organic molecule that is only synthesized by oxygen-evolving phototrophs, including some cyanobacteria and all green algae and plants (Threlfall and Whistance, 1971
; Collins and Jones, 1981; Sakuragi and Bryant, 2006). The conservation of -tocopherol synthesis during the evolution of oxygenic photosynthetic organisms suggests that this molecule performs one or more critical functions. Because -tocopherol is also an essential dietary component, most of our knowledge of tocopherol functions has been obtained from studies in animals, animal cell cultures, and artificial membranes. Studies in these systems have shown that tocopherols scavenge and quench various reactive oxygen species and lipid oxidation by-products, which would otherwise propagate lipid peroxidation chain reactions in membranes (Kamal-Eldin and Appelqvist, 1996). In addition to these antioxidant functions, several other functions have been reported in mammals. These functions, which are independent of the antioxidant activity of tocopherols and are termed nonantioxidant functions, include transcriptional regulation and modulation of signaling pathways (Chan et al., 2001; Azzi et al., 2002; Ricciarelli et al., 2002; Rimbach et al., 2002).
Tocopherol functions have not yet been clearly defined in oxygenic phototrophs, but it is believed that they likely include some or all of the functions reported in animals, as well as other functions possibly specific to photosynthetic organisms. For example, recent studies with tocopherol-deficient mutants of Arabidopsis (Arabidopsis thaliana) demonstrated that tocopherols provide protection against propagation of lipid peroxidation in dormant and germinating seeds and thus are essential for seed longevity and seedling development (Sattler et al., 2004). -Tocopherol has been proposed to protect PSII under high light-induced oxidative stress conditions in the green alga Chlamydomonas reinhardtii (Trebst et al., 2002). Furthermore, we have previously demonstrated that tocopherol-deficient mutants of Synechocystis sp. PCC 6803 grow poorly when challenged with oxidative stress induced by the combination of polyunsaturated fatty acids and high light illumination (Maeda et al., 2005). Therefore, it seems clear that an antioxidant role of -tocopherol is conserved among the oxygenic phototrophs.
The biosynthesis of -tocopherol in cyanobacteria occurs as shown in Figure 1
. Insertional inactivation of the genes encoding each enzyme of the pathway has resulted in a series of mutants in which the content and composition of tocopherol species vary. For example, the slr0089 mutant accumulates only 
-tocopherol (Shintani and DellaPenna, 1998), the sll0418 mutant accumulates 30% of the wild-type level of -tocopherol and a small amount of 
-tocopherol (Shintani et al., 2002; Cheng et al., 2003), whereas the slr1736 mutant lacks all tocopherols (Collakova and DellaPenna, 2001). In light of the established antioxidant activity of -tocopherol, one would expect that a loss or reduction of -tocopherol would lead to an obvious phenotypic difference between the wild-type and mutant strains. Intriguingly, however, the tocopherol-deficient slr1736 mutant was reported to grow similarly to the wild type under both photoautotrophic and photomixotrophic conditions (Collakova and DellaPenna, 2001). These results suggest that -tocopherol is dispensable for the survival of Synechocystis sp. PCC 6803 under the conditions tested (Collakova and DellaPenna, 2001).
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-tocopherol is essential for the normal physiology of the cyanobacterium Synechocystis sp. PCC 6803. Tocopherol mutants exhibited enhanced photosynthetic activities when grown under photoautotrophic conditions, whereas they lost photosynthetic activity after 24 h and were unable to grow under photomixotrophic conditions (in Glc-containing media). These results demonstrate that -tocopherol is essential for the survival of Synechocystis sp. PCC 6803 under photomixotrophic conditions and suggest a role for -tocopherol in the regulation of photosynthesis in this cyanobacterium. Further analyses led to the conclusion that oxidative stress is not the major cause of the lethality in cells grown photomixotrophically and that -tocopherol plays a regulatory role in photosynthesis and macronutrient metabolism in Synechocystis sp. PCC 6803 that is independent of its antioxidant properties. |
RESULTS |
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TOPABSTRACTRESULTSDISCUSSIONMATERIALS AND METHODSLITERATURE CITED |
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Isogenic mutants deficient in tocopherol biosynthesis were constructed in our laboratory wild-type strain (see "Materials and Methods"). Genomic DNAs extracted from each of the previously isolated tocopherol-deficient mutants (Shintani and DellaPenna, 1998; Collakova and DellaPenna, 2001; Shintani et al., 2002) were used for transformation, and the resulting mutants were selected under photoautotrophic growth conditions on the basis of their resistance to kanamycin. Complete segregation of each mutant allele was confirmed by PCR analysis (Fig. 2A
). Table I
shows the tocopherol content of each homozygous mutant. The tocopherol content of the mutants was similar to that reported previously and further confirmed the targeted gene inactivations (Shintani and DellaPenna, 1998; Collakova and DellaPenna, 2001; Cheng et al., 2003). The growth rates of the wild type and mutants were indistinguishable under photoautotrophic growth conditions in liquid B-HEPES medium with 3% (v/v) CO2 at various light intensities (Fig. 2B, 50 µmol photons m–2 s–1; 
5 and 300 µmol photons m–2 s–1, data not shown). The data demonstrate that -tocopherol is not required for the growth of Synechocystis sp. PCC 6803 under photoautotrophic conditions, which is consistent with previous studies (Collakova and DellaPenna, 2001; Dähnhardt et al., 2002; Maeda et al., 2005).
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Cells grown under photoautotrophic conditions were diluted into fresh B-HEPES medium containing 5 mM Glc, and growth was monitored under photomixotrophic conditions. The mutants grew similarly to the wild type during the initial 12 to 24 h, but all mutants stopped growing after about 24 h in the presence of Glc (Fig. 2, C and D). After 72 h, the mutants had completely lost viability and could not form colonies even on Glc-free medium (data not shown). The ultrastructure of the slr1736 mutant cells was dramatically different from that of the wild type when both were grown photomixotrophically. The thylakoid membrane surfaces of the mutant appeared smoother than those of the wild type, and numerous electron-dense oval objects, whose biochemical nature is not yet known, can be seen between thylakoid membranes (compare Fig. 3, C and D). Under these conditions, the PSII activity in the mutant cells was completely lost by 24 h, whereas the wild type maintained similar PSII activity during the course of measurements (Table II). Furthermore, in the slr1736 mutant grown under photomixotrophic conditions, no carboxysomes were detectable in thin-section micrographs (see example in Fig. 3D). These results demonstrate that -tocopherol is essential for survival as well as for maintenance of PSII activity and carboxysomes in Synechocystis sp. PCC 6803 under photomixotrophic conditions.
Oxidative Stress Is Unlikely to Be the Cause of Glc Lethality in Tocopherol Mutants
Light-dependent inactivation of photosynthesis, termed photoinhibition, is often observed under a variety of environmental stresses, including high-intensity light (Allakhverdiev et al., 1999; Hideg et al., 2000; Trebst et al., 2002; for review, see Aro et al., 1993, and refs. therein). Under such conditions, the PsbA protein, a polypeptide that forms a subunit of the PSII core complexes, is rapidly degraded and PSII activity is lost. Given the established role of -tocopherol as an antioxidant and the report that it protects PSII from photoinhibition in C. reinhardtii (Trebst et al., 2002), we hypothesized that the altered photosynthetic activities and growth capacities in the tocopherol mutants under photomixotrophic conditions resulted from elevated oxidative stress due to the loss of -tocopherol. However, immunologically detectable PsbA protein levels were essentially identical for the wild type and mutants grown both photoautotrophically and photomixotrophically (Fig. 4A
). Furthermore, the level of immunologically detectable PsbO, a 33-kD protein closely associated with the tetra-manganese cluster of the PSII oxygen evolution complex (Ferreira et al., 2004), was also essentially identical for the wild-type and mutant cells grown both photoautotrophically and photomixotrophically (Fig. 4A). These results indicate that inactivation of PSII in the tocopherol mutants is not due to damage and degradation of the PsbA and PsbO proteins.
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; Huang et al., 2002; Ushimaru et al., 2002). Similar and low levels of sodB transcripts were detected in the wild-type and slr1736 mutant cells grown photoautotrophically (Fig. 4B, 0 h). Following a shift to photomixotrophic growth conditions, sodB transcript levels increased gradually and similarly in both the wild type and the slr1736 mutant (Fig. 4B, at 4–24 h). These results indicate that the slr1736 mutant is unlikely to be experiencing oxidative stress beyond that which occurs in wild-type cells. Furthermore, as shown in Figure 4, C and D, the cessation of growth observed for the tocopherol mutants under photomixotrophic conditions occurs independently of light intensity within the range from approximately 5 µmol photons m–2 s–1 to 300 µmol photons m–2 s–1. Taken together, these results are not consistent with the hypothesis that oxidative stress causes the inactivation of PSII and growth inhibition observed for the tocopherol mutants under photomixotrophic conditions.
Effect of pH on Glc-Induced Lethality
It is noteworthy that the effect of Glc was dependent on the pH value of the growth medium. The pH in standard B-HEPES medium typically shifted within 48 h from a value of 8.0 to a value between 7.0 and 7.3 due to the supply of 3% CO2 (v/v). Therefore, the possibility that pH influences the Glc-induced lethality was tested using a modified B-HEPES medium (B-HEPES40, containing 40 mM HEPES) that maintained the pH of the culture within ±0.1 pH units for the duration of the growth experiment. Under photoautotrophic conditions, the mutants grew similarly to the wild type at all pH values, indicating that the pH shift has little impact on mutants under these conditions (Fig. 5A ). Under photomixotrophic conditions, however, the mutants grew similarly to the wild type at pH 8.0 and 7.6, whereas their growth stopped after 24 h at pH 7.2 and below (Fig. 5A). The PSII activity of the slr1736 mutant was higher than the wild type at all pH values under photoautotrophic growth conditions, consistent with the results presented in Table II. In contrast, under photomixotrophic conditions, PSII activity was pH dependent and completely lost at pH 7.0 and below (Fig. 5B). These results demonstrate that Glc sensitivity and PSII inactivation of tocopherol mutants are pH dependent and occur at approximately pH 7.2 and below.
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). It has been reported that the cytoplasmic pH value of Synechocystis sp. PCC 6803 is neutral or slightly alkaline (pH 6.9–7.5), depending on growth conditions (Katoh et al., 1996). Therefore, it was hypothesized that the combination of the neutral medium pH and increased intracellular pH due to Glc import and metabolism led to a compromised membrane electrochemical potential, thereby abating ATP synthesis and growth in the tocopherol mutant. This possibility was tested by using electron transport chain inhibitors to disrupt electron transport and hence the development of the membrane electrochemical potential. 3-(3',4'-Dichlorophenyl)-1,1-dimethylurea blocks the input of electrons into the photosynthetic electron transport chain by inhibiting PSII activity; methyl viologen withdraws electrons from the membrane electron transport chain on the acceptor side of PSI, whereas cyanide inhibits cytochrome c oxidase. Regardless of the inhibitors used, the slr1736 mutant showed similar Glc-induced lethality as observed in the absence of the inhibitors (data not shown). These results suggest that Glc toxicity is probably not associated with increased electron flux through the electron transport chain or with an altered membrane potential.
Altered Macronutrient Metabolism in Tocopherol Mutants
How does Glc cause the death of the tocopherol mutants if not by means of oxidative stress or by a modification of electron flux through the electron transport chain? As shown above, under photoautotrophic conditions, the tocopherol mutants exhibited an enhanced photosynthetic activity and elevated total sugar content (Fig. 3B; Table III). We hypothesized that such elevation of the intracellular carbon flux would alter the balance between carbon and other macronutrients and that Glc metabolism would exacerbate this metabolic imbalance, perhaps to a level that could impair growth.
It is noteworthy that the tocopherol mutants appeared pale and chlorotic (greenish-yellow) when grown under photomixotrophic conditions. In cyanobacteria, chlorosis is often associated with macronutrient deprivation, such as nitrogen, carbon, sulfur, iron, and phosphate starvation, because of a rapid degradation of phycobiliproteins (PBPs), light-harvesting antenna proteins that can serve as a reserve of fixed carbon and nitrogen (for review, see Grossman et al., 1994). Analysis of the PBP content revealed that tocopherol mutants contained only 35% of the wild-type level of PBPs after 24 h under photomixotrophic conditions (Fig. 6
). Under these conditions, the abundance of the cpcA transcript, which encodes the -subunit of phycocyanin, also decreased to undetectable levels (Fig. 7A
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), whereas the sbpA transcript encodes an inducible high-affinity, periplasmic sulfate-binding protein (Laudenbach and Grossman, 1991). The nblA and sbpA transcripts have previously been shown to accumulate in response to nitrogen and sulfate limitation, respectively, in Synechocystis sp. PCC 6803 (Laudenbach and Grossman, 1991; Collier and Grossman, 1994; Richaud et al., 2001). Under photoautotrophic growth conditions, the slr1736 mutant accumulated slightly higher levels of these transcripts in comparison to the wild type (Fig. 7A). Under photomixotrophic growth conditions, the slr1736 mutant accumulated substantially higher levels of the nblA and sbpA transcripts after 4 h (Fig. 7A). These data suggest that the slr1736 mutant is sensing and responding to macronutrient stress and that this stress is greatly accentuated under photomixotrophic conditions. One response is increased PBP degradation in the slr1736 mutant under photomixotrophic conditions.
It is known that the expression of alternative sigma factors is induced in response to various stresses, including macronutrient limitation for carbon (sigB and sigH; Caslake et al., 1997; Wang et al., 2004) and nitrogen (sigE; Muro-Pastor et al., 2001). As shown in Figure 7B, the transcript levels of these sigma factors were indeed altered in the slr1736 mutant. For example, the transcript levels of sigB, sigC, and sigE in the slr1736 mutant appeared slightly higher than those of the wild-type control under photoautotrophic growth conditions, and they increased further (by 4 h) in response to Glc treatment (Fig. 7B). These results suggest that the tocopherol mutants are experiencing and transcriptionally responding subtly to macronutrient starvation under photoautotrophic growth conditions and indicate that they are experiencing severe macronutrient starvation related to carbon and nitrogen under photomixotrophic growth conditions. The transcript level for sigI in the slr1736 mutant did not vary significantly from the wild-type control, whereas sigD transcript levels for the mutant and the wild type were highly variable and not reliably reproducible. Interestingly, transcript levels of sigA and sigG in the slr1736 mutant gradually decreased to undetectable levels under photomixotrophic growth conditions. sigA and sigG have been shown to be essential for the survival of this cyanobacterium (Caslake and Bryant, 1996; Huckauf et al., 2000). Therefore, these results indicate that the substantial reduction in the sigA and sigG transcripts combined with the severe macronutrient starvation response led to the cessation of growth in the tocopherol mutants under photomixotrophic growth conditions at pH 7.0.
Transcript Levels of Inorganic Carbon Metabolism Genes
Given the dramatic differences in carbon assimilation between the tocopherol mutants and wild type under both photoautotrophic and photomixotrophic conditions (Fig. 3; Table III), the abundance of genes involved in inorganic carbon (Ci) metabolism was also investigated by reverse transcription (RT)-PCR. In the slr1736 mutant, the transcript levels of carboxysome genes, including rbcL, ccmK1, and ccmL (encoding the large subunit of Rubisco [Pierce et al., 1989] and carboxysome shell proteins [Price et al., 1993], respectively), were identical to those in the wild type under photoautotrophic growth conditions. In contrast, these transcripts gradually decreased to undetectable levels in the slr1736 mutant under photomixotrophic growth conditions, whereas those in the wild type were unaffected (Fig. 7C). As shown by electron micrographs (Fig. 3D), these results are consistent with the loss of carboxysomes in the slr1736 mutant under photomixotrophic growth conditions in the slr1736 mutant. Similarly, the transcript levels of ndhF4, encoding a subunit of the constitutive low-affinity CO2 uptake transporter (Shibata et al., 2001), were not affected under photoautotrophic growth conditions, whereas they gradually decreased to undetectable levels under photomixotrophic conditions in the slr1736 mutant. ndhF3, ndhR, and sbtA encode a subunit of the low CO2-inducible high-affinity CO2 uptake complex, a repressor of ndhF3, and the sodium-dependent bicarbonate transporter, respectively (Klughammer et al., 1999; Shibata et al., 2001, 2002). The transcript levels of these genes were constitutively lower in the slr1736 mutant as compared to the wild type under both photoautotrophic and photomixotrophic growth conditions (Fig. 7D). These results demonstrate that the abundance of Ci gene transcripts is differentially regulated in the slr1736 mutant as compared with the wild type.
A pmgA Mutant Also Shows pH-Dependent Lethality under Photomixotrophic Growth Conditions
A previous study identified the pmgA gene as a locus responsible for the survival of Synechocystis sp. PCC 6803 under photomixotrophic growth conditions (Hihara and Ikeuchi, 1997). Although the underlying mechanism is not completely understood, pmgA has been suggested to play a role in the regulation of Glc metabolism and photosynthesis in Synechocystis sp. PCC 6803 (Hihara and Ikeuchi, 1997). Therefore, a pmgA mutant was constructed in the same wild-type genetic background as the tocopherol mutants (see "Materials and Methods"), and the growth of this mutant was compared to the wild type and the slr1736 mutant under photoautotrophic and photomixotrophic growth conditions at both pH 7.0 and 8.0. Under photoautotrophic conditions at both pH values, the pmgA mutant grew similarly to both the wild type and the slr1736 mutant (Fig. 8A
). Under photomixotrophic growth conditions at pH 7.0, growth of the pmgA mutant ceased by 24 h, whereas it continued to grow at pH 8.0 (Fig. 8B). This pH-dependent growth defect was identical to that observed for the slr1736 mutant under photomixotrophic conditions (Fig. 8B). Previously, the pmgA mutant was also shown to have higher photosynthetic activity under photoautotrophic conditions (Hihara and Ikeuchi, 1998), suggesting that, like the slr1736 mutant, the pmgA mutant possesses enhanced photosynthetic capacity under photoautotrophic conditions. Therefore, total sugar content of the pmgA mutant was measured under photoautotrophic and photomixotrophic conditions. The pmgA mutant accumulated twice as much total sugar as the wild type under both photoautotrophic and photomixotrophic growth conditions (Table III), which is very similar to the results observed for the slr1736 mutant (Table III; see above). The striking similarities between the slr1736 and pmgA mutants lead us to propose that -tocopherol and PmgA may function in the same signal transduction pathway and participate in the regulation of the photosynthetic activity and macronutrient homeostasis in Synechocystis sp. PCC 6803.
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DISCUSSION |
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TOPABSTRACTRESULTSDISCUSSIONMATERIALS AND METHODSLITERATURE CITED |
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). We observed that all of the previously isolated tocopherol mutants (Shintani and DellaPenna, 1998; Collakova and DellaPenna, 2001; Shintani et al., 2002) showed colony morphologies that are highly variable with respect to their size and pigmentation when grown under photomixotrophic conditions (data not shown). Inhomogeneous colony morphology typically indicates genotypic heterogeneity within a given population. It is important to note that these mutants were originally isolated and maintained under photomixotrophic conditions (Shintani and DellaPenna, 1998; Collakova and DellaPenna, 2001; Shintani et al., 2002), which we now know to be lethal for tocopherol biosynthetic mutants. Therefore, it is highly plausible that the previously isolated populations of tocopherol mutants contain secondary suppressor mutations that were selected for under continuous photomixotrophic conditions. We conclude that the authentic tocopherol mutants described here are Glc sensitive and that -tocopherol is essential for survival of Synechocystis sp. PCC 6803 under photomixotrophic growth conditions at pH values below approximately 7.4.
Due to its antioxidant properties in biological membranes, functions of -tocopherol are typically discussed in connection with oxidative stress (Kamal-Eldin and Appelqvist, 1996). Interestingly, in C. reinhardtii, an 80% reduction in -tocopherol levels, due to combined herbicide and high light treatments (1,500 µmol photon m–2 s–1), resulted in the complete loss of PSII activity with concomitant degradation of the D1 (PsbA) protein (Trebst et al., 2002). This suggests that -tocopherol plays an antioxidant role in protecting the structural integrity of PSII during oxidative stress in this green alga. Thus, we initially hypothesized that PSII inactivation in tocopherol mutants under photomixotrophic growth conditions was also related to oxidative stress. However, several lines of evidence indicate that this is not the case. First, the PsbA protein level in tocopherol mutants was not altered, although PSII activity was completely lost (Fig. 4A). Second, the Glc-sensitive phenotype of tocopherol mutants was light independent and occurred at a wide range of light intensities (approximately 5–300 µmol photons m–2 s–1; Fig. 4, C and D). Third, sodB transcript levels, an oxidative stress marker, were identical between the slr1736 and wild type under both photoautotrophic and photomixotrophic growth conditions (Fig. 4B). Last, the deleterious effects of Glc on the slr1736, sll0418, and slr0089 mutants were virtually indistinguishable despite the varying compositions and amounts of tocopherols accumulated in each mutant (Table I; Figs. 2, C and D, and 5A). Should -tocopherol function solely as a bulk antioxidant, an inverse correlation of susceptibility to Glc and tocopherol content would reasonably be expected. This was not observed, however, and therefore we conclude that Glc-induced PSII inactivation and growth inhibition of tocopherol mutants are not associated with oxidative stress or D1-mediated photoinhibition. Instead, we propose that, in addition to protecting Synechocystis sp. PCC 6803 membranes from peroxidation (Maeda et al., 2005), -tocopherol also plays a nonantioxidant role in the survival of Synechocystis sp. PCC 6803 under photomixotrophic growth conditions at pH 7.0.
Nonantioxidant roles of -tocopherol are not without precedent. Studies in animal systems have demonstrated nonantioxidant roles for -tocopherol, including modulation of signaling pathways and transcriptional regulation (Chan et al., 2001; Azzi et al., 2002; Ricciarelli et al., 2002). For example, -tocopherol has been shown to modulate the phosphorylation state of protein kinase C in rat smooth-muscle cells by influencing protein phosphatase 2A activity (Ricciarelli et al., 1998). It has also been demonstrated that -tocopherol affects the expression of genes encoding liver collagen I, -tocopherol transfer protein, and -tropomyosin collagenase (Yamaguchi et al., 2001; Azzi et al., 2002; Rimbach et al., 2002). Similarly, the loss of -tocopherol in the slr1736 mutant constitutively or conditionally altered the abundance of several transcripts, including those encoding components of Ci, nitrogen, and sulfur metabolism (Fig. 7). Loss of -tocopherol also resulted in elevated photosynthetic activity in cells grown photoautotrophically as shown by increased PSII activity and total sugar and glycogen content (Tables II and III; Figs. 3B and 5B). These data are consistent with -tocopherol playing a role in the regulation of photosynthesis and macronutrient metabolism—a role that is independent of its antioxidant properties in Synechocystis sp. PCC 6803.
What is the underling mechanism by which -tocopherol, a small secondary metabolite, could affect such cellular processes on a global scale? In searching for an answer, we focused on the pmgA gene, which was previously shown to be essential for the survival of Synechocystis sp. PCC 6803 under photomixotrophic growth conditions (Hihara and Ikeuchi, 1997). An analysis of conserved domains (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) revealed that the primary structure of PmgA is similar to that of RsbW/RsbT in Bacillus subtilis (E value = 6 x e–25), which is a Ser-Thr kinase that acts in the signal transduction cascade that regulates the activity of SigB, a stress-responsive sigma factor in this bacterium (Price, 2000). The pmgA mutant showed remarkable similarity to the slr1736 mutant under both photoautotrophic and photomixotrophic conditions. These included increased levels of total cellular sugars under both growth conditions (Table III), higher oxygen evolution activity than the wild type under photoautotrophic conditions (Hihara and Ikeuchi, 1998), and nearly identical pH-dependent sensitivity to the presence of Glc (Fig. 8). These combined results demonstrate that both -tocopherol and PmgA are required for the appropriate regulation of photosynthesis and carbon homeostasis in Synechocystis sp. PCC 6803. One possibility is that -tocopherol and PmgA are both necessary components of a not yet fully characterized signal transduction cascade whose disruption leads to Glc lethality in Synechocystis sp. PCC 6803. Recent studies have shown that the His kinase Hik31 is involved in Glc sensing and Glc-induced lethality (Kahlon et al., 2006), whereas the His kinase Hik8 is involved in Glc metabolism and heterotrophic growth in Synechocystis sp. PCC 6803 (Singh and Sherman, 2005). Alternatively, -tocopherol may indirectly influence the activities and functions of the regulatory proteins or proteins involved in Glc metabolism, particularly those associated with the membranes, by affecting membrane integrity (Wang and Quinn, 2000). These possibilities remain to be examined in future studies.
Such a control mechanism for optimal activity of photosynthesis is essential for the normal physiology of Synechocystis sp. PCC 6803. We showed here that the slr1736 mutant accumulated the nblA1-nblA2 and sbpA transcripts slightly higher than the wild type even under photoautotrophic conditions (Fig. 7A), suggesting that the mutant is already perceiving a macronutrient stress response related to nitrogen and sulfur under photoautotrophic conditions. It is important to note that this did not affect the growth rates or the PBP content of the tocopherol mutants, perhaps because this level of stress is moderate and thus tolerated under photoautotrophic conditions. After transfer to photomixotrophic growth conditions, the intracellular carbon flux increased further as exemplified by the increased total sugar content in the tocopherol mutants (Table III). One could imagine this would inevitably exacerbate the altered macronutrient homeostasis in the slr1736 mutant. Indeed, under these conditions, the nblA1-2, sbpA, and sigE transcript levels increased dramatically (Fig. 7, A and B), which parallels the decrease in PBP content and the cpcA transcript level (Figs. 6 and 7A). As a result, the tocopherol mutants showed severe chlorosis and growth defects under these conditions (Fig. 2, C and D).
Interestingly, the elevated photosynthetic rate observed for the tocopherol mutants eventually ceased after cells were transferred to photomixotrophic growth conditions. PSII activity was completely lost, no carboxysomes were detectable, and rbcL and other Ci gene transcript levels decreased substantially by 24 h under these conditions (Table II; Figs. 3D and 7C). It is well documented in higher plants that the activity of photosynthesis is negatively regulated by the accumulation of carbohydrates. One aspect of such regulation is triggered by hexoses and their metabolites, which function as signaling molecules and regulate photosynthetic gene expression (for review, see Koch, 1996; Sheen et al., 1999; and refs. therein). Specifically, in Chenopodium and maize (Zea mays), the addition of Glc induces a large transcriptional down-regulation of rbcS (encoding the small subunit of Rubisco; Krapp et al., 1993; Jang and Sheen, 1994), whereas in Arabidopsis the level of the OE33 transcript, encoding the 33-kD oxygen-evolving protein, is subject to Glc repression (Zhou et al., 1998). Therefore, it is plausible that a functionally analogous sugar repression mechanism exists and regulates Ci gene transcription in Synechocystis sp. PCC 6803. Consistent with these ideas, a Glc-sensitive mutant lacking Hik31 has recently been shown to lack glucokinase activity and, correspondingly, a glucokinase mutant cannot grow in the presence of Glc (Kahlon et al., 2006).
In summary, our efforts in reisolating and characterizing tocopherol mutants under photoautotrophic conditions have yielded new insights into the roles and functions for -tocopherol in Synechocystis sp. PCC 6803. The results described here demonstrate that -tocopherol is essential for the normal physiology of Synechocystis sp. PCC 6803 and suggest that, in addition to its role as an antioxidant, -tocopherol plays a role in regulating photosynthesis and macronutrient homeostasis that is independent of this antioxidant activity. It is important to note that maize and potato (Solanum tuberosum) plants, which are defective in tocopherol cyclase activity and are thus tocopherol deficient, also exhibit large alterations in carbohydrate homeostasis due to impaired sugar metabolism/transport (Provencher et al., 2001; Hofius et al., 2004). Although no biochemical or mechanistic explanation exists for this common phenotype between plants and cyanobacteria, it seems plausible that a function for -tocopherol in the regulation of macronutrient homeostasis is conserved between the two groups of oxygenic phototrophs.
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MATERIALS AND METHODS |
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TOPABSTRACTRESULTSDISCUSSIONMATERIALS AND METHODSLITERATURE CITED |
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Isolation of the original slr1736, sll0418, and slr0089 mutants under photomixotrophic growth conditions has been described previously (Shintani and DellaPenna, 1998; Collakova and DellaPenna, 2001; Shintani et al., 2002). A Glc-tolerant wild-type strain of Synechocystis sp. PCC 6803 (Williams, 1988) was used in this study for transformation and isolation of the tocopherol mutants in the absence of Glc (see "Results"). B-HEPES medium, pH 8.0, was used for selection, maintenance, and growth measurements of the wild type and mutants. This medium was prepared by supplementing BG-11 medium (Stanier et al., 1971) with 4.6 mM HEPES-KOH and 18 mg L–1 ferric ammonium citrate. B-HEPES40 medium, a modified B-HEPES medium containing 40 mM HEPES to provide greater buffering strength, was used in some experiments that require greater control of the medium pH during growth. The wild type was maintained on solid B-HEPES medium containing 1.5% (w/v) agar and 5 mM Glc, and the photoautotrophically selected tocopherol mutants were maintained on solid B-HEPES medium containing 1.5% (w/v) agar, 50 µg kanamycin mL–1, and, importantly, no Glc. For determination of growth characteristics, late-exponential phase cultures were diluted into fresh liquid B-HEPES medium to OD730 nm = approximately 0.05 cm–1. The diluted cultures were grown at 32°C with continuous bubbling with air containing 1% or 3% (v/v) CO2. The OD730 nm was monitored to measure growth. The medium was supplemented with 5 mM Glc for photomixotrophic growth conditions. The growth light intensity was 50 µmol photons m–2 s–1, unless otherwise specified.
Construction and Isolation of Mutants
The wild type was transformed with genomic DNA extracted from the previously isolated slr1736, sll0418, and slr0089 mutants (see above). Segregation of mutant alleles from wild-type alleles was carried out in the absence of Glc and in the presence of 50 µg kanamycin mL–1. Segregation was verified by PCR analysis. Oligonucleotide primers used for PCR analysis were as follows: slr1736 forward primer (5'-GGCTTCTCCTACCCGGAATTCTACTTCCTG-3'), slr1736 reverse primer (5'-GCTTTCTAAGTGTACATCTAGACTCCGCCA-3'), sll0418 forward primer (5'-ATGCCCGAGTATTTGCTTCTGCC-3'), sll0418 reverse primer (5'-GCACTGCTTTGAACATACCGAAG-3'), slr0089 forward primer (5'-TCTACCGGAAATTGCCAACTACCA-3'), and slr0089 reverse primer (5'-CCTAGGAGATTGTGGACTTCAA-3'). The pmgA gene was amplified by PCR using forward primer (5'-TTCTCTGTGCCGAAAGCTTCTATG-3') and reverse primer (5'-CACCATGGTGGCGAATTCAGCC-3'). The amplified DNA fragments were digested with HindIII and EcoRI and ligated with pUC19 that had been digested with the same enzymes. An XbaI fragment of pMS266, containing the aacC1 gene that confers gentamicin resistance, was inserted into the unique SpeI site within the pmgA coding region. The resulting plasmid construct was linearized after digestion with EcoRI and used to transform wild-type Synechocystis sp. PCC 6803 cells. Transformants were selected on solid medium B-HEPES at pH 8.0 in the presence of 20 µg mL–1 gentamicin at room temperature under moderate light intensity (approximately 50 µE m–2 s–1). PCR analyses of the transformants were performed with the same primer pairs described above.
Oxygen Evolution Measurements
Cells grown under photoautotrophic or photomixotrophic conditions for 24 h were harvested by centrifugation at 8,000g at room temperature and resuspended in a 25 mM HEPES buffer, pH 7.0, to obtain an OD730 nm = 1.0 cm–1. Oxygen evolution was measured immediately after the addition of 1 mM 1,4-benzoquinone and 0.8 mM K3Fe(CN)6 to the cell suspension. The excitation light intensity was approximately 3 mmol photons m–2 s–1. The oxygen concentration was measured polarographically with a Clark-type electrode as described previously (Sakamoto and Bryant, 1998).
Estimation of Relative PBP Content
The relative PBP content of cells was determined by a minor modification of the method of Zhao and Brand (1989). Cells were harvested by centrifugation at 8,000g for 6 min and pellets were resuspended in 25 mM HEPES buffer, pH 7.0, to obtain cell suspensions (2 mL) with OD730 nm = 0.5 cm–1. These suspensions (1.0 mL) were heated at 100°C for 1 min. The OD635 nm and OD730 nm were recorded for unheated and heated samples, and the values were then inserted into the following equation: relative PBP content = (
OD635 nm – OD730 nm)/OD730 nm·unheated, where OD indicates ODunheated sample – ODheated sample.
SDS-PAGE and Immunoblotting
Cells were grown under photoautotrophic conditions to the midexponential phase or under photomixotrophic conditions for 24 h, harvested as described above, and resuspended in 25 mM HEPES buffer, pH 7.0, to achieve OD730 nm = 100 cm–1. Cells were disrupted using an equal volume of glass beads and a home-built bead beater; cold cell suspensions were vigorously shaken four times for 30 s, interrupted by 30-s intervals on ice. An aliquot (10 µL) of each sample was mixed with an equal volume of loading buffer; the mixture was incubated at 65°C for 20 min and applied onto a discontinuous SDS-polyacrylamide gel with 10% (w/v) acrylamide in the separating gel as described (Schägger and van Jagow, 1987). Prof. Eva-Mari Aro kindly provided antibodies raised against amino acids 234 to 242 of the PsbA protein of Synechocystis sp. PCC 6803. Prof. Robert Burnap kindly provided antibodies raised against the PsbO protein of Synechocystis sp. PCC 6803. After electrophoresis, proteins were transferred to a nitrocellulose membrane. Proteins were detected by immunoblotting by using enhanced chemiluminescence (Amersham Biosciences), according to the manufacturer's specifications.
Isolation of Total RNA and RT-PCR Analyses
Total RNA was isolated and purified from cells using the mini-to-midi RNA isolation kit (Invitrogen). The RNA samples were purified again after DNase digestion. The RNAs obtained were adjusted to a final concentration of 50 ng RNA µL–1 and stored at –80°C until used. Transcripts were amplified and detected by using the one-step RT-PCR kit (Qiagen) in the presence of the RNase inhibitor RNAsin (Promega) with target-specific oligonucleotide primers. The sequences of the primers used for each of the indicated genes will be made available upon request.
Transmission Electron Microscopy
Cells grown under photoautotrophic and photomixotrophic conditions for 24 h were harvested and immediately fixed overnight at 4°C in a 2.5% (v/v) glutaraldehyde solution prepared in 0.1 M cacodylate buffer, pH 7.4. After secondary fixation in a 1% (w/v) osmium tetroxide solution in the cacodylate buffer, the cells were stained with uranyl acetate (2% w/v), followed by dehydration in the following concentrations of ethanol: 50% (v/v), 70% (v/v), 90% (v/v), 95% (v/v) ethanol in water followed by two washes in 100% (v/v) ethanol. The samples were then embedded in Spurr's resin and polymerized overnight at 60°C. Thin sections (approximately 50- to 60-nm thickness) were stained with 2% (v/v) uranyl acetate before examination under a JEM 1200 EXII transmission electron microscope (JEOL).
Total Sugar Assay
Cells were harvested as described above and washed and resuspended in distilled water to achieve the same OD730 nm. The total sugar content of each cell suspension was determined by a previously described colorimetric assay (Dubois et al., 1956). The total sugar content was calculated relative to the A435 nm for the wild-type cells grown under photoautotrophic and photomixotrophic conditions.
Analysis of Tocopherol Content
The tocopherol content of the wild-type and mutant strains was analyzed as described previously (Cheng et al., 2003).
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ACKNOWLEDGMENTS |
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Received January 1, 2006; returned for revision March 10, 2006; accepted March 10, 2006.
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FOOTNOTES |
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2 Present address: Department of Plant Biology, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK–1871 Frederiksberg C, Denmark.
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: Donald A. Bryant (dab14{at}psu.edu).
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.074765.
* Corresponding author; e-mail dab14{at}psu.edu; fax 617–738–7664.
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-Tocopherol Plays a Role in Photosynthesis and Macronutrient Homeostasis of the Cyanobacterium Synechocystis sp. PCC 6803 That Is Independent of Its Antioxidant Function
