Effects of tocopherol deficiency
In Synechocystis, α–tocopherol has been proposed to have specific (probably regulatory) homeostatic roles beyond its antioxidant activity. A Synechocystis HPT-deficient mutant (Figure S1) was reported to be extremely glucose-sensitive (Sakuragi et al., 2006). However, our Δhpt strain is only slightly compromised under photomixotrophic conditions. The discrepancy between the data is resolved if the pH of the growth medium is considered. The previously described mutant (slr1736 ) was inhibited by added glucose at pH approximately 7.2 and lower, whereas at higher pH (e.g. 7.6 and 8.0), the mutant grew similarly to the WT (Sakuragi et al., 2006). In our experiments, the cultures were started at an initial pH approximately 8.0, not buffered and aerated with ambient air. The pH rose gradually to approximately 9.0 during growth until the stationary phase was reached. Good photomixotrophic growth was also observed by other researchers for an analogous mutant under a similar aeration regime with no buffer added (Sakuragi, 2004). The initial pH of their medium was also 8.0; hence, the growth conditions were similar to ours (Sakuragi, 2004). However, if the medium was bubbled with 3% CO2-enriched air and not buffered, the pH eventually decreased to approximately 7.0. Consequently, growth was inhibited, with concomitant strong bleaching of the culture (Sakuragi, 2004). However, if under the same conditions, the pH was kept at the initial value (8.0) using HEPES buffer, the mutant displayed persistent growth and phenotype (Sakuragi, 2004; Sakuragi et al., 2006). Thus, the cultivation conditions used in our study were permissive for the Δhpt strain. Under photoautotrophic conditions, pH does not influence the Synechocystis strains that lack α–tocopherol, e.g., slr1736, slr0089 and slr0090 (Sakuragi, 2004; Sakuragi et al., 2006).
Nevertheless, adverse effects of α–tocopherol deficiency, such as reduced PBS and PSI contents, were observed in the Δhpt strain grown photomixotrophically, even at high pH (Figures 1 and 2, and Table 1). These observations are consistent with results obtained by other researchers: in the presence of glucose at pH 8.0, the HPT-deficient mutant used by Sakuragi (2004) showed a pale green–yellow coloration, as did our Δhpt strain, due to slight de-pigmentation, particularly decreased PBS content (Figure 1). Thus, the present study corroborates previous data indicating that α–tocopherol may play specific homeostatic roles in Synechocystis (Sakuragi, 2004; Sakuragi et al., 2006). Consequently, although ΔchlP accumulates α–tocotrienol (Shpilyov et al., 2005), which also has a high antioxidant potency (Yoshida et al., 2003), loss of α–tocopherol was hypothesized to be critical in the mutant. Thus, one may expect the phenotypes of ΔchlP and Δhpt to be very similar, given that both strains lack α–tocopherol.
However, the comparative analyses revealed specific features for the ΔchlP mutant. The most striking trait is loss of photoautotrophic growth. Glucose appears to provide an additional energy/carbon source to maintain steady-state levels (through high rates of re-synthesis) of PSI and PSII in ΔchlP (Shpilyov et al., 2005). In contrast, the stability of PSI/II in Δhpt is comparable to that of the WT, as deduced from its robust photoautotrophic growth. Additionally, the 77 K fluorescence emission spectra imply that both PSI and PSII in ΔchlP are structurally perturbed, which is not observed in Δhpt (Figure 2).
The slightly increased light sensitivity of Δhpt (Figures 3-6) cannot be interpreted unambiguously. A possible reason is the absence of α–tocopherol as an antioxidant. Alternatively, this and other specific traits of the strain, e.g. somewhat different PBS, PSI and carotenoid contents (Figures 1-4 and Table 1), may be a side effect of glucose in a tocopherol-less background. Clarification of this issue requires further investigations beyond the scope of the present study.
The present study revealed extremely fast degradation of PSI and PSII induced by strong light in ΔchlP (Figures 3, 5 and 6). Evidently, α–tocopherol deficiency is not the determining factor in this regard, as, under the same conditions, Δhpt displayed only a slightly increased PSI instability in comparison to the WT (Figure 5). Furthermore, the abundant myxoxanthophyll in ΔchlP cells grown under standard light (Figure 4) indicates that the mutant experiences photo-oxidative stress, even under moderate illumination. This observation is corroborated by the more rapid degradation of PSI/II in the mutant under light of 40 μmol photons m−2 sec−1 than in darkness (Shpilyov et al., 2005). Under stronger irradiation (e.g. 500 μmol photons m−2 sec−1), photo-oxidative stress is further aggravated in the mutant, leading to very fast destruction of PSI and PSII. This type of enhanced light sensitivity was not observed in Δhpt and the other Synechocystis strains that lack vitamin E (Dähnhardt et al., 2002; Savidge et al., 2002; Sattler et al., 2003; Sakuragi, 2004; Sakuragi et al., 2006).
Thus, the present data indicate that both PSI and PSII are structurally altered in ΔchlP, unstable and very vulnerable to photodegradation, and these traits are not due to α-tocopherol deficiency in the mutant.
Effects of phylloquinone deficiency
The function of phylloquinone (vitamin K1) (Table S1) in the A1 site of PSI has been extensively explored for more than two decades. Approaches such as reconstitution of the A1 site with foreign quinones in vitro as well as in vivo were very informative. These studies revealed that diverse benzo-, naphtho- and anthraquinones – and even so-called ‘quinonoids’ – bind to the A1 site and function as efficient redox co-factors (Table S1) (reviewed by Ikegami et al., 2000; Itoh et al., 2001; Johnson and Golbeck, 2004; Srinivasan and Golbeck, 2009). Even the tail-less phylloquinone analog menadione (vitamin K3) (Table S1) may occupy the A1 site in the correct orientation and sustain A0→FX electron transfer in vitro (Iwaki and Itoh, 1989, 1994; Kumazaki et al., 1994; reviewed by Ikegami et al., 2000; Itoh et al., 2001). Thus, the ‘head’ group of a quinone (phylloquinone in most oxygenic phototrophs; see below also) is the main factor determining its binding, coordination and redox activity in PSI. However, the ‘tail’ is assumed to improve the quinone binding affinity through hydrophobic interaction with proteins (Iwaki and Itoh, 1989, 1994; Kumazaki et al., 1994; reviewed by Ikegami et al., 2000; Itoh et al., 2001), but the chemical nature of the tail appears not to be critical, as different long-chain isoprenoids, or even non-branched alkyl substituents, may also bind efficiently to the A1 site (Biggins, 1990; Srinivasan and Golbeck, 2009). For the phylloquinone-deficient Synechocystis mutants (menA, B, D and E), it has been shown in vivo that plastoquinone–9 (Table S1) functions at the A1 site but with diminished efficiency due to the more oxidizing redox potential (Johnson et al., 2000; Semenov et al., 2000; Zybailov et al., 2000; reviewed by Johnson and Golbeck, 2004; Srinivasan and Golbeck, 2009). Additionally, incorporation of plastoquinone into PSI leads to a decrease in PSI content (Johnson et al., 2000). Together, both effects result in a reduction of the whole ETC capacity by approximately 40% (Johnson et al., 2000; own observations). However, the capability for photoautotrophic growth is not abolished in these mutants (Johnson and Golbeck, 2004; Srinivasan and Golbeck, 2009; own observations). Altogether, the available data demonstrate that PSI possesses a considerable capacity to accommodate quinones of various structure and size at the A1 site (Table S1).
The incorporation of phylloquinone into PSI requires neither enzymatic activity nor de novo protein synthesis, and is not accompanied by disassembly/re-assembly of PSI in vitro (reviewed by Ikegami et al., 2000; Itoh et al., 2001) and in vivo (Johnson et al., 2001). Moreover, PSI containing a variant quinone, which may be considerably different in size and/or binding affinity, or even with an empty quinone-binding pocket, remains relatively stable in vitro (reviewed by Ikegami et al., 2000; Itoh et al., 2001) as well as in vivo (Johnson et al., 2000, 2001). The available data indicate that – in contrast to the role of Chl a in biogenesis of PSI (and PSII) (Eichacker et al., 1992, 1996; Adamska et al., 2001) – phylloquinone does not determine assembly and stabilization of PSI, at least not in cyanobacteria such as Synechocystis (Johnson and Golbeck, 2004; Srinivasan and Golbeck, 2009).
So far, the nature of the quinone in PSI of the ΔchlP mutant remains to be established. However, several lines of evidence suggest that this may be the geranylgeranylated phylloquinone analog menaquinone–4 (vitamin K2(20)) (Figure S1 and Table S1). First, there is no GGR activity in the mutant (Shpilyov et al., 2005). Hence, formation of (phytylated) phylloquinone is impossible (Figure S1). Second, if synthesis of the tailed vitamin K is interrupted at the MenA stage (Figure S1) or earlier, only plastoquinone–9 occupies the A1 site in the respective Synechocystis mutants (Johnson and Golbeck, 2004; Srinivasan and Golbeck, 2009). Third, the ΔchlP mutant possesses a fully functional ETC (Shpilyov et al., 2005) indicative of a highly active PSI, which appears to exclude the presence of plastoquinone–9 at the A1 site. Therefore, PSI in ΔchlP appears to contain a quinone with a redox capacity comparable to that of phylloquinone, but not phylloquinone itself.
According to the phylloquinone biosynthetic pathway, menaquinone–4 is indeed such a candidate (Figure S1). It possesses the same head group as phylloquinone and differs from phylloquinone only by three additional double bonds in the tail moiety, which appear not to be critical (see above). Indeed, in vitro reconstitution studies established menaquinone–4 to be a fully functional analog of phylloquinone, i.e. displaying comparable binding and electron-transfer properties at the A1 site (Iwaki and Itoh, 1989; Biggins, 1990; Iwaki and Itoh, 1991, 1994; Kumazaki et al., 1994; reviewed by Ikegami et al., 2000; Itoh et al., 2001). Furthermore, menaquinone–4 synthesis and efficient operation in PSI have been demonstrated using isolated spinach chloroplasts (Kaiping et al., 1984) and GGR-deficient rice mutants (Shibata et al., 2004b). Finally, menaquinone–4 was found to function at the A1 site in several oxygenic phototrophs, which, however, use phytylated Chls (Table S1).
Irrespective of the quinone species in PSI in the ΔchlP mutant, this cannot be the reason for PSI instability, as the quinone is not a factor influencing PSI assembly and stabilization, as mentioned above. 77 K fluorescence emission spectra of an analogous menA mutant indicated only depletion in PSI content, with no sign of structural alterations (Johnson et al., 2000). Moreover, the instability of PSII in ΔchlP is certainly not related to the quinone present in PSI, as the biogenesis and function of PSII are independent of those of PSI in cyanobacteria, as deduced from data obtained using phylloquinone-deficient (Johnson et al., 2000) and PSI-lacking Synechocystis mutants (Shen et al., 1993; Wu and Vermaas, 1995).
Hence, as in the case of tocopherol, impaired phylloquinone synthesis also cannot be the origin of the instability and high vulnerability of PSI and especially PSII to photodegradation in the ΔchlP mutant.
Requirement for chlorophyll phytylation
Active ETCs in GGR-deficient mutants of oxygenic phototrophs (Shibata et al., 2004a,b; Shpilyov et al., 2005) indicate that geranylgeranylated electron transfer co-factors, i.e. Chl aGG, pheophytin aGG and menaquinone–4 (see above), are functional. However, when assembled with Chl aGG, PSI and PSII complexes become unstable and tend to degrade spontaneously (i.e. in complete darkness; Shpilyov et al., 2005). The geranylgeranyl residue is more rigid than a phytyl residue due to three additional double bonds. This increased rigidity probably perturbs the association of ChlsGG with apoproteins, possibly also disturbing the interaction of protein subunits with each other. Reaction centre preparations from a GGR-deficient mutant of the purple bacterium Rhodobacter capsulatus synthesizing bacteriochlorophyll (BChl) aGG were also found to be much less stable than similar preparations from WT cells (Bollivar et al., 1994). Thus, phytylation of (B)Chls appears to be generally important for the stability of pigment–protein complexes among chlorophototrophs, with the exception of one purple bacterium, Rhodospirillum rubrum (see below), and heliobacteria. The latter accumulate BChl g with a fully unsaturated C15 isoprenoid (farnesyl) tail (Madigan, 2006). It should be noted that heliobacteria only grow photoheterotrophically or even heterotrophically (Madigan, 2006).
From an evolutionary perspective, (B)Chl tail saturation has apparently become more critical for oxygenic photosynthesis. For example, GGR-deficient mutants of anoxygenic photosynthetic bacteria are still capable of photoautotrophic growth, although less efficiently (Bollivar et al., 1994; Addlesee and Hunter, 1999; Harada et al., 2008a). Moreover, depending on growth stage and conditions, these organisms naturally accumulate various amounts (6–30%) of BChls with unsaturated tail moieties (Bollivar et al., 1994; Addlesee and Hunter, 1999; Mizoguchi et al., 2006; Harada et al., 2008ab). The purple bacterium Rhodospirillum rubrum even accumulates only BChl aGG – although bacteriopheophytin a in its reaction centre is phytylated due to the activity of a special bacteriopheophytin aGG reductase (Addlesee and Hunter, 2002). In striking contrast, Chls with only partially saturated tails never occur in the mature photosynthetic apparatus of oxygenic phototrophs (Tamiaki et al., 2007), and GGR deficiency in these organisms is lethal (Henry et al., 1986; Shibata et al., 2004a; Shpilyov et al., 2005).
Remarkably, similar light sensitivity as in the cyanobacterial ΔchlP mutant was observed in GGR-deficient plants, e.g. tobacco (Tanaka et al., 1999) and rice (Shibata et al., 2004a,b). The likely explanation is that perturbed binding and spatial orientation of Chl aGG in the pigment–protein complexes impedes interactions and thus efficient excitation energy transfer among Chls and possibly also to carotenoids. In turn, this may lead to the build-up of triplet excited states of Chls (3Chl*), leading to the generation of destructive reactive oxygen species (particularly 1O2) upon illumination. This is apparently the reason for aggravated photo-oxidative stress and the high vulnerability of pigment–protein complexes to light-induced degradation in GGR-deficient mutants of oxygenic phototrophic organisms. Thus, beyond stabilizing photosynthetic pigment–protein complexes by Chl phytylation, the second important role of the GGR enzyme in oxygenic phototrophs appears to be to ensure the optimal coordination of Chl molecules in these complexes that is necessary for efficient utilization of light energy and thus protection from photo-oxidative stress in an oxygenic environment.