The Synechocystis model of stress: from molecular chaperones to membranes

Plant Physiol. Biochem., 1999, 37 ( 1 ), 1 - 12

Review

The Synechocystis model of stress: From molecular chaperones to membranes

Attila Glatz l, lmre Vass 2, Dmitry A. Los 3, Lfiszl6 Vigh 1.

t Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, P.O.B. 521, 6701 Szeged, Hungary. 2 Institute of Plant Biology, Biological Research Centre, Hungarian Academy of Sciences, P.O.B. 521, 6701 Szeged, Hungary. 3 Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya str. 35, 127276 Moscow, Russian Federation.

* Author to whom correspondence should be addressed (fax +36 62 432048; e-mail [email protected])

(Received August 20, 1998; accepted November 9, 1998)

Abstract - A highly conserved and exquisitely regulated cellular response to most stress conditions, such as cold, heat and visible or UV-light, is common to all organisms. Cells that have been pre-exposed to a sublethal dose of these stimuli can acquire a transient resistance against the killing effect of a subsequent stress. Specific membrane lipids and stress proteins, many of them molecular chaperones, play a fundamental role in these acclimation processes. According to the presently discussed model, cellular membranes are not simply the primary sites of stress damage, they are also able to sense extreme environmental changes and to activate stress-defence genes remotely as a consequence of a rapid modification in their physical state and microdomain organisation. Underlying the interdependence of the redox sensory pathway and membrane fluidity, sudden changes in membrane order can be signaled to gene expression via the light requiring redox sensory pathway. The photosynthetic Synecho- cystis PCC 6803 has several features that render it a particularly suitable model for such studies. While the general assembly and lipid composition of its thylakoid is similar to that of higher plants, it can easily be transformed by foreign DNA. Nonethe- less, its whole genome has been determined and has become available via the internet. © Elsevier, Paris

Desaturase / light stress / membrane physical state / molecular chaperone / photoinhibition and UV-B damage / redox sensory pathway / temperature stress and adaptation

HSP, heat-shock protein / PQ, plastoquinone / PSII, photosystem II / sHSP, small heat-shock protein

1. I N T R O D U C T I O N

Living organisms have the capability to adapt to sudden changes in their environment. Since most of these stimuli (temperature up- and downshift, UV-B light, etc.) have detrimental effects on cells, they had to develop adequate protective systems. In the last decade, intensive research has been carried out so as to understand the mechanisms that protect cells against stress-induced damages enabling them to sur- vive under adverse conditions. For instance, upon heat shock, proteins tend to misfold which can lead not only to functional loss, but also to their aggregation. Molecular chaperones, especially the DnaK (Hsp70) and chaperonin (Hsp60) families, could efficiently

help to avoid this dangerous situation (for recent reviews see [5, 49]). According to a recent model, the small heat-shock proteins (sHSPs) are also able to act as molecular chaperones in conjunction with other types o f chaperones mostly by serving as an efficient initial rescue component within heat stressed cells ([5], Goloubinoff et al. unpubl.).

Proteins are not the sole target o f different stress- induced damages. Considering that several essential processes (signal transduction, energy production, etc.) are tightly coupled to cellular membranes, it could be predicted, that irreversible disorganisation o f membranes leads ultimately to cell death. It is also well established that the composit ion and the physical order of membranes is strongly affected by the envi-

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2 A. Glatz et al.

ronmental temperature, indicating that biomembranes belong to the key cellular components that have an important role in the adaptation and stress response of different organisms [74]. This minireview is intended to highlight the contribution of molecular chaperones and membranes to stress response and adaptation.

The unicellular photosynthetic organism, Syn- echocystis sp. PCC 6803, has several features that make this strain particularly suitable for studying stress response at the molecular level. There is unequivocal evidence that in higher plant cells exposed to heat stress, the photosynthetic apparatus is irreversibly damaged prior to impairment of other cel- lular functions [3]. The general assembly of photosyn- thetic membranes in cyanobacteria is similar to that of higher plants; therefore, Synechocystis 6803 might serve as a powerful model for studying the molecular mechanisms of stress response and long-term adapta- tion [37, 71, 73]. Being naturally competent, this cyanobacterium can easily be transformed and foreign DNA integrates into the genome at a high frequency [77]. Recently, the complete nucleotide sequence of the Synechocystis 6803 genome has been determined [27] and the collection of the annotated data (Cyano- base) is available via the internet. Moreover, a useful promoter-probing vector has been constructed [43], and the recently published 'plasmid shuffling' method allows the manipulation and mutational analysis of essential genes in this organism [53].

2. HEAT S H O C K A N D C H A P E R O N E S

2.1. The chaperonins

In contrast to most bacteria, Synechocystis 6803 contains two groEL-type genes: one is organised in a groESL operon [38] and the other is a cpn60 gene which possesses no groES in its neighbouring region [8]. Northern analysis showed that these genes are activated when cells are subjected to elevated tempera- tures. Primer extension studies revealed that the tran- ,scriptional startpoints of both chaperonin genes are located within a highly conserved inverted repeat present in the upstream region of both genes [11, 12], It is well documented that this element - originally designated as CIRCE (controlling IR of chaperone expression [80]) - works as an operator. Binding of the repressor protein (HrcA) could abolish the expression of these genes under non heat-shock conditions. In many bacteria, the hrcA gene is part of an operon con- taining genes encoding for the dnaK-type of chaper- ones. This kind of transcriptional regulation seems to

be more widely spread in eubacteria ([59], and refer- ences therein) than the c32-type described for Escheri- chia coli (for a review see [4]). It is likely, that CIRCE also acts as an operator of both chaperonin genes in Synechocystis 6803. Similarly, a search in Cyanobase [27] uncovered the existence of an ORF displaying significant homology to other eubacterial HrcA pro- teins [12]. Interestingly, neither dnaK nor any other heat-shock genes are present near the predicted hrcA gene, indicating that transcriptional regulation of chaperonins in Synechocystis 6803 might differ from what is seen in other eubacteria [59].

Keeping in mind the extreme stress-sensitivity of the photosynthetic apparatus, we have initiated studies on the inducibility of chaperonin genes under various tem- perature and light conditions. Interestingly, the two groEL-type genes displayed altered expression pattern under certain conditions despite the presence of the common CIRCE element. Surprisingly, the cpn60 gene did not respond to sublethal heat treatment when cells were preincubated and exposed to heat stress in the dark. Although the groESL operon produced elevated mRNA level under the same conditions, the rate of induction was significantly lower than that observed in the light. Both chaperonin genes proved to be 'light-sensitive': they showed increased transcriptional activity when Synechocystis 6803 cells were moved from dark to light, even in the absence of heat shock. A pronounced differ- ence could also be observed between the induction of the two chaperonin genes when cells were transferred from light to dark and subjected to thermal stress. While the activation of the groESL operon remained unaffected (compared to the result obtained when cells were moved from dark to light and heat treated), the cpn60 gene did not show such a strong induction [12]. We observed a similar phenomenon when Synechocystis cells were treated with the photosynthetic inhibitor diuron. These results indicate, that light conditions and/or the function- ality of the photosynthetic apparatus have great influ- ence on chaperonin gene expression. One might speculate whether the stability of the mRNAs is affected during the different treatments or whether the existence of additional transcriptional regulatory mechanism(s) is responsible for the altered expression pattern of the groEL-related genes. Although cpn60 mRNA is less sta- ble under heat-shock conditions (in light) than the groESL message (Glatz et al., unpubl.), attempts are made to explore (or rule out) the presence of hitherto unidentified regulatory mechanism(s).

In a previous study, it has been shown that sublethal heat stress enhances the synthesis of at least four types

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Membranes and chaperones 3

(i.e. Hsp70, -60, - 17 and - 14) of heat-shock proteins in Synechocystis 6803 [36]. The protein products of the chaperonin genes have also been resolved using two dimensional SDS-PAGE and identified by N-terminal sequencing. It was demonstrated that significant amounts of both GroEL and Cpn60 are associated to the thylakoid membranes under both normal and ele- vated temperatures [33]. Given that purified E. coli GroEL can bind to model membranes and remain functionally active [63], one might speculate that chaperonins play an important role in the protection of the photosynthetic apparatus [32, 33].

2.2. DnaK homologues

Compared to the GroEL-GroES 'chaperon machine', much less is known about the dnaK-type genes and their products in Synechocystis 6803. It has been suggested that the transcriptional regulation of dnaK [8] is different from that of chaperonin genes, since dnaK was activated at significantly higher temperatures [11]. Heavy metal (Cd 2+) treatment proved to be a strong inducer of dnaK while chapero- nin genes remained uninduced under these conditions [11 ]. Recently, it has been shown that two additional dnaK homologues are present in the genome of Syne- chocystis. Four dnaJs and a grpE analogue open read- ing frames have also been identified [27]. It was shown in another cyanobacterial strain, Synechococcus PCC 7942, that one of the three DnaKs is associated to thylakoid membranes [50]. This result might indicate, that not only chaperonins, but the DnaK family could also be involved in the protection of thylakoid mem- branes.

2.3. Hspl7, the small heat-shock protein homologue in Synechocystis

Of the several classes of HSPs, the small heat-shock proteins (sHSPs) are the least characterised in terms of their regulation and function. Members of the sHSP family in photosynthetic organisms share characteris- tic C-terminal sequences that have also been con- served in alfa-crystallin proteins, sHSPs assemble into large globular complexes which may vary depending on temperature and other conditions. Whereas high levels of sHSPs correlate with acquired stress resis- tance, the precise mechanism by which sHSPs confer stress resistance remained hitherto unclear [35]. In Synechocystis, a sHSP, Hspl7, with significant homol- ogy to a higher plant class I sHSPs was recently identi- fied and shown to be encoded by a 'membrane fluidity

gene'. In fact, transcription of hspl 7 was strongly cor- related with the actual physical order of thylakoid membranes [21 ]. Studies are in progress in our labora- tories to understand how thylakoid physical state exerts a transcriptional control on hspl7. Like several members of sHSPs which have been reported to asso- ciate reversibly with thylakoid membranes in parallel with increased resistance to light and heat stress [16], a large pool of Synechocystis Hspl7 also became membrane bound under temperature stress [21]. Our preliminary in vitro studies indicate that the purified Synechocystis 6803 Hspl7 can bind heat-denatured proteins and prevent protein aggregation. Moreover, Hspl7 can promote protein refolding after stress, in collaboration with DnaK/DnaJ/GrpE chaperones and ATP. Thus, it is tempting to speculate that Hspl 7 plays the two complementary roles of stabilising concomi- tantly stress-labile membranes and aggregation-prone labile proteins.

3. COLD STRESS AND DESATURASES

3.1. Glycerolipids and fatty acids of cyanobacteria

Cyanobacterial cells resemble chloroplasts of higher plants in terms of cellular and membrane structure, and glycerolipid composition [30]. The major glycero- lipid components of both plasma membranes and thylakoid membranes of cyanobacteria are monogalac- tosyl diacylglycerol, digalactosyl diacylglycerol, sulphoquinovosyl diacylglycerol, and phosphatidyl- glycerol. Monogalactosyl diacylglycerol contributes about 50 % of total glycerolipids. The ratio between them differs depending on the strain and growth condi- tions. In addition to these four major lipids, cyanobac- terial cells contain a minor one, monoglucosyl diacylglycerol, at the level of 1% of total glycerolip- ids [52].

Glycerolipids form bilayers and provide the neces- sary structural background for the insertion and func- tioning of membrane proteins. The physical properties of glycerolipids depend predominantly on the degree of unsaturation of the fatty acids that are esterified to the glycerol backbone of the lipids. Therefore, the molecular motion or fluidity of the membranes is remarkably controlled by the changes in unsaturation of membrane lipid fatty acids [9, 20, 54, 70]. The level of unsaturation of membrane lipid fatty acids is altered by changes in the growth temperature of the organism. Such temperature-induced changes in the unsaturation of fatty acids are explained in terms of the regulation

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of membrane fluidity that is necessary for the proper functioning of biological membranes [9]. When the fluidity of the membrane is reduced by a decrease in temperature, plants and cyanobacteria respond by introducing double bonds into the fatty acids of lipids, so that membranes return to a more fluid state [63, 71 ]. The low temperature-induced increase in the level of unsaturated fatty acids was observed and described for a number of cyanobacterial strains, including Syn- echocystis 6803 [51]. In general, the downward shift of temperature leads to the accumulation of polyunsat- urated fatty acids, such as 18:2, 18:3 and 18:4, whereas the level of monounsaturated fatty acids, 18:1, drops [75]. Highly specific enzymes, fatty acid desaturases, are responsible for the introduction of double bonds into the chains of fatty acids of mem- brane lipids [62].

3.2. Temperature-dependent expression of desaturase genes

The cyanobacterium Synechocystis 6803 has four distinct membrane-bound acyl-lipid desaturases, each of which catalyses the desaturation at positions A9 [56], A12 [76], A6 [55] and o3 [57] of C18 fatty acids esterified at the sn-1 position of the glycerol moiety. The order of the desaturation is very strict: the first double bond is introduced by the A9 desaturase (18:0 ---> 18:1a9); the second by the A12 desaturase (18:1 A9

18:Ux9't2); and the third by the A6 desaturase (18:2 A9A2 --~ 18:2~6'9'12). The expression of the 03 desaturase is detected only at low temperatures and leads to the synthesis of 18:3 A9'12'~°3 and 18:4 ~6'9'12't°3 fatty acids as well.

The level of the mRNA for A9 desaturase does not change with the fluctuations of temperature. This gene is transcribed at a very high level at 34 °C, compared to other desaturase genes. It is co-transcribed with the gene located downstream which encodes the acetyl- CoA synthetase [41]. The very high level of gene expression for A9 desaturase and the failure to disrupt the gene [61] support the primary importance of the A9 desaturase for the proper order of membrane lipids, where the balance between saturated and unsaturated fatty acids is critical.

The levels of mRNAs for the genes encoding A6, A12, and 03 desaturases increase 10-15-fold upon the shift in temperature from 34 to 22 °C [40, 41]. These mRNAs are barely detectable at temperatures above 30 °C, but become abundant within 30 min of a down- ward shift in temperature. Low temperature-induced up-regulation of transcription of the genes for A12 and

m3 desaturases was demonstrated using the luciferase reporter gene, which was fused to the promoters of the corresponding genes. A 5-8-fold increase in lumines- cence from the reporter was observed upon lowering the temperature from 35 to 25 °C. Hence, these desat- urase genes may be considered as real cold-inducible genes [41].

It is well known that desaturation of fatty acids is a light-dependent process [75]. Activation of the gene for A12 desaturase at low temperatures was detected only in the light, but never in the dark [39]. The inhib- itor of photosynthesis, diuron, also inhibits transcrip- tion of this gene [40]. The same effects were observed for the gene encoding for 0)3 desaturase (Los, unpubl. results). It is tempting to speculate that photosynthetic energy is essential for the expression of desaturase genes.

The half-life of the A9 desaturase mRNA is about 10 min at normal and low temperatures. Lifetimes of mRNAs for temperature-inducible desaturases, A6 and A12, increased from about 2 min at 34 °C to about 20 min at 22 °C. The half-life of the o3 desaturase mRNA at 34°C was less than l m i n and it was approximately 10 rain at 22 °C [41].

Thus, an increase in the stability of these mRNAs explains in part their accumulation at low tempera- tures. It is likely that the elevated level of these three mRNAs after the downward shift in temperature was the result of the synergistic effects of the activation of transcription and the increase in the stability of the mRNAs. By contrast, the expression of the A9 desatu- rase gene was not associated with changes in either the rate of transcription or the stability of the mRNA [41].

3.3. Desaturase enzymes and their subcellular iocalisation

The western blotting data confirmed the conclusions that were obtained at the mRNA level, i.e. A9 desatu- rase is expressed independently of temperature, whereas the protein levels of A12, A6, and o3 desatu- rases increase with the drop in temperature.

Recently, all four desaturases have been localised in Synechocystis 6803 cells by western blotting and immuno-gold microscopy approaches [47]. It was found that the desaturases are located in both plasma and thylakoid membranes. It implies that not only the plasma membrane, but also the thylakoid membrane are the sites of fatty acid desaturation. This finding may be of primary importance for understanding the mechanisms of adaptation of the photosynthetic mem- branes of cyanobacteria to the changing environment.

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3.4. Role of desaturases in adaptation to low temperatures

The roles of individual desaturases in low-tempera- ture adaptation were recently studied in Synechocystis 6803 mutants that were created using site-directed car- tridge mutagenesis of the desaturases genes [61]. It was shown that disruption of A6 and o~3 desaturases genes (A6-, o)3-, and A6-/o)3- mutants) did not change the physical properties of the membrane lipids, and did not alter the ability of the organism to grow and to sur- vive at low temperatures. In contrast, in the double mutant, A12-/A6-, that synthesised only monosatu- rated fatty acids, 18:1A9, the fluid to gel phase-transi- tion point of the membrane lipids shifted up remarkably in comparison to wild-type cells. The lat- ter mutations caused the low temperature-sensitive phenotype, and made the mutant extremely sensitive to low temperature photoinhibition [61]. Thus, the ability of Synechocystis 6803 to synthesise dienoic fatty acids may be referred to as the ultimate condition for surviv- ing low temperatures. It is notable that, in contrast to the above findings of Tasaka et al. [61], we recently obtained an elevated, rather than reduced, thylakoid membrane fluidity in this double mutant by using the DPH anisotropy technique (Trrrk, pers. comm.). Fur- ther studies are in progress to investigate this apparent contradiction in results obtained in the two laborato- ries.

4. STRESS RESPONSES IN THE PHOTOSYNTHETIC APPARATUS

The thylakoid membrane and the embedded redox complexes of the photosynthetic apparatus, especially the light energy-converting photosystem II (PSII), are very sensitive targets to various environmental stress factors. Among these, the most important are: supra- and suboptimal temperatures, light in the visible and ultraviolet spectral range, heavy metals, various air pollutants and a large class of herbicides which act on the electron transport in PSII.

4.1. High temperature stress and adaptation in thylakoid membranes

Thermal acclimation by Synechocystis 6803 cells by upshifting the growth temperature from 22 to 36 °C afforded significant protection to otherwise lethal tem- peratures using Synechocystis cells. Threshold temper- atures at which the efficiency of PSII became impaired

overlapped with characteristic temperatures above which heat treatment caused a severe decrease in cell survival [37]. Long-term heat hardening of the photo- synthetic apparatus has been correlated with a reduc- tion in the level of lipid unsaturation and an elevation of the protein-to-lipid ratio in parallel with an overall increase in microviscosity of thylakoid membranes [21, 73]. These tendencies are also known to act towards the prevention of the formation of non-bilayer lipid phase [20]. Changes in growth temperature mark- edly influenced the size of protein-immobilised lipid pool [71 ]. It is still questionable whether the level of lipid unsaturation, usually highly reduced upon growth temperature increase in most systems [20], is a com- ponent of thylakoid thermal stability. As in Arabidop- sis mutants deficient in specific polyunsaturated fatty acids [22], enhancement of thermal stability was observed following in situ catalytic hydrogenation of pea thylakoids [70]. In contrast, the photosynthetic machinery in the Fad6/desA::Km r mutant of Syn- echocystis, which does not contain any polyunsatu- rated lipid molecules, exhibited a small but distinct decrease in heat tolerance compared to wild-type and Fad6 mutant [15]. It was shown, however, that whereas these cyanobacterial mutants can withstand large decreases in the amount of polyunsaturated fatty acids, a compensatory response is triggered in order to restore the optimal physical state of the photosynthetic membranes (Trr~k et al., unpubl.). This finding is in full accordance with the capability of cyanobacteria to rapidly readjust membrane fluidity induced either by temperature shift, nitrate starvation or metabolic inhib- itors [13, 14].

It is clearly emerging that cell membranes, when exposed to an abrupt increase in temperature, undergo an immediate reorganisation through changes in the membrane's physical structure. A rapid, non-lethal heat exposure of Synechocystis induced the enhance- ment of PSII thermotolerance in parallel with HSP synthesis and an increased thylakoid molecular order. By using fluorescence lifetime distribution technique, we have also shown that heat pretreatment resulted in modifications in the environmental heterogeneity of the fluorophore diphenylhexatriene. The membrane reorganisation occurs in parallel with the association of GroEL-type chaperonins to the thylakoid [32, 33] and the appearance of highly saturated monoglucosyl diacylglycerol, a precursor of the major lipid compo- nent monogalactosyl diacylglycerol, in the membrane. The thermoprotection caused by heat adaptation together with characteristic changes in membrane

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physical state operate only in light, while precondi- tioning occurring in darkness is ineffective (T6r6k et al., unpubl.). This finding is in accordance with the light dependent transcriptional control of the two GroEL analogues in Synechocystis cells [12]. Based on a recently proposed model, the chaperonin-lipid interaction together with the retailoring of lipid mole- cular species may act as a rapid and effective adap- tive tool to stabilise thermally-stressed membranes [63]. Furthermore, the modulation of the membrane's physical order may repress transcription of heat- shock genes in the heat-adapted state explaining the known temporary induction of the stress response. Thus, such 'cross-talk' between the 'heat-shock sensor membrane' and heat-shock response suggests the existence of a as yet unknown feedback mechanism for heat-shock gene regulation and possibly also oper- ative for other genes regulated by signalling cascades [21, 74].

4.2. Organisation and function of photosystem II under normal and stress conditions

Due to the unique oxygen-evolving function, which is the source of the Earth's oxidising atmosphere, and also to its stress sensitivity, understanding the structure and function of PSII has been in the forefront of research in the last decade. This area gained a signifi- cant impetus with the development of molecular- genetic techniques in routine laboratory procedures, as well as with the identification of the photo-bet- erotrophic cyanobacterium Synechocystis 6803 as an excellent model organism for these studies [77]. Gene inactivation or deletion have been applied to the study of the function of a large number of PSII protein sub- units. The most important examples are (encoding genes are in parenthesis): the D1 (psbA) and D2 (psbD) core proteins of the reaction centre, the inner chlorophyll binding antenna proteins CP47 (psbB) and CP43 (psbC), the o~-subunit of cytochrome b-559 (psbE), the 9-kDa phosphoprotein (psbH), a 5-kDa subunit of the reaction centre core (psbK) and the 33- kDa extrinsic subunit (psbO) that stabilises the cata- lytic Mn cluster of water oxidation (see [67] for a review). Various combinations of site-directed mutagenesis made possible the identification of the redox-active amino acid residues and ligands of redox cofactors in the D1 and D2 proteins [67]. More recently, the application of in vitro random mutagene- sis helped to mimic the evolutionary acclimation of PSII to various environmental conditions, such as high light intensity [31, 48].

4.3. Photoinhibition and UV-B damage: defence mechanisms against light stress

The environmental conditions which can act as stress factors for plant cells include light in the photo- synthetically active, visible and in the ultraviolet spec- tral range. Although it may look surprising, visible light which is the primary driving force of photosyn- thesis can be damaging for the photosynthetic appara- tus. Excess light, that can not be utilised by the photosynthetic electron transport, impairs the function of PSII and damages its protein structure, mainly D 1 and to a lesser extent D2 subunits, in a process called photoinhibition [2, 65]. Intact cells are able to restore light-induced damage by operating a protein repair cycle which can be conveniently studied in cyanobac- teria. The main steps of this process are (a) the removal of damaged D 1 subunit from the membrane, (b) de novo synthesis of new D 1 copies, (c) incorpora- tion of D1 into the membrane and (d) reassembly of functional PSII complexes [2]. Considering that chap- eronins can facilitate protein folding and insertion into the membrane, it is very likely that, in the complex process of D1 protein repair, chaperonins play an important role which could be an interesting area of future research. It is also important to note that the efficiency of this repair cycle is affected by the lipid composition of the thylakoid membrane. In a Syn- echocystis 6803 mutant (Fad6/desA':Km r) in which the second double bound is eliminated from all lipid classes, the recovery of oxygen-evolving activity is largely retarded at low temperatures [15]. It appears therefore, that decreasing the level of polyunsaturated fatty acids affects the repair cycle primarily at the incorporation of new D1 into the membrane [28]. The gene-engineered alteration of lipid unsaturation in Fad6/desA::Kmr is acting as a trigger for additional changes in thylakoid fluidity. Accordingly, factors not related directly to lipid unsaturation may also be responsible for the modified repair cycle (T6r6k, pers. comm.).

Besides visible light, the photosynthetic apparatus is also affected by the ultraviolet components of sunlight. Ecologically, the most important is the so-called ultra- violet-B (UV-B, 280-320 nm) region, whose intensity recently increased at the Earth's surface due to the depletion of its specific absorber, the stratospheric ozone layer. Even though UV-B light has multiple tar- gets in plant cells, such as nucleic acids, aromatic amino acids, whole proteins, quinones, pigments and fatty acids, the PSII complex is a highly UV-sensitive site [64]. It appears that the specific UV-sensitivity of

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PSII, which is 200-300 times higher than that caused by visible light, is related to its unique oxygen-evolv- ing complex [66]. Besides impairment of the electron transport, UV-B light also damages the protein struc- ture of PSII, which affects the D1 and D2 subunits to similar extent. Recent studies indicate that Synechocys- tis 6803 cells are able to restore UV-B induced damage of PSII via a protein repair cycle, which proceeds by the de novo synthesis of D 1 and D2 proteins, as occurs in the case of photodamage by visible light [58].

In order to elucidate the mechanical details of the protein repair cycle, which functions as a very impor- tant defence mechanism against photodamage, it is crucial to understand the regulation of the psbA and psbD genes which are highly conserved in all photo- synthetic organisms [60]. In Synechocystis 6803, the psbA gene has three copies [25]. Two of them (psbA2 and psbA3) encode identical D1 proteins at the amino acid level, but their 5"-untranslated regions are largely different. In contrast, the third copy (psbA1) has a dif- ferent amino acid sequence and does not seem to be expressed [25, 60]. The psbD gene has two copies (psbD1 and psbD2) which encode identical D2 polypeptides, but their non-translated 5'-regions are again different [60, 77]. The expression of both genes is strongly light dependent. At low or moderate visible light intensities, more than 90 % of the psbA tran- scripts are from psbA2. Shifting to high light results in an increase in overall psbA message level, with a somewhat higher enhancement of psbA3 than psbA2 levels [46]. These findings indicate that an important step of enhanced D1 protein synthesis as a response to light-damage is regulated at the transcriptional level in cyanobacteria. According to very recent data, UV-B light specifically increases the psbA3 message level, which may indicate a particular role for the psbA3 gene in UV-B damage defence [44]. The expression of both psbD genes is enhanced by visible light; how- ever, no obvious differences were observed in the light response of the two psbD transcripts in Synechocystis 6803 [45]. It is of note that, in another cyanobacte- rium, Synechococcus sp. PCC 7942, which also has two psbD genes encoding identical D2 proteins, the expression of the psbD2 gene is rapidly enhanced at high light intensities [6]. So far few data are available for the UV-B effect on the psbD gene in Synechocystis, but the induction of fast D2 turnover in higher plants [24] makes it very likely that one or both psbD copies are UV-B inducible in this cyanobacterium. Indeed, a specific UV-B induction of the psbD2 gene was very recently observed (Vass et al., pers. comm.).

When utilising Synechocystis 6803 as a model organism for studies of stress effects on the thylakoid membrane, the sometimes important differences between cyanobacteria and higher plants also have to be taken into consideration. In contrast to cyanobacte- ria, which have small multigene families of psbA and psbD, in higher plants, these reside as single genes in the chloroplast genome. Thus, the regulatory and/or defence role of the different psbA and psbD gene cop- ies against light-induced damage in cyanobacteria may have evolved into other mechanisms in higher plants which regulate the expression of single genes. How- ever, the lessons learned from studies on Synechocystis 6803 can also be very useful in elucidating the details of light responses of the psbA and psbD genes in eukaryotic organisms.

5. THE CENTRAL ROLE OF MEMBRANE PHYSICAL STATE IN THE REGULATION

OF STRESS-DEFENCE GENES

The decrease in ambient temperature leads to changes in the membrane fluidity in the organism [9]. The following observable event is the enhanced expression of the desaturase genes [40], which leads to the accelerated synthesis of desaturases ([47], Los et al., unpubl.) and allows the adoptive desaturation of the fatty acids of membrane lipids [29]. Finally, the membrane fluidity recovers which enables the recov- ery of the photosynthetic activity of the cyanobacterial and plant cells [15, 62]. The questions remaining are: (a) what is the primary sensor of the temperature change; and (b) how is the signal transferred from a sensor to the desaturase genes, in other words, what is the triggering mechanism for the low-temperature induced expression of desaturase genes?

To answer these questions, homogeneous catalytic hydrogenation was applied to the permeaplasts of Syn- echocystis 6803. This technique, applied first to cyano- bacteria by using a water-soluble Pd-catalyst [68], allows the controlled in situ hydrogenation of unsatur- ated fatty acids in membrane lipids [26]. Hydrogena- tion has apparently no other effect than the ordering of the membrane interior and the alteration of membrane fluidity [19, 54, 69]. By hydrogenating the plasma membrane of the cyanobacterium at normal (36 °C) temperature, the level of unsaturated lipids was reduced, followed by the rapid activation of transcrip- tion of the desA gene for A12 desaturase [72]. Since transcription of the desaturase gene is enhanced with a decrease in temperature, and hydrogenation of plasma

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8 A. Glatz et al.

membrane (actually a change in membrane fluidity) can effectively regulate the expression of the desA gene, it is most likely that the low temperature-induced transcription of the desA gene is also regulated by the molecular order of the plasma membrane. These find- ings suggested that the first signal in the biological per- ception of low temperature is the change in the plasma membrane's physical state [42, 62, 72, 74].

A major trigger for heat-shock protein induction is thought to be the cytoplasmic accumulation of aber- rant or partially denatured protein. Recently, direct evidences have been obtained that, under heat-shock conditions, perturbance of membrane structure causes transduction of a signal that induces transcription of heat-shock genes [7]. Altering the molecular order of membranes by administrating aliphatic alkanols, arachidonic acid, certain prostaglandins, drugs or exogenous phospholipase A2 [74] was shown to trig- ger a heat-shock response, further supporting the hypothesis that membranes are an ideal location for primary stress sensors in cells. We have documented that altered growth temperature resulted in changes in the temperature threshold at which the formation of HSPs were initiated [37], as seen in other systems [10], which points to membranes as a sensor of heat- shock response in Synechocystis. In accordance, addi- tion of benzylalcohol, a well-known membrane fluid- ising agent, dramatically reduced the temperature range at which dnaK and the novel 'fluidity gene' hspl7 are activated by heat in Synechocystis 6803 cells. It is notable that groEL analogues displayed dif- ferent sensitivity in their transcriptional activation upon perturbation of thylakoid membrane order [21]. Since the interaction of active protein-folding GroESL oligomers with lipid membranes resulted in an ele- vated molecular order, we assume that this modulation may lead to a simultaneous down-regulation of the transcriptional activity of heat-shock genes [63].

The primary trigger for enhanced D 1 and D2 protein turnover - acting as a defence response against photo- damage of the photosynthetic apparatus - is light itself. However, an increasing body of evidence indi- cates that signal transduction from light absorption to protein synthesis is mediated by a redox signalling pathway which couples photosynthetic electron trans- port to gene expression [1, 23]. The primary redox sensor has not been identified yet, but the mobile pool of plastoquinone (PQ) molecules, which shuttle elec- trons inside the lipid bilayer of the thylakoid mem- brane, is expected to be a key player in the redox sensory pathway. The redox level of the PQ pool is set

by the balance of its reduction by electrons originating from PSII and its oxidation by PSI via the cytochrome b/f complex. The former process depends on light intensity, whereas the latter is limited by the tempera- ture dependent rate of enzymatic reactions of ATP synthesis and carbon assimilation. In addition, the dif- fusion rate of PQ molecules in the lipid bilayer is mod- ulated by changes in thylakoid membrane's physical state attained either by temperature alteration or mem- brane perturbants such as cholesterol and benzyl alco- hol [78, 79]. As a consequence, the reduction level of the PQ pool, and thus redox signalling, is affected by light intensity and temperature in a complex manner. The main tendency is that, at low temperatures, where the enzymatic processes are slower and membrane flu- idity is decreased, lower light intensities are needed to reach the same reduction level in the PQ pool than at higher temperatures. Thus, temperature dependent changes in the physical state of the membrane can be signalled to gene expression via the light requiring redox sensory pathway. This idea is also in accordance with the need of light for desaturase gene induction and the light-dark transition controlled expression of chaperonins in Synechocystis 6803 [12, 40]. Excess photosynthetically active light leads to the formation of highly reactive oxygen species, such as singlet oxy- gen and hydroxyl radicals [18] which can induce membrane damage by attacking double bounds of fatty acids. These effects are also expected to feed back signals for stress gene expression via the path- way which senses the physical state of the membrane.

Interdependence between the redox sensory path- way, as well as membrane physical state and gene expression, is expected to occur not only for visible, but also for ultraviolet light. The main damaging effect of UV-B is manifested at the donor side of PSII [66], which limits electron flow into the PQ pool. In addi- tion, UV-B can directly damage the plastoquinone molecules. Both of these effects can interfere with redox signalling through the PQ pool. UV-B radiation can also photochemically attack double bonds of unsaturated fatty acids [34]. In addition, free radicals, which can induce lipid peroxidation leading to the destruction of unsaturated fatty acids, are also pro- duced by UV-B light [17]. Consequently, induction of both desaturases and molecular chaperones could be a defence response against these membrane damaging effects of UV-B light. In conclusion, the physical state of membranes appears to play a central role in direct or indirect regulation of cold-, heat- and light stress genes. This hypothesis is summarised infigure 1.

Plant Physiol. Biochem.

M e m b r a n e s a n d c h a p e r o n e s 9

Thflakoid membrane

Plasma membrane

PSII V is ib le /UV-B light

Te" " - -

Stress-gene expression Redox sensor 3" PSII RC proteins

JL • Te - Desaturases

Heat shock proteins PSI • y e •

Enzyme .4- Tempera ture reactions

"F lu id i ty" ls t rueture

J i r:" . . . .

Figure 1. The plasma and thylakoid membranes as potential stress sensors controlling the expression of stress-defence genes.

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