Salt stress inhibits photosystems II and I in cyanobacteria

REVIEW

Salt stress inhibits photosystems II and I in cyanobacteria

Suleyman I. Allakhverdiev Æ Norio Murata

Received: 12 May 2008 / Accepted: 12 July 2008 / Published online: 1 August 2008

� Springer Science+Business Media B.V. 2008

Abstract Recent studies of responses of cyanobacterial

cells to salt stress have revealed that the NaCl-induced

decline in the photosynthetic activities of photosystems II

and I involves rapid and slow changes. The rapid decreases in

the activities of both photosystems, which occur within a few

minutes, are reversible and are associated with osmotic

effects, which induce the efflux of water from the cytosol

through water channels and rapidly increase intracellular

concentrations of salts. Slower decreases in activity, which

occur within hours, are irreversible and are associated with

ionic effects that are due to the influx of Na+ and Cl- ions

through K+(Na+) channels and, probably, Cl- channels,

with resultant dissociation of extrinsic proteins from

photosystems. In combination with light stress, salt stress

significantly stimulates photoinhibition by inhibiting repair

of photodamaged photosystem II. Tolerance of photosys-

tems to salt stress can be enhanced by genetically engineered

increases in the unsaturation of fatty acids in membrane

lipids and by intracellular synthesis of compatible solutes,

such as glucosylglycerol and glycinebetaine. In this review,

we summarize recent progress in research on the effects of

salt stress on photosynthesis in cyanobacteria.

Keywords Cyanobacteria � Membrane lipids �Photosystem II � Photosystem I � Salt stress � Tolerance

Abbreviations

BQ 1,4-Benzoquinone

CCCP Carbonyl cyanide m-chlorophenylhydrazone

Chl Chlorophyll

CSA p-Chloromercuriphenyl-sulfonic acid

DCIP 2,6-Dichlorophenolindophenol

DCMU 3-(30,40-Dichlorophenyl)-1,1-dimethylurea

DPC Diphenylcarbazide

FCCP Carbonyl cyanide p-trifluoro-methoxyphenyl-

hydrazone

GG Glucosylglycerol

MV Methylviologen

PSII Photosystem II

PSI Photosystem I

ROS Reactive oxygen species

Introduction

Photosynthesis converts light energy into chemical energy

via electron transport through two photosystems, namely,

photosystem II (PSII) and photosystem I (PSI). PSII and,

sometimes, PSI are particularly sensitive to environmental

stress (Powles 1984; Aro et al. 1993; Hagemann and Erd-

mann 1997; Adir et al. 2003; Allakhverdiev and Murata

2004; Nishiyama et al. 2005, 2006; Murata et al. 2007;

Mohanty et al. 2007; Takahashi and Murata 2008). Since

the efficiency and productivity of photosynthesis are clo-

sely related to the activities of the two photosystems,

considerable attention has been paid to the effects of

environmental stress on these photosystems.

Cyanobacteria provide suitable model systems for studies

of the effects of environmental stress on photosynthesis since

these prokaryotes perform oxygenic photosynthesis using a

photosynthetic apparatus similar to that found in chloroplasts

S. I. Allakhverdiev

Institute of Basic Biological Problems, Russian Academy of

Sciences, Pushchino, Moscow Region 142290, Russia

N. Murata (&)

National Institute for Basic Biology, Okazaki 444-8585, Japan

e-mail: [email protected]

123

Photosynth Res (2008) 98:529–539

DOI 10.1007/s11120-008-9334-x

of higher plants and algae (Pfenning 1978; Oquist et al.

1995). Moreover, cyanobacterial cells can easily be exposed

directly to defined stress conditions in culture (Joset et al.

1996; Hagemann and Erdmann 1997) and they are able to

acclimate to a wide range of environmental stresses (Nishida

and Murata 1996; Hagemann and Erdmann 1997). The

cyanobacterial genes whose expression is enhanced by salt

stress have been identified. Levels of expression of 28 genes

increase more than threefold in response to salt stress spe-

cifically, while those of 11 genes increase specifically in

response to osmotic stress, and those of 34 genes are

increased both by salt stress and by osmotic stress (Kanesaki

et al. 2002). The products of half of these genes are hypo-

thetical proteins, whose functions have not been

characterized to date. Homologs of the salt-induced genes

found in Synechocystis sp. PCC 6803 (hereafter referred to as

Synechocystis) are similarly regulated by salt stress in higher

plants, confirming that this cyanobacterium provides a good

model for investigations of the responses of photosynthetic

organisms to salt stress (Bohnert et al. 2001). However, the

anti-oxidant systems in cyanobacteria differ from those in

higher plants (Demmig-Adams and Adams 1992, 2002;

Asada 2006) and we need to take this observation into

account, especially under conditions of strong light and salt

stress, when anti-oxidant systems are very important.

Nonetheless, using cyanobacterial cells, we can study the

direct effects of salt stress on the photosynthetic machinery,

and are confident that we should be able to apply the results

obtained in cyanobacteria to higher plants.

The basic physiological responses of cyanobacterial cells

to salt stress have been investigated in great detail (for review

of early works, see Hagemann and Erdmann 1997). The

responses occur in three phases. The first phase is observed

after a sudden increase in the ambient concentration of NaCl,

and it includes the influx of Na+ and Cl- ions, within sec-

onds, into the cytoplasm. The second phase, which occurs

within an hour, is characterized by replacement of Na+ ions

by K+ ions, leading to a decline in the toxic effects of high

concentrations of Na+ ions. The third and longest phase lasts

for several hours. This phase is important for the acclimation

of cells to elevated concentrations of ions. During this phase,

the synthesis or uptake of compatible solutes occurs to mit-

igate the toxic effects of salts and to conserve the structures

of complex proteins and cell membranes (Reed et al. 1985),

and also the cyclic electron transport via PSI is activated

(Joset et al. 1996; Hibino et al. 2000. Over a longer period of

time, namely, several days, salt stress inhibits cell division

(Ferjani et al. 2003).

Cyanobacteria have evolved a variety of protective

mechanisms that allow them to acclimate to unfavorable

salt-stress conditions, to survive, and to proliferate. Mech-

anisms of tolerance to salt stress have also been investigated

in detail, with emphasis on the role of Na+ /H+ antiporters

(Allakhverdiev et al. 1999, 2000b; Inaba et al. 2001), water

and ionic channels (Allakhverdiev et al. 2000a, b), the

synthesis of compatible solutes (Hagemann and Erdmann

1997; Hayashi and Murata 1998; Chen and Murata 2002)

and of salt stress-induced proteins (Bhagwat and Apte 1989;

Hagemann et al. 1990, 1991), and membrane-lipid com-

position (Allakhverdiev et al. 1999, 2001).

In this review, we focus on the effects of salt (NaCl)

stress on photosystems in cyanobacteria, the mechanisms

of tolerance to salt stress, and in particular, the inhibition of

the repair of PSII by salt stress. We also discuss the role of

membrane lipids and compatible solutes in the tolerance of

cyanobacterial cells to salt stress.

Osmotic stress decreases the cytoplasmic volume

and inhibits PSI and PSII reversibly

The effects of osmotic stress on the cytoplasmic volume

and the photosynthetic machinery have been studied

extensively in Synechococcus sp. PCC 7942 (hereafter

referred to as Synechococcus) and in Synechocystis. When

Synechocystis cells are incubated in the presence of 0.5 M

sorbitol, the cytoplasmic volume decreases to 30–50% of

the original volume in \5 min (Kanesaki et al. 2002;

Shapiguzov et al. 2005), indicating that shrinkage of the

cytoplasm occurs instantaneously. The Synechocystis gen-

ome contains the apqZ gene for a water channel (Kaneko

et al. 1996). In a Synechocystis cells with a mutant aqpZ

gene, osmotic stress had no effect at all on cytoplasmic

volume (Shapiguzov et al. 2005).

In Synechococcus, osmotic stress due to 1.0 M sorbitol

decreased the cytoplasmic volume to 50% of the original

value in\10 min (Fig. 1a). The presence of a water-channel

blocker, p-chloromercuriphenyl-sulfonic acid (CSA), sig-

nificantly limited the decrease in cytoplasmic volume in

terms of both rate and extent (Fig. 1a). Under normal con-

ditions, the cytoplasmic volume returned to the original

volume after removal of the osmotic stress, but CSA mark-

edly inhibited such recovery (Allakhverdiev et al. 2000a).

These observations indicate that water channels participate

in the efflux and influx of water across the plasma membrane

and in the shrinkage and swelling of the cytoplasm.

The effects of osmotic stress on the activities of PSII and

PSI in intact cells were examined by monitoring the light-

dependent evolution and uptake of oxygen, respectively, in

Synechococcus (Fig. 2a). Within 2 h, osmotic stress due to

1.0 M sorbitol depressed the oxygen-evolving activity of

PSII to 40% of the original value and decreased the electron-

transport activity of PSI to 70% of the original value

(Fig. 2a). By contrast, an examination of the effects of sor-

bitol on the PSII- and PSI-mediated transport of electrons in

vitro revealed that, in isolated thylakoid membranes, the

530 Photosynth Res (2008) 98:529–539

123

presence of 1.0 M sorbitol actually stabilized the oxygen-

evolving machinery (Allakhverdiev et al. 2000a).

Shrinkage of the cytoplasm during the incubation of

Synechococcus cells with 1.0 M sorbitol was complete

within \5 min, indicating that shrinkage occurred more

rapidly than the inactivation of PSI and PSII, which was

complete in 2 h. Thus, the first event after the exposure of

cells to osmotic stress might be the efflux of water through

water channels in the plasma membrane, with a resultant

rapid increase in the intracellular concentration of ions.

Then, PSI and PSII are inactivated only gradually (All-

akhverdiev et al. 2000a).

Salt stress decreases the cytoplasmic volume but only to

a small extent

Previously, the terms ‘‘salt stress’’ and ‘‘osmotic stress’’

were used indiscriminately in many cases. For example, in

investigations of the effects of osmotic stress, NaCl was

frequently used instead of an osmolyte (see, for example,

Hagemann and Erdmann 1997). However, recent studies

have clearly demonstrated that salt stress and osmotic

stress act as different stimuli in biological cells. Osmotic

stress due to 0.5 M sorbitol is very effective in shrinking

Synechocystis cells, whereas salt stress due to 0.5 M NaCl

does not shrink cells as effectively (Kanesaki et al. 2002).

Moreover, osmotic stress (0.5 M sorbitol) and salt stress

(0.5 M NaCl) induce the expression of different sets of

genes in Synechocystis (Paithoonrangsarid et al. 2004;

Shoumskaya et al. 2005). In this context, it is very

important to evaluate the osmotic aspects of salt stress.

Changes in cytoplasmic volume during incubation with

high concentrations of NaCl were examined by monitoring

signals due to the electron paramagnetic resonance of a

water-soluble spin probe (Blumwald et al. 1984; Allakh-

verdiev et al. 2000b). When Synechococcus cells were

incubated in a medium that contained 0.5 M NaCl (the

standard concentration for normal growth is 0.02 M), the

cytoplasmic volume decreased by 20% in \1 h and then

decreased gradually to 55% of the initial volume in 10 h

(Fig. 1b). In the presence of the Na+-channel blocker

phenytoin, the cytoplasmic volume decreased by about

55% in \1 h and then to 45% of the original volume

(Fig. 1b). The presence of both phenytoin and CSA sup-

pressed the decrease in cytoplasmic volume in response to

salt stress (Fig. 1b).

The time courses of changes in cytoplasmic volume due

to 0.5 M NaCl in the presence of the Na+-channel blocker

alone (phenytoin) or of both phenytoin and the water-

channel blocker (CSA) together were similar to those due to

1.0 M sorbitol in the absence of CSA and in its presence,

respectively (Fig. 1a, b). These observations suggest that

NaCl might act as an osmolyte when Na+ channels are not

functional but that NaCl acts as a weak osmolyte when Na+

channels are functional. Thus, it is very likely that Na+ ions

flow into the cytoplasm through Na+ channels and Cl- ions

probably do so through Cl- channels. The genome of

Fig. 1 Changes in the cytoplasmic volume of Synechococcus cells

during incubation under osmotic stress and salt stress. (a) Effects of a

water-channel blocker, p-chloromercuriphenyl-sulfonic acid (CSA),

on sorbitol-induced changes in cytoplasmic volume. Synechococcuscells were incubated with 1.0 M sorbitol in the presence of 100 lM

CSA (4) and in its absence (�; control). (b) Effects of a Na+-

channel blocker (phenytoin) and a water-channel blocker (CSA) on

NaCl-induced changes in cytoplasmic volume. Cells were incubated

with 0.5 M NaCl in the presence of phenytoin (5), of phenytoin plus

CSA (e), or in their absence (�; control). At designated times,

aliquots were withdrawn, and the cytoplasmic volume was deter-

mined by electron paramagnetic resonance with a water-soluble spin

probe (see text). The cytoplasmic volume that corresponded to 100%

was 0.75 ± 0.05 fL. Each point and bar represents the average ± SE

of results from four independent experiments. Adapted from Allakh-

verdiev et al. 2000a, b)

Photosynth Res (2008) 98:529–539 531

123

Synechocystis includes three genes, sll0993, sll0536, and

slr5078, that encode a K+ channel protein and its homologs,

as well as sll1864 that encodes a putative Cl- channel

protein (Kaneko et al. 1996). The genome of Synechococcus

elongatus PCC 6301 (which is very similar to Synecho-

coccus sp. PCC 7942) includes the genes syc0846, syc1209,

and syc2428 that encode a K+ channel protein and its

homologs, as well as syc0045 that encodes a putative Cl-

channel protein (http://bacteria.kazusa.or.jp/cyanobase/

cyano.html). It is very likely that K+ channels allow the

passage of Na+ ions (Nakamura et al. 1998).

Rapid and slow phases of the inactivation of PSI

and PSII

We investigated the osmotic effects and the ionic effects of

salt stress, respectively, on both the oxygen-evolving

activity of PSII and the electron-transport activity of PSI in

Synechococcus cells (Allakhverdiev et al. 2000a, b). As

mentioned above, exposure of Synechococcus cells to salt

stress due to 0.5 M NaCl results in the rapid efflux of water

from the cytoplasm, but the effect is not as extreme as that

of 1.0 M sorbitol. The loss of water from the cytosol would

be expected to increase the intracellular concentrations of

ions and to lead to the reversible inactivation of the oxy-

gen-evolving machinery (Allakhverdiev et al. 2000b), as

observed during osmotic stress due to sorbitol. By contrast,

the ionic effect of 0.5 M NaCl is caused by the influx of

Na+ ions through K+(Na+) channels and the resultant

increases in the intracellular concentrations of Na+ ions

and counterpart anions, which are mostly Cl- ions. These

changes result in the irreversible inactivation of the oxy-

gen-evolving machinery (Allakhverdiev et al. 2000b).

Thus, salt stress appears to be much more damaging to the

oxygen-evolving machinery than osmotic stress.

The inactivation of PSI and PSII in Synechococcus cells

by 0.5 M NaCl included rapid and slow phases (Fig. 2b), in

contrast to the effect of osmotic stress due to 1.0 M sor-

bitol, which only had a rapid phase (Fig. 2a). When the

duration of incubation of Synechococcus cells in the pres-

ence of 0.5 M NaCl was short, for example, 0.5 h, and only

the rapid decline was observed, PSII activity was fully

restored upon the removal of the salt stress. However,

when the duration of incubation was extended to 5 h, no

recovery was observed (Allakhverdiev et al. 2000b).

The rapid phase of the NaCl-induced inactivation of

PSII and PSI appeared to correspond to the time course of

Fig. 2 Changes in the activities

of PSI and PSII in intact cells of

Synechococcus during

incubation under osmotic stress

and salt stress. Cells were

incubated in the presence of

1.0 M sorbitol (a) or 0.5 NaCl

(b). At designated times,

aliquots were withdrawn and the

activity of PSII was monitored

in terms of the light-dependent

evolution of oxygen in the

presence of 1.0 mM BQ, and

the activity of PSI was

monitored in terms of the light-

dependent uptake of oxygen in

the presence of 15 mM DCMU,

0.1 mM DCIP, 5 mM sodium

ascorbate, and 0.1 mM MV.

The oxygen-evolving activity of

PSII that corresponded to 100%

was 594 ± 38 lmol O2 mg-1

Chl h-1. The oxygen-uptake

activity of PSI that

corresponded to 100% was

312 ± 46 lmol O2 mg-1 Chl

h-1. Each point and bar

represents the average ± SE of

results from four independent

experiments. Adapted and

modified from Allakhverdiev

et al. (2000a, b)

532 Photosynth Res (2008) 98:529–539

123

osmotic stress-induced inactivation (Fig. 2a), suggesting

that the rapid reversible decline in the activities of PSII and

PSI might have been caused by the osmotic effect of NaCl.

The slow and irreversible phase appeared to be specific to

ionic effects since the presence of a specific blocker of Na+

channels protected both PSII and PSI against the NaCl-

induced slow inactivation but not against the rapid inacti-

vation (Allakhverdiev et al. 2000b). In the presence of the

Na+ channel blocker, the NaCl-induced inactivation

resembled the sorbitol-induced inactivation. These obser-

vations suggest that Na+ and/or Cl- ions damage the

machinery that is necessary for the repair of PSII after

NaCl-induced inactivation.

Possible sites of inactivation of PSII and PSI by NaCl

Incubation of Synechococcus cells in the presence of 0.5 M

NaCl for 5 h inactivates both oxygen evolution in PSII and

electron transport in PSI (Allakhverdiev et al. 2000b).

Examination of the light-dependent quenching of chloro-

phyll fluorescence due to the reduction of pheophytin in

intact cells demonstrated that the site of inactivation is the

electron-donating side of PSII. Furthermore, incubation of

Synechococcus cells in the presence of NaCl did not

damage QA, pheophytin, and P680 but blocked the trans-

port of electrons from water to P680. These observations

indicate that the photochemical reaction center is not

involved in the NaCl-induced inactivation of PSII (All-

akhverdiev et al. 2000b).

In thylakoid membranes isolated from either Synecho-

cystis (Allakhverdiev et al. 1999) or Synechococcus

(Allakhverdiev et al. 2000b), the transport of electrons

from water to 2,6-dichlorophenolindophenol (DCIP), but

not from diphenylcarbazide (DPC) to DCIP, was sup-

pressed by the presence of 0.5 M NaCl. Since DPC donates

electrons to P680 (Yamashita and Butler 1969; Izawa

1980; Trebst 1980), it seems likely that salt stress inacti-

vates the oxygen-evolving machinery (Kuwabara and

Murata 1983; Miyao and Murata 1983; Murata and Miyao

1985).

We also examined the effects of salt stress on the

activity of PSI by monitoring the uptake of oxygen by

intact cells in the presence of DCIP, sodium ascorbate,

methyl viologen (MV), and 3-(30,40-dichlorophenyl)-1,1-

dimethylurea (DCMU) (Allakhverdiev et al. 2000b). In this

system, electrons are transported from the reduced form of

DCIP to MV through plastocyanin, P700, phylloquinone

(vitamin K1), and iron sulfur centers (Izawa 1980; Golbeck

1994). Incubation of Synechococcus cells with 0.5 M NaCl

suppressed the reduction of P700+ (Allakhverdiev et al.

2001). Since P700+ is reduced by plastocyanin, it seems

likely that the association of plastocyanin with the PSI

complex was disturbed by the presence of NaCl (Allakh-

verdiev et al. 2000b, 2001).

Salt-induced inactivation depends on the activity

and synthesis of Na+/H+ antiporters

The Synechocystis genome includes five genes for putative

Na+/H+ antiporters (Inaba et al. 2001). The Na+/H+

antiport system is responsible for maintaining the intra-

cellular concentration of Na+ ions at a certain low level.

The tolerance of Synechocystis cells to salt stress is related

to the activity of Na+/H+ antiporters, and moreover,

incubation of Synechocystis or Synechococcus cells with

NaCl inactivates the Na+/H+ antiport system in these

cyanobacterial cells (Allakhverdiev et al. 1999, 2000b).

Such inactivation might be due to inhibition of the syn-

thesis of proteins that are involved in the Na+/H+ antiport

system. Thus, the inactivation of Na+/H+ antiporters might

be related to the ionic effect of NaCl on the inactivation of

PSI and PSII (Allakhverdiev et al. 2000b). The inactivation

might be due to inhibition of the synthesis of the proteins

that are required for the function of Na+/H+ antiporters.

By contrast, water channels are predominantly respon-

sible for the hyperosmotic stress-induced inactivation of

PSII and PSI by sorbitol, as well as for some aspects of the

rapid phase of the NaCl-induced inactivation of PSII and

PSI (Allakhverdiev et al. 2000a, b). The rapid inactivation

is reversible, and protein synthesis is not required for the

recovery of the activities of PSI and PSII from the rapid

inactivation (Allakhverdiev et al. 2000b). It seems likely

that the Na+/H+ antiport system is involved in the rapid

and reversible inactivation of PSI and PSII and in the

recovery of their activities.

A hypothetical scheme for the salt-induced inactivation

of electron transport in PSI and PSII

Figure 3 shows a hypothetical scheme that might explain

the NaCl-induced inactivation of the photosynthetic

machinery. K+(Na+) channels, Cl- channels, water chan-

nels, and Na+/H+ antiporters are located on the plasma

membrane. The oxygen-evolving machinery of the PSII

complex is located on the luminal side of thylakoid

membranes. In cyanobacteria, this machinery is stabilized

by three extrinsic proteins, namely, PsbO (33-kD protein),

PsbV (cytochrome c550), and PsbU (Shen et al. 1998; Ni-

shiyama et al. 1999). Cyt c550 and PsbU are loosely bound

to the donor side of the core complex of PSII (Nishiyama

et al. 1997, 1999). These proteins are easily dissociated

from the cyanobacterial PSII complex in the presence of

elevated concentrations of NaCl (Shen et al. 1992, 1998).

Photosynth Res (2008) 98:529–539 533

123

When the extracellular concentrations of Na+ and Cl- ions

increase, water molecules in the intracellular space rapidly

leave the cell through water channels, whereas Na+ and

Cl- ions enter the cytoplasm through K+(Na+) channels

and Cl- channels, respectively, with resultant increases in

the cytosolic concentrations of Na+ and Cl- ions. The

Na+/H+ antiport system probably pumps Na+ ions out of

the cell. It seems likely that Na+ ions in the cytosol then

cross the thylakoid membrane to increase the concentration

of Na+ and Cl- ions in the intrathylakoid space (lumen),

although the mechanism of such movement of ions is

unknown. As a result, the extrinsic proteins of PSII dis-

sociate from the complex and the oxygen-evolving

machinery is partially inactivated. When the salt stress is

removed, water molecules enter the cytosol through the

water channels, and Na+ and Cl- ions leave the cytosol

through their respective channels, with resultant decreases

in the cytosolic concentrations of Na+ ions and Cl- ions.

The intrathylakoid concentrations of Na+ ions and Cl- ions

also decrease, and as a result, the integrity of PSII and PSI

is restored by the renewed binding of the extrinsic proteins

to these complexes.

The Na+/H+ antiport system is inactivated during long-

term incubation of cyanobacterial cells with 0.5 M NaCl,

and when it becomes inoperative, resultant increases occur

in the cytosolic concentrations and intrathylakoid concen-

trations of Na+ ions and Cl- ions. Extended incubation in

the presence of 0.5 M NaCl, during which the extrinsic

proteins remain dissociated from the complexes,

inactivates the Mn cluster, which is the catalytic center of

the oxygen-evolving complex.

A similar mechanism has been postulated for the NaCl-

induced inactivation of PSI. An increase in the intra-

thylakoid concentration of Na+ ions might lead to the

dissociation of plastocyanin or cytochrome c553 from the

PSI complex, causing a decrease in the rate of the PSI-

mediated transport of electrons (Allakhverdiev et al.

2000b, 2001).

Role of cellular energization in the recovery

of the activities of PSI and PSII

In most of the studies of the effects of salt stress on

cyanobacterial cells, the effects were examined in dark-

ness. However, cyanobacteria, such as Synechococcus and

Synechocystis, are photoautotrophic organisms, utilizing

light energy to drive energy production and the assimila-

tion of carbon via photosynthesis. It appears that light is an

important factor in the restoration of the activities of PSII

and PSI during incubation of cyanobacterial cells under

stress (Allakhverdiev et al. 2005). When light is applied to

Synechococcus cells that have lost the activities of PSI and

PSII as a result of salt stress in darkness, these activities are

fully restored. Light also restores the activity of the Na+/

H+ antiporters in Synechococcus. Weak light at 70 lE

m-2 s-1 is sufficient to generate ATP, which seems to

support recovery. An uncoupler, either carbonyl cyanide

Fig. 3 A hypothetical model of the NaCl-induced inactivation of PSI

and PSII in cyanobacterial cells. d, Extrinsic proteins of the oxygen-

evolving machinery of PSII, namely, the 33-kD protein, cytochrome

c550, and PsbU; m, a protein associated with the PSI complex,

namely, plastocyanin or cytochrome c553; red circle, water channel;

yellow circle, K+ (Na+) channel; pink circle, Cl- channel; white

circle, Na+/H+ antiporter. Thylakoid lumen and cytoplasm are

indicated by white and green color, respectively. Adapted and

modified from Allakhverdiev et al. (2000b)

534 Photosynth Res (2008) 98:529–539

123

m-chlorophenylhydrazone (CCCP) or carbonyl cyanide p-

trifluoro-methoxyphenylhydrazone (FCCP), each of which

induces the de-energization of cells, prevents the light-

dependent restoration of the activities of PSII, PSI, and

Na+/H+ antiporters (Allakhverdiev et al. 2005). These

observations confirm that the light-dependent energization

of cyanobacterial cells is important for the tolerance of

photosystems to salt stress. This effect of light is com-

pletely eliminated in the presence of lincomycin, an

inhibitor of protein synthesis (Allakhverdiev et al. 2005).

Thus, it seems likely that, when cyanobacterial cells are

exposed to salt stress, protein synthesis is important for the

recovery of the photosystems and the Na+/H+ antiporters.

The data obtained in experiments with Synechococcus

(Allakhverdiev et al. 2005) and Synechocystis (Allakhver-

diev et al. 1999) are in accord with the observations of

Hagemann et al. (1991), who demonstrated a close corre-

lation between the synthesis of proteins and the tolerance to

salt stress in Synechocystis cells. The involvement of pro-

tein synthesis in recovery was confirmed by protein-

labeling experiments. Furthermore, light not only stimu-

lated the overall synthesis of proteins but also induced the

synthesis of several specific proteins, which were absent

under dark conditions where no recovery occurred (All-

akhverdiev et al. 2005).

The presence of exogenous glucose in darkness also

resulted in regeneration of the activity of Na+/H+ anti-

porters. It is likely that exogenously added glucose

activated respiration since a high, but only transient, stim-

ulation of oxygen consumption was observed in darkness

after the addition of glucose to a culture of Synechococcus

cells (Allakhverdiev et al. 2005). Both photosynthesis and

respiration generate energy, in the form of ATP and of

proton gradients across cytoplasmic and thylakoid mem-

branes. The hypothesis that cellular energization in light or

in the presence of glucose in darkness accelerates the

recovery of the activities of PSII, PSI, and Na+/H+ anti-

porters is supported by the actions of ionophores, namely,

CCCP, FCCP, nigericin plus valinomycin, and N,N0-dicy-

clohexyl-carbodiimide (DCCD) each of which inhibits the

synthesis of ATP. The addition of these compounds pre-

vents the recovery of the activities of PSII and PSI in light

and in the presence of exogenous glucose in darkness

(Allakhverdiev et al. 2005), suggesting that the synthesis of

ATP is essential for the recovery.

Unsaturation of fatty acids in membrane lipids

increases the tolerance of PSII to salt stress

The state of thylakoid membranes plays a prominent role in

the tolerance of the photosynthetic machinery to environ-

mental stresses, and in particular, to cold stress (Wada et al.

1990; Murata et al. 1992) and salt stress (Allakhverdiev

et al. 1999, 2001). The ratio of saturated and unsaturated

fatty acids and the phospholipid composition of mem-

branes are critical factors in this regard (Nishida and

Murata 1996; Sakurai et al. 2003, 2007). When photosyn-

thetic organisms are exposed to salt stress, fatty acids of

membrane lipids are desaturated (Huflejt et al. 1990).

Therefore, it is possible that unsaturation of fatty acid in

membrane lipids might affect the tolerance of the photo-

synthetic machinery to salt stress.

Tasaka et al. (1996) isolated a desA-/desD- mutant

strain of Synechocystis, in which the desA and desD genes

for the D12 and D6 desaturases, respectively, had been

inactivated by targeted mutagenesis. The desA-/desD-

mutant cells contained mono-unsaturated, but not polyun-

saturated, fatty acids whereas wild-type cells contain

polyunsaturated fatty acids, such as di-, tri-, and tetra-

unsaturated fatty acids (Wada and Murata 1989). By

comparing these two strains, Tasaka et al. (1996) demon-

strated that polyunsaturated fatty acids are essential for

growth in Synechocystis at low temperatures. We examined

the contribution of the unsaturation of fatty acids in

membrane lipids to the tolerance of Synechocystis cells to

salt stress by comparing desA-/desD- mutant cells with

wild-type cells (Allakhverdiev et al. 1999). The loss of the

oxygen-evolving activity of PSII in the presence of 0.5 M

NaCl was much more rapid in desA-/desD- cells than in

wild-type cells. The oxygen-evolving activity that had been

lost during incubation with 0.5 M NaCl recovered when

cells were transferred to conditions that allowed photo-

synthesis in light or respiration as a result of the addition of

glucose to the medium. This recovery was much faster in

wild-type than in desA-/desD- cells, and in addition, it

was prevented by lincomycin (Allakhverdiev et al. 1999).

The relationship between the unsaturation of fatty acids

and the tolerance of PSII to salt stress was examined after a

genetically engineered increase in the unsaturation of fatty

acids in the membrane lipids of Synechococcus (Wada et al.

1990; Allakhverdiev et al. 2001). Wild-type cells of Syn-

echococcus synthesize saturated and mono-unsaturated

fatty acids, whereas desA+ cells, which had been trans-

formed with the desA gene for the D12 acyl-lipid desaturase

of Synechocystis, synthesized di-unsaturated fatty acids in

addition to mono-unsaturated fatty acids. Loss of the

activities of PSI and PSII and of the Na+/H+ antiport sys-

tem during incubation with 0.5 M NaCl occurred more

slowly in desA+ cells than in wild-type cells. In addition, we

observed that the PSII activity of isolated thylakoid mem-

branes from desA+ cells was more resistant to 0.5 M NaCl

than that of isolated membranes from wild-type cells (All-

akhverdiev et al. 2001). These findings indicate that the

unsaturation of fatty acids in membrane lipids enhances the

tolerance of PSI and PSII to salt stress.

Photosynth Res (2008) 98:529–539 535

123

There are at least five possible explanations for the

enhanced tolerance of PSII to salt stress upon the unsatu-

ration of membrane lipids, as follows: (i) Water channels,

the activity of which is responsible for the rapid phase of the

NaCl-induced inactivation, are located on the plasma

membrane. Therefore, it is quite plausible that their activity

might be affected by the unsaturation of membrane lipids

and by resultant changes in the fluidity of the membrane.

Such effects might explain why the unsaturation of fatty

acids minimized the rapid phase of the NaCl-induced

inactivation; (ii) K+(Na+) channels are located on the

plasma membrane, and their activities might be enhanced

by the unsaturation of fatty acids of membrane lipids. Such

effects might explain why the unsaturation of fatty acids

counteracted the slow phase of NaCl-induced inactivation;

(iii) The Na+/H+ antiport system, which consists of Na+/

H+ antiporter(s) and H+-ATPase(s), is located in the plasma

membrane. The unsaturation of fatty acids in membrane

lipids might activate the Na+/H+ antiport system via

enhanced fluidity of the membrane, resulting in a decrease

in concentrations of Na+ ions in the cytosol; (iv) The un-

saturation of fatty acids might stimulate the synthesis of the

Na+/H+ antiporter(s) and/or H+-ATPase(s). An increase in

the levels of these components of the antiport system might

result in a decrease in the concentration of Na+ ions in the

cytosol, which would tend to protect PSII and PSI against

NaCl-induced inactivation; (v) The repair of PSII includes a

step of insertion of the precursor to the D1 protein into the

membrane. The unsaturation of fatty acids might accelerate

the insertion, resulting in the enhanced activity of repair.

Synergistic actions of salt stress and light stress

Light stress and salt stress are major environmental factors

that limit the efficiency of photosynthesis. However, the

mechanisms of their effects on PSII are different. These

effects of salt stress and light stress on PSII can be sepa-

rately examined much more easily in cyanobacterial cells

than in leaves of higher plants. PSII of Synechocystis is

resistant to salt stress and the light stress, when each is

applied separately at certain moderate levels (Allakhver-

diev et al. 1999, 2002). However, when they are combined,

they inhibit PSII very efficiently. Moreover, this synergistic

effect is due to inhibition of the repair of PSII from light-

induced inactivation (Allakhverdiev et al. 2002). Labeling

of proteins in vivo and Western and Northern blotting

analyses indicated that salt stress inhibited the translation

and, to a much smaller extent, the transcription of the psbA

genes, which encode the D1 protein (Allakhverdiev et al.

2002). Pulse-chase experiments revealed that salt stress

inhibited the synthesis of D1 de novo in Synechococcus

(Ohnishi and Murata 2006).

Recent studies of photoinhibition of PSII in cyanobac-

teria suggest that oxidative stress due to reactive oxygen

species (ROS) inhibits the repair of PSII but does not

stimulate the photodamage to PSII (Nishiyama et al. 2005,

2006; Murata et al. 2007). The site of action of ROS seems

to be the elongation factor G (EFG) of the translation of

gene expression (Kojima et al. 2007). A further study has

suggested that it is likely that various kinds of stress, such

as salt and moderate heat stress, suppress the fixation of

CO2 and, thus, decrease utilization of NADPH that would

stimulate the formation of ROS (Allakhverdiev et al. 2007;

Takahashi and Murata 2008). These ROS, in turn, inhibit

protein synthesis and, thus, the repair of PSII (see also

Murata et al. 2007; Takahashi and Murata 2008). Another

possibility is that salt stress directly inactivates the trans-

lation machinery by dissociation of its subunits. Further

studies are necessary for a full understanding of the

mechanisms responsible for the effects of salt stress on

photoinhibition of photosynthesis.

In tobacco leaves, Al-Taweel et al. (2007) observed that

salt stress had no effect on photodamage to PSII but did

inhibit the repair of photodamaged PSII via inhibition of

protein synthesis in chloroplasts. In higher plants also, salt

stress seemed to inhibit the fixation of CO2 and decreased

utilization of NADPH that would stimulate the formation

of ROS in PSI (see also Takahashi and Murata 2008).

Compatible solutes increase the tolerance

of the photosynthetic machinery to salt stress

Many plants accumulate compatible solutes in response to

salt stress (Chen and Murata 2002). These compatible sol-

utes include proline, betaine, polyols, sugar alcohols, and

soluble sugars (Fulda et al. 1999). The expression of genes

that are involved in the biosynthesis of compatible solutes is

enhanced under salt stress, but not by osmotic stress (Marin

et al. 2006), and concentrations of accumulated compatible

solutes are correlated with the extent of tolerance to salt

stress (Zhu 2002). Glycinebetaine acts as a compatible

solute by stabilizing the quaternary structures of proteins,

cell membranes and, in particular, the oxygen-evolving

complex of PSII (Papageorgiou and Murata 1995).

Synechocystis cells accumulate glucosylglycerol (GG) as

a major compatible solute and they also accumulate traces of

sucrose transiently in response to salt stress. These com-

patible solutes allow cells to tolerate as much as 1.2 M NaCl

(Reed and Stewart 1988). Synechocystis cells synthesize

GG-phosphate from ADP-glucose and glycerol 3-phosphate

in a reaction catalyzed by GG-phosphate synthase (GgpS),

and they dephosphorylate the intermediate GG-phosphate

to yield GG in a reaction catalyzed by GG-phosphate

phosphatase (Hagemann and Erdmann 1994). GgpS and

536 Photosynth Res (2008) 98:529–539

123

GG-phosphate phosphatase are encoded by the ggpS and

stpA genes, respectively (Hagemann et al. 1997). Prior to salt

stress, Synechocystis cells do not synthesize GG. After

incubation of cells in darkness for 20 to 30 h in the presence

of 0.5 or 1.0 M NaCl, Synechocystis cells accumulate GG at

a level as high as 180–200 lg per mg of Chl, suggesting a

protective role for this compound at high concentrations of

salt in light (Hagemann and Erdmann 1994).

Ferjani et al. (2003) demonstrated that the presence of

GG is important in the tolerance of the cell-division

machinery to salt stress. In DggpS mutant cells of Syn-

echocystis, NaCl at 0.45 M inhibited cell division and

significantly increased cell size, whereas in wild-type cells,

cell division and increases in cell size were inhibited at

high concentrations of NaCl, such as 0.8 M. Electron

microscopy revealed that, in DggpS cells, separation of

daughter cells was incomplete, and aborted division could

be recognized by the presence of a structure that resembled

a division ring. The addition of GG to the culture medium

protected the mutant cells from salt stress and reversed the

adverse effects of NaCl on cell division and cell size. These

observations suggest that GG is important for salt tolerance

and, thus, for the proper division of cells under salt stress.

Our group demonstrated that salt stress due to 0.22 M

NaCl represses the repair of photodamaged PSII by

inhibiting the degradation and synthesis of D1 protein in

salt-stressed Synechococcus cells (Ohnishi and Murata

2006). We had previously transformed Synechococcus cells

with the codA gene for choline oxidase, which catalyzes

the synthesis of glycinebetaine from choline (Deshnium

et al. 1995). The transformed cells accumulated glycineb-

etaine at a concentration between 60 and 80 mM. Salt

stress might reduce the integrity and activity of the trans-

lational machinery in Synechococcus cells, and the

synthesis of glycinebetaine in the codA-transformed cells

might counteract the effects of salt stress. In addition, salt

stress might destabilize and glycinebetaine might stabilize

proteases that are required for the degradation of D1 and

other proteins in PSII (Ohnishi and Murata 2006). In this

respect, it is noted that FtsH, a bacterial homolog of such

proteases, is active when it is in a hexameric structure

(Suno et al. 2006), which might be destabilized by salt and

stabilized by the presence of glycinebetaine.

Conclusion

Changes in cytoplasmic volume and in the activities of PSI

and PSII suggest that NaCl has both osmotic and ionic

effects on cyanobacterial cells. The osmotic effect elicits

the efflux of water from the cytoplasm through water

channels and, thus, most probably increases cytoplasmic

concentrations of cations and anions, such as K+ ions.

These increases in concentrations of ions in the cytosol, in

turn, increase the concentrations of ions in the lumen (in-

trathylakoid space) with resultant dissociation of the

extrinsic proteins, which can lead to the reversible inacti-

vation of both PSI and PSII.

The ionic effect is caused by an influx of Na+ and Cl-

ions across the plasma membrane through K+(Na+) and

Cl- channels, respectively, and it increases the concen-

tration of NaCl in the cytosol. NaCl penetrates the

thylakoid membranes by an unknown mechanism to

increase the concentration of NaCl in the lumen. As a

result, the extrinsic proteins dissociate from both PSII and

PSI, with the resultant irreversible inactivation of PSI and

PSII after long-term incubation. The restoration and

recovery of the photosystems and the Na+/H+ antiporters

in salt-stressed cells might be regulated by the synthesis of

ATP and the subsequent synthesis of proteins.

Light and salt stress have a strong synergistic effect on

photoinhibition of PSII. Salt stress inhibits the repair of PSII

from light-induced inactivation. Synthesis of compatible

solutes, such as glucosylglycerol and glycinebetaine, and

the unsaturation of fatty acids in membrane lipids are key

factors in the repair of and the tolerance to salt stress of

PSII. Hence, a full understanding of the molecular basis of

the mechanisms of tolerance of photosystems to salt stress is

essential for the introduction, by plant breeding and genetic

engineering, of salt tolerance in crop plants. A particularly

promising approach in efforts to increase the ability of

plants to tolerate high concentrations of salt is genetic

engineering that increases levels of compatible solutes and

levels of unsaturation of membrane lipids.

Acknowledgments This work was supported, in part, by the

Cooperative Research Program on Stress-Tolerant Plants of the

National Institute for Basic Biology, Japan, and by grants from

the Russian Foundation for Basic Research (Nos. 08-04-00241 and

08-04-91300) and from the Molecular and Cell Biology Programs of

the Russian Academy of Sciences (to S.I.A.).

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