Degradation and release from the thylakoid membrane of Photosystem II subunits after UV-B irradation of the liverwort Conocephalum conicum

The effect of ultraviolet-B (UV-B) radiation on the amount of various Photosystem (PS) II subunits has been studied in the thalloid liverwort Conocephalum conicum. UV-B irradiation led to a drastic decrease of the reaction center proteins D1 and D2 and the outer light harvesting antenna (LHC II). A minor reduction was found in the levels of the CP 43 polypeptide of the inner antenna and the 33, 23 and 16 kDa extrinsic polypeptides of PS II. During UV-B irradiation, the extrinsic polypeptides accumulated in the soluble protein fraction, but D1 and D2 were not dedectable. Streptomycin, but not cycloheximide inhibited the repair process of PS II, indicating that only protein synthesis in the chloroplast is necessary for recovery. This indicates that the extrinsic proteins of PS II dissociate from the membrane during UV-B treatment and reassociate with PS II in the course of the repair process. We conclude that the reaction center core is a target of UV-B radiation in C. concicum. The extrinsic proteins of PS II are not directly affected by UV-B, but their release is the consequence of UV-B-induced degradation of the D1 and D2 proteins.     

Repair of UV-B induced damage of Photosystem II via de novo synthesis of the D1 and D2 reaction centre subunits in Synechocystis sp. PCC 6803

The repair of ultraviolet-B radiation induced damage to the structure and function of Photosystem II was studied in the cyanobacterium Synechocystis sp. PCC 6803. UV-B irradiation of intact Synechocystis cells results in the loss of steady-state oxygen evolution, an effect accompanied by a parallel loss of both D1 and D2 protein subunits of the Photosystem II reaction centre. Transfer of the UV-irradiated cells to normal growth conditions under visible light results in partial recovery of the inhibited oxygen evolving activity and restoration of the lost D1 and D2 proteins. The extent of recovery decreases with increasing degree of damage: after 50% inhibition, the original activity is completely restored within 2 hours. In contrast, after 90–95% inhibition less than half of the original activity is regained during a 4 hour recovery period. The translation inhibitor lincomycin completely blocks the recovery process if added after the UV-B treatment, and accelerates the kinetics of activity loss if added before the onset of UV-B irradiation. Substantial retardation of recovery and acceleration of activity loss is also observed if the very low intensity short wavelength contribution (<290 nm) is not filtered out from the UV-B light source. It is concluded that in intact cells UV-B induced damage of the Photosystem II complex can be repaired. This process is the first example of simultaneous D1 and D2 protein repair in Photosystem II, and considered to function as an important defence mechanism against detrimental UV-B effects in oxygenic photosynthetic organisms. De novo synthesis of the D1 and D2 reaction centre subunits is a key step of the repair process, which itself can also be inhibited by ultraviolet light, especially by the short wavelength UV-C components, or by high doses of UV-B.     

Inhibition of the repair of Photosystem II by oxidative stress in cyanobacteria

The activity of Photosystem II (PS II) is severely restricted by a variety of environmental factors and, under environmental stress, is determined by the balance between the rate of damage to PS II and the rate of the repair of damaged PS II. The effects of oxidative stress on damage and repair can be examined separately, and it appears that, while light can damage PS II directly, oxidative stress acts primarily by inhibiting the repair of PS II. Studies in cyanobacteria have demonstrated that oxidative stress suppresses the de novo synthesis of proteins, in particular, the D1 protein, which is required for the repair of PS II.     

Inhibition of Photosystem II by UV-B Radiation and the Conditions for Recovery in the Liverwort Conocephalum conicum Dum.

Inhibition of photosynthesis by UV-B was investigated in the thalloid liverwort Conocephalum conicum Dum. UV-B irradiance was adjusted to a strength producing 50% inhibition of the rate of photosynthesis during 10 min of irradiation. A linear relationship of the fluorescence terms F_v/F_m of photosystem (PS) II and J_P was observed following a UV-B irradiation. This suggested that PS II was a major site of UV-B-induced damage of photosynthesis. The apparent inhibition of F_v/F_m was much smaller when electron flow to the secondary PS II acceptor Q_B was inhibited by DCMU or when F_v/F_m was measured at 77 K. Apparently, the major target of UV-B effects was electron donation to the PS II reaction center, rather than electron transfer reactions at the PS II acceptor side. The time required for repair of PS II from UV-B-induced damage was light-dependent and minimal at a flux density of 5 μE m^?2 s^?1. Low temperatures and the presence of streptomycin inhibited the repair processes of PS II, indicating that protein synthesis may be involved in the recovery of PS II. The data indicate that UV-B irradiation on bright and cool winter days may be most harmful for photosynthesis of C. conicum. A repeated irradiation of the thalli with UV-B induced tolerance of photosynthesis which was related to an accumulation of pigments with a maximum of absorption around 315 nm.     

The role of the FtsH and Deg proteases in the repair of UV-B radiation-damaged Photosystem II in the cyanobacterium Synechocystis PCC 6803

The photosystem two (PSII) complex found in oxygenic photosynthetic organisms is susceptible to damage by UV-B irradiation and undergoes repair in vivo to maintain activity. Until now there has been little information on the identity of the enzymes involved in repair. In the present study we have investigated the involvement of the FtsH and Deg protease families in the degradation of UV-B-damaged PSII reaction center subunits, D1 and D2, in the cyanobacterium Synechocystis 6803. PSII activity in a DeltaFtsH (slr0228) strain, with an inactivated slr0228 gene, showed increased sensitivity to UV-B radiation and impaired recovery of activity in visible light after UV-B exposure. In contrast, in DeltaDeg-G cells, in which all the three deg genes were inactivated, the damage and recovery kinetics were the same as in the WT. Immunoblotting showed that the loss of both the D1 and D2 proteins was retarded in DeltaFtsH (slr0228) during UV-B exposure, and the extent of their restoration during the recovery period was decreased relative to the WT. However, in the DeltaDeg-G cells the damage and recovery kinetics of D1 and D2 were the same as in the WT. These data demonstrate a key role of FtsH (slr0228), but not the Deg proteases, for the repair of PS II during and following UV-B radiation at the step of degrading both of the UV-B damaged D1 and D2 reaction center subunits.     

The role of the FtsH and Deg proteases in the repair of UV-B radiation-damaged Photosystem II in the cyanobacterium Synechocystis PCC 6803

The photosystem two (PSII) complex found in oxygenic photosynthetic organisms is susceptible to damage by UV-B irradiation and undergoes repair in vivo to maintain activity. Until now there has been little information on the identity of the enzymes involved in repair. In the present study we have investigated the involvement of the FtsH and Deg protease families in the degradation of UV-B-damaged PSII reaction center subunits, D1 and D2, in the cyanobacterium Synechocystis 6803. PSII activity in a ΔFtsH (slr0228) strain, with an inactivated slr0228 gene, showed increased sensitivity to UV-B radiation and impaired recovery of activity in visible light after UV-B exposure. In contrast, in ΔDeg-G cells, in which all the three deg genes were inactivated, the damage and recovery kinetics were the same as in the WT. Immunoblotting showed that the loss of both the D1 and D2 proteins was retarded in ΔFtsH (slr0228) during UV-B exposure, and the extent of their restoration during the recovery period was decreased relative to the WT. However, in the ΔDeg-G cells the damage and recovery kinetics of D1 and D2 were the same as in the WT. These data demonstrate a key role of FtsH (slr0228), but not the Deg proteases, for the repair of PS II during and following UV-B radiation at the step of degrading both of the UV-B damaged D1 and D2 reaction center subunits.     

The interaction of visible and UV-B light during photodamage and repair of Photosystem II

In order to understand the mechanism of photodamage induced by solar radiation under natural conditions, we studied the interaction of visible and ultraviolet-B light in the inactivation and repair of the Photosystem II complex by using oxygen evolution and flash-induced chlorophyll fluorescence measurements. In isolated spinach thylakoids and Synechocystis 6803 cells, in which de novo protein synthesis is blocked by lincomycin, photodamage of Photosystem II by visible and UV-B light is characterized by linear semilogarithmic inactivation curves for both separate and combined illumination protocols. The extent of PS II inactivation obtained after combined illumination can be well simulated by assuming independent damaging events induced by visible and UV-B photons. In intact Synechocystis cells capable of protein repair, simultaneous illumination by visible and UV-B light impairs Photosystem II activity to a smaller extent than expected from the independent damaging events. This protective effect is pronounced at low visible light (130 μE m⁻² s⁻¹), but becomes negligible at high intensities (1300 μE m⁻² s⁻¹). Exposure of intact Synechocystis 6803 cells to direct sunlight leads to a rapid inactivation of PS II, accompanied by the accumulation of donor side inhibited centers. This phenomenon, which shows the impairment of the manganese cluster of water oxidation was not observed when the ultraviolet components of sunlight were filtered out. We conclude that visible and UV-B photons inactivate PS II via non-interacting mechanisms, which affect different target sites. In intact cells, the two spectral regions do interact, and results in synergistically enhanced protein repair capacity when UV-B radiation is accompanied by low intensity visible light, which provides protection against photodamage. However, this ameliorating effect becomes insignificant at high light intensities characteristic of direct sunlight. This revised version was published online in June 2006 with corrections to the Cover Date.     

Evidence for the ultraviolet-B (280–320 nm) radiation induced structural reorganization and damage of photosystem II polypeptides in isolated chloroplasts

Direct evidence for the possible loss of photosystem II (PS II) activity in chloroplasts of Vigna sinensis L. cv. Walp after ultraviolet-B (UV-B, 280–320 nm) radiation treatment was provided by polyacrylamide gel electrophoretic analysis of PS II polypeptides. A 30 min UV-B treatment of chloroplasts caused a 50% loss of PS II activity. The artificial electron donor. Mn²⁺ failed to restore UV-B radiation induced loss of PS II activity, while diphenyl carbazide (DPC) and NH₂OH only partially restored activity. Such a loss in PS II activity was found to be primarily due to a loss of 23 and 33 kDa extrinsic polypeptides. UV-B treatment induced the synthesis of a few polypeptides and a 29 kDa light-harvesting chlorophyll protein.     

Nitric oxide donor-mediated inhibition of phosphorylation shows that light-mediated degradation of photosystem II D1 protein and phosphorylation are not tightly linked

An outcome of the photochemistry during oxygenic photosynthesis is the rapid turn over of the D1 protein in the light compared to the other proteins of the photosystem II (PS II) reaction center. D1 is a major factor of PS II instability and its replacement a primary event of the PS II repair cycle. D1 also undergoes redox-dependent phosphorylation prior to its degradation. Although it has been suggested that phosphorylation modulates D1 metabolism, reversible D1 phosphorylation was reported not to be essential for PS II repair in Arabidopsis. Thus, the involvement of phosphorylation in D1 degradation is controversial. We show here that nitric oxide donors inhibit in vivo phosphorylation of the D1 protein in Spirodela without inhibiting degradation of the protein. Thus, D1 phosphorylation is not tightly linked to D1 degradation in the intact plant.     

Ultraviolet-B Induced Changes in Ultrastructure and D1/D2 Proteins in Cyanobacteria Synechococcus sp. PCC 7942

Effects of ultraviolet-B (UV-B) irradiation on ultrastructure, total cellular protein, and PS2 proteins D1 and D2 of Synechococcus sp. PCC 7942 cells was studied. The scanning electron micrographs showed UV-B radiation induced bending of the cells. The transmission electron micrographs revealed disorganization and shift in thylakoid lamellar structure to one side of the cell. The cellular phycocyanin/chlorophyll ratio decreased with increasing UV-B treatment and due to this the colour of cells turned light-green. No apparent change in total cellular proteins was evident, but the contents of two major proteins of PS2, D1 and D2, showed decline due to UV-B irradiation, although to different extent.     

Accumulation of Pathogenesis-Related Proteins in Tobacco Leaves Irradiated with UV-B

Nicotiana tabacum L. (cv. Petit Havana SR1) were grown under ultraviolet-B (UV-B, 290–320 nm) irradiation, and soluble proteins were extracted from the leaves. Two-dimensional electrophoresis revealed that a minimum of 12 polypeptides were induced by UV-B. Polypeptides which were so abundant as to be detectable by Coomassie brilliant blue staining were then subjected to N-terminal amino acid sequence analyses. Two of the polypeptides were identified as a 23 kDa protein of PS II and 6 as a pathogenesis-related protein 5 (PR-5). Immunoblotting demonstrated that other PR proteins, PR-1 and PR-3 were also induced by UV-B. Salicylic acid (SA), which is an important component of signal transduction that leads to the expression of PR proteins and exhibition of acquired resistance to pathogens, increased in response to exposure to UV-B. In addition, the activity of phenylalanine ammonialyase, which catalyzes the synthesis from phenylalanine of trans-cinnamic acid, the endogenous precursor of SA, was transiently increased by UV-B irradiation. These results suggest that UV-B activates the signal transduction pathway, which is a common step in pathogen infection. Received 8 May 2000/ Accepted in revised form 29 August 2000     

Inhibition of photosynthesis by UV-B exposure and its repair in the bloom-forming cyanobacterium <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis>

The photosynthetic performance of Microcystis aeruginosa FACHB 854 during the process of UV-B exposure and its subsequent recovery under photosynthetic active radiation (PAR) was investigated in the present study. Eight hours UV-B radiation (3.15 W m⁻²) stimulated the increase of photosynthetic pigments content at the early stage of UV-B exposure followed by a significant decline. It suggested that UV-B damage was not an immediate process, and there existed a dynamic balance between damage and adaptation in the exposed cells. Short-term UV-B exposure severely inhibited the photosynthetic capability, but it could restore quickly after being transferred to PAR. Further investigations revealed that the PS II of M. aeruginosa FACHB 854 was more sensitive to UV-B exposure than PS I, and the oxygen-evolving complex of PS II was an important damage target of UV-B. The inhibition of photosynthetic performance caused by UV-B could be recovered to 90.9% of pretreated samples after 20 h exposure at low PAR, but it could not be recovered in the dark as well as under low PAR in the presence of Chloromycetin. It can be concluded that PAR and de novo protein synthesis were essential for the recovery of UV-B-damaged photosynthetic apparatus.     

Inhibition of CO2-fixation and its effect on the activity of Photosystem II,on D1-protein synthesis and phosphorylation

To study the effects of limitations in the Calvin-cycle on Photosystem (PS) II function and on its repair by D1-protein turnover, glycerinaldehyde (DLGA) was applied to 1 h dark-adapted pea leaves via the petiole. The application resulted in a 90% inhibition of photosynthetic oxygen evolution after 90 min illumination at either 120 or 500 µmol m^–2 s^–1. In the control leaves an increase of light-dependent oxygen production to 147 and 171% was observed after 90 min illumination. According to chlorophyll fluorescence quenching analysis the inhibition of photosynthetic electron transport by DLGA led to a substantial increase in the reduction state of the primary quinone acceptor of PS II, QA, and to a rise in membrane energetisation. However, PS II functionality was hardly affected by DLGA at the low light intensity as indicated by the constant high yield of variable fluorescence, Fv/Fm. Only at 500 µmol m^–2 s^–1 a 15% loss of Fv/Fm was observed in the presence of DLGA indicating that inactivated PS II centres had accumulated. The control leaves also showed a slight loss of Fv/Fm which did not affect photosynthetic electron transport due to a faster reoxidation of QA. The relative stability of PS II function in the presence of DLGA could not be ascribed to an increased repair by the rapid turnover of the D1-protein. Radioactive pulse-labelling studies with [14C] leucine in combination with immunological determination of the protein content revealed that both synthesis and degradation of the protein were inhibited in DLGA-treated leaves whereas in the control leaves a stimulation of D1-protein turnover was observed. The changes of D1-protein turnover could be explained by differences in the occupancy state of the QB-binding niche. A relation between the phosphorylation status of the PS II polypeptides and the turnover of the D1-protein could not be established. As shown by radioactive labelling with [32P]i, addition of DLGA led to an increase in the phosphorylation level of the PS II polypeptides D1 and D2 at the low light intensity when compared to the non-treated control. At the higher light intensity the phosphorylation level of the PS II polypeptides in control and DLGA-treated leaves were identical in spite of the substantial differences in D1-protein turnover.     

A new paradigm for the action of reactive oxygen species in the photoinhibition of photosystem II

Inhibition of the activity of photosystem II (PSII) under strong light is referred to as photoinhibition. This phenomenon is due to the imbalance between the rate of photodamage to PSII and the rate of the repair of damaged PSII. Photodamage is initiated by the direct effects of light on the oxygen-evolving complex and, thus, photodamage to PSII is unavoidable. Studies of the effects of oxidative stress on photodamage and subsequent repair have revealed that reactive oxygen species (ROS) act primarily by inhibiting the repair of photodamaged PSII and not by damaging PSII directly. Thus, strong light has two distinct effects on PSII; it damages PSII directly and it inhibits the repair of PSII via production of ROS. Investigations of the ROS-induced inhibition of repair have demonstrated that ROS suppress the synthesis de novo of proteins and, in particular, of the D1 protein, that are required for the repair of PSII. Moreover, a primary target for inhibition by ROS appears to be the elongation step of translation. Inhibition of the repair of PSII by ROS is accelerated by the deceleration of the Calvin cycle that occurs when the availability of CO₂ is limited. In this review, we present a new paradigm for the action of ROS in photoinhibition.     

A new paradigm for the action of reactive oxygen species in the photoinhibition of photosystem II

Inhibition of the activity of photosystem II (PSII) under strong light is referred to as photoinhibition. This phenomenon is due to the imbalance between the rate of photodamage to PSII and the rate of the repair of damaged PSII. Photodamage is initiated by the direct effects of light on the oxygen-evolving complex and, thus, photodamage to PSII is unavoidable. Studies of the effects of oxidative stress on photodamage and subsequent repair have revealed that reactive oxygen species (ROS) act primarily by inhibiting the repair of photodamaged PSII and not by damaging PSII directly. Thus, strong light has two distinct effects on PSII; it damages PSII directly and it inhibits the repair of PSII via production of ROS. Investigations of the ROS-induced inhibition of repair have demonstrated that ROS suppress the synthesis de novo of proteins and, in particular, of the D1 protein, that are required for the repair of PSII. Moreover, a primary target for inhibition by ROS appears to be the elongation step of translation. Inhibition of the repair of PSII by ROS is accelerated by the deceleration of the Calvin cycle that occurs when the availability of CO(2) is limited. In this review, we present a new paradigm for the action of ROS in photoinhibition.     

Reversible phosphorylation and turnover of the D1 protein under various redox states of Photosystem II induced by low temperature photoinhibition

Reversible phosphorylation and turnover of the D1 protein in vivo were studied under low-temperature photoinhibition of pumpkin leaves and under subsequent recovery at low light at 4 °C or 23 °C. The inactivation of PS II and photodamage to D1 were not enhanced during low-temperature photoinhibition when compared to that at room temperature. The PS II repair cycle, however, was completely blocked at 4 °C at the level of D1 degradation. Both the recovery of the photochemical activity of PS II and the degradation of the damaged D1 protein at low light at 23 °C were delayed about 1 hour after low-temperature photoinhibition, suggesting that in addition to the decrease in catalytic turnover of the enzyme, the protease was specifically inactivated in vivo at low temperature. The effect of low temperature on the other regulatory enzymes of PS II repair, protein kinase and phosphatase [Rintamäki et al. (1996) J Biol Chem 271: 14870-14875] was variable. The D1 protein kinase was operational at low temperature while dephosphorylation of the D1 protein seemed to be completely inhibited during low temperature treatment. Under subsequent recovery conditions at low light and 23 °C, the high phosphorylation level of D1 was sustained in leaf discs photoinhibited at low temperature, despite the recovery of the phosphatase activity. This high phosphorylation level of D1 was due to the persistently active kinase. The D1 kinase, previously shown to get activated by reduction of plastoquinone, was, however, found to be maximally active already at relatively low redox state of the plastoquinone pool. We suggest that phosphorylation of PS II centers increases the stability of PS II complexes and concomitantly improves their survival under stress conditions.     

Strategies of ultraviolet-B protection in microscopic algae

Different species of microalgae show a wide range of susceptibility to ultraviolet-B (UV-B) radiation. To identify factors responsible for the UV-B tolerance of some of the algae, we compared 8 species that are highly tolerant to UV-B to 8 species that are highly susceptible. The tolerant species contained substantial amounts of an acetolysis-resistant residue. The residue consists of sporopollenin, a biopolymer of variable chemical composition that occurs in the algal cell walls and absorbs UV-B radiation. The susceptible species contained little or no sporopollenin. We propose that sporopollenin provides protection to the tolerant species by screening the incident UV-B radiation. Previous studies showed that the mycosporine-like amino acids (MAA) also act as effective UV-B screens. Our data indicate that sporopollenin provides a constant protection while MAA are induced by radiation stress and occur with some delay. The tolerant species also differ from the susceptible species in their capacity to repair the reaction centers damaged by UV-B. The tolerant algae became vulnerable to UV-B when protein synthesis needed for repair was blocked by streptomycin. In the susceptible species, streptomycin had no effect during the UV-B stress. The repair deficiency in the susceptible species can be explained either by relatively less effective protein synthesis or by an inhibition of the protein synthesis by UV-B. In the tolerant species, the structures needed for protein synthesis are protected by UV-B screening of sporopollenin and MAA.     

Mathematical modelling of photoinhibition and Photosystem II repair cycle. I. Photoinhibition and D1 protein degradation in vitro and in the absence of chloroplast protein synthesis in vivo

The kinetics of photoinhibition of Photosystem II and D1 protein degradation were studied by applying mathematical modelling to new and published data. The word photoinhibition refers here only to such inhibition of PS II activity that requires chloroplast protein synthesis for recovery. It is shown that acceptor-side photoinhibition in vitro as well as in vivo photoinhibition in higher plants and cyanobacteria in the presence of prokaryotic translation inhibitors follow first-order kinetics. Degradation of damaged D1 protein also fits in a first-order reaction equation with respect to the concentration of photoinhibited PS II centres. It is shown that photoprotective lowering of the ratio of variable to maximum fluorescence can be distinguished from the lowering of this ratio associated with photoinhibition.