Disturbed structure and function of chloroplasts during blocked biosynthesis of 5-aminolevulinic acid in the light

Xantha-702 mutant of cotton (Gossypium hirsutum L.) proved to have blocked synthesis of 5-aminolevulinic acid in the light. Accordingly, mutant leaves accumulated 2–5% chlorophyll of baseline. Mutant plants demonstrated disturbed production of pigment-protein complexes of photosystems I (PSI) and II (PSII) and generation of the chloroplast membrane system blocked at the early stages, largely, at the stages of vesicles and single short thylakoid. The functional activity of the PSI and PSII reaction centers was close to zero. Only the chlorophyll a/b light-harvesting complexes of PSI and PSII with the chlorophyll fluorescence peaks at 728 and 681 nm, respectively, were produced in the xantha-702 mutant. We propose that the genetic block of 5-aminolevunilic acid biosynthesis in the light in the xantha-702 mutant disturbs the formation and activity of the complexes of the reaction centers of PS-I and PS-II and inhibits the development of the whole membrane system of chloroplasts.     

Effects of Drought Stress on the Photosynthesis in Maize

To clarify how the components of the entire photosynthetic electron transport chain in response to drought stress in maize. The activities of photosystem II (PSII), photosystem I (PSI), and the electron transport chain between PSII and PSI of maize were investigated by prompt fluorescence (PF), delayed fluorescence (DF) and 820 nm modulated reflection (MR). Maize (Zea mays L.) plants were subjected to different levels of soil water availability including control, moderate and severe drought stress. A significant decrease in ?E₀, Ψ₀ and PI_ABS was found in maize treated with moderate drought stress. A significant increase in ABS/RC was observed, but there were no significant change in the fast MR phase and the amplitude of DF under moderate drought stress compared to the control. Under severe drought stress, the exchange capacity between Q_A to Q_B, reoxidation capacity of plastoquinol, and the oxidation and re-reduction rates of PC and P₇₀₀ all decreased. These results demonstrated that moderate drought stress reduced the photochemical activity of PSII from Q_A to PQH₂, while the photochemical activity of PSI was unscathed. However, severe drought stress inhibited the entire electron transport chain from the donor side of PSII to PSI-end electron acceptors. In addition, the photochemical activity of PSII is more sensitive to drought stress than PSI.     

Photoinhibition of photosystem I in <Emphasis Type="Italic">Nephrolepis falciformis</Emphasis> depends on reactive oxygen species generated in the chloroplast stroma

We studied how high light causes photoinhibition of photosystem I (PSI) in the shade-demanding fern Nephrolepis falciformis, in an attempt to understand the mechanism of PSI photoinhibition under natural field conditions. Intact leaves were treated with constant high light and fluctuating light. Detached leaves were treated with constant high light in the presence and absence of methyl viologen (MV). Chlorophyll fluorescence and P700 signal were determined to estimate photoinhibition. PSI was highly oxidized under high light before treatments. N. falciformis showed significantly stronger photoinhibition of PSI and PSII under constant high light than fluctuating light. These results suggest that high levels of P700 oxidation ratio cannot prevent PSI photoinhibition under high light in N. falciformis. Furthermore, photoinhibition of PSI in N. falciformis was largely accelerated in the presence of MV that promotes the production of superoxide anion radicals in the chloroplast stroma by accepting electrons from PSI. From these results, we propose that photoinhibition of PSI in N. falciformis is mainly caused by superoxide radicals generated in the chloroplast stroma, which is different from the mechanism of PSI photoinhibition in Arabidopsis thaliana and spinach. Here, we provide some new insights into the PSI photoinhibition under natural field conditions.     

Structural and functional characteristics of photosynthetic apparatus of chlorophyll-containing grape vine tissue

Photosynthesis in tissues under periderm of woody stems and shoots of perennial plants occurs in environment that is very different from the internal environment of leaf chloroplasts. These tissues are characterized by high CO₂ and low O₂ concentrations, more acidic surroundings, besides that only light which have passed through periderm reaches photosynthetic antennas. In contrast to leaves of deciduous plants chlorenchyma tissues of wintering plant organs are exposed to temperature fluctuations during all seasons, that is why the photosynthetic apparatus of woody stems has to be able to adapt to a wide range of environmental temperatures. In order to reveal unique features, which enable photosynthetic apparatus of chlorenchyma cells in woody plant organs to implement biological functions under different light and temperature conditions, we studied photosynthetic tissues of stem cortex in grapevine (Vitis vinifera L.) under normal conditions and after exposure to suboptimal temperatures and high light intensity. Comparative analysis of photosynthetic pigment composition and low-temperature chlorophyll fluorescence emission spectrum of leaves, young shoots and chlorenchyma of lignified shoots revealed relatively high level of chlorophyll b and carotenoids, and high photosystem II (PSII) to photosystem I (PSI) ratio in woody shoots. Analysis of parameters of variable chlorophyll fluorescence revealed high PSII activity in grapevine shoot cortex and demonstrated improved freeze tolerance and higher sensitivity to light of photosynthetic apparatus in grape vine in comparison to leaves. It was shown for the first time that photosynthetic apparatus in chlorenchyma cells of vine undergoes so-called “state-transition”–fast rearrangements leading to redistribution of energy between photosystems. Analysis of fatty acid (FA) compositions of lipids in examined tissues showed that the FA unsaturation index in green tissue of vine is lower than in leaves. A distinct feature of FA compositions of lipids in vine cortex was relatively high level of linoleic acid.     

Increase in the rate of photosynthetic linear electron transport in cyanobacteruim <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 lacking phycobilisomes

Compensating changes in the pigment apparatus of photosynthesis that resulted from a complete loss of phycobilisomes (PBS) were investigated in the cells of a PAL mutant of cyanobacterium Synechocystis sp. PCC 6803. The ratio PBS/chlorophyll calculated on the basis of the intensity of bands in the action spectra of photosynthetic activity of two photosystems in the wild strain was 1: 70 for PSII and 1: 300 for PSI. Taking into consideration the number of chlorophyll molecules per reaction center in each photosystem, these ratios could be interpreted as association of PBS with dimers of PSII and trimers of PSI as well as greater dependence of PSII as compared with PSI on light absorption by PBS. The ratio PSI/PSII determined by photochemical cross-section of the reactions of two photosystems was 3.5: 1.0 for wild strain of Synechocystis sp. PCC 6803 and 0.7: 1.0 for the PAL mutant. A fivefold increase in the relative content of PSII in pigment apparatus corresponds to a 5-fold increase in the intensity of bands at 685 and 695 nm as related to the band of PSI at 726 nm recorded in low-temperature fluorescence spectrum of the PAL mutant. Inhibition of PSII with diuron resulted in a pronounced stimulation of chlorophyll fluorescence in the PAL mutant as compared to the wild strain of Synechocystis sp. PCC 6803; these data suggested an activation of electron transfer between PSII and PSI in the mutant cells. Thus, the lack of PBS in the mutant strain of Synechocystis sp. PCC 6803 was compensated for by the higher relative content of PSII in the pigment apparatus of photosynthesis and by a rise in the rate of linear electron transport.     

Effects of heat stress on photosynthetic electron transport in a marine cyanobacterium <Emphasis Type="Italic">Arthrospira</Emphasis> sp.

Arthrospira (Spirulina) is widely used as human health food and animal feed. In cultures grown outdoors in open ponds, Arthrospira cells are subjected to various environmental stresses, such as high temperature. A better understanding of the effects of high temperature on photosynthesis may help optimize the productivity of Arthrospira cultures. In this study, the effects of heat stress on photosynthetic rate, chlorophyll a fluorescence transients, and photosystem (PS) II, PSI activities in a marine cyanobacterium Arthrospira sp. were examined. Arthrospira cells grown at 25 °C were treated for 30 min at 25 (control), 30, 34, 37, or 40 °C in the dark. Heat stress (30–37 °C) enhanced net photosynthetic O₂ evolution rate. Heat stress caused over-reduction PSII acceptor side, damage of donor side of PSII, decrease in the energetic connectivity of PSII units, and decrease in the performance of PSII. When the temperature changed from 25 to 37 °C, PSII activity decreased, while PSI activity increased, the enhancement of photosynthetic O₂ evolution was synchronized with the increase in PSI activity. When temperature was further increased to 40 °C, it induced a decrease in photosynthetic O₂ evolution rate and a more severe decrease in PSII activity, but an increase in PSI activity. These results suggest that PSI activity was the decisive factor determining the change of photosynthetic O₂ evolution when Arthrospira was exposed to a temperature from 25 to 37 °C, but then, PSII activity became the decisive factor adjusting the change of photosynthetic O₂ evolution when the temperature was increased to 40 °C.     

The Involvement of the Complexes of Photosystems in the Organization of Chloroplast Ultrastructure in Pea Mutants

We studied the involvement of pigment-protein complexes of photosystems (PS) in the development and spatial arrangement of thylakoids in chloroplasts of pea (Pisum sativum L.) leaves. The initial line (cv. Torsdag) and its mutants, chlorotica 2004 displaying primary disturbances in the PSI reaction centers and chlorotica 2014 containing only 50% of chlorophyll and, as a sequence, the reduced amount of all pigment-protein complexes. A proportional decrease in the content of PSI and PSII complexes in the chlorotica 2014 mutant resulted in a partial reduction of the whole chloroplast membrane system, whereas grana and stroma thylakoid regions were well developed. In contrast, a loss of only 20% of chlorophyll and destruction of PSI complexes in the chlorotica 2004 mutant by 50% resulted in the destruction of stroma thylakoid regions and disturbed longitudinal thylakoid and grana orientation. It was concluded that protein-protein interactions in pigment-protein complexes played a key role in the structure of thylakoid membranes and their longitudinal orientation.     

Blue light reduces photosynthetic efficiency of cyanobacteria through an imbalance between photosystems I and II

Several studies have described that cyanobacteria use blue light less efficiently for photosynthesis than most eukaryotic phototrophs, but comprehensive studies of this phenomenon are lacking. Here, we study the effect of blue (450 nm), orange (625 nm), and red (660 nm) light on growth of the model cyanobacterium Synechocystis sp. PCC 6803, the green alga Chlorella sorokiniana and other cyanobacteria containing phycocyanin or phycoerythrin. Our results demonstrate that specific growth rates of the cyanobacteria were similar in orange and red light, but much lower in blue light. Conversely, specific growth rates of the green alga C. sorokiniana were similar in blue and red light, but lower in orange light. Oxygen production rates of Synechocystis sp. PCC 6803 were five-fold lower in blue than in orange and red light at low light intensities but approached the same saturation level in all three colors at high light intensities. Measurements of 77 K fluorescence emission demonstrated a lower ratio of photosystem I to photosystem II (PSI:PSII ratio) and relatively more phycobilisomes associated with PSII (state 1) in blue light than in orange and red light. These results support the hypothesis that blue light, which is not absorbed by phycobilisomes, creates an imbalance between the two photosystems of cyanobacteria with an energy excess at PSI and a deficiency at the PSII-side of the photosynthetic electron transfer chain. Our results help to explain why phycobilisome-containing cyanobacteria use blue light less efficiently than species with chlorophyll-based light-harvesting antennae such as Prochlorococcus, green algae and terrestrial plants.     

The action of lipase on chloroplast membranes: II. Polypeptide patterns of bean galactolipase- and phospholipase A2-treated thylakoid membranes

Thylakoid membranes obtained from bean chloroplasts treated with bean galactolipase or phospholipase A₂ (from Crotalus terr. terr.) showed marked changes in their polypeptide patterns when separated on SDS-PAGE. The obtained results have been discussed with regard to the relationship between chloroplast lipids and polypeptides originating from chlorophyll-protein complexes of bean thylakoids. A coexistence between galactolipids and the peripheral antennae in PS I complex and LHCP³ as well as a conspicuous role of phospholipids in PSI and PSII centre chlorophyll-protein complexes has to be underlined.Abbreviations CP1 chlorophyll a-protein complex of PSI - CPa chlorophyll a-protein complex of PSII - D₁₀ digitonin subchloroplast particles enriched in PSII - D₁₄₄ digitonin subchloroplast particles enriched in PSI - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - LHCP^1–3 light harvesting chlorophyll a/b protein complexes - PAGE polyacrylamide gel electrophoresis - PSI photosystem I - PSII photosystem II - SDS sodium dodecyl sulphate - TCA trichloroacetic acid - Tricine N-Tris-(hydroxymethyl)-methylglycine - Tris Tris-(hydroxymethyl)-aminomethan     

Effects of cycloheximide on photosynthetic abilities,reflectance spectra and fluorescence emission spectra in <Emphasis Type="Italic">Phyllostachys edulis</Emphasis>

Key message

CHX had remarkable inhibition on P. edulis photosynthesis, and the reflectance indexes and F ₆₈₅ / F ₇₃₅ had the potential value for quantifying the effects of antibiotics on trees.

Abstract

To reveal the effects of antibiotics on photosynthesis and provide help for remote sensing the influence of antibiotics on trees, we investigated the effects of cycloheximide (CHX) on Phyllostachys edulis. In CHX treatment, the photosynthetic pigment content in P. edulis was decreased markedly, which led to the increase in the reflectance spectra in visible region. CHX reduced the donor side and acceptor side of photosystem II (PSII), density of reaction centers, quantum production and electron transport in PSII, and raised the dissipation of absorbed light energy. Besides the dissipation, the absorbed light energy can be emitted as fluorescence with two main peaks in the red (685 nm) and far-red (735 nm) region, respectively. In 0.50 mM CHX treatment, a significant decline in the height and area of the peak at 685 nm might result from Chl loss reducing the light absorption and lower photochemical reaction in PSII. When fourth derivative analysis of fluorescence emission spectra was performed, the changes of the peaks at 718, 735 and 750 nm might result from the decline of absorbed solar radiation caused by the reduced pigment content and/or the damages to the PSI. In CHX treatment, a remarkable increase in intercellular CO₂ concentrations and light compensation point and decrease in light saturation point demonstrated that the CO₂ assimilation ability was decreased. Those results suggested that the photosynthesis in trees can be reduced after they were watered with wastewater containing CHX. The reflectance indexes and F ₆₈₅/F ₇₃₅ (H ₆₈₅/H ₇₃₅ and A ₆₈₅/A ₇₃₅) were markedly affected by CHX, demonstrating that they had the potential value for quantifying the effects of antibiotics on trees.

     

The action of lipase on chloroplast membranes: II. Polypeptide patterns of bean galactolipase- and phospholipase A2-treated thylakoid membranes

Thylakoid membranes obtained from bean chloroplasts treated with bean galactolipase or phospholipase A₂ (from Crotalus terr. terr.) showed marked changes in their polypeptide patterns when separated on SDS-PAGE. The obtained results have been discussed with regard to the relationship between chloroplast lipids and polypeptides originating from chlorophyll-protein complexes of bean thylakoids. A coexistence between galactolipids and the peripheral antennae in PS I complex and LHCP³ as well as a conspicuous role of phospholipids in PSI and PSII centre chlorophyll-protein complexes has to be underlined.Abbreviations CP1 chlorophyll a-protein complex of PSI - CPa chlorophyll a-protein complex of PSII - D₁₀ digitonin subchloroplast particles enriched in PSII - D₁₄₄ digitonin subchloroplast particles enriched in PSI - DCMU 3-(3,4-dichlorophenyl)-1, 1-dimethylurea - LHCP^1-3 light harvesting chlorophyll a/b protein complexes - PAGE polyacrylamide gel electrophoresis - PSI photosystem I - PSII photosystem II - SDS sodium dodecyl sulphate - TCA trichloroacetic acid - Tricine N-Tris-(hydroxymethyl)-methylglycine - Tris Tris-(hydroxymethyl)-aminomethan     

Evidence of the supercomplex organization of photosystem II and light-harvesting complexes in <Emphasis Type="Italic">Nannochloropsis granulata</Emphasis>

Diverse light-harvesting complexes (LHCs) have been found in photosynthetic microalgae that originated from secondary endosymbiosis involving primary red algae. However, the associations between LHCs and photosystem I (PSI) and photosystem II (PSII) in these microalgae are not fully understood. Eustigmatophyta is a red algal lineage that appears to have a unique organization in its photosynthetic machinery, consisting of only chlorophyll a and carotenoids that are atypical compared with other closely related groups. In this study, the supramolecular organization of pigment–protein complexes in the eustigmatophyte alga, Nannochloropsis granulata was investigated using Clear Native (CN) PAGE coupled with two-dimensional (2D) SDS-PAGE. Our results showed two slowly migrating green bands that corresponded to PSII supercomplexes, which consisted of reaction centers and LHCs. These green bands were also characterized as PSII complexes by their low temperature fluorescence emission spectra. The protein subunits of the PSII–LHC resolved by 2D CN/SDS-PAGE were analyzed by mass spectrometry, and four different LHC proteins were identified. Phylogenetic analysis of the identified LHC protein sequences revealed that they belonged to four different Lhc groups; (1) stress-related Lhcx proteins, (2) fucoxanthin chlorophyll a/c-binding Lhcf proteins, (3) red-shifted Chromera light-harvesting proteins (Red-CLH), and (4) Lhcr proteins, which are commonly found in organisms possessing red algal plastids. This is the first report showing evidence of a pigment–protein supercomplex consisting of PSII and LHCs, and to identify PSII-associated LHC proteins in Nannochloropsis.     

Linking chloroplast relocation to different responses of photosynthesis to blue and red radiation in low and high light-acclimated leaves of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> (L.)

Low light (LL) and high light (HL)-acclimated plants of A. thaliana were exposed to blue (BB) or red (RR) light or to a mixture of blue and red light (BR) of incrementally increasing intensities. The light response of photosystem II was measured by pulse amplitude-modulated chlorophyll fluorescence and that of photosystem I by near infrared difference spectroscopy. The LL but not HL leaves exhibited blue light-specific responses which were assigned to relocation of chloroplasts from the dark to the light-avoidance arrangement. Blue light (BB and BR) decreased the minimum fluorescence (\(F_{0}^{\prime }\)) more than RR light. This extra reduction of the \(F_{0}^{\prime }\) was stronger than theoretically predicted for \(F_{0}^{\prime }\) quenching by energy dissipation but actual measurement and theory agreed in RR treatments. The extra \(F_{0}^{\prime }\) reduction was assigned to decreased light absorption of chloroplasts in the avoidance position. A maximum reduction of 30% was calculated. Increasing intensities of blue light affected the fluorescence parameters NPQ and q_P to a lesser degree than red light. After correcting for the optical effects of chloroplast relocation, the NPQ responded similarly to blue and red light. The same correction method diminished the color-specific variations in q_P but did not abolish it; thus strongly indicating the presence of another blue light effect which also moderates excitation pressure in PSII but cannot be ascribed to absorption variations. Only after RR exposure, a post-illumination overshoot of \(F_{0}^{\prime }\) and fast oxidation of PSI electron acceptors occurred, thus, suggesting an electron flow from stromal reductants to the plastoquinone pool.     

Different strategies of photoacclimation by two strains of Emiliania huxleyi (Haptophyta)1

Photoacclimation involves the modification of components of the light and dark reactions to optimize photosynthesis following changes in available light. All of the energy required for photosynthesis comes from linear electron transport through PSII and PSI and is dependent upon the amount of light harvested by PSII relative to PSI (a*_PSII and a*_PSI). The amount of light harvested is determined by the effective absorption cross‐sections (σ_PSII, σ_PSI) and cellular contents of the PSII and PSI reaction center complexes (RCII, RCI). Here, we examine the effective absorption cross‐sections and reaction center contents for calcifying (B11) and noncalcifying (B92) strains of the globally important coccolithophorid Emiliania huxleyi (Lohmann) W. H. Hay et H. Mohler when grown under various photon flux densities (PFDs). The two strains displayed different “strategies” of acclimation. As growth PFD increased, B11 preferentially changed σ and the cellular content of chl a per cell over PSU “size” (the total cellular chl a content associated with the reaction center complexes); strain B92 preferentially changed PSU size over the cellular content of reaction complexes. Neither strategy was specifically consistent with the majority of previous studies from other microalgal species. For both strains, cellular light absorption for PSII and PSI was maintained close to unity across the range of growth PFDs since changes of σ_PSII and σ_PSI were reciprocated by those of RCIIs and RCIs per cell. Our results demonstrate a significant adaptive flexibility of E. huxleyi to photoacclimate. Finally, we calculated the amount of chl a associated with either photosystem to consider our interpretations of photoacclimation based on conventional determinations of PSU size.     

Respiratory terminal oxidases alleviate photo-oxidative damage in photosystem I during repetitive short-pulse illumination in <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

Oxygenic phototrophs are vulnerable to damage by reactive oxygen species (ROS) that are produced in photosystem I (PSI) by excess photon energy over the demand of photosynthetic CO₂ assimilation. In plant leaves, repetitive short-pulse (rSP) illumination produces ROS to inactivate PSI. The production of ROS is alleviated by oxidation of the reaction center chlorophyll in PSI, P700, during the illumination with the short-pulse light, which is supported by flavodiiron protein (FLV). In this study, we found that in the cyanobacterium Synechocystis sp. PCC 6803 P700 was oxidized and PSI was not inactivated during rSP illumination even in the absence of FLV. Conversely, the mutant deficient in respiratory terminal oxidases was impaired in P700 oxidation during the illumination with the short-pulse light to suffer from photo-oxidative damage in PSI. Interestingly, the other cyanobacterium Synechococcus sp. PCC 7002 could not oxidize P700 without FLV during rSP illumination. These data indicate that respiratory terminal oxidases are critical to protect PSI from ROS damage during rSP illumination in Synechocystis sp. PCC 6803 but not Synechococcus sp. PCC 7002.     

Photosystem electron-transport capacity and light-harvesting antenna size in maize chloroplasts

Spectrophotometric and kinetic measurements were applied to yield photosystem (PS) stoichiometries and the functional antenna size of PSI, PSII_α, and PSII_β in Zea mays chloroplasts in situ. Concentrations of PSII and PSI reaction centers were determined from the amplitude of the light-induced absorbance change at 320 and 700 nm, which reflect the photoreduction of the primary electron acceptor Q of PSII and the photooxidation of the reaction center P700 of PSI, respectively. Determination of the functional chlorophyll antenna size (N) for each photosystem was obtained from the measurement of the rate of light absorption by the respective reaction center. Under the experimental conditions employed, the rate of light absorption by each reaction center was directly proportional to the number of light-harvesting chlorophyll molecules associated with the respective photosystem. We determined N_P700 = 195, N_α = 230, N_β = 50 for the number of chlorophyll molecules in the light-harvesting antenna of PSI, PSII_α, and PSII_β, respectively. The above values were used to estimate the PSII/PSI electron-transport capacity ratio (C) in maize chloroplasts. In mesophyll chloroplasts C > 1.4, indicating that, under green actinic excitation when Chl a and Chl b molecules absorb nearly equal amounts of excitation, PSII has a capacity to turn over electrons faster than PSI. In bundle sheath chloroplasts C < 1, suggesting that such chloroplasts are not optimally poised for linear electron transport and reductant generation.     

Quality Control of Photosystem II: THYLAKOID UNSTACKING IS NECESSARY TO AVOID FURTHER DAMAGE TO THE D1 PROTEIN AND TO FACILITATE D1 DEGRADATION UNDER LIGHT STRESS IN SPINACH THYLAKOIDS*

Photosystem II is vulnerable to light damage. The reaction center-binding D1 protein is impaired during excessive illumination and is degraded and removed from photosystem II. Using isolated spinach thylakoids, we investigated the relationship between light-induced unstacking of thylakoids and damage to the D1 protein. Under light stress, thylakoids were expected to become unstacked so that the photodamaged photosystem II complexes in the grana and the proteases could move on the thylakoids for repair. Excessive light induced irreversible unstacking of thylakoids. By comparing the effects of light stress on stacked and unstacked thylakoids, photoinhibition of photosystem II was found to be more prominent in stacked thylakoids than in unstacked thylakoids. In accordance with this finding, EPR spin trapping measurements demonstrated higher production of hydroxyl radicals in stacked thylakoids than in unstacked thylakoids. We propose that unstacking of thylakoids has a crucial role in avoiding further damage to the D1 protein and facilitating degradation of the photodamaged D1 protein under light stress.In the chloroplasts of higher plants and green algae, thylakoid membranes are closely associated and stack to form grana. Under electron microscopy, cylindrical grana consisting of 10–20 layers of thylakoids have been observed. They have a diameter of 300–600 nm and are interconnected by lamellae of several hundred nm in length (1, 2). The structure of grana in the chloroplasts of higher plants is well known, but the precise role of grana is incompletely understood. Their possible functions in primary photochemical reactions and subsequent events have been discussed extensively (3–9). Photosystem I (PSI)³ and II (PSII) complexes are segregated from each other in thylakoids, showing lateral heterogeneity in their distribution. The PSII complex is a multisubunit pigment-protein complex responsible for the photochemical oxidation of water and reduction of plastoquinone (8, 10–13). It comprises >25 protein subunits and other low molecular weight cofactors, including chlorophylls, carotenoids, plastoquinones, and manganeses. In the chloroplasts of higher plants, PSII complexes and the associated light-harvesting antenna complex LHCII are not present throughout the thylakoid membranes but are abundant in the grana (2, 14). A densely packed array of PSII complexes in the grana was visualized by electron microscopy (8, 15). Grana formation is more prominent in shade leaves (or shade plants) than in sun leaves (or sun plants), so it has been suggested that enrichment of the PSII·LHCII complex in grana is a strategy of plants to collect excitation energy by PSII under weak light (16). The grana structure probably provides an organized environment for PSII. PSI and ATP synthase are located exclusively in the stroma-exposed thylakoids, including the stroma thylakoids, grana end membranes, and grana margins, because these complexes protrude into the stroma. Cytochrome b₆/f complexes without this protrusion are present uniformly throughout the thylakoids (3). It has been suggested that separation of PSI and PSII complexes on the thylakoids through grana formation is important to prevent “spillover” of excitation energy from PSII to PSI, which lowers photosynthesis efficiency (17).An active PSII complex comprises a homodimer of PSII monomers (13). When thylakoids are exposed to excessive visible light, the PSII dimer dissociates into two monomers (18), but the most significant change takes place inside the monomeric PSII, where the reaction center-binding D1 protein is photodamaged and degraded by specific proteases (19, 20). The photodamage to the D1 protein is a photooxidative process. This is caused by reactive oxygen species (ROS), most probably singlet oxygen (¹O₂) or the hydroxyl radical (HO^•) produced by overreduction of the acceptor side of PSII under excessive illumination or by endogenous cationic radicals, such as the oxidized forms of the primary electron donor P680 and the secondary electron donor Tyr_Z (Tyr¹⁶¹ of D1) to PSII (21). Strong illumination of the grana may readily cause damage to the PSII complexes by ROS and endogenous cationic radicals, because the grana is rich in PSII complexes. Segregation of PSI and PSII in the stacked thylakoids should make the electron transport between PSI and PSII a rate-limiting step in the electron flow, and overexcitation of PSII under these conditions may stimulate ROS production at the acceptor side of PSII. Close association of LHCII with the PSII core complexes should also stimulate ROS generation in the grana. Unstacking of the thylakoids, which is also expected to lead to random distribution of PSI and PSII on the thylakoids and dissociation of the LHCII from the PSII core, may be important to avoid photodamage to PSII.In the proteolysis of the damaged D1 protein in the chloroplasts of higher plants, the N-terminal Thr of the D1 protein is dephosphorylated, and the subsequent degradation produces 23- and 9-kDa fragments as the primary cleavage products (19, 20). The protease(s) and phosphatase(s) involved in these steps are presumably localized in the stroma thylakoids, grana end membranes, and grana margin. Lateral migration of the damaged PSII complexes from the grana to the membrane regions where the damaged PSII complexes are repaired is therefore important for degradation of the D1 protein. Thylakoid unstacking, if it occurs under light stress, should stimulate diffusion of the protein complexes on the thylakoids, thereby stimulating D1 turnover.First, we examined if excessive visible light can induce unstacking of the thylakoids. Second, we studied the effects of strong illumination on stacked and unstacked thylakoids to see if they showed different responses to excessive light. We strongly suggest that unstacking of the thylakoids caused by light stress is necessary to avoid further photodamage to the D1 protein and to facilitate degradation and removal of the photodamaged D1 protein from PSII complexes.     

Examination of chlorophyll fluorescence in sulfur-deprived cells of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

Pulse amplitude modulation fluorimetry was used to assess chlorophyll fluorescence parameters in Chlamydomonas reinhardtii cells during sulfur deprivation. A significant (fourfold) increase in the chlorophyll fluorescence yield (parameters F ₀ and F _m) normalized to the chlorophyll concentration was shown for deprived cells. The chlorophyll content did not change during the deprivation experiments. An analysis of nonphotochemical quenching of chlorophyll fluorescence indicated a considerable modification of the energy deactivation pathways in photosystem II (PSII) of sulfur-deprived cells. For example, starved cells exhibited a less pronounced pH-dependent quenching of excited states and a higher thermal dissipation of excess light energy in the reaction centers of PSII. It was also shown that the photosynthetic apparatus of starved cells is primarily in state 2 and that back transition to state 1 is suppressed. However, these changes cannot cause the discovered elevation of chlorophyll fluorescence intensity (F ₀ and F _m) in the cells under sulfur limitation. The observed increase in the chlorophyll fluorescence intensity under sulfur deprivation may be due to partial dissociation of peripheral light-harvesting complexes from the reaction centers of PSII or a malfunction of the dissipative cycle in PSII, involving cytochrome b ₅₅₉.     

Fluorescence Induction Kinetics in Membrane Preparations of Photosystem II with Heterogeneous Metal Clusters (Mn/Fe) in the Oxygen-Evolving Complex

Transport of electrons in spinach photosystem II (PSII) whose oxygen-evolving complex (OEC) contains heterogeneous metal clusters 2Mn2Fe and 3Mn1Fe was studied by measuring the fluorescence induction kinetics (FIK). PSII(2Mn,2Fe) and PSII(3Mn,1Fe) preparations were produced using Cadepleted PSII membranes (PSII(–Ca)). It was found that FIK in PSII(2Mn,2Fe) membranes is similar in form to FIK in PSII(–Ca) samples, but the fluorescence yield is lower in PSII(2Mn,2Fe). The results demonstrate that, just as in PSII(–Ca) preparations, there is electron transfer from the metal cluster in the OEC to the primary plastoquinone electron acceptor Q_A. They also show that partial substitution of Mn cations with Fe has no effect on the electron transport on the acceptor side of PSII. Thus, these data demonstrate the possibility of water oxidation either by the heterogeneous metal cluster or just by the manganese dimer. We established that FIK in PSII(3Mn,1Fe) preparations are similar in form to FIK in PSII(2Mn,2Fe) membranes but PSII(3Mn,1Fe) is characterized by a slightly higher maximal fluorescence yield, F_max. The electron transfer rate in PSII(3Mn,1Fe) preparations significantly (by a factor of two) increases in the presence of Ca²⁺, whereas Ca²⁺ has hardly any effect on the electron transport in PSII(2Mn,2Fe) membranes. In Mndepleted PSII membranes, FIK reaches its maximum (the so-called peak K), after which the fluorescence yield starts to decrease as the result of two factors: the oxidation of reduced primary plastoquinone Q _A ^? and the absence of electron influx from the donor side of PSII. The replacement of Mn cations by Fe in PSII(?Mn) preparations leads to fluorescence saturation and disappearance of the K peak. This is possibly due to the deceleration of the charge recombination process that takes place between reduced primary electron acceptor Q _A ^? and oxidized tyrosine Y _Z ^+. which is an electron carrier between the OEC and the primary electron donor P680.