Low-temperature Fluorescence Emission Spectra Research Articles

Different impact of high light on the response and recovery of wild type and lut2 mutant of Arabidopsis thaliana at low temperature

Carotenoid composition is important for plant protection against unfavorable environment. In the present study the effect of several days treatment with high light illumination at low temperature (500 µmol photons m−2 s−1 and (12/10 °C) on Arabidopsis thaliana plants—wt and mutant lut2, was investigated. In addition, a comparative study of the ability of wt and lut2 plants to recover after one week return to control conditions was performed. The response of wt and lut2 to both stress factors was evaluated by changes in net photosynthesis, photochemistry and energy distribution and interaction between both photosystems. The alterations in oxygen evolving capacity were evaluated by registration of flash-induced oxygen yields and kinetics of initial oxygen burst. For the first time the effect of exposure at low temperature/high light on the fluidity of thylakoid membranes isolated from wt and lut2 mutant was followed. The analysis of low temperature (77 K) fluorescence emission and excitation spectra revealed that during exposition to both stress factors the antenna size of photosystem I and photosystem II were affected. Lut2 mutant showed higher sensitivity to the inhibitory action of high light at low temperature, with the grana-situated photosystem II centers being more affected. However, lut2 mutant exhibited a higher ability to restore the photosynthetic activity after recovery period under control conditions. Data presented showed that at low temperature, complete replacement of lutein in lut2 mutant by xanthophyll cycle-associated carotenoids did not result in higher protection against high light, but facilitated better recovery during the recovery period.

Formation of prolamellar-body-like ultrastructures in etiolated cyanobacterial cells overexpressing light-dependent protochlorophyllide oxidoreductase in Leptolyngbya boryana.

Protochlorophyllide (Pchlide) reduction is the penultimate step of chlorophyll (Chl) biosynthesis, and is catalyzed by two evolutionarily unrelated enzymes: dark-operative Pchlide oxidoreductase (DPOR) and light-dependent Pchlide oxidoreductase (LPOR). Because LPOR is the sole Pchlide reductase in angiosperms, dark-grown seedlings of angiosperms become etiolated. LPOR exists as a ternary complex of Pchlide-NADPH-LPOR to form paracrystalline prolamellar bodies (PLBs) in etioplasts. Because LPOR is distributed ubiquitously across oxygenic phototrophs including cyanobacteria, it would be important to determine whether cyanobacterial LPOR has the ability to form PLBs. We isolated a DPOR-less transformant ΔchlL/LPORox, carrying a plasmid to overexpress cyanobacterial LPOR in the cyanobacterium Leptolyngbya boryana. The transformant did not produce Chl in the dark and became etiolated with an accumulation of Pchlide and LPOR. Novel PLB-like ultrastructures were observed in etiolated cells, which disappeared during the early stage of the light-dependent greening process. However, the rate of Chl production in the greening process of ΔchlL/LPORox was almost the same as that observed in the control cells, which carried an empty vector. An in vitro LPOR assay of extracts of dark-grown ΔchlL/LPORox cells suggested that the PLB-like structures are deficient in NADPH. Low-temperature fluorescence emission spectra of membrane fractions of the etiolated cells indicated the absence of the photoactive form of Pchlide, which was consistent with the inefficiency of the greening process. Cyanobacterial LPOR exhibited an intrinsic ability to form PLB-like ultrastructures in the presence of the co-accumulation of Pchlide; however, the PLB-like structure differed from the authentic PLB regarding NADPH deficiency.

Evidence of the supercomplex organization of photosystem II and light-harvesting complexes in Nannochloropsis granulata.

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.

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.

Heat- and light-induced detachment of the light harvesting complex from isolated photosystem I supercomplexes

In a previous study, using photosystem I enriched stroma thylakoid membrane vesicles, we have shown that the light harvesting complexes of this photosystem are prone to heat- and light–induced, thermo-optically driven detachment from the supercomplex [43]. We have also shown that the splitting of the supercomplex occurs in a gradual and specific manner, selectively affecting the different constituents of the antenna complexes. Here we further analyse these heat- and light-induced processes in isolated Photosystem I supercomplex using circular dichroism and 77K fluorescence emission spectroscopy and immuno blotting, and obtain further details on the sequence of events of the dissociation process as well as on the thermal stability of the different components.Our absorption and circular dichroism spectroscopy and immuno blotting data show that the dissociation of LHCI from PSI-LHCI supercomplex starts above 50°C. Also, the low temperature fluorescence emission spectra depicts decrease of maximum fluorescence emission at 730nm and an increase of the intensity at 685nm, and about 10nm blue-shifts, from 730 to 720nm and from 685 to 676nm, respectively, indicating the heat (50°C) induced detachment of LHCI from PSI core complexes. The reaction centre proteins are highly stable even at high temperatures. Lhca2 is more heat stable than the other light harvesting protein complexes of PSI, whereas Lhca4 and Lhca3 are rather labile. Combined heat and light treatments significantly enhances the disorganization of PSI-LHCI supercomplexes, indicating a thermo-optic mechanism, which might have significant role under combined heat and light stress conditions.

Altered Energy Transfer Among Phycobiliproteins of CyanobacteriumSynechococcus-6301Under High Temperature Stress

Altered Energy Transfer Among Phycobiliproteins of Cyanobacterium Synechococcus-6301 Under High Temperature Stress. Decrease in phycocyanin absorption and fluorescence emission intensity is observed when intact phycobilisomes are exposed to high temperatures in the range 30-50oC in case of Synechococcus 6301. The shift in the peak position of both fluorescence and absorption spectra clearly indicates the structural alterations in pigment proteins interaction. Low temperature (77K) fluorescence emission spectra clearly demonstrate that the energy transfer from phycocyanin to allophycocyanin is main target for high temperature stress in the cyanobacterium Synechococcus 6301 and there by affects PS-II photochemistry..

FLUORESCENCE EMISSION SPECTRA OF MARINE AND BRACKISH-WATER ECOTYPES OF FUCUS VESICULOSUS AND FUCUS RADICANS (PHAEOPHYCEAE) REVEAL DIFFERENCES IN LIGHT-HARVESTING APPARATUS(1).

The Bothnian Sea in the northerly part of the Baltic Sea is a geologically recent brackish-water environment, and rapid speciation is occurring in the algal community of the Bothnian Sea. We measured low-temperature fluorescence emission spectra from the Bothnian Sea and the Norwegian Sea ecotypes of Fucus vesiculosus L., a marine macroalga widespread in the Bothnian Sea. Powdered, frozen thallus was used to obtain undistorted emission spectra. The spectra were compared with spectra measured from the newly identified species Fucus radicans Bergström et L. Kautsky, which is a close relative of F. vesiculosus and endemic to the Bothnian Sea. The spectrum of variable fluorescence was used to identify fluorescence peaks originating in PSI and PSII in this chl c-containing alga. The spectra revealed much higher PSII emission, compared to PSI emission, in the Bothnian Sea ecotype of F. vesiculosus than in F. radicans or in the Norwegian Sea ecotype of F. vesiculosus. The results suggest that more light-harvesting chl a/c proteins serve PSII in the Bothnian Sea ecotype of F. vesiculosus than in the two other algal strains. Treatment of the Bothnian Sea ecotype of F. vesiculosus in high salinity (10, 20, and 35 practical salinity units) for 1 week did not lead to spectral changes, indicating that the measured features of the Bothnian Sea F. vesiculosus are stable and not simply a direct result of exposure to low salinity.

Early and late plastid development in response to chill stress and heat stress in wheat seedlings

Five-day-old etiolated wheat (Triticum aestivum L.) seedlings were transferred to 7°C (chill stress), 25°C (control), and 42°C (heat stress) and were kept in the dark or light for different time periods. Plastids were isolated from the control and stressed seedlings, and their low-temperature (77K) fluorescence emission spectra were monitored. Most of the Protochlorophyllide (Pchlide) present in heat-stressed etiolated seedlings were in nonphototransformable form. The phototransformable Pchlide (F657) rapidly decreased when 5-day-old etiolated seedlings were transferred to 42°C in the dark for 24h. A flash illumination of 0.2s given to etiolated heat-stressed seedlings resulted in substantial arrest of Shibata shift, while in chill-stress conditions, it was only partially affected. In high temperature, due to disaggregation of polymeric Pchlide-Pchlide oxidoreductase (POR)-nicotinamide adenine dinucleotide phosphate (NADPH) molecules, the conversion of nonphototransformable Pchlide to its phototransformable form is substantially delayed resulting in impaired Shibata shift and belated development of the core antenna CP47 Photosystem II (PSII). Chill stress, however, did not disaggregate the polymeric Pchlide-POR-NADPH molecule-suppressed Pchlide and Chl synthesis and impaired of the assembly of PSII core antenna CP47 that emits F695 and PSI that emits F735. The decreased gene/protein expression and reduced posttranslational import of plastidic proteins, importantly POR in temperature-stressed plants, may be responsible for the delay in conversion of nonphototransformable to phototransformable form of Pchlide and plastid biogenesis.

Sll0396 regulates transcription of the phycocyanin genes in Synechocystis sp. PCC 6803

An olive-green mutant was generated in Synechocystis sp. strain PCC 6803 by inactivation of the sll0396 gene. Whole-cell absorption spectra of the mutant revealed the missing of phycocyanin peak. An investigation of the low-temperature fluorescence emission spectra revealed that the sll0396Ω mutant has a reduced amount of phycocyanin. Western blot analysis showed that the mutant contained less phycocyanin β- and α-subunits and lacked the 30- and 32-kDa linker polypeptides, and northern blot analysis revealed that the transcription of the 1.4-kb cpcBA gene encoding the phycocyanin β- and α-subunits was lower in the mutant. The Sll0396 protein has a DNA-binding motif and shares homology with known response regulators. Our results indicate that Sll0396 plays a regulatory role in the transcription of the phycocyanin genes during phycobilisome synthesis.

PGRL1 Participates in Iron-induced Remodeling of the Photosynthetic Apparatus and in Energy Metabolism in Chlamydomonas reinhardtii

PGRL1 RNA and protein levels are increased in iron-deficient Chlamydomonas reinhardtii cells. In an RNAi strain, which accumulates lower PGRL1 levels in both iron-replete and -starved conditions, the photosynthetic electron transfer rate is decreased, respiratory capacity in iron-sufficient conditions is increased, and the efficiency of cyclic electron transfer under iron-deprivation is diminished. Pgrl1-kd cells exhibit iron deficiency symptoms at higher iron concentrations than wild-type cells, although the cells are not more depleted in cellular iron relative to wild-type cells as measured by mass spectrometry. Thiol-trapping experiments indicate iron-dependent and redox-induced conformational changes in PGRL1 that may provide a link between iron metabolism and the partitioning of photosynthetic electron transfer between linear and cyclic flow. We propose, therefore, that PGRL1 in C. reinhardtii may possess a dual function in the chloroplast; that is, iron sensing and modulation of electron transfer.

Characterization of chlorophyll deficient mutants of pea

A series of 17 nuclear and 1 non-mendelian pea mutants of the chlorotica and chlorina phenotypes were characterized by oxygen evolution capacity of the leaves and polypeptide composition of the photosynthetic membranes. Four chlorina mutants lack oxygen evolution capacity. Changes in the polypeptide pattern of the membranes were detected for 6 of the mutants. The ultrastructure and the photochemical activities of chloroplasts from the non-mendelian mutant and 10 of the nuclear mutants were also investigated. Abnormal membrane organizations were observed in all cases, except for one chlorina mutant. Chloroplasts from 3 mutants that lack oxygen evolution capacity do not possess detectable photosystem (PS) II activity. Altered PS I and PS II activities were observed for the other mutants that were tested. The low temperature fluorescence emission spectrum of leaves from the Chl b- and grana-rich mutant chlorotica-5824 have strongly reduced peaks at 685 and 695 nm, while that of leaves from the Chl b- and grana-deficient mutant chlorina-5535 indicates a reduction in the emission at 685 nm and at the long-wavelength maximum. The long-wavelength band is located at 734 nm for the wild type, 730 nm for chlorotica-5824 and 716 nm for chlorina-5535.

Effect of monogalactosyldiacylglycerol on the interaction between photosystem II core complex and its antenna complexes in liposomes of thylakoid lipids

The non-bilayer lipid monogalactosyldiacylglycerol (MGDG) is the most abundant type of lipid in the thylakoid membrane and plays an important role in regulating the structure and function of photosynthetic membrane proteins. In this study, we have reconstituted the isolated major light-harvesting complexes of photosystem II (PSII) (LHCIIb) and a preparation consisting of PSII core complexes and minor LHCII of PSII (PSIICC) into liposomes that consisted of digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG), with or without MGDG. Transmission electron microscopy and freeze-fracture studies showed unilamellar proteoliposomes, and demonstrated that most of the MGDG is incorporated into bilayer structures. The impact of MGDG on the functional interaction between LHCIIb and PSIICC was investigated by low temperature (77 K) fluorescence emission spectra and the photochemical activity of PSII. The additional incorporation of LHCIIb into liposomes containing PSIICC markedly increased oxygen evolution of PSIICC. Excitation at 480 nm of chlorophyll (Chl) b in LHCIIb stimulated a characteristic fluorescence emission of the Chl a in PSII (684.2 nm), rather than that of the Chl a in LHCIIb (680 nm) in the LHCIIb-PSIICC proteoliposomes, which indicated that the energy was transferred from LHCIIb to PSIICC in liposome membranes. Increasing the percentage of MGDG in the PSIICC-LHCIIb proteoliposomes enhanced the photochemical activity of PSII, due to a more efficient energy transfer from LHCIIb to PSIICC and, thus, an enlarged antenna cross section of PSII.

Structural and Functional Analysis of the Antiparallel Strands in the Lumenal Loop of the Major Light-harvesting Chlorophyll a/b Complex of Photosystem II (LHCIIb) by Site-directed Mutagenesis

The light-harvesting chlorophyll a/b-binding protein of photosystem II (LHCIIb) fulfills multiple functions, such as light harvesting and energy dissipation under different illuminations. The crystal structure of LHCIIb at the near atomic resolution reveals an antiparallel strands structure in the lumenal loop between the transmembrane helices B/C. To study the structural and functional significances of this structure, three amino acids (Val-119, His-120, and Ser-123) in this region have been exchanged to Phe, Leu, and Gly, respectively, and the influence of the mutagenesis on the structure and function of LHCIIb has been investigated. The results are as follows. 1) Circular dichroism spectra of the mutations reveals that the antiparallel strands in the lumenal region are very important for adjusting pigment conformation in the neoxanthin domain of LHCIIb. Although the mutagenesis causes only a slight loss of the Neo binding in the complexes (V119F, 0.09; S123G, 0.19; and H120L, 0.27), it imparts remarkable changes to the pigment conformation. 2) Substituting Ser-123 with Gly results in a higher susceptibility to photodamage, an increased tendency to aggregate, and enhanced fluorescence quenching induced by the medium acidification. These results demonstrate that this antiparallel strands domain plays an important role in regulating the pigment conformation and in adjusting the aggregation and the fluorescence yield of LHCIIb.

Biological variability in the ratios of protochlorophyllide forms in leaves and epicotyls of dark-grown pea ( Pisum sativum L.) seedlings (A statistical method to resolve complex spectra)

Low-temperature (77 K) fluorescence emission spectra of 100 dark-grown pea ( Pisum sativum L.) seedlings of various ages were measured. The spectra of the 100 leaf samples were collected into a separate data group and those of epicotyls formed another one. This group was divided into three sub-groups as spectra of uppermost, middle and lowermost 3 cm sections. Further sub-groups were formed on the basis of the ages of the plants. The spectra were normalized to their total integral values (within the 580–780 nm region) then the AVERAGE (arithmetic mean function) and AVEDEV (average of the absolute deviations of data points of their mean function) spectra were calculated. Very sharp bands were found in the AVEDEV spectra. Even the strongly overlapped 629 and 636 nm emission bands appeared as separate peaks, due to the decrease of their half-bandwidth values in the AVEDEV function. Both types of spectra were resolved into Gaussian components. The results showed that the variabilities of the 633 and 655 nm protochlorophyllide forms were similar in the leaves. In epicotyls, the protochlorophyllide forms had different variabilities, especially in the middle sections. The most variable was the amplitude of the 636 nm band and the variabilities of the 629 and 655 nm bands were smaller but still remarkable. The calculation of AVEDEV spectra is an effective method to study the biological variability and spectral resolution of biological samples containing chromophores with multiple spectral properties.

Fluorescence Lifetimes of Protochlorophyllide in Plants with Different Proportions of Short-wavelength and Long-wavelength Protochlorophyllide Spectral Forms¶

Abstract Dark-grown leaves of maize (Zea mays), wheat (Triticum aestivum), wild-type pea (Pisum sativum) and its light-independent photomorphogenesis mutant (lip1) have different proportions of protochlorophyllide (Pchlide) forms as revealed by low-temperature fluorescence emission spectra. Four discrete spectral forms of Pchlide, with emission peaks around 633, 640, 656 and 670 nm, could be distinguished after Gaussian deconvolution. In maize and wheat the 656 nm component was the most prominent, whereas for wild-type pea and its lip1 mutant, the 633 and 640 nm components contributed mostly to the fluorescence emission spectra. For the fluorescence lifetimes measured at 77 K a double exponential model was the most adequate to describe the Pchlide fluorescence decay not only for the Pchlide650–656 form but also for the short-wavelength Pchlide forms. A fast component in the range 0.3–0.8 ns and a slow component in the range 5.1–7.1 ns were present in all samples, but the values varied, depending on specie...

High salt stress in wheat leaves causes retardation of chlorophyll accumulation due to a limited rate of protochlorophyllide formation

When exposed to salt stress, leaves from dark‐grown wheat seedlings (Triticum aestivum, cv. Giza 168) showed reduced accumulation of chlorophyll during irradiation. To elucidate the mechanism behind salt‐influenced reduction of chlorophyll biosynthesis, we have investigated the effect of salt stress on the spectral forms of Pchlide, the phototransformation of Pchlide to Chlide, the Shibata shift, the regeneration of Pchlide and the accumulation of Pchlide from 5‐aminolevulinic acid (ALA). We found that the phototransformation of Pchlide to Chlide was not affected by salt stress. The blue shift (Shibata shift) of newly formed Chlide was delayed both after flash irradiation and in continuous light. The reformation of Pchlide in darkness after a flash irradiation or after a period of 3‐h irradiation was retarded in the salt‐treated leaves. However, after a 20‐h dark period, Pchlide was reformed even in salt‐treated leaves but the formation of short‐wavelength Pchlide was suppressed. Compared to controls, salt treatment also reduced the amount of Pchlide accumulated in leaves floated on ALA. The increase in the low temperature fluorescence emission spectrum at 735 nm, which occurred gradually during several hours of irradiation with continuous light in control leaves, was completely suppressed in salt‐treated leaves. It is concluded that salt stress inhibits chlorophyll accumulation partly by reducing the rate of porphyrin formation but, as discussed, also by a possible reduction in the formation of chlorophyll‐binding proteins.

Impairment of the photosynthetic apparatus by oxidative stress induced by photosensitization reaction of protoporphyrin IX

Treatment with the herbicide acifluorfen-sodium (AF-Na), an inhibitor of protoporphyrinogen oxidase, caused an accumulation of protoporphyrin IX (Proto IX) , light-induced necrotic spots on the cucumber cotyledon within 12–24 h, and photobleaching after 48–72 h of light exposure. Proto IX-sensitized and singlet oxygen ( 1O 2)-mediated oxidative stress caused by AF-Na treatment impaired photosystem I (PSI), photosystem II (PSII) and whole chain electron transport reactions. As compared to controls, the F v/ F m (variable to maximal chlorophyll a fluorescence) ratio of treated samples was reduced. The PSII electron donor NH 2OH failed to restore the F v/ F m ratio suggesting that the reduction of F v/ F m reflects the loss of reaction center functions. This explanation is further supported by the practically near-similar loss of PSI and PSII activities. As revealed from the light saturation curve (rate of oxygen evolution as a function of light intensity), the reduction of PSII activity was both due to the reduction in the quantum yield at limiting light intensities and impairment of light-saturated electron transport. In treated cotyledons both the Q (due to recombination of Q A − with S 2) and B (due to recombination of Q B − with S 2/S 3) band of thermoluminescence decreased by 50% suggesting a loss of active PSII reaction centers. In both the control and treated samples, the thermoluminescence yield of B band exhibited a periodicity of 4 suggesting normal functioning of the S states in centers that were still active. The low temperature (77 K) fluorescence emission spectra revealed that the F 695 band (that originates in CP-47) increased probably due to reduced energy transfer from the CP47 to the reaction center. These demonstrated an overall damage to the PSI and PSII reaction centers by 1O 2 produced in response to photosensitization reaction of protoporphyrin IX in AF-Na-treated cucumber seedlings.

Sorbitol regulates energy transfer from allophycocyanin to the terminal emitter within phycobilisomes in Synechocystis sp

The effects of sorbitol on energy transfer of phycobilisomes (PBSs) in vivo were investigated in a chlN deletion mutant of Synechocystis sp. PCC 6803. When the mutant was grown in the dark, it contained intact and functional PBSs but essentially no chlorophyll or photosystems. Therefore, the structural and functional changes of the mutant PBSs in vivo can be detected by measurement of low temperature (77 K) and room temperature fluorescence emission spectra. Our results, for the first time, demonstrate that sorbitol decreases the energy transfer from allophycocyanin to the terminal emitter, indicating the site for osmotic regulation of excitation transfer in PBSs.

Changes in the energy distribution in mutant thylakoid membranes of pea with modified pigment content. II. Changes due to magnesium ions concentration

Low-temperature (77 K) steady-state chlorophyll fluorescence emission spectra, room temperature fluorescence and light scattering of thylakoid membranes isolated from pea mutants were studied as a function of Mg 2+ concentration. The mutants have modified pigment content and altered structural organization of the pigment-protein complexes, distinct surface electric properties and functions. The analysis of the 77 K emission spectra revealed that Mg 2+-depletion of the medium caused not only an increased energy flow toward photosystem I in all investigated membranes but also changes in the quenching of the fluorescence, most probably by internal conversion. The results indicated that the macroorganization of the photosynthetic apparatus of mutants at supramolecular level (distribution and segregation of two photosystems in thylakoid membranes) and at supermolecular level (stacking of photosystem II supercomplexes) required different Mg ion concentrations. The data confirmed that the segregation of photosystems and the stacking of thylakoid membranes are two distinct phenomena and elucidated some features of their mechanisms. The segregation is initiated by changes in the lateral microorganization of light harvesting complexes II, their migration (repulsion from photosystem I) and subsequent separation of the two photosystems. Most likely 3D aggregation and formation of macrodomains, containing only photosystem II antenna complexes, play a certain precursory role for the increasing degree of the membrane stacking and the energy coupling between the light harvesting complexes II and the core complexes of photosystem II in the frame of photosystem II supercomplexes.

Autotrophic cells of the Synechocystis psbH deletion mutant are deficient in synthesis of CP47 and accumulate inactive PS II core complexes

Cells of the psbH deletion mutant IC7 of the cyanobacterium Synechocystis PCC 6803 grown in the absence of glucose contain strongly reduced levels of chlorophyll when compared with cells grown in the presence of glucose, or compared with wild-type (WT) cells. Low-temperature fluorescence emission spectra revealed decreased content of both active PS II (Photosystem II) and PS I (Photosystem I) complexes. Analysis of thylakoid membrane complexes of IC7 by native electrophoresis showed a similar set of chlorophyll-proteins, namely a PS II core complex and trimeric and monomeric PS II complexes, as in WT. However, in contrast to WT, the (35)S-methionine protein labeling pattern of the mutant exhibited no preferential labeling of the D1 protein in the PS II core complexes, and the labeled D1 and D2 proteins accumulated predominantly in the PS II reaction center lacking CP47. The results show that in autotrophically grown cells of the psbH deletion mutant, selective D1 turnover is inhibited and synthesis of CP47 becomes a limiting step in the PS II assembly.