Unveiling large charge transfer character of PSII in an iron-deficient cyanobacterial membrane: A Stark fluorescence spectroscopy study.

In this work, we applied Stark fluorescence spectroscopy to an iron-stressed cyanobacterial membrane to reveal key insights about the electronic structures and excited state dynamics of the two important pigment-protein complexes, IsiA and PSII, both of which prevail simultaneously within the membrane during iron deficiency and whose fluorescence spectra are highly overlapped and hence often hardly resolved by conventional fluorescence spectroscopy. Thanks to the ability of Stark fluorescence spectroscopy, the fluorescence signatures of the two complexes could be plausibly recognized and disentangled. The systematic analysis of the SF spectra, carried out by employing standard Liptay formalism with a realistic spectral deconvolution protocol, revealed that the IsiA in an intact membrane retains almost identical excited state electronic structures and dynamics as compared to the isolated IsiA we reported in our earlier study. Moreover, the analysis uncovered that the excited state of the PSII subunit of the intact membrane possesses a significantly large CT character. The observed notably large magnitude of the excited state CT character may signify the supplementary role of PSII in regulative energy dissipation during iron deficiency.

A different perspective for nonphotochemical quenching in plant antenna complexes.

Light-harvesting complexes of plants exert a dual function of light-harvesting (LH) and photoprotection through processes collectively called nonphotochemical quenching (NPQ). While LH processes are relatively well characterized, those involved in NPQ are less understood. Here, we characterize the quenching mechanisms of CP29, a minor LHC of plants, through the integration of two complementary enhanced-sampling techniques, dimensionality reduction schemes, electronic calculations and the analysis of cryo-EM data in the light of the predicted conformational ensemble. Our study reveals that the switch between LH and quenching state is more complex than previously thought. Several conformations of the lumenal side of the protein occur and differently affect the pigments' relative geometries and interactions. Moreover, we show that a quenching mechanism localized on a single chlorophyll-carotenoid pair is not sufficient but many chlorophylls are simultaneously involved. In such a diffuse mechanism, short-range interactions between each carotenoid and different chlorophylls combined with a protein-mediated tuning of the carotenoid excitation energies have to be considered in addition to the commonly suggested Coulomb interactions.

Excitation energy transfer in the far-red absorbing violaxanthin/vaucheriaxanthin chlorophyll a complex from the eustigmatophyte alga FP5.

This work highlights spectroscopic investigations on a new representative of photosynthetic antenna complexes in the LHC family, a putative violaxanthin/vaucheriaxanthin chlorophyll a (VCP) antenna complex from a freshwater Eustigmatophyte alga FP5. A representative VCP-like complex, named as VCP-B3 was studied with both static and time-resolved spectroscopies with the aim of obtaining a deeper understanding of excitation energy migration within the pigment array of the complex. Compared to other VCP representatives, the absorption spectrum of the VCP-B3 is strongly altered in the range of the chlorophyll a Qy band, and is substantially red-shifted with the longest wavelength absorption band at 707 nm at 77 K. VCP-B3 shows a moderate xanthophyll-to-chlorophyll a efficiency of excitation energy transfer in the 50-60% range, 20-30% lower from comparable VCP complexes from other organisms. Transient absorption studies accompanied by detailed data fitting and simulations support the idea that the xanthophylls that occupy the central part of the complex, complementary to luteins in the LHCII, are violaxanthins. Target analysis suggests that the primary route of xanthophyll-to-chlorophyll a energy transfer occurs via the xanthophyll S1 state.

The extraordinary longevity of kleptoplasts derived from the Ross Sea haptophyte Phaeocystis antarctica within dinoflagellate host cells relates to the diminished role of the oxygen-evolving Photosystem II and to supplementary light harvesting by mycosporine-like amino acid/s.

The haptophyte Phaeocystis antarctica and the novel Ross Sea dinoflagellate that hosts kleptoplasts derived from P. antarctica (RSD; R.J. Gast et al., 2006, J. Phycol. 42 233-242) were compared for photosynthetic light harvesting and for oxygen evolution activity. Both chloroplasts and kleptoplasts emit chlorophyll a (Chl a) fluorescence peaking at 683nm (F683) at 277K and at 689 (F689) at 77K. Second derivative analysis of the F689 band at 77K revealed two individual contributions centered at 683nm (Fi-683) and at 689 (Fi-689). Using the p-nitrothiophenol (p-NTP) treatment of Kobayashi et al. (Biochim. Biophys. Acta 423 (1976) 80-90) to differentiate between Photosystem (PS) II and I fluorescence emissions, we could identify PS II as the origin of Fi-683 and PS I as the origin of Fi-689. Both emissions could be excited not only by Chl a-selective light (436nm) but also by mycosporine-like amino acids (MAAs)-selective light (345nm). This suggests that a fraction of MAAs must be proximal to Chls a and, therefore, located within the plastids. On the basis of second derivative fluorescence spectra at 77K, of p-NTP resolved fluorescence spectra, as well as of PSII-driven oxygen evolution activities, PS II appears substantially less active (~1/5) in dinoflagellate kleptoplasts than in P. antarctica chloroplasts. We suggest that a diminished role of PS II, a known source of reactive oxygen species, and a diminished dependence on nucleus-encoded light-harvesting proteins, due to supplementary light-harvesting by MAAs, may account for the extraordinary longevity of RSD kleptoplasts.

Responses to iron limitation are impacted by light quality and regulated by RcaE in the chromatically acclimating cyanobacterium Fremyella diplosiphon.

Photosynthetic organisms adapt to environmental fluctuations of light and nutrient availability. Iron is critical for photosynthetic organismal growth, as many cellular processes depend upon iron cofactors. Whereas low iron levels can have deleterious effects, excess iron can lead to damage, as iron is a reactive metal that can result in the production of damaging radicals. Therefore, organisms regulate cellular iron levels to maintain optimal iron homeostasis. In particular, iron is an essential factor for the function of photosystems associated with photosynthetic light-harvesting complexes. Photosynthetic organisms, including cyanobacteria, generally respond to iron deficiency by reduced growth, degradation of non-essential proteins and in some cases alterations of cellular morphology. In response to fluctuations in ambient light quality, the cyanobacterium Fremyella diplosiphon undergoes complementary chromatic adaptation (CCA). During CCA, phycobiliprotein composition of light-harvesting antennae is altered in response to green light (GL) and red light (RL) for efficient utilization of light energy for photosynthesis. We observed light-regulated responses to iron limitation in F. diplosiphon. RL-grown cells exhibited significant reductions in growth and pigment levels, and alterations in iron-associated proteins, which impact the accumulation of reactive oxygen species under iron-limiting conditions, whereas GL-grown cells exhibited partial resistance to iron limitation. We investigated the roles of known CCA regulators RcaE, RcaF and RcaC in this light-dependent iron-acclimation response. Through comparative analyses of wild-type and CCA mutant strains, we determined that photoreceptor RcaE has a central role in light-induced oxidative stress associated with iron limitation, and impacts light-regulated iron-acclimation responses, physiologically and morphologically.

A different sequence of events than previously reported leads to arsenic-induced damage in Ceratophyllum demersum L.

Arsenic (As) is a common pollutant, and still many questions remain concerning As toxicity mechanisms under environmentally relevant conditions in plants. Here we investigated thresholds and interactions of various proposed As toxicity mechanisms. Experiments were done under environmentally pertinent conditions in the rootless aquatic macrophyte Ceratophyllum demersum L., a model for plant shoots. Arsenic (provided as As(v)) inhibited plant metabolism at much lower concentrations and with a different sequence of events than previously reported. The first observed effect of toxicity was a decrease in pigment concentration, it started even at 0.5 µM As. In contrast to toxic metals, no inhibition of the photosystem II reaction centre (PSIIRC; measured as Fv/Fm) was found at sublethal As concentrations. Instead, the decrease in light harvesting pigments caused a less efficient exciton transfer towards the PSIIRC. At higher As concentrations this led to increased non-photochemical quenching (NPQ) by light harvesting complex II (LHCII). Afterwards, photosynthetic electron transport decreased, but the increase in starch content indicated stronger inhibition of starch consumption than production. At lethal As concentration, photosynthesis was completely inhibited, its malfunction caused oxidative stress and not the other way round as reported previously. Photosynthesis was inhibited before any sign of oxidative stress was observed. Elevated phosphate drastically shifted thresholds of lethal As effects, not only by the known uptake competition but also by modifying uptake regulation and intracellular processes.

Overexpression of a proton-coupled vacuolar glucose exporter impairs freezing tolerance and seed germination.

Arabidopsis vacuoles harbor, besides sugar transporter of the TMT-type, an early response to dehydration like 6 (ERDL6) protein involved in glucose export into the cytosol. However, the mode of transport of ERDL6 and the plant's feedback to overexpression of its activity on essential properties such as, for example, seed germination or freezing tolerance, remain unexplored. Using patch-clamp studies on vacuoles expressing AtERDL6 we demonstrated directly that this carrier operates as a proton-driven glucose exporter. Overexpression of BvIMP, the closest sugar beet (Beta vulgaris) homolog to AtERDL6, in Arabidopsis leads surprisingly to impaired seed germination under both conditions, sugar application and low environmental temperatures, but not under standard conditions. Upon cold treatment, BvIMP overexpressor plants accumulated lower quantities of monosaccharides than the wild-type, a response in line with the reduced frost tolerance of the transgenic Arabidopsis plants, and the fact that cold temperatures inhibits BvIMP transcription in sugar beet leaves. With these findings we show that the tight control of vacuolar sugar import and export is a key requisite for cold tolerance and seed germination of plants.

Photoprotection in the green tidal alga Ulva prolifera: role of LHCSR and PsbS proteins in response to high light stress.

Ulva prolifera, an intertidal macroalga, has to adapt to wide variations in light intensity, making this species particularly rewarding for studying the evolution of photoprotective mechanisms. Intense light induced increased non-photochemical quenching (NPQ) and stimulated de-epoxidation of xanthophyll cycle components, while DTT-treated samples had lower NPQ capacity, indicating that the xanthophyll cycle must participate in photoprotection. In this work, we found that the PsbS-related NPQ was maintained in U. prolifera. According to analysed gene expression, both LhcSR and psbS were up-regulated in high light, suggesting that these two genes are light-induced. LHCSR and PsbS proteins were present at different light intensities and accumulated under high light conditions, and PsbS concentrations were higher than LHCSR, showing that the NPQ mechanism of U. prolifera is more dependent on PsbS protein concentration. Moreover, the level of both LHCSR and PsbS proteins was high even in the darkness, and neither the transcript level nor protein content of LhcSR and psbS genes varied significantly following short-term exposure to intense light. These findings suggest that this alga can modulate NPQ levels through regulation of the xanthophyll cycle and concentrations of PsbS and/or LHCSR.

Nerium oleander indirect leaf photosynthesis and light harvesting reductions after clipping injury or Spodoptera eridania herbivory: high sensitivity to injury.

Variable indirect photosynthetic rate (P(n)) responses occur on injured leaves after insect herbivory. It is important to understand factors that influence indirect P(n) reductions after injury. The current study examines the relationship between gas exchange and chlorophyll a fluorescence parameters with injury intensity (% single leaf tissue removal) from clipping or Spodoptera eridania Stoll (Noctuidae) herbivory on Nerium oleander L. (Apocynaceae). Two experiments showed intercellular [CO(2)] increases but P(n) and stomatal conductance reductions with increasing injury intensity, suggesting non-stomatal P(n) limitation. Also, P(n) recovery was incomplete at 3d post-injury. This is the first report of a negative exponential P(n) impairment function with leaf injury intensity to suggest high N. oleander leaf sensitivity to indirect P(n) impairment. Negative linear functions occurred between most other gas exchange and chlorophyll a fluorescence parameters with injury intensity. The degree of light harvesting impairment increased with injury intensity via lower (1) photochemical efficiency indicated lower energy transfer efficiency from reaction centers to PSII, (2) photochemical quenching indicated reaction center closure, and (3) electron transport rates indicated less energy traveling through PSII. Future studies can examine additional mechanisms (mesophyll conductance, carbon fixation, and cardenolide induction) to cause N. oleander indirect leaf P(n) reductions after injury.

Functions of the water soluble chlorophyll-binding protein in plants.

Functional aspects of water soluble chlorophyll-binding protein (WSCP) in plants were investigated during the courses of leaf senescence, chlorophyll biogenesis, stress response and photoprotection. The cDNA sequence encoding WSCP from cauliflower was cloned into a binary vector to facilitate Agrobacterium tumefaciens mediated transformation of Nicotiana tabacum. The resultant transgenic tobacco plants overexpressed the CauWSCP gene under the control of a 35S-promoter. Analyses of protein and pigment contents indicate that WSCP overexpression does not enhance chlorophyll catabolism in vivo, thus rendering a role of WSCP in Chl degradation unlikely. Accumulation of higher levels of protochlorophyllide in WSCP overexpressor plants corroborates a proposed temporary storage and carrier function of WSCP for chlorophyll and late precursors. Although WSCP overexpressor plants did not show significant differences in non-photochemical quenching of chlorophyll fluorescence, they are characterized by significantly lower zeaxanthin accumulation and peroxidase activity at different light intensities, even at high light intensities of 700-900µmol photons m(-2)s(-1). These results suggest a photoprotective function of the functional chlorophyll binding-WSCP tetramer by shielding of chlorophylls from molecular oxygen.

Isolation of the elusive supercomplex that drives cyclic electron flow in photosynthesis.

Photosynthetic light reactions establish electron flow in the chloroplast's thylakoid membranes, leading to the production of the ATP and NADPH that participate in carbon fixation. Two modes of electron flow exist-linear electron flow (LEF) from water to NADP(+) via photosystem (PS) II and PSI in series and cyclic electron flow (CEF) around PSI (ref. 2). Although CEF is essential for satisfying the varying demand for ATP, the exact molecule(s) and operational site are as yet unclear. In the green alga Chlamydomonas reinhardtii, the electron flow shifts from LEF to CEF on preferential excitation of PSII (ref. 3), which is brought about by an energy balancing mechanism between PSII and PSI (state transitions). Here, we isolated a protein supercomplex composed of PSI with its own light-harvesting complex (LHCI), the PSII light-harvesting complex (LHCII), the cytochrome b(6)f complex (Cyt bf), ferredoxin (Fd)-NADPH oxidoreductase (FNR), and the integral membrane protein PGRL1 (ref. 5) from C. reinhardtii cells under PSII-favouring conditions. Spectroscopic analyses indicated that on illumination, reducing equivalents from downstream of PSI were transferred to Cyt bf, whereas oxidised PSI was re-reduced by reducing equivalents from Cyt bf, indicating that this supercomplex is engaged in CEF (Supplementary Fig. 1). Thus, formation and dissociation of the PSI-LHCI-LHCII-FNR-Cyt bf-PGRL1 supercomplex not only controlled the energy balance of the two photosystems, but also switched the mode of photosynthetic electron flow.

An internal antisense RNA regulates expression of the photosynthesis gene isiA.

Small regulatory noncoding RNAs exist in both eukaryotic and prokaryotic organisms. Most of these RNA transcripts are trans-encoded RNAs with short and only partial antisense complementarity to their target RNAs, which regulate gene expression by modifying mRNA stability and translation. In contrast, reports on the function of cis-encoded, perfectly complementary antisense RNAs in eubacteria are rare. Cyanobacteria respond to iron deficiency by expressing IsiA (iron stress-induced protein A), which forms a giant ring structure around photosystem I. Here, we show that this process is controlled by IsrR (iron stress-repressed RNA), a cis-encoded antisense RNA transcribed from the isiA noncoding strand. Artificial overexpression of IsrR under iron stress causes a strongly diminished number of IsiA-photosystem I supercomplexes, whereas IsrR depletion results in premature expression of IsiA. The coupled degradation of IsrR/isiA mRNA duplexes appears to be a reversible switch that can respond to environmental changes. IsrR is the only RNA known so far to regulate a photosynthesis component.

A chlorophyll a/b-binding protein homolog that is induced by iron deficiency is associated with enlarged photosystem I units in the eucaryotic alga Dunaliella salina.

Adaptation of the halotolerant alga Dunaliella salina to iron deprivation involves extensive changes of chloroplast morphology, photosynthetic activities, and induction of a major 45-kDa chloroplast protein termed Tidi. Partial amino acid sequencing of proteolytic peptides suggested that Tidi resembles chlorophyll a/b-binding proteins which compose light-harvesting antenna complexes (LHC) (Varsano, T., Kaftan, D., and Pick, U. (2003) J. Plant Nutr. 26, 2197-2210). Here we show that Tidi shares the highest amino acid sequence similarity with light-harvesting I chlorophyll a/b-binding proteins from higher plants but has an extended proline-rich N-terminal domain. The accumulation of Tidi is reversed by iron supplementation, and its level is inversely correlated with photosystem I (PS-I) reaction center proteins. In native gel electrophoresis, Tidi co-migrates with enlarged PS-I-LHC-I super-complexes. Single particle electron microscopy analysis revealed that PS-I units from iron-deficient cells are larger (31 and 37 nm in diameter) than PS-I units from control cells (22 nm). The 77 K chlorophyll fluorescence emission spectra of isolated complexes suggest that the Tidi-LHC-I antenna are functionally coupled to the reaction centers of PS-I. These findings indicate that Tidi acts as an accessory antenna of PS-I. The enlargement of PS-I antenna in algae and in cyanobacteria under iron deprivation suggests a common limitation that requires rebalancing of the energy distribution between the two photosystems.

Photosystem II assembly in CP47 mutant of Synechocystis sp. PCC 6803 is dependent on the level of chlorophyll precursors regulated by ferrochelatase.

Accumulation of chlorophyll and expression of the chlorophyll (Chl)-binding CP47 protein that serves as the core antenna of photosystem II are indispensable for the assembly of a functional photosystem II. We have characterized the CP47 mutant with an impaired photosystem II assembly and its two spontaneous pseudorevertants with their much improved photoautotrophic growth. The complementing mutations in these pseudorevertants were previously mapped to the ferrochelatase gene (1). We demonstrated that complementing mutations dramatically decrease ferrochelatase activity in pseudorevertants and that this decrease is responsible for their improved photoautotrophic growth. Photoautotrophic growth of the CP47 mutant was also restored by in vivo inhibition of ferrochelatase by a specific inhibitor. The decrease in ferrochelatase activity in pseudorevertants was followed by increased steady-state levels of Chl precursors and Chl, leading to CP47 accumulation and photosystem II assembly. Similarly, supplementation of the CP47 mutant with the Chl precursor Mg-protoporphyrin IX increased the number of active photosystem-II centers, suggesting that synthesis of the mutated CP47 protein is enhanced by an increased Chl availability in the cell. The probable role of ferrochelatase in the regulation of Chl biosynthesis is discussed.

Watching the components of photosynthetic bacterial membranes and their in situ organisation by atomic force microscopy.

The atomic force microscope has developed into a powerful tool in structural biology allowing information to be acquired at submolecular resolution on the protruding structures of membrane proteins. It is now a complementary technique to X-ray crystallography and electron microscopy for structure determination of individual membrane proteins after extraction, purification and reconstitution into lipid bilayers. Moving on from the structures of individual components of biological membranes, atomic force microscopy has recently been demonstrated to be a unique tool to identify in situ the individual components of multi-protein assemblies and to study the supramolecular architecture of these components allowing the efficient performance of a complex biological function. Here, recent atomic force microscopy studies of native membranes of different photosynthetic bacteria with different polypeptide contents are reviewed. Technology, advantages, feasibilities, restrictions and limits of atomic force microscopy for the acquisition of highly resolved images of up to 10 A lateral resolution under native conditions are discussed. From a biological point of view, the new insights contributed by the images are analysed and discussed in the context of the strongly debated organisation of the interconnected network of membrane-associated chlorophyll-protein complexes composing the photosynthetic apparatus in different species of purple bacteria.

Phosphorylation-dephosphorylation of light-harvesting complex II as a response to variation in irradiance is thiol sensitive and thylakoid sufficient: modulation of the sensitivity of the phenomenon by a peripheral component.

Downregulation of phosphorylation of chlorophyll a/b-binding proteins (LHCII) of the photosystem II at high irradiance could only be demonstrated with leaf discs but not in isolated thylakoids. The present view suggests this phenomenon to be regulated by stromal thioredoxin. Here, we show that high-light inactivation of LHCII phosphorylation can be reproduced in isolated thylakoids and have explained the apparent absence of inactivation in vitro to be due to the derepressed activity of a peripheral kinase. We investigated this phenomenon with Arachis hypogea thylakoids prepared with (Th:A) or without (Th:B) tricine, where tricine is known for removing peripheral proteins from thylakoids. While LHCII remained phosphorylated at high irradiance in Th:B, the response of Th:A mimicked Arachis leaflets where LHCII was transiently phosphorylated with irradiance. LHCII phosphorylation in Th:A was sensitive to thiol reducing conditions, but in Th:B, the phenomenon became insensitive to thiol reduction following illumination. Washing Th:B with tricine made them resemble Th:A, and conversely, Th:A reconstituted with the Tricine extract resembled Th:B with respect to both irradiance response and thiol sensitivity. In vitro phosphorylation reactions indicated a thiol insensitive kinase activity to be present in the Tricine extract that was capable of phosphorylating histone H1 as well as purified LHCII. This peripherally associated kinase activity explained the sustenance of LHCII phosphorylation as well as its thiol insensitivity at high irradiance in Th:B thylakoids. Contrary to the current view, our results clearly show that irradiance dependent phosphorylation and dephosphorylation of LHCII is a thylakoid sufficient phenomenon, although it remained open to regulation by thiol redox state modulation.