Examination of chlorophyll fluorescence decay kinetics in sulfur deprived algae Chlamydomonas reinhardtii

Chlorophyll fluorescence decay kinetics was measured in sulfur deprived cells of green alga Chlamydomonas reinhardtii with a home made picosecond fluorescence laser spectrometer. The measurements were carried out on samples either shortly adapted to the dark ('Fo conditions') or treated to reduce Qa ('Fm conditions'). Bi-exponential fitting of decay kinetics was applied to distinguish two components one of them related to energy trapping (fast component) and the other to charge stabilization and recombination in PS 2 reaction centers (slow component). It was found that the slow component yield increased by 2.0 and 1.2 times when measured under 'Fo' and 'Fm conditions', respectively, in sulfur deprived cells as compared to control ones. An additional rapid rise of the slow component yield was observed when incubation was carried out in a sealed bioreactor and cell culture turned to anaerobic conditions. The obtained results strongly indicate the existence of the redox control of PS 2 activity during multiphase adaptation of C. reinhardtii to sulfur deficiency stress. Probable mechanisms responsible for the observed increased recombinant fluorescence yield in starved cells are discussed.     

Nonphotochemical quenching of chlorophyll fluorescence in Chlamydomonas reinhardtii

Unlike plants, Chlamydomonas reinhardtii shows a restricted ability to develop nonphotochemical quenching upon illumination. Most of this limited quenching is due to state transitions instead of DeltapH-driven high-energy state quenching, qE. The latter could only be observed when the ability of the cells to perform photosynthesis was impaired, either by lowering temperature to approximately 0 degrees C or in mutants lacking RubisCO activity. Two main features were identified that account for the low level of qE in Chlamydomonas. On one hand, the electrochemical proton gradient generated upon illumination is apparently not sufficient to promote fluorescence quenching. On the other hand, the capacity to transduce the presence of a DeltapH into a quenching response is also intrinsically decreased in this alga, when compared to plants. The possible mechanism leading to these differences is discussed.     

Protein and chlorophyll in photosystem II probed by infrared spectroscopy

The infrared spectra of photosystem II (PS II) enriched submembrane fractions isolated from spinach are obtained in water and in heavy water suspension Other spectra are obtained after a photooxidation reaction was performed on PS II to bleach the pigments. The water bands are removed by computer subtraction and the amide bands (A, B, I, II, and III) of the protein are identified. Computer enhancement techniques are used to narrow the bandwidth of the bands that the weak chlorophyll bands, buried in the much stronger protein bands, can be observed. Comparing the spectra of native and photooxidized PS II pr in water and in heavy water, we determine that three polypeptide domains are present in the native material. The first domain, which contains 22% of th is situated in the peripheral region of the PS II system. The polypeptides in this region are unfolded and devoid of chlorophyll. The second domain con of the polypeptides, is more organized, and contains the chlorophylls. The third domain has an alpha-helix configuration, does not contain chlorophyll, a affected by the photooxidation reaction or by the proton/deuteron exchange. Three different types of chlorophyll organisation are identified: two have carbonyl groups non-bonded, differing from one another only in their hydrophobic milieux; the third is weakly bonded to another unidentified group. Other forms of chlorophyll organisation are present but could not be observed because their absorption is buried in the protein amide I band.     

Examination of chlorophyll fluorescence in sulfur-deprived cells of Chlamydomonas reinhardtii

Modulated fluorometry (PAM) was applied for probing the photosynthesis in cells of C. reinhardtii during sulfur deprivation. A significant (up to a fourfold) increase in chlorophyll fluorescence yield (parameters F(o) and F(m)) normalized to chlorophyll concentration was shown for deprived cells. An analysis of nonphotochemical quenching of chlorophyll fluorescence indicated a considerable modification of the energy deactivation pathways in PS II of sulfur-deprived cells. Thus, starved cells exhibited a lower deltapH-dependent quenching of excited states and a higher thermal dissipation of excess light energy in reaction centers of PS II, as well as the transition of the photosynthetic apparatus primarily to state 2. However, these changes cannot cause the elevation of chlorophyll fluorescence in the cells under sulfur limitation. The phenomenon observed may be due to a partial dissociation of light-harvesting complexes from reaction centers of PS II and/or dysfunction of the dissipative cycle in PS II with cytochrome b559 as an intermediate.     

Characterization of chlorophyll a/b proteins of photosystem I from Chlamydomonas reinhardtii.

In this study we have isolated the chlorophyll a/b-binding proteins from a photosystem I preparation of the green alga Chlamydomonas reinhardtii and characterized them by N-terminal sequencing, fluorescence, and absorption spectroscopy and by immunochemical means. The results indicate that in this organism, the light-harvesting complex of photosystem I (LHCI) is composed of at least seven distinct polypeptides of which a minimum number of three are shown to bind chlorophyll a and b. Both sequence homology and immunological cross-reactivity with other chlorophyll-binding proteins suggest that all of the LHCI polypeptides bind pigments. Fractionation of LHCI by mildly denaturing methods showed that, in contrast to higher plants, the long wavelength fluorescence emission typical of LHCI (705 nm in C. reinhardtii) cannot be correlated with the presence of specific polypeptides, but rather with changes in the aggregation state of the LHCI components. Reconstitution of both high aggregation state and long wavelength fluorescence emission from components that do not show these characteristics confirm this hypothesis.     

Characterization of the wave phenomenon of flash-induced chlorophyll fluorescence in Chlamydomonas reinhardtii

Flash-induced chlorophyll fluorescence relaxation is a powerful tool to monitor the reoxidation reactions of the reduced primary quinone acceptor, Q_A^? by Q_B and the plastoquinone (PQ) pool, as well as the charge recombination reactions between the donor and acceptor side components of Photosystem II (PSII). Under certain conditions, when the PQ pool is highly reduced (e.g. in microaerobic conditions), a wave phenomenon appears in the fluorescence relaxation kinetics, which reflects the transient reoxidation and re-reduction of Q_A^? by various electron transfer processes, which in cyanobacteria is mediated by NAD(P)H dehydrogenase (NDH-1). The wave phenomenon was also observed and assigned to the operation of type 2 NAD(P)H dehydrogenase (NDH-2) in the green alga Chlamydomonas reinhardtii under hydrogen-producing conditions, which required a long incubation of algae under sulphur deprivation (Krishna et al. J Exp Bot 70 (21):6321–6336, 2019). However, the conditions that induce the wave remained largely uncharacterized so far in microalgae. In this work, we investigated the wave phenomenon in Chlamydomonas reinhardtii under conditions that lead to a decrease of PSII activity by applying hydroxylamine treatment, which impacts the donor side of PSII in combination with a strongly reducing environment of the PQ pool (microaerobic conditions). A similar wave phenomenon could be induced by photoinhibitory conditions (illumination with strong light in the presence of the protein synthesis inhibitor lincomycin). These results indicate that the fluorescence wave phenomenon is activated in green algae when the PSII activity decreases relative to Photosystem I (PS I) activity and the PQ pool is strongly reduced. Therefore, the fluorescence wave could be used as a sensitive indicator of altered intersystem electron transfer processes, e.g. under stress conditions.

     

Donor side capacity of Photosystem II probed by chlorophyll a fluorescence transients

The chlorophyll a fluorescence transient measured under high light shows a typical O-J-I-P polyphasic rise. However, under certain stress situations such as heat or drought stress, a rapid phase with a maximum around 300 µs has been observed and called K (Guissé et al. (1995a) Arch Sci Genève 48: 147–160). Here, we show that under various conditions, the appearance of the K-step and the following dip, as well as the lowered maximum fluorescence level (FM) attainable, can be explained by an imbalance between the electron flow leaving the RC to the acceptor side and the electron flow coming to the RC from the donor side. This leads to a stable oxidation of the secondary electron donor, the tyrosine Z (YZ), and possibly to the accumulation of P680+. In the case of heat stress, we confirm that this situation is caused by an inhibition of electron donation to YZ, which is due to a damaged oxygen evolving complex (OEC). Finally, we present a model which includes the OEC, YZ, P680, QA and QB which is in good agreement with the experimental data. The appearance of the K-step, under natural conditions, can now be used as a convenient stress indicator and specifically attributed to a damage on the electron donor side.     

Molecular remodeling of photosystem II during state transitions in Chlamydomonas reinhardtii

State transitions, or the redistribution of light-harvesting complex II (LHCII) proteins between photosystem I (PSI) and photosystem II (PSII), balance the light-harvesting capacity of the two photosystems to optimize the efficiency of photosynthesis. Studies on the migration of LHCII proteins have focused primarily on their reassociation with PSI, but the molecular details on their dissociation from PSII have not been clear. Here, we compare the polypeptide composition, supramolecular organization, and phosphorylation of PSII complexes under PSI- and PSII-favoring conditions (State 1 and State 2, respectively). Three PSII fractions, a PSII core complex, a PSII supercomplex, and a multimer of PSII supercomplex or PSII megacomplex, were obtained from a transformant of the green alga Chlamydomonas reinhardtii carrying a His-tagged CP47. Gel filtration and single particles on electron micrographs showed that the megacomplex was predominant in State 1, whereas the core complex was predominant in State 2, indicating that LHCIIs are dissociated from PSII upon state transition. Moreover, in State 2, strongly phosphorylated LHCII type I was found in the supercomplex but not in the megacomplex. Phosphorylated minor LHCIIs (CP26 and CP29) were found only in the unbound form. The PSII subunits were most phosphorylated in the core complex. Based on these observations, we propose a model for PSII remodeling during state transitions, which involves division of the megacomplex into supercomplexes, triggered by phosphorylation of LHCII type I, followed by LHCII undocking from the supercomplex, triggered by phosphorylation of minor LHCIIs and PSII core subunits.     

Purification of highly active oxygen-evolving photosystem II from Chlamydomonas reinhardtii

A method is described for the isolation and purification of active oxygen-evolving photosystem II (PS II) membranes from the green alga Chlamydomonas reinhardtii. The isolation procedure is a modification of methods evolved for spinach (Berthold et al. 1981). The purity and integrity of the PS II preparations have been assesssed on the bases of the polypeptide pattern in SDS-PAGE, the rate of oxygen evolution, the EPR multiline signal of the S₂ state, the room temperature chlorophyll a fluorescence yield, the 77 K emission spectra, and the P700 EPR signal at 300 K. These data show that the PS II characteristics are increased by a factor of two in PS II preparations as compared to thylakoid samples, and the PS I concentration is reduced by approximately a factor ten compared to that in thylakoids.Abbreviations BSA bovine serum albumin - Chl chlorophyll - DCBQ 2,6-dichloro-p-benzoquinone - DCMU (diuron) 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DMQ 2,5-dimethyl-p-benzoquinone - EDTA ethylenediamine tetraacetic acid - EPR electron paramagnetic resonance - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - MES 2-[N-Morpholino]ethanesulfonic acid - OEE oxygen evolving enhancer - PS II photosystem II - SDS-PAGE sodium dedocyl sulfate polyacrylamide gel electrophoresis     

Studies on chloroplast development in Chlamydomonas reinhardtii II. Effects of interposed darkness on chlorophyll synthesis

Effects of an inserted dark incubation on light-induced chlorophyllsynthesis in dark grown Chlamydomonai reinhardtii y-1 cellswere studied. Chlorophyll synthesis in cells with the interposeddark incubation proceeded faster than that in cells withoutthe dark incubation when it was inserted within 2.5 hr afterthe onset of illumination. Within this limit, the longer theinitial illumination given, the shorter was the length of darkincubation required to obtain a maximum rate of chlorophyllsynthesis. However, when the dark incubation was provided laterthan 2.5 hr, the rate of subsequent chlorophyll synthesis wasreduced. Since cells responded to the dark treatment in differentmanners before and after the 2.5 hr point, this time was designatedas the transition point. This 2.5 hr period corresponds to thelength of the regular lag phase in chlorophyll synthesis undercontinuous illumination. Based on these results, the nature of the previously postulatedpromoting factor (P-factor) in chlorophyll synthesis is discussed. (Received June 13, 1972; )     

The chlorophyll-a/b proteins of photosystem II in Chlamydomonas reinhardtii

We have adapted the procedure for the isolation of PSII membranes from higher plants (D.A. Berthold et al., 1981, FEBS Lett. 134, 231–234) to the green algae Chlamydomonas reinhardtii. The chlorophyll (Chl)-binding proteins from this PSII preparation have been further separated into single Chl-binding polypeptides and characterized spectroscopically. Seven single polypeptides were shown to bind Chl a and Chl b. In particular, we demonstrate that polypeptides p9, p10 and p22, which had not been previously shown to bind Chl a and b, have characteristics similar to those of CP29, CP26 and CP24 from higher plants. We note, however, that p9 and p10 are phosphorylatable in C. reinhardtii, at variance with CP29 and CP26 from higher plants. Our data support the notion that the PSII antenna systems in C. reinhardtii and in higher plants are very similar. Therefore, studies on the organization and regulation of light-harvesting processes in C. reinhardtii may provide information of general relevance for both green algae and higher plants.Abbreviations Chl chlorophyll - IEF isoelectrofocusing - LHC light harvesting complex - MW molecular weight - PAGE polyacrylamide gel electrophoresis - PS photosystem - RC reaction centre - SDS sodium dodecylsulfate We thank Dr. J. Olive (Institut Jacques Monod, Paris, France) for the electron-microscopy analysis, C. de Vitry (Institut de Biologie Physico-Chimique, Paris, France) for the kind gift of a PSII RC preparation and P. Dainese and M.L. Di Paolo (Universitá di Padova, Padova, Italy) for helpfull discussions. Professor Strasser and Elizbeth Scwartz (Université de Genova, Genova, Switzerland) are thanked for assistance in taking low-temperature fluorescence emission spectra. Roberto Bassi was recipient of a short-term fellowship from the European Molecular Biology Organization fellowship, during the early phases of the work.     

Effect of dichromate on photosystem II activity in xanthophyll-deficient mutants of Chlamydomonas reinhardtii

The photosystem II activity and energy dissipation was investigated when algal Chlamydomonas reinhardtii genotypes were exposed to dichromate toxicity effect. The exposure during 24 h to dichromate effect of two C. reinhardtii mutants having non-functional xanthophylls cycle, as npq1 zeaxanthin deficient and npq2 zeaxanthin accumulating, induced inhibition of PSII electron transport. After dichromate-induced toxicity, PSII functions of C. reinhardtii mutants were investigated under different light intensities. To determine dichromate toxicity and light intensity effect on PSII functional properties we investigated the change of energy dissipation via PSII electron transport, non-photochemical regulated and non-regulated energy dissipation according to Kramer et al. (Photosynth Res 79:209–218, 2004). We showed the dependency between dichromate toxicity and light-induced photoinhibition in algae deficient in xanthophyll cycle. When algal mutants missing xanthophylls cycle were exposed to dichromate toxicity and to high light intensity energy dissipation via non-regulated mechanism takes the most important pathway reaching the value of 80%. Therefore, the mutants npq1 and npq2 having non-functional xanthophylls cycle were more sensitive to dichromate toxic effects.     

Turnover of thylakoid photosystem II proteins during photoinhibition of Chlamydomonas reinhardtii

The turnover of photosystem-II proteins during photoinhibition was analyzed in the green alga Chlamydomonas reinhardtii. Changes in the amount of photosystem II core complex polypeptides D1, D2, 44 kDa and 51 kDa, the antennae-CP-29 and light-harvesting-complex-II polypeptides and the water-oxidizing complex polypeptides of 30 kDa, 23 kDa and 16 kDa were monitored by a variety of techniques. Only the D1 and D2 polypeptides were found to turnover during photoinhibition when cells were exposed to ten fold photosynthesis-saturating light (2500 W/m2 for 90 min) at 25 degrees C. While 80% of photosystem-II activity was lost, a reduction of only 20% was observed in the total amount of D1 and D2 proteins. However, inhibition of chloroplast translation by chloramphenicol during photoinhibition resulted in the loss of about 60% of the D1 and 40% of the D2 proteins, as demonstrated by Western blotting and dot blotting of isolated thylakoids, quantitative analysis of immunogold-labeled whole-cell thin sections, and chase of radioactively prelabelled proteins during photoinhibition. We propose that the light-dependent turnover of the D1 protein is a protective mechanism against photoinhibition as far as the removal and replacement of D1 is compatible with the photoinactivation incurred by photosystem II. At light intensities at which the rate of D1 removal becomes limiting, loss of photosystem-II activity exceeds the turnover of D1 and the stability of the D2 protein is impaired as well.     

Mg‐dechelatase is involved in the formation of photosystem II but not in chlorophyll degradation in Chlamydomonas reinhardtii

The STAY‐GREEN (SGR) gene encodes Mg‐dechelatase which catalyzes the conversion of chlorophyll (Chl) a to pheophytin (Pheo) a. This reaction is the first and most important regulatory step in the Chl degradation pathway. Conversely, Pheo a is an indispensable molecule in photosystem (PS) II, suggesting the involvement of SGR in the formation of PSII. To investigate the physiological functions of SGR, we isolated Chlamydomonas sgr mutants by screening an insertion‐mutant library. The sgr mutants had reduced maximum quantum efficiency of PSII (F_v/F_m) and reduced Pheo a levels. These phenotypes were complemented by the introduction of the Chlamydomonas SGR gene. Blue Native polyacrylamide gel electrophoresis and immunoblotting analysis showed that although PSII levels were reduced in the sgr mutants, PSI and light‐harvesting Chl a/b complex levels were unaffected. Under nitrogen starvation conditions, Chl degradation proceeded in the sgr mutants as in the wild type, indicating that ChlamydomonasSGR is not required for Chl degradation and primarily contributes to the formation of PSII. In contrast, in the Arabidopsis sgr triple mutant (sgr1 sgr2 sgrL), which completely lacks SGR activity, PSII was synthesized normally. These results suggest that the Arabidopsis SGR participates in Chl degradation while the ChlamydomonasSGR participates in PSII formation despite having the same catalytic property.     

Effect of dibromothymoquinone on chlorophyll a fluorescence in Chlamydomonas reinhardtii cells incubated in complete or sulfur-depleted medium

The influence of dibromothymoquinone on chlorophyll fluorescence was studied in Chlamydomonas reinhardtii cells using PAM and PEA fluorometers. The reagent affected differently control cells incubated in complete medium and S-starved cells. Thus, the fluorescence yield in the control essentially increased in the presence of dibromothymoquinone, which can be due to the inactivation of light-harvesting complex II protein kinase, followed by the suppression of membrane transition from high-fluorescence state 1 to low-fluorescence state 2. On the contrary, S-starved cells with membranes in state 2 showed a lower fluorescence yield in the presence of dibromothymoquinone than without it. The JIP test of OJIP fluorescence transients suggests that dibromothymoquinone inhibits both light-harvesting complex II kinase and photosynthetic electron transport when added to control, while in starved cells, it acts predominantly as an electron acceptor.     

Changes in chlorophyll a fluorescence in Euglena gracilis and Chlamydomonas reinhardtii after exposure to wood-ash

Recycling of wood-ash to boreal forests has been suggested to prevent depletion of essential soil nutrient or reduce the negative effects of acidification of surface waters. The aim of this investigation was to study the effects of different concentrations of wood-ash (5, 10 and 12.5 g l⁻¹ diluted in cultivating medium) on chlorophyll a fluorescence in Euglena gracilis and Chlamydomonas reinhardtii under laboratory conditions. The green alga C. reinhardtii was more susceptible to wood-ash solutions than the flagellate E. gracilis. Two different forms of wood-ash solutions were tested. In the first solution no adjustment of pH was made and after 7 days of incubation with wood-ash the pH for the different wood-ash concentrations (5, 10 and 12.5 g l⁻¹) were 8, 9 and 11, respectively. In the second solution, the pH was adjusted to 7. The results show that no negative effect on fluorescence yield (F_v/F_m), relative electron transport rate (ETR), photochemical quenching (qP) or non-photochemical quenching (NPQ) was observed in E. gracilis. In contrast, C. reinhardtii displayed strong inhibition at concentrations of 10 and 12.5 g l⁻¹ with non-adjusted pH. The negative effects of high pH on photochemical activity in C. reinhardtii could either be related to (1) the destruction of the ΔpH across the thylakoid membranes or (2) other parts in the photosynthetic systems that are negatively affected by changing pH. The results indicate that elevated pH levels due to wood-ash application could be an environmental stress factor to phytoplankton communities and may lead to loss of diversity among primary producers in aquatic ecosystems. If wood-ash application was to become general practice in or near aquatic ecosystems a rapid change in pH induced by wood-ash must be avoided.     

Evidence that the variable chlorophyll fluorescence in Chlamydomonas reinhardtii is not recombination luminescence

Room temperature single photon timing measurements on intact, Chlamydomonas reinhardtii cells at low excitation energies have been analysed using a four exponential kinetic model. Closing the PSII reaction centres produced two major variable lifetime and two minor constant lifetime components. The yield of each component mirrored the changes in lifetime. Such observations indicate the presence of well-connected PSII centres favoring excitation energy transfer. A Chlamydomonas mutant lacking PSII reaction centre proteins exhibited decay components equivalent to those seen at F_M in the wild-type. A titration of in vivo fluorescence, in both the mutant and wild-type algae, using DNB, produced decay components similar to those seen on opening PSII reaction centres. Such observations indicate that the luminescence hypothesis for the origin of the long-lived lifetime component is not the case.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1, 1-dimethyl urea - DNB m,Dinitrobenzene - PSII photosystem II - RCII PSII recation centre - I^- reduced pheophytin - Q_A primary stable electron ecceptor of PSII - Ch1 chlorophyl1 - LHCII light harvesting Ch1a/b protein complex of PSII - F_O initial fluorescence level - F_M maximum fluorescence level - F_V variable fluorescence (F_M-F_O) - ps picosecond - ns nanosecond     

Revised assignment of room-temperature chlorophyll fluorescence emission bands in single living cells of Chlamydomonas reinhardtii

Room temperature (RT) microspectrofluorimetry in vivo of single cells has a great potential in photosynthesis studies. In order to get new information on RT chlorophyll fluorescence bands, we analyzed the spectra of Chlamydomonas reinhardtii mutants lacking fundamental proteins of the thylakoid membrane and spectra of photoinhibited WT cells. RT spectra of single living cells were characterized thorough derivative analyses and Gaussian deconvolution. The results obtained suggest that the dynamism in LHCII assembly could be sufficient to explain the variations in amplitudes of F680 (free LHCII), F694 (LHCII-PSII) and F702 (LHCII aggregates); F686 was assigned to the PSII core. Based on the revised assignments and on the variations observed, we discuss the meaning of the two fluorescence emission ratios F680/(F686 + F694) and F702/(F686 + F694), showing that these are sensitive parameters under moderate photoinhibition. In the most photoinhibited samples, the RT spectra tended to degenerate, showing characteristics of mutants that are partly depleted in PSII.