Photosynthesis Research (v.137, #2)

Klaus Apel (1942–2017): a pioneer of photosynthesis research by Jean-David Rochaix; Chanhong Kim; Wiebke Apel (153-159).
We present here a Tribute to Klaus Apel (1942–2017), a photosynthesis pioneer—an authority on plant molecular genetics—in five parts. The first section is a prologue. The second section deals with a chronological discussion of Apel’s research life, prepared by the editor Govindjee; it is based on a website article at the Boyce Thompson Institute (BTI) by Patricia Waldron (https://btiscience.org/explore-bti/news/post/bti-says-goodbye-klaus-apel/), as approved for use here by Keith C. Hannon and David Stern of BTI. The third section, which focuses on Apel’s pioneering work on singlet oxygen-mediated EXECUTER-dependent signaling in plants, is written by two of us (J-DR and CK). The fourth section includes three selected reminiscences, one from BTI and two from ETH (Eidgenössische Technische Hochschule). This tribute ends with section five, which is a very brief presentation of Klaus Apel’s personal life, by Wiebke Apel.
Keywords: Photosynthesis; Chlorophyll synthesis; Light stress; Photoprotection; Protochlorophyllide; Reactive oxygen species; Singlet oxygen; Retrograde signaling

Remembering John M. Olson (1929–2017) by Robert E. Blankenship; Daniel C. Brune; Jon C. Olson (161-169).
Here we provide reflections of and a tribute to John M. Olson, a pioneering researcher in photosynthesis. We trace his career, which began at Wesleyan University and the University of Pennsylvania, and continued at Utrech in The Netherlands, Brookhaven National Laboratory, and Odense University in Denmark. He was the world expert on pigment organization in the green photosynthetic bacteria, and discovered and characterized the first chlorophyll-containing protein, which has come to be known as the Fenna–Matthews–Olson (FMO) protein. He also thought and wrote extensively on the origin and early evolution of photosynthesis. We include personal comments from Brian Matthews, Raymond Cox, Paolo Gerola, Beverly Pierson and Jon Olson.
Keywords: Green sulfur bacteria; Chlorosome; Fenna–Matthews–Olson protein; FMO protein

Site, trigger, quenching mechanism and recovery of non-photochemical quenching in cyanobacteria: recent updates by Ravi R. Sonani; Alastair Gardiner; Rajesh P. Rastogi; Richard Cogdell; Bruno Robert; Datta Madamwar (171-180).
In the original publication, under the subtitle Recovery: fluorescence recovery protein (FRP), paragraph 4 the text section enclosed in quotation marks does not occur in one of the original publications cited (Sluchanko et al. 2017a, b).Cyanobacteria exhibit a novel form of non-photochemical quenching (NPQ) at the level of the phycobilisome. NPQ is a process that protects photosystem II (PSII) from possible highlight-induced photo-damage. Although significant advancement has been made in understanding the NPQ, there are still some missing details. This critical review focuses on how the orange carotenoid protein (OCP) and its partner fluorescence recovery protein (FRP) control the extent of quenching. What is and what is not known about the NPQ is discussed under four subtitles; where does exactly the site of quenching lie? (site), how is the quenching being triggered? (trigger), molecular mechanism of quenching (quenching) and recovery from quenching. Finally, a recent working model of NPQ, consistent with recent findings, is been described.
Keywords: Cyanobacteria; Non-photochemical quenching; Orange carotenoid protein; Phycobilisome; Fluorescence recovery protein

Correction to: Site, trigger, quenching mechanism and recovery of non-photochemical quenching in cyanobacteria: recent updates by Ravi R. Sonani; Alastair Gardiner; Rajesh P. Rastogi; Richard Cogdell; Bruno Robert; Datta Madamwar (181-182).
In the original publication, under the subtitle Recovery: fluorescence recovery protein (FRP), paragraph 4 the text section enclosed in quotation marks does not occur in one of the original publications cited (Sluchanko et al. 2017a, b).

Plants photosynthesis-related traits are co-regulated to capture light and CO2 to optimize the rate of CO2 assimilation (A). The rising CO2 often benefits, but potassium (K) deficiency adversely affects A that contributes to the majority of plant biomass. To evaluate mechanisms of photosynthetic limitations and adaptations, soybean was grown under controlled conditions with an adequate (control, 5.0 mM) and two K-deficient (moderate, 0.50 and severe, 0.02 mM) levels under ambient (aCO2; 400 µmol mol−1) and elevated CO2 (eCO2; 800 µmol mol−1). Results showed that under severe K deficiency, pigments, leaf absorption, processes of light and dark reactions, and CO2 diffusion through stomata and mesophyll were down co-regulated with A while light compensation point increased and photorespiration, alternative electron fluxes, and respiration were up-regulated. However, under moderate K deficiency, these traits were well co-regulated with the sustained A without any obvious limitations amid ≈ 50% reduction in leaf K level. Primary mechanism of K limitation to A was either biochemical processes (L b ≈ 60%) under control and moderate K deficiency or the CO2 diffusion limitations (D L ≈ 70%) with greater impacts of mesophyll than stomatal pathways under severe K deficiency. The eCO2 increased D L while lessened the L b under K deficiency. Adaptation strategies to severe K deficiency included an enhanced K utilization efficiency (KUE), and reduction of photosystem II excitation pressure by decreasing photosynthetic pigments, light absorption, and photochemical quenching while increasing photorespiration and alternative electron fluxes. The eCO2 also stimulated A and KUE when K deficiency was not severe. Thus, plants responded to K deficiency by a coordinated regulation of photosynthetic processes to optimize A, and eCO2 failed to alleviate the D L in severely K-deficient plants.
Keywords: Alternative electron sink; Carboxylation; CO2 diffusion; Glycine max ; Photochemistry; Photorespiration

Diatoms account for about 40% of primary production in highly productive ecosystems. The development of a new generation of fluorometers has made it possible to improve estimation of the electron transport rate from photosystem II, which, when coupled with the carbon incorporation rate enables estimation of the electrons required for carbon fixation. The aim of this study was to investigate the daily dynamics of these electron requirements as a function of the diel light cycle in three relevant diatom species and to apprehend if the method of estimating the electron transport rate can lead to different pictures of the dynamics. The results confirmed the species-dependent capacity for photoacclimation under increasing light levels. Despite daily variations in the photosynthetic parameters, the results of this study underline the low daily variability of the electron requirements estimated using functional absorption of the photosystem II compared to an estimation based on a specific absorption cross section of chlorophyll a. The stability of the electron requirements throughout the day would suggest it is potentially possible to estimate high-frequency primary production by using autonomous variable fluorescence measurements from ships-of-opportunity or moorings, without taking potential daily variation in this parameter into consideration, but this result has to be confirmed on natural phytoplankton assemblages. The results obtained in this study confirm the low electron requirements of diatoms to perform photosynthesis, and suggest a potential additional source of energy for carbon fixation, as recently described in the literature for this class.
Keywords: Phytoplankton; Photosynthesis; PAM; 13C incorporation; Electron transport rate (ETR)

The role of charge-transfer states in the spectral tuning of antenna complexes of purple bacteria by Michele Nottoli; Sandro Jurinovich; Lorenzo Cupellini; Alastair T. Gardiner; Richard Cogdell; Benedetta Mennucci (215-226).
The LH2 antenna complexes of purple bacteria occur, depending on light conditions, in various different spectroscopic forms, with a similar structure but different absorption spectra. The differences are related to point changes in the primary amino acid sequence, but the molecular–level relationship between these changes and the resulting spectrum is still not well understood. We undertook a systematic quantum chemical analysis of all the main factors that contribute to the exciton structure, looking at how the environment modulates site energies and couplings in the B800–850 and B800–820 spectroscopic forms of LH2. A multiscale approach combining quantum chemistry and an atomistic classical embedding has been used where mutual polarization effects between the two parts are taken into account. We find that the loss of hydrogen bonds following amino acid changes can only explain a part of the observed blue-shift in the B850 band. The coupling of excitonic states to charge-transfer states, which is different in the two forms, contributes with a similar amount to the overall blue-shift.
Keywords: Excitonic model; Antenna complex; Quantum chemistry

We address a challenge in the engineering of proteins to redirect electron transfer pathways, using the bacterial photosynthetic reaction centre (RC) pigment–protein complex. Direct electron transfer is shown to occur from the QA quinone of the Rhodobacter sphaeroides RC containing a truncated H protein and bound on the quinone side to a gold electrode. In previous reports of binding to the quinone side of the RC, electron transfer has relied on the use of a soluble mediator between the RC and an electrode, in part because the probability of QB quinone reduction is much greater than that of direct electron transfer through the large cytoplasmic domain of the H subunit, presenting a ~ 25 Å barrier. A series of C-terminal truncations of the H subunit were created to expose the quinone region of the RC L and M proteins, and all truncated RC H mutants assembled in vivo. The 45M mutant was designed to contain only the N-terminal 45 amino acid residues of the H subunit including the membrane-spanning α-helix; the mutant RC was stable when purified using the detergent N-dodecyl-β-d-maltoside, contained a near-native ratio of bacteriochlorophylls to bacteriopheophytins, and showed a charge-separated state of $${{ ext{P}}^{ ext{+}}}{{ ext{Q}}_{ ext{A}}^-}$$ P+QA- . The 45M-M229 mutant RC had a Cys residue introduced in the vicinity of the QA quinone on the newly exposed protein surface for electrode attachment, decreasing the distance between the quinone and electrode to ~ 12 Å. Steady-state photocurrents of up to around 200 nA/cm2 were generated in the presence of 20 mM hydroquinone as the electron donor to the RC. This novel configuration yielded photocurrents orders of magnitude greater than previous reports of electron transfer from the quinone region of RCs bound in this orientation to an electrode.
Keywords: Rhodobacter sphaeroides ; Reaction centre; Truncated H protein; Electron transfer; Photocurrent; Bio-photovoltaic; Solar cell

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.
Keywords: P700 oxidation; Reactive oxygen species; Terminal oxidase; Oxygen

Dissecting the individual contribution of conserved cysteines to the redox regulation of RubisCO by María Jesús García-Murria; Hemanth P. K. Sudhani; Julia Marín-Navarro; Manuel M. Sánchez del Pino; Joaquín Moreno (251-262).
Oxidation of the cysteines from ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) leads to inactivation and promotes structural changes that increase the proteolytic sensitivity and membrane association propensity related to its catabolism. To uncover the individual role of the different cysteines, the sequential order of modification under increasing oxidative conditions was determined using chemical labeling and mass spectrometry. Besides, site-directed RubisCO mutants were obtained in Chlamydomonas reinhardtii replacing single conserved cysteines (Cys84, Cys172, Cys192, Cys247, Cys284, Cys427, Cys459 from the large and sCys41, sCys83 from the small subunit) and the redox properties of the mutant enzymes were determined. All mutants retained significant carboxylase activity and grew photoautotrophically, indicating that these conserved cysteines are not essential for catalysis. Cys84 played a noticeable structural role, its replacement producing a structurally altered enzyme. While Cys247, Cys284, and sCys83 were not affected by the redox environment, all other residues were oxidized using a disulfide/thiol ratio of around two, except for Cys172 whose oxidation was distinctly delayed. Remarkably, Cys192 and Cys427 were apparently protective, their absence leading to a premature oxidation of critical residues (Cys172 and Cys459). These cysteines integrate a regulatory network that modulates RubisCO activity and conformation in response to oxidative conditions.
Keywords: Cysteine network; Oxidative inactivation; Redox regulation; Ribulose 1,5-bisphosphate carboxylase/oxygenase; RubisCO catabolism; Site-directed cysteine substitution

The intracellular distribution of inorganic carbon fixing enzymes does not support the presence of a C4 pathway in the diatom Phaeodactylum tricornutum by Daniela Ewe; Masaaki Tachibana; Sae Kikutani; Ansgar Gruber; Carolina Río Bártulos; Grzegorz Konert; Aaron Kaplan; Yusuke Matsuda; Peter G. Kroth (263-280).
Diatoms are unicellular algae and important primary producers. The process of carbon fixation in diatoms is very efficient even though the availability of dissolved CO2 in sea water is very low. The operation of a carbon concentrating mechanism (CCM) also makes the more abundant bicarbonate accessible for photosynthetic carbon fixation. Diatoms possess carbonic anhydrases as well as metabolic enzymes potentially involved in C4 pathways; however, the question as to whether a C4 pathway plays a general role in diatoms is not yet solved. While genome analyses indicate that the diatom Phaeodactylum tricornutum possesses all the enzymes required to operate a C4 pathway, silencing of the pyruvate orthophosphate dikinase (PPDK) in a genetically transformed cell line does not lead to reduced photosynthetic carbon fixation. In this study, we have determined the intracellular location of all enzymes potentially involved in C4-like carbon fixing pathways in P. tricornutum by expression of the respective proteins fused to green fluorescent protein (GFP), followed by fluorescence microscopy. Furthermore, we compared the results to known pathways and locations of enzymes in higher plants performing C3 or C4 photosynthesis. This approach revealed that the intracellular distribution of the investigated enzymes is quite different from the one observed in higher plants. In particular, the apparent lack of a plastidic decarboxylase in P. tricornutum indicates that this diatom does not perform a C4-like CCM.
Keywords: C4 photosynthesis; Chloroplast; Green fluorescent protein (GFP); Carboxylation; Decarboxylation

Absolute quantification of selected photosynthetic electron transfer proteins in Chlamydomonas reinhardtii in the presence and absence of oxygen by Denitsa Nikolova; Claudia Heilmann; Susan Hawat; Philipp Gäbelein; Michael Hippler (281-293).
The absolute amount of plastocyanin (PC), ferredoxin-NADP+-oxidoreductase (FNR), hydrogenase (HYDA1), and ferredoxin 5 (FDX5) were quantified in aerobic and anaerobic Chlamydomonas reinhardtii whole cells using purified (recombinant) proteins as internal standards in a mass spectrometric approach. Quantified protein amounts were related to the estimated amount of PSI. The ratios of PC to FNR to HYDA1 to FDX5 in aerobic cells were determined to be 1.4:1.2:0.003:0. In anaerobic cells, the ratios changed to 1.1:1.3:0.019:0.027 (PC:FNR:HYDA1:FDX5). Employing sodium dithionite and methyl viologen as electron donors, the specific activity of hydrogenase in whole cells was calculated to be 382 ± 96.5 μmolH2 min−1 mg−1. Importantly, these data reveal an about 70-fold lower abundance of HYDA1 compared to FNR. Despite this great disproportion between both proteins, which might further enhance the competition for electrons, the alga is capable of hydrogen production under anaerobic conditions, thus pointing to an efficient channeling mechanism of electrons from FDX1 to the HYDA1.
Keywords: Plastocyanin; Hydrogenase; Ferredoxin-NADP+-oxidoreductase; Absolute quantification; Ferredoxin 5; Chlamydomonas reinhardtii

15N photo-CIDNP MAS NMR analysis of reaction centers of Chloracidobacterium thermophilum by Jeremias C. Zill; Zhihui He; Marcus Tank; Bryan H. Ferlez; Daniel P. Canniffe; Yigal Lahav; Peter Bellstedt; A. Alia; Igor Schapiro; John H. Golbeck; Donald A. Bryant; Jörg Matysik (295-305).
Photochemically induced dynamic nuclear polarization (photo-CIDNP) has been observed in the homodimeric, type-1 photochemical reaction centers (RCs) of the acidobacterium, Chloracidobacterium (Cab.) thermophilum, by 15N magic-angle spinning (MAS) solid-state NMR under continuous white-light illumination. Three light-induced emissive (negative) signals are detected. In the RCs of Cab. thermophilum, three types of (bacterio)chlorophylls have previously been identified: bacteriochlorophyll a (BChl a), chlorophyll a (Chl a), and Zn-bacteriochlorophyll a′ (Zn-BChl a′) (Tsukatani et al. in J Biol Chem 287:5720–5732, 2012). Based upon experimental and quantum chemical 15N NMR data, we assign the observed signals to a Chl a cofactor. We exclude Zn-BChl because of its measured spectroscopic properties. We conclude that Chl a is the primary electron acceptor, which implies that the primary donor is most likely Zn-BChl a′. Chl a and 81-OH Chl a have been shown to be the primary electron acceptors in green sulfur bacteria and heliobacteria, respectively, and thus a Chl a molecule serves this role in all known homodimeric type-1 RCs.
Keywords: Chlorophototrophy; Reaction centers; Chloracidobacterium thermophilum ; 15N-MAS NMR; Photo-CIDNP; Zn-BChl a

Spectrally decomposed dark-to-light transitions in Synechocystis sp. PCC 6803 by Alonso M. Acuña; Pascal van Alphen; Filipe Branco dos Santos; Rienk van Grondelle; Klaas J. Hellingwerf; Ivo H. M. van Stokkum (307-320).
Photosynthetic activity and respiration share the thylakoid membrane in cyanobacteria. We present a series of spectrally resolved fluorescence experiments where whole cells of the cyanobacterium Synechocystis sp. PCC 6803 and mutants thereof underwent a dark-to-light transition after different dark-adaptation (DA) periods. Two mutants were used: (i) a PSI-lacking mutant (ΔPSI) and (ii) M55, a mutant without NAD(P)H dehydrogenase type-1 (NDH-1). For comparison, measurements of the wild-type were also carried out. We recorded spectrally resolved fluorescence traces over several minutes with 100 ms time resolution. The excitation light was at 590 nm so as to specifically excite the phycobilisomes. In ΔPSI, DA time has no influence, and in dichlorophenyl-dimethylurea (DCMU)-treated samples we identify three main fluorescent components: PB–PSII complexes with closed (saturated) RCs, a quenched or open PB–PSII complex, and a PB–PSII ‘not fully closed.’ For the PSI-containing organisms without DCMU, we conclude that mainly three species contribute to the signal: a PB–PSII–PSI megacomplex with closed PSII RCs and (i) slow PB → PSI energy transfer, or (ii) fast PB → PSI energy transfer and (iii) complexes with open (photochemically quenched) PSII RCs. Furthermore, their time profiles reveal an adaptive response that we identify as a state transition. Our results suggest that deceleration of the PB → PSI energy transfer rate is the molecular mechanism underlying a state 2 to state 1 transition.
Keywords: Cyanobacteria; Spectrally resolved fluorometry; Singular value decomposition; Time-resolved spectroscopy; Plastoquinone pool; Cyclic electron flow

Uphill energy transfer in photosystem I from Chlamydomonas reinhardtii. Time-resolved fluorescence measurements at 77 K by Wojciech Giera; Sebastian Szewczyk; Michael D. McConnell; Kevin E. Redding; Rienk van Grondelle; Krzysztof Gibasiewicz (321-335).
Energetic properties of chlorophylls in photosynthetic complexes are strongly modulated by their interaction with the protein matrix and by inter-pigment coupling. This spectral tuning is especially striking in photosystem I (PSI) complexes that contain low-energy chlorophylls emitting above 700 nm. Such low-energy chlorophylls have been observed in cyanobacterial PSI, algal and plant PSI–LHCI complexes, and individual light-harvesting complex I (LHCI) proteins. However, there has been no direct evidence of their presence in algal PSI core complexes lacking LHCI. In order to determine the lowest-energy states of chlorophylls and their dynamics in algal PSI antenna systems, we performed time-resolved fluorescence measurements at 77 K for PSI core and PSI–LHCI complexes isolated from the green alga Chlamydomonas reinhardtii. The pool of low-energy chlorophylls observed in PSI cores is generally smaller and less red-shifted than that observed in PSI–LHCI complexes. Excitation energy equilibration between bulk and low-energy chlorophylls in the PSI–LHCI complexes at 77 K leads to population of excited states that are less red-shifted (by ~ 12 nm) than at room temperature. On the other hand, analysis of the detection wavelength dependence of the effective trapping time of bulk excitations in the PSI core at 77 K provided evidence for an energy threshold at ~ 675 nm, above which trapping slows down. Based on these observations, we postulate that excitation energy transfer from bulk to low-energy chlorophylls and from bulk to reaction center chlorophylls are thermally activated uphill processes that likely occur via higher excitonic states of energy accepting chlorophylls.
Keywords: Photosystem I; Light-harvesting complex I; Chlamydomonas reinhardtii ; Time-resolved fluorescence; Excitation energy transfer; Red chlorophylls