Photosynthesis Research (v.137, #1)

Shmuel Malkin (1934–2017) by Stephen K. Herbert; Yona Siderer; Govindjee (1-15).
We present here the life and work of Shmuel Malkin (1934–2017), an accomplished scientist and a gifted musician who touched the lives of many around the world. His early scientific work addressed the dynamics of light harvesting and electron transport in photosynthesis. Later, he used photoacoustic and photothermal methodologies to explore all aspects of photosynthesis. As a musician, Shmuel played the piano often for family and friends but after his formal retirement, he produced a body of original musical compositions, many of which were performed publicly. Throughout his life, Shmuel was a caring and deeply thoughtful man, respected and loved by colleagues, family, and friends. This tribute presents a summary of Shmuel’s work as well as remembrances written by his wife, Nava Malkin, their son, Eyal Malkinson, and many of his colleagues: Michael Havaux from France; Sandra and Marcel Jansen from Ireland; David Cahen, Marvin Edelmann, Joop and Onnie de Graaf, Jonathan Gressel, Uri Pick, Yona Siderer, and Elisha Tel-Or from Israel; Ulrich Schreiber from Germany; James Barber and Alison Telfer from the UK; Govindjee, Stephen Herbert and Thomas Sharkey from the USA. Minnie Ho and Iris Malkin of the USA wrote contributions about Shmuel’s music.
Keywords: Bessel Kok; Hebrew University; Photoacoustic; Photosynthesis; Research Institute for Advanced Studies; Weizmann Institute of Science

The dynamic and efficient coordination of primary photosynthetic reactions with leaf energization and metabolism under a wide range of environmental conditions is a fundamental property of plants involving processes at all functional levels. The present historical perspective covers 60 years of research aiming to understand the underlying mechanisms, linking major breakthroughs to current progress. It centers on the contributions of Ulrich Heber who had pioneered novel concepts, fundamental methods, and mechanistic understanding of photosynthesis. An important first step was the development of non-aqueous preparation of chloroplasts allowing the investigation of chloroplast metabolites ex vivo (meaning that the obtained results reflect the in vivo situation). Later on, intact chloroplasts, retaining their functional envelope membranes, were isolated in aqueous media to investigate compartmentation and exchange of metabolites between chloroplasts and external medium. These studies elucidated metabolic interaction between chloroplasts and cytoplasm during photosynthesis. Experiments with isolated intact chloroplasts clarified that oxygenation of ribulose-1.5-bisphosphate generates glycolate in photorespiration. The development of non-invasive optical methods enabled researchers identifying mechanisms that balance electron flow in the photosynthetic electron transport system avoiding its over-reduction. Recording chlorophyll a (Chl a) fluorescence allowed one to monitor, among other parameters, thermal energy dissipation by means of ‘nonphotochemical quenching’ of the excited state of Chl a. Furthermore, studies both in vivo and in vitro led to basic understanding of the biochemical mechanisms of freezing damage and frost tolerance of plant leaves, to SO2 tolerance of tree leaves and dehydrating lichens and mosses.
Keywords: Chlorophyll a fluorescence; Chloroplast; Cyclic electron transport; Lichen; Light scattering; Carbon metabolism; Photorespiration; Photosynthesis; Xanthophylls

Binding of pigments to the cyanobacterial high-light-inducible protein HliC by Mahendra Kumar Shukla; Manuel J. Llansola-Portoles; Martin Tichý; Andrew A. Pascal; Bruno Robert; Roman Sobotka (29-39).
Cyanobacteria possess a family of one-helix high-light-inducible proteins (HLIPs) that are widely viewed as ancestors of the light-harvesting antenna of plants and algae. HLIPs are essential for viability under various stress conditions, although their exact role is not fully understood. The unicellular cyanobacterium Synechocystis sp. PCC 6803 contains four HLIPs named HliA–D, and HliD has recently been isolated in a small protein complex and shown to bind chlorophyll and β-carotene. However, no HLIP has been isolated and characterized in a pure form up to now. We have developed a protocol to purify large quantities of His-tagged HliC from an engineered Synechocystis strain. Purified His-HliC is a pigmented homo-oligomer and is associated with chlorophyll and β-carotene with a 2:1 ratio. This differs from the 3:1 ratio reported for HliD. Comparison of these two HLIPs by resonance Raman spectroscopy revealed a similar conformation for their bound β-carotenes, but clear differences in their chlorophylls. We present and discuss a structural model of HliC, in which a dimeric protein binds four chlorophyll molecules and two β-carotenes.
Keywords: Synechocystis ; HLIPs; HliC; Raman spectroscopy; Chlorophyll; β-Carotene

Cell size has implications for the package effect in photon absorption as well as for metabolic scaling of metabolism. In this study, we have avoided species-related differences by using isolates of the marine planktonic diatom Coscinodiscus granii with cells of different sizes and grown at different light intensities to investigate their energy allocation strategies. To make full use of incident light, several fold variations in cellular chlorophyll a content were employed across cell size. This modulation of pigment-related light absorbance was deemed effective as similar light absorbing capacities were found in all treatments. Unexpected low values of O2 evolution rate at the highest irradiance level of 450 μmol photons m−2 s−1 were found in medium and large cells, regardless of more photons being absorbed under these conditions, suggesting the operation of alternative electron flows acting as electron sinks. The growth rate was generally larger at higher irradiance levels except for the large cells, in which growth slowed at 450 μmol photons m−2 s−1, suggesting that larger cells achieved a balance between growth and photoprotection by sacrificing growth rate when exposed to high light. Although the ratio of carbon demand to rates of uncatalysed CO2 diffusion to the cell surface reached around 20 in large cells grown under higher irradiance, the carbon fixation rate was not lowered, due to the presence of a highly effective carbon dioxide concentrating mechanism.
Keywords: Diatom; Photosynthesis; Photoacclimation; Photoprotection; Photochemical efficiency of Photosystem I; Cell size

UMP kinase activity is involved in proper chloroplast development in rice by Fei Chen; Guojun Dong; Xiaohui Ma; Fang Wang; Yanli Zhang; Erhui Xiong; Jiahuan Wu; Huizhong Wang; Qian Qian; Limin Wu; Yanchun Yu (53-67).
Isolation of leaf-color mutants is important in understanding the mechanisms of chloroplast biogenesis and development. In this study, we identified and characterized a rice (Oryza sativa) mutant, yellow leaf 2 (yl2), exhibiting pale yellow leaves with a few longitudinal white stripes at the early seedling stage then gradually turning yellow. Genetic analyses revealed that YL2 encodes a thylakoid membrane-localized protein with significant sequence similarity to UMP kinase proteins in prokaryotes and eukaryotes. Prokaryotic UMP kinase activity was subsequently confirmed, with YL2 deficiency causing a significant reduction in chlorophyll accumulation and photochemical efficiency. Moreover, YL2 is also light dependent and preferentially expressed in green tissues. Chloroplast development was abnormal in the yl2 mutant, possibly due to reduced accumulation of thylakoid membranes and a lack of normal stroma lamellae. 2D Blue-Native SDS-PAGE and immunoblot analyses revealed a reduction in several subunits of photosynthetic complexes, in particular, the AtpB subunit of ATP synthase, while mRNA levels of corresponding genes were unchanged or increased compared with the wild type. In addition, we observed a significant decrease (ca. 36.3%) in cpATPase activity in the yl2 mutant compared with the wild type. Taken together, our results suggest that UMP kinase activity plays an essential role in chloroplast development and regulating cpATPase biogenesis in rice.
Keywords: Chloroplast development; Photosynthetic complexes; Rice (Oryza sativa L.); Thylakoid biogenesis; UMP kinase; Yellow leaf mutant

In higher plant chloroplasts, the plastid-encoded RNA polymerase (PEP) consists of four catalytic subunits and numerous nuclear-encoded accessory proteins, including pTAC10, an S1-domain-containing protein. In this study, pTAC10 knockout lines were characterized. Two ptac10 mutants had an albino phenotype and severely impaired chloroplast development. The pTAC10 genomic sequence fused to a four-tandem MYC tag driven by its own promoter functionally complemented the ptac10-1 mutant phenotype. pTAC10 was present in both the chloroplast stroma and thylakoids. Two-dimensional blue native polyacrylamide gel electrophoresis (BN-PAGE), and immunoblotting assays showed that pTAC10:MYC co-migrates with one of the PEP core subunits, RpoB. A comprehensive investigation of the plastid gene expression profiles by quantitative RT-PCR revealed that, compared with wild-type plants, the abundance of PEP-dependent plastid transcripts is severely decreased in the ptac10-1 mutant, while the amount of plastid transcripts exclusively transcribed by NEP either barely changes or even increases. RNA blot analysis confirmed that PEP-dependent chloroplast transcripts, including psaB, psbA and rbcL, substantially decrease in the ptac10-1 mutant. Immunoblotting showed reduced accumulation of most chloroplast proteins in the ptac10 mutants. These data indicate the essential role of pTAC10 in plastid gene expression and plastid development. pTAC10 interacts with chloroplast-targeted casein kinase 2 (cpCK2) in vitro and in vivo and can be phosphorylated by Arabidopsis cpCK2 in vitro at sites Ser95, Ser396 and Ser434. RNA-EMSA assays showed that pTAC10 is able to bind to the psbA, atpE and accD transcripts, suggesting a non-specific RNA-binding activity of pTAC10. The RNA affinity of pTAC10 was enhanced by phosphorylation and decreased by the amino acid substitution Ser434-Ala of pTAC10. These data show that pTAC10 is essential for plastid gene expression in Arabidopsis and that it can be phosphorylated by cpCK2.
Keywords: Arabidopsis; pTAC10; Plastid gene expression; RNA-binding; CK2; Phosphorylation

Time-resolved FTIR difference spectroscopy has been used to study photosystem I (PSI) particles with three different benzoquinones [plastoquinone-9 (PQ), 2,6-dimethyl-1,4-benzoquinone (DMBQ), 2,3,5,6-tetrachloro-1,4-benzoquinone (Cl4BQ)] incorporated into the A1 binding site. If PSI samples are cooled in the dark to 77 K, the incorporated benzoquinones are shown to be functional, allowing the production of time-resolved (P700+A1 −P700A1) FTIR difference spectra. If samples are subjected to repetitive flash illumination at room temperature prior to cooling, however, the time-resolved FTIR difference spectra at 77 K display contributions typical of the P700 triplet state (3P700), indicating a loss of functionality of the incorporated benzoquinones, that occurs because of double protonation of the incorporated benzoquinones. The benzoquinone protonation mechanism likely involves nearby water molecules but does not involve the terminal iron–sulfur clusters FA and FB. These results and conclusions resolve discrepancies between results from previous low-temperature FTIR and EPR studies on similar PSI samples with PQ incorporated.
Keywords: Photosystem I; Electron transfer; Photosynthesis; Phylloquinone; Benzoquinone; Time-resolved FTIR

Structural integrity of Synechocystis sp. PCC 6803 phycobilisomes evaluated by means of differential scanning calorimetry by Nia Petrova; Svetla Todinova; Hajnalka Laczko-Dobos; Tomas Zakar; Sindhujaa Vajravel; Stefka Taneva; Zoltan Gombos; Sashka Krumova (95-104).
Phycobilisomes (PBSs) are supramolecular pigment–protein complexes that serve as light-harvesting antennae in cyanobacteria. They are built up by phycobiliproteins assembled into allophycocyanin core cylinders (ensuring the physical interaction with the photosystems) and phycocyanin rods (protruding from the cores and having light-harvesting function), the whole PBSs structure being maintained by linker proteins. PBSs play major role in light-harvesting optimization in cyanobacteria; therefore, the characterization of their structural integrity in intact cells is of great importance. The present study utilizes differential scanning calorimetry and spectroscopy techniques to explore for the first time, the thermodynamic stability of PBSs in intact Synechocystis sp. PCC 6803 cells and to probe its alteration as a result of mutations or under different growth conditions. As a first step, we characterize the thermodynamic behavior of intact and dismantled PBSs isolated from wild-type cells (having fully assembled PBSs) and from CK mutant cells (that lack phycocyanin rods and contain only allophycocyanin cores), and identified the thermal transitions of phycocyanin and allophycocyanin units in vitro. Next, we demonstrate that in intact cells PBSs exhibit sharp, high amplitude thermal transition at about 63 °C that strongly depends on the structural integrity of the PBSs supercomplex. Our findings implicate that calorimetry could offer a valuable approach for the assessment of the influence of variety of factors affecting the stability and structural organization of phycobilisomes in intact cyanobacterial cells.
Keywords: Phycobilisomes; Thermal stability; Differential scanning calorimetry; Synechocystis sp. PCC 6803; Cyanobacterial mutants

Low light (LL) and high light (HL)-acclimated plants of A. thaliana were exposed to blue (BB) or red (RR) light or to a mixture of blue and red light (BR) of incrementally increasing intensities. The light response of photosystem II was measured by pulse amplitude-modulated chlorophyll fluorescence and that of photosystem I by near infrared difference spectroscopy. The LL but not HL leaves exhibited blue light-specific responses which were assigned to relocation of chloroplasts from the dark to the light-avoidance arrangement. Blue light (BB and BR) decreased the minimum fluorescence ($$F_{0}^{prime }$$ F0′ ) more than RR light. This extra reduction of the $$F_{0}^{prime }$$ F0′ was stronger than theoretically predicted for $$F_{0}^{prime }$$ F0′ quenching by energy dissipation but actual measurement and theory agreed in RR treatments. The extra $$F_{0}^{prime }$$ F0′ reduction was assigned to decreased light absorption of chloroplasts in the avoidance position. A maximum reduction of 30% was calculated. Increasing intensities of blue light affected the fluorescence parameters NPQ and qP to a lesser degree than red light. After correcting for the optical effects of chloroplast relocation, the NPQ responded similarly to blue and red light. The same correction method diminished the color-specific variations in qP but did not abolish it; thus strongly indicating the presence of another blue light effect which also moderates excitation pressure in PSII but cannot be ascribed to absorption variations. Only after RR exposure, a post-illumination overshoot of $$F_{0}^{prime }$$ F0′ and fast oxidation of PSI electron acceptors occurred, thus, suggesting an electron flow from stromal reductants to the plastoquinone pool.
Keywords: Blue light response; Cyclic electron transport; Leaf optics; P700; Photoinhibition; Phototropin

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

Natively oxidized amino acid residues in the spinach cytochrome b 6 f complex by Ryan M. Taylor; Larry Sallans; Laurie K. Frankel; Terry M. Bricker (141-151).
The cytochrome b 6 f complex of oxygenic photosynthesis produces substantial levels of reactive oxygen species (ROS). It has been observed that the ROS production rate by b 6 f is 10–20 fold higher than that observed for the analogous respiratory cytochrome bc 1 complex. The types of ROS produced (O2 •−, 1O2, and, possibly, H2O2) and the site(s) of ROS production within the b 6 f complex have been the subject of some debate. Proposed sources of ROS have included the heme b p , PQ p •− (possible sources for O2 •−), the Rieske iron–sulfur cluster (possible source of O2 •− and/or 1O2), Chl a (possible source of 1O2), and heme c n (possible source of O2 •− and/or H2O2). Our working hypothesis is that amino acid residues proximal to the ROS production sites will be more susceptible to oxidative modification than distant residues. In the current study, we have identified natively oxidized amino acid residues in the subunits of the spinach cytochrome b 6 f complex. The oxidized residues were identified by tandem mass spectrometry using the MassMatrix Program. Our results indicate that numerous residues, principally localized near p-side cofactors and Chl a, were oxidatively modified. We hypothesize that these sites are sources for ROS generation in the spinach cytochrome b 6 f complex.
Keywords: Cytochrome b 6 f complex; Mass spectrometry; Reactive oxygen species; Spinach