Göran Samuelsson’s research while affiliated with Umeå University and other places

Chlamydomonas reinhardtii is a unicellular green alga that can survive at a wide range of inorganic carbon (Ci) concentrations by regulating the activity of a CO2-concentrating mechanism (CCM) as well as other cellular functions. Under CO2 limited conditions, C. reinhardtii cells display a wide range of adaptive responses including changes in photosynthetic electron transport, mitochondria localization in the cells, the structure of the pyrenoid starch sheath, and primary metabolism. In addition to these functional and structural changes, gene and protein expression are also affected. Several physiological aspects of the CO2 response mechanism have been studied in detail. However, the regulatory components (transcription factors and transcriptional regulators) involved in this process are not fully characterized. Here we report a comprehensive analysis of the C. reinhardtii nuclear proteome using liquid chromatography electrospray ionization spectrometry (LC-ESI-MS). The study aims to identify the proteins that govern adaptation to varying CO2 concentrations in Chlamydomonas. The nuclear proteome of C. reinhardtii cells grown in the air at high (5%) and low (0.04%) CO2 concentrations were analyzed. Using this approach, we identified 1378 proteins in total, including 90 putative transcription factors and 27 transcriptional regulators. Characterization of these new regulatory components could shed light on the molecular mechanisms underlying acclimation to CO2 stress.

The PsbO protein is an essential extrinsic subunit of photosystem II, the pigment‐protein complex responsible for light‐driven water splitting. Water oxidation in photosystem II supplies electrons to the photosynthetic electron transfer chain and is accompanied by proton release and oxygen evolution. While the electron transfer steps in this process are well defined and characterized, the driving forces acting on the liberated protons, their dynamics, and their destiny are all largely unknown. It was suggested that PsbO undergoes proton‐induced conformational changes and forms hydrogen bond networks that ensure prompt proton removal from the catalytic site of water oxidation, i.e. the Mn4CaO5 cluster. This work reports the purification and characterization of heterologously expressed PsbO from green algae Chlamydomonas reinhardtii and two isoforms from the higher plant Solanum tuberosum (PsbO1 and PsbO2). A comparison to the spinach PsbO reveals striking similarities in intrinsic protein fluorescence and CD spectra, reflecting the near‐identical secondary structure of the proteins from algae and higher plants. Titration experiments using the hydrophobic fluorescence probe ANS revealed that eukaryotic PsbO proteins exhibit acid‐base hysteresis. This hysteresis is a dynamic effect accompanied by changes in the accessibility of the protein’s hydrophobic core and is not due to reversible oligomerization or unfolding of the PsbO protein. These results confirm the hypothesis that pH‐dependent dynamic behavior at physiological pH ranges is a common feature of PsbO proteins and causes reversible opening and closing of their β‐barrel domain in response to the fluctuating acidity of the thylakoid lumen. This article is protected by copyright. All rights reserved.

Vyacheslav Vasilevich (V.V.) Klimov (or Slava, as most of us called him) was born on January 12, 1945 and passed away on May 9, 2017. He began his scientific career at the Bach Institute of Biochemistry of the USSR Academy of Sciences (Akademy Nauk (AN) SSSR), Moscow, Russia, and then, he was associated with the Institute of Photosynthesis, Pushchino, Moscow Region, for about 50 years. He worked in the field of biochemistry and biophysics of photosynthesis. He is known for his studies on the molecular organization of photosystem II (PSII). He was an eminent scientist in the field of photobiology, a well-respected professor, and, above all, an outstanding researcher. Further, he was one of the founding members of the Institute of Photosynthesis in Pushchino, Russia. To most, Slava Klimov was a great human being. He was one of the pioneers of research on the understanding of the mechanism of light energy conversion and of water oxidation in photosynthesis. Slava had many collaborations all over the world, and he is (and will be) very much missed by the scientific community and friends in Russia as well as around the World. We present here a brief biography and some comments on his research in photosynthesis. We remember him as a friendly and enthusiastic person who had an unflagging curiosity and energy to conduct outstanding research in many aspects of photosynthesis, especially that related to PSII.

In oxygenic photosynthesis, light energy is stored in the form of chemical energy by converting CO2 and H2O into carbohydrates. The light-driven oxidation of water that provides the electrons and protons for the subsequent CO2 fixation takes place in photosystem II (PSII). Recent studies show that in higher plants HCO3- increases PSII activity by acting as a mobile acceptor of the protons produced by PSII. In the green alga Chlamydomonas reinhardtii a luminal carbonic anhydrase, CrCAH3, (Cr), was suggested to improve proton removal from PSII, possibly by rapid reformation of HCO3- from CO2. In this study we investigated the interplay between PSII and CrCAH3 by membrane-inlet mass spectrometry (MIMS) and X-ray crystallography. MIMS measurements showed that CrCAH3 was most active at the slightly acidic pH values prevalent in the thylakoid lumen under illumination. Two crystal structures of CrCAH3 in complex with either acetazolamide or phosphate ions were determined at 2.6 and 2.7 Å resolution, respectively. CrCAH3 is a dimer at pH 4.1 that is stabilized by swapping of the N-terminal arms, a feature not previously observed in α-type carbonic anhydrases. The structure contains a disulfide bond and redox-titration of CrCAH3 function with dithiothreitol suggested a possible redox regulation of the enzyme. The stimulating effect of CrCAH3 and CO2/HCO3- on PSII activity was demonstrated by comparing the flash-induced oxygen-evolution pattern of wt and CrCAH3-less PSII preparations. We showed that CrCAH3 has unique structural features that allow this enzyme to maximize PSII activity at low pH and CO2 concentration. Copyright © 2015, American Society of Plant Biologists.

In our previous research, we showed that low Cd concentration increases the effectiveness of the processes leading to activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). This stimulation was dependent on carbonic anhydrase (CA) activity and resulted in protecting Rubisco activity against Cd toxicity. The aim of the present paper was to test whether this mechanism has any influence on light activation of photosynthesis during the first 2 h of illumination. Both the “activation mechanism” of plant response to Cd-stress conditions and its full efficiency at low Cd concentration were confirmed. The CA-dependent light activation of Rubisco at low Cd level was correlated with accelerated attaining of the maximum Rubisco activity by these plants. The amount of Rubisco was also Cd- and timedependent and varied from continuous accumulation in control plants till reaching the maximum level within 30 minutes for the high Cd concentration. An increase in CA activity that was found to be parallel to the decrease of the amount of CA suggested activation of the enzyme by low Cd concentration

Significance Photosynthesis by cyanobacteria, algae, and plants sustains life on Earth by oxidizing water to the O 2 we breathe and by converting CO 2 into biomass we eat, burn, or use otherwise. Although O 2 production and CO 2 reduction are functionally and structurally well separated in photosynthetic organisms, there is a long debated role of CO 2 / in water oxidation. Here we demonstrate that acts as mobile acceptor and transporter of protons produced by photosystem II, and that depletion of leads to a reversible down-regulation of O 2 production. These findings add a previously unidentified component to the regulatory networks in higher plants, algae, and cyanobacteria and conclude the long quest for the function of CO 2 / in photosynthetic water oxidation.

Over 40 years ago, Joliot et al. (Photochem Photobiol 10:309-329, 1969) designed and employed an elegant and highly sensitive electrochemical technique capable of measuring O2 evolved by photosystem II (PSII) in response to trains of single turn-over light flashes. The measurement and analysis of flash-induced oxygen evolution patterns (FIOPs) has since proven to be a powerful method for probing the turnover efficiency of PSII. Stemler et al. (Proc Natl Acad Sci USA 71(12):4679-4683, 1974), in Govindjee's lab, were the first to study the effect of "bicarbonate" on FIOPs by adding the competitive inhibitor acetate. Here, we extend this earlier work by performing FIOPs experiments at various, strictly controlled inorganic carbon (Ci) levels without addition of any inhibitors. For this, we placed a Joliot-type bare platinum electrode inside a N2-filled glove-box (containing 10-20 ppm CO2) and reduced the Ci concentration simply by washing the samples in Ci-depleted media. FIOPs of spinach thylakoids were recorded either at 20-times reduced levels of Ci or at ambient Ci conditions (390 ppm CO2). Numerical analysis of the FIOPs within an extended Kok model reveals that under Ci-depleted conditions the miss probability is discernibly larger (by 2-3 %) than at ambient conditions, and that the addition of 5 mM HCO3 (-) to the Ci-depleted thylakoids largely restores the original miss parameter. Since a "mild" Ci-depletion procedure was employed, we discuss our data with respect to a possible function of free or weakly bound HCO3 (-) at the water-splitting side of PSII.

Supporting Material and Methods. (DOCX)

Accumulation of GS-CFP-2A in vacuole like structures when co-expressed with ARF1 (T31N). Bright field (A) and confocal (B) images of Arabidopsis protoplast transiently expressing GS-CFP-2A-ARF1 (T31N). In addition to the phenotype shown in Figure 5G–H, some protoplasts showed CFP fluorescence in vacuole like structures as shown here. Bars = 5 µm. (TIF)