Characterization of the Key Step for Light-driven Hydrogen Evolution in Green Algae

Under anaerobic conditions, several species of green algae perform a light-dependent hydrogen production catalyzed by a special group of [FeFe] hydrogenases termed HydA. Although highly interesting for biotechnological applications, the direct connection between photosynthetic electron transport and hydrogenase activity is still a matter of speculation. By establishing an in vitro reconstitution system, we demonstrate that the photosynthetic ferredoxin (PetF) is essential for efficient electron transfer between photosystem I and HydA1. To investigate the electrostatic interaction process and electron transfer between PetF and HydA1, we performed site-directed mutagenesis. Kinetic analyses with several site-directed mutagenesis variants of HydA1 and PetF enabled us to localize the respective contact sites. These experiments in combination with in silico docking analyses indicate that electrostatic interactions between the conserved HydA1 residue Lys³⁹⁶ and the C terminus of PetF as well as between the PetF residue Glu¹²² and the N-terminal amino group of HydA1 play a major role in complex formation and electron transfer. Mapping of relevant HydA1 and PetF residues constitutes an important basis for manipulating the physiological photosynthetic electron flow in favor of light-driven H₂ production.     

Novel insights into the function of LHCSR3 in Chlamydomonas reinhardtii

Light is essential for photosynthesis but excess light is hazardous as it may lead to the formation of reactive oxygen species. Photosynthetic organisms struggle to optimize light utilization and photosynthesis while minimizing photo-oxidative damage. Hereby light to heat dissipation via specialized proteins is a potent mechanism to acclimate toward excess light. In the green alga Chlamydomonas reinhardtii the expression of an ancient light-harvesting protein LHCSR3 enables cells to dissipate harmful excess energy. Herein we summarize newest insights into the function of LHCSR3 from C. reinhardtii.     

The possible role of extra magnesium and nitrogen supply to alleviate stress caused by high irradiation and temperature in lemon trees

Plant and Soil - The aims of this study were to (i) assess how the P concentration in leaves of common Australian native plant species varies across a broad geographic region that includes...     

Control of Hydrogen Photoproduction by the Proton Gradient Generated by Cyclic Electron Flow in Chlamydomonas reinhardtii

Hydrogen photoproduction by eukaryotic microalgae results from a connection between the photosynthetic electron transport chain and a plastidial hydrogenase. Algal H₂ production is a transitory phenomenon under most natural conditions, often viewed as a safety valve protecting the photosynthetic electron transport chain from overreduction. From the colony screening of an insertion mutant library of the unicellular green alga Chlamydomonas reinhardtii based on the analysis of dark-light chlorophyll fluorescence transients, we isolated a mutant impaired in cyclic electron flow around photosystem I (CEF) due to a defect in the Proton Gradient Regulation Like1 (PGRL1) protein. Under aerobiosis, nonphotochemical quenching of fluorescence (NPQ) is strongly decreased in pgrl1. Under anaerobiosis, H₂ photoproduction is strongly enhanced in the pgrl1 mutant, both during short-term and long-term measurements (in conditions of sulfur deprivation). Based on the light dependence of NPQ and hydrogen production, as well as on the enhanced hydrogen production observed in the wild-type strain in the presence of the uncoupling agent carbonyl cyanide p-trifluoromethoxyphenylhydrazone, we conclude that the proton gradient generated by CEF provokes a strong inhibition of electron supply to the hydrogenase in the wild-type strain, which is released in the pgrl1 mutant. Regulation of the trans-thylakoidal proton gradient by monitoring pgrl1 expression opens new perspectives toward reprogramming the cellular metabolism of microalgae for enhanced H₂ production.     

PGRL1 Participates in Iron-induced Remodeling of the Photosynthetic Apparatus and in Energy Metabolism in Chlamydomonas reinhardtii

PGRL1 RNA and protein levels are increased in iron-deficient Chlamydomonas reinhardtii cells. In an RNAi strain, which accumulates lower PGRL1 levels in both iron-replete and -starved conditions, the photosynthetic electron transfer rate is decreased, respiratory capacity in iron-sufficient conditions is increased, and the efficiency of cyclic electron transfer under iron-deprivation is diminished. Pgrl1-kd cells exhibit iron deficiency symptoms at higher iron concentrations than wild-type cells, although the cells are not more depleted in cellular iron relative to wild-type cells as measured by mass spectrometry. Thiol-trapping experiments indicate iron-dependent and redox-induced conformational changes in PGRL1 that may provide a link between iron metabolism and the partitioning of photosynthetic electron transfer between linear and cyclic flow. We propose, therefore, that PGRL1 in C. reinhardtii may possess a dual function in the chloroplast; that is, iron sensing and modulation of electron transfer.     

Biochemical and Structural Studies of the Large Ycf4-Photosystem I Assembly Complex of the Green Alga Chlamydomonas reinhardtii

Ycf4 is a thylakoid protein essential for the accumulation of photosystem I (PSI) in Chlamydomonas reinhardtii. Here, a tandem affinity purification tagged Ycf4 was used to purify a stable Ycf4-containing complex of >1500 kD. This complex also contained the opsin-related COP2 and the PSI subunits PsaA, PsaB, PsaC, PsaD, PsaE, and PsaF, as identified by mass spectrometry (liquid chromatography–tandem mass spectrometry) and immunoblotting. Almost all Ycf4 and COP2 in wild-type cells copurified by sucrose gradient ultracentrifugation and subsequent ion exchange column chromatography, indicating the intimate and exclusive association of Ycf4 and COP2. Electron microscopy revealed that the largest structures in the purified preparation measure 285 × 185 Å; these particles may represent several large oligomeric states. Pulse-chase protein labeling revealed that the PSI polypeptides associated with the Ycf4-containing complex are newly synthesized and partially assembled as a pigment-containing subcomplex. These results indicate that the Ycf4 complex may act as a scaffold for PSI assembly. A decrease in COP2 to 10% of wild-type levels by RNA interference increased the salt sensitivity of the Ycf4 complex stability but did not affect the accumulation of PSI, suggesting that COP2 is not essential for PSI assembly.     

Light‐dependent N‐terminal phosphorylation of LHCSR3 and LHCB4 are interlinked in Chlamydomonas reinhardtii

Phosphorylation dynamics of LHCSR3 were investigated in Chlamydomonas reinhardtii by quantitative proteomics and genetic engineering. LHCSR3 protein expression and phosphorylation were induced in high light. Our data revealed synergistic and dynamic N‐terminal LHCSR3 phosphorylation. Phosphorylated and nonphosphorylated LHCSR3 associated with PSII‐LHCII supercomplexes. The phosphorylation status of LHCB4 was closely linked to the phosphorylation of multiple sites at the N‐terminus of LHCSR3, indicating that LHCSR3 phosphorylation may operate as a molecular switch modulating LHCB4 phosphorylation, which in turn is important for PSII‐LHCII disassembly. Notably, LHCSR3 phosphorylation diminished under prolonged high light, which coincided with onset of CEF. Hierarchical clustering of significantly altered proteins revealed similar expression profiles of LHCSR3, CRX, and FNR. This finding indicated the existence of a functional link between LHCSR3 protein abundance and phosphorylation, photosynthetic electron flow, and the oxidative stress response.     

Functional proteomics of circadian expressed proteins from Chlamydomonas reinhardtii

In this study, functional proteomics was successfully applied for the characterization of circadian expressed, basic proteins. For this purpose, we have chosen the green model alga Chlamydomonas reinhardtii since its entire nuclear genome is available and it is ideally suited for biochemical enrichment procedures. Proteins from cells harvested during subjective day and night were heparin affinity purified. They were separated by two-dimensional gel electrophoresis suited for basic proteins and analyzed after tryptic digestion by electrospray ionization mass spectrometry. We can show for the first time that the expressions of a protein disulfide isomerase-like protein and a tetratricopeptide repeat protein change in a circadian manner. Interestingly, both proteins are known to be interaction partners in multiprotein complexes including RNA binding proteins.     

A retrospective analysis of the clinical case records of 'autistic psychopaths' diagnosed by Hans Asperger and his team at the University Children's Hospital,Vienna

To date, it is questionable whether the diagnostic criteria for Asperger syndrome (AS) as stated by ICD-10 or DSM-IV still reflect Asperger's original account of 'autistic psychopathy' (AP) from the 1940s. The present study examined 74 clinical case records of children with AP diagnosed by Hans Asperger and his team at the Viennese Children's Clinic and Asperger's private practice between 1950 and 1986. The characteristic features of the children are outlined, including reasons for referral, parental background, behavioural problems, cognitive functioning, communication and interests. Results show that the patients of Asperger described in our study represent a subgroup of children with very high intellectual functioning, specific circumscribed interests and talents but impaired social, communication and motor skills. Sixty-eight percent of the sample met ICD-10 criteria for AS, while 25% fulfilled the diagnostic criteria for autism. Implications for the diagnosis of AS are discussed.     

Comparison of the subunit compositions of the PSI-LHCI supercomplex and the LHCI in the green alga Chlamydomonas reinhardtii

Although the light-harvesting chlorophyll protein complex I (LHCI) of photosystem I (PSI) is intimately associated with the PSI core complex and forms the PSI-LHCI supercomplex, the LHCI is normally synthesized in PSI-deficient mutants. In this paper, we compared the subunit compositions of the PSI-LHCI supercomplex and the LHCI by immunoblot analysis and two-dimensional gel electrophoresis combined with mass spectrometry. The PSI-LHCI supercomplex and the LHCI were purified by sucrose density gradient centrifugation and (diethylamino)ethyl column chromatography from n-dodecyl-beta-D-maltoside-solubilized thylakoids of the wild-type and DeltapsaB mutant of the green alga Chlamydomonas reinhardtii. The PSI-LHCI supercomplex contained all of the nine Lhca polypeptides (Lhca1-9) that are detected in wild-type thylakoids. In contrast, the LHCI retained only six Lhca polypeptides, whereas Lhca3 and two minor polypeptides, Lhca2 and Lhca9, were lost during the purification procedure. Sucrose density gradient centrifugation showed that the purified LHCI retains an oligomeric structure with an apparent molecular mass of 300-400 kDa. We therefore concluded that Lhca2, Lhca3, and Lhca9 are not required for the stable oligomeric structure of the LHCI and that the association of these polypeptides in the LHCI is stabilized by the presence of the PSI core complex. Finally, we discuss the possible localization and function of Lhca polypeptides in the LHCI.