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In photosynthetic eukaryotes, thousands of proteins are translated in the cytosol and imported into the chloroplast through the concerted action of two translocons-termed TOC and TIC-located in the outer and inner membranes of the chloroplast envelope, respectively. The degree to which the molecular composition of the TOC and TIC complexes is conserved over phylogenetic distances has remained controversial. Here, we combine transcriptomic, biochemical, and genetic tools in the green alga Chlamydomonas (Chlamydomonas reinhardtii) to demonstrate that, despite a lack of evident sequence conservation for some of its components, the algal TIC complex mirrors the molecular composition of a TIC complex from Arabidopsis thaliana. The Chlamydomonas TIC complex contains three nuclear-encoded subunits, Tic20, Tic56, and Tic100, and one chloroplast-encoded subunit, Tic214, and interacts with the TOC complex, as well as with several uncharacterized proteins to form a stable supercomplex (TIC-TOC), indicating that protein import across both envelope membranes is mechanistically coupled. Expression of the nuclear and chloroplast genes encoding both known and uncharacterized TIC-TOC components is highly coordinated, suggesting that a mechanism for regulating its biogenesis across compartmental boundaries must exist. Conditional repression of Tic214, the only chloroplast-encoded subunit in the TIC-TOC complex, impairs the import of chloroplast proteins with essential roles in chloroplast ribosome biogenesis and protein folding and induces a pleiotropic stress response, including several proteins involved in the chloroplast unfolded protein response. These findings underscore the functional importance of the TIC-TOC supercomplex in maintaining chloroplast proteostasis.

In the green alga Chlamydomonas reinhardtii, hydrogen production is catalyzed via the [FeFe]-hydrogenases HydA1 and HydA2. The electrons required for the catalysis are transferred from ferredoxin (FDX) towards the hydrogenases. In the light, ferredoxin receives its electrons from photosystem I (PSI) so that H2 production becomes a fully light-driven process. HydA1 and HydA2 are highly O2 sensitive; consequently, the formation of H2 occurs mainly under anoxic conditions. Yet, photo-H2 production is tightly coupled to the efficiency of photosynthetic electron transport and linked to the photosynthetic control via the Cyt b6f complex, the control of electron transfer at the level of photosystem II (PSII) and the structural remodeling of photosystem I (PSI). These processes also determine the efficiency of linear (LEF) and cyclic electron flow (CEF). The latter is competitive with H2 photoproduction. Additionally, the CBB cycle competes with H2 photoproduction. Consequently, an in-depth understanding of light-driven H2 production via photosynthetic electron transfer and its competition with CO2 fixation is essential for improving photo-H2 production. At the same time, the smart design of photo-H2 production schemes and photo-H2 bioreactors are challenges for efficient up-scaling of light-driven photo-H2 production. [ABSTRACT FROM AUTHOR]

The photosystem I (PSI) of the green alga Chlamydomonas reinhardtii associates with 10 light-harvesting proteins (LHCIs) to form the PSI-LHCI complex. In the context of state transitions, two LHCII trimers bind to the PSAL, PSAH and PSAO side of PSI to produce the PSI-LHCI-LHCII complex. In this work, we took advantage of chemical crosslinking of proteins in conjunction with mass spectrometry to identify protein–protein interactions between the light-harvesting proteins of PSI and PSII. We detected crosslinks suggesting the binding of LHCBM proteins to the LHCA1-PSAG side of PSI as well as protein–protein interactions of LHCSR3 with LHCA5 and LHCA3. Our data indicate that the binding of LHCII to PSI is more versatile than anticipated and imply that LHCSR3 might be involved in the regulation of excitation energy transfer to the PSI core via LHCA5/LHCA3. [ABSTRACT FROM AUTHOR]

Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the 120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella.

In this work, a modular data processing workflow for single-particle (sp) and single-cell (sc) inductively coupled plasma-mass spectrometry (ICP-MS) is presented. To achieve more reliable detection thresholds, a special focus is placed on the parameter estimation of Gaussian and Poisson distributions that describe the background (BG) signal. For Gaussian models, the widely used iterative outlier test was improved by an algorithm that adjusts the test for different BG levels by incrementing the test factor. Through careful evaluation, the standard deviation of the experimental sc- and sp-ICP-MS data was applied as a robust measure of the convergence quality of the test. In addition, the outlier analysis was separated from the subsequent event detection more strictly than it is often reported. Importantly, a data-dependent decision criterion based on Gaussian and Poisson modeling was developed to effectively address extra-Poisson variance in experimental data. In the second part, a gate filter was developed to reduce the amount of excess false-positive events in sc-ICP-MS. To that end, a secondary filter based on the signal peak height is used to remove rare false-positive events without affecting the signal intensity of the events that are detected correctly. Two approaches, based on a numerical approximation via the detection limit, and critical values of the Gaussian and Poisson distribution are presented to calculate the gate filter level. Possible sources of false-positive events, some of which are specific to sc-ICP-MS, are discussed. The combined processing workflow was applied to analyze the distribution of six endogenous elements in Chlamydomonas reinhardtii cell populations. The gate filter corrected the cell number concentration by up to 44% (22% on average), and mass per cell by up to 30% (17% on average). [ABSTRACT FROM AUTHOR]

Rovibrationally resolved spectra of the Nj=22, Ka=0←1 transition and of the Nj=23, Ka=0←0 and Ka=1←0 transitions of the hydrogen‐bonded (HF)2 have been measured in the near infrared range near 1.3 μm by cw‐diode laser cavity ring‐down spectroscopy in a pulsed supersonic slit jet expansion. The spectroscopic assignment and analysis provided an insight into the dynamics of these highly‐excited vibrational states, in particular concerning the predissociation of the hydrogen bond and the tunneling process of the hydrogen bond switching. Together with our previously analyzed spectra of the Nj=21 and Nj=22 components, the mode‐specific dynamics in all three components of this triad can now be compared. In the N=2 triad, the HF‐stretching vibration is excited by two quanta with similar excitation energy, but the quanta are distributed in three different ways, which has a distinct influence on the dynamics. The observed band centers and tunneling splittings are in agreement with our recent calculations on the (HF)2 potential energy hypersurface SO‐3, resolving the long‐standing discussion about the symmetry ordering of polyad levels in this overtone region. The results are also discussed in relation to the general questions of non‐statistical reaction dynamics of polyatomic molecules and clusters and in relation to quasi‐adiabatic channel above barrier tunneling. [ABSTRACT FROM AUTHOR]