Biology subjects

Wollman, F.-A.

Publications and source records attributed to Wollman, F.-A..

11 recordsLinked to original sources

bioRxiv2025

Searching α-solenoid proteins involved in organellar gene expression

In photosynthetic eukaryotes of the green lineage, the expression of the chloroplast genome is mainly regulated post-transcriptionally, by RNA-binding proteins encoded in the nuclear genome (OTAF for Organelle Trans-Acting Factor). Most of those identified to date belong to two families of -solenoid proteins - the pentatrico-peptide repeat (PPR) and octatrico-peptide repeat (OPR) families - and interact with specific sequences on their target mRNAs through a domain composed of repeated motifs, allowing their maturation, splicing, editing, stabilization and translation activation. To identify new OTAFs, we developed three approaches for annotating - solenoid proteins targeted to the chloroplast or the mitochondria. One to identify distant homologs of existing OTAF families, and two others (decision tree and random forest classifiers) to identify new OTAF families. The combined approaches efficiently retrieve previously annotated OTAFs in 2 model organisms. It identified 1067 OPR proteins and 4983 PPR proteins in 43 proteomes of Archaeplastida. Our analysis also identified putative proteins composed of both OPR and PPR domains. Finally, our results identified 3300 other -solenoid candidates which are likely to participate as new regulators of organelle gene expression. In particular, we identified new candidates in species in which the regulatory mechanisms of plastid gene expression are still understudied, such as in the glaucophyte Cyanophora paradoxa and the red alga Porphyridium purpureum. We thus provide valuable new tools to decipher the repertoire of OTAF, as well as new candidates for experimental characterization in the entire eukaryotic tree of life.

evolutionary biology↗

bioRxiv2024

Complexome profiling of the Chlamydomonas psb28 mutant reveals THYLAKOID ENRICHED FRACTION 5 as an early photosystem II assembly factor

Several auxiliary factors are required for the assembly of photosystem (PS) II, one of which is Psb28. While the absence of Psb28 in cyanobacteria has little effect on PSII assembly, we show here that the Chlamydomonas psb28-null mutant is severely impaired in PSII assembly, showing drastically reduced PSII supercomplexes, dimers and monomers, while overaccumulating RCII, CP43mod and D1mod. The mutant had less PSI and more Cytb6f and showed fewer thylakoid stacks and distorted chloroplast morphology. Complexome profiling of the psb28 mutant revealed that TEF5, the homolog of Arabidopsis PSB33/LIL8, co-migrated particularly with RCII. TEF5 also interacted with PSI. A Chlamydomonas tef5 null mutant is also severely impaired in PSII assembly and overaccumulates RCII and CP43mod. RC47 was not detectable in the light-grown tef5 mutant. Our data suggest a possible role for TEF5 in facilitating the assembly of CP47mod into RCII. Both the psb28 and tef5 mutants exhibited decreased synthesis of CP47 and PsbH, suggesting negative feedback regulation possibly exerted by the accumulation of RCII and/or CP43mod in both mutants. The strong effects of missing auxiliary factors on PSII assembly in Chlamydomonas suggest a more effective protein quality control system in this alga than in land plants and cyanobacteria. One-sentence summaryThe Chlamydomonas psb28 mutant is severely impaired in PSII assembly which via complexome profiling allowed identifying TEF5 as a novel PSII assembly factor that likely facilitates CP47 assembly. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plcell/pages/General-Instructions) is: Michael Schroda (m.schroda@rptu.de).

plant biology↗

bioRxiv2024

The high-light-tolerant desert alga Chlorella ohadii is protected from oxidative stress by a non-photochemical quenching-independent mechanism

Non-photochemical quenching (NPQ) mechanisms are crucial for protecting photosynthesis from photoinhibition in plants, algae, and cyanobacteria, and their modulation is a long-standing goal for improving photosynthesis and crop yields. The current work demonstrates that Chlorella ohadii, a green micro-alga that thrives in the desert under high light intensities which are fatal to many photosynthetic organisms, does not perform nor require NPQ to protect photosynthesis under constant high light. Instead of dissipating excess energy, it minimizes its uptake by eliminating the photosynthetic antenna of photosystem II. In addition it accumulates antioxidants that neutralize harmful reactive oxygen species (ROS) and ramps up cyclic electron flow around PSI. These NPQ-independent responses proved efficient in preventing ROS accumulation and reducing oxidative damage to proteins in high-light-grown cells.

plant biology↗

bioRxiv2022

FtsH Protease Inactivation Allows Accumulation of Aberrant Photosystem II in a Chlamydomonas Rubredoxin Mutant

The assembly of photosystem II (PSII) requires the participation of assembly proteins that facilitate the step-wise association of its protein and pigment components into a functional complex capable of oxidizing water and reducing plastoquinone. We previously identified one such factor, the membrane-bound rubredoxin RBD1, but its precise role remains unknown in part due to the inability of the 2pac mutant strain of Chlamydomonas reinhardtii, which lacks RBD1, to accumulate PSII. Here, we show that decreased PSII accumulation in 2pac is due to increased proteolytic degradation. Inactivating the thylakoid membrane FtsH protease in the 2pac mutant background led to an increase in the abundance of PSII subunits and their integration into higher molecular weight complexes, including PSII dimers, capable of sustaining photoautotrophic growth. Dark- and low light-grown 2pac ftsh1-1 both accumulated a 23-kD fragment of the D1 protein, a marker typically associated with structural changes resulting from photodamage or photoinhibition. We introduced a HIS-tagged version of the PsbH protein into the 2pac ftsh1-1 background to purify and examine PSII. We found no detectable changes with respect to cofactor composition relative to the wild-type, leading to us to propose a model in which RBD1 promotes the proper folding of D1, possibly via delivery or reduction of the non-heme iron during PSII assembly. Our results demonstrate that introduction of the ftsh1-1 mutation into mutants defective in the biogenesis of thylakoid membrane complexes can allow for the accumulation and study of aberrant complexes that would otherwise be degraded due to their high protease sensitivity.

plant biology↗

bioRxiv2021

Retracing the evolution from antimicrobial to targeting peptides

We asked what peptide features govern targeting to the mitochondria versus the chloroplast using antimicrobial peptides as a starting point. This approach was inspired by the endosymbiotic hypothesis that organelle-targeting peptides derive from antimicrobial amphipathic peptides delivered by the host cell, to which organelle progenitors became resistant. To explore the molecular changes required to convert antimicrobial into targeting peptides, we expressed a set of 13 antimicrobial peptides in Chlamydomonas reinhardtii. Peptides were systematically modified to test distinctive features of mitochondrial and chloroplast targeting peptides, and we assessed their targeting potential by following the intracellular localization and maturation of a Venus fluorescent reporter used as cargo protein. Mitochondrial targeting can be achieved by some unmodified antimicrobial peptide sequences. Targeting to both organelles is improved by replacing Lysines with Arginines. Chloroplast targeting is enabled by the presence of flanking unstructured sequences, additional constraints consistent with chloroplast endosymbiosis having occurred in a cell that already contained mitochondria. If indeed targeting peptides evolved from antimicrobial peptides, required modifications imply a temporal evolutionary scenario with an early exchange of cationic residues, and a late acquisition of chloroplast specific motifs.

evolutionary biology↗

bioRxiv2021

Complexome profiling on the lpa2 mutant reveals insights into PSII biogenesis and new PSII associated proteins

We have identified the homolog of LOW PSII ACCUMULATION 2 (LPA2) in Chlamydomonas. A Chlamydomonas lpa2 mutant grew slower in low light and was hypersensitive to high light. PSII maximum quantum efficiency was reduced by 38%. Synthesis and stability of newly made PSII core subunits D1, D2, CP43, and CP47 were not impaired. Complexome profiling revealed that in the mutant CP43 was reduced to [~]23%, D1, D2, and CP47 to [~]30% of wild-type levels, while small PSII core subunits and components of the oxygen evolving complex were reduced at most by factor two. PSII supercomplexes, dimers, and monomers were reduced to 7%, 26%, and 60% of wild-type levels, while RC47 was increased [~]6-fold. Our data indicate that LPA2 acts at a step during PSII assembly without which PSII monomers and especially further assemblies become intrinsically unstable and prone to degradation. Levels of ATP synthase and LHCII were 29% and 27% higher in the mutant than in the wild type, whereas levels of the cytochrome b6f complex were unaltered. While the abundance of PSI core subunits and antennae hardly changed, LHCI antennae were more disconnected in the lpa2 mutant, presumably as an adaptive response to reduce excitation of PSI. The disconnection of LHCA2,9 together with PSAH and PSAG was the prime response, but independent and additional disconnection of LHCA1,3-8 along with PSAK occurred as well. Finally, based on co-migration profiles, we identified three novel putative PSII associated proteins with potential roles in regulating PSII complex dynamics, assembly, and chlorophyll breakdown. One-sentence summaryWe provide evidence that the Chlamydomonas LPA2 homolog acts at a step in PSII biogenesis without which PSII monomers and further assemblies become unstable and prone to degradation.

plant biology↗

bioRxiv2020

The state of oligomerization of Rubisco controls the rate of LSU translation in Chlamydomonas reinhardtii

Ribulose 1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) is a key enzyme for photosynthesis-driven life on Earth. While present in all photosynthetic organisms, its most prominent form is a hetero-oligomer in which a Small Subunit (SSU) stabilizes the core of the enzyme built from Large Subunits (LSU), yielding, after a chaperone-assisted multistep assembly, a LSU8SSU8 hexadecameric holoenzyme. Here we use Chlamydomonas reinhardtii, and a combination of site-directed mutants, to dissect the multistep biogenesis pathway of Rubisco in vivo. We identify assembly intermediates, in two of which LSU is associated with the RAF1 chaperone. Using genetic and biochemical approaches we further unravel a major regulation process during Rubisco biogenesis which places translation of its large subunit under the control of its ability to assemble with the small subunit, by a mechanism of Control by Epistasy of Synthesis (CES). Altogether this leads us to propose a model where the last assembly intermediate, an octameric LSU8-RAF1 complex which delivers LSU to SSU to form the Rubisco enzyme, converts to a key regulator form able to exert a negative feed-back on the initiation of translation of LSU, when SSU is not available.

plant biology↗

bioRxiv2020

An antimicrobial origin of targeting peptides to endosymbiotic organelles

Mitochondria and chloroplasts emerged from primary endosymbiosis. Most proteins of the endosymbiont were subsequently expressed in the nucleo-cytosol of the host and organelle-targeted via the acquisition of N-terminal presequences, whose evolutionary origin remains enigmatic. Using a quantitative assessment of their physico-chemical properties, we show that organelle targeting peptides, which are distinct from signal peptides targeting other subcellular compartments, group with a subset of antimicrobial peptides. We demonstrate that extant antimicrobial peptides target a fluorescent reporter to either the mitochondria or the chloroplast in the green alga Chlamydomonas reinhardtii and, conversely, that extant targeting peptides still display antimicrobial activity. Thus, we provide strong computational and functional evidence for an evolutionary link between organelle-targeting and antimicrobial peptides. Our results support the view that resistance of bacterial progenitors of organelles to the attack of host antimicrobial peptides has been instrumental in eukaryogenesis and in emergence of photosynthetic eukaryotes.

evolutionary biology↗

bioRxiv2019

Molecular characterization of Chlamydomonas reinhardtii telomeres and telomerase mutants

Telomeres are repeated sequences found at the end of the linear chromosomes of most eukaryotes and are required for chromosome integrity. They shorten with each cell division because of the end-replication problem. Expression of the reverse transcriptase telomerase allows for extension of telomeric repeats to counteract telomere shortening. Although Chlamydomonas reinhardtii, a photosynthetic unicellular green alga, is widely used as a model organism in photosynthesis and flagella research, and for biotechnological applications, the biology of its telomeres has not been investigated in depth. Here, we show that the C. reinhardtii (TTTTAGGG)n telomeric repeats are mostly non-degenerate and that the telomeres form a protective structure, ending with a 3' overhang. While telomere size and length distributions are stable under various standard growth conditions, they vary substantially between 12 genetically close reference strains. Finally, we identify CrTERT, the gene encoding the catalytic subunit of telomerase and show that mutants of this gene display an \"ever shortening telomere\" phenotype and eventually enter replicative senescence, demonstrating that telomerase is required for long-term maintenance of telomeres in C. reinhardtii.

plant biology↗

bioRxiv2017

Cyclic electron flow in Chlamydomonas reinhardtii

Cyclic electron flow (CEF), one of the major alternative electron transport pathways to the primary linear electron flow (LEF) in chloroplasts has been discovered in the middle of the last century. It is defined as a return of the reductants from the acceptor side of the Photosystem I (PSI) to the pool of its donors via the cytochrome b6f, and has proven essential for photosynthesis. However, despite many efforts aimed at its characterisation, the pathway and regulation of CEF remain equivocal, and its physiological significance remains to be properly defined. Here we use novel spectroscopic approaches to measure CEF in transitory conditions in the green alga Chlamydomonas reinhardtii. We show that CEF operates at the same maximal rate regardless of the oxygen concentration, and that the latter influences LEF, rather than CEF in vivo, which questions the recent hypotheses about the CEF supercomplex formation. We further reveal that the pathways proposed for CEF in the literature are inconsistent with the kinetic information provided by our measurements. We finally provide cues on the regulation of CEF by light.

plant biology↗

bioRxiv2017

Photosynthesis is heavily chlororespiration-sensitive under fluctuating light conditions

Photosynthesis needs to adjust to dynamically changing light intensities in order to maximize its efficiency, notably by the employment of alternative electron pathways. One of them is chlororespiration - initially described in Chlamydomonas reinhardtii. This electron transfer pathway, found in all photosynthetic lineages, consists of a reduction of plastoquinone (PQ) through an NAD(P)H:PQ oxidoreductase and quinol (PQH2) oxidation by Plastid Terminal Oxidase, PTOX. Hence, chlororespiration constitutes an electron pathway potentially antagonistic to the linear photosynthetic electron flow from H2O to CO2. However, the limited flow chlororespiratory enzymes can sustain suggests that their relative contribution, at least in the light and in steady-state conditions, is insubstantial. Here, we focused on the involvement of PTOX in Chlamydomonas reinhardtii during transitions from dark to light and vice versa. We show that the kinetics of redox relaxation of the chloroplast in the dark was greatly affected when PTOX2, the major plastoquinol oxidase in Chlamydomonas, is lacking. Importantly, we show that this has a direct physiological relevance, as the growth of a PTOX2-lacking mutant is markedly slower in intermittent light. The latter can be rationalized in terms of a decreased flux sustained by photosystem II due to a redox limitation at the acceptor side of the PSI during the illumination periods. We finally show that the long-term regulation of cyclic electron flow around PSI is strongly affected in the PTOX2 mutant, substantiating an important role of chlororespiration in the maintenance of chloroplast redox balance.

plant biology↗