Genome Comparisons between Botrytis fabae and the Closely Related Gray Mold Fungus Botrytis cinerea Reveal Possible Explanations for Their Contrasting Host Ranges.

While Botrytis cinerea causes gray mold on many plants, its close relative, Botrytis fabae, is host-specifically infecting predominantly faba bean plants. To explore the basis for its narrow host range, a gapless genome sequence of B. fabae strain G12 (BfabG12) was generated. The BfabG12 genome encompasses 45.0 Mb, with 16 chromosomal telomere-to-telomere contigs that show high synteny and sequence similarity to the corresponding B. cinerea B05.10 (BcB0510) chromosomes. Compared to BcB0510, it is 6% larger, due to many AT-rich regions containing remnants of transposable elements, but encodes fewer genes (11,420 vs. 11,707), due to losses of chromosomal segments with up to 20 genes. The coding capacity of BfabG12 is further reduced by nearly 400 genes that had been inactivated by mutations leading to truncations compared to their BcB0510 orthologues. Several species-specific gene clusters for secondary metabolite biosynthesis with stage-specific expression were identified. Comparison of the proteins secreted during infection revealed high similarities, including 17 phytotoxic proteins that were detected in both species. Our data indicate that evolution of the host-specific B. fabae occurred from an ancestral pathogen with wide host range similar to B. cinerea and was accompanied by losses and degeneration of genes, thereby reducing its pathogenic flexibility.

The impact of light and thioredoxins on the plant thiol-disulfide proteome.

Thiol-based redox regulation is a crucial posttranslational mechanism to acclimate plants to changing light availability. Here, we conducted a biotin switch-based redox proteomics study in Arabidopsis (Arabidopsis thaliana) to systematically investigate dynamics of thiol-redox networks in response to temporal changes in light availability and across genotypes lacking parts of the thioredoxin (Trx) or NADPH-Trx-reductase C (NTRC) systems in the chloroplast. Time-resolved dynamics revealed light led to marked decreases in the oxidation states of many chloroplast proteins with photosynthetic functions during the first 10 min, followed by their partial reoxidation after 2 to 6 h into the photoperiod. This involved f, m, and x-type Trx proteins showing similar light-induced reduction-oxidation dynamics, while NTRC, 2-Cys peroxiredoxins, and Trx y2 showed an opposing pattern, being more oxidized in light than dark. In Arabidopsis trxf1f2, trxm1m2, or ntrc mutants, most proteins showed increased oxidation states in the light compared to wild type, suggesting their light-dependent dynamics were related to NTRC/Trx networks. While NTRC deficiency had a strong influence in all light conditions, deficiencies in f- or m-type Trxs showed differential impacts on the thiol-redox proteome depending on the light environment, being higher in constant or fluctuating light, respectively. The results indicate plant redox proteomes are subject to dynamic changes in reductive and oxidative pathways to cooperatively fine-tune photosynthetic and metabolic processes in the light. The importance of the individual elements of the NTRC/Trx networks mediating these responses depend on the extent of light variability, with NTRC playing a crucial role to balance protein-redox states in rapidly fluctuating light.

Degradation of FATTY ACID EXPORT PROTEIN1 by RHOMBOID-LIKE PROTEASE11 contributes to cold tolerance in Arabidopsis.

Plants need to acclimate to different stresses to optimize growth under unfavorable conditions. In Arabidopsis (Arabidopsis thaliana), the abundance of the chloroplast envelope protein FATTY ACID EXPORT PROTEIN1 (FAX1) decreases after the onset of low temperatures. However, how FAX1 degradation occurs and whether altered FAX1 abundance contributes to cold tolerance in plants remains unclear. The rapid cold-induced increase in RHOMBOID-LIKE PROTEASE11 (RBL11) transcript levels, the physical interaction of RBL11 with FAX1, the specific FAX1 degradation after RBL11 expression, and the absence of cold-induced FAX1 degradation in rbl11 loss-of-function mutants suggest that this enzyme is responsible for FAX1 degradation. Proteomic analyses showed that rbl11 mutants have higher levels of FAX1 and other proteins involved in membrane lipid homeostasis, suggesting that RBL11 is a key element in the remodeling of membrane properties during cold conditions. Consequently, in the cold, rbl11 mutants show a shift in lipid biosynthesis toward the eukaryotic pathway, which coincides with impaired cold tolerance. To test whether cold sensitivity is due to increased FAX1 levels, we analyzed FAX1 overexpressors. The rbl11 mutants and FAX1 overexpressor lines show superimposable phenotypic defects upon exposure to cold temperatures. Our re-sults show that the cold-induced degradation of FAX1 by RBL11 is critical for Arabidop-sis to survive cold and freezing periods.

Competition co-immunoprecipitation reveals the interactors of the chloroplast CPN60 chaperonin machinery.

The functionality of all metabolic processes in chloroplasts depends on a balanced integration of nuclear- and chloroplast-encoded polypeptides into the plastid's proteome. The chloroplast chaperonin machinery is an essential player in chloroplast protein folding under ambient and stressful conditions, with a more intricate structure and subunit composition compared to the orthologous GroEL/ES chaperonin of Escherichia coli. However, its exact role in chloroplasts remains obscure, mainly because of very limited knowledge about the interactors. We employed the competition immunoprecipitation method for the identification of the chaperonin's interactors in Chlamydomonas reinhardtii. Co-immunoprecipitation of the target complex in the presence of increasing amounts of isotope-labelled competitor epitope and subsequent mass spectrometry analysis specifically allowed to distinguish true interactors from unspecifically co-precipitated proteins. Besides known substrates such as RbcL and the expected complex partners, we revealed numerous new interactors with high confidence. Proteins that qualify as putative substrate proteins differ from bulk chloroplast proteins by a higher content of beta-sheets, lower alpha-helical conformation and increased aggregation propensity. Immunoprecipitations targeted against a subunit of the co-chaperonin lid revealed the ClpP protease as a specific partner complex, pointing to a close collaboration of these machineries to maintain protein homeostasis in the chloroplast.

Proteomics and constraint-based modelling reveal enzyme kinetic properties of Chlamydomonas reinhardtii on a genome scale.

Metabolic engineering of microalgae offers a promising solution for sustainable biofuel production, and rational design of engineering strategies can be improved by employing metabolic models that integrate enzyme turnover numbers. However, the coverage of turnover numbers for Chlamydomonas reinhardtii, a model eukaryotic microalga accessible to metabolic engineering, is 17-fold smaller compared to the heterotrophic cell factory Saccharomyces cerevisiae. Here we generate quantitative protein abundance data of Chlamydomonas covering 2337 to 3708 proteins in various growth conditions to estimate in vivo maximum apparent turnover numbers. Using constrained-based modeling we provide proxies for in vivo turnover numbers of 568 reactions, representing a 10-fold increase over the in vitro data for Chlamydomonas. Integration of the in vivo estimates instead of in vitro values in a metabolic model of Chlamydomonas improved the accuracy of enzyme usage predictions. Our results help in extending the knowledge on uncharacterized enzymes and improve biotechnological applications of Chlamydomonas.

TurboID reveals the proxiomes of Chlamydomonas proteins involved in thylakoid biogenesis and stress response.

In Chlamydomonas (Chlamydomonas reinhardtii), the VESICLE-INDUCING PROTEIN IN PLASTIDS 1 and 2 (VIPP1 and VIPP2) play roles in the sensing and coping with membrane stress and in thylakoid membrane biogenesis. To gain more insight into these processes, we aimed to identify proteins interacting with VIPP1/2 in the chloroplast and chose proximity labeling (PL) for this purpose. We used the transient interaction between the nucleotide exchange factor CHLOROPLAST GRPE HOMOLOG 1 (CGE1) and the stromal HEAT SHOCK PROTEIN 70B (HSP70B) as test system. While PL with APEX2 and BioID proved to be inefficient, TurboID resulted in substantial biotinylation in vivo. TurboID-mediated PL with VIPP1/2 as baits under ambient and H2O2 stress conditions confirmed known interactions of VIPP1 with VIPP2, HSP70B, and the CHLOROPLAST DNAJ HOMOLOG 2 (CDJ2). Proteins identified in the VIPP1/2 proxiomes can be grouped into proteins involved in the biogenesis of thylakoid membrane complexes and the regulation of photosynthetic electron transport, including PROTON GRADIENT REGULATION 5-LIKE 1 (PGRL1). A third group comprises 11 proteins of unknown function whose genes are upregulated under chloroplast stress conditions. We named them VIPP PROXIMITY LABELING (VPL). In reciprocal experiments, we confirmed VIPP1 in the proxiomes of VPL2 and PGRL1. Our results demonstrate the robustness of TurboID-mediated PL for studying protein interaction networks in the chloroplast of Chlamydomonas and pave the way for analyzing functions of VIPPs in thylakoid biogenesis and stress responses.

One-helix protein 2 is not required for the synthesis of photosystem II subunit D1 in Chlamydomonas.

In land plants and cyanobacteria, co-translational association of chlorophyll (Chl) to the nascent D1 polypeptide, a reaction center protein of photosystem II (PSII), requires a Chl binding complex consisting of a short-chain dehydrogenase (high chlorophyll fluorescence 244 [HCF244]/uncharacterized protein 39 [Ycf39]) and one-helix proteins (OHP1 and OHP2 in chloroplasts) of the light-harvesting antenna complex superfamily. Here, we show that an ohp2 mutant of the green alga Chlamydomonas (Chlamydomonas reinhardtii) fails to accumulate core PSII subunits, in particular D1 (encoded by the psbA mRNA). Extragenic suppressors arose at high frequency, suggesting the existence of another route for Chl association to PSII. The ohp2 mutant was complemented by the Arabidopsis (Arabidopsis thaliana) ortholog. In contrast to land plants, where psbA translation is prevented in the absence of OHP2, ribosome profiling experiments showed that the Chlamydomonas mutant translates the psbA transcript over its full length. Pulse labeling suggested that D1 is degraded during or immediately after translation. The translation of other PSII subunits was affected by assembly-controlled translational regulation. Proteomics showed that HCF244, a translation factor which associates with and is stabilized by OHP2 in land plants, still partly accumulates in the Chlamydomonas ohp2 mutant, explaining the persistence of psbA translation. Several Chl biosynthesis enzymes overaccumulate in the mutant membranes. Partial inactivation of a D1-degrading protease restored a low level of PSII activity in an ohp2 background, but not photoautotrophy. Taken together, our data suggest that OHP2 is not required for psbA translation in Chlamydomonas, but is necessary for D1 stabilization.

Botrytis hypersensitive response inducing protein 1 triggers noncanonical PTI to induce plant cell death.

According to their lifestyle, plant pathogens are divided into biotrophic and necrotrophic organisms. Biotrophic pathogens exclusively nourish living host cells, whereas necrotrophic pathogens rapidly kill host cells and nourish cell walls and cell contents. To this end, the necrotrophic fungus Botrytis cinerea secretes large amounts of phytotoxic proteins and cell wall-degrading enzymes. However, the precise role of these proteins during infection is unknown. Here, we report on the identification and characterization of the previously unknown toxic protein hypersensitive response-inducing protein 1 (Hip1), which induces plant cell death. We found the adoption of a structurally conserved folded Alternaria alternata Alt a 1 protein structure to be a prerequisite for Hip1 to exert its necrosis-inducing activity in a host-specific manner. Localization and the induction of typical plant defense responses by Hip1 indicate recognition as a pathogen-associated molecular pattern at the plant plasma membrane. In contrast to other secreted toxic Botrytis proteins, the activity of Hip1 does not depend on the presence of the receptor-associated kinases BRI1-associated kinase 1 and suppressor of BIR1-1. Our results demonstrate that recognition of Hip1, even in the absence of obvious enzymatic or pore-forming activity, induces strong plant defense reactions eventually leading to plant cell death. Botrytis hip1 overexpression strains generated by CRISPR/Cas9 displayed enhanced infection, indicating the virulence-promoting potential of Hip1. Taken together, Hip1 induces a noncanonical defense response which might be a common feature of structurally conserved fungal proteins from the Alt a 1 family.

A Cyanophage MarR-Type Transcription Factor Regulates Host RNase E Expression during Infection.

The marine picocyanobacterium Prochlorococcus contributes significantly to global primary production, and its abundance and diversity is shaped in part by viral infection. Here, we identified a cyanophage-encoded MarR-type transcription factor that induces the gene expression of host Prochlorococcus MED4 endoribonuclease (RNase) E during phage infection. The increase in rne transcript levels relies on the phage (p)MarR-mediated activation of an alternative promoter that gives rise to a truncated yet enzymatically fully functional RNase E isoform. In this study, we demonstrate that pMarR binds to an atypical activator site downstream of the transcriptional start site and that binding is enhanced in the presence of Ca2+ ions. Furthermore, we show that dimeric pMarR interacts with the α subunit of RNA polymerase, and we identified amino acid residues S66, R67, and G106, which are important for Ca2+ binding, DNA binding, and dimerization of pMarR, respectively.

Fatty acid export (FAX) proteins contribute to oil production in the green microalga Chlamydomonas reinhardtii.

In algae and land plants, transport of fatty acids (FAs) from their site of synthesis in the plastid stroma to the endoplasmic reticulum (ER) for assembly into acyl lipids is crucial for cellular lipid homeostasis, including the biosynthesis of triacylglycerol (TAG) for energy storage. In the unicellular green alga Chlamydomonas reinhardtii, understanding and engineering of these processes is of particular interest for microalga-based biofuel and biomaterial production. Whereas in the model plant Arabidopsis thaliana, FAX (fatty acid export) proteins have been associated with a function in plastid FA-export and hence TAG synthesis in the ER, the knowledge on the function and subcellular localization of this protein family in Chlamydomonas is still scarce. Among the four FAX proteins encoded in the Chlamydomonas genome, we found Cr-FAX1 and Cr-FAX5 to be involved in TAG production by functioning in chloroplast and ER membranes, respectively. By in situ immunolocalization, we show that Cr-FAX1 inserts into the chloroplast envelope, while Cr-FAX5 is located in ER membranes. Severe reduction of Cr-FAX1 or Cr-FAX5 proteins by an artificial microRNA approach results in a strong decrease of the TAG content in the mutant strains. Further, overexpression of chloroplast Cr-FAX1, but not of ER-intrinsic Cr-FAX5, doubled the content of TAG in Chlamydomonas cells. We therefore propose that Cr-FAX1 in chloroplast envelopes and Cr-FAX5 in ER membranes represent a basic set of FAX proteins to ensure shuttling of FAs from chloroplasts to the ER and are crucial for oil production in Chlamydomonas.

Multiple knockout mutants reveal a high redundancy of phytotoxic compounds contributing to necrotrophic pathogenesis of Botrytis cinerea.

Botrytis cinerea is a major plant pathogen infecting more than 1400 plant species. During invasion, the fungus rapidly kills host cells, which is believed to be supported by induction of programmed plant cell death. To comprehensively evaluate the contributions of most of the currently known plant cell death inducing proteins (CDIPs) and metabolites for necrotrophic infection, an optimized CRISPR/Cas9 protocol was established which allowed to perform serial marker-free mutagenesis to generate multiple deletion mutants lacking up to 12 CDIPs. Whole genome sequencing of a 6x and 12x deletion mutant revealed a low number of off-target mutations which were unrelated to Cas9-mediated cleavage. Secretome analyses confirmed the loss of secreted proteins encoded by the deleted genes. Infection tests with the mutants revealed a successive decrease in virulence with increasing numbers of mutated genes, and varying effects of the knockouts on different host plants. Comparative analysis of mutants confirmed significant roles of two polygalacturonases (PG1, PG2) and the phytotoxic metabolites botrydial and botcinins for infection, but revealed no or only weak effects of deletion of the other CDIPs. Nicotiana benthamiana plants with mutated or silenced coreceptors of pattern recognition receptors, SOBIR1 and BAK1, showed similar susceptibility as control plants to infection by B. cinerea wild type and a 12x deletion mutant. These results raise doubts about a major role of manipulation of these plant defence regulators for B. cinerea infection. Despite the loss of most of the known phytotoxic compounds, the on planta secretomes of the multiple mutants retained substantial phytotoxic activity, proving that further, as yet unknown CDIPs contribute to necrosis and virulence. Our study has addressed for the first time systematically the functional redundancy of fungal virulence factors, and demonstrates that B. cinerea releases a highly redundant cocktail of proteins to achieve necrotrophic infection of a wide variety of host plants.

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

While the composition and function of the major thylakoid membrane complexes are well understood, comparatively little is known about their biogenesis. The goal of this work was to shed more light on the role of auxiliary factors in the biogenesis of photosystem II (PSII). Here we have identified the homolog of LOW PSII ACCUMULATION 2 (LPA2) in Chlamydomonas. A Chlamydomonas reinhardtii lpa2 mutant grew slower in low light, was hypersensitive to high light, and exhibited aberrant structures in thylakoid membrane stacks. Chlorophyll fluorescence (Fv/Fm) was reduced by 38%. Synthesis and stability of newly made PSII core subunits D1, D2, CP43, and CP47 were not impaired. However, complexome profiling revealed that in the mutant CP43 was reduced to ~23% and D1, D2, and CP47 to ~30% of wild type levels. Levels of PSI and the cytochrome b6f complex were unchanged, while levels of the ATP synthase were increased by ~29%. PSII supercomplexes, dimers, and monomers were reduced to ~7%, ~26%, and ~60% of wild type levels, while RC47 was increased ~6-fold and LHCII by ~27%. We propose that LPA2 catalyses a step during PSII assembly without which PSII monomers and further assemblies become unstable and prone to degradation. The LHCI antenna was more disconnected from PSI in the lpa2 mutant, presumably as an adaptive response to reduce excitation of PSI. From the co-migration profiles of 1734 membrane-associated proteins, we identified three novel putative PSII associated proteins with potential roles in regulating PSII complex dynamics, assembly, and chlorophyll breakdown.

A longer isoform of Stim1 is a negative SOCE regulator but increases cAMP-modulated NFAT signaling.

Alternative splicing is a potent modifier of protein function. Stromal interaction molecule 1 (Stim1) is the essential activator of store-operated Ca2+ entry (SOCE) triggering activation of transcription factors. Here, we characterize Stim1A, a splice variant with an additional 31 amino acid domain inserted in frame within its cytosolic domain. Prominent expression of exon A is found in astrocytes, heart, kidney, and testes. Full-length Stim1A functions as a dominant-negative regulator of SOCE and ICRAC, facilitating sequence-specific fast calcium-dependent inactivation and destabilizing gating of Orai channels. Downregulation or absence of native Stim1A results in increased SOCE. Despite reducing SOCE, Stim1A leads to increased NFAT translocation. Differential proteomics revealed an interference of Stim1A with the cAMP-SOCE crosstalk by altered modulation of phosphodiesterase 8 (PDE8), resulting in reduced cAMP degradation and increased PIP5K activity, facilitating NFAT activation. Our study uncovers a hitherto unknown mechanism regulating NFAT activation and indicates that cell-type-specific splicing of Stim1 is a potent means to regulate the NFAT signalosome and cAMP-SOCE crosstalk.

In Vivo Structure-Function Analysis and Redox Interactomes of Leishmania tarentolae Erv.

Import and oxidative folding of proteins in the mitochondrial intermembrane space differ among eukaryotic lineages. While opisthokonts such as yeast rely on the receptor and oxidoreductase Mia40 in combination with the Mia40:cytochrome c oxidoreductase Erv, kinetoplastid parasites and other Excavata/Discoba lack Mia40 but have a functional Erv homologue. Whether excavate Erv homologues rely on a Mia40 replacement or directly interact with imported protein substrates remains controversial. Here, we used the CRISPR-Cas9 system to generate a set of tagged and untagged homozygous mutants of LTERV from the kinetoplastid model parasite Leishmania tarentolae. Modifications of the shuttle cysteine motif of LtErv were lethal, whereas replacement of clamp residue Cys17 or removal of the kinetoplastida-specific second (KISS) domain had no impact on parasite viability under standard growth conditions. However, removal of the KISS domain rendered parasites sensitive to heat stress and led to the accumulation of homodimeric and mixed LtErv disulfides. We therefore determined and compared the redox interactomes of tagged wild-type LtErv and LtErvΔKISS using stable isotope labeling by amino acids in cell culture (SILAC) and quantitative mass spectrometry. While the Mia40-replacement candidate Mic20 and all but one typical substrate with twin Cx3/9C-motifs were absent in both redox interactomes, we identified a small set of alternative potential interaction partners with putative redox-active cysteine residues. In summary, our study reveals parasite-specific intracellular structure-function relationships and redox interactomes of LtErv with implications for current hypotheses on mitochondrial protein import in nonopisthokonts. IMPORTANCE The discovery of the redox proteins Mia40/CHCHD4 and Erv1/ALR, as well as the elucidation of their relevance for oxidative protein folding in the mitochondrial intermembrane space of yeast and mammals, founded a new research topic in redox biology and mitochondrial protein import. The lack of Mia40/CHCHD4 in protist lineages raises fundamental and controversial questions regarding the conservation and evolution of this essential pathway. Do protist Erv homologues act alone, or do they use the candidate Mic20 or another protein as a Mia40 replacement? Furthermore, we previously showed that Erv homologues in L. tarentolae and the human pathogen L. infantum are not only essential but also differ structurally and mechanistically from yeast and human Erv1/ALR. Here, we analyzed the relevance of such structural differences in vivo and determined the first redox interactomes of a nonopisthokont Erv homologue. Our data challenge recent hypotheses on mitochondrial protein import in nonopisthokonts.

Parkinson mice show functional and molecular changes in the gut long before motoric disease onset.

BACKGROUND: There is increasing evidence that Parkinson's disease (PD) might start in the gut, thus involving and compromising also the enteric nervous system (ENS). At the clinical onset of the disease the majority of dopaminergic neurons in the midbrain is already destroyed, so that the lack of early biomarkers for the disease represents a major challenge for developing timely treatment interventions. Here, we use a transgenic A30P-α-synuclein-overexpressing PD mouse model to identify appropriate candidate markers in the gut before hallmark symptoms begin to manifest. METHODS: Based on a gait analysis and striatal dopamine levels, we defined 2-month-old A30P mice as pre-symptomatic (psA30P), since they are not showing any motoric impairments of the skeletal neuromuscular system and no reduced dopamine levels, but an intestinal α-synuclein pathology. Mice at this particular age were further used to analyze functional and molecular alterations in both, the gastrointestinal tract and the ENS, to identify early pathological changes. We examined the gastrointestinal motility, the molecular composition of the ENS, as well as the expression of regulating miRNAs. Moreover, we applied A30P-α-synuclein challenges in vitro to simulate PD in the ENS. RESULTS: A retarded gut motility and early molecular dysregulations were found in the myenteric plexus of psA30P mice. We found that i.e. neurofilament light chain, vesicle-associated membrane protein 2 and calbindin 2, together with the miRNAs that regulate them, are significantly altered in the psA30P, thus representing potential biomarkers for early PD. Many of the dysregulated miRNAs found in the psA30P mice are reported to be changed in PD patients as well, either in blood, cerebrospinal fluid or brain tissue. Interestingly, the in vitro approaches delivered similar changes in the ENS cultures as seen in the transgenic animals, thus confirming the data from the mouse model. CONCLUSIONS: These findings provide an interesting and novel approach for the identification of appropriate biomarkers in men.

Vernalization Alters Sink and Source Identities and Reverses Phloem Translocation from Taproots to Shoots in Sugar Beet.

During their first year of growth, overwintering biennial plants transport Suc through the phloem from photosynthetic source tissues to storage tissues. In their second year, they mobilize carbon from these storage tissues to fuel new growth and reproduction. However, both the mechanisms driving this shift and the link to reproductive growth remain unclear. During vegetative growth, biennial sugar beet (Beta vulgaris) maintains a steep Suc concentration gradient between the shoot (source) and the taproot (sink). To shift from vegetative to generative growth, they require a chilling phase known as vernalization. We studied sugar beet sink-source dynamics upon vernalization and showed that before flowering, the taproot underwent a reversal from a sink to a source of carbohydrates. This transition was induced by transcriptomic and functional reprogramming of sugar beet tissue, resulting in a reversal of flux direction in the phloem. In this transition, the vacuolar Suc importers and exporters TONOPLAST SUGAR TRANSPORTER2;1 and SUCROSE TRANSPORTER4 were oppositely regulated, leading to the mobilization of sugars from taproot storage vacuoles. Concomitant changes in the expression of floral regulator genes suggest that these processes are a prerequisite for bolting. Our data will help both to dissect the metabolic and developmental triggers for bolting and to identify potential targets for genome editing and breeding.

Overexpression of Sedoheptulose-1,7-Bisphosphatase Enhances Photosynthesis in Chlamydomonas reinhardtii and Has No Effect on the Abundance of Other Calvin-Benson Cycle Enzymes.

The productivity of plants and microalgae needs to be increased to feed the growing world population and to promote the development of a low-carbon economy. This goal can be achieved by improving photosynthesis via genetic engineering. In this study, we have employed the Modular Cloning strategy to overexpress the Calvin-Benson cycle (CBC) enzyme sedoheptulose-1,7-bisphosphatase (SBP1) up to threefold in the unicellular green alga Chlamydomonas reinhardtii. The protein derived from the nuclear transgene represented ∼0.3% of total cell protein. Photosynthetic rate and growth were significantly increased in SBP1-overexpressing lines under high-light and elevated CO2 conditions. Absolute quantification of the abundance of all other CBC enzymes by the QconCAT approach revealed no consistent differences between SBP1-overexpressing lines and the recipient strain. This analysis also revealed that the 11 CBC enzymes represent 11.9% of total cell protein in Chlamydomonas. Here, the range of concentrations of CBC enzymes turned out to be much larger than estimated earlier, with a 128-fold difference between the most abundant CBC protein (rbcL) and the least abundant (triose phosphate isomerase). Accordingly, the concentrations of the CBC intermediates are often but not always higher than the binding site concentrations of the enzymes for which they act as substrates. The enzymes with highest substrate to binding site ratios might represent good candidates for overexpression in subsequent engineering steps.

VIPP2 interacts with VIPP1 and HSP22E/F at chloroplast membranes and modulates a retrograde signal for HSP22E/F gene expression.

VIPP proteins aid thylakoid biogenesis and membrane maintenance in cyanobacteria, algae, and plants. Some members of the Chlorophyceae contain two VIPP paralogs termed VIPP1 and VIPP2, which originate from an early gene duplication event during the evolution of green algae. VIPP2 is barely expressed under nonstress conditions but accumulates in cells exposed to high light intensities or H2 O2 , during recovery from heat stress, and in mutants with defective integration (alb3.1) or translocation (secA) of thylakoid membrane proteins. Recombinant VIPP2 forms rod-like structures in vitro and shows a strong affinity for phosphatidylinositol phosphate. Under stress conditions, >70% of VIPP2 is present in membrane fractions and localizes to chloroplast membranes. A vipp2 knock-out mutant displays no growth phenotypes and no defects in the biogenesis or repair of photosystem II. However, after exposure to high light intensities, the vipp2 mutant accumulates less HSP22E/F and more LHCSR3 protein and transcript. This suggests that VIPP2 modulates a retrograde signal for the expression of nuclear genes HSP22E/F and LHCSR3. Immunoprecipitation of VIPP2 from solubilized cells and membrane-enriched fractions revealed major interactions with VIPP1 and minor interactions with HSP22E/F. Our data support a distinct role of VIPP2 in sensing and coping with chloroplast membrane stress.

Identification of Chloroplast Envelope Proteins with Critical Importance for Cold Acclimation.

The ability of plants to withstand cold temperatures relies on their photosynthetic activity. Thus, the chloroplast is of utmost importance for cold acclimation and acquisition of freezing tolerance. During cold acclimation, the properties of the chloroplast change markedly. To provide the most comprehensive view of the protein repertoire of the chloroplast envelope, we analyzed this membrane system in Arabidopsis (Arabidopsis thaliana) using mass spectrometry-based proteomics. Profiling chloroplast envelope membranes was achieved by a cross comparison of protein intensities across the plastid and the enriched membrane fraction under both normal and cold conditions. We used multivariable logistic regression to model the probabilities for the classification of an envelope localization. In total, we identified 38 envelope membrane intrinsic or associated proteins exhibiting altered abundance after cold acclimation. These proteins comprise several solute carriers, such as the ATP/ADP antiporter nucleotide transporter2 (NTT2; substantially increased abundance) or the maltose exporter MEX1 (substantially decreased abundance). Remarkably, analysis of the frost recovery of ntt loss-of-function and mex1 overexpressor mutants confirmed that the comparative proteome is well suited to identify key factors involved in cold acclimation and acquisition of freezing tolerance. Moreover, for proteins with known physiological function, we propose scenarios explaining their possible roles in cold acclimation. Furthermore, spatial proteomics introduces an additional layer of complexity and enables the identification of proteins differentially localized at the envelope membrane under the changing environmental regime.

The NADH Dehydrogenase Nde1 Executes Cell Death after Integrating Signals from Metabolism and Proteostasis on the Mitochondrial Surface.

The proteolytic turnover of mitochondrial proteins is poorly understood. Here, we used a combination of dynamic isotope labeling and mass spectrometry to gain a global overview of mitochondrial protein turnover in yeast cells. Intriguingly, we found an exceptionally high turnover of the NADH dehydrogenase, Nde1. This homolog of the mammalian apoptosis inducing factor, AIF, forms two distinct topomers in mitochondria, one residing in the intermembrane space while the other spans the outer membrane and is exposed to the cytosol. The surface-exposed topomer triggers cell death in response to pro-apoptotic stimuli. The surface-exposed topomer is degraded by the cytosolic proteasome/Cdc48 system and the mitochondrial protease Yme1; however, it is strongly enriched in respiratory-deficient cells. Our data suggest that in addition to their role in electron transfer, mitochondrial NADH dehydrogenases such as Nde1 or AIF integrate signals from energy metabolism and cytosolic proteostasis to eliminate compromised cells from growing populations.