Fungal unspecific peroxygenases (UPOs) are biocatalysts of outstanding interest. Providing access to novel UPOs using a modular secretion system was the central goal of this work. UPOs represent an enzyme class, catalysing versatile oxyfunctionalisation reactions on a broad substrate scope. They are occurring as secreted, glycosylated proteins bearing a haem-thiolate active site and solely rely on hydrogen peroxide as the oxygen source. Fungal peroxygenases are widespread throughout the fungal kingdom and hence a huge variety of UPO gene sequences is available. However, the heterologous production of UPOs in a fast-growing organism suitable for high throughput screening has only succeeded once—enabled by an intensive directed evolution campaign. Here, we developed and applied a modular Golden Gate-based secretion system, allowing the first yeast production of four active UPOs, their one-step purification and application in an enantioselective conversion on a preparative scale. The Golden Gate setup was designed to be broadly applicable and consists of the three module types: i) a signal peptide panel guiding secretion, ii) UPO genes, and iii) protein tags for purification and split-GFP detection. We show that optimal signal peptides could be selected for successful UPO secretion by combinatorial testing of 17 signal peptides for each UPO gene. The modular episomal system is suitable for use in Saccharomyces cerevisiae and was transferred to episomal and chromosomally integrated expression cassettes in Pichia pastoris. Shake flask productions in Pichia pastoris yielded up to 24 mg/L secreted UPO enzyme, which was employed for the preparative scale conversion of a phenethylamine derivative reaching 98.6 % ee. Our results demonstrate a rapid workflow from putative UPO gene to preparative scale enantioselective biotransformations.

Proteome remodeling is a fundamental adaptive response and proteins in complex and functionally related proteins are often co-expressed. Using a deep sampling strategy we define Arabidopsis thaliana tissue core proteomes at around 10,000 proteins per tissue and absolutely quantify (copy numbers per cell) nearly 16,000 proteins throughout the plant lifecycle. A proteome wide survey of global post translational modification revealed amino acid exchanges pointing to potential conservation of translational infidelity in eukaryotes. Correlation analysis of protein abundance uncovered potentially new tissue and age specific roles of entire signaling modules regulating transcription in photosynthesis, seed development and senescence and abscission. Among others, the data suggest a potential function of RD26 and other NAC transcription factors in seed development related to desiccation tolerance as well as a possible function of Cysteine-rich Receptor-like Kinases (CRKs) as ROS sensors in senescence. All of the components of ribosome biogenesis factor (RBF) complexes were co-expressed tissue and age specifically indicating functional promiscuity in the assembly of these little described protein complexes in Arabidopsis. Treatment of seedlings with flg22 for 16 hours allowed us to characterize proteome architecture in basal immunity in detail. The results were complemented with parallel reaction monitoring (PRM) targeted proteomics, phytohormone, amino acid and transcript measurements. We obtained strong evidence of suppression of jasmonate (JA) and JA-Ile levels by deconjugation and hydroxylation via IAA-ALA RESISTANT3 (IAR3) and JASMONATE-INDUCED OXYGENASE 2 (JOX2) under the control of JASMONATE INSENSITIVE 1 (MYC2). This previously unknown regulatory switch is another part of the puzzle of the as yet understudied role of JA in pattern triggered immunity. The extensive coverage of the Arabidopsis proteome in various biological scenarios presents a rich resource to plant biologists that we make available to the community.

The Arabidopsis (Arabidopsis thaliana) calmodulin-binding transcription activator3 (CAMTA3) is a repressor of immunity-related genes but an activator of cold-induced or general stress-responsive genes in plants. Post-transcriptional or posttranslational mechanisms have been proposed to control CAMTA3 functions in different stress responses. Here, we show that treatment with the bacterial flg22 elicitor induces CAMTA3 phosphorylation, which is accompanied by its destabilization and nuclear export. Two flg22-responsive mitogen-activated protein kinases (MAPKs), MPK3 and MPK6, directly phosphorylate CAMTA3, with the phospho-sites contributing to CAMTA3 degradation and suppression of downstream target gene expression. However, the flg22-induced nuclear export and phospho-mobility shift can still be observed for the CAMTA3 phospho-null variant of the MAPK-modified sites, suggesting additional flg22-responsive kinases might be involved. Taken together, we propose that flg22-induced CAMTA3 depletion facilitates de-repression of downstream defense target genes, which involves phosphorylation, increased protein turnover, and nucleo-cytoplasmic trafficking.

Mitogen‐activated protein kinase (MAPK) cascades are key signalling modules of plant defence responses to pathogen‐associated molecular patterns (PAMPs, e.g. bacterial flg22 peptide). The Tandem Zinc Finger Protein 9 (TZF9) is an RNA‐binding protein that is phosphorylated by two PAMP‐responsive MAPKs, MPK3 and MPK6. We mapped the major phosphosites in TZF9 and showed their importance for controlling in vitro RNA‐binding activity, in vivo flg22‐induced rapid disappearance of TZF9‐labelled processing body‐like structures and TZF9 protein turnover. Microarray analysis showed a strong discordance between transcriptome (total mRNA) and translatome (polysome‐associated mRNA) in the tzf9 mutant, with more mRNAs associated to ribosomes in the absence of TZF9. This suggests that TZF9 may sequester and inhibit translation of subsets of mRNAs. Fittingly, TZF9 physically interacts with poly(A)‐binding protein 2 (PAB2), a hallmark constituent of stress granules – a site for stress‐induced translational stalling/arrest. TZF9 even promotes stress granule assembly in the absence of stress. Hence, MAPKs may control defence gene expression post‐transcriptionally through release from translation arrest within TZF9‐PAB2‐containing RNA granules or perturbing PAB2 functions in translation control (e.g. in the mRNA closed‐loop model of translation).

For centuries, crop plants have represented the basis of the daily human diet. Among them, cereals and legumes, accumulating oils, proteins, and carbohydrates in their seeds, distinctly dominate modern agriculture, thus play an essential role in food industry and fuel production. Therefore, seeds of crop plants are intensively studied by food chemists, biologists, biochemists, and nutritional physiologists. Accordingly, seed development and germination as well as age- and stress-related alterations in seed vigor, longevity, nutritional value, and safety can be addressed by a broad panel of analytical, biochemical, and physiological methods. Currently, functional genomics is one of the most powerful tools, giving direct access to characteristic metabolic changes accompanying plant development, senescence, and response to biotic or abiotic stress. Among individual post-genomic methodological platforms, proteomics represents one of the most effective ones, giving access to cellular metabolism at the level of proteins. During the recent decades, multiple methodological advances were introduced in different branches of life science, although only some of them were established in seed proteomics so far. Therefore, here we discuss main methodological approaches already employed in seed proteomics, as well as those still waiting for implementation in this field of plant research, with a special emphasis on sample preparation, data acquisition, processing, and post-processing. Thereby, the overall goal of this review is to bring new methodologies emerging in different areas of proteomics research (clinical, food, ecological, microbial, and plant proteomics) to the broad society of seed biologists.

Casein kinase 2 is a ubiquitous protein kinase that has puzzled researchers for several decades because of its pleiotropic activity. Here, we set out to identify the in vivo targets of plastid casein kinase 2 (pCK2) in Arabidopsis thaliana. Survey phosphoproteome analyses were combined with targeted analyses with wild-type and pck2 knockdown mutants to identify potential pCK2 targets by their decreased phosphorylation state in the mutant. To validate potential substrates, we complemented the pck2 knockdown line with tandem affinity tag (TAP)-tagged pCK2 and found it to restore growth parameters, as well as many, but not all, putative pCK2-dependent phosphorylation events. We further performed a targeted analysis at the end-of-night to increase the specificity of target protein identification. This analysis confirmed light-independent phosphorylation of several pCK2 target proteins. Based on the aforementioned data, we define a set of in vivo pCK2-targets that span different chloroplast functions, such as metabolism, transcription, translation and photosynthesis. The pleiotropy of pCK2 functions is also manifested by altered state transition kinetics during short-term acclimation and significant alterations in the mutant metabolism, supporting its function in photosynthetic regulation. Thus, our data expand our understanding on chloroplast phosphorylation networks and provide insights into kinase networks in the regulation of chloroplast functions.

BackgroundSkin ageing is associated with structure-functional changes in the extracellular matrix, which is in part caused by proteolytic degradation. Since cysteine cathepsins are major matrix protein-degrading proteases, we investigated the age-dependent expression of elastolytic cathepsins K, S, and V in human skin, their in vitro impact on the integrity of the elastic fibre network, their cleavage specificities, and the release of bioactive peptides.MethodsCathepsin-mediated degradation of human skin elastin samples was assessed from young to very old human donors using immunohistochemical and biochemical assays, scanning electron microscopy, and mass spectrometry.ResultsElastin samples derived from patients between 10 and 86 years of age were analysed and showed an age-dependent deterioration of the fibre structure from a dense network of thinner fibrils into a beaded and porous mesh. Reduced levels of cathepsins K, S, and V were observed in aged skin with a predominant epidermal expression. Cathepsin V was the most potent elastase followed by cathepsin K and S. Biomechanical analysis of degraded elastin fibres corroborated the destructive activity of cathepsins. Mass spectrometric determination of the cleavage sites in elastin revealed that all three cathepsins predominantly cleaved in hydrophobic domains. The degradation of elastin was efficiently inhibited by an ectosteric inhibitor. Furthermore, the degradation of elastin fibres resulted in the release of bioactive peptides, which have previously been associated with various pathologies.ConclusionCathepsins are powerful elastin-degrading enzymes and capable of generating a multitude of elastokines. They may represent a viable target for intervention strategies to reduce skin ageing.

Cullin RING-type E3 ubiquitin ligases SCFTIR1/AFB1-5 and their AUX/IAA targets perceive the phytohormone auxin. The F-box protein TIR1 binds a surface-exposed degron in AUX/IAAs promoting their ubiquitylation and rapid auxin-regulated proteasomal degradation. Here, by adopting biochemical, structural proteomics and in vivo approaches we unveil how flexibility in AUX/IAAs and regions in TIR1 affect their conformational ensemble allowing surface accessibility of degrons. We resolve TIR1·auxin·IAA7 and TIR1·auxin·IAA12 complex topology, and show that flexible intrinsically disordered regions (IDRs) in the degron’s vicinity, cooperatively position AUX/IAAs on TIR1. We identify essential residues at the TIR1 N- and C-termini, which provide non-native interaction interfaces with IDRs and the folded PB1 domain of AUX/IAAs. We thereby establish a role for IDRs in modulating auxin receptor assemblies. By securing AUX/IAAs on two opposite surfaces of TIR1, IDR diversity supports locally tailored positioning for targeted ubiquitylation, and might provide conformational flexibility for a multiplicity of functional states.

Proteome remodeling is a fundamental adaptive response, and proteins in complexes and functionally related proteins are often co-expressed. Using a deep sampling strategy we define core proteomes of Arabidopsis thaliana tissues with around 10 000 proteins per tissue, and absolutely quantify (copy numbers per cell) nearly 16 000 proteins throughout the plant lifecycle. A proteome-wide survey of global post-translational modification revealed amino acid exchanges pointing to potential conservation of translational infidelity in eukaryotes. Correlation analysis of protein abundance uncovered potentially new tissue- and age-specific roles of entire signaling modules regulating transcription in photosynthesis, seed development, and senescence and abscission. Among others, the data suggest a potential function of RD26 and other NAC transcription factors in seed development related to desiccation tolerance as well as a possible function of cysteine-rich receptor-like kinases (CRKs) as ROS sensors in senescence. All of the components of ribosome biogenesis factor (RBF) complexes were found to be co-expressed in a tissue- and age-specific manner, indicating functional promiscuity in the assembly of these less-studied protein complexes in Arabidopsis. Furthermore, we characterized detailed proteome remodeling in basal immunity by treating Arabidopsis seeldings with flg22. Through simultaneously monitoring phytohormone and transcript changes upon flg22 treatment, we obtained strong evidence of suppression of jasmonate (JA) and JA-isoleucine (JA-Ile) levels by deconjugation and hydroxylation by IAA-ALA RESISTANT3 (IAR3) and JASMONATE-INDUCED OXYGENASE 2 (JOX2), respectively, under the control of JASMONATE INSENSITIVE 1 (MYC2), suggesting an unrecognized role of a new JA regulatory switch in pattern-triggered immunity. Taken together, the datasets generated in this study present extensive coverage of the Arabidopsis proteome in various biological scenarios, providing a rich resource available to the whole plant science community.