SummaryProcessing by proteases irreversibly regulates the fate of plant proteins and hampers the production of recombinant proteins in plants, yet only few processing events have been described in agroinfiltrated Nicotiana benthamiana, which has emerged as the main transient protein expression platform in plant science and molecular pharming.Here, we used in‐gel digests and mass spectrometry to monitor the migration and topography of 5040 plant proteins within a protein gel. By plotting the peptides over the gel slices, we generated peptographs that reveal where which part of each protein was detected within the protein gel. These data uncovered that 60% of the detected proteins have proteoforms that migrate at lower than predicted molecular weights, implicating extensive proteolytic processing. This analysis confirms the proteolytic removal and degradation of autoinhibitory prodomains of most but not all proteases, and revealed differential processing within pectinemethylesterase and lipase families. This analysis also uncovered intricate processing of glycosidases and uncovered that ectodomain shedding might be common for a diverse range of receptor‐like kinases. Transient expression of double‐tagged candidate proteins confirmed processing events in vivo. This large proteomic dataset implicates an elaborate proteolytic machinery shaping the proteome of N. benthamiana.

The new farnesyl pyrophosphate (FPP) derivative with a shifted olefinic double bond from C6‐C7 to C7‐C8 is accepted and converted by the sesquiterpene cyclases protoilludene synthase (Omp7) as well as viridiflorene synthase (Tps32). In both cases, a so far unknown germacrene derivative was found to be formed, which we name “germacrene F”. Both cases are examples in which a modification around the central olefinic double bond in FPP leads to a change in the mode of initial cyclization (from 1→11 to 1→10). For Omp7 a rationale for this behaviour was found by carrying out molecular docking studies. Temperature‐dependent NMR experiments, accompanied by NOE studies, show that germacrene F adopts a preferred mirror‐symmetric conformation with both methyl groups oriented in the same directions in the cyclodecane ring.

The economic and ecologically important genus Eucalyptus is rich in structurally diverse specialized metabolites. While some specialized metabolite classes are highly prevalent across the genus, the cyanogenic glucoside prunasin is only produced by c. 3% of species. To investigate the evolutionary mechanisms behind prunasin biosynthesis in Eucalyptus, we compared de novo assembled transcriptomes, together with online resources between cyanogenic and acyanogenic species. Identified genes were characterized in vivo and in vitro. Pathway characterization of cyanogenic Eucalyptus camphora and Eucalyptus yarraensis showed for the first time that the final glucosylation step from mandelonitrile to prunasin is catalyzed by a novel UDP-glucosyltransferase UGT87. This step is typically catalyzed by a member of the UGT85 family, including in Eucalyptus cladocalyx. The upstream conversion of phenylalanine to mandelonitrile is catalyzed by three cytochrome P450 (CYP) enzymes from the CYP79, CYP706, and CYP71 families, as previously shown. Analysis of acyanogenic Eucalyptus species revealed the loss of different ortholog prunasin biosynthetic genes. The recruitment of UGTs from different families for prunasin biosynthesis in Eucalyptus demonstrates important pathway heterogeneities and unprecedented dynamic pathway evolution of chemical defense within a single genus. Overall, this study provides relevant insights into the tremendous adaptability of these long-lived trees.

Silyl ether protecting groups are important tools in organic synthesis, ensuring selective reactions of hydroxyl functional groups. Enantiospecific formation or cleavage could simultaneously enable the resolution of racemic mixtures and thus significantly increase the efficiency of complex synthetic pathways. Based on reports that lipases, which today are already particularly important tools in chemical synthesis, can catalyze the enantiospecific turnover of trimethylsilanol (TMS)-protected alcohols, the goal of this study was to determine the conditions under which such a catalysis occurs. Through detailed experimental and mechanistic investigation, we demonstrated that although lipases mediate the turnover of TMS-protected alcohols, this occurs independently of the known catalytic triad, as this is unable to stabilize a tetrahedral intermediate. The reaction is essentially non-specific and therefore most likely completely independent of the active site. This rules out lipases as catalysts for the resolution of racemic mixtures alcohols through protection or deprotection with silyl groups.

Macrocyclization of peptides is typically used to fix specific bioactive conformations and improve their pharmacological properties. Recently, macrobicyclic peptides have received special attention owing to their capacity to mimic protein structures or be key components of peptide-drug conjugates. Here, we describe the development of novel synthetic strategies for two distinctive types of peptide macrobicycles. A multicomponent macrocyclo-dimerization approach is introduced for the production of interconnected β-turns, allowing two macrocyclic rings to be formed and dimerized in one pot. Also, an on-resin double stapling strategy is described for the assembly of lactam-bridged macrobicycles with stable tertiary folds.

Heterodimeric complexes incorporating the lipase-li ke proteins EDS1 wi th PAD4 or SAG101 are central hubs in plant innate immunity. EDS1 functions encompass signal relay from TIR domain-containing intracellular NLR-type immune receptors (TNLs) towards RPW8-type helper NLRs (RNLs) and, in A. thaliana, bolstering of signaling and resistance mediated by cell-s u r face pattern recognition receptors (PRRs). Increasing evidence points to the activation of EDS1 complexes by small molecule binding. •We used CRISPR/Cas-generated mutant lines and agroinfiltration-based complementation assays to interrogate functions of EDS1 complexes in N. benthamiana. •We do not detect impaired PRR signaling in N. benthamiana lines deficient in EDS1 complexes or RNLs. Intriguingly, in assays monitoring functions of SlEDS1-NbEDS1 complexes in N. benthamiana, mutations within the SlEDS1 catalytic triad can abolish or enhance TNL immunity. Furthermore, nuclear EDS1 accumulation is sufficient for N. benthamianaTNL (Roq1) immunity.•Reinforcing PRR signaling in Arabidopsis might be a derived function of the TNL/EDS1 immune sector. Although Solanaceae EDS1 functionally depends on catalytic triad residues in some contexts, our data do not support binding of a TNL-derived small molecule in the triad environment. Whether and how nuclear EDS1 activity connects to membrane pore-f orming RNLs remains unknown.

Arabidopsis seeds release large capsules of mucilaginous polysaccharides, which are shaped by an intricate network of cellulosic microfibrils. Cellulose synthase complexes are guided by the microtubule cytoskeleton, but it is unclear which proteins mediate this process in the seed coat epidermis. Using reverse genetics, we identified IQ67 DOMAIN 9 (IQD9) and KINESIN LIGHT CHAIN-RELATED 1 (KLCR1) as two highly expressed genes during seed development and comprehensively characterized their roles in cell wall polysaccharide biosynthesis. Mutations in IQD9 as well as in KLCR1 lead to compact mucilage capsules with aberrant cellulose distribution, which can be rescued by transgene complementation. IQD9 physically interacts with KLCR1 and localizes to cortical MTs to maintain their organization in SCE cells. IQD9 as well as a previously identified TONNEAU1 (TON1) RECRUITING MOTIF 4 (TRM4) protein act to maintain cellulose synthase velocity. Our results demonstrate that IQD9, KLCR1 and TRM4 are MT-associated proteins that are required for seed mucilage architecture. This study provides the first direct evidence that members of the IQD, KLCR and TRM families have overlapping roles in cell wall biosynthesis. Therefore, SCE cells provide an attractive system to further decipher the complex genetic regulation of polarized cellulose deposition.

Terpene synthase-mediated biotransformation of eleven synthetic sulfur- or oxygen-containing non-natural prenyl diphosphates resulted in the formation of five novel terpenoids and analogues. Uniquely, they trap intermediate steps and form heterocycles or compounds with alkyne side chains. Computational modelling differentiates convertible from inconvertible substrates and thereby provides an understanding of the detailed molecular mechanism of terpene cyclases. Two terpene cyclases were used as biocatalytic tool, namely, limonene synthase from Cannabis sativa (CLS) and 5-epi-aristolochene synthase (TEAS) from Nicotiana tabacum. They showed significant substrate flexibility towards non-natural prenyl diphosphates to form novel terpenoids, including core oxa- and thia-heterocycles and alkyne-modified terpenoids. We elucidated the structures of five novel monoterpene-analogues and a known sesquiterpene-analogue. These results reflected the terpene synthases′ ability and promiscuity to broaden the pool of terpenoids with structurally complex analogues. Docking studies highlight an on-off conversion of the unnatural substrates.

Hemicellulosic polysaccharides built of b-1,4-linked mannose units have been found throughout the plant kingdom and have numerous industrial applications. Here, I review recent advances in the biosynthesis and modification of plant b-mannans. These matrix polymers can associate with cellulose bundles to impact the mechanical properties of plant fibers or biocomposites. In certain algae, mannan microfibrils even replace cellulose as the dominant structural component of the cell wall. Conversely, patterned galactoglucomannan found in Arabidopsis thaliana seed mucilage significantly modulates cell wall architecture and abiotic stress tolerance despite its relatively low content. I also discuss the subcellular requirements for b-mannan biosynthesis, the increasing number of carbohydrate-active enzymes involved in this process, and the players that continue to be puzzling. I discuss how cellulose synthase-like enzymes elongate (gluco)mannans in orthogonal hosts and highlight the discoveries of plant enzymes that add specific galactosyl or acetyl decorations. Hydrolytic enzymes such as endo-b-1,4-mannanases have recently been involved in a wide range of biological contexts including seed germination, wood formation, heavy metal tolerance, and defense responses. Synthetic biology tools now provide faster tracks to modulate the increasingly-relevant mannan structures for improved plant traits and bioproducts.

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