UFMylation involves the covalent modification of substrate proteins with UFM1 (Ubiquitin-fold modifier 1) and is important for maintaining ER homeostasis. Stalled translation triggers the UFMylation of ER-bound ribosomes and activates C53-mediated autophagy to clear toxic polypeptides. C53 contains noncanonical shuffled ATG8-interacting motifs (sAIMs) that are essential for ATG8 interaction and autophagy initiation. However, the mechanistic basis of sAIM-mediated ATG8 interaction remains unknown. Here, we show that C53 and sAIMs are conserved across eukaryotes but secondarily lost in fungi and various algal lineages. Biochemical assays showed that the unicellular alga Chlamydomonas reinhardtii has a functional UFMylation pathway, refuting the assumption that UFMylation is linked to multicellularity. Comparative structural analyses revealed that both UFM1 and ATG8 bind sAIMs in C53, but in a distinct way. Conversion of sAIMs into canonical AIMs impaired binding of C53 to UFM1, while strengthening ATG8 binding. Increased ATG8 binding led to the autoactivation of the C53 pathway and sensitization of Arabidopsis thaliana to ER stress. Altogether, our findings reveal an ancestral role of sAIMs in UFMylation-dependent fine-tuning of C53-mediated autophagy activation.

Roots are highly plastic organs enabling plants to adapt to a changing below-ground environment. In addition to abiotic factors like nutrients or mechanical resistance, plant roots also respond to temperature variation. Below the heat stress threshold, Arabidopsis thaliana seedlings react to elevated temperature by promoting primary root growth, possibly to reach deeper soil regions with potentially better water saturation. While above-ground thermomorphogenesis is enabled by thermo-sensitive cell elongation, it was unknown how temperature modulates root growth. We here show that roots are able to sense and respond to elevated temperature independently of shoot-derived signals. This response is mediated by a yet unknown root thermosensor that employs auxin as a messenger to relay temperature signals to the cell cycle. Growth promotion is achieved primarily by increasing cell division rates in the root apical meristem, depending on de novo local auxin biosynthesis and temperature-sensitive organization of the polar auxin transport system. Hence, the primary cellular target of elevated ambient temperature differs fundamentally between root and shoot tissues, while the messenger auxin remains the same.

The rapid annotation and identification by mass spectrometry techniques of flavonoids remains a challenge, due to their structural diversity and the limited availability of reference standards. This study applies a workflow to characterize two isoflavonoids, the orobol-C-glycosides analogs, using high-energy collisional dissociation (HCD)- and collision-induced dissociation (CID)-type fragmentation patterns, and also to evaluate the antioxidant effects of these compounds by ferric reducing antioxidant power (FRAP), 2,2′-azino-bis(3-ethylbenzothiazolin acid) 6-sulfonic acid (ABTS), and 2,2-diphenyl-1-picrylhydrazyl (DPPH) methods. By the CID-type fragmentation, in positive mode and at all high-resolution mass spectrometry (HRMS) multiple stage, there were shown differences in the annotation of the compounds, mainly concerning some ratios of relative abundance. At CID-MS2 20 eV, the compounds could be efficiently characterized, because they present distinct base peaks [M + H]+ and [M + H–H2O]+ for the orobol-8-C- and orobol-6-C-glycoside, respectively. Similarly, by the HCD-type fragmentation, in HRMS2 stage, differences between orobol analogs in both mode of ionization were observed. However, the HR HCD-MS2 at 80 eV, in positive mode, generated more ions and each isomer presented different base peaks ions, [0,2X]+ for the orobol-8-C-glycoside and [0,3X]+ for the orobol-6-C-glycoside. By the DPPH, the 8-C-derivative showed a very close value compared with the standard rutin and, in the ABTS method, a higher radical-scavenging activity. In both methods, the EC50 of orobol-8-C-glycoside was almost twice better compared with orobol-6-C-glycoside. In FRAP, both C-glycosides showed a good capacity as Fe+3 reducing agents. We could realize that combined MS techniques, highlighting the positive mode of ionization, can be used to evaluate the isoflavones analogs being useful to differentiate between the isomeric flavones; therefore, these data are important to mass spectrometry dereplication studies become more efficient.

Long‐chain ferulic acid esters, such as eicosyl ferulate (1), show a complex and analytically valuable fragmentation behavior under negative‐ion electrospay collision‐induced dissociation ((‐)‐ESI‐CID) mass spectrometry, as studied by use of a high‐resolution (Orbitrap) mass spectrometer. In a strong contrast to the very simple fragmentation of the [M + H]+ ion, which is discussed briefly, the deprotonated molecule, [M ‐ H]‐, exhibits a rich secondary fragmentation chemistry. It first loses a methyl radical (MS2) and the ortho‐quinoid [M ‐ H ‐ Me]‐• radical anion thus formed then dissociates by loss of an extended series of neutral radicals, CnH2n+1• (n = 0‐16) from the long alkyl chain, in competition with the expulsion of CO and CO2 (MS3). The further fragmentation (MS4) of the [M ‐ H ‐ Me ‐ C3H7]‐ ion, discussed as an example, and the highly specific losses of alkyl radicals from the [M ‐ H ‐ Me ‐ CO]‐• and [M ‐ H ‐ Me ‐ CO2]‐• ions provide some mechanistic and structural insights.

Identification and structural determination of small molecules by mass spectrometry is an important step in chemistry and biochemistry. However, the chemically realistic annotation of a fragment ion spectrum can be a difficult challenge. We developed ChemFrag, for the detection of fragmentation pathways and the annotation of fragment ions with chemically reasonable structures. ChemFrag combines a quantum chemical with a rule‐based approach. For different doping substances as test instances, ChemFrag correctly annotates fragment ions. In most cases, the predicted fragments are chemically more realistic than those from purely combinatorial approaches, or approaches based on machine learning. The annotation generated by ChemFrag often coincides with spectra that have been manually annotated by experts. This is a major advance in peak annotation and allows a more precise automatic interpretation of mass spectra.

The cullin‐RING E3 ligases (CRLs) regulate diverse cellular processes in all eukaryotes. CRL activity is controlled by several proteins or protein complexes, including NEDD8, CAND1, and the CSN. Recently, a mammalian protein called Glomulin (GLMN) was shown to inhibit CRLs by binding to the RING BOX (RBX1) subunit and preventing binding to the ubiquitin‐conjugating enzyme. Here, we show that Arabidopsis ABERRANT LATERAL ROOT FORMATION4 (ALF4) is an ortholog of GLMN. The alf4 mutant exhibits a phenotype that suggests defects in plant hormone response. We show that ALF4 binds to RBX1 and inhibits the activity of SCFTIR1, an E3 ligase responsible for degradation of the Aux/IAA transcriptional repressors. In vivo, the alf4 mutation destabilizes the CUL1 subunit of the SCF. Reduced CUL1 levels are associated with increased levels of the Aux/IAA proteins as well as the DELLA repressors, substrate of SCFSLY1. We propose that the alf4 phenotype is partly due to increased levels of the Aux/IAA and DELLA proteins.

Representative compounds with a 1,3‐dihydroxybenzene substructure belonging to different important polyphenol classes (stilbenes, flavones, isoflavones, flavonols, flavanones, flavanols, phloroglucinols, anthraquinones and bisanthraquinones) were investigated based on detailed high‐resolution tandem mass spectrometry measurements with an Orbitrap system under negative ion electrospray conditions. The mass spectral behaviour of these compound classes was compared among each other not only with respect to previously described losses of CO, CH2CO and C3O2 but also concerning the loss of CO2 and successive specific fragmentations. Furthermore, some unusual fragmentations such as the loss of a methyl radical during mass spectral decomposition are discussed. The obtained results demonstrate both similarities and differences in their mass spectral fragmentation under MSn conditions, allowing a characterization of the corresponding compound type.

Mass spectrometry (MS) is an important analytical technique for the detection and identification of small compounds. The main bottleneck in the interpretation of metabolite profiling or screening experiments is the identification of unknown compounds from tandem mass spectra.Spectral libraries for tandem MS, such as MassBank or NIST, contain reference spectra for many compounds, but their limited chemical coverage reduces the chance for a correct and reliable identification of unknown spectra outside the database domain.On the other hand, compound databases like PubChem or ChemSpider have a much larger coverage of the chemical space, but they cannot be queried with spectral information directly. Recently, computational mass spectrometry methods and in silico fragmentation prediction allow users to search such databases of chemical structures.We present a new strategy called MetFusion to combine identification results from several resources, in particular, from the in silico fragmenter MetFrag with the spectral library MassBank to improve compound identification. We evaluate the performance on a set of 1062 spectra and achieve an improved ranking of the correct compound from rank 28 using MetFrag alone, to rank 7 with MetFusion, even if the correct compound and similar compounds are absent from the spectral library. On the basis of the evaluation, we extrapolate the performance of MetFusion to the KEGG compound database.

A series of prevailing prenylated furanocoumarins from leaves of Dorstenia gigas and Dorstenia foetida (Moraceae) were investigated by liquid chromatography/electrospray tandem mass spectrometry. The mass spectral behavior of the furanocoumarins under positive ion electrospray conditions is discussed using both an ion trap and a triple quadrupole system. It is demonstrated that both methods represent valuable tools not only for the rapid classification of this type of compounds, but also with respect to their substitution pattern.

MassBank is the first public repository of mass spectra of small chemical compounds for life sciences (<3000 Da). The database contains 605 electron‐ionization mass spectrometry(EI‐MS), 137 fast atom bombardment MS and 9276 electrospray ionization (ESI)‐MSn data of 2337 authentic compounds of metabolites, 11 545 EI‐MS and 834 other‐MS data of 10 286 volatile natural and synthetic compounds, and 3045 ESI‐MS2 data of 679 synthetic drugs contributed by 16 research groups (January 2010). ESI‐MS2 data were analyzed under nonstandardized, independent experimental conditions. MassBank is a distributed database. Each research group provides data from its own MassBank data servers distributed on the Internet. MassBank users can access either all of the MassBank data or a subset of the data by specifying one or more experimental conditions. In a spectral search to retrieve mass spectra similar to a query mass spectrum, the similarity score is calculated by a weighted cosine correlation in which weighting exponents on peak intensity and the mass‐to‐charge ratio are optimized to the ESI‐MS2 data. MassBank also provides a merged spectrum for each compound prepared by merging the analyzed ESI‐MS2 data on an identical compound under different collision‐induced dissociation conditions. Data merging has significantly improved the precision of the identification of a chemical compound by 21–23% at a similarity score of 0.6. Thus, MassBank is useful for the identification of chemical compounds and the publication of experimental data.