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.

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.

Mechanistically, nonhost resistance of Arabidopsis thaliana against the oomycete Phytophthora infestans is not well understood. Besides PEN2 and PEN3, which contribute to penetration resistance, no further components have been identified so far. In an ethylmethane sulphonate–mutant screen, we mutagenized pen2-1 and screened for mutants with an altered response to infection by P. infestans. One of the mutants obtained, enhanced response to Phytophthora infestans6 (erp6), was analyzed. Whole-genome sequencing of erp6 revealed a single nucleotide polymorphism in the coding region of the kinase domain of At1g08720, which encodes the putative MAPKKK ENHANCED DISEASE RESISTANCE1 (EDR1). We demonstrate that three independent lines with knock-out alleles of edr1 mount an enhanced response to P. infestans inoculation, mediated by increased salicylic acid signaling and callose deposition. Moreover, we show that the single amino acid substitution in erp6 causes the loss of in vitro autophosphorylation activity of EDR1. Furthermore, growth inhibition experiments suggest a so-far-unknown involvement of EDR1 in the response to the pathogen-associated molecular patterns flg22 and elf18. We conclude that EDR1 contributes to the defense response of A. thaliana against P. infestans. Our data position EDR1 as a negative regulator in postinvasive nonhost resistance.

Transient infiltrations in tobacco are commonly used in plant studies, but the host response to different disarmed Agrobacterium strains is not fully understood. The present study shows that pretreatment with disarmed Agrobacterium tumefaciens GV3101 primes the defense response to subsequent infection by Pseudomonas syringae in Nicotiana tabacum. The presence of a trans-zeatin synthase (tzs) gene in strain GV3101 may be partly responsible for the priming response, as the tzs-deficient Agrobacterium sp. strain LBA4404 only weakly imparts such responses. Besides inducing the expression of defense-related genes like PR-1 and NHL10, GV3101 pretreatment increased the expression of tobacco mitogen-activated protein kinase (MAPK) pathway genes like MEK2, WIPK (wound-induced protein kinase), and SIPK (salicylic acid-induced protein kinase). Furthermore, the GV3101 strain showed a stronger effect than the LBA4404 strain in activating phosphorylation of the tobacco MAPK, WIPK and SIPK, which presumably prime the plant immune machinery. Lower doses of exogenously applied cytokinins increased the activation of MAPK, while higher doses decreased the activation, suggesting a balanced level of cytokinins is required to generate defense response in planta. The current study serves as a cautionary warning for plant researchers over the choice of Agrobacterium strains and their possible consequences on subsequent pathogen-related studies.

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.

Simultaneous mutation of two WRKY-type transcription factors, WRKY18 and WRKY40, renders otherwise susceptible wild-type Arabidopsis plants resistant towards the biotrophic powdery mildew fungus Golovinomyces orontii. Resistance in wrky18 wrky40 double mutant plants is accompanied by massive transcriptional reprogramming, imbalance in salicylic acid (SA) and jasmonic acid (JA) signaling, altered ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) expression, and accumulation of the phytoalexin camalexin. Genetic analyses identified SA biosynthesis and EDS1 signaling as well as biosynthesis of the indole-glucosinolate 4MI3G as essential components required for loss-of-WRKY18 WRKY40–mediated resistance towards G. orontii. The analysis of wrky18 wrky40 pad3 mutant plants impaired in camalexin biosynthesis revealed an uncoupling of pre- from postinvasive resistance against G. orontii. Comprehensive infection studies demonstrated the specificity of wrky18 wrky40–mediated G. orontii resistance. Interestingly, WRKY18 and WRKY40 act as positive regulators in effector-triggered immunity, as the wrky18 wrky40 double mutant was found to be strongly susceptible towards the bacterial pathogen Pseudomonas syringae DC3000 expressing the effector AvrRPS4 but not against other tested Pseudomonas strains. We hypothesize that G. orontii depends on the function of WRKY18 and WRKY40 to successfully infect Arabidopsis wild-type plants while, in the interaction with P. syringae AvrRPS4, they are required to mediate effector-triggered immunity.

The importance of phytohormone balance is increasingly recognized as central to the outcome of plant–pathogen interactions. Next to their well-known developmental role, brassinosteroids (BR) were recently found to be involved in plant innate immunity. In this study, we examined the role of BR in rice (Oryza sativa) innate immunity during infection with the root-knot nematode Meloidogyne graminicola, and we studied the inter-relationship with the jasmonate (JA) pathway. Exogenous epibrassinolide (BL) supply at low concentrations induced susceptibility in the roots whereas high concentrations of BL enforced systemic defense against this nematode. Upon high exogenous BL supply on the shoot, quantitative reverse-transcription polymerase chain reaction (qRT-PCR) confirmed a strong feedback inhibitory effect, leading to reduced BR biosynthesis in the root. Moreover, we demonstrate that the immune suppressive effect of BR is at least partly due to negative cross-talk with the JA pathway. Mutants in the BR biosynthesis or signaling pathway accumulate slightly higher levels of the immediate JA-precursor 12-oxo-phytodienoic acid, and qRT-PCR data showed that the BR and JA pathway are mutually antagonistic in rice roots. Collectively, these results suggest that the balance between the BR and JA pathway is an effective regulator of the outcome of the rice–M. graminicola interaction.

The barley pathogen Rhynchosporium commune secretes necrosis-inducing proteins NIP1, NIP2, and NIP3. Expression analysis revealed that NIP1 transcripts appear to be present in fungal spores already, whereas NIP2 and NIP3 are synthesized after inoculation of host plants. To assess the contribution of the three effector proteins to disease development, deletion mutants were generated. The development of these fungal mutants on four barley cultivars was quantified in comparison with that of the parent wild-type strain and with two fungal strains failing to secrete an “active” NIP1 avirulence protein, using quantitative polymerase chain reaction as well as microscopic imaging after fungal green fluorescent protein tagging. The impact of the three deletions varied quantitatively depending on the host genotype, suggesting that the activities of the fungal effectors add up to produce stronger growth patterns and symptom development. Alternatively, recognition events of differing intensities may be converted into defense gene expression in a quantitative manner.