Mapping the chemical space of compounds to chemical structures remains a challenge in metabolomics. Despite the advancements in untargeted liquid chromatography-mass spectrometry (LC-MS) to achieve a high-throughput profile of metabolites from complex biological resources, only a small fraction of these metabolites can be annotated with confidence. Many novel computational methods and tools have been developed to enable chemical structure annotation to known and unknown compounds such as in silico generated spectra and molecular networking. Here, we present an automated and reproducible Metabolome Annotation Workflow (MAW) for untargeted metabolomics data to further facilitate and automate the complex annotation by combining tandem mass spectrometry (MS2) input data pre-processing, spectral and compound database matching with computational classification, and in silico annotation. MAW takes the LC-MS2 spectra as input and generates a list of putative candidates from spectral and compound databases. The databases are integrated via the R package Spectra and the metabolite annotation tool SIRIUS as part of the R segment of the workflow (MAW-R). The final candidate selection is performed using the cheminformatics tool RDKit in the Python segment (MAW-Py). Furthermore, each feature is assigned a chemical structure and can be imported to a chemical structure similarity network. MAW is following the FAIR (Findable, Accessible, Interoperable, Reusable) principles and has been made available as the docker images, maw-r and maw-py. The source code and documentation are available on GitHub. The performance of MAW is evaluated on two case studies. We found that MAW can improve candidate ranking by integrating spectral databases with annotation tools like SIRIUS which contributes to an efficient candidate selection procedure. The results from MAW are also reproducible and traceable, compliant with the FAIR guidelines. Taken together, MAW could greatly facilitate automated metabolite characterization in diverse fields such as clinical metabolomics and natural product discovery.

DNA double strand breaks (DSBs) are lethal threats that need to be repaired. Although many of the proteins involved in the early steps of DSB repair have been characterized, recent reports indicate that damage induced long and small RNAs also play an important role in DSB repair. Here, using a Nicotiana benthamiana transgenic line originally designed as a reporter for targeted knock-ins, we show that DSBs generated by Cas9 induce the transcription of long stable RNAs (damage-induced long RNAs - dilRNAs) that are translated into proteins. Using an array of single guide RNAs we show that the initiation of transcription takes place in the vicinity of the DSB. Single strand DNA nicks are not able to induce transcription, showing that cis DNA damage-induced transcription is specific for DSBs. Our results support a model in which a default and early event in the processing of DSBs is transcription into RNA which, depending on the genomic and genic context, can undergo distinct fates, including translation into protein, degradation or production of small RNAs. Our results have general implications for understanding the role of transcription in the repair of DSBs and, reciprocally, reveal DSBs as yet another way to regulate gene expression.

Protein engineering through directed evolution and (semi-)rational approaches has been applied successfully to optimize protein properties for broad applications in molecular biology, biotechnology, and biomedicine. The potential of protein engineering is not yet fully realized due to the limited screening throughput hampering the efficient exploration of the vast protein sequence space. Data-driven strategies have emerged as a powerful tool to leverage protein engineering by providing a model of the sequence-fitness landscape that can exhaustively be explored in silico and capitalize on the high diversity potential offered by nature However, as both the quality and quantity of the inputted data determine the success of such approaches, the applicability of data-driven strategies is often limited due to sparse data. Here, we present a hybrid model that combines direct coupling analysis and machine learning techniques to enable data-driven protein engineering when only few labeled sequences are available. Our method achieves high performance in predicting a protein’s fitness based on its sequence regardless of the number of sequences-fitness pairs in the training dataset. Besides reducing the computational effort compared to state-of-the-art methods, it outperforms them for sparse data situations, i.e., 50 − 250 labeled sequences available for training. In essence, the developed method is auspicious for data-driven protein engineering, especially for protein engineers who have only access to a limited amount of data for sequence-fitness landscape modeling.

The shape of tomato fruits is closely correlated to microtubule organization and the activity of microtubule associated proteins (MAP), but insights into the mechanism from a cell biology perspective are still largely elusive. Analysis of tissue expression profiles of different microtubule regulators revealed that functionally distinct classes of MAPs are highly expressed during fruit development. Among these, several members of the plant-specific MAP70 family are preferably expressed at the initiation stage of fruit development. Transgenic tomato lines overexpressing SlMAP70 produced elongated fruits that show reduced cell circularity and microtubule anisotropy, while SlMAP70 loss-of-function mutant showed an opposite effect with flatter fruits. Microtubule anisotropy of fruit endodermis cells exhibited dramatic rearrangement during tomato fruit development, and SlMAP70-1 is likely implicated in cortical microtubule organization and fruit elongation throughout this stage by interacting with SUN10/SlIQD21a. The expression of SlMAP70 (or co-expression of SlMAP70 and SUN10/SlIQD21a) induces microtubule stabilization and prevents its dynamic rearrangement, both activities are essential for fruit shape establishment after anthesis. Together, our results identify SlMAP70 as a novel regulator of fruit elongation, and demonstrate that manipulating microtubule stability and organization at the early fruit developmental stage has a strong impact on fruit shape.

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.

Wound healing is a fundamental property of plants and animals that requires recognition of cellular damage to initiate regeneration. In plants, wounding activates a defense response via the production of jasmonic acid and a regeneration response via the hormone auxin and several ethylene response factor (ERF) and NAC domain-containing protein (ANAC) transcription factors. To better understand how plants recognize damage and initiate healing, we searched for factors upregulated during the horticulturally relevant process of plant grafting and found four related DNA binding with one finger (DOF) transcription factors, HIGH CAMBIAL ACTIVITY2 (HCA2), TARGET OF MONOPTEROS6 (TMO6), DOF2.1, and DOF6, whose expression rapidly activated at the Arabidopsis graft junction. Grafting or wounding a quadruple hca2, tmo6, dof2.1, dof6 mutant inhibited vascular and cell-wall-related gene expression. Furthermore, the quadruple dof mutant reduced callus formation, tissue attachment, vascular regeneration, and pectin methylesterification in response to wounding. Wcalluscallus found that activation of DOF gene expression after wounding required auxin, but hormone treatment alone was insufficient for their induction. However, modifying cell walls by enzymatic digestion of cellulose or pectin greatly enhanced TMO6 and HCA2 expression, whereas genetic modifications to the pectin or cellulose matrix using the PECTIN METHYLESTERASE INHIBITOR5 overexpression line or korrigan1 mutant altered TMO6 and HCA2 expression. Changes to the cellulose or pectin matrix were also sufficient to activate the wound-associated ERF115 and ANAC096 transcription factors, suggesting that cell-wall damage represents a common mechanism for wound perception and the promotion of tissue regeneration.Graphical abstract

Arbuscular mycorrhizal (AM) symbiosis modulates plant‐herbivore interactions. Still, how it shapes the overall plant defence strategy and the mechanisms involved remain unclear. We investigated how AM symbiosis simultaneously modulates plant resistance and tolerance to a shoot herbivore, and explored the underlying mechanisms. Bioassays with Medicago truncatula plants were used to study the effect of the AM fungus Rhizophagus irregularis on plant resistance and tolerance to Spodoptera exigua herbivory. By performing molecular and chemical analyses, we assessed the impact of AM symbiosis on herbivore‐triggered phosphate (Pi)‐ and jasmonate (JA)‐related responses. Upon herbivory, AM symbiosis led to an increased leaf Pi content by boosting the mycorrhizal Pi‐uptake pathway. This enhanced both plant tolerance and herbivore performance. AM symbiosis counteracted the herbivore‐triggered JA burst, reducing plant resistance. To disentangle the role of the mycorrhizal Pi‐uptake pathway in the plant\'s response to herbivory, we used the mutant line ha1‐2, impaired in the H+‐ATPase gene HA1, which is essential for Pi‐uptake via the mycorrhizal pathway. We found that mycorrhiza‐triggered enhancement of herbivore performance was compromised in ha1‐2 plants. AM symbiosis thus affects the defence pattern of M. truncatula by altering resistance and tolerance simultaneously. We propose that the mycorrhizal Pi‐uptake pathway is involved in the modulation of the plant defence strategy.

With a favored taste and various bioactivities, coffee has been consumed as a daily beverage worldwide. The current study presented a multi-faceted comparative metabolomics approach dissecting commercially available coffee products in the Middle East region for quality assessment and functional food purposes using NMR and GC/MS platforms. NMR metabolites fingerprinting led to identification of 18 metabolites and quantification (qNMR) of six prominent markers for standardization purposes. An increase of β-ethanolamine (MEA) reported for the first time, 5-(hydroxymethyl) furfural (5-HMF), concurrent with a reduction in chlorogenic acid, kahweol, and sucrose levels post roasting as revealed using multivariate data analyses (MVA). The diterpenes kahweol and cafestol were identified in green and roasted Coffea arabica, while 16-O-methyl cafestol in roasted C. robusta. Moreover, GC/MS identified a total of 143 metabolites belonging to 15 different chemical classes, with fructose found enriched in green C. robusta versus fatty acids abundance, i.e., palmitic and stearic acids in C. arabica confirming NMR results. These potential results aided to identify novel quality control attributes, i.e., ethanolamine, for coffee in the Middle East region and have yet to be confirmed in other coffee specimens.

A protocol for synthesizing triazole-containing pyrazolines and pyrazoles selectively using trifluoromethylated 5-(1,2,3-triazol-1-yl)enones as starting materials, is reported. The selectivity of the reaction was controlled by the nature of the hydrazine or derivative used: free hydrazines furnished the 1,5-regiosiomer exclusively in yields up to 98%, whereas protected hydrazines provided the 1,3-regioisomer in yields up to 77%. To demonstrate the synthetic versatility of the triazole-based enone, reactions with other unsymmetrical dinucleophiles (hydroxylamine hydrochloride and S-methyl isothiourea sulfates) allowed the selective preparation of triazole-containing isoxazoline and pyrimidine rings.

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.