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Publikation

Frey, M.; Bathe, U.; Meink, L.; Balcke, G. U.; Schmidt, J.; Frolov, A.; Soboleva, A.; Hassanin, A.; Davari, M. D.; Frank, O.; Schlagbauer, V.; Dawid, C.; Tissier, A.; Combinatorial biosynthesis in yeast leads to over 200 diterpenoids Metab. Eng. 82, 193-200, (2024) DOI: 10.1016/j.ymben.2024.02.006

Diterpenoids form a diverse group of natural products, many of which are or could become pharmaceuticals or industrial chemicals. The modular character of diterpene biosynthesis and the promiscuity of the enzymes involved make combinatorial biosynthesis a promising approach to generate libraries of diverse diterpenoids. Here, we report on the combinatorial assembly in yeast of ten diterpene synthases producing (+)-copalyldiphosphate-derived backbones and four cytochrome P450 oxygenases (CYPs) in diverse combinations. This resulted in the production of over 200 diterpenoids. Based on literature and chemical database searches, 162 of these compounds can be considered new-to-Nature. The CYPs accepted most substrates they were given but remained regioselective with few exceptions. Our results provide the basis for the systematic exploration of the diterpenoid chemical space in yeast using sequence databases.
Publikation

Cherevatskaya, M.; Cherepanov, I.; Kalganova, N.; Erofeeva, N.; Romanovskaya, E.; Frolov, A.; Bilova, T.; Moiseev, S.; Wessjohann, L. A.; Sydnone imines as a new class of promising plant growth and stress tolerance modulators—A first experimental structure–activity overview Stresses 4, 133-154, (2024) DOI: 10.3390/stresses4010008

Due to the oncoming climate changes, various environmental stresses (drought, salinity, heavy-metals, low or high temperatures, etc.) might dramatically affect crop yields and the quality of produced foods. Therefore, to meet the growing food demand of the human population, improvement of stress tolerance of the currently cultured crops is required. The knowledge of the molecular underlying mechanisms provides a versatile instrument to correct plant metabolism via chemical tools and to thereby increase their adaptive potential. This will preserve crop productivity and quality under abiotic stress conditions. Endogenously produced nitric oxide (NO) is one of the key signaling factors activating stress tolerance mechanisms in plants. Thus, the application of synthetic NO donors as stress-protective phytoeffectors might support maintaining plant growth and productivity under stressful conditions. Sydnone imines (sydnonimines) are a class of clinically established mesoionic heterocyclic NO donors which represent a promising candidate group for such phytoeffectors. Therefore, here, we provide an overview of the current progress in the application of sydnone imines as exogenous NO donors in plants, with a special emphasis on their potential as herbicides as well as herbicide antidotes, growth stimulants and stress protectors triggering plant tolerance mechanisms. We specifically address the structure–activity relationships in the context of the growth modulating activity of sydnone imines. Growth stimulating or antidote effects are typical for 4-α-hydroxybenzyl derivatives of sydnone imines containing an alkyl substituent in position N-3. The nature of the substituent of the N-6 atom has a significant influence on the activity profile and the intensity of the effect. Nevertheless, further investigations are necessary to establish reliable structure–activity relationships (SAR). Consequently, sydnone imines might be considered promising phytoeffector candidates, which are expected to exert either protective effects on plants growing under unfavorable conditions, or herbicidal ones, depending on the exact structure.
Publikation

Kappen, J.; Manurung, J.; Fuchs, T.; Vemulapalli, S. P. B.; Schmitz, L. M.; Frolov, A.; Agusta, A.; Muellner-Riehl, A. N.; Griesinger, C.; Franke, K.; Wessjohann, L. A.; Challenging structure elucidation of lumnitzeralactone, an ellagic acid derivative from the Mangrove Lumnitzera racemosa Mar. Drugs 21, 242, (2023) DOI: 10.3390/md21040242

The previously undescribed natural product lumnitzeralactone (1), which represents a derivative of ellagic acid, was isolated from the anti-bacterial extract of the Indonesian mangrove species Lumnitzera racemosa Willd. The structure of lumnitzeralactone (1), a proton-deficient and highly challenging condensed aromatic ring system, was unambiguously elucidated by extensive spectroscopic analyses involving high-resolution mass spectrometry (HRMS), 1D 1H and 13C nuclear magnetic resonance spectroscopy (NMR), and 2D NMR (including 1,1-ADEQUATE and 1,n-ADEQUATE). Determination of the structure was supported by computer-assisted structure elucidation (CASE system applying ACD-SE), density functional theory (DFT) calculations, and a two-step chemical synthesis. Possible biosynthetic pathways involving mangrove-associated fungi have been suggested.
Publikation

Degtyaryov, E.; Pigolev, A.; Miroshnichenko, D.; Frolov, A.; Basnet, A. T.; Gorbach, D.; Leonova, T.; Pushin, A. S.; Alekseeva, V.; Dolgov, S.; Savchenko, T.; 12-Oxophytodienoate reductase overexpression compromises tolerance to Botrytis cinerea in hexaploid and tetraploid wheat Plants 12, 2050, (2023) DOI: 10.3390/plants12102050

12-Oxophytodienoate reductase is the enzyme involved in the biosynthesis of phytohormone jasmonates, which are considered to be the major regulators of plant tolerance to biotic challenges, especially necrotrophic pathogens. However, we observe compromised tolerance to the necrotrophic fungal pathogen Botrytis cinerea in transgenic hexaploid bread wheat and tetraploid emmer wheat plants overexpressing 12-OXOPHYTODIENOATE REDUCTASE-3 gene from Arabidopsis thaliana, while in Arabidopsis plants themselves, endogenously produced and exogenously applied jasmonates exert a strong protective effect against B. cinerea. Exogenous application of methyl jasmonate on hexaploid and tetraploid wheat leaves suppresses tolerance to B. cinerea and induces the formation of chlorotic damages. Exogenous treatment with methyl jasmonate in concentrations of 100 µM and higher causes leaf yellowing even in the absence of the pathogen, in agreement with findings on the role of jasmonates in the regulation of leaf senescence. Thereby, the present study demonstrates the negative role of the jasmonate system in hexaploid and tetraploid wheat tolerance to B. cinerea and reveals previously unknown jasmonate-mediated responses.
Publikation

Farag, M. A.; Abdelwareth, A.; Zayed, A.; Eissa, T. F.; Dokalahy, E.; Frolov, A.; Wessjohann, L. A.; A comparative metabolomics approach for egyptian mango fruits classification based on UV and UPLC/MS and in relation to its antioxidant effect Foods 11, 2127, (2022) DOI: 10.3390/foods11142127

Mango (Mangifera indica L.) is a tropical climacteric fruit that encompasses a myriad of metabolites mediating for its nutritive value, unique taste, flavor, and medicinal uses. Egypt is among the top mango producers worldwide, albeit little characterization has been made toward its fruits’ chemical composition. This study aims to assess metabolites difference via comparative profiling and fingerprinting of Egyptian mango in context to its cultivar (cv.) type and/or growth province. To achieve such goal, hyphenated chromatographic techniques (UPLC/MS) and UV spectroscopy were employed and coupled to multivariate data analysis for Egyptian mango fruits’ classification for the first time. UPLC/MS led to the detection of a total of 47 peaks identified based on their elution times and MS data, belonging to tannins as gallic acid esters, flavonoids, xanthones, phenolic acids and oxylipids. UV/Vis spectra of mango fruits showed similar absorption patterns mostly attributed to the phenolic metabolites, i.e., gallic acid derivatives and phenolic acids showing λmax at ca. 240 and 270 nm. Modeling of both UPLC/MS and UV data sets revealed that cv. effect predominated over geographical origin in fruits segregation. Awees (AS) cv. showed the richest phenolic content and in agreement for its recognition as a premium cv. of mango in Egypt. Results of total phenolic content (TPC) assay revealed that AS was the richest in TPC at 179.1 mg GAE/g extract, while Langara from Ismailia (LI) showed the strongest antioxidant effect at 0.41 mg TE/g extract. Partial least square modeling of UV fingerprint with antioxidant action annotated gallates as potential contributor to antioxidant effect though without identification of exact moieties based on UPLC/MS. The study is considered the first-time investigation of Egyptian mango to aid unravel phytoconstituents responsible for fruits benefits using a metabolomics approach.
Publikation

Balarynová, J.; Klčová, B.; Sekaninová, J.; Kobrlová, L.; Cechová, M. Z.; Krejčí, P.; Leonova, T.; Gorbach, D.; Ihling, C.; Smržová, L.; Trněný, O.; Frolov, A.; Bednář, P.; Smýkal, P.; The loss of polyphenol oxidase function is associated with hilum pigmentation and has been selected during pea domestication New Phytol. 235, 1807–1821, (2022) DOI: 10.1111/nph.18256

Seed coats serve as protective tissue to the enclosed embryo. As well as mechanical there are also chemical defence functions. During domestication, the property of the seed coat was altered including the removal of the seed dormancy. We used a range of genetic, transcriptomic, proteomic and metabolomic approaches to determine the function of the pea seed polyphenol oxidase (PPO) gene. Sequencing analysis revealed one nucleotide insertion or deletion in the PPO gene, with the functional PPO allele found in all wild pea samples, while most cultivated peas have one of the three nonfunctional ppo alleles. PPO functionality cosegregates with hilum pigmentation. PPO gene and protein expression, as well as enzymatic activity, was downregulated in the seed coats of cultivated peas. The functionality of the PPO gene relates to the oxidation and polymerisation of gallocatechin in the seed coat. Additionally, imaging mass spectrometry supports the hypothesis that hilum pigmentation is conditioned by the presence of both phenolic precursors and sufficient PPO activity. Taken together these results indicate that the nonfunctional polyphenol oxidase gene has been selected during pea domestication, possibly due to better seed palatability or seed coat visual appearance.
Publikation

Leonova, T.; Ihling, C.; Saoud, M.; Frolova, N.; Rennert, R.; Wessjohann, L. A.; Frolov, A.; Does filter-aided sample preparation provide sufficient method linearity for quantitative plant shotgun proteomics? Front. Plant Sci. 13, 874761, (2022) DOI: 10.3389/fpls.2022.874761

Due to its outstanding throughput and analytical resolution, gel-free LC-based shotgun proteomics represents the gold standard of proteome analysis. Thereby, the efficiency of sample preparation dramatically affects the correctness and reliability of protein quantification. Thus, the steps of protein isolation, solubilization, and proteolysis represent the principal bottleneck of shotgun proteomics. The desired performance of the sample preparation protocols can be achieved by the application of detergents. However, these compounds ultimately compromise reverse-phase chromatographic separation and disrupt electrospray ionization. Filter-aided sample preparation (FASP) represents an elegant approach to overcome these limitations. Although this method is comprehensively validated for cell proteomics, its applicability to plants and compatibility with plant-specific protein isolation protocols remain to be confirmed. Thereby, the most important gap is the absence of the data on the linearity of underlying protein quantification methods for plant matrices. To fill this gap, we address here the potential of FASP in combination with two protein isolation protocols for quantitative analysis of pea (Pisum sativum) seed and Arabidopsis thaliana leaf proteomes by the shotgun approach. For this aim, in comprehensive spiking experiments with bovine serum albumin (BSA), we evaluated the linear dynamic range (LDR) of protein quantification in the presence of plant matrices. Furthermore, we addressed the interference of two different plant matrices in quantitative experiments, accomplished with two alternative sample preparation workflows in comparison to conventional FASP-based digestion of cell lysates, considered here as a reference. The spiking experiments revealed high sensitivities (LODs of up to 4 fmol) for spiked BSA and LDRs of at least 0.6 × 102. Thereby, phenol extraction yielded slightly better recoveries, whereas the detergent-based method showed better linearity. Thus, our results indicate the very good applicability of FASP to quantitative plant proteomics with only limited impact of the protein isolation technique on the method’s overall performance.
Publikation

Leonova, T.; Shumilina, J.; Kim, A.; Frolova, N.; Wessjohann, L.; Bilova, T.; Frolov, A.; Agar-based polyethylene glycol (PEG) infusion model for pea (Pisum sativum L.) — perspectives of translation to legume crop plants Biol. Commun. 67, 236-244, (2022) DOI: 10.21638/spbu03.2022.309

Due to the oncoming climate changes water deficit represents one of the most important abiotic stressors which dramatically affects crop productivity worldwide. Because of their importance as the principal source of food protein, legumes attract a special interest of plant scientists. Moreover, legumes are involved in symbiotic association with rhizobial bacteria, which is morphologically localized to root nodules. These structures are critical for fixation of atmospheric nitrogen and highly sensitive to drought. Therefore, new drought-tolerant legume cultivars need to be developed to meet the growing food demand. However, this requires a comprehensive knowledge of the molecular mechanisms behind the plant stress response. To access these mechanisms, adequate and reliable drought stress models need to be established. The agar-based polyethylene glycol (PEG) infusion model allows a physiologically relevant reduction of soil water potential (Ψw), although it is restricted to seedlings and does not give access to proteomics and metabolomics studies. Earlier, we successfully overcame this limitation and optimized this model for mature Arabidopsis plants. Here we make the next step forward and address its application to one of the major crop legumes — pea. Using a broad panel of physiological and biochemical markers, we comprehensively prove the applicability of this setup to legumes. The patterns of drought-related physiological changes are well-interpretable and generally resemble the stress response of plants grown in soil-based stop-watering models. Thus, the proposed model can be efficiently used in the study of stress-related metabolic adjustment in green parts, roots and root nodules of juvenile and flowering plants.
Publikation

Hegazi, N. M.; Khattab, A. R.; Frolov, A.; Wessjohann, L. A.; Farag, M. A.; Authentication of saffron spice accessions from its common substitutes via a multiplex approach of UV/VIS fingerprints and UPLC/MS using molecular networking and chemometrics Food Chem. 367, 130739, (2022) DOI: 10.1016/j.foodchem.2021.130739

Saffron is a spice revered for its unique flavor and health attributes often subjected to fraudulence. In this study, molecular networking as a visualization tool for UPLC/MS dataset of saffron and its common substitutes i.e. safflower and calendula (n = 21) was employed for determining genuineness of saffron and detecting its common substitutes i.e. safflower and calendula. Saffron was abundant in flavonol-O-glycosides and crocetin esters versus richness of flavanones/chalcones glycosides in safflower and cinnamates/terpenes in calendula. OPLS-DA identified differences in UPLC/MS profiles of different saffron accessions where oxo-hydroxy-undecenoic acid-O-hexoside was posed as saffron authentication marker and aided in discrimination between Spanish saffron of high quality from its inferior grade i.e. Iranian saffron along with crocetin di-O-gentiobiosyl ester and kaempferol-O-sophoroside. Kaempferol-O-neohesperidoside and N,N,N,-p-coumaroyl spermidine were characteristic safflower metabolites, whereas, calendulaglycoside C and di-O-caffeoyl quinic acid were unique to calendula. UV/VIS fingerprint spectral regions of picrocrocin (230–260 nm) and crocin derivatives (400–470 nm) were posed as being discriminatory of saffron authenticity and suggestive it can replace UPLC/MS in saffrom quality determination.
Publikation

Soboleva, A.; Frolova, N.; Bureiko, K.; Shumilina, J.; Balcke, G. U.; Zhukov, V. A.; Tikhonovich, I. A.; Frolov, A.; Dynamics of Reactive Carbonyl Species in Pea Root Nodules in Response to Polyethylene Glycol (PEG)-Induced Osmotic Stress Int. J. Mol. Sci. 23, 2726, (2022) DOI: 10.3390/ijms23052726

Drought dramatically affects crop productivity worldwide. For legumes this effect is especially pronounced, as their symbiotic association with rhizobia is highly-sensitive to dehydration. This might be attributed to the oxidative stress, which ultimately accompanies plants’ response to water deficit. Indeed, enhanced formation of reactive oxygen species in root nodules might result in up-regulation of lipid peroxidation and overproduction of reactive carbonyl compounds (RCCs), which readily modify biomolecules and disrupt cell functions. Thus, the knowledge of the nodule carbonyl metabolome dynamics is critically important for understanding the drought-related losses of nitrogen fixation efficiency and plant productivity. Therefore, here we provide, to the best of our knowledge, for the first time a comprehensive overview of the pea root nodule carbonyl metabolome and address its alterations in response to polyethylene glycol-induced osmotic stress as the first step to examine the changes of RCC patterns in drought treated plants. RCCs were extracted from the nodules and derivatized with 7-(diethylamino)coumarin-3-carbohydrazide (CHH). The relative quantification of CHH-derivatives by liquid chromatography-high resolution mass spectrometry with a post-run correction for derivative stability revealed in total 194 features with intensities above 1 × 105 counts, 19 of which were down- and three were upregulated. The upregulation of glyceraldehyde could accompany non-enzymatic conversion of glyceraldehyde-3-phosphate to methylglyoxal. The accumulation of 4,5-dioxovaleric acid could be the reason for down-regulation of porphyrin metabolism, suppression of leghemoglobin synthesis, inhibition of nitrogenase and degradation of legume-rhizobial symbiosis in response to polyethylene glycol (PEG)-induced osmotic stress effect. This effect needs to be confirmed with soil-based drought models.
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