Tatiana Leonova

St. John’s wort is a medicinal plant of significant pharmaceutical importance, yet its metabolic composition and biological activity vary considerably depending on genetic and environmental factors. Understanding the interplay between genotype and growing conditions is essential for ensuring reproducible therapeutic properties of herbal raw materials. This study aimed to comprehensively evaluate the effects of genotype and growing location on the metabolite profiles and antibacterial/antioxidant activities of H. perforatum. Seven accessions from the VIR collection were grown in two climatically distinct regions of Russia: the temperate zone (Pushkin, Leningrad Region) and a warm region (Maykop, Republic of Adygea). Methanolic extracts were analyzed using LC-MS/MS, yielding 89 annotated metabolites belonging to 8 major compound classes. Antibacterial activity was assessed against Gram-positive (Staphylococcus aureus, Micrococcus luteus) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa) strains, while antioxidant capacity was evaluated using DPPH and TEAC assays. PCA identified cultivation location (36.7%) and genotype (14.8%) as the major sources of metabolic variation, with a significant location × cultivar interaction detected for 70.2% of all analysed features. Naphthodianthrones and procyanidins consistently accumulated in the shoots of H. perforatum plants grown in the temperate region, while flavonoids and acylphloroglucinols exhibited genotype-dependent responses. Extracts from the plants grown in Pushkin showed more pronounced antibacterial activity, particularly against M. luteus and E. coli, and higher antioxidant capacity compared to the Maykop-grown plants, correlating with elevated levels of bioactive compounds. Genotype k-14 displayed the most pronounced metabolic plasticity. The metabolite profiles and bioactivity of H. perforatum are associated with complex genotype-environment interactions. These findings highlight the importance of considering both genetic background and cultivation location for the production of standardized herbal materials with more consistent pharmacological properties.

Due to oncoming climate changes, drought is recognized as one of the most important factors of crop yield losses. Water stress differentially affects plant longevity and productivity depending on the ontogenesis step. Specifically, drought occurring at the reproductive stage can reduce crop productivity and the quality of harvested seeds. Therefore, breeding new cultivars preserving both productivity and seed quality under drought conditions is urgently mandatory. However, no straightforward and reliable markers of drought tolerance are established for this ontogenesis step so far. It is generally unclear whether genetic differences between genotypes known as drought-tolerant and drought-sensitive can be traced in performance of the progeny mature seeds and their metabolic responses after rewatering. Therefore, here we address physiological, reproductive and seed metabolic responses in 29 pea genotypes varying in the degree of documented drought tolerance. The plants were subjected to transient irrigation withdrawal during pod formation. Based on the physiological data, metabolomes of nine most contrasting genotypes were profiled by GC-MS. The results revealed strong genotype-dependency of the stress-induced metabolic shifts, which only partly matched the physiological data. Thus, only complementation of physiological plant and seed traits with metabolic data might give access to interpretation of drought tolerance in grain legumes.

Fine-tuning of the immune response plays a key role in legume-rhizobial symbiosis. Rhizobial Nod factors can suppress the defense responses during symbiosis, but the possible mechanisms of such regulation remain poorly understood. Here, we observe that Nod factors effectively suppress the expression of genes encoding defense markers (WRKYs, PRs, PALs), the reactive oxygen species (ROS) formation, and reduce the content of pattern recognition receptor (PRR) LYK9 induced by treatment with deacetylated chitooligosaccharide CO8-DA in pea roots. Since PRR LYK9 may recognize both chitin/COs and peptidoglycan, it likely plays an important role in the activation of defense responses during rhizobial inoculation. To identify potential regulators through which Nod factors suppress the immune response in plants during symbiosis with rhizobia, proteome and transcriptome analyses were performed. This allowed identifying several potential candidates activated by Nod factors, such as superoxide dismutase and catalase enzymes, which prevent excessive ROS accumulation and the development of oxidative stress. We also found ubiquitin ligases and ubiquitin-conjugating enzymes that may target PRRs activated in response to rhizobial inoculation. LYK9 degradation via ubiquitinylation was shown to prevent a hypersensitive response in plants. Nod factors activate enzymes involved in jasmonic acid biosynthesis, which in turn activates the transcription factor ABR1, suppressing the abscisic acid-induced responses and decreasing the immune response. Finally, we showed that LysM-receptor-like kinases PsLYK11/MtLYK11, probable homologs of Arabidopsis AtLYK3 in pea and Medicago, are involved in regulation of the immune response.

Due to oncoming climate changes, droughts, high salinity, extreme temperatures became quite common stressors universally occurring in the most of terrestrial habitats. Expansions of these changes are often accompanied with strong herbivore attacks. Due to the outstanding impact of these factors on sustainable agriculture, since several last decades, the biochemistry and molecular biology of plant stress response remains in the focus of the research interest worldwide. Thus, bottom-up proteomics became a versatile tool of plant research in general and of stress biology in particular. As plant-derived materials are recognized as an extremely complex matrix, which is rich in polysaccharides, polyphenols and hardly water-soluble proteins, their proteome is typically analyzed by gel-based techniques. However, recent advances in sample preparation techniques (first of all — protein solubilization and digestion) allowed establishment of gel-free methods for plant-derived samples. Implementation of high-throughput nano-flow reversed phase-high performance liquid chromatography coupled on-line to electrospray ionization mass spectrometry (nanoRP-HPLC-ESI-MS) gave access to data-rich datasets giving high protein identification rates. Moreover, high reproducibility of HPLC allows highly sensitive and precise quantification. Therefore, over the recent decade, shotgun proteomics became the method of choice in the study of adaptive stress responses of plant proteome. Here we address the bottom-up shotgun proteomics strategy in plant biology and discuss its application to the study of plant stress response. We also discuss the main steps of the plant proteome analysis pipeline and address emerging problems and future perspectives.

Drought poses a significant challenge to the sustainable development of modern agriculture and to the achievement of high crop yields. Water deficit causes osmotic stress and triggers plant physiological responses characterized by reduced water potential, diminished stomatal conductance, and decreased photosynthetic efficiency. Long-term adaptation to osmotic stress entails intricate metabolic rearrangements, leading to the accumulation of osmoprotectants, activation of antioxidant systems, and increased biosyntheses of stress-protective proteins. The severity and duration of drought, along with plant genotype and developmental stage, influence the plant response to stress, consequently affecting crop yield and quality. Particularly in the context of legumes, which are crucial for human and animal nutrition, understanding adaptive strategies to water deficit is essential for the cultivation of drought-resistant genotypes, primarily because these crops predominantly thrive in semi-arid regions. Proteomics and metabolomics approaches, in turn, serve as valuable tools, offering critical insights into the molecular dynamics governing plant responses to drought stress. Furthermore, the use of reliable drought simulation models is imperative for the effective evaluation of legume response to water scarcity, aiding the cultivation of drought-tolerant varieties. This review highlights the perspectives of utilizing different osmotic stress models to investigate proteome and metabolome alteration within seeds of food legumes.

In light of recent climate change, with its rising temperatures and precipitation changes, we are facing the need to increase the valuable crop’s tolerance against unfavorable environmental conditions. Emmer wheat is a cereal crop with high nutritional value. We investigated the possibility of improving the stress tolerance of emmer wheat by activating the synthesis of the stress hormone jasmonate by overexpressing two genes of the jasmonate biosynthetic pathway from Arabidopsis thaliana, ALLENE OXIDE SYNTHASE (AtAOS) and OXOPHYTODIENOATE REDUCTASE 3 (AtOPR3). Analyses of jasmonates in intact and mechanically wounded leaves of non-transgenic and transgenic plants showed that the overexpression of each of the two genes resulted in increased wounding-induced levels of jasmonic acid and jasmonate-isoleucine. Against all expectations, the overexpression of AtAOS, encoding a chloroplast-localized enzyme, does not lead to an increased level of the chloroplast-formed 12-oxo-phytodienoic acid (OPDA), suggesting an effective conversion of OPDA to downstream products in wounded emmer wheat leaves. Transgenic plants overexpressing AtAOS or AtOPR3 with increased jasmonate levels show a similar phenotype, manifested by shortening of the first and second leaves and elongation of the fourth leaf, as well as increased tolerance to osmotic stress induced by the presence of the polyethylene glycol (PEG) 6000.

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.

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 (Ψ_w), 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. agar-based PEG infusion model, biochemical stress markers, drought, osmotic stress, oxidative stress, physiological stress markers, polyethylene glycol (PEG)

Summary: 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.