Tatiana Bilova

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.

During drought, numerous compounds accumulate in plant tissues, but their physiological roles remain unclear – they may function as osmolytes, osmoprotectants, or merely arise as by-products of stress-induced metabolic shifts. We developed an experimental approach to link accumulation patterns with specific functions, using Scots pine ( Pinus sylvestris L.) saplings subjected to water deprivation and subsequent rewatering as a model system. We monitored changes in relative water content (RWC) and osmotic adjustment dynamics, employed untargeted primary metabolite profiling for preliminary screening of compounds correlated with water status, and performed quantitative GC-MS and LC-MS analyses of selected metabolites. Major inorganic cations (K⁺, Ca²⁺, Mg²⁺) were also quantified to assess their potential roles. Our results revealed that tryptophan, valine, and lysine – though generally present in low abundance – exhibited selective accumulation under severely reduced RWC (≤ 70%), suggesting their involvement as osmoprotectants. Major organic acids, particularly shikimic acid, showed trends consistent with osmotic adjustment. Notably, neither sucrose nor inorganic cations appeared to function as primary osmolytes in this context. The proposed approach offers a viable strategy for identifying compounds involved in plant adaptation to water deficit, with potential applications in breeding programs aimed at improving drought tolerance. Highlights: An approach to identify osmolytes and osmoprotectants was implemented Accumulation of Trp, Val and Lys was consistent with their role in osmoprotection Osmotic adjustment relied predominantly on organic acids than on inorganic ions Monosaccharides but not sucrose correlates with changes in needle water status

Decades ago, the introduction of GC-MS marked a significant advancement in primary plant metabolite studies. Here, in our review, we will delve into critical aspects of the workflow, spanning the selection of an analytical platform, sample preparation, analytical acquisition, and data processing and interpretation. The exceptional separation capabilities of GC, characterized by remarkable chromatographic resolution, render it ideal for analysis of the complex plant metabolome, including the separation of isomeric compounds. The diversity of analytical platforms allows the investigation of plant metabolomes using targeted and non-targeted approaches. GC-MS, equipped with efficient extraction methods and reliable derivatization protocols for semi- and non-volatile compounds, enables qualitative and quantitative analysis of these molecules. The stability of derivatives forms the foundation for the robustness and reproducibility of GC-MS methods, and their mass spectra provide characteristic fragments for confident identification and sensitive quantification of individual metabolites. There has been key progress in the advancement of GC-MS approaches to studying plant metabolism. However, the presence of artifacts during GC-MS analysis, particularly during derivatization, is a challenge that requires careful validations, which frequently necessitate additional investigations. The feasible solutions that were achieved to overcome the limitations in GC-MS-based studies are a particular focus of the present discussion.

Innovative developments of GC-MS over the last two decades made this methodology a powerful tool for profiling a broad range of volatile metabolites and non-volatile ones of non-polar, semi-polar and even polar nature after appropriate derivatization. Indeed, the high potential of GC-MS in the analysis of low molecular weight metabolites involved in essential cellular functions (energy production, metabolic adjustment, signaling) made it the method of choice for the life and plant scientists. However, despite these advances, due to their intrinsic thermal lability, multiple classes of hydrophilic low-molecule weight metabolites (like nucleotides, sugar phosphates, cofactors, CoA esters) are unsuitable under the high-temperature conditions of the split–splitless (SSL) injection and GC separation, which makes the analysis of such compounds by GC-MS challenging. Therefore, to ensure comprehensive coverage of the plant metabolome, the GC-MS-based metabolomics platform needs to be efficiently combined with other metabolomics techniques and instrumental strategies. Moreover, to get a deeper insight into dynamics of plant cell metabolism in response to endogenic and exogenic clues, integration of the metabolomics data with the output obtained from other post-genomics techniques is desired. Therefore, here, we overview different strategies for the integration of the GC-MS-based metabolite profiling output with the data, acquired by other metabolomics techniques in terms of the multi-platform metabolomics approach. Further, we comprehensively discuss the implementation of the GC-MS-based metabolomics in multi-omics strategies and the data integration strategies behind this. This approach is the promising strategy, as it gives deep and multi-level insight into physiological processes in plants in the systems biology context, with consideration of all levels of gene expression. However, multiple challenges may arise in the way of integrating data from different omics technologies, which are comprehensively discussed in this review.

Metabolomics, which is typically referred to as the post-genomic methodology addressing low-molecular-weight metabolites, became a powerful tool in post-genomic research over the last two decades. Indeed, the state-of-the-art metabolomics relies on several well-established complementary platforms—nuclear magnetic resonance (NMR) spectroscopy, liquid and gas chromatography coupled on-line with mass spectrometry (LC- and GC-MS, respectively), and capillary electrophoresis–mass spectrometry (CE-MS). Among them, GC-MS represents one of the oldest and most well-established techniques currently employed in the metabolomics of volatile compounds and non-volatiles—polar low-molecular-weight metabolites, which can be efficiently converted in volatile form by comprehensive derivatization of polar functional groups. Currently, GC-MS is established as the principal analytical method for characterizing primary plant metabolism, although other methods also contribute significantly to determining the complete metabolite profile. Therefore, here, we address the role of GC-MS in plant metabolomics and its potential for the profiling of low-molecular-weight metabolites. Further, we comprehensively review the methods of sample preparation with special emphasis on extraction and derivatization approaches, which are currently employed to improve the method performance and its metabolome coverage.

Species Pseudomonas brassicacearum was described both as plant growth-promoting (PGP) rhizobacterium and necrotrophic opportunistic phytopathogen. An important PGP property of this rhizobacterium is the activity of 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which reduces abiotic stress in plants. Tomatoes are an important but drought-sensitive agricultural crop. Growth and physiological and biochemical responses of well-watered and water-limited tomatoes to such kind of rhizobacteria were addressed using Ps. brassicacearum Am3 or its ACC deaminase deficient mutant T8-1. The counts of the introduced P. brassicacearum Am3 and T8-1 in the tomato rhizosphere at the end of experiment were similar under both water regimes. The applied drought and inoculations did not yield differences in the content of water, chlorophyll, indole-3-acetic and indole-3-lactic acids in leaves. Drought reduced stomatal conductance and inhibited shoot growth, whereas only wild type Am3 maintained shoot biomass on the level of well-watered plants. Strain Am3 lowered difference in the contents of thiobarbithuric acid reactive substances (expressed as malondialdehyde equivalents) between well-watered and droughted plants. This strain also decreased abscisic acid content in roots, as well as hydrogen peroxide, salicylic and indole-carboxylic acid contents in leaves of water-limited plants. The mutant T8-1 decreased content of abscisic and indole-3-lactic acids in roots and abscisic and indole-3-carboxylic acids in leaves of water-limited plants. Thus, under water-limitation Ps. brassicacearum Am3 having high ACC deaminase activity exhibited the properties of PGPR and stimulated plant growth, but the deficiency of ACC deaminase in T8-1 redirected the action of bacteria towards saprophytic or even deleterious effects.

Decades ago, the introduction of GC-MS marked a significant advancement in primary plant metabolites studies. Here, in Part 2 of the review, we will delve into critical aspects of the workflow, spanning the selection of an analytical platform, sample preparation, analytical acquisition, and data processing and interpretation. The exceptional separation capabilities of GC, characterized by remarkable chromatographic resolution, rendered it ideal for analysis of the complex plant metabolome, including the separation of isomeric compounds. The diversity in analytical platforms allows the investigation of plant metabolomes using targeted and non-targeted approaches. GC-MS, equipped with efficient extraction methods and reliable derivatization protocols for semi- and non-volatile compounds, enables qualitative and quantitative analysis of these molecules. The stability of derivatives forms the foundation for the robustness and reproducibility of GC-MS methods, and their mass spectra provide characteristic fragments for confident identification and sensitive quantification of individual metabolites. Ongoing advancements in all stages have elevated GC-MS-based metabolomics to an indispensable tool for unraveling the intricacies of plant metabolism. However, it is imperative to conduct careful validations and address potential artifacts at each step, especially derivatization, and further investigations of the aroused problems are needed.

Innovative developments of GC-MS over the last two decades made this methodology a powerful tool for profiling a broad range of volatile metabolites and non-volatile ones of non-polar, semi-polar and even polar nature after appropriate derivatization. Indeed, the high potential of GC-MS in the analysis of low molecular weight metabolites involved in essential cellular functions (energy production, metabolic adjustment, signaling) made it the method of choice for the life and plant scientists. However, despite these advances, due to their intrinsic thermal lability, multiple classes of hydrophilic low-molecule weight metabolites (like nucleotides, sugar phosphates, cofactors, CoA esters) are unsuitable under the high temperature conditions of the SSL injection and GC separation, that makes analysis of such compounds by GC-MS challenging. Therefore, to ensure comprehensive coverage of the plant metabolome, the GC-MS-based metabolomics platform needs to be efficiently combined with other metabolomics techniques and instrumental strategies. Moreover, to get a deeper insight into dynamics of plant cell metabolism in response to endogenic and exogenic clues, integration of the metabolomics data with the output obtained from other post-genomics techniques is desired. Therefore, here we overview different strategies for integration of the GC-MS-based metabolite profiling output with the data, acquired by other metabolomics techniques in terms of the multi-platform metabolomics approach. Further, we comprehensively discuss the implementation of the GC-MS-based metabolomics in multi-omics strategies and the data integration strategies behind this. We are convinced that this approach is the strategy of future, as it gives deep and multi-level insight into physiological processes in plants in the systems biology context with consideration of all levels of gene expression. However, multiple challenges may arise in the way of integrating data from different omics technologies, which are comprehensively discussed in this review.

Drought is a major threat to global food security, with climate change enhancing its severity. Living organisms employ various adaptation strategies against loss of water, with desiccation tolerance representing the most extreme ones. The present study provides an analysis of desiccation response mechanisms in Pisum sativum L., focusing on the early seedling development. The experiments were carried out on 3-day-old seedlings with radicle up to 20 mm and similar seedlings with cotyledons removed, each subjected to the 'desiccation and rehydration' treatment. We assessed stress responses using growth and electrolyte leakage tests, and by measuring ascorbate content and lipid peroxidation products. The results demonstrated that while all desiccated seedlings experienced oxidative stress, seedlings with cotyledons exhibited superior recovery capacity, e.g. maintaining membrane integrity, ascorbate status and the ability to form the adventitious roots. We also monitored the ABA-dependent stress response by registering expression of several genes associated with "response to water deprivation" (ABI3, ABI4, ABI5, HVA22, PER1, LEA14, RD22-1/2/3, LTI65, and LTP4). Intrudingly, 'desiccation and rehydration' treatment resulted in the 40-fold up-regulation of ABI5 in hypocotyls of seedlings both with removed and intact cotyledons and the 108-fold up-regulation of this gene only in roots of seedlings with intact cotyledons. We propose that the post-germination stage represents a ‘resilience window’ where embryonic protective mechanisms remain partially active while the plant prepares for autotrophic growth. Herewith, the role of cotyledons extends beyond their classical role as nutrient reserves by coordinating both developmental and stress responses under conditions of severe water stress.

Zinc excess (Zn stress) could lead to deleterious effects in plants such as enhanced ROS production, inhibition of photosynthetic machinery, and impairment of nutrient uptake. Hence, we aimed to investigate the complexity of metabolic responses to Zn stress in Amaranthus caudatus young and mature leaves, as well as in roots by means of proteomics. Our previous metabolomics research has indicated potential involvement of gluconate and salicylate in Zn tolerance mechanisms. However, proteomics study of metabolic adjustments underlying Zn stress tolerance can give additional insight to the issue, as a lot of enzymes are known to be affected by the excess of transitional metals. The results obtained through bottom-up proteomics were complementary to our earlier metabolomics data and, furthermore, enlightened other important details in the metabolic response of A. caudatus plants to the applied Zn stress. In particular, the significant involvement of redox-related enzymes was shown, especially for the roots, and their possible interactions with salicylate and jasmonate signaling could be proposed. Furthermore, Zn²⁺-induced changes in roots and young leaves strongly affected sugar metabolism, enhanced protein quality control system, while mature leaves were characterized by remarkable decrease in subunits of photosynthetic electron transport complexes. Thus, this work emphasizes massive metabolic reprogramming aimed to reinforce root defense responses while supporting young leaves with sugar metabolites. Mass spectrometry proteomics data are available via ProteomeXchange with identifier PXD069557.