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.

In recent decades, heavy metal pollution has become a significant environmental stress factor. Plants are characterized by high biochemical plasticity and can adjust their metabolism to ensure survival under a changing environment. Here we report, to our knowledge, the first gas chromatography-mass spectrometry (GC-MS)-based metabolomics study of Zn-induced stress responses in Amaranthus caudatus plants. The study was performed with root and leaf aqueous methanolic extracts after their lyophilization and sequential derivatization with methoxylamine hydrochloride and N-methyl-N-(trimethylsilyl)trifluoroacetamide. In total, 419 derivatives were detected in the samples, and 144 of them could be putatively annotated. The metabolic shifts in seven-week-old A. caudatus plants in response to a seven-day treatment with 300 µmol/L ZnSO₄·7H₂O in nutrient solution were organ-specific and more pronounced in roots. Most of the responsive metabolites were up-regulated and dominated by sugars and sugar acids. The revealed effects could be attributed to the involvement of these metabolites in osmotic regulation, antioxidant protection and Zn²⁺ complexation. A 59-fold up-regulation of gluconic acid in roots distinctly indicated enhanced glucose oxidation due to oxidative stress upon the Zn treatment. Gluconic acid might be further employed in Zn²⁺ complexation. Pronounced Zn-induced up-regulation of salicylic acid in roots and shoots suggested a key role of this hormone in stress signaling and activation of Zn stress tolerance mechanisms. Overall, our study provides the first insight into the general trends of Zn-induced biochemical rearrangements and main adaptive metabolic shifts in A. caudatus.

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.

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.

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.

It is known that the progressive climate changes and environmental pollution prevalent all over the world lead to increasingly frequent and prolonged episodes of stress, and it challenges plants to adapt to changing adverse conditions. Given the significant crop losses caused by challenging environments, protecting plants from the destructive effects of these factors has become one of the most pressing challenges in modern agricultural biology and plant physiology. One of the approaches to protect plants from stressors in modern agrobiotechnology is the use of nitric oxide (NO) donors, which are chemicals that can release NO in plant cells and stimulate their germination, growth, biomass accumulation, and protection against external factors. Studying their mechanism of action at the molecular level is essential for understanding the targets of NO action, as well as for developing new phytoeffectors. A multiomics approach, along with physiological data, is often the method of choice. In this review, we compare widely used NO donors (e.g., sodium nitroprusside, S-nitrosoglutathione, NONOates) in terms of chemical properties, NO-release kinetics, stability, and suitability for different application methods (seed treatment, foliar spray, root application). Finally, we highlight recent advances from proteomics, metabolomics, and transcriptomics that have enabled system-wide identification of NO-responsive pathways and candidate targets. By integrating physiological, biochemical, and multiomics data, this review provides a framework for understanding NO-mediated stress tolerance and for the rational design of new NO-based phytoeffectors.