Drought is one of the major stress factors affecting the growth and development of plants. In this context, drought-related losses of crop plant productivity impede sustainable agriculture all over the world. In general, plants respond to water deficits by multiple physiological and metabolic adaptations at the molecular, cellular, and organism levels. To understand the underlying mechanisms of drought tolerance, adequate stress models and arrays of reliable stress markers are required. Therefore, in this review we comprehensively address currently available models of drought stress, based on culturing plants in soil, hydroponically, or in agar culture, and critically discuss advantages and limitations of each design. We also address the methodology of drought stress characterization and discuss it in the context of real experimental approaches. Further, we highlight the trends of methodological developments in drought stress research, i.e., complementing conventional tests with quantification of phytohormones and reactive oxygen species (ROS), measuring antioxidant enzyme activities, and comprehensively profiling transcriptome, proteome, and metabolome.

Drought is one of the major stress factors affecting growth and development of plants. In this context, drought-related losses of crop plant productivity impede sustainable agriculture all over the world. In general, plants responses to water deficit by multiple physiological and metabolic adaptations at the molecular, cellular and organism levels. To understand the underlying mechanisms of drought tolerance, adequate stress models and arrays of reliable stress markers are required. Therefore, in this review we comprehensively address currently available models of drought stress, based on culturing plants in soil, hydroponic or agar culture. These experimental setups give access to different aspects of plant response to drought, like decrease of tissue water potential, reduction of stomata conductance and photosynthesis efficiency, accumulation of low-molecular weight solutes (metabolic adjustment) and drought protective proteins. Till now, this pattern of markers was successfully extended to the methods of enzyme chemistry, molecular biology and omics techniques. Thus, conventional tests can be efficiently complemented by determination of phytohormone and reactive oxygen species (ROS) contents, activities of antioxidant enzymes, as well as comprehensive profiling of transcriptome, proteome and metabolome.

Glycation is a non-enzymatic post-translational modification of proteins, formed by the reaction of reducing sugars and α-dicarbonyl products of their degradation with amino and guanidino groups of proteins. Resulted early glycation products are readily involved in further transformation, yielding a heterogeneous group of advanced glycation end products (AGEs). Their formation is associated with ageing, metabolic diseases, and thermal processing of foods. Therefore, individual glycation adducts are often considered as the markers of related pathologies and food quality. In this context, their quantification in biological and food matrices is required for diagnostics and establishment of food preparation technologies. For this, exhaustive protein hydrolysis with subsequent amino acid analysis is the strategy of choice. Thereby, multi-step enzymatic digestion procedures ensure good recoveries for the most of AGEs, whereas tandem mass spectrometry (MS/MS) in the multiple reaction monitoring (MRM) mode with stable isotope dilution or standard addition represents “a gold standard” for their quantification. Although the spectrum of quantitatively assessed AGE structures is continuously increases, application of untargeted profiling techniques for identification of new products is desired, especially for in vivo characterization of anti-glycative systems. Thereby, due to a high glycative potential of plant metabolites, more attention needs to be paid on plant-derived AGEs.

Type 2 diabetes mellitus (T2DM) is one of the most widely spread metabolic diseases. Because of its asymptomatic onset and slow development, early diagnosis and adequate glycaemic control are the prerequisites for successful T2DM therapy. In this context, individual amino acid residues might be sensitive indicators of alterations in blood glycation levels. Moreover, due to a large variation in the half-life times of plasma proteins, a generalized biomarker, based on multiple glycation sites, might provide comprehensive control of the glycemic status across any desired time span. Therefore, here, we address the patterns of glycation sites in highly-abundant blood plasma proteins of T2DM patients and corresponding age- and gender-matched controls by comprehensive liquid chromatography-mass spectrometry (LC-MS). The analysis revealed 42 lysyl residues, significantly upregulated under hyperglycemic conditions. Thereby, for 32 glycation sites, biomarker behavior was demonstrated here for the first time. The differentially glycated lysines represented nine plasma proteins with half-lives from 2 to 21 days, giving access to an integrated biomarker based on multiple protein-specific Amadori peptides. The validation of this biomarker relied on linear discriminant analysis (LDA) with random sub-sampling of the training set and leave-one-out cross-validation (LOOCV), which resulted in an accuracy, specificity, and sensitivity of 92%, 100%, and 85%, respectively.

Protein glycation is a ubiquitous non-enzymatic post-translational modification, formed by reaction of protein amino and guanidino groups with carbonyl compounds, presumably reducing sugars and α-dicarbonyls. Resulting advanced glycation end products (AGEs) represent a highly heterogeneous group of compounds, deleterious in mammals due to their pro-inflammatory effect, and impact in pathogenesis of diabetes mellitus, Alzheimer’s disease and ageing. The body of information on the mechanisms and pathways of AGE formation, acquired during the last decades, clearly indicates a certain site-specificity of glycation. It makes characterization of individual glycation sites a critical pre-requisite for understanding in vivo mechanisms of AGE formation and developing adequate nutritional and therapeutic approaches to reduce it in humans. In this context, proteomics is the methodology of choice to address site-specific molecular changes related to protein glycation. Therefore, here we summarize the methods of Maillard proteomics, specifically focusing on the techniques providing comprehensive structural and quantitative characterization of glycated proteome. Further, we address the novel break-through areas, recently established in the field of Maillard research, i.e., in vitro models based on synthetic peptides, site-based diagnostics of metabolism-related diseases (e.g., diabetes mellitus), proteomics of anti-glycative defense, and dynamics of plant glycated proteome during ageing and response to environmental stress.

Protein glycation is usually referred to as an array of non-enzymatic post-translational modifications formed by reducing sugars and carbonyl products of their degradation. The resulting advanced glycation end products (AGEs) represent a heterogeneous group of covalent adducts, known for their pro-inflammatory effects in mammals, and impacting on pathogenesis of metabolic diseases and ageing. In plants, AGEs are the markers of tissue ageing and response to environmental stressors, the most prominent of which is drought. Although water deficit enhances protein glycation in leaves, its effect on seed glycation profiles is still unknown. Moreover, the effect of drought on biological activities of seed protein in mammalian systems is still unstudied with respect to glycation. Therefore, here we address the effects of a short-term drought on the patterns of seed protein-bound AGEs and accompanying alterations in pro-inflammatory properties of seed protein in the context of seed metabolome dynamics. A short-term drought, simulated as polyethylene glycol-induced osmotic stress and applied at the stage of seed filling, resulted in the dramatic suppression of primary seed metabolism, although the secondary metabolome was minimally affected. This was accompanied with significant suppression of NF-kB activation in human SH-SY5Y neuroblastoma cells after a treatment with protein hydrolyzates, isolated from the mature seeds of drought-treated plants. This effect could not be attributed to formation of known AGEs. Most likely, the prospective anti-inflammatory effect of short-term drought is related to antioxidant effect of unknown secondary metabolite protein adducts, or down-regulation of unknown plant-specific AGEs due to suppression of energy metabolism during seed filling.