Environmental stress is one of the major factors reducing crop productivity. Due to the oncoming climate changes, the effects of drought and high light on plants play an increasing role in modern agriculture. These changes are accompanied with a progressing contamination of soils with heavy metals. Independent of their nature, environmental alterations result in development of oxidative stress, i.e. increase of reactive oxygen species (ROS) contents, and metabolic adjustment, i.e. accumulation of soluble primary metabolites (amino acids and sugars). However, a simultaneous increase of ROS and sugar concentrations ultimately results in protein glycation, i.e. non-enzymatic interaction of reducing sugars or their degradation products (α-dicarbonyls) with proteins. The eventually resulting advanced glycation end-products (AGEs) are known to be toxic and pro-inflammatory in mammals. Recently, their presence was unambiguously demonstrated in vivo in stressed Arabidopsis thaliana plants. Currently, information on protein targets, modification sites therein, mediators and mechanisms of plant glycation are being intensively studied. In this chapter, we comprehensively review the methodological approaches for plant glycation research and discuss potential mechanisms of AGE formation under stress conditions. On the basis of these patterns and additional in vitro experiments, the pathways and mechanisms of plant glycation can be proposed.
- Advanced glycation end-products (AGEs)
- Environmental stress
1.1. Environmental stress, ROS and protein glycation
1.1.1. Environmental stress and ROS formation
Environmental stress is one of the major factors reducing the productivity of crop plants all over the world . Drought, high light, salinity and increased heavy metal soil contents, as well as extreme temperature, represent its important manifestations . On the physiological level, extreme environmental conditions ultimately results in decrease in the CO2 assimilation rate and in growth inhibition . Simultaneous accumulation of reduced equivalents results in an overload of the chloroplast and mitochondrial electron transport chains and enhanced production of the reactive oxygen species (ROS), i.e. singlet oxygen (1O2), superoxide radical anion (O2⋅–), peroxide ion (O22–), hydrogen peroxide (H2O2), various hydroperoxides and hydroxide radical (OH⋅) . When ROS production overwhelms their detoxication, oxidative stress develops .
Thus, transfer of electrons to molecular oxygen (O2) from ubisemiquinone in mitochondria and thylakoid membrane-bound primary electron acceptor of photosystem I (PSI) in chloroplasts yields O2⋅– (and, when further reduction occurs, O22–), further converted to H2O2 by superoxide dismutase (SOD) activity (predominantly Mn- and CuZn-SOD in mitochondria and chloroplasts, respectively) [6,7]. The radical oxygen species can abstract protons from (bis-)allylic methylenes of polyunsaturated fatty acids (PUFAs) . The subsequent capture of O2 molecule by the resulting carbon-centered radical yields a peroxyl radical, that is able to initiate a chain reaction of lipid peroxidation . The PUFAs can be directly attacked by the protonated form of O2⋅– (HO2⋅) , thus the content of lipid hydroperoxides is one of the most reliable markers of oxidative stress.
The hydroperoxides can be easily involved in the Fenton reaction, i.e. transition metal ion-mediated reduction, yielding OH⋅, i.e. one of the most short-living and toxic ROS, directly and irreversibly modifying lipids, proteins and nucleic acids . The metal ions oxidized during Fenton reaction are reduced
1.1.2. Protein glycation
In the first step of this process (usually termed “early glycation”), reducing sugars, aldoses and ketoses reversibly interact with amino groups resulting in the very labile N/O-acetal intermediates: aldoamines and ketoamines, respectively (Figure 1). These compounds easily condense yielding aldimines and ketoimines (Schiff bases), which undergo Amadori  and Heyns  rearrangements, variants of the acyloin shift. Amadori rearrangement involves proton transfer from C1 to C2 via the enol/enamine intermediate yielding
These early glycation products, as well as free sugars, readily autoxidize (by the mechanisms similar to those described for free sugars) with formation of highly reactive α-dicarbonyl compounds (presumably glyoxal, methylglyoxal and various osones) – potent reactive intermediates of advanced glycation . Depending on the structure of the carbohydrate moiety involved in this degradation, i.e. free sugars, or protein-bound early glycation products, two principle advanced glycation pathways, namely “oxidative glycosylation” and “glycoxidation”, respectively, are distinguished [14,22,23]. The interaction of α-dicarbonyls with lysyl amino and arginyl guanidino side chain groups results in formation of so-called advanced glycation end-products (AGEs) – protein Maillard reaction compounds accumulating during thermal processing of food (Figure 2) , but also endogenously, e.g. under the conditions of persisting hyperglycemia.
1.1.3. Advanced Glycation End-products (AGEs)
AGEs represent a highly heterogenic group of compounds, varying greatly in their stability. Thus, the term “advanced glycation end-products” is, to high extent, conventional: some AGEs are still reactive and can be easily involved in further reactions . In the past few decades, several AGEs were comprehensively characterized (Figure 2). Among lysine-derived modifications,
Upon their absorption in human intestine, AGEs interact with endothelial and macrophage pattern recognition receptors for AGEs (e.g. RAGEs) and trigger NF-κB-mediated expression of pro-inflammatory species (e.g. adhesion molecules, including vascular cell adhesion molecule-1 and intercellular adhesion molecule-1)  and foster the development of inflammatory diseases – e.g. atherosclerosis and type 2 diabetes mellitus . AGEs of different chemical structure and origin, most often CML, pentosidine and hydroimidazolones, are known to be the ligands of RAGEs and to trigger inflammatory response .
Surprisingly, other reports showed mammalian serum and urinary concentrations of AGEs to be independent from dietary intake of thermally processed foods . Moreover, the levels of CML and fluorescent AGEs in the plasma of vegetarian individuals were higher in comparison to those in the omnivorous individuals , even though the vegetarian diet had lower contents of lysine- and arginine-containing proteins. Remarkably, this effect was stronger in plasma of long-term vegetarians . These facts indicate a high relevance of protein glycation (both early and advanced) in plants. Obviously, this explains the presence of multiple efficient anti-glycative enzymatic systems, like glyoxalase I and II , ribulosamine/erythrulosamine 3-kinase , acylamino acid-releasing enzyme .
Recently, Bechtold and co-workers reported an increase in the total contents of individual AGE classes upon the application of experimental environmental stress . Thus, it is obvious that environmental changes are accompanied with enhanced generation of AGEs in plant tissues. In other words, due to the continuously altering growth conditions, AGEs might accumulate in plants during their life span, causing stress-related changes of the plant proteome and its physiological state. Important to note, that due to the dramatically different metabolic background (i.e. other patterns of carbohydrates, as well as high contents of potential antioxidants and carbonyl traps), pathways of glycation in plants may differ from those described in mammalians. However, no information about the proteins and biochemical pathways affected by such glycation reactions (i.e. its structural and functional patterns) was available until very recently. The most recent studies from our labs on the protein glycation patterns of model plants in the absence and presence of environmental stresses, as well as the impact of protein glycation in plant ageing, are added to this chapter.
2. Methodological approaches for the study of protein glycation in plants
2.1. Proteomics in plant glycation research
Recently, using liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach, Bechtold and co-workers estimated the total levels of individual AGE classes in
2.2. Protein isolation and proteolysis
Depending on the target protein fraction (soluble or total), aqueous (aq.), extraction can be performed in the absence and presence of phenol, respectively . In the first case, however, even the extracts of green parts contain high amounts of soluble metabolites that might inhibit the activity of proteases used for digestion. Such incompleteness of proteolysis can be observed as well, when proteins are extracted from plant parts rich in anti-nutritive (i.e. protease inhibiting or denaturing) phenolics like insoluble condensed tannins in seeds . Therefore, the extracts can be purified by gel filtration chromatography and/or ultrafiltration using Centricon or Vivaspin centrifugation devices  prior to the determination of protein concentration. Alternatively, the proteins can be isolated by phenol extraction. In this case, phenolics contaminants can be removed by addition of 1–5% of soluble or insoluble polyvinylpyrrolidone . While purified aqueous extracts can be easily digested by proteases in the presence of only deoxycholate as a denaturizing agent , the dried proteins isolated with phenol (containing also the fraction of hydrophobic membrane proteins) can be reconstituted only in the presence of both chaotropic compounds (urea, thiourea) and strong detergents. Conventional detergents, such as sodium dodecyl sulfate (SDS) or 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), co-elute with proteolytic peptides and disturb ESI. This can be avoided by application of the detergents which do not impact protease activity and can be easily destroyed upon the digest. For example,
The completeness of the digestion can be controlled by the SDS-polyacrylamide gel electrophoresis (PAGE). Performing SDS-PAGE additionally prior to proteolysis provides the opportunity to validate the Bradford assay results by relative quantification of total lane densities and, if necessary, normalize the results of the LC-MS-based label-free quantification. It is important to stress that the use of chaotropic agents, acid-labile detergents and strong acids for their cleavage ultimately require RP-based solid phase extraction (SPE) after completion of the digest and verification of its completeness by SDS-PAGE.
2.3. LC-MS analysis of glycated peptide mixtures
The proteolytic digests are, typically, complex mixtures. Therefore, for successful detection and identification of their components (i.e. peptides), adequate chromatographic and mass separation techniques need to be applied. The selection of a strategy for LC-MS analysis (in terms of throughput, sensitivity, separation efficiency and reproducibility) depends on the aims of particular research. In the absolute majority of cases, LC-MS analyses rely on so-called data-dependent acquisition experiments (DDA) . These complex experiments comprise survey MS scan (typically performed in Orbitrap- or TOF mass analyzer), and multiple dependent ones – MS/MS, relying on linear ion trap (LIT) or QqTOF analysis [56,57]. Modern instruments provide a possibility for introduction of the second dependent scan. Thus, hybrid LTQ-Orbitrap instruments support multi-step activation (MSA) experiments, comprising an additional MS/MS scan with a low-energy collision-induced dissociation (CID) . The modification-specific neutral losses, appearing under these conditions, trigger a high-energy dependent MS/MS scan providing rich structural information. In the past decade, these experiments were successfully applied to glycated tryptic digests using Amadori-specific losses of two and three waters, as well as additional formaldehyde molecule .
In the most straightforward way, DDA experiments are performed without additional analytical procedures preceding a RP-HPLC separation – so-called shotgun proteomics. However, as well as other PTMs, glycative and glycoxidative modifications are low abundant. Hence, their ionization might be suppressed by highly abundant species. To avoid this, early glycated tryptic peptides can be selectively enriched by boronic acid affinity chromatography (BAC) before LC-MS/MS analysis . However, such enrichment is not possible for the whole fraction of advanced glycated peptides due to their structural heterogeneity. Therefore, these low-abundant species need to be directly detected in conventional data-dependent acquisition (DDA) experiments. As the DDA algorithm relies on the MS/MS analysis of the most intense signals in each time segment, this type of experiments suffers from so-called undersampling, i.e. missed fragmentation of low-abundant quasi-molecular ions . Because of this reason, shotgun proteomics is not a desired strategy for the analysis of PTMs.
Thus, the number of co-eluting peptides in DDA analyses needs to be reduced to increase the coverage of the AGE-modified proteome. This can be addressed by three approaches:
The generalized analytical strategy might comprise both qualitative and quantitative approaches, i.e. identification of glycated peptides in DDA experiments with their subsequent label-free quantification in additional full-scan MS experiments (Figure 4). For identification of glycation sites, early glycated peptides can be selectively enriched , while the analysis of AGE-containing species might rely on two-dimensional liquid chromatography (LC × LC) . BAC is a well-established analytical tool to enrich Amadori and Heyns products from mammalian tissues (predominantly plasma) . However, for application to the study of plant glycation, this method requires some optimization. Thus, the protein extract must be effectively washed prior to digestion (e.g. by ultrafiltration) to remove the co-extracted carbohydrate-related metabolites (mono-, oligosaccharides, sugar esters and glycosides of (poly)phenolics). Due to their
Pre-fractionation is usually introduced as an (relative to the RP) orthogonal separation procedure – most often cation exchange or hydrophilic interaction chromatography (HILIC) [62,64]. This approach essentially decreases sample complexity, facilitating, thereby, the fragmentation of low-abundant species. Similarly, fractionation can be applied on the MS level. Thus, simultaneously formed quasi-molecular ions (i.e. originating from the co-eluting species) can be fractioned by their
Early glycated tryptic peptides can be annotated in high-resolution Orbitrap-MS experiments by the
The physiological role of glycation can be assessed by the system biology software tools. Thus, for the grouping of AGE-modified proteins by their functions, the mapping software MapMan (Max-Planck Institute of Molecular Plant Physiology, Potsdam-Golm, Germany, http://mapman.gabipd.org) can be used. The functional annotation of proteins might give insight in biological effects of AGEs in plants and provide the material for future biological studies. Thus, the proteomic data can be complemented by the result of transcriptional analysis and determination of enzymatic activities. Afterwards, the functional role of glycation in respect of particular proteins can be confirmed by the experiments with corresponding mutants.
In vivoglycation of plant proteins
3.1. Plant protein glycation patterns
The possibility of plant protein glycation is considered since the beginning of the past decade, when Yamauchi and co-workers proposed the formation of AGEs as one of the possible mechanisms underlying inactivation of ribulose bisphosphate carboxylase/oxidase (RuBisCO) by high light . Thereby, they proposed ascorbic acid as a possible precursor of AGEs. Indeed, this highly abundant compound in plant tissues can easily autoxidize and is recognized, therefore, as a potent glycation agent . However, besides ascorbic acid, photosynthetically active leaf tissues contain high amounts of highly reactive pentoses, tetroses and trioses, as well as their phosphorylated forms, that might be even more reactive . Probably, these sugars could be an important factor of light-dependent glycation. Recently, Bechtold and co-workers provided
Implementation of the proteomic approach resulted in identification of proteins involved in glycation and exact modification sites therein. This strategy allowed identification of several hundreds of polypeptides containing early glycation moieties. Interestingly, the number of modification sites was higher in
Surprisingly, in plant proteins, the numbers of AGE-modified residues are essentially higher in comparison to the early glycated sites: approximately three- and seven-fold differences were observed for
It was shown in mammalians that the proteins controlling gene expression (e.g. transcription factors or the molecules involved in protein metabolism) can be the targets of glycation . The same was demonstrated for plants. This might indicate the involvement of AGE formation in the regulation of gene expression on the levels of transcription and protein biosynthesis. This can be explained by the role of protein degradation in AGE metabolism and high representation of arginyl residues in the transcription factors that makes these molecules highly amenable to interaction with α-dicarbonyls .
3.2. Protein glycation and environmental stress
3.2.1. Experimental models for the study of plant glycation
The main environmental stresses the plant can encounter in its habitat are high irradiation, contamination with heavy metals or metalloids and drought. It is necessary to take into account that drought is a general manifestation accompanying water deficit and is characteristic for the response to some other environmental factors, like high salt contents in soil or extremely low or high temperatures . Obviously, for the study of any stress-specific response, selection of an appropriate model is of the principle importance. In this context, the researcher needs to be able to define all stress parameters by the selected experimental setup. This can be relatively easily achieved for a high light stress by using a phytotron equipped with the lamps providing required light intensity and complete climate control. In this case, a soil model can be applied (Figure 6) . However, this approach does not provide the conditions equal for all plants, when a heavy metal stress is applied. That is why, growth of plants in aqueous (aq.) culture with a subsequent addition of a heavy metal salt to a growth medium seems to be a more adequate solution  (Figure 6).
As far as the drought stress is concerned, the water deficit can be modeled both in soil and in aqueous systems. According to Boyer , this water deficit can be expressed as a decrease of water potential (Ψw), i.e., as a difference of water potentials in a solution and pure water divided by partial molar volume of water. However, the soil-based approaches do not allow experimental definition of Ψw, as in this case it depends from the water absorption by plant. In aqueous culture, osmotically active substances, like commonly used sorbitol or polyethylene glycol (PEG), can affect the function of root epidermis . Because of this, agar-based PEG infusion model, introduced in the past decade by van der Weele and co-workers , and additionally optimized to mature plants (Figure 6) seems to be more suited for this purpose. The confirmation of the stress development might rely on the determination of native leaf fluorescence , hydrogen peroxide contents  and some further parameters.
3.2.2. Stress-related glycation of plant proteins
Light is one of the most essential factors determining plant growth, development and survival . However, a continuous exposure of plants to the high doses of sun irradiation might exceed the capacity of the mesophyll photosynthetic apparatus and trigger development of the oxidative stress . In the context of the protein Maillard chemistry, discussed above, it is logical to assume that enhanced ROS and monosaccharide production would lead to the increase of AGE formation in plant green tissues. As was proposed earlier, such modifications of the RuBisCO polypeptide chains might impact inactivation of the enzyme with high light . Similar mechanisms might accompany the development of drought and metal stress. Moreover, the tissue metal-scavenging mechanisms include activation of the enzyme phytochelatinsynthase, requiring GSH as a substrate . Hence, glyoxalase system, critical for detoxication of methylglyoxal (MGO) and utilizing GSH as a substrate as well , can not perform efficient scavenging of this dicarbonyl under stress conditions, which might stimulate enhanced glycation by MGO in the proteins of metal-treated plants.
The effect of stress on the formation of AGEs differs on the qualitative and quantitative levels. Thus, in most cases, relatively low number of stress-specific glycation sites can be detected in the proteins of stressed plants, and such sites are representing mostly the molecules involved in transcription and protein degradation (i.e. those known to be upregulated under stress conditions) . This could be explained by the activation of some unknown enzymatic systems eliminating either AGE precursors, or AGE-modified proteins, or their early glycated precursors, i.e. Amadori and Heyns compounds. Indeed, activation of the glyoxalase system (comprising enzymes glyoxalase I and II) is well documented during environmental stress . Additionally, although in mammals advanced glycation decreases the rates of proteolysis, in plants it can be not the case, that was confirmed by
Compared to the qualitative alterations, the quantitative stress-related changes in glycated proteome are much more pronounced. Thus, several AGE classes were at least two-fold increased even after short application of light stress . This tendency could be followed on the level of individual glycation sites. However, changes in representation of a certain AGE moiety need to be verified on the level of the abundance of corresponding protein, i.e. the abundance of specific modification sites need to be considered together with the data on gene expression on the RNA and protein levels. Moreover, the changes in protein degradation rates need to be taken into account.
As was demonstrated in the pioneer study of Bechtold and co-workers, stress-related upregulation of early glycation products is much stronger in comparison to AGEs , which was confirmed on the level of individual glycation sites. Thus, stress conditions lead to the considerable increase of the contents of Amadori and Heyns adducts, while the response on the level of advanced glycation is much lower. Moreover, stress-specific AGEs are dominated by α-dicarbonyl-derived products, and only few could originate from Amadori or Heyns products. The negligible role of early glycation products in AGE formation additionally supports the existence of powerful Amadori/Heyns product-degrading enzymatic mechanisms in plant tissues. Moreover, it might indicate the presence of a potent stress-inducible anti-glycation system scavenging or/and reducing α-dicarbonyls. This assumption can be supported by the absence of changes in the carbonylated proteome, as well as glyoxal and methylglyoxal contents throughout the stress development. Thus, scavenging of these advanced glycation intermediates by the amino functioned metabolites might be the most probable scenario.
In vitromodeling of plant glycation reactions
Due to their high photosynthetic activity, green parts of plants are characterized with high contents of carbohydrates. Thus, for
To address this question,
To conclude, protein glycation is a common post-translational modification in plants. Despite this, essential differences in comparison to mammalian glycation patterns were observed. Thus, glycation patterns are strongly dominated by AGEs, while the number of Amadori-modified lysyl residues is at least one order of magnitude lower compared to human plasma proteome. Moreover, individual AGE-modified sites are not represented by their Amadori/Heyns counterparts. It indicates autoxidation of free sugars rather than glycoxidation (i.e. AGE formation from early glycated products – one of the main glycation mechanisms in mammalians) as the major pathway of advanced glycation in plants. Environmental stress considerably affects glycation patterns, mostly on the quantitative level. However, due to the high heterogeneity of potential plant glycation agents, a high variability of glycation pathways and mechanisms can be expected. To clarify these pathways, simple
We thank the “Deutsche Forschungsgemeinschaft” (DFG, FR-3117/2-1) for financial support to AF, GP, DB and TB, the Ernst-Schering Foundation to UG, the Deutscher Akademischer Austauschdienst (DAAD Program “Dmitry Mendeleev”) to ET, TB and EL, St. Petersburg University (grant No. 126.96.36.1994) to NO and EL are gratefully acknowledged, as well as the state of Saxony-Anhalt and SKWP (Agrochemical Institute Piesteritz) to LAW. This work was conducted under the auspices of the Science Campus Plant Based Bioeconomy (www.sciencecampus-halle.de).