**1. Introduction**

#### **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 [1]. Drought, high light, salinity and increased heavy metal soil contents, as well as extreme temperature, represent its important manifestations [2]. On the physiological level, extreme environmental conditions ultimately results in decrease in the CO2 assimilation rate and in growth inhibition [3]. 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 (1 O2), superoxide radical anion (O2 ⋅–), peroxide ion (O2 2–), hydrogen peroxide (H2O2), various hydroperoxides and hydroxide radical (OH<sup>⋅</sup> ) [4]. When ROS production overwhelms their detoxication, oxidative stress develops [5].

Thus, transfer of electrons to molecular oxygen (O2) from ubisemiquinone in mitochondria and thylakoid membrane-bound primary electron acceptor of photosystem I (PSI) in chloro‐ plasts yields O2 ⋅– (and, when further reduction occurs, O2 2–), 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) [8]. 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 [9]. The PUFAs can be directly attacked by the protonated form of O2 ⋅– (HO2 ⋅ ) [10], 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 ionmediated reduction, yielding OH<sup>⋅</sup> , i.e. one of the most short-living and toxic ROS, directly and irreversibly modifying lipids, proteins and nucleic acids [11]. The metal ions oxidized during Fenton reaction are reduced *in vivo* by cellular antioxidants or O2 ⋅– (Haber-Weiss reaction), that considerably increases the production of OH<sup>⋅</sup> [12]. As environmental stress is accompanied with a strong upregulation of mono- and oligosaccharides in all plant tissues [13], metalcatalyzed oxidation of sugars (so-called monosaccharide autoxidation) [14] also might be enhanced under the conditions of oxidative stress. The resulting products – hydroxycarbonyls and α-oxocarbonyls – are the potent protein modification agents and can induce essential changes in their structure and function [15]. These reactions, termed protein glycation, i.e. modification of proteins by carbonyl compounds (carbohydrates and α-oxocarbonyls), is believed to be an important factor in stress-related protein damage [16].

#### *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 [17] and Heyns [18] rearrangements, variants of the acyloin shift. Amadori rearrangement involves proton transfer from C1 to C2 via the enol/enamine intermediate yielding *N*-substituted 1 amino-deoxy-ketoses, the Amadori products (Figure 1A). Similarly, in the case of ketoamines, in course of Heyns rearrangement [18], a proton migrates from C2 to C1 forming 2-aminodeoxyaldosyl adducts, often referred to as Heyns products (Figure 1B) [19]. Both Amadori and Heyns products are termed as "early glycation products", the first relatively stable intermedi‐ ates of glycation [20].

**Figure 1.** Formation of N/O-acetal and Schiff intermediates with subsequent Amadori rearrangement (A), and keta‐ mine intermediates with subsequent Heyns rearrangement (B, not all intermediates shown).

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 [21]. Depending on the structure of the carbohydrate moiety involved in this degradation, i.e. free sugars, or protein-bound early glycation prod‐ ucts, 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 accumulat‐ ing during thermal processing of food (Figure 2) [24], but also endogenously, e.g. under the conditions of persisting hyperglycemia.

#### *1.1.3. Advanced Glycation End-products (AGEs)*

**1. Introduction**

anion (O2

hydroxide radical (OH<sup>⋅</sup>

stress develops [5].

plasts yields O2

protonated form of O2

*1.1.2. Protein glycation*

reliable markers of oxidative stress.

mediated reduction, yielding OH<sup>⋅</sup>

**1.1. Environmental stress, ROS and protein glycation**

296 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

production of the reactive oxygen species (ROS), i.e. singlet oxygen (1

⋅– (and, when further reduction occurs, O2

Environmental stress is one of the major factors reducing the productivity of crop plants all over the world [1]. Drought, high light, salinity and increased heavy metal soil contents, as well as extreme temperature, represent its important manifestations [2]. On the physiological level, extreme environmental conditions ultimately results in decrease in the CO2 assimilation rate and in growth inhibition [3]. Simultaneous accumulation of reduced equivalents results in an overload of the chloroplast and mitochondrial electron transport chains and enhanced

Thus, transfer of electrons to molecular oxygen (O2) from ubisemiquinone in mitochondria and thylakoid membrane-bound primary electron acceptor of photosystem I (PSI) in chloro‐

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) [8]. 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 [9]. The PUFAs can be directly attacked by the

The hydroperoxides can be easily involved in the Fenton reaction, i.e. transition metal ion-

irreversibly modifying lipids, proteins and nucleic acids [11]. The metal ions oxidized during

with a strong upregulation of mono- and oligosaccharides in all plant tissues [13], metalcatalyzed oxidation of sugars (so-called monosaccharide autoxidation) [14] also might be enhanced under the conditions of oxidative stress. The resulting products – hydroxycarbonyls and α-oxocarbonyls – are the potent protein modification agents and can induce essential changes in their structure and function [15]. These reactions, termed protein glycation, i.e. modification of proteins by carbonyl compounds (carbohydrates and α-oxocarbonyls), is

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

2–), hydrogen peroxide (H2O2), various hydroperoxides and

) [10], thus the content of lipid hydroperoxides is one of the most

, i.e. one of the most short-living and toxic ROS, directly and

[12]. As environmental stress is accompanied

) [4]. When ROS production overwhelms their detoxication, oxidative

O2), superoxide radical

2–), further converted to H2O2 by

⋅– (Haber-Weiss reaction), that

*1.1.1. Environmental stress and ROS formation*

⋅–), peroxide ion (O2

⋅– (HO2 ⋅

considerably increases the production of OH<sup>⋅</sup>

Fenton reaction are reduced *in vivo* by cellular antioxidants or O2

believed to be an important factor in stress-related protein damage [16].

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

**Figure 2.** AGEs detected *in vivo*: GD-HI, glyoxal-derived dihydroxyimidazolidine; MGD-HI, methylglyoxal-derived di‐ hydroxyimidazolidine; MG-H, methylglyoxal-derived hydroimidazolone; Glarg, glyoxal-derived hydroimidazolone; CML, N<sup>ε</sup> -carboxymethyllysine; CEL, N<sup>ε</sup> -carboxyethyllysine; GLAP, glyceraldehyde-derived pyridinium compound.

are still reactive and can be easily involved in further reactions [25]. In the past few decades, several AGEs were comprehensively characterized (Figure 2). Among lysine-derived modifi‐ cations, *N*<sup>ε</sup> -(carboxymethyl)lysine (CML) [26], *N*<sup>ε</sup> -(carboxyethyl)lysine (CEL) [27], ε-(2 formyl-5-hydroxymethyl-pyrrolyl)-*L*-norleucine (pyrraline) [28] and glyceraldehyde-derived pyridinium compound (GLAP) [29] are the best-characterized. Not less attention was paid to the modifications of arginine. Thus, Schwarzenbolz and coworkers reported 1-(4-amino-4 carboxybutyl)2-imino-5-oxo-imidazolidine (Glarg) as a product of the reaction of arginine with glyoxal [30] and yielding *N<sup>δ</sup>* -carboxyethylarginine (CMA) upon hydrolysis at 37°C [31]. Methylglyoxal was shown to form isomeric methylglyoxal-derived hydroimidazolones (MG-Hs) with *N*<sup>δ</sup> -(5-methyl-4-oxo-5-hydroimidazolinone-2-yl)-*L*-ornithine (MG-H1) as the major isomer [32]. Hydrolysis of 2-amino-5-(2-amino-4-hydro-4-methyl-5-imidazolon-1-yl)pentano‐ ic acid (MG-H3) yields carboxyethyl-*L*-arginine (CEA) [33]. Sequential modification of arginine with two methylglyoxal molecules results in *N*<sup>δ</sup> -(5-hydroxy-4,6-dimethylpyrimidine-2-yl)-*L*ornithine (argpyrimidine, Argpyr) [34] and *N*<sup>δ</sup> -(4-carboxy-4,6-dimethyl-5,6-dihydroxy-1,4,5,6 tetrahydropyrimidine-2-yl)-*L*-ornithine (tetra-hydroargpyrimidine, TH-Argpyr) [35].

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) [36] and foster the development of inflam‐ matory diseases – e.g. atherosclerosis and type 2 diabetes mellitus [37]. 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 [38].

Surprisingly, other reports showed mammalian serum and urinary concentrations of AGEs to be independent from dietary intake of thermally processed foods [39]. Moreover, the levels of CML and fluorescent AGEs in the plasma of vegetarian individuals were higher in comparison to those in the omnivorous individuals [40], 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 [41]. 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 [42], ribulosamine/erythrulosamine 3-kinase [43], acylamino acid-releasing enzyme [44].

Recently, Bechtold and co-workers reported an increase in the total contents of individual AGE classes upon the application of experimental environmental stress [16]. 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 antiox‐ idants 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.
