*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 [72]. 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) [73]. 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 [74] (Figure 6).

**Figure 6.** Experimental models of environmental stress.

As far as the drought stress is concerned, the water deficit can be modeled both in soil and in aqueous systems. According to Boyer [75], 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 [72]. Because of this, agar-based PEG infusion model, introduced in the past decade by van der Weele and co-workers [76], 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 [77], hydrogen peroxide contents [16] and some further parameters.

#### *3.2.2. Stress-related glycation of plant proteins*

representation of arginyl residues in the transcription factors that makes these molecules

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 [72]. 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) [73]. 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

As far as the drought stress is concerned, the water deficit can be modeled both in soil and in aqueous systems. According to Boyer [75], this water deficit can be expressed as a decrease of

highly amenable to interaction with α-dicarbonyls [71].

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

*3.2.1. Experimental models for the study of plant glycation*

**3.2. Protein glycation and environmental stress**

more adequate solution [74] (Figure 6).

**Figure 6.** Experimental models of environmental stress.

Light is one of the most essential factors determining plant growth, development and survival [78]. 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 [7]. 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 [67]. Similar mechanisms might accompany the development of drought and metal stress. Moreover, the tissue metal-scavenging mechanisms include activation of the enzyme phyto‐ chelatinsynthase, requiring GSH as a substrate [79]. Hence, glyoxalase system, critical for detoxication of methylglyoxal (MGO) and utilizing GSH as a substrate as well [80], 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) [81]. 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 [82]. Additionally, although in mammals advanced glycation decreases the rates of proteolysis, in plants it can be not the case, that was confirmed by *in vitro* experiments [67]. Thus, acylamino acid-releasing enzyme might impact in plant protein degradation pathways [44]. However, the pathways, involved in enhanced degradation of glycated proteins in plants still need to be studied. Finally, Amadori and Heyns products can be degraded via phosphorylation pathway [43]. Besides, the possibility of the further reactions of AGEs to form new unknown products also needs to be considered [83].

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 [16]. 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 upre‐ gulation of early glycation products is much stronger in comparison to AGEs [16], 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 mecha‐ nisms 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.

#### **3.3.** *In vitro* **modeling of plant glycation reactions**

Due to their high photosynthetic activity, green parts of plants are characterized with high contents of carbohydrates. Thus, for *B. napus* leaf tissue, these contents were more than 1 μmol/ g f.w. for the major sugars (glucose, fructose, sucrose, Asc and DHA) and were in the range of 10–60 nmol/g f.w. for such compounds as glucose-6-phosphate, ribose and arabinose that are known to highly-reactive [84,85]. Hence, metabolic background of plant glycation differs essentially from that reported for mammals. Thus, animal tissues are rich in glucose (that is known to be one of the weakest glycation agents), while the contents of more reactive sugars are negligible [86]. This strong prevalence of one potential glycation agents dramatically affects the mammalian glycation patterns, which are strongly dominated by the protein fructosa‐ mines, i.e. glucose-derived Amadori compounds [64]. In contrast, photosynthetically active tissues are rich in trioses, tetroses and pentoses as well, that might affect the early glycation patterns. Moreover, these highly reactive carbohydrates might be much more susceptible to autoxidation, i.e. represent potent precursors of α-dicarbonyls and, hence, AGEs. Obviously, the ability of certain sugars to act as a glycation agent depends not only on its tissue content but also on its ability to react with proteins. However, the reactivities of individual plant carbohydrates towards protein lysyl and arginyl residues, as well as their glycation potential, are still unknown.

To address this question, *in vitro* glycation models based on synthetic peptides were estab‐ lished recently [24,83]. Ideally, such model peptides are N-terminally protected and contain only one residue (lysine or arginine, typically in mid position) susceptible to glycation. Essential advantage of such systems is their relative simplicity: the products can be separated and analyzed by tandem mass spectrometry (MSn). Evaluation of peptide tandem mass spectra can be complemented by adequate methods of carbohydrate and α-dicarbonyl analysis [87,88]. These peptide models can be potentially applied to the assessment of reactivity of individual glycation agents (i.e. sugars and dicarbonyls) and determination of their glycation potential. Such analyses might rely on integration of selected XICs (characteristic for individual glycation products) at specific retention times. This would give a possibility for screening of plant sugars for their ability to form certain Amadori/Heyns compounds and AGEs. This would essentially impact in understanding of plant glycation pathways.
