**3.** *In vivo* **glycation 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 [67]. Thereby, they proposed ascorbic acid as a possible precursor of AGEs. Indeed, this highly abundant compound in plant tissues can easily autoxidize and is recog‐ nized, therefore, as a potent glycation agent [68]. However, besides ascorbic acid, photosyn‐ thetically 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 [69]. Probably, these sugars could be an important factor of light-dependent glycation. Recently, Bechtold and co-workers provided *in vivo* confirmation of this assumption: the authors found that tissue fructosyl lysine contents (determined by LC-MS/MS in exhaustive enzymatic protein hydro‐ lyzates) are approximately four-fold higher in the day time in comparison to the dark period. It was not, however, the case for AGEs – just minor changes in their contents were observed during the day.

species is the absence of the neutral losses accompanying parent and fragment ions. Addi‐ tionally, the glycation state of peptides can be confirmed by characteristic fragments in the low *m/z* range [61,66]. This information can be further used for label-free quantification of indi‐ vidual AGE-modified peptides and, hence, specific glycation sites. The quantification typically relies on the integration of the annotated signals and qualitative comparisons of obtained peak areas. Thereby, the peak areas can be normalized to the signals of unmodified peptides in the same sample or in quality controls (QC). The peak integration can be performed by means of

**Figure 5.** Annotation of Amadori peptides by nanoUPLC-ESI-LTQ-Orbitrap-MS/MS DDA experiments. Reversed phase ESI-Orbitrap-MS total ion chromatogram and the segment of the extracted ion chromatogram (insert) for *m/z* 736.39 ± 0.02 in the tR range of 30–35 min (A) and an ESI-LTQ-MS/MS spectrum at *m/z* 736.4 corresponding to the [M +3H]3+ ion of the peptide VFDEFKAmPLVEEPQNLIK (B) acquired at 32.5 min of the same DDA experiment. Pyrylium b

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 deter‐ mination of enzymatic activities. Afterwards, the functional role of glycation in respect of particular proteins can be confirmed by the experiments with corresponding mutants.

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

the vendor software packages – Xcalibur Quan Browser or LCquan.

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

and y fragment ions are marked with asterisk [61].

**3.** *In vivo* **glycation of plant proteins**

**3.1. Plant protein glycation patterns**

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 *Arabidopsis thaliana* in comparison to its close relative *Brassica napus*. Thus, glycation patterns might vary between species, although in both cases they are dominated by triose- and tetrose-derived products, accompanied with less abundant groups of pentose-modified sites, while hexose-derived modifications (typically the most represen‐ tative in mammals) were less abundant.

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 *A. thaliana* and *B. napus* proteome, respectively. It is the principle difference from glycation in mammals: though thousands of early glycated proteins were identified in human plasma and red blood cell membranes [64,70], only several dozens were proved to be AGEmodified [61,66]. Interestingly, the AGE modification sites in plant proteins are not accompa‐ nied with their early glycated counterparts, and are mostly originating from glyoxal and methylglyoxal. This situation differs drastically from the observations done with mammals. Indeed, several confirmed AGE sites in blood proteins (at least those representing the major plasma polypeptide human serum albumin, HSA) resembled the early glycated residues, indicating glycoxidation as an important pathway of AGE formation *in vivo*. The absence of such glycation sites in plant proteome clearly indicates the early glycation products as unlikely precursors of AGEs in plants. In this context, oxidative glycosylation rather than glycoxidation might be the predominant AGE formation pathway in plants. Remarkably, the number of early glycated lysyl residues was not only absolutely but also relatively (in comparison to the number of AGE-modified sites) lower in *B. napus*, than in *A. thaliana*. Most probably, it indicates higher activities of deglycation enzymes in the former plant.

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 [71]. 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 [71].

#### **3.2. Protein glycation and environmental stress**
