Protein Glycation in Plants—An Under-Researched Field with Much Still to Discover

International Journal of Molecular Sciences

Review Protein Glycation in Plants—An Under-Researched Field with Much Still to Discover

Naila Rabbani 1,*, Maryam Al-Motawa 2,3 and Paul J. Thornalley 2,3,*

1 Department of Basic Medical Science, College of Medicine, QU Health, Qatar University, Doha P.O. Box 2713, Qatar 2 Diabetes Research Center, Qatar Biomedical Research Institute, Hamad Bin Khalifa University, Qatar Foundation, Doha P.O. Box 34110, Qatar; [email protected] 3 College of Health and Life Sciences, Hamad Bin Khalifa University, Qatar Foundation, Doha P.O. Box 34110, Qatar * Correspondence: [email protected] (N.R.); [email protected] (P.J.T.); Tel.: +974-7479-5649 (N.R.); +974-7090-1635 (P.J.T.) Received: 9 April 2020; Accepted: 28 May 2020; Published: 30 May 2020

Abstract: Recent research has identiﬁed glycation as a non-enzymatic post-translational modiﬁcation of proteins in plants with a potential contributory role to the functional impairment of the plant proteome. Reducing sugars with a free aldehyde or ketone group such as glucose, fructose and galactose react with the N-terminal and lysine side chain amino groups of proteins. A common early-stage glycation adduct formed from glucose is Nε-fructosyl-lysine (FL). Saccharide-derived reactive dicarbonyls are arginine residue-directed glycating agents, forming advanced glycation endproducts (AGEs). A dominant dicarbonyl is methylglyoxal—formed mainly by the trace-level degradation of triosephosphates, including through the Calvin cycle of photosynthesis. Methylglyoxal forms the major quantitative AGE, hydroimidazolone MG-H1. Glucose and methylglyoxal concentrations in plants change with the developmental stage, senescence, light and dark cycles and also likely biotic and abiotic stresses. Proteomics analysis indicates that there is an enrichment of the amino acid residue targets of glycation, arginine and lysine residues, in predicted functional sites of the plant proteome, suggesting the susceptibility of proteins to functional inactivation by glycation. In this review, we give a brief introduction to glycation, glycating agents and glycation adducts in plants. We consider dicarbonyl stress, the functional vulnerability of the plant proteome to arginine-directed glycation and the likely role of methylglyoxal-mediated glycation in the activation of the unfolded protein response in plants. The latter is linked to the recent suggestion of protein glycation in sugar signaling in plant metabolism. The overexpression of glyoxalase 1, which suppresses glycation by methylglyoxal and glyoxal, produced plants resistant to high salinity, drought, extreme temperature and other stresses. Further research to decrease protein glycation in plants may lead to improved plant growth and assist the breeding of plant varieties resistant to environmental stress and senescence—including plants of commercial ornamental and crop cultivation value.

Keywords: glycation; advanced glycation end products (AGEs); methylglyoxal; glyoxalase; dicarbonyl stress; unfolded protein response; Arabidopsis; Brassica; crops

1. Protein Glycation in Plants: Three Papers Setting the Scene In 2009, we published the ﬁrst report on the steady-state levels of protein oxidation, nitration and glycation adducts in cytosolic protein extracts from leaves of Arabidopsis thaliana (thale cress) [1]. The cytosolic protein contents of the early-stage glycation adduct, Nε-fructosyl-lysine (FL), and eight advanced glycation endproducts (AGEs) were presented. This highlighted an aspect of the proteome

Int. J. Mol. Sci. 2020, 21, 3942; doi:10.3390/ijms21113942 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 3942 2 of 16 of higher plants that is intuitive but had been little investigated: proteins are glycated in plants. In 2014, Takagi et al. showed that methylglyoxal (MG)—a precursor of the major quantitative AGE, hydroimidazolone MG-H1—is formed during photosynthesis in chloroplasts isolated from leaves of spinach [2]. In 2017, Bilova et al. published a proteomics study identifying proteins modiﬁed by AGEs in Arabidopsis thaliana [3]. These studies revealed that higher plants produce glycating agents as a part of their vital photosynthetic metabolism, and the proteome of plants is continually subjected to glycation forming early glycation adducts and AGEs [4]. Further research in this area is, therefore, important to improve understanding of plant growth, resistance to environmental stress and senescence—including for varieties of commercial importance for ornamental and crop production. Below, in this review, we give a brief introduction to glycation, glycating agents and glycation adducts, and, following recent developments in mammalian glycation, we consider dicarbonyl stress, the susceptibility of the plant proteome to functional inactivation by arginine-directed glycation and the role of glycation in the activation of the unfolded protein response (UPR) in plants [5,6]. The latter is likely linked to the recent suggestion of the involvement of protein glycation in sugar signaling in plant metabolism [7]. We limit our coverage to higher plants.

2. Glycation: The Maillard Reaction Protein glycation is the non-enzymatic reaction of simple reducing sugars and related saccharide derivatives with proteins in a complex series of sequential and parallel pathways called the Maillard reaction [8]. Reducing sugars have an aldehyde or ketone group by which reactions occur with the protein substrate, typically on N-terminal and lysine side chain amino groups. In higher plants, examples of simple reducing sugars involved in protein glycation are glucose, fructose and galactose. Glucose reacts with the N-terminal amino groups and lysine residue side chain amino groups of proteins to form an initial Schiﬀ’s base, which undergoes an Amadori rearrangement to form fructosamines: Nα-1-deoxyfructosyl N-terminal amino groups and Nε-fructosyl-lysine (FL). The reactivity of reducing sugars with N-terminal amino groups is usually faster than that with lysine side chain amino groups because of the lower pKa of the former, but lysine side chains are present at higher concentrations than N-termini in the plant proteome. Some lysine side chain amino groups may also be activated towards glycation by interaction with neighboring cationic lysine and arginine residues, decreasing the pKa of the lysine sidechain amino group target [9]. Sucrose, a non-reducing sugar, is the main vehicle for sugar transport in plants. This fact greatly decreases the risk of glycation of the plant proteome by sugar in transit in the plant body (roots, stems and leaves) (44). Common saccharide derivatives studied in glycation reactions are phosphorylated glycolytic intermediates, such as glucose-6-phosphate (G6P) and ribose-5-phosphate (R5P)—the latter an intermediate of the pentosephosphate pathway and Calvin cycle of photosynthesis in plants [10]. They react with and modify the N-terminal and lysine side chain amino groups of proteins. There are also reactive dicarbonyl saccharide derivatives—such as glyoxal, MG and 3-deoxyglucosone (3-DG) [11]. Dicarbonyl metabolites are arginine-directed glycating agents, forming predominantly hydroimidazolone derivatives such as methylglyoxal-derived hydroimidazolone MG-H1 and related structural isomers, and analogous hydroimidazolones from glyoxal and 3-DG [12,13]. For the reaction of glyoxal with arginine, the ring-opened rearrangement of the initial dihydroxyimidazolidine to Nω-carboxymethyl-arginine (CMA) is favored [14]. Glyoxal reacts with lysine residues to form the AGE Nε-carboxymethyl-lysine (CML). CML is mainly formed by the oxidative degradation of FL [15], with formation also by the glycation of lysine residues by ascorbic acid [16]. Glyoxal is formed in lipid peroxidation and the slow oxidative degradation of monosaccharides and proteins glycated by glucose [11]. MG is mainly formed by the trace-level degradation of triosephosphates, glyceraldehyde-3-phosphate (GA3P) and dihydroxyacetonephosphate (DHAP) [17]. GA3P is an intermediate in photosynthesis, and GA3P and DHAP are intermediates in glycolysis, gluconeogenesis and glyceroneogenesis in lipid synthesis. 3-DG is formed by the enzymatic and non-enzymatic degradation of proteins glycated by glucose [11,18]. Int. J. Mol. Sci. 2020, 21, 3942 3 of 16

Glycating agents and glycation adduct residues in proteins have been detected in plants. Glycation has been in studied in plants of both research interest and commercial ornamental and crop relevance [1,3,19–24]. The levels of glycating agents and glycation adduct residues in proteins vary with light and dark cycles, the stage of development and environmental stresses [1,24]. The molecular structures, metabolic source and likely functional significance of glycation adducts in plants are summarized in Table2. There are also enzymes of anti-glycation defense that suppress protein glycation in plant tissues: the glyoxalase system, which metabolizes glyoxal and MG and thereby suppresses the formation of related AGEs [25]; aldoketo reductases (AKRs) that also metabolize glyoxal, MG and likely 3-DG [26]; and ribulosamine/erythrulosamine 3-kinase—a putative protein-repair enzyme that deglycates proteins glycated by R5P [10]. There is also an acylamino acid-releasing enzyme that degrades glycated proteins [27]. Glycation adducts in plant proteins are formed by a slow in situ rate of protein glycation. They are removed by the degradation of glycated proteins by cellular proteolysis. Some glycation adducts are repaired by deglycation enzymes [10,27] and also by the slow spontaneous reversal of the protein glycation, where glycation adducts have moderate stability [12,13,28]. The levels of glycation adducts measured, therefore, are the steady-state levels maintained by these conflicting processes. The steady-state levels of protein glycation adducts are influenced by light, the stage of development, the season, environmental stresses, nutrients and other factors influential for plant metabolism [1] (Table1). Protein glycation has been implicated in the deterioration of plant seeds in storage [ 29]. The presence of enzymes in plants that suppress protein glycation and repair glycated proteins [25,30]—the enzymatic defense system against glycation [31]—suggests that glycation of the plant proteome poses a threat to plant physiology and growth.

Table 1. Eﬀect of growth conditions on protein glycation in Arabidopsis thaliana.

Growth Condition Effect on Protein Glycation of Glycating Reference Daylight to dark Early glycation adduct, FL: 3 mmol/mol lys (daylight entry), [1] growth cycle increasing to 10 mmol/mol lys (dark entry). Glycation adducts detected: CML, CMA, FL, G-H1 and MG-H1. Protein targets: a core group of 112 proteins, including chloroplast ATP synthase (β-subunit) and phosphoglycerate kinase. Glycated protein abundances were similar in heat, light and drought stresses. Diurnal period, Glycated proteins with altered abundance were: light stress—2 (RPI3 heat, light and and TPI, decreased); heat stress—1 (TPI, decreased); diurnal [22] drought variation—8 (ASP5, FTSH2 and RAN3, increased; AOC2, BAS1, CORI3, OASB, PRK, PRXQ and PURA, decreased); and drought stress 17 (A2, GSA2 and P83484, increased; CAT2, CICDH, CTIMC, CYP18-4, FBP, GGAT1, GLU1, LOX2, P25697, PER34, RBCS-1A, RBCS-3B, TGG2 and TL29, decreased). AGEs increased: G-H1 (0.14 to 0.35 mmol/mol arg) and CML (0.77 to Excess light stress [1] 1.65 mmol/mol lys). Major glycation adducts detected: CML, CMA and G-H1; 785 glycation sites detected on 724 proteins—33 and 62 glycation sites were unique for control and osmotically stressed plants, respectively. Abundance changes of AGE-modified proteins under osmotic stress Osmotic stress (range—2-fold decrease to 27-fold increase): 12 proteins involved in [19] lipid metabolism, DNA supercoils and methylation; protein ubiquitination and degradation; energy metabolism; cell organization and development; cell wall formation; and the regulation of transcription and stress. MG-H1 and CEL-modified proteins detected by immunoblotting and Ammonium NH + 4 immunoassays; 15% increase in CEL in ammonium NH +-grown [38] salts 4 plants compared with those in nitrate NO3−-grown control plants. Int. J. Mol. Sci. 2020, 21, 3942 4 of 16

Int. J. Mol. Sci. 2020, 21, 3942 4 of 18 Int. J. Mol. Sci. 2020, 21, 3942 4 of 18 Int. J. Mol. Sci. 2020, 21, 3942 4 of 18 Int. J. Mol. Sci. 2020, 21, 3942 4 of 18 Int. J. Mol. Sci. 2020, 21, 3942 Table 2. Early-stage glycation adducts and advanced glycation endproducts. 4 of 18 Table 1. Early-stage glycation adducts and advanced glycation endproducts. Table 1. Early-stage glycation adducts and advanced glycation endproducts. Glycating AgentTable 1. Early-stage glycation adducts and advanced Comment glycation endproducts. Glycating Agent Table 1. Early-stage glycation adducts and advanced glycation Comment endproducts. Glycating Agent Comment GlycatingH AgentO Formed by the degradation of reducing Comment sugars, glycated proteins, GlycatingH AgentO Formed by the degradation of reducing suga Commentrs, glycated proteins, nucleotides and lipid H Formednucleotides by the anddegradation lipid peroxidation of reducing suga [11,32rs,]. glycated Metabolized proteins, by nucleotides the and lipid H O Formedperoxidation by the [11,32]. degradation Metabolized of reducing by the suga glyoxalasers, glycated system proteins, [5]. Glyoxal nucleotides is present and inlipid solution O peroxidationFormedglyoxalase by the system[11,32]. degradation Metabolized [5]. Glyoxal of reducing by is the present suga glyoxalasers, in glycated solution system proteins, mainly [5]. Glyoxal nucleotides as mono- is present and in lipid solution H O Formedperoxidationmainly as by mono- the [11,32]. degradation and Metabolized di-hydrates of reducing [33].by the suga glyoxalasers, glycated system proteins, [5]. Glyoxal nucleotides is present and in lipid solution H mainlyperoxidationand di-hydrates as mono- [11,32]. and [33 Metabolizeddi-hydrates]. [33]. by the glyoxalase system [5]. Glyoxal is present in solution GlyoxalO peroxidationmainly as mono- [11,32]. and Metabolized di-hydrates [33].by the glyoxalase system [5]. Glyoxal is present in solution HGlyoxalO mainly as mono- and di-hydrates [33]. Glyoxal mainly as mono- and di-hydrates [33]. CHGlyoxal3 CHGlyoxal3 O 3 O FormedFormed mainly mainly by bythe thetrace-level trace-level degradation degradation of GA3P of and GA3P DHAP and [17]. Relatively high flux CH3 O Formed mainly by the trace-level degradation of GA3P and DHAP [17]. Relatively high flux 3 O FormedreactiveDHAP mainlydicarbonyl. [17]. Relatively by the Metabolized trace-level high ﬂux bydegradation the reactive glyoxalase dicarbonyl.of GA3P system and [5]. MetabolizedDHAP MG is[17]. present Relatively by in solution high flux mainly as O reactiveFormed dicarbonyl.mainly by the Metabolized trace-level bydegradation the glyoxalase of GA3P system and [5]. DHAP MG is[17]. present Relatively in solution high fluxmainly as H O Formedreactivemono-the glyoxalase and mainlydicarbonyl. di-hydrates by system the Metabolized trace-level [33]. [5]. Precursor MG bydegradation is the present of glyoxalase the major inof solutionGA3P syAGE,stem and MG-H1. mainly[5]. DHAP MG asis[17]. present mono- Relatively in solution high flux mainly as H O mono-reactive and dicarbonyl. di-hydrates Metabolized [33]. Precursor by the of glyoxalase the major AGE,system MG-H1. [5]. MG is present in solution mainly as H O reactivemono-and di-hydrates and dicarbonyl. di-hydrates [Metabolized33]. [33]. Precursor Precursor by the of of theglyoxalase the major major AGE,syAGE,stem MG-H1. [5]. MG is present in solution mainly as Methylglyoxal (MG) MethylglyoxalMethylglyoxalH (MG) (MG) mono- and di-hydrates [33]. Precursor of the major AGE, MG-H1. Methylglyoxal (MG) MethylglyoxalO (MG)O MethylglyoxalHO H O (MG)O HO H O O H O O FormedFormed byby the the degradation degradation of reducing of reducing sugars sugars and glycated and glycated proteins. Additionally formed by the HO H Formed by the degradation of reducing sugars and glycated proteins. Additionally formed by the HOCH2 H enzymaticproteins. repair Additionally of FL [34]. formed Metabo bylized the by enzymatic aldoketo reductases repair of [5]. FL 3-DG [34]. is present in solution as HOCH2 H H enzymaticFormed by repair the degradation of FL [34]. ofMetabo reducinglized sugars by aldoketo and glycated reductases proteins. [5]. 3-DG Additionally is present formed in solution by the as 2 H H H H aMetabolized complex mixture by aldoketoof cyclic he reductasesmiacetals and [5]. hemiketals 3-DG is present[33]. in solution HOCH2 H OHH H aenzymatic complex mixturerepair of of FL cyclic [34]. heMetabomiacetalslizedlized and byby aldoketoaldoketohemiketals reductasesreductases [33]. [5].[5]. 3-DG3-DG isis presentpresent inin solutionsolution asas 2 H OHH H as a complex mixture of cyclic hemiacetals and hemiketals [33]. H H H a complex mixture of cyclic hemiacetals and hemiketals [33]. 3-Deoxyglucosone3-DeoxyglucosoneH OHH (3-DG)(3-DG) a complex mixture of cyclic hemiacetals and hemiketals [33]. 3-Deoxyglucosone3-Deoxyglucosone (3-DG) (3-DG) 3-DeoxyglucosoneGlycation Adduct (3-DG) Comment - 3-DeoxyglucosoneGlycation Adduct (3-DG) Comment CO2- Glycation AdductOH Comment CO2- Glycation+ AdductOH Comment CO2 Glycation+ AdductOH Comment HC (CH- ) NH+ HCCO2(CH- 2) 4 NH 2 OH CO2 2 4 + 2 OH O HC (CH+ 2)4 NH+ 2 O Early-stage glycation adduct [1]. Formed from glucose non-enzymatically and exposure to HCNH3+(CH ) NH HO O Early-stage glycation adduct [1]. Formed from glucose non-enzymatically and exposure to HCNH3(CH+ 2)4 NH2 HO Early-stage glycation adduct [1]. Formed from glucose non-enzymatically and exposure to NH3 2 4 2 HO O OH increasedEarly-stage glucose glycation concentration. adduct Repaired [1]. Formed intracellularly from glucose by fructosamine 3-phosphokinase [35]. + OH OH increasedEarly-stage glucose glycation concentration. adduct [1]. RepairedFormed from intracellularly glucoseglucose non-enzymaticallynon-enzymatically by fructosamine 3-phosphokinaseandand exposureexposure toto [35]. NH3+ HO OH OH non-enzymatically and exposure to increased glucose concentration. 3 HO OH OH increased glucose concentration. Repaired intracellularly by fructosamine 3-phosphokinase [35]. Nε-(1-Deoxy-D-fructos-1-yl)lysineOH (FL) increased glucose concentration. Repaired intracellularly by fructosamine 3-phosphokinase [35]. Nε-(1-Deoxy-D-fructos-1-yl)lysine (FL)OH Repaired intracellularly by fructosamine 3-phosphokinase [35]. Nε-(1-Deoxy-D-fructos-1-yl)lysine (FL)OH - Nε-(1-Deoxy-D-fructos-1-yl)lysineCO2- (FL) Nε-(1-Deoxy-D-fructos-1-yl)lysineCO2- NH CH (FL)3 CO2 NH CH3 A major quantitative arginine-derived AGE formed from MG. Influenced by the rate of the HC (CH- ) NH NH CH3 A major quantitative arginine-derived AGE formed from MG. Influenced by the rate of the HCCO2(CH- 2) 3 NH HCH A major quantitative arginine-derived AGE formed from MG. Influenced by the rate of the HCCO2(CH2)3 NH NH HCH3 formation of MG, rate of metabolism of MG by Glo1 of the glyoxalase system and cellular + 2 3 NHN H 3 formationAA major major quantitative of quantitative MG, rate arginine-derived of metabolism arginine-derived of AGE MG byformed AGE Glo1 formed fromof the MG. glyoxalase from Influenced MG. system by theand rate cellular of the HCNH3+(CH ) NH N Aformation major quantitative of MG, rate arginine-derived of metabolism of AGE MG formedby Glo1 from of the MG. glyoxalase Influenced system by the and rate cellular of the HCNH3(CH+ 2)3 NH N OH proteolysis. Major AGE in Arabidopsis thaliana. Implicated in protein misfolding and, in excess, NH3 2 3 OH proteolysis.formationInﬂuenced of Major MG, by the rateAGE rate of in metabolism ofArabidopsis the formation ofthaliana. MG by of Implicated MG,Glo1 of rate the in of glyoxalase protein metabolism misfolding system of and and, cellular in excess, + N O formationproteolysis. of Major MG, rate AGE of in metabolism Arabidopsis of thaliana. MG by Implicated Glo1 of the in glyoxalase protein misfolding system and and, cellular in excess, MG-derivedNH3+ hydroimidazoloneN activation of the UPR. MG-derivedNH3 hydroimidazoloneO activationproteolysis.MG by Glo1 of Major the of UPR. theAGE glyoxalase in Arabidopsis system thaliana. and Implicated cellular proteolysis. in protein misfolding Major and, in excess, MG-derived (MG-H1) hydroimidazoloneO proteolysis.activation of Major the UPR. AGE in Arabidopsis thaliana. Implicated in protein misfolding and, in excess, MG-derived (MG-H1) hydroimidazolone activationArabidopsis of the UPR. thaliana. MG-derived (MG-H1) hydroimidazolone activationAGE in of the UPR. Implicated in protein misfolding and, Int. J. Mol. Sci. 2020 , 21 , 3942 (MG-H1) 5 of 18 Int. J. Mol. Sci. 2020 , 21 , 3942 (MG-H1) in excess, activation of the UPR. 5 of 18 Int. J. Mol. Sci. 2020, 21, 3942 5 of 18

- CO2- CO2- + A major quantitative lysine-derived AGE. Formed by the oxidative CO2 + - A major quantitative lysine-derived AGE. Formed by the oxidative degradation of FL (major), HC (CH2)4 NH+ 2 CH2 CO2- A major quantitative lysine-derived AGE. Formed by the oxidative degradation of FL (major), HC (CH2)4 NH2 CH2 CO2- glycationAdegradation major quantitative by glyoxal of FL and lysine-derived (major), by ascorbic glycation AGE.acid (u byFormedsually glyoxal minor). by the and oxidative Increased by ascorbic degradation by light acid stress of inFL Arabidopsis (major), HC +(CH2)4 NH2 CH2 CO2 NH3+ glycation(usually by minor). glyoxal Increased and by ascorbic by light acid stress (usually in Arabidopsisminor). Increased thaliana. by lightThe stress in Arabidopsis NH3+ thaliana.glycation The by CML/FLglyoxal and ratio by is ascorbic a marker acid of oxidative(usually minor). stress. Increased by light stress in Arabidopsis NH3 thaliana. The CML/FL ratio is a marker of oxidative stress. Nε-Carboxymethyl-lysine (CML) thaliana.CML/FL The ratio CML/FL is a markerratio is a of marker oxidative of oxidative stress. stress. Nε-Carboxymethyl-lysine (CML) Nε-Carboxymethyl-lysine+ - (CML) H3N+ CH CO2- H3N+ CH CO2- H3N CH CO2 (CH2)4 (CH2)4 (CH2)4 N - MajorMajor quantitative quantitative crosslink crosslink formed formed in protein in protein glycation glycation [36]. Produced [36]. from the degradation of N N CO2- Major quantitative crosslink formed in protein glycation [36]. Produced from the degradation of H N N CO2- FLMajor residues quantitative with a proximatecrosslink formed arginine in residu proteine. glycationContent in [36]. plant Produced proteins from is unknown. the degradation of H N NH (CH ) CO Produced from the degradation of FL residues with a proximate H 2 3 CH 2 FL residues with a proximate arginine residue. Content in plant proteins is unknown. HO NH (CH2)3 CH FLarginine residues residue. with a proximate Content arginine in plant residu proteinse. Content is unknown. in plant proteins is unknown. HO N NH (CH2)3 NHCH + HO N 3+ H H N NH3+ H OH H GlucosepaneNH3 H OH H Glucosepane -OH Glucosepane CO2- N CO2- N HCCO2(CH ) NH N HC (CH2)3 NH + HC (CH+ 2)3 NH HN + - Low-level pentose sugar-derived glycation crosslink and intense fluorophore. Considered to NH3+ 2 3 HN N+ CO2- Low-levelLow-level pentose pentose sugar-derived sugar-derived glycation glycation crosslink crosslink and intense and fluorophore. intense Considered to NH3+ HN N CO2- reflectLow-level pentosephosphate pentose sugar-derived pathway glycation activity [37]. crosslink and intense fluorophore. Considered to NH3 N(CH ) CHCO2 reflectﬂuorophore. pentosephosphate Considered pathway to reﬂect activity pentosephosphate [37]. pathway (CH2)4 CH reflect pentosephosphate pathway activity [37]. (CH2)4 CH + Pentosidine 2 4 NH3+ activity [37]. Pentosidine NH3+ Pentosidine NH3

3. Glycation in Arabidopsis thaliana Studies of protein glycation in Arabidopsis thaliana under normal growth and stress conditions are summarized in Table1. Protein glycation was studied in Arabidopsis thaliana by assaying protein glycation adducts in cytosolic protein extracts, using the reference method of liquid

Int. J. Mol. Sci. 2020, 21, 3942 5 of 16 chromatography-tandem mass spectrometry (LC-MS/MS). This involves the prior exhaustive enzymatic hydrolysis of proteins and quantitation of glycation adducts, glycated amino acids, by stable isotopic dilution analysis LC-MS/MS [13,39,40]. Under basal conditions, the mean extent of protein modification by major glycation adducts was: FL residues, 26%; MG-H1 residues, 4%; and CML residues, 3% [1]. There was a relatively high content of FL residues in plant protein on entering the daylight period—ca. 3 mmol/mol lys. This glycation adduct increased during and beyond the daylight period, when photosynthesis leads to a 10-fold increase in the glucose concentration of the leaf tissue [41] (Table1). The increase in FL residue content reflects this increased exposure to glucose. The CML residue content of plant protein was relatively high, 0.35–0.71 mmol/mol lys, and showed a similar trend to the change in the FL residue content. This may be due to the high content of FL residues and ascorbic acid [42]—precursors of CML residue formation [15,16]. Excess light stress also increased the CML and G-H1 residue content of total leaf protein by ca. 2-fold. CML and G-H1 adduct residues are formed by the glycation of proteins with glyoxal, which may relate to periods of increased lipid peroxidation and formation of glyoxal [32]. MG-derived MG-H1 residues were the AGE of highest content in the Arabidopsis thaliana proteome at ca. 2 mmol/mol arg [1]. Other MG-derived AGE residue contents, Nε-(1-carboxyethyl)lysine (CEL) and methylglyoxal-derived lysine dimer MOLD, were ca. 10-fold and 400-fold lower than MG-H1. All the AGE residues, except for CML, tended to show oscillatory diurnal behavior, where the maxima of residue contents occurred in the middle of the light and dark periods and lower levels of AGE residue contents occurred between these times [1]. In recent studies, the boronate affinity enrichment method was used to identify proteins in Arabidopsis thaliana modified by FL residues, with protein identification by high mass resolution Orbitrap mass spectrometry proteomics. One hundred and twelve glycated proteins were identified [22]. FL modification and the location of the glycation sites in retained proteins were detected through mass spectrometric detection and the fragmentation of peptides in tryptic digests using high sensitivity mass proteomics, with a mass increment of lysine residues (+162 Da). In this approach, however, there is typically a mean peptide sequence coverage of ca. 25%, and, therefore, some glycation adducts are missed [43]. CML, CMA, G-H1 and MG-H1 residues were also detected in the retained proteins—although these adducts do not bind to boronate affinity columns [12,13,44]. A core group of 112 proteins were identified as glycated, including chloroplast ATP synthase (β-subunit) and phosphoglycerate kinase; 90% of the glycated proteins were of chloroplast origin [22]. The abundances of most of these glycated proteins were similar in experiments investigating the effects of heat, light and drought stresses. The numbers of abundance changes of glycated proteins in stress conditions were: light stress, 2; heat stress, 1; diurnal variation, 8; and drought conditions, 17 [22] (Table1). Proteins modified by early-stage glycation adducts and AGEs were examined in a proteomics study. AGE residues were detected at 96 different sites in 71 proteins, with age-dependent changes. Unique age-related proteins modified by AGEs (AGE, sequence location) in 9- and 12-week-old plants and pathways involved were: β-carbonic anhydrase-2, chloroplastic (CMA, R202) and ACT domain containing protein ACR9 (MG-H1, R395)—involved in amino acid metabolism; putative fucosyl transferase-7 (CML, K393 and K394)—involved in cell wall biosynthesis; tetratricopeptide repeat-like superfamily protein (CEL, R83; MG-H1, R86)—involved in the oxidative stress response; and CEL in two uncharacterized proteins. Homology modeling revealed glutamyl and aspartyl residues in close proximity (less than 0.5 nm) to these sites in three aging-specific and eight differentially glycated proteins, four of which were modified in catalytic domains [3]. The protein domains of plant proteins susceptible to glycation have not been widely studied. In mammalian proteins, the domains susceptible to glycation by MG were the tailless complex polypeptide-1 (TCP-1) and GroEL protein domains of chaperonins, the 14-3-3 domain, the α/β subunits of the proteasome, class I and class II aminoacyl-tRNA synthetases, actin and Rossmann-like α/β/α sandwich fold [6]. Similar domains in plant proteins may be susceptible to glycation by MG. Int. J. Mol. Sci. 2020, 21, 3942 6 of 16

A comprehensive study of the changes in proteins modified by AGEs of Arabidopsis thaliana in osmotic stress was reported [19]. Plants were grown from seeds and at 6 weeks and then transferred to new growth medium with and without polyethylenglycol-8000 to provide drought stress and the accumulation of osmolytes, amino acids and carbohydrates. After the application of this osmotic stress for 3 days, changes in 31 stress-specific and 12 differentially AGE-modified proteins reflecting AGEs at 56 different sites were found [19]. Monosaccharide autoxidation [45] was proposed as the main stress-related glycation mechanism, and glyoxal, as the major glycation agent in plants subjected to drought [19].

4. Dicarbonyl Stress in Plants Dicarbonyl stress is the abnormal accumulation of reactive dicarbonyl metabolites leading to increased protein glycation and is linked to cell and tissue dysfunction, aging and disease [5]. MG is a key dicarbonyl metabolite contributing to dicarbonyl stress in plants, unavoidably formed by the non-enzymatic degradation of triosephosphates. It is formed at relatively high flux compared to other dicarbonyl metabolites, ca. 0.1% of glucose metabolism, and has high reactivity with proteins [5]. It is precursor of the major AGE in plant proteins quantitatively, MG-H1 [24,46]. We determined the content of glyoxal, MG and 3-DG of leaves of Brassica oleracea plants by the reference analytical method of stable isotopic dilution analysis LC-MS/MS [24]. We studied the leaf dicarbonyl content at three stages of development (days post-seeding): cotyledons (6 days), first fully developed mature leaves (30 days) and mature plants (65 days). The glyoxal content of cotyledons was ca. 0.4 nmol/g fresh weight (ca. 0.4 µM), and it was similar at 30 days but increased 2-fold at 65 days. The MG content of cotyledons was ca. 3 nmol/g fresh weight (ca. 3 µM), and it was similar at 30 days but increased ca. 33% at 65 days. The 3-DG content of cotyledons was ca. 8 nmol/g fresh weight (ca. 0.8 µM); it increased to ca. 2-fold at 30 days and then decreased to levels similar to those at the cotyledon stage at 65 days (Table3). The markedly higher estimates of MG published previously of ca. 50 nmol/g fresh weight [47] were overestimates likelydue to triosephosphate degradation to MG during pre-analytical processing [48]. The mathematical metabolic modeling of in situ glycation in physiological tissues predicts the steady-state cellular concentrations of MG as 1–4 µM[24] The reactivity towards protein glycation with respect to glucose of glyoxal, MG and 3-DG is ca. 5000, 20,000 and 200 times higher, respectively, so MG is expected to be the major dicarbonyl glycating agent in the leaves of Brassica oleracea. The mature 65 day Brassica plant appears to have been suffering dicarbonyl stress. The increase and later decrease in 3-DG content may reflect the mobilization of glucose metabolism, declining in the later stages of maturity; cf. measurements of glucose and fructose with plant development [49]. The siRNA silencing of Glo1 and accumulation of MG in other species produced an accelerated aging phenotype, whereas the overexpression of Glo1 increased longevity and produced resistance to metabolic dysfunction in aging [50,51]. Therefore, increased MG glycation may impair plant proteome integrity and vitality in older plants and provide a cue for senescence. The prolonged use of ammonium salts as the sole nitrogen source to plants may result in physiological and morphological disorders leading to decreased plant growth. This is a worldwide problem, constraining crop production. It is common example of abiotic stress [52]. The effect of this on dicarbonyl stress in Arabidopsis thaliana was investigated. Changes in the activities of glycolytic enzymes increased the formation and concentration of MG. The excessive accumulation of MG, dicarbonyl stress, produced increased MG-derived AGEs. Dicarbonyl stress may contribute to ammonium toxicity symptoms in Arabidopsis thaliana and the ammonium salt impairment of crop plant growth [38]. Int. J. Mol. Sci. 2020, 21, 3942 7 of 16 Int. J. Mol. Sci. 2020, 21, 3942 9 of 18 Int. J. Mol. Sci. 2020, 21, 3942 9 of 18 Int. J. Mol. Sci. 2020, 21, 3942 9 of 18 TableTable 3. Reac 3. Reactivetive dicarbonyl dicarbonyl glycating glycating agents agents in Brassica in Brassica oleracea oleracea duringduring development development.. Table 3. Reactive dicarbonyl glycating agents in Brassica oleracea during development. Table 3. Reactive dicarbonyl glycating agents in Brassica oleracea during development. DicarbonylDicarbonyl Metabolite Metabolite Days Dicarbonyl Metabolite Days Post-Sowing Plant Plant Appearance Appearance (nmol/g(nmolDicarbonyl Fresh/g Fresh Weight; Weight; Metabolite Mean Mean ± SD,SD, n = 6) Post-SowingDays Post-Sowing Plant Appearance (nmol/g Fresh Weight; Mean± ± SD, n = 6) Days Post-Sowing Plant Appearance (nmol/gGlyoxal Fresh Weight; MG Mean ± SD, 3-DGn = 6) GlyoxalGlyoxal MG MG 3-DG3-DG Glyoxal MG 3-DG

6 0.38 ± 0.04 2.90 ± 0.81 0.76 ± 0.29 6 6 0.38 0.040.38 ± 0.04 2.90 0.81 2.90 ± 0.81 0.76 0.760.29 ± 0.29 6 0.38 ±± 0.04 2.90 ± 0.81± 0.76 ± 0.29±

30 0.46 ± 0.12 3.47 ± 1.21 1.80 ± 1.05 * 30 0.46 ± 0.12 3.47 ± 1.21 1.80 ± 1.05 * 3030 0.460.46 ± 0.120.12 3.47 3.47 ± 1.211.21 1.80 ± 1.80 1.05 *1.05 * ± ± ±

65 0.81 ± 0.32 **,OO 4.08 ± 0.27 * 0.49 ± 0.23 O 65 0.81 ± 0.32 **,OO 4.08 ± 0.27 * 0.49 ± 0.23 O 65 0.81 0.81± 0.320.32 **,OO 4.08 ± 0.27 * 0.49 ± 0.23 O 65 ± 4.08 0.27 * 0.49 0.23 O **,OO ± ±

B. oleracea leaves were from broccoli cv. GDDH33, a well characterized doubled haploid breeding line derived from cv. Green Duke, was sown into F2 compost. The leaves from six plants were removed and flash frozen in liquid nitrogen and stored at 20 ◦C until analysis. The dicarbonyl contents in the leaves were determined by stable isotopic dilution analysis− LC-MS/MS [24]. Briefly, plant leaf (ca. 10 mg fresh weight) was homogenized in 13 13 5 % trichloroacetic acid with 0.3 % azide to inhibit peroxidase. Internal standards ([ C3]MG, [ C2]glyoxal and 13 [ C6]3-DG, 2 pmol) were added, mixed and centrifuged (10,000 g, 10 min, 4 ◦C). Supernatants were derivatized with 1,2-diaminobenzene and analyzed by LC-MS/MS. Significance: * and **, P < 0.05 and P < 0.01, with respect to 6 days; and O and OO, P < 0.05 and P < 0.01, with to respect 30 days; Student’s t-test. Data on MG estimation were published previously [24].

5. Enzymatic Defense Against Glycation—The Glyoxalase System and Aldoketo Reductases 5. Enzymatic Defense Against Glycation—The Glyoxalase System and Aldoketo Reductases 5.The Enzymatic glyoxalase Defense pathway Against catalyzes Glycation—The the conversion Glyoxalase of MG to D-lactate.System and It isAldoketo a two-step, Reductases enzymatic 5. Enzymaticpathway:The glyoxalase Defense glyoxalase pathwayAgainst 1 (Glo1) Glycation—Thecatalyzes catalyzes the the conversion conversion Glyoxalase of ofMG System the to hemithioacetal D-lactate. and Aldoketo It is formed a two-step, Reductases non-enzymatically enzymatic The glyoxalase pathway catalyzes the conversion of MG to D-lactate. It is a two-step, enzymatic pathway:Thefrom glyoxalase MG glyoxalase and reducedpathway 1 (Glo1) glutathione catalyzes catalyzes the (GSH) conversion the to S-D-lactoylglutathioneconversion of MG to ofD-lactate. the hemithioacetal It (SLG); is a two-step, and glyoxalase formed enzymatic 2non- (Glo2) pathway: glyoxalase 1 (Glo1) catalyzes the conversion of the hemithioacetal formed non- pathway:enzymaticallycatalyzes glyoxalase the from hydrolysis 1MG (Glo1) and of SLGreducedcatalyzes to D-lactate, glutathionethe conversion reforming (GSH) of theto the GSHS-D-lactoylglutathione hemithioacetal consumed in theformed Glo1-catalyzed(SLG); non- and enzymatically from MG and reduced glutathione (GSH) to S-D-lactoylglutathione (SLG); and enzymaticallyglyoxalasestep [53 –255 (Glo2)from] (Figure MGcatalyzes1 ).and There thereduced ishydrolysis a further glutathione proteinof SLG to called(GSH) D-lactate, glyoxalase-3 to S-D-lactoylglutathione reforming (Glo3) the [GSH56], butconsumed (SLG); concern andin remains the glyoxalase 2 (Glo2) catalyzes the hydrolysis of SLG to D-lactate, reforming the GSH consumed in the glyoxalaseGlo1-catalyzedon its 2 functional (Glo2) step catalyzes attribution[53–55] the (Figure ashydrolysis a glyoxalase 1). There of SLG is involved a tofurther D-lactate, in protein MG reforming metabolism called glyoxalase-3 the physiologically GSH consumed (Glo3) due [56], in tothe but its low Glo1-catalyzed step [53–55] (Figure 1). There is a further protein called glyoxalase-3 (Glo3) [56], but Glo1-catalyzedconcerncatalytic remains e ffistepciency. [53–55]on Catalyticits (Figurefunctional effi 1).ciency Thereattribution is is defined a further as by a theprotein glyoxalase specificity called involvedconstantglyoxalase-3 kcatin / K(Glo3)MGM [ 57metabolism ].[56], The but kcat /K M concern remains on its functional attribution as a glyoxalase involved in MG metabolism concernphysiologicallyvalues remains for Arabidopsis dueon toits itsfunctional thaliana low catalyticGlo1 attribution (isoform efficiency. 2,as accountingCatalytica glyoxalase efficiency for >involved99% is of defined Glo1 in MG activity) by themetabolism specificity and Glo3 are physiologically10 1 1due to its low6 catalytic1 1efficiency. Catalytic efficiency is defined by the specificity physiologicallyconstant1.1 10kcat/Kmin dueM [57].− toM The−its andlow kcat 3.4/KcatalyticM values10 minefficiency. for− ArabidopsisM− , respectivelyCatalytic thaliana efficiency [ 56Glo1,58]; (isoform ca.is defined 3100-fold 2, accounting by higherthe specificity for for Glo1 >99% than cat M cat M ×constant k /K [57]. The ×k /K10 values−1 for−1 Arabidopsis6 thaliana−1 −Glo11 (isoform 2, accounting for >99% constantof Glo1Glo3. k activity)cat The/KM proteomic[57]. and The Glo3 kcat abundances are/KM 1.1 values x 10 offor minGlo1 ArabidopsisM and and Glo3 thaliana3.4 in× 10Arabidopsis Glo1 min (isoformM , thaliana respectively 2, accountingwere [56,58]; 1140 for and ca >99%. 300 3100- ppm, of Glo1 activity) and Glo3 are 1.1 x 1010 min−1M−1 and 3.4 × 106 min−1M−1, respectively [56,58]; ca. 3100- of foldGlo1respectively higher activity) for and [Glo159 ],Glo3 ca.than 4-foldare Glo3. 1.1 higher x The 1010 prot formin Glo1eomic−1M−1 than and abundances Glo3.3.4 × 10 The6 min of substrate Glo1−1M−1 ,and respectively of Glo1Glo3 isin the Arabidopsis [56,58]; hemithioacetal ca. thaliana3100- (HA) fold higher for Glo1 than Glo3. The proteomic abundances of Glo1 and Glo3 in Arabidopsis thaliana foldwere higheradduct 1140 for ofand MGGlo1 300 with thanppm, reduced Glo3. respectively The glutathione prot [59],eomic ca (GSH),. 4-foldabundances whereas higher of thefor Glo1 substrateGlo1 and than Glo3 of Glo3. Glo3 in TheArabidopsis is MG; substrate the concentrationthaliana of Glo1 were 1140 and 300 ppm, respectively [59], ca. 4-fold higher for Glo1 than Glo3. The substrate of Glo1 wereis the of1140 GSHhemithioacetal and in 300Arabidopsis ppm, (HA) respectively thaliana adductis of [59], ca. MG 0.4ca with. mM4-fold r [educed60 higher], and glutathione for the Glo1 equilibrium than (GSH), Glo3. constant whereas The substrate for the HA substrate of formation Glo1 of is is the1 hemithioacetal (HA) adduct of MG with reduced glutathione (GSH), whereas the substrate of is Glo3the333 hemithioacetal is MMG;− [the61], concentration leading (HA) adduct to an of HA of GSH MG/MG in with concentrationArabidopsis reduced thaliana glutathione ratio ofis ca. 0.17. (GSH), 0.4Taking mM whereas [60], these and the factors the substrate equilibrium into accountof Glo3 is MG; the concentration of GSH in Arabidopsis thaliana is ca. 0.4 mM [60], and the equilibrium Glo3constant (is3100 MG; for the4 HA concentration0.17 formation), the ratio is of of333 GSH the M− rate1in [61], Arabidopsis of leading metabolism tothaliana an ofHA/MG MGis ca. in 0.4 concentration situ mM by [60], Glo1 and/Glo3 ratio the is of equilibrium ca. 0.17. 2100, Taking or only constant× × for HA formation is 333 M−1 [61], leading to an HA/MG concentration ratio of 0.17. Taking constanttheseca. factors 0.05%for HA ofinto formation MG account is metabolized is (3100333 M x− 1 4[61],by x 0.17), Glo3. leading the Therefore, to ratio an HA/MGof Glo3the rate doesconcentration of not metabolism contribute ratio ofof significantly MG0.17. inTaking situ toby MG these factors into account (3100 x 4 x 0.17), the ratio of the rate of metabolism of MG in situ by theseGlo1/Glo3 metabolismfactors isinto ca. in account2100, plants or under(3100only cax physiological .4 0.05%x 0.17), of theMG conditions ratio is metabolized of the and rate may byofhave metabolismGlo3. a diTherefore,fferent, of MG as Glo3 yet in unidentified,situdoes by not Glo1/Glo3 is ca. 2100, or only ca. 0.05% of MG is metabolized by Glo3. Therefore, Glo3 does not Glo1/Glo3contributefunction. is significantlyca There. 2100, is or also toonly MG a protein ca metabolism. 0.05% called of MG glyoxalase-4in plan is metabolizedts under with physiological no by kinetic Glo3. characteristicsTherefore, conditions Glo3 and reported, maydoes have not which a contribute significantly to MG metabolism in plants under physiological conditions and may have a contributedifferent,appears significantly as to yet have unidentified, a roleto MG in themetabolism fu metabolismnction. Therein plan of highists also under concentrations a protein physiological called of exogenous conditionsglyoxalase-4 MG and with (10 may mM) no have kinetic [62 ].a This different, as yet unidentified, function. There is also a protein called glyoxalase-4 with no kinetic different,characteristicsphysiological as yet unidentified,reported, relevance which remains function. appears to beThere evaluatedto have is also a withrole a protein in the the markedly metabolismcalled glyoxalase-4 lower of levels high of withconcentrations MG no levels kinetic found of in characteristics reported, which appears to have a role in the metabolism of high concentrations of characteristicsexogenousplants—see MG reported, Table(10 mM)3. which [62]. Thisappears physiological to have a rele rolevance in the remains metabolism to be evaluated of high concentrations with the markedly of exogenous MG (10 mM) [62]. This physiological relevance remains to be evaluated with the markedly exogenouslower levels MG of (10 MG mM) levels [62]. found This physiological in plants—see rele Tablevance 3. remains to be evaluated with the markedly lower levels of MG levels found in plants—see Table 3. lower levels of MG levels found in plants—see Table 3. Int. J. Mol. Sci. 2020, 21, 3942 8 of 16 Int. J. Mol. Sci. 2020, 21, 3942 10 of 18

Figure 1. The glyoxalase system. Shown is the metabolism of methylglyoxal to D-lactate. Glyoxal is metabolizedFigure 1. The similarly glyoxalase to glycolate.system. Shown is the metabolism of methylglyoxal to D-lactate. Glyoxal is metabolized similarly to glycolate. The presence of the glyoxalase system in plants has been known for many years [25], where it was initiallyThe linked presence to plantof the cell glyoxalase growth [system63]. Later, in plants it emerged has been that theknown overexpression for many years of Glo1 [25], and where Glo2 it inwas tobacco initially plants linked provided to plant increasedcell growth tolerance [63]. Late tor, high it emerged exogenous that MG the andoverexpression high salinity of stress Glo1 [and30]. GenomicGlo2 in tobacco analysis plants identified provided 19 potential increased Glo1 tolerance and four to high Glo2 exogenous proteins in MG rice and and high 22 Glo1 salinity and stress nine Glo2[30]. Genomic proteinsin analysisArabidopsis identified thaliana 19. Thepotential expression Glo1 profilesand four di Glo2ffered proteins in response in rice to abioticand 22 stressesGlo1 and in dininefferent Glo2 tissues proteins and in during Arabidopsis various thaliana stages. of The vegetative expression and profiles reproductive differed development in response [64 ].to Aabiotic more recentstresses study in different identified tissues 40% Glo1and during activity various and 10% stages Glo2 of activity vegetative in the and chloroplasts reproductive of spinach development [65]. In Arabidopsis[64]. A more thaliana recent, understudy highidentified CO2 concentrations,40% Glo1 activity where and photosynthesis 10% Glo2 activity and in the the formation chloroplasts of MG of arespinach increased, [65]. In Glo1 Arabidopsis and Glo2 thaliana expression, under and high activities CO2 concentrations, were increased. where This identifies photosynthesis the function and the of theformation glyoxalase of MG system are in increased, plants as one Glo1 fundamental and Glo2 toex plantpression biochemistry, and activities providing were protection increased. against This endogenousidentifies the dicarbonylfunction of stress the glyo [65xalase]. The system increased in plants expression as one of fundamental Glo1 was involved to plant in biochemistry, the adaptive responseproviding of protection wild type againstArabidopsis endogenous thaliana dicarbonyland the ascorbate-deficient stress [65]. The increased mutant vtc2-2 expressionto prolonged of Glo1 exposurewas involved to high in lightthe adaptive intensity response [66], now of seen wild as duetype to Arabidopsis protecting againstthaliana risk and of the photosynthesis-linked ascorbate-deficient dicarbonylmutant vtc2-2 stress. to prolonged exposure to high light intensity [66], now seen as due to protecting against risk ofThere photosynthesis-l are AKRs ininked higher dicarbonyl plants [67 stress.]. These were investigated in Oryza sativa (Asian rice) for theirThere role in are anti-glycation AKRs in higher defense. plants AKR [67] isoform-1. These ofwere rice investigated (OsAKR1) was in inducedOryza sativa by abscisic (Asian acidrice) and for varioustheir role stress in anti-glycation treatments, whereas defense. two AKR other isoform-1 AKR genes of rice were (OsAKR1) moderately was stress-inducible. induced by abscisic OsAKR1 acid is anand NAPDH-dependent various stress treatments, reductase whereas with catalytic two othe activityr AKR towards genes MG were and moderately malondialdehyde, stress-inducible. the latter formedOsAKR1 in lipidis an peroxidation. NAPDH-dependent The heterologous reductase expression with ofcatalytic OsAKR1 activity in transgenic towards tobacco MG plants and producedmalondialdehyde, increased the tolerance latter formed to oxidative in lipid stress peroxidation. generated by The methylviologen heterologous and expression improved of resistanceOsAKR1 toin hightransgenic temperature. tobacco Transgenic plants produced tobacco plants increased also exhibited tolerance higher to oxidative AKR activity stress and generated accumulated by lessmethylviologen MG in their leavesand improved than the wild resistance type plants, to high both intemperature. the presence Transgenic and absence tobacco of heat stress.plants These also resultsexhibited suggest higher OsAKR1 AKR activity may also and have accumulated a role in cytoprotection less MG in their against leaves dicarbonyl than the stress wild in type plants plants, [26]. both in the presence and absence of heat stress. These results suggest OsAKR1 may also have a role 6.in Glycationcytoprotection in Plants—Considerations against dicarbonyl stress for in Crops plants and [26]. Other Commercial Aspects Brassica oleracea is an economically and nutritionally important species of plant that is the product of domestication6. Glycation in with Plants—Considerations limited genetic diversity for comparedCrops and to Other its wild Commercial ancestral relatives. Aspects The species exists in severalBrassica diff erentoleracea cultivated is an economically forms—cabbage, and cauliflower,nutritionally broccoli important and Brusselsspecies sprouts—andof plant that alsois the in itsproduct wild formof domestication distributed alongwith limited the European genetic Atlanticdiversity seaboard compared and to throughoutits wild ancestral the Mediterranean relatives. The area.species The exists reduced in several genetic different base of domesticated cultivated forms—cabbage,B. oleracea makes cauliflower, it difficult to broccoli find new and variants Brussels that contributesprouts—and towards also in phenotypes its wild form capable distributed of resisting along stress the European that are neededAtlantic to seaboard respond and to the throughout local and globalthe Mediterranean challenges of area. food The security reduced [68 ].genetic base of domesticated B. oleracea makes it difficult to find

Int. J. Mol. Sci. 2020, 21, 3942 9 of 16

Self-incompatibility (the rejection of “self”-pollen) is a reproductive barrier preventing inbreeding, to promote outcrossing and hybrid vigor [69]. Glo1 is a stigma compatibility factor required for pollination to occur and is targeted by the self-incompatibility system. Decreased Glo1 expression reduced compatibility, and the overexpression of Glo1 in self-incompatible Brassica napus stigmas resulted in the partial breakdown of the self-incompatibility response, suggesting MG-modified proteins may produce a response leading to pollen rejection [70]. A copy number increase of the GLO1 gene has been explored in Brassica plants [71]. In future, crop varieties having functional increased copy numbers of GLO1 may be assessed for improved cross-breeding and improved growth and rigor in maturity and at harvest. Recombinant human proteins may be expressed in plant-based systems. Examples include human serum albumin and protein immunogenic epitopes for vaccine development and production [72,73]. Recombinant human serum albumins (HSAs) were produced in Oryza sativa. LC-MS analysis identified a greater number of hexose-glycated arginine and lysine residues on HSA produced in Oryza sativa. There was supplier-to-supplier and lot-to-lot variability in the degree of glycation. Glycation influenced the presence of oligomeric species and tertiary structure. This may have further implications for the use of HSA as a therapeutic product in Oryza sativa [72]. The relevance of this glycation is not yet clear but if it impairs the function of HSA—such as ligand binding and esterase activity [74]. It may bring into doubt the use of plant-produced albumin for clinical applications.

7. Why Is Glycation Potentially Damaging to Plants? Glycation in plant proteins is found at relatively low levels, estimated at 26 mol% for FL residues and 4 mol% for MG-H1 residues [1]. Glycation is particularly damaging if it occurs on amino acids in the functional domains of proteins and if modification produces the loss or change of the charge of the target amino acid [75]. To assess the probability of glycation sites being in functional domains in the plant proteome, we applied sequence-based receptor binding domain (RBD) analysis [76] to the proteome of Arabidopsis thaliana. In optimized format, RBD analysis involves a plot of the mean hydrophobicity against the mean dipole moment of a window of five amino acid residues moved sequentially along the sequence of a protein (with a gyration angle between two consecutive residues in the sequence of 100◦ assumed). This approach had 80% accuracy when validated against a database of known interacting proteins [76]. The outcome of the application of RBD analysis to the proteome of Arabidopsis thaliana is given herein for the first time in Table4. The prediction of the proportion of amino acid residues of the RBD region or functional domain suggests that the amino acid residue targets of glycation, lysine and arginine residues, are enriched in functional domains—2.1-fold and 3.7-fold, respectively. By contrast, the amino acid targets of oxidative damage are depleted in functional domains: enrichment—cys, 0.8; met, 0.7; tyr, 0.8; and trp, 0.6. The plant proteome thereby is predicted to be relatively resistant to oxidative functional impairment [77,78]. The glycation of lysine by glucose to form FL residues leads to the retention of sidechain charge, whereas the glycation of arginine by MG produces MG-H1 residues and a loss of charge. Since arginine residues are often in functional domains for salt bridge electrostatic interaction with other proteins, enzyme substrates and nucleic acids, the formation of MG-H1 often produces protein inactivation and misfolding [6,75]. The plant proteome is, therefore, susceptible to glycation by MG at functional domains. This is likely an important physiological source of misfolded proteins and substrates for the UPR in plants [79]. Int. J. Mol. Sci. 2020, 21, 3942 10 of 16

Table 4. Receptor binding domain (RBD) analysis of the proteome of Arabidopsis thaliana.

Amino Count Acid Proteome RBD % AA in Proteome % AA in RBD Fold Enrichment Ala 463,770 25,941 6.5 3.3 0.5 Arg 380,640 150,922 5.3 19.5 3.7 Asn 317,995 44,745 4.4 5.8 1.3 Asp 384,200 52,528 5.3 6.8 1.3 Cys 130,271 10,915 1.8 1.4 0.8 Gln 250,179 38,180 3.5 4.9 1.4 Glu 474,124 70,661 6.6 9.1 1.4 Gly 473,373 30,225 6.6 3.9 0.6 His 160,243 20,712 2.2 2.7 1.2 Ile 392,264 8682 5.5 1.1 0.2 Leu 697,276 28,075 9.7 3.6 0.4 Lys 449,328 101,031 6.3 13.0 2.1 Met 164,360 11,802 2.3 1.5 0.7 Phe 314,311 8387 4.4 1.1 0.2 Pro 341,009 29,637 4.7 3.8 0.8 Ser 636,209 67,405 8.9 8.7 1.0 Thr 369,142 36,395 5.1 4.7 0.9 Trp 90,588 5539 1.3 0.7 0.6 Tyr 209,664 17,971 2.9 2.3 0.8 Val 487,953 15,139 6.8 2.0 0.3 Total: 7,186,899 774,892 100 100 Amino acid count and RBD analysis applied to 15,938 reviewed protein sequences from the UniProt Knowledgebase (UniProtKB; www.uniprot.org).

8. Role of Dicarbonyl Stress in the Unfolded Protein Response in Plants Recent studies in mammalian cells suggest that the MG modification of proteins produces misfolding and activation of the UPR [6]. There is an analogous UPR system in plants. It is activated in response to cold, drought, heavy metals and light stress. It preserves proteostasis and protects against the inhibition of photosynthesis [79]. The UPR in plants remains to be fully characterized [6,80]. As in mammalian systems, the inhibitor of enzymatic glycosylation, tunicamycin, has often been used to activate the UPR in the endoplasmic reticulum (ER) or induce ER stress. The physiological activators of unfolded proteins are different, often linked to spontaneous modification such as oxidative damage and MG-derived AGE formation. The latter is particularly damaging to protein structure because it produces a loss of charge, modifications are often in folded and highly structured functional domains, and MG modification also inactivates chaperonins, which catalyze the correct folding of proteins [6]. The link between dicarbonyl stress and the unfolded protein response in plants now deserves investigation.

9. Conclusions Protein glycation is an unavoidable part of plant metabolism and proteotoxicity, contributing to the damaging eﬀects of excess light, environmental and other stresses in plants. Glycation by glucose and MG produces major early-stage glycation adducts and AGEs, respectively. The levels of these glycating agents and related glycation adducts change with the developmental stage, senescence, light and dark cycles and also biotic and abiotic stresses. Proteomics analysis suggests the susceptibility of the plant proteome to functional inactivation by glycation—particularly glycation on arginine residues by MG. Dicarbonyl stress is an abnormal metabolic state, developing in mature plants during normal growth and cultivation. It may be linked to plant self-incompatibility, impaired plant vitality, pre-mature senescence and sensitivity to abiotic stress—including salinity, drought, extreme temperature and the prolonged use of ammonium salts. Metabolically, dicarbonyl stress is a driver of the increased formation of misfolded proteins and activation of the UPR. Crop breeding for increased functional Int. J. Mol. Sci. 2020, 21, 3942 13 of 18

plants during normal growth and cultivation. It may be linked to plant self-incompatibility, impaired Int. J. Mol. Sci.plant2020 vitality,, 21, 3942 pre-mature senescence and sensitivity to abiotic stress—including salinity, drought, 11 of 16 extreme temperature and the prolonged use of ammonium salts. Metabolically, dicarbonyl stress is a driver of the increased formation of misfolded proteins and activation of the UPR. Crop breeding for GLO1 geneincreased copy numbers functional in GLO1 plants gene may copy produce numbers varieties in plants resistant may produce to dicarbonyl varieties stress, resistant abiotic to stresses and senescencedicarbonyl with stress, improved abiotic breeding stresses andand growthsenescence characteristics—including with improved breeding plantsand growth of commercial ornamentalcharacteristics—including and crop cultivation plants value. of Metabolic commercialdrivers, ornamental dicarbonyl and crop cultivation metabolite, value. and Metabolic the consequences drivers, dicarbonyl metabolite, and the consequences of and a strategy for the resolution of of and a strategydicarbonyl for stress the in resolution plants are summarized of dicarbonyl in Figure stress 2. in plants are summarized in Figure2.

Figure 2. MetabolicFigure 2. Metabolic drivers, drivers, pathophysiological pathophysiological eeffectsﬀects an andd a strategy a strategy for the for resolution the resolution of dicarbonyl of dicarbonyl stress in plants.stress in plants.

Author Contributions: N.R. and P.J.T. wrote the manuscript, P.J.T. did the dicarbonyl analysis for Table 2 and Author Contributions:M.A.-M. computedN.R. the and proteome P.J.T. wrotestatistics the for manuscript,Table 4. All authors P.J.T. read did and the appr dicarbonyloved the analysismanuscript. for All Table 1 and M.A.-M. computedauthors have the read proteome and agreed statistics to the published for Table version4. All of authorsthe manuscript. have read and agreed to the published version of the manuscript.Funding: We thank the Qatar Foundation for a PhD studentship to M.A.-M. and funding for P.J.T.’s research and Qatar University for funding research for N.R. Funding: We thank the Qatar Foundation for a PhD studentship to M.A.-M. and funding for P.J.T.’s research and Qatar UniversityAcknowledgments: for funding We research thank Dr forPeter N.R. G Walley for cultivation and sample collection for assay of dicarbonyl glycating agents in Brassica oleracea. Acknowledgments: We thank Peter G Walley for cultivation and sample collection for assay of dicarbonyl glycating agentsConflicts in ofBrassica Interest: oleracea. We have no conflicts of interest. Conﬂicts ofAbbreviations Interest: We have no conﬂicts of interest. AGEs, advanced glycation endproducts; AbbreviationsAKR, aldoketoreductase; CEL, Nε-(1-carboxyethyl)lysine; AGEs advanced glycation endproducts; AKR aldoketoreductase; CEL Nε-(1-carboxyethyl)lysine; CMA Nω-carboxymethylarginine; CML Nε-carboxymethyl-lysine; 3-DG 3-deoxyglucosone; DHAP dihydroxyacetonephosphate; ER endoplasmic reticulum; FL Nε-fructosyl-lysine; GA3P glyceraldehyde-3-phosphate; G-H1 glyoxal-derived hydroimidazolone, Nδ-(5-hydro-4-imidazolon-2-yl)ornithine; Glo1 glyoxalase 1; Glo2 glyoxalase 2; Int. J. Mol. Sci. 2020, 21, 3942 12 of 16

G6P glucose-6-phosphate; GSH reduced glutathione; HA hemithioacetal; HAS human serum albumin; LC-MS/MS liquid chromatography-tandem mass spectrometry; MG methylglyoxal; MG-H1 methylglyoxal-derived hydroimidazolone, Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine; MOLD methylglyoxal-derived lysine dimer, 1,3-di(Nε-lysino)-4-methyl-imidazolium; OsAKR1 aldoketo reductase isoform-1 of Oryza sativa; RBD receptor binding domain; R5P ribose-5-phosphate; SLG S-D-lactoylglutathione; UPR unfolded protein response.

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