Quantitative measurement of specific biomarkers for protein oxidation, nitration and glycation in Arabidopsis leaves

Quantitative measurement of specific biomarkers for proteinoxidation, nitration and glycation in Arabidopsis leaves

Ulrike Bechtold1,*, Naila Rabbani1,2, Philip M. Mullineaux1 and Paul J. Thornalley1,2

1Department of Biological Sciences, University of Essex, Colchester CO4 3SQ, UK, and2Clinical Sciences Research Institute, Warwick Medical School, University of Warwick, University Hospital,

Coventry CV2 2DX, UK

Received 14 January 2009; revised 13 March 2009; accepted 7 April 2009.

*For correspondence (fax +44 1206 872595; e-mail [email protected]).

SUMMARY

Higher plants are continually exposed to reactive oxygen and nitrogen species during their lives. Together with

glucose and reactive dicarbonyls, these can modify proteins spontaneously, leading to protein oxidation,

nitration and glycation. These reactions have the potential to damage proteins and have an impact on

physiological processes. The levels of protein oxidation, nitration and glycation adducts were assayed, using

liquid chromatography coupled with tandem mass spectrometry, in total leaf extracts over a diurnal cycle and

when exposed to conditions that promote oxidative stress. Changes in the levels of oxidation, glycation and

nitration adducts were found between the light and dark phases under non-stress conditions. A comparison

between wild-type plants and a mutant lacking peptide methionine sulfoxide reductase (pmsr2-1) showed

increased protein oxidation, nitration and glycation of specific amino acid residues during darkness in pmsr2-1.

Short-term excess light exposure, which promoted oxidative stress, led to increased protein glycation,

specifically by glyoxal. This suggested that any increased oxidative damage to proteins was within the repair

capacity of the plant. The methods developed here provide the means to simultaneously detect a range of

protein oxidation, nitration and glycation adducts within a single sample. Thus, these methods identify a range

of biomarkers to monitor a number of distinct biochemical processes that have an impact on the proteome and

therefore the physiological state of the plant.

INTRODUCTION

The proteome of plants is continually subjected to attack by

reactive oxygen species (ROS) such as superoxide anion

radicals, hydrogen peroxide (H2O2), singlet oxygen and the

hydroxyl radicals. Amino acid residues undergo oxidative

modifications, with sulfur-containing amino acids being

particularly susceptible. Cysteine thiols are oxidized to glu-

tathione-mixed disulfides, sulfenic and sulfinic acid deriva-

tives, while methionine residues are oxidized to methionine

sulfoxide (MetSO, Figure 1). Tyrosine residues are oxidized

to dityrosine (Figure 1), and tryptophan residues are oxi-

dized to N-formylkynurenine (NFK, Figure 1) (Simat and

Steinhart, 1998; Droge, 2002; Møller and Kristensen, 2006).

Oxidative modification may change and impair the function

of the protein, and target oxidized proteins for proteasomal

or lysosomal destruction (Davies, 1993; Grune et al., 1997).

Oxidative damage to proteins has been implicated in chan-

ges in gene expression, growth and development, and

senescence (Lander et al., 1997; Wautier and Schmidt, 2004;

Feechan et al., 2005; Unterluggauer et al., 2008). The enzy-

matic and non-enzymatic anti-oxidant systems of plants

suppress oxidative damage to proteins, but repair of pro-

teins is also an indispensable function in plants (Bechtold

et al., 2004; Romero et al., 2004). For example, plant peptide

MetSO reductase (PMSR) removes MetSO residues in pro-

teins by reducing them to methionine (Boschi-Muller et al.,

2005; Hansel et al., 2005; Weissbach et al., 2005), and it has

been shown previously that loss of one isoform of PMSR in

Arabidopsis thaliana had a detrimental effect on both

metabolism and plant growth (Bechtold et al., 2004).

The plant proteome is also exposed continually to reactive

nitrogen species (RNS) such as nitric oxide (NO), peroxy-

nitrite and related RNS. These are highly reactive signalling

molecules that rapidly diffuse and permeate membranes

(Durner et al., 1998; Neill et al., 2003). In plants, peroxynitrite

has generally been regarded as the side product of a

scavenging reaction to control levels of NO (Van Camp

et al., 1998). In animals, peroxynitrite is a strong oxidizing

agent and hence a precursor of the oxidation adducts

ª 2009 The Authors 1Journal compilation ª 2009 Blackwell Publishing Ltd

The Plant Journal (2009) doi: 10.1111/j.1365-313X.2009.03898.x

MetSO, dityrosine and NFK (Pryor and Squadrito, 1995;

Kuhn et al., 2004). Peroxynitrite is also a nitrating agent,

forming 3-nitrotyrosine (3-NT) residues (Figure 1), a charac-

teristic signature of protein nitration damage. Recently,

production of peroxynitrite and nitration of proteins in

response to pathogen infection was also detected in plant

G-H1 MG-H1

HC

CO

NH

HN

NNH(CH2)3

H

H

O

HC

CO

NH

HN

NNH

CH3

H

O

(CH2)3

HN

N

NH

CH2

H

O

HOCH2(CHOH)2

HC

CO

NH

(CH2)3

3DG-H1

Argpyrimidine (fluorophore)

HC

CO

NH N

N

CH3

CH3

OHNH(CH2)3

MetSO

CO

HC

NH

CH2 OH

NO2

3-NT

N

HNH

HC

CO

CH2

O

O

H

NFK (fluorophore)

Dityrosine

S CH3

O

HC

CO

NH

(CH2)2

CML CEL

HC

CO

NH

NH2CH2CO2–(CH2)4

MOLDGOLD

+HC

CO

NH

CH

CO

NH

N N (CH2)4

CH3

(CH2)4+

DOLD

C

CH3

CO2–

HHC

CO

NH

NH2(CH2)4

CH2

OH

CH2

OH

HC COHN

CH

HN CO

FL

+CO

CH

NH

O

HO

OH

NH2

OH

OH

(CH2)4

HC

CO

NH

HN

NH2+

NH(CH2)3

CH2 CO2–

CMA

Oxidationmarkers

Nitrationmarker

Glycationmarkers

HC

CO

NH

CH

CO

NH

N N (CH2)4(CH2)4+

CH

CO

NH

HC

CO

NH

N N (CH2)4(CH2)4

H2C

(CHOH)2CH2OH

+ +

Figure 1. Molecular structures of protein oxidation, nitration and glycation adduct residues determined in Arabidopsis thaliana leaves in this study.

2 Ulrike Bechtold et al.

ª 2009 The AuthorsJournal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), doi: 10.1111/j.1365-313X.2009.03898.x

tissue proteins by immunoblotting (Saito et al., 2006;

Valderrama et al., 2007).

A further potential type of spontaneous modification of

the plant proteome is glycation. Glycation of proteins

involves a complex series of parallel and sequential reac-

tions collectively called the Maillard reaction (Ahmed and

Thornalley, 2007). Important physiological glycating agents

include glucose and physiological dicarbonyl metabolites,

particularly glyoxal, methylglyoxal (MG) and 3-deoxygluco-

sone (Thornalley et al., 1999). Early-stage glycation reac-

tions with glucose lead to the formation of fructosamine

derivatives such as Ne-fructosyl-lysine (FL, Figure 1) and

fructosyl-modified N-terminal amino acid residues. Later-

stage reactions form many further adducts, collectively

called advanced glycation end products (AGEs). Dicarbonyl

metabolites react with proteins to principally form arginine-

derived hydroimidazolones and the lysine-derived AGEs

Ne-carboxymethyl-lysine (CML) and Ne-carboxyethyl-lysine

(CEL). Some AGEs also form cross-linked proteins such as

the lysine dimers, glyoxal-derived lysine dimer (GOLD),

methylglyoxal-derived lysine dimer (MOLD) and 3-deosy-

glurosone-derived lysine dimer (DOLD) (Figure 1) and

lysine/arginine-derived fluorescent cross-linked pentosidine

(Figure 1) (Thornalley, 2006). In animals, there are a number

of enzymatic defences against glycation, catalysing the

repair of early glycation adducts and preventing the forma-

tion of AGEs (Thornalley, 2003). Homologues of these

enzymes have been found in higher plants, including

ribulosamine/erythrulosamine 3-kinase, which catalyses

the repair of early glycation adducts (Fortpied et al., 2005),

glyoxalase 1 and aldoketo reductases, which prevent for-

mation of AGEs by metabolism of dicarbonyl metabolites

(Jez and Penning, 2001; Yadav et al., 2005), and the acyla-

mino acid-releasing enzyme, which degrades glycated

proteins (Yamauchi et al., 2003). The presence of these

enzymatic ‘repair’ systems in plants suggests that glycation

of the plant proteome is suppressed enzymatically and that

failure of this suppression may pose a threat to plant

function and survival. Indeed, resistance of plants to stress

conditions was found in tobacco plants over-expressing

glyoxalase 1 (Singla-Pareek et al., 2003).

There is growing evidence that ROS/RNS-induced protein

modifications are of physiological relevance in plants.

Nitrosylated and glutathionylated proteins are formed dur-

ing defence responses (Saito et al., 2006; Romero-Puertas

et al., 2008) in response to chemical oxidants (Dixon et al.,

2005) and nitric oxide donors (Lindermayr et al., 2005).

These modifications are not only specific but also reversible,

and thus are important in regulating protein activity, stability

and function. Importantly, the assays used to detect oxida-

tive modifications generally only detect a single modifica-

tion, either directly by immunodetection or indirectly by

labelling the modified residue, followed by gel electropho-

resis and mass spectrometry (Rinaalducci et al., 2008;

Vandelle and Delledonne, 2008). However, immunodetec-

tion of 3-NT residues lacks specificity, and an over-estima-

tion of 3-NT residue content by 50–100-fold using these

methods has been found to be common in physiological

samples. Thus specific and sensitive measurement of 3-NT

residues by tandem mass spectrometry is required as an

alternative (Rabbani and Thornalley, 2008a).

Application of mass spectrometric techniques may meet

the demand for assays for ‘biomarkers’ of oxidative and

nitrosative stress in plants tissue (Corpas et al., 2007). If such

biomarker assays are to be applied in the study of stress

responses in plants, an in-depth analysis of the production of

proteins modified by oxidation, nitration or glycation under

various physiological conditions is of great importance. In

this report, we describe the application of high-pressure

liquid chromatography coupled to tandem mass spectrom-

etry (LC/MS-MS), combined with stable isotopic calibration,

to detect and quantify protein oxidation, nitration and

glycation adduct residues in protein extracts of Arabidopsis.

This technique allows the simultaneous quantification of a

variety of protein modifications in a single plant extract, to

give a complete profile. The aim of this study was to develop

protocols suitable for detection of the various protein

modifications in Arabidopsis leaf tissue, and to assess the

effect of various environmental conditions on the distribu-

tion and concentration of these protein modifications. To

establish these procedures, protein adduct profiles during a

light/dark cycle in wild-type plants and in a mutant defective

in the repair enzyme peptide MetSO reductase (pmsr2-1;

Bechtold et al., 2004) were analysed. This mutant has already

been characterized as having higher protein oxidation levels

and turnover of proteins when grown under a short-day (8 h)

photoperiod (Bechtold et al., 2004). In addition, protein

adducts were analysed in plants subjected to excess light

stress, which led to photo-oxidative stress. These data reveal

that short stress treatments on wild-type plants result in a

different range of specific modified peptide adduct biomar-

kers to those encountered in a diurnal cycle, indicating

specificity of their accumulation in response to certain

physiological states of the plant.

RESULTS

Detection of markers of protein glycation, oxidation and

nitration in Arabidopsis thaliana leaves

Exhaustive enzymatic hydrolysates of plant protein were

analysed for 15 markers of protein damage by oxidation,

nitration and glycation. Thirteen adduct residues were

detected; only argpyrimidine and DOLD residues were below

the limit of detection (<2 and <0.5 pmol mg)1 protein,

respectively). Examples of LC-MS/MS multiple reaction-

monitoring chromatograms are shown in Figure 2. MetSO

was a major oxidative adduct residue in Arabidopsis,

Protein damage in Arabidopsis 3

ª 2009 The AuthorsJournal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), doi: 10.1111/j.1365-313X.2009.03898.x

whereas the contents of NFK and dityrosine were intermedi-

ate and low, respectively, compared with MetSO (Table 1).

The levels of 3-NT residues in wild-type Arabidopsis were

generally 100–1000 times higher than those in mammalian

tissues (Ahmed et al., 2005b; Rabbani and Thornalley, 2008a).

Diurnal variation in the oxidation, nitration and glycation

profiles of the leaf proteome

Generally, the diurnal variation of amino acids content in

Arabidopsis leaf protein was relatively small (Table 1).

Markers of protein damage, however, showed variations

between <2- and 4-fold, with MetSO residues showing the

most marked variation (Table 1). The time courses of chan-

ges in markers of protein damage in Arabidopsis wild-type

and the pmsr2-1 mutant are shown in Figures 3 and 4. In the

wild-type plants, the MetSO residue content of leaf protein

increased fourfold during the daylight period and decreased

rapidly on entering and throughout the dark period (Fig-

ure 3a). A similar diurnal variation was found in the pmsr2-1

mutant, although there was a 4.4 mmol mol)1 Met (P < 0.05,

Kolmogorov–Smirnov test) increase in the MetSO residue

0

50 000

100 000

150 000

4 6 8 10 12

4 6 8 10 12 4 6 8 10 12

4 6 8 10 12Retention time (min)

Res

po

nse

(co

un

ts) MetSO

0

20 000

40 000

60 000

Retention time (min)

Res

po

nse

(co

un

ts) [2H3]MetSO

0

20 000

40 000

60 000

22 23 24 25 26 22 23 24 25 26

22 24 26 28 3022 24 26 28 30Retention time (min)

Res

po

nse

(co

un

ts) 3-NT

0

50 000

100 000

150 000

Retention time (min)

Res

po

nse

(co

un

ts)

[2H3]3-NT

0

2000

4000

6000

8000

Retention time (min)

Res

po

nse

(co

un

ts) CML

0

2000

4000

6000

8000

Retention time (min)

Res

po

nse

(co

un

ts) [13C6]CML

0

10 000

20 000

30 000

40 000

Retention time (min)

Res

po

nse

(co

un

ts)

MG-H1

0

25 000

50 000

75 000

100 000

Retention time (min)

Res

po

nse

(co

un

ts) [15N2]MG-H1

(a) (b)

(c) (d)

(e) (f)

(g) (h)

Figure 2. Specimen analytical chromatograms

for determination of protein glycation, oxidation

and nitration adducts by LC-MS/MS in Arabid-

opsis thaliana leaves.

(a,b) MetSO (a) and [2H3]-methyl-MetSO (b)

(50 pmol).

(c,d) 3-NT (c) and [2H3]-ring-3-NT (d) (10 pmol).

(e,f) CML (e) and [13C6]-CML (f) (10 pmol).

(g,h) MG-H1 (g) and [15N2]MG-H1 (h) (50 pmol).

Chromatographic conditions are described in

Experimental procedures.

4 Ulrike Bechtold et al.

ª 2009 The AuthorsJournal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), doi: 10.1111/j.1365-313X.2009.03898.x

content in the dark in comparison to wild-type. There were

significant increases in other protein damage markers in the

pmsr2-1 mutant in the dark compared with the wild-type

control: NFK, 1.6-fold (Figure 3b, P < 0.01); dityrosine, 2.2-

fold (Figure 3c, P < 0.01); 3-NT, 1.5-fold (Figure 3d, P < 0.05);

G-H1, 2.3-fold (Figure 4c, P < 0.01); CMA, 1.7-fold (Figure 4d,

P < 0.05). Dityrosine and 3-NT residue contents also showed

a tendency to increase in the light and decrease in the dark.

Comparing values for the middle of the light period (13 h

time point) and the middle of the dark period (0 h time

point), dityrosine and 3-NT residue contents showed 4- and

1.7-fold increases, respectively, during the light in wild-type

plants in comparison to the dark period. However, this

diurnal variation in dityrosine and 3-NT contents was not

observed in pmsr2-1.

Glycation adduct residues are also a major feature of

damage in the proteome of Arabidopsis. There was a

progressive increase in the FL residue content of wild-type

plant proteins through the light period, continuing into and

reaching a maximum in the dark period. The content of CML

residues showed a similar trend (Figure 4a,b). In the pmsr2-1

mutant, FL residue content changed similarly to the wild-

type, except that it was increased by 2–5 mmol mol)1 Lys. In

contrast, for CML residue content, there was a ninefold

increase in pmsr2-1 compared to the wild-type at the middle

of the light period (Figure 4b). No other marker of protein

damage showed a similar marked increase in the pmsr2-1

mutant at this time point, although MetSO and CMA residue

contents were also at a maximum at this time point in

Table 1 Detection level of various protein modifications in total leafprotein extracts of Arabidopsis thaliana

Analyte residue

Diurnal variation

Minimum–maximum

Foldvariation

Oxidation marker residues

Dityrosine (mmol mol)1 Tyr) 0.004–0.012 3MetSO (mmol mol)1 Met) 10.9–44.1 4NFK (mmol mol)1 Trp) 0.39–0.64 2Nitration markerNitrotyrosine (mmol mol)1 Tyr) 0.43–0.75 2Glycation marker residues

FL (mmol mol)1 Lys) 3.0–11.2 4G-H1 (mmol mol)1 Arg) 0.10–0.30 3MG-H1 (mmol mol)1 Arg) 1.84–2.23 <23DG-H ((mmol mol)1 Arg) 0.24–0.65 3CMA (mmol mol)1 Arg) 0.43–0.54 <2CML (mmol mol)1 Lys) 0.35–0.71 2CEL (mmol mol)1 Lys) 0.19–0.39 2GOLD (mmol mol)1 Lys) 0.0006–0.0008 <2MOLD (mmol mol)1 Lys) 0.005–0.009 2Amino acid residues

Lys (nmol mg)1 protein) 211–348 <2Arg (nmol mg)1 protein) 158–236 <2Met (nmol mg)1 protein) 56–83 <2Tyr (nmol mg)1 protein) 64– 90 <2Trp (nmol mg)1 protein) 101–150 <2

Total leaf protein extract was prepared from 5-week-old plants. Thelevel of detection is expressed as the minimum and maximum levelsdetected throughout a diurnal cycle in terms of mmol per mol ofunmodified amino acid.

0

10

20

30

40

50

Diurnal time (h)

Met

SO

(m

mo

l mo

l met

–1)

0.0

0.2

0.4

0.6

0.8

1.0

8 13 16 0 68 13 16 0 6

8 13 16 0 6 8 13 16 0 6

Diurnal time (h)

NF

K (

mm

ol m

ol t

rp–1

)

0.000

0.005

0.010

0.015

Diurnal time (h)

Dit

yro

sin

e (m

mo

l mo

l tyr

–1)

0.0

0.2

0.4

0.6

0.8

1.0

Diurnal time (h)

3-N

T (

mm

ol m

ol t

yr–1

)

(a) (b)

(c) (d)

Figure 3. Protein oxidation and nitration adduct

residues in total leaf extracts of wild-type and

pmsr2-1 throughout a diurnal cycle.

Plant tissue was harvested from 5-week-old

plants over a 24 h period, and total protein

extracts were subjected to enzymatic hydrolysis

followed by LC/MS-MS.

(a) MetSO, (b) NFK, (c) dityrosine and (d) 3-NT.

The open and closed bars on top of the garphs

indicate the light and dark periods, respectively.

Key: black squares, wild-type control; open

squares, pmsr2-1 mutant. Data are means �SEM (n = 3).

Protein damage in Arabidopsis 5

ª 2009 The AuthorsJournal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), doi: 10.1111/j.1365-313X.2009.03898.x

both wild-type and pmsr2-1 (Figure 3a and Figure 4d).

MG-derived MG-H1 residues were the AGE that showed

the highest content in the Arabidopsis proteome, at approx-

imately 2 mmol mol)1 Arg (Figure 4f). The contents of other

MG-derived AGE residue, CEL (Figure 4e) and MOLD (Fig-

ure 4h), were approximately 10- and 400-fold lower than

this, respectively. All the AGE residues, except for CML,

tended to show oscillatory diurnal behaviour, whereby

maximal residue contents occurred in the middle of the

light and dark periods. The G-H1 and CMA residue contents

of the pmsr2-1 mutant tended to be higher than the wild-

type control in both the light and dark periods (Figure 4c,d).

The content of other AGE residues, however, was little

changed in pmsr2-1 compared to the wild-type control.

Exposure to short-term excess light stress

Exposure to short-term excess light stress leads to the over-

reduction of components of photosynthetic electron trans-

port, and therefore to increases in thermal dissipation and

potentially damage of the light-harvesting complex of pho-

tosystem II (PSII) (Baker, 2008). Inhibition of photosynthetic

electron transport can result in the production of ROS in the

chloroplast, mainly via the reduction of O2 in the Mehler

0.0000

0.0005

0.0010

8 13 16 0 6Diurnal time (h)

GO

LD

(m

mo

l mo

l lys

–1)

0

5

10

15

8 13 16 0 6Diurnal time (h)

FL

(m

mo

l mo

l lys

–1)

0

2

4

6

8

8 13 16 0 6Diurnal time (h)

CM

L (

mm

ol m

ol l

ys–1

)

0.0

0.1

0.2

0.3

0.4

0.5

Diurnal time (h)

G-H

1 (m

mo

l mo

l arg

–1)

0.0

0.5

1.0

1.5

8 13 16 0 68 13 16 0 6Diurnal time (h)

CM

A (

mm

ol m

ol a

rg–1

)

0.0

0.2

0.4

0.6

Diurnal time (h)

CE

L (

mm

ol m

ol l

ys–1

)

0

1

2

3

8 13 16 0 68 13 16 0 6Diurnal time (h)

MG

-H1

(mm

ol m

ol a

rg–1

)

0.000

0.005

0.010

8 13 16 0 6Diurnal time (h)

MO

LD

(m

mo

l mo

l lys

–1)

(a) (b)

(c) (d)

(e) (f)

(g) (h)

Figure 4. Protein glycation adduct residues in

total leaf extracts of wild-type and pmsr2-1

throughout a diurnal cycle.

Plant tissue was harvested from 5-week-old

plants over a 24 h period, and total protein

extracts were subjected to enzymatic hydrolysis,

followed by LC/MS-MS. (a) FL, (b) CML, (c) G-H1,

(d) CMA, (e) CEL, (f) MG-H1, (g) GOLD, (h) MOLD.

The open and closed bars on top of the graphs

indicate the light and dark periods, respectively.

Key: black squares, wild-type control; open

squares, pmsr2-1 mutant. Data are means �SEM (n = 3).

6 Ulrike Bechtold et al.

ª 2009 The AuthorsJournal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), doi: 10.1111/j.1365-313X.2009.03898.x

reaction and the accumulation of singlet oxygen (Barber and

Andersson, 1992; Asada, 1999; Fryer et al., 2003; Flors et al.,

2006). The excess light stress applied led to a decrease in

two maximum efficiency of PSII from 0.751 � 0.023 to

0.515 � 0.029 (P < 0.001), indicating that photo-oxidative

damage has occurred in PSII reaction centres. This was

further supported by a 3–4-fold increase in foliar H2O2 levels

(Figure 5a; P < 0.05). Excess light stress increased the G-H1

and CML residue contents of total leaf protein by approxi-

mately twofold (Figure 5b, P < 0.05). Interestingly, there was

no significant effect on any other marker of protein damage.

DISCUSSION

In this study, we obtained quantitative estimates of

glycation, oxidation and nitration adducts in a foliar protein

extract of Arabidopsis thaliana. The estimates reveal that

there is significant damage to the plant proteome as a result

of early and advanced glycation, oxidation and nitration

processes, with evidence of changes in levels throughout a

diurnal cycle. The proteome contents of these markers of

protein damage are maintained at these low levels by

enzymatic and non-enzymatic defences against glycation,

oxidation and nitration. Based on the mean amino acid

residue length of proteins in the Arabidopsis proteome (406

residues) and the mean amino acid contents of the proteome

(2.46% Met, 6.38% Lys, 5.43% Arg, 2.86% Tyr and 1.27% Trp),

a mean of 26% of proteins are predicted to contain a FL

residue, 10% of proteins are predicted to contain a

MetSO residue, 4% of proteins are predicted to contain

a MG-H1 residue, 3% of proteins are predicted to contain a

CML residue, 0.7% of proteins are predicted to contain a

3-NT residue, and 0.3% of proteins are predicted to contain

a NFK residue. The protein contents of the oxidation markers

MetSO, dityrosine and NFK, and of most glycation adducts,

are similar to those in mammalian cellular protein extracts

(Thornalley et al., 2003; Ahmed et al., 2005b), except those

of FL and CML are approximately 10-fold higher, and that of

3-NT is approximately 20-fold higher than in mammalian

protein extracts.

MetSO is a major oxidation marker in physiological

systems, and has been shown to be the predominant

oxidation marker in Arabidopsis leaves (Table 1). The pro-

tein content of MetSO is controlled and suppressed by

peptide MetSO reductases. In plants, there are five class A

and nine class B PMSR isoforms in Arabidopsis (Sadanan-

dom et al., 2000; Rouhier et al., 2006). In wild-type Arabid-

opsis, PMSR2 expression is increased during a 16 h dark

period, and is abolished in the pmsr2-1 null mutant (Bech-

told et al., 2004). This resulted in increased oxidative stress

and damage to lipids and proteins. In pmsr2-1, the accumu-

lation of MetSO residues was only slightly higher on exiting

the dark period compared to the wild-type (Figure 3a).

However, recovery from oxidative and nitrosative damage

during the dark period was slowed or abolished, leading to

increased protein damage in pmsr2-1 (Figure 3a–d). The

absence of PMSR2 may therefore lead to functional impair-

ment of redox-active and anti-oxidant defences, resulting in

increased formation and/or decreased inactivation of ROS

and RNS. The lowered enzyme activity of a number of anti-

oxidant enzymes (data not shown) and the increased H2O2

levels (Bechtold et al., 2004) during the dark period in

pmsr2-1 support this argument. The oxidation and reduction

of MetSO may therefore be an indirect mechanism to

alleviate oxidative stress, thus preventing further oxidative

damage to proteins. In the mutant, the loss of ability to cycle

methionine residues increases total protein oxidation and

protein turnover, with profound consequences for carbon

metabolism, leading to increased H2O2 production and lipid

peroxidation (Bechtold et al., 2004). These reactive mole-

cules lead to a variety of other protein modifications as

shown in Figures 3 and 4. Therefore, in wild-type plants, the

maintenance of reduced methionine residues may indirectly

0.0

0.5

1.0

1.5

G-H1 CML

AG

E r

esid

ue

(mm

ol m

ol a

rg o

r ly

s–1)

0

20

40

60

80

100

120

140

160

180

LL EL

nm

ol g

fwt–1

(a)

(b)

Figure 5. H2O2 levels (a) and protein glycation adduct residues (b) of leaf

extracts of wild-type Arabidopsis thaliana under excess light. Key: white bars,

low light; black bars, excess light. Data are means � SEM (n = 3); mmol mol)1

Arg for G-H1 and mmol mol)1 Lys for CML. *P < 0.05 and **P < 0.001 with

respect to the low-light control.

Protein damage in Arabidopsis 7

ª 2009 The AuthorsJournal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), doi: 10.1111/j.1365-313X.2009.03898.x

contribute to protein damage. Due to the increased protein

turnover observed in pmsr2-1 and redundancy in the PMSR

gene families (Romero et al., 2004, 2006), the absolute levels

of the measured protein modifications may be under-

estimated. This would explain the apparently low, but

significant difference, in MetSO between wild-type and

mutant at the end of the dark period (Figure 3a). The MetSO

residue content in chloroplast proteins was previously

estimated to be approximately 20–30% of total methionine

residues (200–300 mmol mol)1 Met) (Romero et al., 2004). In

this study, the estimate of MetSO residue content in total

plant proteins is approximately 10% of this. This means that

chloroplast proteins bear the brunt of oxidative damage

compared with other plant proteins. PMSRA2 in the cytosol

and PMSRA4 in the plastid are up-regulated by oxidative

stress conditions, especially by high light stress, indicating

the requirement for repair mechanisms during oxidative

stress conditions (Romero et al., 2006). This is further

supported by the fact that loss of the plastidial isoforms of

PMSR confers increased susceptibly to oxidative stress,

whereas over-expression renders plants resistant to oxida-

tive stress (Romero et al., 2004). However, despite the

presence of a family of PMSR isozymes, the oxidative

damage to methionine residues increased markedly during

the light period, and coincided with an increase of dityrosine

and 3-NT in wild-type (Figure 3c,d). This suggests a constant

oxidative burden on the proteome mainly during the light

period. Tyrosine radicals in particular are formed in D1 and

D2 proteins of the PSII reaction centre, and play a major role

during charge separation as the primary oxidant for the

oxygen-evolving complex (Barry et al., 1990; Hoganson and

Babcock, 1992). The tyrosine radical is essential for the

function of photosynthetic light reactions (Tang et al., 1996),

and, as an intermediate in the formation of dityrosine, may

be the reason for increased dityrosine levels in leaf protein

during the light period. Furthermore, tyrosyl radical residues

may also trap NO, leading to the formation of 3-NT residues

(Gunther et al., 2002), and potentially contributing to the

increase in the 3-NT residue content of leaf protein. Trypto-

phan residues are also a target for oxidative damage. We

have detected increased NFK levels in pmsr2-1 during the

dark period and at the beginning of the light period

(Figure 3b). NFK residues were previously detected by 2D

gel analysis and tandem mass spectrometry in peptides

from rice leaf and potato tuber mitochondria undergoing

oxidative stress. Interestingly, 17 proteins were detected as

targets for tryptophan oxidation that are either redox-active

or involved in redox processes, and therefore closely

associated with oxidative stress (Møller and Kristensen,

2006).

Advanced glycation end products are a heterogeneous

group of protein modifications. These modifications are

permanent, and therefore require replenishment by unmodi-

fied proteins. AGEs are usually found at low levels in normal

individuals, but can accumulate to significantly higher levels

during certain diseases and ageing (Thornalley, 2005;

Ahmed and Thornalley, 2007). Recognition of AGEs within

cells by specific receptors results in oxidative stress by

production of H2O2 via NADPH oxidases, triggering a MAP

kinase signaling cascade leading to activation of redox-

regulated transcription factors (Yan et al., 1994; Lander

et al., 1997). In plants, very little is known about protein

glycation. Susceptibility of plant proteins to glycation has

been reported in vitro. For example, ribulose-1,5-bisphos-

phate carboxylase/oxygenase (Rubisco) activity is sensitive

to glycation by ascorbic acid, forming CML residues (Ya-

mauchi et al., 2002). Additionally, protein glycation has also

been implicated in the deterioration of plants seeds in

storage (Murthy and Sun, 2000). In Arabidopsis, FL occurred

at a very high level compared to other AGEs, especially on

entering the light period, and continued to increase until the

middle of the dark period (Figure 4a). This corresponds to

the period of photosynthesis and an approximately 10-fold

increase in glucose concentration in leaf tissue (Bechtold

et al., 2004). Interestingly, the foliar concentration of glucose

was increased in pmsr2-1 by up to 50% (Bechtold et al.,

2004), which corresponds to the increase in the FL residue

content of leaf protein in pmsr2-1 (Figure 4a). The content of

CML residues in pmsr2-1 protein extracts was also high

(Figure 4b). This may be due to the high content of FL

residues and ascorbic acid (Conklin et al., 1997), which are

precursors of CML residue formation (Ahmed et al., 1986;

Dunn et al., 1990).

Exposure of Arabidopsis plants to excess light stress led

to reversible photo-inhibition of photosynthesis, an increase

in hydrogen peroxide (Figure 5a), and an increase in G-H1

and CML only (Figure 5b). Excess light stress has previously

been shown to produce H2O2 mainly in the chloroplasts of

veinal tissue (Fryer et al., 2003). Therefore, protein damage

under excess light stress may be limited to certain tissue

types and subcellular compartments. Extraction of whole-

leaf tissue may dilute weak signals of protein damage, which

could explain the lack of diversity of protein damage under

excess light stress, but could also reflect the high repair

capacity within chloroplasts where H2O2 originates. There-

fore, the level of detection may vary depending on the

type of stress, compartmentalization and tissue specificity.

On the other hand, increased proteome damage adducts

formed by glyoxal suggests that sources of glyoxal, such as

autoxidation of glucose or lipid peroxidaton (Rabbani and

Thornalley, 2008b), may be important mediators of func-

tional impairment during excess light stress. This indicates a

direct causal link of biochemical pathways and specific

protein damage during excess light stress, highlighting the

sensitivity of this method and its application for the devel-

opment of protein damage biomarkers. Genetic modifica-

tion of components of the plant’s defence against glyoxal

glycation has been shown to protect against environmental

8 Ulrike Bechtold et al.

ª 2009 The AuthorsJournal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), doi: 10.1111/j.1365-313X.2009.03898.x

stress, particularly for components of the glyoxalase system

(Thornalley, 1993; Singla-Pareek et al., 2003).

In summary, in this paper, we describe methods that can

quantify specific damage to the proteome of Arabidopsis by

glycation, oxidation and nitration. The presence of AGEs,

oxidation and nitration products in non-stressed plants, their

diurnal pattern, and the selectivity towards certain adducts

during stress suggests that protein modifications may not

merely be unintentional damage. There is clear evidence

that protein modifications in plants occur under normal

growth conditions. Thus even so-called ‘constitutive’ mod-

ifications may play an important role in mechanisms to

regulate activity through protein abundance. The ability to

exactly determine and quantify various types of protein

damage has a clear advantage, as the underlying biochem-

ical pathways/mechanisms responsible for specific damage

can be identified and associated with environmental condi-

tions. The pmsr2-1 mutant provides an insight into how a

disturbance in the balance of oxidation and reduction of

proteins can lead to a general increase in protein damage,

and how plants have developed mechanisms to fine-tune

the oxidative load of proteins. This paper highlights the

importance of developing accurate tools to analyse profiles

of multiple protein damage in plants. The described method

has wide-ranging applications for investigation of the

involvement of protein modifications in a number of

physiological processes, especially in response to stress

and development.

EXPERIMENTAL PROCEDURES

Plant material and growth conditions

Plants of Arabidopsis thaliana ecotype Col-0 (wild-type) and a var-iant with a null mutation in PMSR2 (pmsr2-1) were grown undershort-day conditions (8 h light/16 h dark) at 22�C, 65% relativehumidity, and 150 lmol photons m)2 sec)1 light. The pmsr2-1mutant has been previously restored to wild-type by complemen-tation with a genomic PMSR2 fragment (Bechtold et al., 2004). Five-week-old plants were harvested into liquid nitrogen at various timesduring the day. The plants were subjected to excess light stress(10-fold higher than growth light conditions) 2 h after the onset ofthe light period for 1 h duration. The low-light control was kept inthe controlled environment as described above. Immediately afterexcess light stress, chlorophyll fluorescence measurements wereperformed to assess the effect of photo-damage on the efficiency ofphotosynthetic electron transport (Barbagallo et al., 2003).

Hydrogen peroxide assay

At the end of the stress treatment, leaf material was harvested fromplants treated with excess light and low light. Leaf material (100 mg)was extracted on ice in 100 mM HCl. The extract was centrifuged(11 270 g, 4�C, 10 min), and the supernatant (500 ll) was elutedthrough charcoal (500 ll of a 1:3 w/v charcoal/water mixture in amini-column, with forced flow using a 1 ml syringe). The resultingsupernatant was used for determination of the concentration of

hydrogen peroxide using Amplex� Ultra Red (Molecular Probes,http://www.invitrogen.com) and horseradish peroxidase. The pro-duct of the reaction, resorufin, was detected spectrophotometricallyat 571 nm, and the result was calibrated against a standard curve ofknown concentrations of hydrogen peroxide (0–10 nmol).

Preparation of samples for LC-MS/MS

All steps of the protein extraction were performed under nitrogen toavoid autoxidation during sample processing. Total leaf protein wasextracted on ice in 1 ml of HEPES buffer at pH 7.5. The samples werecentrifuged (4�C, 18 900 g, 30 min), and protein in the supernatantwas concentrated by microspin ultrafiltration (12 kDa cut-off,VectaSpin filter; Whatman, http://www.whatman.com). The result-ing protein concentrate was assayed by the Bradford method, andan aliquot (100 lg) was digested by exhaustive sequential enzy-matic hydrolysis with pepsin, pronase E, aminopeptidase andprolidase as described previously (Ahmed et al., 2002). Thishydrolysate was used for assay of markers of protein glycation,oxidation and nitration.

Quantification of protein glycation, oxidation and nitration

adduct residues by LC-MS/MS

The following glycation, oxidation and nitration adducts and aminoacids were quantified by LC-MS/MS: protein glycation adducts(argpyrimidine, CML, CEL, 3DG-H, dityrosine, DOLD, FL, G-H1,GOLD, MG-H1 and MOLD), oxidation markers (NFK, dityrosine andMetSO), the nitration marker 3-NT (Figure 1), and amino acids Lys,Arg, Met, Trp and Tyr. Normal isotopic abundance and stable iso-tope-substituted calibration standards were prepared as describedpreviously (Thornalley et al., 2003). NFK was prepared by formyla-tion of kynurenine (Fukunaga et al., 1982), and [15N2]NFK was pre-pared by oxidation of [15N2]Trp with H2O2 and purification byreverse-phase HPLC (Simat and Steinhart, 1998). Samples wereassayed by LC-MS MS by the method described previously(Thornalley et al., 2003) with modifications (Ahmed et al., 2005a),and detection conditions for NFK (parent mass 237.0 Da, fragmentmass 191.0 Da, collision energy 12 eV) and [15N2]NFK (parent mass239.0 Da, fragment mass 193.0 Da, collision energy 12 eV). Glyca-tion, oxidation and nitration adduct residues of plant proteinextracts were detected in exhaustive enzymatic digests (50 lgprotein equivalent) by electrospray positive ionization.

LC-MS/MS multiple reaction monitoring

The detection response was normalized to the responses of relatedisotopic internal standards, and calibrated by assay of authenticanalytical standards. Samples were assayed by LC-MS/MS using a2690 separation module with a Quattro Ultima triple quadrupolemass spectrometric detector (Waters-Micromass, http://www.waters.com/). The ionization source temperature was 120�C, and thedesolvation gas temperature 350�C. The cone gas and desolvationgas flow rates were 150 and 550 l h)1, respectively. The capillaryvoltage was 3.55 kV and the cone voltage was 80 V. Argon gas(0.27 Pa) was used in the collision cell. Programmed molecular ionand fragment ion masses and collision energies were optimized to�0.1 Da and �1 eV, respectively, for multiple reaction monitoringdetection of analytes. The amounts of internal standard used were10 nmol for amino acids, 250 pmol for FL, and 10–50 pmol for AGEsand oxidation and nitrosation biomarkers. LC-MS/MS data analysiswas performed using the WATERS MASSLYNX program (version 4.0).

Protein damage in Arabidopsis 9

ª 2009 The AuthorsJournal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), doi: 10.1111/j.1365-313X.2009.03898.x

Bioinformatics

Data on the genome and proteome of Arabidopsis thaliana wereobtained from the European Bioinformatics Institute (http://www.ebi.ac.uk).

Statistical analysis

Results are given as means � SD or SEM for 3–5 determinations, asindicated. Statistical tests were all two-sided (£0.05). The signifi-cance of differences between wild-type and pmsr2-1 or excess-lightand low-light treatments was determined using Student’s t test andthe Mann–Whitney U test, respectively. All statistical analyses wereperformed using the SPSS statistical software package (version 15.0,http://www.spss.com).

ACKNOWLEDGEMENTS

This project was supported by a Royal Society Research grant (2005/R1) awarded to U.B., and a grant from the University of Essex toP.M.M. N.R. and P.J.T. thank the Wellcome Trust for support for ourresearch on protein glycation, oxidation and nitration research.

REFERENCES

Ahmed, N. and Thornalley, P.J. (2007) Advanced glycation end-products: what is their relevance to diabetic complications? Dia-betes Obes. Metab. 9, 233–245.

Ahmed, M.U., Thorpe, S.R. and Baynes, J.W. (1986) Identificationof N epsilon-carboxymethyllysine as a degradation product offructoselysine in glycated protein. J. Biol. Chem. 261, 4889–4894.

Ahmed, N., Argirov, O.K., Minhas, H.S., Cordeiro, C.A. and Thorn-

alley, P.J. (2002) Assay of advanced glycation endproducts(AGEs): surveying AGEs by chromatographic assay with deriva-tization by 6-aminoquinolyl-N-hydroxysuccinimidyl-carbamateand application to Ne-carboxymethyl-lysine- and Ne-(1-carboxy-ethyl) lysine-modified albumin. Biochem. J. 364, 1–14.

Ahmed, N., Ahmed, U., Thornalley, P.J., Hager, K., Fleischer, G.A.

and Munch, G. (2005a) Protein glycation, oxidation and nitrationadduct residues and free adducts of cerebrospinal fluid in Alz-heimer’s disease and link to cognitive impairment. J. Neurochem.92, 255–263.

Ahmed, N., Babaei-Jadidi, R., Howell, S.K., Beisswenger, P.J. and

Thornalley, P.J. (2005b) Degradation products of proteins dam-aged by glycation, oxidation and nitration in clinical type 1 dia-betes. Diabetologia 48, 1590–1603.

Asada, K. (1999) The water–water cycle in chloroplasts: scavengingof active oxygens and dissipation of excess photons. Annu. Rev.Plant Physiol. Plant Mol. Biol. 50, 601–639.

Baker, N.R. (2008) Chlorophyll fluorescence: a probe of photosyn-thesis in vivo. Annu. Rev. Plant Biol. 59, 89–113.

Barbagallo, R.P., Oxborough, K., Pallett, K.E. and Baker, N.R. (2003)Rapid, non-invasive screening for perturbations of metabolismand plant growth using chlorophyll fluorescence imaging. PlantPhysiol. 132, 485–493.

Barber, J. and Andersson, B. (1992) Too much of a good thing: lightcan be bad for photosynthesis. Trends Biochem. Sci. 17, 61–66.

Barry, B.A., el Deeb, M.K., Sandusky, P.O. and Babcock, G.T. (1990)Tyrosine radicals in photosystem II and related model com-pounds. Characterization by isotopic labeling and EPR spectros-copy. J. Biol. Chem. 265, 20139–20143.

Bechtold, U., Murphy, D.J. and Mullineaux, P.M. (2004) Arabidopsispeptide methionine sulfoxide reductase2 prevents cellular oxi-dative damage in long nights. Plant Cell 16, 908–919.

Boschi-Muller, S., Olry, A., Antoine, M. and Branlant, G. (2005) Theenzymology and biochemistry of methionine sulfoxide reducta-ses. Biochim. Biophys. Acta 1703, 231–238.

Conklin, P.L., Pallanca, J.E., Last, R.L. and Smirnoff, N. (1997)L-ascorbic acid metabolism in the ascorbate-deficient Arabidop-sis mutant vtc1. Plant Physiol. 115, 1277–1285.

Corpas, F.J., del Rio, L.A. and Barroso, J.B. (2007) Need of bio-markers of nitrosative stress in plants. Trends Plant Sci. 12,436–438.

Davies, K.J.A. (1993) Protein modification by oxidation and the roleof proteolytic enzymes. Biochem. Soc. Trans. 21, 346–352.

Dixon, D.P., Skipsey, M., Grundy, N.M. and Edwards, R. (2005)Stress-induced protein S-glutathionylation. Plant Physiol. 138,2233–2244.

Droge, W. (2002) Free radicals in the physiological control of cellfunction. Physiol. Rev. 82, 47–95.

Dunn, J.A., Ahmed, M.U., Murtiashaw, M.H., Richardson, J.M.,

Walla, M.D., Thorpe, S.R. and Baynes, J.W. (1990) Reaction ofascorbate with lysine and protein under autoxidizing conditions:formation of Ne-(carboxymethyl)lysine by reaction betweenlysine and products of autoxidation of ascorbate. Biochemistry29, 10964–10970.

Durner, J., Wendehenne, D. and Klessig, D.F. (1998) Defense geneinduction in tobacco by nitric oxide, cyclic GMP, and cyclic ADP-ribose. Proc. Natl Acad. Sci. USA 95, 10328–10333.

Feechan, A., Kwon, E., Yun, B.W., Wang, Y., Pallas, J.A. and Loake,

G.J. (2005) A central role for S-nitrosothiols in plant diseaseresistance. Proc. Natl Acad. Sci. USA 102, 8054–8059.

Flors, C., Fryer, M.J., Waring, J., Reeder, B., Bechtold, U., Mullineaux,

P.M., Nonell, S., Wilson, M.T. and Baker, N.R. (2006) Imaging theproduction of singlet oxygen in vivo using a new fluorescentsensor, Singlet Oxygen Sensor Green�. J. Exp. Bot. 57, 1725–1734.

Fortpied, J., Gemayel, R., Stroobant, V. and Van Schaftingen, E.

(2005) Plant ribulosamine/erythrulosamine 3-kinase, a putativeprotein-repair enzyme. Biochem. J. 388, 795–802.

Fryer, M.J., Ball, L., Oxborough, K., Karpinski, S., Mullineaux, P.M.

and Baker, N.R. (2003) Control of Ascorbate Peroxidase 2expression by hydrogen peroxide and leaf water status duringexcess light stress reveals a functional organisation of Arabid-opsis leaves. Plant J. 33, 691–705.

Fukunaga, Y., Katsuragi, Y., Izumi, T. and Sakiyama, F. (1982) Flu-orescence characteristics of kynurenine and N’-formylkynure-nine. Their use as reporters of the environment of tryptophan 62in hen egg-white lysozyme. J. Biochem. 92, 129–141.

Grune, T., Reinheckel, T. and Davies, K.J.A. (1997) Degradation ofoxidized proteins in mammalian cells. FASEB J. 11, 526–534.

Gunther, M.R., Sturgeon, B.E. and Mason, R.P. (2002) Nitric oxidetrapping of the tyrosyl radical-chemistry and biochemistry. Tox-icology 177, 1–9.

Hansel, A., Heinemann, S.H. and Hoshi, T. (2005) Heterogeneity andfunction of mammalian MSRs: enzymes for repair, protection andregulation. Biochim. Biophys. Acta 1703, 239–247.

Hoganson, C.W. and Babcock, G.T. (1992) Protein-tyrosyl radicalinteractions in photosystem II studied by electron spin resonanceand electron nuclear double resonance spectroscopy: compari-son with ribonucleotide reductase and in vitro tyrosine. Bio-chemistry 31, 11874–11880.

Jez, J.M. and Penning, T.M. (2001) The aldo-keto reductase (AKR)superfamily: an update. Chem. Biol. Interact. 130, 499–525.

Kuhn, D.M., Sakowski, S.A., Sadidi, M. and Geddes, T.J. (2004)Nitrotyrosine as a marker for peroxynitrite-induced neurotoxicity:

10 Ulrike Bechtold et al.

ª 2009 The AuthorsJournal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), doi: 10.1111/j.1365-313X.2009.03898.x

the beginning or the end of the end of dopamine neurons?J. Neurochem. 89, 529–536.

Lander, H.M., Tauras, J.M., Ogiste, J.S., Hori, O., Moss, R.A. and

Schmidt, A.M. (1997) Activation of the receptor for advancedglycation end products triggers a p21ras-dependent mitogen-activated protein kinase pathway regulated by oxidant stress.J. Biol. Chem. 272, 17810–17814.

Lindermayr, C., Saalbach, G. and Durner, J. (2005) Proteomic iden-tification of S-nitrosylated proteins in Arabidopsis. Plant Physiol.137, 921–930.

Møller, I.M. and Kristensen, B.K. (2006) Protein oxidation in plantmitochondria detected as oxidized tryptophan. Free Radic. Biol.Med. 40, 430–435.

Murthy, U.M. and Sun, W.Q. (2000) Protein modification by Ama-dori and Maillard reactions during seed storage: roles of sugarhydrolysis and lipid peroxidation. J. Exp. Bot. 51, 1221–1228.

Neill, S.J., Desikan, R. and Hancock, J.T. (2003) Nitric oxide signal-ling in plants. New Phytol. 159, 11–35.

Pryor, W.A. and Squadrito, G.L. (1995) The chemistry of peroxyni-trite: a product from the reaction of nitric oxide with superoxide.Am. J. Physiol. 268, L699–L722.

Rabbani, N. and Thornalley, P.J. (2008a) Assay of 3-nitrotyrosine intissues and body fluids by liquid chromatography with tandemmass spectrometric detection. Methods Enzymol. 440, 337–359.

Rabbani, N. and Thornalley, P.J. (2008b) The dicarbonyl proteome:proteins susceptible to dicarbonyl glycation at functional sites inhealth, aging, and disease. Ann. NY Acad. Sci. 1126, 124–127.

Rinalducci, S., Murgiano, L. and Zolla, L. (2008) Redox proteomics:basic principles and future perspectives for the detection of pro-tein oxidation in plants. J. Exp. Bot., 59, 3781–3801.

Romero, H.M., Berlett, B.S., Jensen, P.J., Pell, E.J. and Tien, M.

(2004) Investigations into the role of the plastidial peptidemethionine sulfoxide reductase in response to oxidative stress inArabidopsis. Plant Physiol. 136, 3784–3794.

Romero, H.M., Pell, E.J. and Tien, M. (2006) Expression profileanalysis and biochemical properties of the peptide methioninesulfoxide reductase A (PMSRA) gene family in Arabidopsis. PlantSci. 170, 705–714.

Romero-Puertas, M.C., Campostrini, N., Matte, A., Righetti, P.G.,

Perazzolli, M., Zolla, L., Roepstorff, P. and Delledonne, M. (2008)Proteomic analysis of S-nitrosylated proteins in Arabidopsisthaliana undergoing hypersensitive response. Proteomics 7,1459–1469.

Rouhier, N., Santos, C., Tarrago, L. and Rey, P. (2006) Plant methi-onine sulfoxide reductase A and B multigenic families. Photo-synth. Res. 89, 247–262.

Sadanandom, A., Poghosyan, Z., Fairbairn, D.J. and Murphy, D.J.

(2000) Differential regulation of plastidial and cytosolic isoformsof peptide methionine sulfoxide reductase in Arabidopsis. PlantPhysiol. 123, 255–263.

Saito, S., Yamamoto-Katou, A., Yoshioka, H., Doke, N. and Kawakita,

K. (2006) Peroxynitrite generation and tyrosine nitration in defenseresponses in tobacco BY-2 cells. Plant Cell Physiol. 47, 689–697.

Simat, T.J. and Steinhart, H. (1998) Oxidation of free tryptophan andtryptophan residues in peptides and proteins. J. Agric. Food.Chem. 46, 490–498.

Singla-Pareek, S.L., Reddy, M.K. and Sopory, S.K. (2003) Geneticengineering of the glyoxalase pathway in tobacco leads toenhanced salinity tolerance. Proc. Natl Acad. Sci. USA 100,14672–14677.

Tang, X.S., Zheng, M., Chisholm, D.A., Dismukes, G.C. and Diner,

B.A. (1996) Investigation of the differences in the local proteinenvironments surrounding tyrosine radicals YzÆ and YdÆ in pho-tosystem II using wild-type and the D2-Tyr160Phe mutant ofSynechocystis 6803. Biochemistry, 35, 1475–1484.

Thornalley, P.J. (1993) The glyoxalase system in health and disease.Mol. Aspects Med. 14, 287–371.

Thornalley, P.J. (2003) Glyoxalase I – structure, function and a crit-ical role in the enzymatic defence against glycation. Biochem.Soc. Trans. 31, 1343–1348.

Thornalley, P.J. (2005) Glycation free adduct accumulation in renaldisease: the new AGE. Pediatr. Nephrol. 20, 515–522.

Thornalley, P.J. (2006) Quantitative screnning of protein glycation,oxidation, and nitration adducts by LC-MS/MS: protein damage indiabetes, uremia, cirrhosis, and Alzheimer’s disease. In RedoxProteomics (Dalle-Donne, I., Scaloni, A. and Butterfield, D.A.,eds). Wiley: Hoboken, NJ, pp. 681–727.

Thornalley, P.J., Langborg, A. and Minhas, H.S. (1999) Formation ofglyoxal, methylglyoxal and 3-deoxyglucosone in the glycation ofproteins by glucose. Biochem. J. 344, 109–116.

Thornalley, P.J., Battah, S., Ahmed, N., Karachalias, N., Agalou, S.,

Babaei-Jadidi, R. and Dawnay, A. (2003) Quantitative screening ofadvanced glycation endproducts in cellular and extracellularproteins by tandem mass spectrometry. Biochem. J. 375, 581–592.

Unterluggauer, H., Micutkova, L., Lindner, H., Sarg, B., Hernebring,

M., Nystrom, T. and Jansen-Durr, P. (2009) Identification of Hsc70as target for AGE modification in senescent human fibroblasts.Biogerontology, 10, 299–309.

Valderrama, R., Corpas, F.J., Carreras, A., Fernandez-Ocana, A.,

Chaki, M., Luque, F., Gomez-Rodriguez, M.V., Colmenero-Varea,

P., del Rio, L.A. and Barroso, J.B. (2007) Nitrosative stress inplants. FEBS Lett. 581, 453–461.

Van Camp, W., Van Montagu, M. and Inze, D. (1998) H2O2 and NO:redox signals in disease resistance. Trends Plant Sci. 3, 330–334.

Vandelle, E. and Delledonne, M. (2008) Methods for nitric oxidedetection during plant–pathogen interactions. Methods Enzymol.437, 575–594.

Wautier, J.L. and Schmidt, A.M. (2004) Protein glycation: a firm linkto endothelial cell dysfunction. Circ Res. 95, 233–238.

Weissbach, H., Resnick, L. and Brot, N. (2005) Methionine sulfoxidereductases: history and cellular role in protecting against oxida-tive damage. Biochim. Biophys. Acta, 1703, 203–212.

Yadav, S.K., Singla-Pareek, S.L., Reddy, M.K. and Sopory, S.K.

(2005) Transgenic tobacco plants overexpressing glyoxalaseenzymes resist an increase in methylglyoxal and maintain higherreduced glutathione levels under salinity stress. FEBS Lett. 579,6265–6271.

Yamauchi, Y., Ejiri, Y. and Tanaka, K. (2002) Glycation by ascorbicacid causes loss of activity of ribulose-1,5-bisphosphate carbox-ylase/oxygenase and its increased susceptibility to proteases.Plant Cell Physiol. 43, 1334–1341.

Yamauchi, Y., Ejiri, Y., Toyoda, Y. and Tanaka, K. (2003) Identifica-tion and biochemical characterization of plant acylamino acid-releasing enzyme. J. Biochem. (Tokyo) 134, 251–257.

Yan, S.D., Schmidt, A.M., Anderson, G.M., Zhang, J., Brett, J., Zou,

Y.S., Pinsky, D. and Stern, D. (1994) Enhanced cellular oxidantstress by the interaction of advanced glycation end productswith their receptors/binding proteins. J. Biol. Chem. 269, 9889–9897.

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