ORIGINAL PAPER

Changes in expression of proteins involved in alleviationof Fe-deficiency by sulfur nutrition in Brassica napus L.

Sowbiya Muneer • Bok Rye Lee • Dong Won Bae •

Tae Hwan Kim

Received: 27 February 2013 / Revised: 17 June 2013 / Accepted: 18 June 2013

� Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2013

Abstract In order to characterize the significance of sulfur

(S) nutrition in protein expression under iron (Fe)-deficient

conditions, gel-based proteomic analysis was performed

with the leaves of Brassica napus exposed to S and Fe

combined treatments: sufficient in S and Fe (?S/?Fe, con-

trol), sufficient S but Fe deprived (?S/-Fe), deprived S but

sufficient Fe (-S/?Fe), and deprived S and Fe (-S/-Fe).

The resulting data showed that 15 proteins were down-reg-

ulated due to production of oxidative damage as indicated by

H2O2 and O2-1 localizations and due to leaf chlorosis in

leaves in S-deprived leaves either in presence (-S/?Fe) or

absence of Fe (-S/-Fe), whereas these down-regulated

proteins were well expressed in the presence of S (?S/-Fe)

compared to control (?S/?Fe). In addition, two proteins

were up-regulated under S-deprived condition in presence

(-S/?Fe) and absence of (-S/-Fe) Fe. The functional

classification of these identified proteins was estimated that

40 % of the proteins belong to chloroplast precursor, and

rest of the proteins belongs to hypothetical proteins, RNA

binding, secondary metabolism and unknown proteins. On

the other hand, five protein spots from S deprived (-S/?Fe)

and ten spots from Fe deprived (-S/-Fe) conditions were

absent, whereas they were well expressed in presence of S

(?S/-Fe) compared to control plants (?S/?Fe). These

results suggest that sulfur nutrition plays an important role in

alleviating protein damage in Fe-deficient plants and adap-

tation to Fe-deficiency in oilseed rape.

Keywords Brassica napus � Fe-deficiency � Proteomics �S-deficiency

Abbreviations

2DE Two-dimensional gel electrophoresis

SDS-PAGE Sodium dodecyl sulfate polyacrylamide

gel electrophoresis

MALDI-TOF Matrix-assisted laser desorption/ionization-

time of flight

IPG Immobilized pH gradient

IAA Iodoacetamide

DTT Dithiothreitol

Introduction

Iron (Fe) is an important mineral nutrient for the growth and

development of plants, Fe-deficiency has been often

appeared in the crop plants growing in alkaline and calcar-

eous soil (Lindsay and Schwab 1982). The uptake of Fe and

its mobilization is often limited because it is mainly present

as insoluble Fe(III) precipitates. Fe-deficiency is a common

abiotic stress especially for many photosynthetic organisms

in higher plants (Muneer et al. 2012). Fe deficiency leads to

Communicated by J.-H. Liu.

T. H. Kim: Deceased.

S. Muneer � T. H. Kim (&)

Department of Animal Science, Institute of Agricultural Science

and Technology, College of Agriculture and Life Science,

Chonnam National University, Buk-Gwangju,

P.O. Box 205, Gwangju 500-600, Korea

e-mail: grassl@chonnam.ac.kr

B. R. Lee

Environmental Friendly Agricultural Bio-material Research

Team, College of Agriculture, Chonnam National University,

Gwangju 500-757, Korea

D. W. Bae

Central Instrument Facility, Biomaterial Analytical Lab,

Gyeongsang National University, Jinju 660-701, Korea

123

Acta Physiol Plant

DOI 10.1007/s11738-013-1336-4

decrease in amount of photosynthetic pigments (Spiller and

Terry 1980; Morales et al. 1998), which causes leaf chlorosis

and even necrosis. A general feature of Fe deficiency is a

reduction of protein synthesis in the chloroplasts (Shetty and

Miller 1966). It has been well documented in many plant

species and lower organism that Fe deficiency resulted in a

degradation of chloroplast proteins such as light harvesting

phycobilisomes (Guikema and Sherman 1983). The loss of

other important photosynthetic proteins such as PSI and PSII

has been also observed under Fe-deficient condition (Tim-

perio et al. 2007; Muneer et al. 2012). These are accompa-

nied with the decrease in all membrane components

including electron carrier in photosynthetic electron trans-

port chain (Spiller and Terry 1980).

Sulfur (S) is essential mineral for production of crop yield

and is a main component of amino acids such as cysteine and

methionine, oligopeptides such as GSH and PCs, vitamins

and other co-factors such as biotic, CoA and large number of

secondary products (Leustek and Saito 1999). In particular,

several experimental evidences reported that S-nutrition is

involved in the Fe acquisition mechanism (Astolfi et al.

2012; Zuchi et al. 2012). The availability of S is associated

with the production and release of high-affinity chelating

compounds such as phytosiderophores present in barley

roots (Astolfi et al. 2012). High S supply leads to the

induction of Fe and S concentration in the leaves of durum

wheat plants (Zuchi et al. 2012). The positive roles of

S-nutrition in Fe acquisition could be explained by an

increased production of phytosiderophores and nicotian-

amine (Astolfi et al. 2012) with strategy II plants (gramina-

ceous monocotyledonous species), because methionine is

S-containing amino acid required for the synthesis of

S-adenosylmethionine which is a common precursor of

molecules as nicotianamine, phytosiderophores, ethylene

and polyamines (Hesse and Hoefgen 2003). Similarly, Zuchi

et al. (2009) showed that S-nutrition could improve the

capacity of Fe utilization by increasing the activity of Fe(III)-

chelate reductase and the expression of Fe2? transporter gene

(LeIRT1) with strategy I in tomato plants. It has been well

documented that maintaining Fe homeostasis is an important

physiological factor in building prosthetic groups such as

heme and Fe–S clusters, and in assembling them into apo-

proteins, which are major components of plant metabolism

(Briat et al. 2010). Fe–S clusters are co-factors of proteins

that function in vital processes such as photosynthesis, res-

piration, S and N metabolism, plant hormone and coenzyme

synthesis (Balk and Pilon 2011).

On the other hand, in recent decades, the use of low

S-containing fertilizers and low input of sulfur from

atmospheric deposition on the soil has increased the inci-

dence of S-deficiency in oilseed rape (McGrath and Zhao

1996), which is a high S-demanding plant (McGrath and

Zhao 1996; Blake-Kalff et al. 2000).

Thus the characterization of the proteins expressed by S

and Fe combined treatment deserves to be a good strategy

for the understanding of the possible roles of S-nutrition in

alleviating Fe deficiency stress. In this study, a gel based

proteomic approach and a functional classification of the

identified proteins was performed for the leaves of B. napus

after 10 days of S and Fe combined treatments.

Materials and methods

Plant material and treatment

Surface-sterilized seeds of B. napus L. cv. Mosa were ger-

minated in wet filter paper in the Petri dishes at 30 �C in the

dark for 3 days. Four seedlings were transplanted and then

thinned to two after 2 weeks. The seedlings were grown in 3

L plastic pots with hydroponic nutrient solution containing

(mM for the macro elements): 1.0 NH4NO3; 0.4 KH2PO4; 3.0

CaCl2; 1.5 MgSO4; 0.15 K2HPO4; 0.2 Fe–Na EDTA; and

(lM for the micro elements): 14 H3BO3; 5.0 MnSO4�H2O,

3.0 ZnSO4�7H2O; 0.7 CuSO4�5H2O; 0.7 (NH4)6MO7O24; 0.1

COCl2. The nutrient solution was continuously aerated and

renewed every 5 days. Natural light was supplemented with

200 lmol m-2 S-1 at the canopy height for 16 h day-1.

Eight-week-old plants were divided in four groups with three

replications to receive different treatments: sufficient in S

and Fe (?S/?Fe, control), sufficient S but Fe deprived

(?S/-Fe), deprived S but sufficient Fe (-S/?Fe), and

deprived S and Fe (-S/-Fe). For the S-deficient Hoagland

media, SO42- containing salts were replaced by equimolar

amount of Cl-(of K?, Mn2?, Zn2?, Cu2?) salts. After

10 days of treatment, leaves were separated by the order of

ontogenic appearance, which was designated as leaf number

(i.e. giving leaf No. 1 for the oldest leaf). In this study, mature

leaves which are the leaf ranks numbered 4 and 5 were

considered.

H2O2 and O2- localization in situ

To visualize H2O2 localized in leaf tissues, leaf discs from

the four treatments after 10 days were excised and

immersed in a 1 % solution of 3,30-diaminobenzidine

(DAB) in Tris–HCl buffer (pH 6.5), vacuum-infiltrated for

5 min and then incubated at room temperature for 16 h in

the absence of light. Leaves were illuminated until

appearance of brown spots characteristic of the reaction of

DAB with H2O2. Leaves were bleached by immersing in

boiling ethanol to visualize the brown spots and were

photographed under microscope (Leica DM4000; Leica,

Wetzlar, Germany) at 409 magnification.

For the visualization of O2-, leaf discs were immersed

in a 0.1 % solution of nitroblue tetrazolium (NBT) in

Acta Physiol Plant

123

K-phosphate buffer (pH 6.4), containing 10 mM Na-azide,

and were vacuum-infiltrated for 5 min and illuminated

until appearance of dark spots, characteristic of blue for-

mazan precipitate. After bleaching in boiling ethanol, the

leaf samples were photographed as described above.

Protein extraction

One gram of leaf tissues was homogenized in phosphate

buffer (pH 7.6) containing 40 mM tris, 0.07 % bME (beta

mercapto ethanol), 2 % PVP (Polyvinylpyrrolidone) and

1 % TritonX 100 at 4 �C using chilled pestle and mortar on

ice. The homogenates were centrifuged at 15,000 rpm for

15 min, and proteins were precipitated with 10 % TCA/

acetone overnight at -20 �C. The resultant precipitate was

centrifuged at 15,000 rpm for 15 min and washed with 80 %

chilled acetone containing 2 mM EDTA and 0.07 % ßME.

The proteins’ pellet was solubilized in solubilization buffer

containing 9 M urea, 2 M thiourea, 4 % CHAPS, 2 % Tri-

tonX100, 50 mM DTT and 0.2 % ampholine (pH 4–7).

Two-dimensional polyacrylamide gel electrophoresis

Three hundred micrograms of proteins was separated by

2-DE in the first dimension by isoelectric focusing on

11 cm IPG strip (pI 4–7) and the second dimension by

SDS-PAGE on Protean II unit (Bio-Rad, Hercules, USA)

according to method given by Qureshi et al. (2010). For

first dimension the isoelectric focusing gel consisted of

8 M urea, 3.5 % polyacrylamide, 2 % Nonidet P-40, 2 %

Bio-Lyte, ammonium persulfate and tetramethylenedi-

amine. Electrophoresis was carried out at 200 V for

30 min, followed by 400 V for 16 h and 600 V for 1 h.

After separation, SDS-PAGE as the second dimension was

performed using 12 % polyacrylamide gel. The gels were

stained with Coomassie brilliant blue R.

Image acquisition and data analysis

The gels obtained from three biological replicates of each

treatment were used for image acquisition and data anal-

ysis. Spot detection, spot measurement, back ground sub-

traction and spot matching were performed using PD-Quest

software (Bio-Rad Hercules, V8.0.1, CA, USA) in auto-

matic spot detection mode to review the annotations of

spots statistically using one way ANOVA analysis auto-

matically by the software. One of the gels was selected as a

reference gel for spot matching. The amount of protein spot

was expressed as the volume of spot, which was defined as

the sum of intensity of all the pixels that make up the spot.

In order to correct variability derived from CBB staining

and to reflect the quantitative variation in intensity of

protein spots, the spot volumes were normalized as a

percentage of the total volume in all spots in the gel. The

resulting data from image analysis were referred to PD-

Quest software for querying protein spots that showed

quantitative and qualitative variation. The pI and Mr of

each protein were determined using 2-DE markers (Sigma).

Protein digestion by trypsin

The differentially expressed protein spots were excised

manually from the gels, and washed with distilled water three

times by a centrifugation for 1,100 rpm at 22–24 �C for

10 min. The protein spots were treated with 50 % acetoni-

trile after washing and centrifuged for 10 min at 22–24 �C at

1,100 rpm. The protein spots were then treated with 1 M

DTT and 50 mM ammonium carbonate, and incubated for

45 min at 38 �C. After incubation protein spots were treated

with IAA and 55 mM ammonium carbonate, centrifuged

again for 30 min at 22–24 �C at 1,100 rpm and were vacuum

dried for 15 min. The protein spot was then treated with

trypsin and were kept at 37 �C overnight before running on

MALDI-TOF for peptide identification.

Analysis of digested proteins using MALDI-TOF

Protein digestion with trypsin was done manually as

described above. The generated peptides were purified using

NuTip C-18 columns (Glygen Corp., Columbia, MD, USA).

The resulting peptides were added to a-cyano-4-hydroxyci-

namic acid matrix and dried onto a plate for analysis using a

matrix-assisted laser desorption ionization time-of-flight

(MALDI-TOF) MS (Voyager- DE RP, Applied Biosystems).

The calibration of MALDI-TOF was external and data were

collected in the reflector mode. Data were searched on the

internet using an in-house licensed Mascot search engine

(version 2.2.18, Matrix Science, Ltd., London, UK,

www.matrixscience.com) against all entries in viridiplanate.

Carbamidomethylation of cysteines was set as a fixed mod-

ification and oxidation of methionines was set as a variable

modification with report top as 20 maximum probability hits

and 1–2 missed cleavages. The identified proteins were

matched with other species from NBCI data base to observe

matched number of peptides. Trypsin was specified as the

proteolytic enzyme and one missed cleavage was allowed.

Identified proteins with a peptide mass fingerprint were

denoted as having an unambiguous identification.

Results

Effect of S/Fe combined treatment on morphology

S and Fe deprivation leads to severe chlorosis and necrosis

characterized by yellowing of leaves and necrosis on

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123

margins (Fig. 1, indicated by black arrows). S- supply

reduces the negative effects and retains the green color in

leaves.

Production of reactive oxygen species (ROS)

Accumulation of oxidative stress was visualized in situ in

the form of H2O2 and O2- by histochemical methods. For

visualizing H2O2 accumulation, the DAB reaction which is

based on the formation of brownish parts as a result of

H2O2 presence was used. S-deprived leaves exhibited

highly enhanced brownish staining indicated by white

arrows (Fig. 2a). The staining was not increased in the

?S/-Fe leaves compared to controls. Production of O2-

was studied by a reaction with nitroblue tetrazolium

(NBT), which is reduced by O2- giving rise to dark brown

spots of blue formazan. In the both -S/?Fe and -S/-Fe

leaves, dark brown spot areas were widespread (Fig. 2d)

while very little difference in spot number was seen

between controls and ?S/-Fe leaves.

Differentially expressed proteins in leaves by 2DE

After separation of proteins on 2DE, staining with CBB,

the protein spots obtained from S and Fe combined

treatments were evaluated by comparing that of control

(?S/?Fe) with the PD-Quest software with three replica-

tions. Two hundred proteins were reproducibly detected on

2D gels by S and Fe combined treatments. Fifteen protein

spots (spot 1–15) were down-regulated under S-deprived

condition regardless of Fe presence (-S/?Fe) or absence

(-S/-Fe) (Fig. 3b, c) indicated with red arrows with

respect to the control. However, these down-regulated pro-

teins were well expressed in the presence of S (?S/-Fe).

In addition, two proteins (spot 16 and spot 17) were

up-regulated under S-deprived conditions in presence

(-S/?Fe) and absence of Fe (-S/-Fe), but the expression

was down to control level in the presence of S (?S/-Fe)

compared to control (Fig. 3d, indicated with red arrows).

The enlarged and focused images of the differentially

expressed proteins by the treatments along with fold

changes are presented in Fig. 4.

Analogously five spots, indicated with purple arrows on

the control gel (?S/?Fe, Fig. 3a), were absent under Fe-

deficient condition in the absence of S (-S/?Fe), and ten

spots, indicated with blue arrows on the control gel, were

disappeared under Fe-deprived condition in the absence of

S (-S/-Fe). However, most of these disappeared spots

were expressed in the presence of S under Fe deprivation

(?S/-Fe) as indicated with green arrows in Fig. 3d.

Fig. 1 Morphological changes

in leaves of four S/Fe combined

treatments: sufficient in S and

Fe (?S/?Fe, control), deprived

S and Fe (-S/-Fe), deprived S

but sufficient Fe (-S/?Fe), and

sufficient S but deprived Fe

(?S/-Fe) for 10 days.

Morphology of leaves was

observed visually showing

chlorosis and necrosis

(indicated with black arrows)

Acta Physiol Plant

123

Identification of differentially expressed proteins

in leaves

Fifteen proteins down-regulated under Fe-deprived in the

absence of S (-S/-Fe) and two proteins up-regulated in

the Fe-deprived in the presence of S (?S/-Fe) were

identified by MALDI-TOF–MS (Table 1). The six proteins

related to photosynthesis were down-regulated in presence

(-S/?Fe) and absence of iron (-S/-Fe) within a range

from 1.15-fold to 17.0-fold (Fig. 4). These proteins were

identified as cytochrome (spot 7), beta-ketoacyl-acyl car-

rier protein synthase (spot 8, 9), putative chloroplast inner

envelope protein (spot 14), protein XAP5 CIRCADIAN

TIMEKEEPER-like isoform 19 (spot 12), and putative

R2R3 MYB transcription factor (spot 3). However, these

proteins were well expressed, representing 1.03-fold up to

1.36-fold change (Fig. 4b) in presence of sulfur (?S/-Fe)

under Fe deprivation condition.

Besides photosynthetic proteins, five other down-regulated

proteins in presence (-S/?Fe) and absence of Fe (-S/-Fe)

were related to other metabolites and were identified as

nucleotide-binding site leucine-rich repeat protein (spot 11),

UPF0678 fatty acid-binding protein-like protein At1g79260

(spot 13), target of FKB12/rapamycin complex (spot 15),

+S+Fe -S-Fe -S+Fe +S-Fe

40X 40X 40X40X

(a)H

2O

2

(b)O

2

•−1

Treatment

Fig. 2 Representative images

of H2O2 (a) and O2-1 (b)

localization in leaf tissue after

10 days of S/Fe combined

treatments. Brownish parts and

dark brown spot (indicated with

white arrows) prove H2O2 and

O2-1 accumulation (color figure

online)

14.4

21.5

31.0

45.0

66.2

97.4MK 4 7 4 7

14.4

21.5

31.0

45.0

66.2

97.4MK

16

17

1 2

3 45

6 7

8 9 10

(a) (b)

121314

15

(c) (d)

11

1 23 4

5

6 7

89 10

11

121314

15a b

cd e

a b

c

d

f

g

hjk

o

f

g

hi

k

m

n

o

e

16

17

1 23 4

56 7

89

10

11

16

17

1 23 4

56 7

89

10

11

121314

15

16

17

1213

Fig. 3 2D references images of

four S/Fe combined treatments:

a sufficient in S and Fe (?S/

?Fe, control), b deprived S and

Fe (-S/-Fe), c deprived S but

sufficient Fe (-S/?Fe;), and

d sufficient S but deprived Fe

(?S/-Fe) in leaves of B. napus

L. Proteins were separated in

the first dimension on a

nonlinear IPG strips, pH 4–7

and in 2nd dimension on 12 %

polyacrylamide SDS-gel.

Quantitative image represents

differentially expressed

proteins. Blue and purple

arrows indicate absence of

protein spots in -S/Fe and -S/

?Fe respective gels, Green

arrows indicate restoring of

protein spots with respect to that

of control. Red arrows indicate

comparison of protein spots to

each other identified by

MALDI-TOF (color figure

online)

Acta Physiol Plant

123

whereas they were well expressed in sulfur nutrient plants in

absence of Fe (?S/-Fe). In particular, two proteins which

were identified as ethylene receptor (spot 16) and (?)-neo-

menthol dehydrogenase (spot 17) up-regulated by 2.08-fold

and 2.50-fold, respectively, in the absence of sulfur (-S/-Fe),

but only 1.17-fold and 1.50-fold in the presence of sulfur under

Fe-deprivation (?S/-Fe).

Six other down-regulated proteins from S/Fe deprived

leaves (-S/-Fe) were identified as hypothetical proteins

(spot 1 and spot 6), unknown proteins (spot 5 and spot

10), predicted protein (spot 2) and uncharacterized protein

(spot 4).

The all identified proteins were classified by Beven et al.

(1998) and were grouped into five classes (Fig. 5) as 40 %

chloroplast precursor, 10 % secondary metabolism, 10 %

unknown proteins, 10 % hypothetical proteins, and 10 %

RNA binding related metabolite proteins.

Discussion

S- or Fe-deprived leaves exhibited visible chlorosis, char-

acterized by the yellowish and chlorotic and sometimes

necrotic spots (indicated by black arrows) as well as

(b) Spot -S/-Fe -S/+Fe +S/-Fe Spot -S/-Fe -S/+Fe +S/-Fe

1 -2.67 -2.00 -1.10 10 -1.48 -1.41 -1.152 -2.06 -1.83 -1.18 11 -3.00 -2.14 -1.673 -4.30 -2.53 -1.48 12 -17.00 -2.62 -1.364 -5.00 -3.75 -1.50 13 -17.00 -2.62 -1.36 5 -4.22 -3.17 -1.26 14 -1.62 -1.31 -1.036 -1.38 -1.32 -1.16 15 -1.39 -1.34 -1.087 -1.43 -1.15 -1.11 16 +2.08 +1.75 +1.17 8 -1.69 -1.74 -1.26 17 +2.50 +1.60 +1.509 -1.77 -1.67 -1.22

(a) Fig. 4 Enlarged images of

differentially expressed proteins

(a) and relative intensities

expressed as fold change in

relative to control (b) after

10 days of S/Fe combined

treatments, sufficient in S and

Fe (?S/?Fe, control), deprived

S and Fe (-S/-Fe), deprived S

but sufficient Fe (-S/?Fe;), and

sufficient S but deprived Fe

(?S/-Fe). The relative

intensity was quantified by PD-

Quest software, where ‘?’ and

‘-‘ refer up- and down-

regulation, respectively,

compared to control

Acta Physiol Plant

123

prominent wrinkles in the lamina and curling of the leaf

margins (Fig. 1). The leaf chlorosis caused by Fe-defi-

ciency was more severe in the absence of S. Leaf chlorosis

may be due to the loss of chlorophyll especially under Fe-

deficient condition. Such a net decrease in chlorophyll

contents is attributed to the well-known Fe requirement for

the formation of precursor molecules, d-aminolevulinic

acid and protochlorophyllide (Marschner et al. 1986). More

severe chlorosis in S-deprived leaves certainly attributed to

an additional loss of chlorophyll content, because S is an

integral constituent of two S-containing amino acids, cys-

teine and glutathione, which both act as structural and

functional elements of proteins (Droux 2004). In addition,

the present study showed that Fe-deficiency in the absence

of S increased the accumulation of H2O2 and O2- (Fig. 2)

which is referred as the induction of oxidative stress,

whereas the accumulation of these oxyradicals was largely

reduced in the presence of S. Oxidative stress under Fe-

deficiency might be primarily due to Fe leakage from

impaired multiple protein complexes (Briat et al. 2007) and

from co-factors of antioxidant enzymes (Szacilowski et al.

2005). These findings led to hypothesize that Fe-depriva-

tion results in a proteolytic damage which in turn induces

oxidative stress (or vice versa), and that adequate

S-availability might alleviate the deleterious impacts of Fe

deprivation on photosynthetic organelles by supplying S in

building prosthetic group Fe–S clusters which are co-fac-

tors of proteins involved in vital processes such as photo-

synthesis, respiration, S and N metabolism, plant hormone

and coenzyme synthesis (Balk and Pilon 2011).

Indeed, the proteomic analysis showed that S- and/or Fe-

deprivation reduced protein spots in number (Fig. 3). The

Table 1 Identification of differentially expressed proteins under S/Fe combined treatment in leaves of B. napus identified by MALDI-TOF

Spot Acession

number

Homology Coverage

(%)

Matched

peptides

Mascot

score

Mr

value

Ther.pI/

exp.pI

Species

1 gi|255541682 Conserved hypothetical protein 27 106 55 42,672 6.6/5.0 Ricinus communis

2 gi|224064404 Predicted protein 41 64 58 16,795 7.0/5.6 Populus trichocarpa

3 gi|37987865 Putative R2R3 MYB transcription factor 62 35 37 6,503 9.8/4.3 Lolium multiflorum

4 gi|212274435 Uncharacterized protein LOC100191698 17 93 65 57,411 9.5/6.0 Zea mays

5 gi|297741965 Un named protein product 72 31 58 4,895 9.2/6.5 Vitis vinifera

6 gi|222631403 Hypothetical protein OsJ_18351 15 94 50 69,640 8.3/6.8 Oryza sativa

7 gi|195653535 Cytochrome P450 CYP71C36 18 100 52 60,439 7.3/7.0 Zea mays

8 gi|351721843 Beta-ketoacyl-acyl carrier protein

synthase III

20 82 53 41,844 6.6/4.3 Glycine max

9 gi|351721843 Beta-ketoacyl-acyl carrier protein

synthase III

20 82 53 41,844 6.6/4.4 Glycine max

10 gi|41052663 Unknown protein 48 50 60 10,858 5.4/5.6 Oryza sativa

11 gi|379068466 Nucleotide-binding site leucine-rich

repeat protein

27 73 68 30,937 5.0/5.8 Rhododendron

formosanum

12 gi|359475047 Protein XAP5 CIRCADIAN

TIMEKEEPER-like isoform 1

25 86 61 39,153 6.2/6.5 Vitis vinifera

13 gi|357130717 UPF0678 fatty acid-binding protein-like

protein At1g79260

31 67 54 23,108 9.3/5.6 Brachypodium

distachyon

14 gi|159885628 Putative chloroplast inner envelope protein 31 51 49 18,163 7.1/5.0 Hordeum vulgare

15 gi|159468810 Target of FKB12/rapamycin complex 34 69 59 22,169 4.8/4.6 Chlamydomonas

reinhardtii

16 gi|11935116 Ethylene receptor 13 88 60 70,587 6.7/7.0 Carica papaya

17 gi|357460053 (?)-neomenthol dehydrogenase 23 71 66 32,472 6.3/6.9 Medicago truncatula

Hypothetical proteins

30%

RNA binding 10%Chloroplast

precursor40%

unknown protein10%

Secondary metabolism

10%

Fig. 5 Functional classification of identified proteins from leaves

analyzed by MALDI-TOF–MS as described by Beven et al. (1998)

Acta Physiol Plant

123

loss of protein spots was observed under S-deficiency

mostly in the absence of Fe (10 spots, Fig. 2b) and less in

the presence of Fe (5 spots, Fig. 3c), while it was largely

restored in the presence of S even under Fe-deficient

conditions (Fig. 3d). The loss of protein spots might be due

to progressive depletion of biochemical pathways associ-

ated with signal transduction and gene regulation in par-

ticular manner involved in protein synthesis (Pandey et al.

2006; Choudhary et al. 2009) and might be associated with

an excessive production of ROS which leads to incorrect

folding or assembly of proteins, and consequent protein

degradation (Luo et al. 2002). Recently, we estimated that

S-deficiency resulted in strong decrease of incorporation of

newly absorbed NO3- and SO4

2- into proteins by 33.6 and

67.5 %, respectively, in young leaves of oilseed rape (Lee

et al. 2013). This suggests that S-nutrition retains the loss

of proteins in leaves by activating the induction of sulfate

and/or nitrate transporter mechanism (Abdallah et al. 2010)

and by increasing de novo synthesis of proteins in

Fe-deficient leaves. In the S-deprived leaves (-S/?Fe and

-S/-Fe), 15 proteins were down-regulated, but these

down-regulated proteins were well expressed in the pres-

ence of S (?S/-Fe) (Fig. 3). The loss of protein (down-

regulation) in S- and/or Fe-deprived leaves may reflect an

overall reduction of biosynthesis of chloroplast-targeted

proteins. Given that the chloroplast as the primary orga-

nelle to be affected by abiotic stress, we observed in an

accompanied work with BN-PAGE of integral thylakoid

proteins that a strong reductions of PSI (RCI ? LHCI),

PSII, RuBisCO, cyt b6/f, LHCII trimer complex were

observed under Fe-deficient condition in the absence of S

and that the loss of most thylakoidal protein complexes

under Fe-deficiency was largely restored in the presence of

S (data not shown). Forty percent of differently expressed

proteins by S and Fe combined treatment was identified to

be related to chloroplast precursor such as putative R2R3

MYB transcription factor spot 3), cytochrome (spot 7),

beta-ketoacyl-acyl carrier protein synthase III (spot 8 and

9), protein XAP5 CIRCADIAN TIMEKEEPER-like iso-

form 1 (spot 12), and putative chloroplast inner envelope

protein (spot 14) (Table 1). These proteins were down-

regulated in S-deprived leaves (more severely in the

absence of Fe), whereas they were expressed to the control

level in the S-supplied leaves (Fig. 3). This may be caused

by a stronger decline in S compounds in the leaves rather

than that in Fe content. This suggests that the consequent

low availability of S may limit the activity of (4Fe–4S) and

(2Fe–2S) clusters which are involved in chloroplast

membranes (Imsande 1999) especially in oilseed rape

which is a high S-demanding plant. It can be thus con-

cluded that down-regulation of chloroplast precursor pro-

teins in Fe-deprived leaves in the absence of S is probably

due to proteolytic loss of photosystems (Landry and Pell

1993; Briat et al. 2007) and the light harvesting pigments

such as chlorophyll and carotenoids. In addition, some

proteins related to RNA binding protein and secondary

metabolism were differentially expressed by S and Fe

combined treatment (Table 1). Interestingly, ethylene

receptor (spot 16) was 1.75- and 2.0-fold up-regulated,

respectively, in S-deprived leaves (-S/?Fe and -S/-Fe),

whereas the expression of this protein in the presence of S

was 1.17-fold decreased to the control level. This result is

consistent with the finding of Zuchi et al. (2009) who

showed an increase in ethylene production in Fe-deficient

plants, with a lesser extent in the absence of S with strategy

I, in tomato plants. The lack of increase in ethylene pro-

duction in S-deficient might be due to inadequate supply of

reduced S to sustain the ethylene production because eth-

ylene is synthesized from methionine (Hesse and Hoefgen

2003). It was reported that ethylene regulates the expres-

sion of many genes involved in Fe acquisition, transport,

and homeostasis (Romera and Alcantara 2004). Given that

ethylene production is increased by ammonia accumulation

in plant tissue (Feng and Barker 1993), our results seem to

support this hypothesis: as the severity of S- and Fe-defi-

ciency increased, protein synthesis would likely decline

leading to ammonia accumulation in the plant (Kim et al.

2004; Nikiforova et al. 2005) and ethylene production

would be increased.

These results indicate that S supply under Fe-deficient

condition has a role in regulating the biosynthesis of eth-

ylene which is a main component involved in Fe uptake

(Douchkov et al. 2002; Romera and Alcantara 2004) in

oilseed rape, one of strategy I plants which do not release

phytosidrophores. Taken together, the results indicate that

S-nutrition has a more influential role in modulating the

protein expression to alleviate deleterious impacts of Fe-

deficiency in the leaves of oilseed rape plants.

Author contribution S. Muneer and T.H. Kim designed

the experiments. S. Muneer carried out all the experiments

and wrote the paper under guidance of Prof. T.H. Kim.

T.H. Kim interpreted the data and drafted the manuscript.

B.R. Lee performed chemical analysis and critical correc-

tion. D.W. Bae performed MALDI-TOF analysis.

Acknowledgments This study was financially supported by Chon-

nam National University, 2012.

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