Environmental and Experimental Botany 74 (2011) 245254

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Environmental and Experimental Botany

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Respiratory activity, energy and redox status in sulphur-decient bean plants

Izabela M. Juszczuk , Monika Ostaszewska
Institute of Experimental Plant Biology and Biotechnology, University of Warsaw, Miecznikowa 1, 02-096 Warsaw, Poland

a r t i c l e

i n f o

Article history:
Received 21 January 2011
Received in revised form 15 June 2011
Accepted 16 June 2011
Keywords:
Complex I
Energy status
Internal NAD(P)H dehydrogenase type II
Redox homeostasis
Respiration
Sulphur deciency

a b s t r a c t
Sulphur (S) is an essential nutrient that due to its chemistry plays important roles in many metabolic
processes. S-decient bean plants (Phaseolus vulgaris L. cv. Zota saxa) showed decreased sulphate concentrations and sulphur to nitrogen ratios in the leaves and roots, less chloroplastic pigments and lower
dry matter production. Phenotypic effects of S deciency appeared as depressed shoot growth, paling
and curling of the youngest leaves, chlorotic and/or necrotic spots on the leaf surface. Our results show
that S deciency changes mitochondrial function, cellular energy status and redox homeostasis. ATP production in bean leaf and root mitochondria was lower as the result of decreased activity of Complex
I. Increased activities of internal NADH dehydrogenases (NDin ) may at least partially compensate for
Complex I impairment. External NADH dehydrogenases (NDex ) activities, as well as protein level and
capacity of alternative oxidase (AOX), did not change in S-decient bean plants. Total ATP concentration
severely decreased in leaf and root tissues. Pyridine nucleotide level was changed in S-decient bean
plants, NAD(H) pool became more reduced in leaf and root tissues whereas NADP(H) pool was more oxidized in the leaves. Our ndings indicate that exible function of plant mitochondrial respiratory chain
could be an important target during adaptations to S deciency.
2011 Elsevier B.V. All rights reserved.

1. Introduction
Sulphur (S) is a ninth least abundant essential macronutrient for
the plant cell and thus its uptake and homeostasis are tightly regulated (Droux, 2004; Saito, 2004). S is taken up as sulphate (SO4 2 ) by
roots and transported to the shoots. The metabolically useful form
of sulphur for a plant is sulphide (S2 ), which comes from the reduction of SO4 2 in the chloroplasts. Then, sulphide is incorporated into
cysteine (Cys), which becomes the rst carbon/nitrogen-reduced
sulphur product and serves as a sulphur donor in the plant cell
(Hesse et al., 2004; Davidian and Kopriva, 2010). Sulphur, being
a constituent of cysteine and methionine and a variety of other
metabolites (e.g. glutathione, phytochelatins, ferredoxin, thioredoxin) as well as forming clusters with Fe, plays a role in crucial
processes in the plant cell, such as biosynthesis, assembly and
activity regulation of proteins, antioxidant defence, toxin tolerance,
photosynthesis and respiration (Droux, 2004; Nikiforova et al.,
2004; Saito, 2004; Davidian and Kopriva, 2010).

Abbreviations: AOX, alternative oxidase; BSA, bovine serum albumin; DW,

dry weight; DTT, dithiothreitiol; EDTA, etylenediamine tetraacetic acid; FW, fresh
weight; GSH, glutathione (reduced form); MOPS, 3-(N-morfolino) propanesulfonic
acid; NDex , external NAD(P)H dehydrogenases; NDin , internal NAD(P)H dehydrogenases; PAR, photosynthetically active radiation; PVP, polivinylpyrrolidone; ROS,
reactive oxygen species; SHAM, salicylhydroxamic acid.
Corresponding author. Tel.: +48 22 5543008; fax: +48 22 5542022.
E-mail address: izabelaj@biol.uw.edu.pl (I.M. Juszczuk).
0098-8472/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.envexpbot.2011.06.006

Sulphate uptake by root cell transporters is ATP-dependent and

its reduction to sulphide requires eight electrons and about twice
as much energy as nitrate reduction (Hell, 1997). Reduction of
sulphate is generally believed to take place in green and nongreen plastids, where redox potential is provided by thioredoxin
and ferredoxin, while non-photosynthetic reduction depends on
NADPH. A small proportion of sulphate could be reduced by plastids in heterotrophic tissues (Hell et al., 1997). Due to the presence
of enzymes for cysteine synthesis in the cytosol, plastids and
mitochondria, it was assumed that cysteine can be synthesized
in all three compartments (Kawashima et al., 2005). Plants synthesize glutathione (GSH) in a sequence of two steps that both
consume ATP (Noctor et al., 2002). Sulphur-rich amino acid and GSH
biosynthesis requires ATP and C-skeletons, that both come from
mitochondria. Thus, the function of the electron transport chain
and the Krebs Cycle link mitochondrial activity with S metabolism
(Kopriva and Rennenberg, 2004).
Plant mitochondria, apart from the ATP biosynthesis, coordinate
redox homeostasis of the plant cell in response to developmental
changes as well as variable and/or stressful environmental conditions (Rhoads and Subbaiah, 2007; Sweetlove et al., 2007). This is
due to functional plasticity of plant mitochondria as compared to
the animal counterpart. The uniqueness of the electron transport
chain of plant mitochondria relays on the presence of additional
internal and external NAD(P)H dehydrogenases (NDin/ex ) that drive
electrons to ubiquinone bypassing Complex I, and of the alternative oxidase (AOX), which directly reduces oxygen omitting the
cytochrome pathway Complexes III and IV (Rasmusson et al., 2008).

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Intensive oxidative metabolism of the mitochondrial respiratory

chain results in the constitutive production of reactive oxygen
species (ROS) in Complexes I and III (Mller, 2001). The activities of NDin/ex and AOX decrease the efciency of ATP production
and mitochondrial ROS formation and thus allow plant growth due
to the possibility of substrate oxidation under a wide spectrum of
developmental and environmental challenges (Escobar et al., 2006;
Rasmusson et al., 2008).
Sulphur availability is now a limiting factor in plant cultivation in many regions of the world. S-decient areas are becoming
widespread due to both reduction of sulphur dioxide (SO2 ) emission caused by strict atmospheric input regulation and changes
in agronomic practices, such as use of S-free fertilizers (Vestreng
et al., 2007). Sulphur deciency in certain soils affects plant growth,
vigour and resistance to stress (Knop et al., 2007; Kruse et al.,
2007). In plants grown under depleted sulphur supply, a decrease of
biomass, protein level, chlorophylls and cellular S/C/N ratio imbalance were observed providing evidence for a general reduction of
metabolic activity (Nikiforova et al., 2005). The effects of sulphur
deciency on various metabolic processes in the plant cell have
been the subject of intensive studies for a long time (reviewed
by Lewandowska and Sirko, 2008). Most studies regard to the
oxidative stress responses, since sulphur deciency caused serious
imbalance in concentration of cysteine and glutathione, which are
main antioxidants in the plant cell apart from ascorbate (Nikiforova

et al., 2003, 2004, 2006; Wawrzynska

et al., 2005). There is no information about changes in mitochondrial respiration relating to the
energy and redox status of S-decient plants. However, expression proling of Arabidopsis genes indicates that majority of genes
belonging to the category dened as energy were down-regulated
under sulphur starvation (Nikiforova et al., 2004). The declines of
the transcript levels were observed for genes encoding enzymes of
tricarboxylic acid cycle and glycolysis (Nikiforova et al., 2005).
Sulphur metabolic pathways in plants depend on the mitochondrial function, but activity of the respiratory complexes and
other mitochondrial proteins is modulated by the redox status
of cysteine. Sulphydryl/thiol (SH), a strong nucleophilic (electron donating) group present in cysteine molecule determines the
structure and/or function of proteins changing its redox state in
a reversible reaction of sulphydryldisulphide (SS) interchange
(Buchanan and Balmer, 2005; Yi et al., 2010). An epitomic mitochondrial protein is AOX, which activity is strongly dependent
on redox state of two cysteine residues in the catalytic center
(Crichton et al., 2010). Enzymatic complexes of the plant mitochondrial respiratory chain require sulphur not only as integral
elements of the thiol groups but also for the assembly of several different ironsulphur (FeS) clusters. Multiple 2Fe2S and
4Fe4S clusters have been localized in Complex I subunits (Lin
et al., 1995). In Complex II three types of FeS clusters have been
characterized: 2Fe2S, 3Fe4S and 4Fe4S, but Complex III contains only one 2Fe2S cluster (Vigani et al., 2009 and references
therein).
This study describes changes in respiratory activities, energy
and redox status of bean leaf and root tissue induced by S deciency and shows that mitochondrial activity is compromised by
a lack of S. As the major site of sulphate uptake is the root, but
the major site of reduction and biosynthetic need is the leaf, we
have chosen both tissues to establish a systemic response of mitochondrial respiration to sulphur deprivation. Presented data cast
new light on the role of plant mitochondria in S deciency. Since,
in contrast to Complex I, additional NDin/ex do not contain FeS
clusters and their activity is usually up-regulated under stress conditions (Rasmusson et al., 2008), they could bypass and/or support
Complex I in nucleotide oxidation in mitochondria of S-decient
plants. The implications of increased NDin/ex engagement in respiration comprise lower ATP level and changes in cellular redox

homeostasis in S-decient plants. Thus, the plasticity of mitochondrial respiratory metabolism could allow plant acclimation to the
limitation of this macronutrient.
2. Materials and methods
2.1. Plant material and growth conditions
Bean seeds (Phaseolus vulgaris L. cv. Zota saxa) were germinated in darkness and 5-day-old seedlings were transferred
to 6-l boxes with complete Knop nutrient medium (control)
or without sulphate (S, S-decient) (chloride salts were substituted for all sulphate salts), ushed with air. Cotyledons
were removed when the seedlings were 7-day-old. The nutrient solution was supplemented every day and changed every 4
days. Plants were grown under a 16-h photoperiod at 150 mol
quanta m2 s1 PAR (daylight and warm white 1:1; LF-40W; Pia,
Poland) day/night temperature of 25/20 C and 60/70% relative
humidity. Leaves or roots from 19-day-old control or S-decient
plants were used in experiments always after 4 h of the light
period.
2.2. Mineral concentrations
Sulphur (S) and sulphate (SO4 2 ) were estimated in leaf or root
tissue samples that were dried at 50 C up to the equal weights and
then homogenized with a mechanistic mortar (Retsch, Germany)
at 5000 rpm. To determine total S concentrations, 1 g of plant tissue was mineralized in a mixture of HNO3 and HClO4 (4:1, w/w)
for 1.5 h (rst 30 min at 80 C and then 1 h at 120 C). Sulphate concentration was estimated after digestion of 5 g of plant tissue in
50 ml of 2% CH3 COOH and 1 h of continuous vortexing. Elemental
contents were determined by Inductively Coupled Plasma Optical
Emission Spectrometry, ICP-OES (Thermo Elemental IRIS Advantage, USA). The analysis was performed at Warsaw University of Life
Sciences, in a collaboration with an authorized Laboratory of Forest
Environment Chemistry of Forest Research Institute, Poland. Nitrogen (N) concentration was estimated in leaf or root tissue digest,
according to Dumas method using CN analyser (LECO TruSpecTM
CN, USA). Iron (Fe) concentration was assessed in the whole roots
and leaves of control and S-decient plants. Roots were washed
at 4 C in deionized water for 5 min and, for removing unbound
and weakly bound metal from the apoplast, additionally desorbed
in 100 mM CaCl2 for 10 min, then in 100 mM EDTA, pH 8.0 for
10 min and again washed in water for 10 min. Plant samples were
dried at 55 C for 4 days, and dry biomass was determined. Dried
plant material was mineralized in 65% HNO3 and 39% H2 O2 (9:1,
v/v) in a closed system microwave mineralizer (Milestone Ethos
900, Milestone, Bergamo, Italy) as described in Wojas et al. (2009).
Iron concentration was measured using a ame atomic absorption
spectrophotometer (Thermo Scientic iCE 3000 Series). Certied reference material (Virginia tobacco leaves CTA-VTL-2) was
included in the analysis.
2.3. Chloroplastic pigments extraction and determination
The weighed samples of 100 mg fresh leaf tissue were homogenized with 25 ml of 80% (v/v) acetone. The homogenate was
ltered and centrifuged at 5000 g for 10 min. The supernatant was separated and the absorbances were read out on
Schimadzu UV-260 spectrophotometer. Chlorophyll a showed
the maximum absorbance at 662 nm, chlorophyll b at 646 nm
and total carotenoids at 470 nm. The amount of these pigments was calculated according to the formulas of Lichtenthaler
(1987).

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247

2.4. Isolation of mitochondria

2.7. Pyridine nucleotide estimations

Mitochondria were isolated from 30 to 40 g of leaves or 70 to

80 g of roots. Plant tissue was ground, using a cold mortar and
pestle, in 300 ml of homogenization medium, pH 7.4, containing:
0.45 M mannitol, 30 mM MOPS, 5 mM EDTA, 5 mM DTT, 0.6% (w/v)
PVP-40, 10 mM ascorbate and 1% (w/v) BSA. The homogenate was
ltered through a 80 m miracloth and mitochondria were isolated
by differential centrifugations: 1500 g for 10 min and 9600 g
for 20 min (Eppendorf 5804R). The pellet was suspended in 20 ml
of the homogenization medium and centrifuged again at 1000 g
for 10 min and 9600 g for 20 min. Resulting pellet was resuspended in 4 ml of the homogenization medium and mitochondria
were puried on a discontinuous Percoll gradient using a modied
method of Nishimura et al. (1982). The discontinuous gradient, pH
7.2, was composed of 4.5 ml 60% (v/v) Percoll, 6 ml 45% (v/v) Percoll, 12 ml (leaves) or 6 ml (roots) 28% (v/v) Percoll and 6 ml 10%
(v/v) Percoll, all in: 0.45 M mannitol, 20 mM MOPS, 0.1% (w/v) BSA
and 1% (w/v) PVP-25 and kept at 4 C until used. The crude mitochondrial fraction was carefully layered on top of gradients and
centrifuged at 7000 g (Beckman Avanti, angle rotor F0650) for
1 h 40 min (leaves) or 40 min (roots). The mitochondrial fraction
appeared at the interface between 28% and 45% (v/v) Percoll layers.
To obtain puried mitochondria, the mitochondrial fraction was
collected, washed in a medium, pH 7.2, containing: 0.45 M mannitol, 10 mM MOPS, 1 mM EDTA, 0.5% (w/v) BSA and centrifuged at
9600 g for 25 min. The pellet was suspended in about 150 l of
washing medium. Mitochondrial integrity was calculated by comparison of cytochrome c oxidase (EC 1.9.3.1) activity measured in
isotonic medium, rst in the absence and then in the presence
of 0.04% (w/v) Triton X-100 to disrupt mitochondrial membranes
(Wigge and Gardestrm, 1987). NAD-malic enzyme (EC 1.1.1.39)
activity was determined in isolated mitochondria as described by
Day et al. (1984).

Determination of pyridine nucleotides was performed in leaf

or root tissue extracts. About 500 mg of frozen leaf powder was
homogenized in a precooled mortar with 2.5 ml of 0.1 M HCl (oxidized forms) or 0.1 M NaOH (reduced forms of nucleotides) in 50%
ethanol. The homogenate was centrifuged at 10,000 g for 10 min
at 4 C. Alkaline extracts were heated at 60 C for 15 min and then
cooled on ice. The supernatants were neutralized and centrifuged
at 10,000 g for 10 min at 4 C. Pyridine nucleotides in whole tissue extracts were determined by the method of Lechevallier et al.
(1977) as described by Juszczuk and Rychter (1997).

2.5. Respiratory measurements

Oxygen uptake by isolated mitochondria was measured with a
Clark-type oxygen electrode (Oxygraph and Oxygraph Plus Software, Hansatech, Norfolk, UK) at 25 C in a nal volume of 0.5 ml of
the reaction medium, pH 7.2, containing: 0.45 M mannitol, 10 mM
Na-phosphate buffer, 5 mM MgCl2 , 10 mM KCl, 0.1% (w/v) BSA,
and 50100 g of mitochondrial protein. Malate (10 mM) in the
presence of 2 mM glutamate and 0.5 mM NAD+ , succinate (20 mM)
with 0.2 mM ATP, and NADH (1 mM) were used as substrates to
stimulate respiration, and 80 mM ADP was added when appropriate. Complex I activity (with malate as a substrate) was inhibited
by the addition of 1 M rotenone. The cytochrome pathway was
inhibited with 800 mM KCN in the presence of 1 mM pyruvate
and 10 mM DTT to activate the alternative pathway. The alternative pathway was inhibited with 750 mM salicylhydroxamic
acid (SHAM).
2.6. Determination of adenylates
Concentrations of ATP and ADP were measured in leaf or root tissue extracts. Plant tissue was collected and immediately frozen in
liquid nitrogen. Portions of about 2050 mg of frozen tissue powder
were homogenized for maximum 3 min with 300 l of 3% trichloric acid in precooled eppendorf adapters of a mixer-mill (MM 200,
Retsch, Germany). The homogenate was centrifuged at 10,000 g
for 10 min at 4 C. The supernatant was stored at 20 C. ADP was
converted to ATP by pyruvate kinase (Roche). The ATP concentration was assayed luminometrically by the luciferinluciferase
reaction (Ludin, 2000), using a commercial ATP kit SL (Biothema).

2.8. Protein SDSPAGE and immunodetection

The samples of mitochondrial proteins were suspended in onefourth of the volume of the loading buffer, pH 6.8, containing:
0.3 M TrisHCl, 50% (v/v) glycerol, 0.02% (w/v) bromophenol blue
and 5% (w/v) SDS, 500 mM DTT and boiled for 5 min. The portions of 10 g of mitochondrial protein per lane were separated
on 12% SDSPAGE and transferred to nitrocellulose membrane
using a Bio-Rad wet blotting apparatus. The following antisera were
used: mice monoclonal antiserum against AOX from Sauromatum
guttatum (Elthon et al., 1989; kindly provided by Prof. P. Gardestrm, Ume, Sweden), 1:100-diluted; antiserum against outer
mitochondrial membrane porin, voltage-dependent anion channel
(VDAC) from Arabidopsis thaliana (Agrisera), 1:5000-diluted. Antimouse (Bio-Rad) or anti-rabbit IgG-HRP (Bio-Rad) were used as
secondary antibodies, 1:3000- and 1:35,000-diluted, respectively.
Visualization was performed with a chemiluminescent reagent system (SigmaAldrich) using Kodak developing reagents for X-ray
lms. Films were scanned and band intensities were estimated as
the total volume of optical density after the background subtracting using Quantity One 4.6.2. software (Bio-Rad). Data from the
representative Western blots were taken for the analysis. In each
experiment, values of relative protein amount were calculated as
the percentage of the band density determined for Control plants
(set to 100%).
2.9. NAD kinase activity determination
Frozen leaf or root tissue powder was homogenized with 50 mM
phosphate buffer, pH 7.6 and centrifuged at 15,000 g for 15 min at
4 C. The supernatant was used for enzyme assays within 6 h. NAD
kinase (EC 2.7.1.23) activity was estimated according to Hayashi
et al. (2005) as described by Szal et al. (2008).
2.10. Protein determination
Soluble protein concentration was determined by the Bradford
(1976) method using BSA as a standard.
2.11. Statistical analyses
All presented results are the mean of n replicates (n = 312)
with standard deviation (SD). The signicance of the differences
compared to the control was tested using the Students t test. Different letters (ab or, if needed, cd ) indicate signicant differences at
P < 0.05.
3. Results
3.1. Symptoms of S deciency
In S-decient plants growth depression became apparent after
15 days of initiating the starvation. Generally the growth of the

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Fig. 1. Visible effects of sulphur deciency on growth and morphology of bean plants grown in hydroponic culture. Growth of the shoots of S-decient bean plants affected
by S deprivation in comparison with control plants (A), morphology of the leaves of S-sufcient and S-decient plants (B), shoot height and root length comparison (C), and
close-up of young leaves of S-decient plant showing loss of the green pigmentation, chlorotic and/or necrotic spots, curling of the margins and wrinkles in leaf lamina (D).
White arrows indicate diagnosed chlorosis () or necrosis ( ) of lamina tissues.

shoots was more affected than of the roots. Shoots were smaller
in S-decient bean plants (Fig. 1), but root length did not signicantly differ (Table 1) even if some of the roots in the experimental
population were visibly longer as compared to control plants
(Fig. 1C). The leaf size was reduced (Table 1) and the lamina of
the leaves showed wrinkles and curling of the margins forming
the characteristic structures (Fig. 1D). The youngest leaves became
pale, they presented loss of the green pigmentation and turned

Table 1
Growth parameters of 19-day-old bean plants grown in full Knop nutrient medium
(Control) or without sulphate (S). Means of 69 replicates with SD as indicated.
WWC, weight water content. Data values for same parameters having different
superscript letters are statistically signicant at ab P < 0.05.
Leaves

Roots

Control

Control

FW (g)
DW (g)
WWC (%)
Leaf area (cm2 )
Root length (cm)

8.76a 0.76
0.89a 0.06
89.84 1.34
80a 4

4.66b 0.34
0.46b 0.02
90.77 1.55
56b 3

2.43 0.13
0.18 0.01
92.59 1.67
169 14

2.67 0.19
0.24 0.01
92.10 1.98
191 19

Shoot/root ratio

2.59a 0.26

1.24b 0.13

completely yellow (Fig. 1D). The chlorotic and/or necrotic spots

were visible on the leaf surface (Fig. 1D). The older leaves of Sdecient bean plants were not much affected visually by sulphur
deprivation, but they were always smaller than in control plants
(Fig. 1).
The fresh and dry weights of the leaves decreased by about
48% in S plants, whereas the root fresh and dry weights
did not change (Table 1). The growth of the whole shoot was
affected by S deprivation. The lower shoot dry matter production compared to root ultimately decreased shoot to root dry
mass ratio by over 50% (Table 1). The developmental changes of
the leaves of S-starved bean plants resulted in the decreased leaf
area whereas root length remained similar (Table 1). Sulphurdecient plants did not show changed water status, calculated
as (FW DW)/FW 100%, neither in the leaves nor in the roots
(Table 1).

3.2. Effect of S deciency on total S, SO4 2 , N, Fe, soluble protein

and non-protein thiol levels
To conrm the metabolic status of the plants starving from sulphur deciency, the total concentrations of elemental sulphur (S)

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249

Table 2
Elemental sulphur (S), sulphate (SO4 2 ), soluble protein, nitrate (N), non-protein thiols concentrations, sulphur/nitrogen (S/N) ratio and iron (Fe) level in the leaves (young
and old) and roots of 19-day-old bean plants grown in full Knop nutrient medium (Control) or without sulphate (S). Means of 412 replicates with SD as indicated. Data
values for same parameters having different superscript letters are statistically signicant at ab P < 0.05.
Leaves

Roots
S

Control

S (mg g1 DW)
SO4 2 (mg g1 DW)
Protein (% FW)
N (mg g1 DW)
Thiols (nmol g1 FW)
S/N ratio
Fe (g g1 DW)

Young

Old

Young

Old

2.29a 0.09
0.35a 0.006
2.0a 0.1
59.6 6
150a 12
0.038a
0.07 0.01

2.36a 0.11
0.39a 0.02
1.9b 0.08
57.7 7
142a 14
0.040a
0.12 0.03

0.89b 0.06
0.09b 0.001
0.9b 0.07
56.9 8
86b 11
0.016b
0.07 0.01

1.65b 0.12
0.22b 0.01
1.1a 0.06
54.3 7
112b 13
0.030b
1.13 0.04

and sulphate (SO4 2 ) were measured in the leaves and roots of

these plants. Moreover, the level of S and SO4 2 was measured
in the old and young leaves separately. In 19-day-old sulphurdecient plants the level of elemental sulphur in the young leaves
was by about 60% lower than in control plants. Less evident changes
were observed for S concentration in the old leaves of these plants
(Table 2). The concentrations of sulphate in the old and young leaves
of S plants were about 43% and 74% lower, respectively, as compared to control plants (Table 2). In the roots, the level of elemental
S was lower by 58% in S plants. The concentration of sulphate in
the roots of 19-day-old S-decient plants was 68% lower than in Ssufcient plants (Table 2). Total N concentration did not change in
the leaves or roots of S-decient plants, but since the total S concentration severely decreased, the decline of S/N ratio was observed in
all tissues, especially in the young leaves (Table 2). Fe concentration
did not signicantly change in the leaves, but its level decreased by
about 50% in the roots of S-decient plants (Table 2). Soluble protein
concentrations in the young and old leaves as well as in the roots of
S-decient bean plants was only about half of the value for the control plants (Table 2). Concentration of non-protein thiols was also
considerably decreased by S deprivation in both leaves and roots.
In the young leaves of S-decient bean plants thiol concentration
decreased by 43%, but in the old leaves only by 21%. Root thiols
content in S plants was lower by as much as 60% as compared to
control (Table 2).

3.3. Chloroplastic pigments

In the leaves of S-decient plants, chlorophyll a, chlorophyll
b and carotenoids were decreased signicantly. There was a
marked increase in the ratio of carotenoids/chlorophylls in Sdecient plants, while chlorophyll a/b ratio was not affected
(Table 3).

(mg g1 FW)

Leaves
S

Control
Chl a
Chl b
Car
Chl a/b ratio
Car/Chl ratio

3.85a
1.43a
0.54a
2.52a
0.10b

0.23
0.09
0.05
0.21
0.01

1.02b
0.46b
0.21b
2.22a
0.14a

0.13
0.03
0.01
0.18
0.02

1.98a 0.08
0.71a 0.01
2.1a 0.09
49.7 6
183a 10
0.040a
3.74a 0.67

0.84b 0.01
0.23b 0.01
1.1b 0.08
48.9 7
75b 9
0.017b
1.88b 0.44

3.4. Oxygen consumption and AOX capacity in isolated

mitochondria
In order to obtain an overview of the respiratory activity, we
measured the O2 consumption by mitochondria isolated from the
leaves or roots of bean plants. Percoll-puried mitochondria of sulphur sufcient plants had high respiration rates and ADP/O ratios
(Table 4). The respiratory rates of root mitochondria were always
higher than those of leaf mitochondria, and differences in oxygen
uptake between S and control leaf and root mitochondria were
similar and substrate-independent. The constitutive cultivation of
bean plants without sulphate in the growing medium resulted in a
decrease of the total respiration and in the ADP/O ratios when either
malate or succinate, but not NADH, were used as respiratory substrates (Table 4). Malate oxidation was lower in S leaf and root
mitochondria despite the NAD-malic enzyme activity being 40%
and 30% higher in S-decient than in control plants, respectively.
The inhibition of Complex I by rotenone during malate oxidation
indicates that internal NADH dehydrogenase (NDin ) activity was
doubled in both leaf and root mitochondria of S bean plant as
Table 4
Respiratory state 3 activities of mitochondria isolated from young leaves or roots
of 19-day-old bean control or sulphur-decient plants. Substrate concentrations
were 10 mM malate with 2 mM glutamate and 0.5 mM NAD+ , 20 mM succinate
with 0.2 mM ATP, 1 mM NADH and 0.08 mM ADP. Complex I was inhibited by 1 M
rotenone. The capacities (maximum activities in vitro) of AOX (nmol O2 mg1 protein min1 ), COX (nmol cyt c mg1 protein min1 ) and NAD-malic enzyme (nmol
NADH mg1 protein min1 ) were determined as described in Section 2. AOX capacity was measured in the presence of 0.8 mM KCN, 1 mM pyruvate and 10 mM DTT
and was inhibited by 0.75 mM SHAM. Depending on the substrate, 50100 g of
mitochondrial protein was used for the respiratory measurements. Data are presented as mean values SD and are averages from 3 mitochondrial isolations. *In
AOX-activated conditions. Statistically signicant differences at ab P < 0.05. ND, non
detected.
Leaves

Roots
S

Control
Table 3
Chlorophyll a (Chl a), chlorophyll b (Chl b) and carotenoids (Car) concentrations,
chlorophyll a/b (Chl a/b) and carotenoids/chlorophyll (Car/Chl) ratios in the young
leaves of 19-day-old bean plants grown in full Knop nutrient medium (Control) or
without sulphate (S). Means of 69 replicates with SD as indicated. Data values for
same parameters having different superscript letters are statistically signicant at
ab
P < 0.05.

Control

Oxygen consumption (nmol O2 mg

Total respiration
199a 6
Malate
56b 5
Malate + rotenone
Succinate
315a 22
253 27
NADH
ADP/O
Malate
Succinate
NADH
AOX*
Malate
Succinate
NADH
COX
NAD-malic enzyme

2.3a 0.4
1.5a 0.2
1.2 0.2
49
ND
136
497a
83a

8
10
37
12

Control

protein min
b

73
123a
153b
228

4
10
34
35

2.0b 0.5
1.3b 0.3
1.1 0.2
43
ND
128
343b
50b

7
9
26
9

)
269a
67b
385a
323

18
6
51
32

2.4a 0.1
1.6a 0.1
1.5 0.2
117
167
206
697a
67a

13
16
13
61
8

139b
145a
217b
293

11
9
49
34

2.0b 0.1
1.3b 0.1
1.5 0.4
120
152
224
585b
45b

14
11
19
54
6

250

I.M. Juszczuk, M. Ostaszewska / Environmental and Experimental Botany 74 (2011) 245254

in S-decient plants. In root extracts of sulphur-decient plants,

the level of ATP decreased by similar amount as in the leaves, but
no signicant differences in ADP levels were observed between the
groups. However, root tissue ATP/ADP ratio and the total adenylate
pool (ATP + ADP) were lower by about 20% and 30%, respectively,
in S than in control plants (Fig. 3B).
3.6. Pyridine nucleotide status

Fig. 2. Immunodetection of AOX and VDAC proteins in mitochondria isolated from

the leaves and roots of 19-day-old control and sulphur-decient (S) bean plants.
Mitochondrial proteins (10 g) were separated by SDSPAGE and immunoblotted
with specic monoclonal antibodies combined with a chemiluminescence detection system (as described in Section 2). Values of AOX protein abundance, indicated
above the protein bands, were obtained by densitometry of AOX vs. VDAC bands
of the Western blots. Molecular mass (kDa) of proteins are indicated on the left.
Representative results are shown.

compared to control (Table 4). When NADH was used as a respiratory substrate, the intensity of its oxidation did not differ in leaf
and root mitochondria isolated from S and control bean plants
(Table 4). This indicates that S deciency does not inuence the
activity of the external NADH dehydrogenase (NDex ).
In the presence of KCN there were no changes in substrate oxidation in leaf and root mitochondria of S and control plants. In
control plants, the level of AOX capacity amounted to about 2565%
of the total leaf or root mitochondrial respiration, depending on the
substrate. In S-decient leaf or root mitochondria, the AOX capacity
did not change but amounted to about 6086% of the total respiration determined in mitochondria of S-decient plants (Table 4).
The similar AOX capacity observed under sulphur deprivation was
accompanied by almost equal abundance of AOX protein, relative
to the values obtained prior to stress (Fig. 2). COX capacities were
always lower by about 30% in S leaf and root mitochondria as
compared to the unstressed control plants (Table 4).
3.5. Leaf and root ATP and ADP concentration
ATP and ADP concentration was measured in plant extracts and
expressed per FW. Leaf ATP concentration during the light period
was about 30% lower in S than in control plants (Fig. 3A). In leaf
extracts, ADP concentration was higher in S-decient plants by
about 40% than in S-sufcient plants, which may indicate lowered
phosphorylation efciency. These changes inuenced the total
adenylate content (ATP + ADP), which was signicantly lower in S
plants as compared to control (Fig. 3). Leaf ATP/ADP ratio during the
light period decreased from 3.5 0.32 in unstressed to 1.83 0.19

In bean leaf and root tissue extracts prepared from plants after
45 h of the light period, the total pyridine nucleotide concentrations were changed in S plants as compared to control and
there were differences between the pools of specic nucleotides
(Fig. 4AD). In the leaves of S-decient plants, the concentration of total NAD(H) was lower due to a marked decrease in
NAD, whereas NADH level increased (Fig. 4A). In the roots of S
plants the total NAD(H) was higher, mainly due to an increase in
NADH (Fig. 4B). The changes in the levels of NADH and NAD led
to the signicant increase in NADH/NAD ratios, by about 50% in
the leaves and roots (Fig. 4A and B). NADP(H) pool in the leaves
of S plants increased by over 50% as compared to the control,
mainly due to the increase in NADP resulting in two times lower
ratio of NADPH/NADP than in control plants (Fig. 4C). Opposite
to the leaves, in the roots there was a decrease in total NADP(H)
because of the decrease in NADP concentration and this reected
almost twice as higher NADPH/NADP ratio as compared to control
(Fig. 4D).
4. Discussion
Our analysis of bean plants under sulphur deciency stress
provided one of the rst studies, in which mitochondrial respiration, energy, and redox status have been characterized.
As sulphur-related metabolites represent an integral part of
plant metabolism with multiple interactions, sulphur deciency
induces a number of adaptive responses, which must be coordinated. Plant mitochondria, while exibly regulating redox
homeostasis in the cell, control the overall cellular metabolism,
which thereby might effectively adapt to the variable environmental cues (Rhoads and Subbaiah, 2007; Foyer and Noctor,
2009).
4.1. Phenotypic, growth and biochemical prole of
sulphur-starved bean plants
Bean plants subjected to constitutive sulphur deciency present
well-known specic morphological and biochemical symptoms of

Fig. 3. ATP and ADP concentrations in the leaves (A) and roots (B) of 19-day-old control and S-decient bean plants. Means from 3 to 9 replicates SD. Statistically signicant
differences at ab,cd P < 0.05.

I.M. Juszczuk, M. Ostaszewska / Environmental and Experimental Botany 74 (2011) 245254

251

Fig. 4. Pyridine nucleotide concentrations in the leaves (A and C) and roots (B and D) of 19-day-old control and S-decient bean plants. NAD(P)H/NAD(P) ratios are given in
the tables as insets to the graphs. Values are means from 3 to 6 replicates SD as indicated on the graphs. In the tables all SD values do not exceed 2%. Statistically signicant
differences at ab,cd P < 0.05.

S deciency. Among these symptoms, pale and unevenly expanded

youngest leaves with bleached and/or chlorotic and sometimes
even necrotic spots as well as prominent wrinkles in the lamina
tissues and curling of the leaf margins (Fig. 1), were particularly typical. In S-decient bean plants, severe decrease of total sulphur and
sulphate concentrations in young than in old leaf tissue (Table 2)
further supports that the symptoms resulted from the deciency
of this macronutrient. Moreover, chlorotic and necrotic spots were
analysed by microscopy observations and veried for microbiological contaminations or insect activity (data not shown). These results
indicate that chlorosis and/or necroses on the leaves appear primarily due to S deciency. The old leaves of bean plants were almost
morphologically unchanged by S deciency (Fig. 1). A poor mobility of S from older to young parts of a developing plant has been
often observed in sulphur-starved plants (Hawkesford and De Kok,
2006). Indeed, a relatively higher severity of S deciency effects in
the young leaves have been previously reported in stressed maize
(Astol et al., 2003), barley (Astol et al., 2006), tomato (Zuchi et al.,
2009) and mulberry (Tewari et al., 2010) plants.
The pale appearance of young leaves in S-decient bean plants
was a consequence of the lower concentration of chlorophylls a
and b in the leaf tissues (Table 3). Since S is an integral constituent of two S-rich amino acids, cysteine and methionine,
which both act as structural and functional elements of proteins
(Droux, 2004), the decline of chloroplastic pigments in the young
leaves may be the result of an overall decrease of biosynthesis of
chloroplast-targeted proteins. A lower soluble protein concentration was detected in the leaves of examined bean plants (Table 2),

but also in the other plant species subjected to lack of sulphur or

low S supply (Nikiforova et al., 2005; Tewari et al., 2010). Moreover, an important methionine derivative, S-adenosylomethionine
(SAM) acts as a cofactor during chlorophyll biosynthesis (Roje,
2006) and its availability may limit this process under S decient conditions as it was observed in Arabidopsis (Nikiforova et al.,
2005). Depressed chlorophyll biosynthesis was accompanied by the
degradation of the chloroplast structure in the leaves of S bean
plants (data not shown). This observation suggests that the photosynthetic apparatus in S-decient bean plants can be disrupted.
All of the above data combined with the lower cellular NADPH
level (Fig. 4C) and dry matter content (Table 2) in leaf tissues of S
bean plants may indicate disturbances in photosynthetic electron
transport chain reactions and CO2 xation. In S-decient Arabidopsis, a lower biomass production, protein concentration, and
chlorophylls content were found together with affected photosynthesis (Wulff-Zottele et al., 2010), particularly with the
perturbations in the expression of genes encoding enzymes of the
Calvin cycle (Nikiforova et al., 2005). Lower S supply decreased photosynthesis in tomato leaves due to signicantly declined protein
and chlorophyll content (Xu et al., 1996).
Bleaching of S bean leaves as well as the presence of the
chlorotic and necrotic spots and degraded chloroplasts might also
represent symptoms of oxidative stress. It is well documented
that S deciency results in lower concentrations of glutathione
(Nikiforova et al., 2005 and references therein), one of the most
important non-enzymatic antioxidant (Rouhier et al., 2008). A signicant increase of carotenoids/chlorophylls ratio in S-decient

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I.M. Juszczuk, M. Ostaszewska / Environmental and Experimental Botany 74 (2011) 245254

bean plants may be an adaptation to the oxidative stress. Similar

increase of carotenoids/chlorophylls ratio was noted in mulberry
plants as a response to imbalanced H2 O2 concentration in leaf tissues under S deciency (Tewari et al., 2010).
The major part of sulphate and nitrogen taken up by the roots
is incorporated into amino acids and/or used for protein biosynthesis, thus the ow of these two macronutrients must be strictly
coordinated and balanced (Carfagna et al., 2011 and references
therein). Sulphur deciency in plant tissues resulting in concomitant impairment of protein biosynthesis leads to an excess of
reduced N (Prosser et al., 2001; Nikiforova et al., 2006). The signicant decrease of S/N ratio in S bean plant tissues reects only
the lower S concentration in the leaf and root bean tissues but
not increased N uptake since N level did not change (Table 2).
This indicates that the equilibrium of S/N in bean plants under
sulphur deciency is shifted and N tends to accumulate. Low S/N
ratio might be accountable for the negative effect of S deciency on
photosynthesis and growth of bean plants. The S/N imbalance has
been detected in tomato leaves (Xu et al., 1996), sugar beet shoots
(Thomas et al., 2000), spinach leaves (Prosser et al., 2001) and Arabidopsis leaves (Nikiforova et al., 2003, 2005) under S deciency.
S deciency resulted in a signicant decrease of shoot growth
and dry matter production and thus shoot/root ratio of bean plants
(Table 1). Plants require energy and sugars for the growth and
development. We did not estimate sugar content in the tissues of
sulphur-decient bean plants, but ATP level decreased much more
severely in leaf (48%) than in root tissue (20%) (Fig. 3), which
may indicate decreased photosynthesis. Then, lower availability of
energy in the leaf tissue of S bean plants might restrict shoot
growth in comparison to roots, where ATP concentration probably covered the energy demand for similar growth as in control
plants. To our best knowledge, ATP concentration has never been
measured in the leaves of any S-decient plants, but effects of the
decline in ATP level on root growth were observed in S-decient
broad beans, pea and alfalfa plants (Pacyna et al., 2006; Scherer
et al., 2008). In the roots of S-decient Vicia faba, ATP concentration decreased, while the inuence of S supply on ADP and AMP
levels was less pronounced (Pacyna et al., 2006). In S-decient bean
root cells ATP can be used extensively in sulphate uptake from the
soil by ATP-dependent transporters. The level of photosynthetically
produced pyridine nucleotides can limit shoot growth of S bean
plants since NADPH/NADP ratio in S-decient bean leaf tissue was
only half of the value for control plants (Fig. 4C). The concentrations
of NADPH, a reducing power produced during the light-dependent
phase of photosynthesis, decreased in S-starved leaves (Fig. 4C),
so NADPH supply to the Calvin cycle and sugar biosynthesis was
potentially lower as compared to control.
The biomass production decreased in S bean leaf and root
tissues but we did not observe changes in weight water content
(WWC) as compared to control plants (Table 1). This is in contrary to what was previously observed in tomato (Xu et al., 1996)
and mulberry (Tewari et al., 2010); in those plants water content
increased under S deciency. Complex changes of water relationships are species-specic in plant tissues under stress conditions
and this might explain the different WWC of the bean vs. other
plants subjected to S deciency.
4.2. Shift in mitochondrial function and energy status
In S-decient bean plants, mitochondrial function changed due
to the lower activity of Complex I as compared to control (Table 4).
Sulphur, forming clusters with Fe in Complex I subunits (Lin et al.,
1995), takes part in the input of electrons to the mitochondrial respiratory chain. Since Fe concentration did not change in the leaves
of S-decient bean plants, we suppose that it is not Fe but rather S
availability which limits the formation of Complex I FeS clusters

and therefore may affect Complex I activity (Table 4). However,

in the roots of S bean plants, the level of Fe severely decreased
(Table 2) and thus lower content of both Fe and S (Table 2) may be
associated with impairment of Complex I (Table 4). Iron deciency
in sycamore cells caused large decrease of some of FeS mitochondrial clusters, but the activity of Complex I remained unaffected
(Pascal and Douce, 1993). By contrast, activities of all respiratory
chain complexes were depressed in cucumber roots under Fe deciency (Vigani et al., 2009).
We analysed the activity of NADH dehydrogenases type II, that
are known to partially compensate for Complex I function in stress
conditions, allowing for operation of the Krebs cycle and avoiding
overreduction of cytosol and mitochondria (Escobar et al., 2006;
Rasmusson et al., 2008). External NADH dehydrogenase (NDex )
activity remained unchanged when the capacity of Complex I was
decreased in mitochondria isolated from the leaves and roots of S
bean plants (Table 4). While the NDex are able to directly oxidise
NAD(P)H from the cytosol, the NADH oxidation by internal NADH
dehydrogenase (NDin ) is regulated by the concentration of NADH in
the mitochondrial matrix (Mller and Palmer, 1982). Higher NADH
level is required for operation of alternative NADH dehydrogenases
because they have lower afnity for NADH than Complex I (Mller,
2001). Since Complex I activity was lower in the leaves and roots of
S plants (Table 4), NADH level probably tends to accumulate in the
mitochondrial matrix, inducing NDin to perform NADH oxidation.
Indeed, we observed increased malate oxidation in the presence
of Complex I inhibitor, rotenone, in S-decient bean mitochondria (Table 4). Both NDin and NDex do not contain FeS clusters
(Rasmusson et al., 2008) and less S ions are required (as a part of
cysteine and methionine residues) for NDin and NDex assembly as
compared to Complex I. Since alternative NDin and NDex belong to
stress-activated enzymes (Escobar et al., 2006; Rasmusson et al.,
2008), their biosynthesis under S deciency can be preferentially
sustained by low S availability. In Fe-decient cucumber mitochondria, NDin and NDex were strongly induced when Complex I activity
was limited by the availability of Fe ions (Vigani et al., 2009; Vigani
and Zocchi, 2010).
In S-decient bean plants Complex I was not completely inhibited and can still partially contribute to oxygen consumption and
ATP synthesis, but energy status decreased as reected by ATP level
in leaf and root S bean tissues (Fig. 3). In vitro measurements of
the respiratory activity of mitochondria isolated from S-decient
plants indicated lower capacity for ATP generation (ADP/O ratio)
with malate being a substrate for Complex I (Table 4). Oxidation
of the substrates by Complex II, NDex or NDin did not show the
changes in ADP/O ratios between S-decient and control mitochondria (Table 4). In phosphate-decient bean root mitochondria the
efciency of ATP production was always lower and substrate independent (Juszczuk et al., 2001). In S bean leaf and root extracts, the
total adenylate pool was lower than in control plants (Fig. 3). This
is probably due to the disturbances in photosynthetic apparatus in
the leaves (Fig. 1, Table 2) and decreased Complex I activity (Table 4)
in both leaves and roots of S-decient bean plants. Lower total
adenylate level and ATP concentration was observed in the roots
of phosphate decient bean plants (Rychter et al., 1992) having
decreased mitochondrial respiration (Juszczuk et al., 2001).
Cytochrome c oxidase activity was always lower in S-decient
bean plants (Table 4). The similar effect was observed in bean
root mitochondria of plants subjected to phosphate deciency. In
those plants alternative oxidase (AOX) protein level and capacity
increased (Juszczuk et al., 2001). It is intriguing that sulphur deprivation, accompanied by sulphur deciency in bean plant tissues,
changed neither AOX activity measured in vitro (capacity) as CNresistant respiration (Table 4), nor AOX protein level (Fig. 2). AOX
activity measured in vivo (Guy et al., 1989) is relatively independent of AOX protein levels and capacity. Therefore it is impossible

I.M. Juszczuk, M. Ostaszewska / Environmental and Experimental Botany 74 (2011) 245254

253

to conclude from AOX capacity about actual participation of AOX in

respiration (Florez-Sarasa et al., 2009). Under several stress conditions AOX protein exists in surplus (Rasmusson et al., 2009). We
think that the unchanged capacity and protein level of AOX in
sulphur-decient bean plants as compared to the control should be
considered in the terms of predicted role of AOX. Sulphur, being part
of two conserved cysteine residues in the catalytic center of alternative oxidase, is essential for the function of the enzyme (Crichton
et al., 2010). In S-decient bean plants, sulphur can be preferentially
directed into AOX biosynthesis since AOX, together with NAD(P)H
dehydrogenases type II, may act against the overreduction of the
electron transport chain (Rasmusson et al., 2008, 2009). Alternative respiratory pathways can minimize oxidative challenge and
protect S-decient bean plants from oxidative stress. Non-protein
thiol content was lower in S-decient bean plants as compared to
control (Table 2). This may indicate a shift in antioxidative defence
system in S-decient bean plants. S starvation results in oxidative
stress gene inductions in Arabidopsis (Nikiforova et al., 2003) and

tobacco (Wawrzynska
et al., 2005) or oxidative stress symptoms in
mulberry (Tewari et al., 2010) plants.

Rychter, 1997) as a result of a lower demand for reduced equivalents due to the great decrease of respiration (Rychter and Mikulska,
1990) and ATP level (Rychter et al., 1992).

4.3. Changes in redox homeostasis in response to sulphur

deciency

Acknowledgements

Pyridine nucleotides, NAD(P)H, need to be re-oxidized to

allow functioning of different metabolic pathways. The increased
NADH/NAD ratios in S bean tissues as compared to control
(Fig. 4 A and B), partially reect the mitochondrial function since
these organelles mainly consume reducing power as NADH. The
lower activity of Complex I in S bean plants (Table 4) can lead
to overreduction of NADH pool in mitochondria. Higher activity
of NAD-malic enzyme also indicates increased concentrations of
mitochondrial NADH as a substrate for higher activity of NDin
(Table 4). We found that in leaf and root extracts of S-decient
bean plants, total NADH concentration increased (Fig. 4A and B), but
NADH comes both from the mitochondrial and cytosolic reactions.
In S-decient bean plants, NADH of mitochondrial and/or cytosolic
origin can be effectively oxidized both by NDin and NDex (Table 4).
The cellular NADPH/NADP ratios decreased in the leaf but
increased in the root extracts of S-decient bean plants. The concentrations of NADP mainly inuence this state since NADP level
increased in the leaves but decreased in the roots (Fig. 4C and
D). We did not measure NADPH level produced by S-decient
bean chloroplasts, and the lower leaf cellular NADPH/NADP ratio
in fact resulted from the combination of redox processes in mitochondria, chloroplasts and cytosol. Since S deciency promotes
oxidative stress symptoms in plant tissues (Tewari et al., 2010),
NADPH could be consumed by NADPH-oxidase, which generates
superoxide anion (Torres and Dangl, 2005) and whose activity
increases under stress conditions (Torres, 2010). Mitochondrial
external NADPH and NADH dehydrogenases both consume NADPH,
also that of chloroplastic origin (Rasmusson et al., 2008). Finally, the
processes consuming NADPH may exceed NADP reduction by the
photosynthetic electron carriers. It is unclear how such a decrease
of NADP concentration is coupled to S deciency in bean roots.
Restricted availability of ATP for ATP-dependent phosphorylating
step of NADP biosynthesis cannot account for this, since in the
leaves NADP formation is enhanced (Fig. 4C), and ATP decline is
even more profound (Fig. 3A). A more likely explanation is the
decreased NAD kinase activity in S-decient bean roots, which
was 320 nmol NADP mg1 protein h1 as compared to 980 nmol
NADP mg1 protein h1 in control plants, whereas in the leaves
it remained unchanged at the level of 950 nmol NADP mg1 protein h1 . In bean plants subjected to another mineral deciency,
that of inorganic phosphate, both NADH/NAD and NADPH/NADP
ratios severely increased in the leaves and roots (Juszczuk and

5. Conclusion
Our results conrm that the availability of S determines mitochondrial respiration and changes cellular energy and redox
homeostasis. We found that bean mitochondria respond to S deciency in the tissues decreasing the efciency of ATP production
as the result of lower Complex I activity. A shift in mitochondrial
functioning allows the oxidation of nucleotides due to increased
activity of additional NDin . Such strategy is extensively observed
in plant species (summarized in Rasmusson et al., 2008) that can
survive stress conditions even if they become smaller, weaker and
give lower yield of biomass from the agricultural point of view. In
this study we prove that exible function of mitochondrial respiratory chain could be an important target during adaptations to S
deciency.

The authors wish to thank Prof. Anna M. Rychter and Dr. Izabella
Koodziejek (University of Warsaw, Faculty of Biology, Institute of
Experimental Plant Biology and Biotechnology) for critical reading of the manuscript; Dr. Wiesaw Szulc (Warsaw University of
Life Sciences, Laboratory of Forest Environment Chemistry of Forest Research Institute, Warsaw, Poland) for sulphur and sulphate
concentration assays; Dr. Anna Barabasz and Anna Wilkowska for
(Unihelp in iron concentration measurements and Maria Waeza
versity of Warsaw, Faculty of Biology, Institute of Experimental
Plant Biology and Biotechnology) for microscopy observations and
remarks.
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