Annals of RSCB
Vol. XV, Issue 1
PHOTOSYNTHESIS AND WATER RELATIONS OF LEAF CELLS
EXPOSED TO SALT STRESS
Cs. Bartha, L. Fodorpataki, Erika Nagy, Zs. Gy. Keresztes,
Gyöngyi Székely, O. Popescu
”BABES-BOLYAI” UNIVERSITY, CLUJ-NAPOCA
Summary
Stress tolerance of plants relies on molecular processes that occur at the cellular level and
are integrated by the complex regulatory network of the multicellular organisms. One of the
mostly widespread environmental stress that impairs the metabolism, development and
productivity of many crop plants is generated by elevated concentrations of sodium chloride in
the soil solution that surrounds the root system. The main objective of the present study is to
investigate physiological and biochemical changes induced in leaf cells of different lettuce
varieties, in order to identify the sources of differential salt tolerance and to perform a selection
of varieties able to survive in the presence of salt stress. The physiological and biochemical
markers used by us to reveal different aspects of salt stress tolerance of lettuce varieties were
energetic efficiency of photochemical reactions in chloroplasts, changes is chlorophylls and
carotenoid pigment content of thylakoid membranes, cell membrane damage due to
peroxidation of unsaturated fatty acids in membrane lipids, the rate of carbon dioxide
assimilation by mesophyll cells, stomatal conductivity during leaf gas exchange, and free
proline content of leaf cells performing osmoregulation. From among the many parameters of
the induced chlorophyll fluorescence related to quantum efficiency of photosynthesis, the
vitality index or relative fluorescence decay (Rfd) was the most sensitive one. The timeresolved dynamics of stomatal conductance and net carbon dioxide assimilation showed that
osmotic stress induced by high salt concentrations is more severe during the first hours after
exposure to salt stress, and the velocity and degree of recovery allows a fine selection of the
more tolerant lettuce varieties. Membrane damage by lipid peroxidation is enhanced only by
high salt concentrations (100 mM) and seems to be proportional with the salt sensitivity of
different lettuce cultivars. Free proline content of young leaf cells increases very much under
salt stress, and the highest concentrations of this osmolyte can be found in cold-sensitive
varieties.
Keywords: chlorophyll fluorescence, membrane lipid peroxidation, photosynthesis, proline,
salt stress, stomatal conductance, vitality index
[email protected]
accumulation or exclusion of Na+ and Clions; control of ion uptake by roots and
transport into leaves (Demidchik and
Maathuis, 2007); compartmentalization of
sodium and chloride ions; synthesis of
compatible solutes; changes in photosynthesis and gas exchange; alteration in
membrane
structure;
induction
of
antioxidative enzymes, other protective
proteins and plant growth regulators. Partial
stomatal closure under high salinity is
induced by the presence of sodium ions in
the apoplast surrounding the guard cells,
causing a reduction in rates of transpiration
Introduction
Salinity is a major environmental
factor limiting plant growth and
productivity. Nearly 20% of the world’s
cultivated area and nearly half of the
world’s irrigated lands are affected by high
salinity (Munns and Tester, 2008). Many
plants develop mechanisms either to
exclude salt from their cells (or at least to
minimize the entry of the salt in
photosynthetic tissues, especially in the
cytoplasm of cells) or to tolerate its
presence within the cells. Mechanisms of
salt
tolerance
include:
selective
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Annals of RSCB
Vol. XV, Issue 1
tolerance or sensitivity that enables
selection of the most suitable varieties for
cultivation on soils with enhanced salinity.
and increase of water use efficiency (Rios
et al., 2008; Fodorpataki and Szigyarto,
2009; Plett et al., 2010) .
Exposure to long-term salt stress
leads to a decreased activity of
antioxidative enzymes, associated with
increased sensitivity. The up-regulation of
antioxi-dant capacity is an early response to
salt stress, and the increased activity of
antioxidants in salt tolerant varieties in
response to salt stress suggests that
scavenging of reactive oxygen species is a
part of the general adaptive strategy of
plants exposed to salinity (Fodorpataki,
2008; Kohler et al., 2009; Oh et al., 2010).
Regarding
biotechnological
strategies to enhance plant salt tolerance,
much effort has been made in the field of
oxidative stress responses, regulation of ion
homeostasis, signalling pathways and
osmoprotectant synthesis. To achieve salt
tolerance, the task is either to prevent or
alleviate the damage, or to re-establish
homeostatic conditions in the new stressful
environment. Identification of molecular
markers linked to salinity tolerance traits
has provided plant breeders a new tool for
selecting varieties with improved tolerance
(Veeranagamallaiah et al., 2008; Ashraf
and Akram, 2009; Plett et al., 2010).
Lettuce is a moderately saltsensitive plant. Its fresh leaves are largely
used in the healthy human alimentation,
being a source of vitamins, mineral ions and
several secondary metabolites, such as
flavonoids. When plants are exposed to
high salinity, the polyphenol content and
the antioxidant properties of the leaves are
enhanced (Llorach et al., 2008; Monteiro et
al., 2007; Mulabagal et al., 2010).
Different environmental stresses
induce the biosynthesis of several healthpromoting phytochemicals (Altunkaya et
al., 2009; Hasaneen et al. 2009; Oh et al.,
2009).
The aim of this study is to reveal
physiological and biochemical changes in
leaf cells of different lettuce varieties
exposed to different degrees of salt stress,
in order to indicate differential salt
Materials and methods
Seven varieties of lettuce (Lactuva
sativa L.) were used: three early (cold
tolerant) cultivars (Arktic King, Parella
Green, Valdor), three late (cold sensitive
and heat tolerant) cultivars (Bowl Red, Oak
Leaf, Paris Island) and an amphitolerant
cultivar (Krolowa). All of these are widely
cultivated in European countries. Seeds
were provided by B & T World Seeds
(France). Seeds were germinated and
seedlings were grown in pots filled with
vermiculate, in a vegetation chamber
(Versatile Environmental Test Chamber),
under controlled condition: a daily
photoperiod with 12 hours of light and 12
hours of darkness, with a relative air
humidity of 75%, a photon flux density of
90 µM m-2s-1, and a temperature of 18°C
during the light period and 16°C during the
dark period. Control plants were supplied
with ¼ strength Hoagland nutrient solution,
while the experimental variants were
watered with identical amounts of ¼
Hoagland solution supplemented with 50
mM, 100 mM and 150 mM NaCl (p. a.). 3
weeks old plants were used for investigation of physiological and biochemical
reactions to salt stress. For gas exchange
measurements, plants were grown with ¼
Hoagland solution, and salt stress was
applied only once, before determinations.
Parameters of induced chlorophyll a
fluorescence were measured with a pulse
amplitude modulated chlorophyll fluorometer (PAM-FMS2, Hansatech). Leaves
were dark adapted for 5 min. The
modulated light was sufficiently weak (0.04
µM m-2s-1) so as not to produce any
significant variable fluorescence. A single
saturating flash (2000 µM m-2s-1 for 0.5 s)
was applied to reach the maximal
fluorescence Fm. After the decline of the
signal, the actinic light was turned on (100
µM m-2s-1) to start the induction kinetics.
The determined parameters were initial
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Annals of RSCB
Vol. XV, Issue 1
µM m-2s-1, air temperature in the leaf
cuvette was 26°C, reference carbon dioxide
concentration was 340 ppm, reference
relative
air
humidity
was
80%.
Measurements of transpiration rate, net
carbon dioxide assimilation, water pressure
deficit in the mesophyll, internal carbon
dioxide concentration of the leaf and
stomatal conductivity were determined 3
hours, 9 hours and 24 hours after the
treatment of 6 weeks old plants with 50
mM, 100 mM and 150 mM NaCl,
respectively (Pinheiro et al., 2008;
Monteiro et al., 2009).
Free proline content in the plant
material was determined by generation of a
coloured product with ninhidrine. 0.1 g of
fresh plant material was homogenized in a
pre-chilled mortar with 3% sulfosalicilic
acid cooled on ice (5 µl for every mg of
plant fresh weight). The extract was
centrifuged for 10 minutes at 20000 g, then
200 µl of 96% acetic acid and 200 µl
ninhidrine solution (containing 2.5% w/v
ninhidrine, 60% v/v 96% acetic acid and
40% v/v of 6 M ortophosphoric acid) was
added to each 100 µl of supernatant. The
samples were incubated in test tubes for 1
hour at 96°C, and after cooling, 1 ml of
toluene was added to extract the reaction
product. The mixtures were stirred, and
when two layers were separated, 2 ml of the
upper layer were transferred in a cuvette.
The concentration of the red product was
determined on the base of its absorbance at
520 nm using toluene as reference. The
praline content was determined using a
calibration curve (Valerio et al., 2007; Li et
al. 2010).
Each determination was made with
4 replicates, then the means and standard
errors were calculated. In data sets with
parametric distribution, significant differrences between treatment means were
determined using the post-ANOVA Fisher
LSD test, while in data sets with nonparametric distribution, significant differences between means were established
with the Kruskal-Wallis test followed by
the Mann-Whitney U-test.
fluorescence F0, maximal fluorescence Fm,
the Fv/Fm ratio (Fv or variable fluorescence being the difference between the
maximal and the initial fluorescence), the
F0/Fv ratio, modulated maximal fluorescence Fm’, steady state fluorescence Fs, the
effective quantum use efficiency (Φ)
representing the ratio (Fm’–Fs)/Fm’, as
well as the vitality index (Rfd) expressed as
the ratio (Fm–Fs)/Fs (Fodorpataki and
Bartha, 2008; Zribi et al., 2009).
Extraction
of
photosynthetic
pigments was performed in darkness with
N,N-dimethyl-formamide. 0.1 g leaf was
immersed
in
4
ml
of
N,Ndimethylformamide and kept for 48 hours
for complete extraction of pigments. The
extracts were centrifuged for 5 min. at 7000
g, and the supernatant was used for
measurement of optical density at 480 nm,
646.8 nm and 663.8 nm. Chlorophyll a,
chlorophyll b and carote-noid pigments
were determined spectro-photometrically
(Pinheiro et al., 2008).
For
determination
of
lipid
peroxidation, 0.2 g of fresh plant material
was ground in a pre-chilled mortar with 2
ml of 10% (w/v) trichloroacetic acid,
containing 0.25% (w/v) 2-thiobarbituric
acid. The homogenate was transferred in a
test tube heated for 45 minutes at 96 ºC,
then cooled quickly in an ice-bath and
centrifuged for 15 minutes at 12000 g and
10ºC. Determination of lipid peroxidation
products designated as thiobarbituric acidreactive substances (consisting mostly in
malondialdehyde) was performed photometrically on the supernatant, by measuring
the absorbance at 532 nm and 600 nm, and
substracting A600 from A532. For calculation
of the concentration of lipid peroxidation
products, the extinction coefficient of 155
mM-1 cm-1 was used (Eraslan et al., 2007;
Li et al., 2010).
Gas exchange parameters of the
fifth youngest leaves of 6 weeks old lettuce
plantlets were measured with a Ciras-2 leaf
gas exchange system (PP Systems),
provided with a PLC6 automatic leaf
cuvette. Photon flux density was set to 500
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Annals of RSCB
Vol. XV, Issue 1
fluorescence were moderately diminished
(Zribi et al., 2009).
Net photosynthetic carbon dioxide
assimilation rate decreases especially in the
first 3 hours after exposure to salt stress,
and this inhibition is proportional with the
salt concentration in all of the investigated
cultivars. While in cold-tolerant cultivars
(e. g. Parella Green, Fig. 2A) a new steady
state level was established 9 and 12 hours
after treatment, in cold-sensitive varieties
(e. g. Bowl Red, Fig. 2B) this rate
continued to decrease between 9 and 12
hours after initiation of salt stress with 100
mM and 150 mM NaCl.
Results and discussion
In vivo induced chlorophyll
fluorescence is a sensitive, non-destructive
tool for the study of environmental impacts
on the primary energy-conversion processes
of photosynthesis, on which the entire
primary biomass production relies. From
the different parameters of induced, nonmodulated
and
pulse
amplification
modulated chlorophyll fluorescence, was
the relative fluorescence decrease (Rfd),
also known as the vitality index (Fig. 1).
10
*
-1
*
*
Ø
*
4
50 mM NaCl
100 mM NaCl
150 mM NaCl
*
2
*
0
Parella Green
12
A
-2
6
Net photosynthetic CO2 assimilation (µM m s )
Vitality index (Fm-Fs)/Fs
8
Paris Island
Arktic King
Oak Leaf
-2
8
Ø
50 mM NaCl
6
100 mM NaCl
150 mM NaCl
4
2
0
3 hours
9 hours
24 hours
8
B
-2
-1
Net photosynthetic CO2 assimilation (µM m s )
Fig. 1. Vitality index deduced from chlorophyll
fluorescence parameters in leaves of 4 varieties
of Lactuca sativa exposed to salt stress. Bars
represent standard errors from means (n = 4),
asterisk indicates significant differences from
control (P < 0.05)
10
The value of this index depends on
the difference between the temporary
maximal fluorescence yield in dark-adapted
samples (Fm) and the steady state
chlorophyll fluorescence level (Fs) in
illuminated leaf cells. A reasonable
explanation for this is that the steady state
fluorescence level of the modulated
chlorophyll fluorescence exhibits the widest
range of changes upon salt stress
conditions, as compared to the other
measured parameters of fluores-cence.
Vitality index is not significantly
decreased by 50 mM NaCl, but it is
strongly diminished by 150 mM NaCl,
especially in the cold-sensitive Oak Leaf
and Paris Island cultivars. At similar salt
concentrations, in tomato leaves the
parameters of non-modulated chlorophyll
fluorescence remained relatively unaffected, but the parameters of modulated
7
6
5
Ø
50 mM NaCl
4
100 mM NaCl
150 mM NaCl
3
2
1
0
3 hours
9 hours
24 hours
Fig. 2. Dynamics of net carbon dioxide
assimilation rate in the leaves of two lettuce
cultivars (A: Parella Green, B: Bowl Red)
exposed to short-term salt stress. Bars represent
standard errors from means (n = 4)
The photosynthetic pigment content
of chloroplasts may indicate stress
conditions induced by sodium chloride,
which inhibits protein synthesis, thus
causing a decrease in the amount of several
enzymes involved in chlorophyll synthesis
and in the mevalonic acid pathway
(Pinheiro et al., 2008). In some lettuce
cultivars (e. g. Krolowa) salt stress inhibits
in the same degree the accumulation of
chlorophylls and carotenoids in the
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Annals of RSCB
Vol. XV, Issue 1
increase lipid peroxidation in any of the
studied lettuce cultivars. At 100 mM NaCl,
some cultivars did not exhibit enhanced
membrane damage, while in others the
degree of lipid peroxidation increased
significantly (Fig. 4).
thylakoid memb-ranes, while in other
varieties chlorophyll content exhibits a
more pronounced decrement, resulting in a
lowered chloro-phylls to carotenoids ratio
in the leaves of salt-stressed plants (Fig. 3).
This ratio may be also influenced by
the generation of extra amounts of reactive
oxygen species in photosynthesizing cells,
which induces accumulation of protective
carotenoids during acclimation of plants to
salt stress (Li et al., 2010).
-1
Lipid peroxidation (MDA in µg g FW)
25
Chlorophylls to Carotenoids molar ratio
5,4
5,2
5,0
4,8
*
*
*
20
*
15
Ø
50 mM NaCl
100 mM NaCl
10
*
5
0
*
Valdor
Ø
4,6
Bowl Red
Krolowa
50 mM NaCl
4,4
Fig. 4. Membrane lipid peroxidation in cells of
3 lettuce cultivars exposed to salt stress (MDA
– malondialdehyde). Bars represent standard
errors from means (n = 4), asterisk indicates
significant differences from control (P < 0.05)
100 mM NaCl
4,2
4,0
3,8
3,6
Valdor
Bowl Red
Krolowa
Fig. 3. Chlorophylls to carotenoids ratio in the
chloroplasts of leaves of 3 lettuce cultivars
exposed to long-term salt stress. Bars represent
standard errors from means (n = 4), asterisk
indicates significant differences from control (P
< 0.05)
Because the osmotic component of
salt stress induces a quick depletion of
water potential in mesophyll cells, one of
the first physiological reactions that enable
salt tolerance is the closure of stomata,
induced by accumulation of abscisic acid in
the guard cells of stomata of the
dehydrating leaves (Fodorpataki and
Szigyarto, 2009). This is the reason why
stomatal
conduc-tance
decreases
significantly during the first 9 hours after
exposure to salt stress. In most cases, this
decrement is followed by a partial recovery
after 9 hours of exposure to 50 mM NaCl.
Recovery is not possible under more severe
salt stress (Fig. 5). The adaptive
significance of this decreased stomatal
conductance is most probably the avoidance
of dehydration by transpiration.
The
negative side-effect of stomatal closure is
the impairment of carbon dioxide
acquisition for photosynthesis, resulting in
an inhibition of biomass production and
growth.
One of the first signs of oxidative
stress caused by increased salinity is peroxidation of unsaturated fatty acids in
membrane lipids, which results in damaged
membrane structure and in formation of
mobile peroxidation products (especially
malondialdehyde) that are very toxic and
amplify the oxidative damage. This is why
any increase in lipid peroxidation is an
early indicator of stress condition
associated with enhanced generation of
reactive
oxygen
species
(hydrogen
peroxide, superoxide and hydroxyl radicals,
singlet oxygen). Because many stress
factors initiate the overproduction of
reactive oxygen species (especially during
the photochemical reactions when absorbed
light energy saturates the photosynthetic
capacity), lipid peroxida-tion is a general
consequence of metabolic disturbances
(Eraslan et al., 2007; Kohler et al., 2009).
Mild salt stress (50 mM NaCl) did not
215
Annals of RSCB
Vol. XV, Issue 1
10000
Free proline content (in % of control)
A
300
-2
-1
Stomatal conductance (mM m s )
350
250
Ø
200
50 mM NaCl
100 mM NaCl
150
150 mM NaCl
100
50
3 hours
9 hours
7000
*
Ø
6000
50 mM NaCl
5000
100 mM NaCl
4000
150 mM NaCl
*
3000
*
2000
1000
*
*
Parella
Green
24 hours
250
*
*
*
*
*
200
150
Ø
50 mM NaCl
100 mM NaCl
150 mM NaCl
100
Paris Island
Arktic King
Oak Leaf
Fig. 6. Proline content of 4 lettuce varieties, as
a molecular marker of salt stress tolerance by
osmoregulation. Bars represent standard errors
from means (n = 4), asterisk indicates
significant differences from control (P < 0.05)
B
-1
-2
8000
0
0
Stomatal conductance (mM m s )
*
9000
50
Conclusions
0
3 hours
9 hours
Vitality index, determined from
parameters of induced, non-modulated and
pulse amplitude modulated chlorophyll
fluores-cence, can be considered a sensitive
indicator of salt sensitivity of lettuce
varieties. This index decreases drastically
especially in the cold sensitive cultivars (e.
g. Paris Island and Oak Leaf), but only
under severe salt stress exerted by 150 mM
NaCl.
Free proline content of cells is a
very useful molecular marker of the
capacity of different lettuce cultivars to
cope with osmotic stress generated by
increased salinity. Proline content increases
especi-ally in the heat tolerant cultivars, and
it seems to be proportional with the degree
of salt stress.
Membrane damage by lipid
peroxidation is generated only at
concentrations of sodium chloride that are
higher than 50 mM, and it occurs only in
the less tolerant cultivars.
Under salt stress, chlorophyll content of
leaf cells decreases in a higher extent than
the amount of carotenoid pigments, and this
results in a decreased chlorophylls to
carotenoids ratio.
Net photosynthetic carbon dioxide
assi-milation decreases mainly during the
first hours after salt treatment, and its
24 hours
Fig. 5. Dynamics of stomatal conductance in
the leaves of two lettuce cultivars (A: Parella
Green, B: Bowl Red) exposed to short-term salt
stress. Bars represent standard errors from
means (n = 4)
In relation to dehydration tolerance
at cellular level, an efficient osmoregulation
is of crucial importance. Free proline is the
most frequent compatible solute in vascular
plants, which accumulates in cells under
osmotic stress (Plett et al., 2010). The
amount of free proline increases
significantly in all of the studied lettuce
cultivars, but the proline concentration
varies in a wide range depending on lettuce
variety and on salt content. Proline accumulation is most pronounced in the late
cultivars (e. g. Paris Island, Oak Leaf),
which are generally more cold-sensitive
than the early ones (e. g. Parella Green,
Arktic King, Fig. 6). Differences
in
proline accumulation indicate different
degrees of tolerance of osmotic stress
induced by high salinity.
216
Annals of RSCB
Vol. XV, Issue 1
subsequent partial recovery occurs in a
higher extent in cold tolerant cultivars.
Recovery
of
the
stomatal
conductivity during the first day after
exposure to salt stress is possible only at
lower concent-rations of sodium chloride,
while severe salt stress impairs transpiration
and carbon dioxide acquisition for a longer
period of time.
Induced chlorophyll fluorescence
and gas exchange parameters are
physiological markers that can be used to
select more salt tolerant lettuce cultivars,
while free proline content and lipid
peroxidation
are
good
biochemical
indicators of salt sensitivity.
freshwater green alga and of duckweed exposed
to salinity and heavy metal stress. In:
Photosynthesis: Energy from the Sun, pp. 14511454, 2008. Edited by J. F. Allen, E. Gantt, J.
H. Golbeck and B. Osmond, Springer,
Dordrecht.
Fodorpataki L., Szigyártó L.: A Növények
Ökofiziológiájának Alapjai, Kriterion, ClujNapoca, pp. 347-364, 2009.
Hasaneen, M. N. A., Younis, M. E., Tourky,
S. M. N.: Salinity-biofertility interactive effects
on growth, carbohyd-rates and photosynthetic
efficiency of Lactuca sativa. Plant Omics J., 2,
60-69, 2009.
Kohler, J., Hernandez, J. A., Caracava, F.,
Roldan, A.: Induction of antioxidant enzymes is
involved in the greater effectiveness of a PGPR
versus AM fungi with respect to increasing the
tolerance of lettuce to severe salt stress.
Environ. Exp. Bot., 65, 245-252, 2009.
Li, G., Wan, S., Zhou, J., Yang, Z., Qin, P.:
Leaf chlorophyll fluorescence, hyper-spectral
reflectance, pigments content, malondialdehyde
and proline accumulation responses of castor
bean (Ricinus communis L.) seedlings to salt
stress levels. Ind. Crops Prod., 31, 13-19, 2010.
Llorach, R., Martinez-Sanchez, A., TomasBarberan, F. A., Gil, M. I., Ferreres, F.:
Characterization of polyphenols and antioxidant
properties of five lettuce varieties and escarole.
Food Chem., 108, 1028-1038, 2008.
Monteiro, M., Santos, C., Mann, R. M.,
Soares, A. M. V. M., Lopes, T.: Evaluation of
cadmium genotoxicity in Lactuca sativa L.
using nuclear microsatellites. Environ. Exp.
Bot., 60, 421-427, 2007.
Monteiro, M. S., Santos, C., Soares, A. M. V.
M., Mann, R. M.: Assessment of biomarkers of
cadmium stress in lettuce. Ecotox. Environ.
Safety, 72, 811-818, 2009.
Mulabagal, V., Ngouajio, M., Nair, A., Zhang,
Y., Gottumukkala, A. L., Nair, M. G.: In vitro
evaluation of red and green lettuce (Lactuca
sativa) for functional food properties. Food
Chem., 118, 300-306, 2010.
Munns, R., Tester, M.: Mechanisms of salinity
tolerance. Annu. Rev. Plant Biol., 59, 651-681,
2008.
Oh, M.-M., Carey, E. E., Rajashekar, C. B.:
Environmental
stresses
induce
healthpromoting phytochemicals in lettuce. Plant
Physiol. Biochem., 47, 578-583, 2009.
Oh, M.-M., Trick, H. N., Rajashekar, C.
B.:Secondary metabolism and antioxidants are
involved in environmental adaptation and stress
Acknowledgement
The authors wish to thank for the
financial support provided from programs
co-financed by The Sectorial Operational
Programme “Human Resources Development, Contract POSDRU 6/1.5/S/3 –
„Doctoral studies: through science towards
society".
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