Journal of Experimental Botany, Vol. 52, No. 355, pp. 351±360, February 2001
In situ and in vitro senescence induced by KCl stress:
nutritional imbalance, lipid peroxidation and antioxidant
metabolism
C.L. Vieira Santos1, A. Campos, H. Azevedo and G. Caldeira
Biology Department, Cell Biology Centre, University of Aveiro, 3800 Aveiro, Portugal
Received 21 August 2000; Accepted 5 September 2000
Abstract
Introduction
Sunflower (Helianthus annuus L. cv. SH222) plants
and calli were exposed to KCl stress for three
weeks. Calli were more tolerant to KCl than plants.
KCl stress decreased NO3 , Mn, Fe and B levels in
whole plants and P, Ca and Mg in shoots. NO3 ,
P, Ca, Mg, Mn, and B levels decreased in 100 mMstressed calli. Chlorophyll content, Fm and (Fm F0)/Fm
ratio decreased in stressed leaves, while F0 increased
only in leaves exposed to severe stress (100 and
150 mM). Membrane permeability and lipid peroxidation increased in plants under all stress conditions and in 100 and 150 mM stressed calli, but
remained unchanged in 25 mM stressed calli. Salt
stress also induced changes relating to antioxidant
enzymes: plants under all stress conditions showed
a decrease in catalase, peroxidase and SOD activities. Calli under moderate stress (25 mM KCl)
showed an increase of catalase, peroxidase and
SOD activities, but the activities of peroxidase and
SOD decreased when calli were exposed to higher
KCl concentrations. The decrease of antioxidant
enzyme activities is in tune with lipid peroxidation
and membrane permeability increases. On the
other hand, calli adapted for 6 months to 100 mM
KCl showed an increase of these enzyme activities
compared to unstressed calli, while MDA production
and membrane permeability were not significantly
affected.
Induced premature senescence is often quanti®ed by
decreases of soluble protein contents, changes of chl au
chl b ratio (Lin and Kao, 1998; Lutts et al., 1996) and
by membrane permeability anduor lipid peroxidation
increases (Dhindsa and Matowe, 1981; Dhindsa et al.,
1981). Lipid peroxidation induced by active AOS is
considered to be an important mechanism of membrane
deterioration (Irigoyen et al., 1992; Shalata and Tal,
1998). Plants have evolved non-enzymatic and enzymatic protection mechanisms that ef®ciently scavenge AOS
and prevent damaging effects of free radicals (Irigoyen
et al., 1992; Shalata and Tal, 1998). Enzymatic protec.
tion is partly performed by SOD that eliminates O2
radicals and by catalase and peroxidases that degrade
H2O2 in¯uencing the level of lipid peroxidation.
Senescence induced by salt stress is also related to the
accumulation of toxic ions or to nutrient depletion. For
example, some nutrients are involved in photosynthesis
and in protein synthesis regulation and their depletion
may lead to serious inhibition of these processes. In
particular, Mg de®ciency may lead to decreases of chlorophyll synthesis and variable ¯uorescence. Another
example is calcium, a second messenger and an important
element in cell wall and in membrane structureustability
(White, 1998), which de®ciency may lead to serious
cell damage. Also, Zn, Cu and Mn de®ciencies were
reported to affect SOD isoenzyme activities (Yu and
Rengel, 1999). Maintenance of membrane integrity and
the selective uptake of essential minerals as well as ion
compartmentation are some of the parameters that
were previously related with salt tolerance acquisition
(Salama et al., 1994).
Key words: Helianthus annuus, KCl stress, mineral deficiencies, oxidative stress, tissue culture, senescence.
1
To whom correspondence should be addressed. Fax: q351 2344 26408. E-mail: [email protected]
Abbreviations: AOS, active oxygen species; BA, benzyladenine; GA3, gibberelic acid; ID, inhibition dose; MDA, malondialdehyde; MS, Murashige and
Skoog medium; NAA, naphthalene acetic acid; RLR, relative leakage ratio; SOD, superoxide dismutase.
Society for Experimental Biology 2001
352
Vieira Santos et al.
In the present report, the effects of KCl stress on
sun¯ower plant and calli growth, nutritional imbalance
and senescence parameters were studied. It is also
proposed a correlation between changes of antioxidant
enzyme activities (SOD, catalase and peroxidase) and
membrane permeability and lipid peroxidation in in vitro
and in situ KCl-stressed cells.
Materials and methods
Plant material and growth conditions
Sun¯ower (Helianthus annuus L. cv. SH222) seeds were supplied
by SCLEPAL, Portugal. Six groups of 5-d-old seedlings
(n ˆ 60 for each group) were grown on aerated Long Ashton
medium (according to Santos and Cadeira, 1999), supplemented, respectively, with 0 mM KCl ( 0.06 MPa), 10 mM
( 0.09 MPa), 25 mM ( 0.14 MPa), 50 mM ( 0.29 MPa),
100 mM ( 0.52 MPa), and 150 mM ( 0.63 MPa). Plants were
grown at 22"2 8C, with light supplied by Osram 36 W lamps
giving an intensity of 98"2 mmol m 2 s 1 and a photoperiod
of 16 h. Plant fresh and dry weights were measured at 0, 3, 8,
15, and 21 d of culture (n ˆ 15).
One-month-old calli, obtained on modi®ed MS (Murashige
and Skoog, 1962) medium (MSmod) supplemented with NAA
0.5 mg dm 3, BA 0.5 mg dm 3 and GA3 0.1 mg dm 3 (according to Santos and Caldeira, 1999), were used for KCl stress
studies. Calli were grown in a growth chamber at 22"1 8C, with
light supplied by Osram 18 W lamps giving an intensity of
93"1 mmol m 2 s 1 and a photoperiod of 16 h. Six groups
of calli ("0.1 g fresh weight) were grown on MSmod
medium containing, respectively, 0 mM ( 0.19 MPa), 10 mM
( 0.21 MPa), 25 mM ( 0.27 MPa), 50 mM ( 0.38 MPa),
100 mM ( 0.57 MPa), and 150 mM ( 0.76 MPa) KCl. Calli
samples (n ˆ 15) from all stress conditions were harvested
for measurement of fresh and dry weights at 0, 3, 8, 15, and
21 d after stress imposition.
Calli that survived to 100 mM KCl, after treatment, were
maintained in this salinity for 6 months.
Determination of salt resistance
Resistance to KCl was estimated as ID50, which is de®ned as the
KCl concentration required in the growth medium to inhibit
dry weight gain of plants or calli by 50%, 2 weeks after salt
exposure.
Mineral analysis
Ion content was determined in roots, shoots and in calli. Dried
tissues were mineralized and NO3 , SO24 and Cl quanti®cation
was performed by a capillary electrophoresis system (Waters
Quanta 4000) (Santos and Caldeira, 1999). For the analysis of
all other elements (B, Ca, Cu, Fe, K, Mg, Mn, P, and Zn) dried
tissues were treated according to Evans and Bucking (Evans
and Bucking, 1976) and elements were determined by Induced
Coupled Plasma Spectroscopy (Jobin Ivon JY70 Plus) according to Santos and Caldeira (Santos and Caldeira, 1999). Results
were averaged from six individuals in two independent assays
with three replicates each.
Determination of MDA concentration and membrane
permeability
Lipid peroxidation was determined by malondialdehyde (MDA)
content (according to Dhindsa and Matowe, 1981): 0.25 g of
tissue samples were homogenized in 5 cm3 trichloroacetic acid
0.1% and centrifuged at 10 000 g for 10 min. The supernatant
was collected and 1 cm3 was mixed with 4 cm3 20% trichloroacetic acid and 0.5% thiobarbituric acid. The mixture was
heated at 95 8C (30 min), quickly cooled and centrifuged at
10 000 g for 10 min. The supernatant was used to determine
MDA concentration at 532 nm.
Membrane permeability was determined by conductivity
(LtuL0) and by UV absorbance (RLR) as described previously
(Lutts et al., 1996): samples of plants and calli were washed in
de-ionized water to remove surface-adhered electrolytes. Tissue
segments were placed in tubes with 10 cm3 de-ionized water
and incubated overnight at 25 8C (85 rpm). Electrolyte leakage
analysis was performed by conductivity of the bath solution
before and after autoclaving. Solute leakage was determined by
measuring the absorbance (280 nm) of the bathing solutions
before and after freezing the tissues (Redmann et al., 1986).
Chlorophyll concentration and fluorescence parameters
in plants
Chlorophyll content was determined from expanded and young
leaves according to Arnon (Arnon, 1949).
Plants were dark adapted for 20 min in a growth chamber at
22"2 8C. Fluorescence was monitored in expanded and young
leaves using a Plant Ef®ciency Analyser (Hansatech Instruments
Ltd., UK) by illuminating leaves with a peak wavelenght
650 nm and a saturating light intensity of 3000 mmol m 2 s 1.
The basal non-variable chlorophyll ¯uorescence (F0) and the
maximal ¯uorescence induction (Fm) were determined and then
the maximum quantum yield of PSII ((Fm F0)uFm) (Maxwell
and Johnson, 2000) was estimated.
Antioxidant enzymes and soluble protein content
Tissue samples (0.5 g) were homogenized at 4 8C in 1 cm3 of
K-phosphate buffer 0.05 M (pH 7.8) containing 0.1 mM
ethylenediamine tetraacetic acid, 5 mM cysteine, 1% polyvinyl
pyrrolidone, and 0.2% Triton X-100 (Olmos et al., 1994).
Homogenates were ®ltered and centrifuged at 8000 g for 15 min
at 4 8C. The supernatant was dialysed at 4 8C for 24 h against
1 dm3 10 mM K-phosphate buffer (pH 7.8) containing 10 mM
leupeptin and 100 mM phenylmethylsulphonyl ¯uoride. The
dialysed samples were centrifuged at 2500 g for 10 min at 4 8C
and used for enzymatic determination (Olmos et al., 1994).
Catalase (EC 1.11.1.6) was determined according to Aebi
(Aebi, 1974). Guaiacol peroxidase was determined (Putter,
1974). For superoxide dismutase (EC 1.15.1.1) samples were
treated, and SOD activity was determined, by the method
described earlier (Osswald et al., 1992). Soluble proteins were
determined according to Bradford (Bradford, 1976).
Fisher and t-tests
Results presented are average means for at least three independent assays with three replicates each. Variance analysis was
performed by Fisher test and the means were statistically tested
using a 2-sided t-test. Statistical signi®cance is assumed at
P-0.05.
Results
Plant and calli growth
KCl stress decreased sun¯ower plant and calli growth
rates (Fig. 1a±c). This decrease was evident not only in
Senescence in KCl-stressed cells 353
shoot and root fresh and dry weights, but also in leaf
areas (data not shown). For concentrations higher than
25 mM, shoots were more affected than roots (Fig. 1a, b).
Stressed leaves were small and chlorotic and soon became
necrotic in plants exposed to 100 and 150 mM KCl.
Plants exposed to 150 mM died 3 weeks after stress
imposition. After 15 d, negative correlation were established between shoot (y ˆ 0.00321 3 0.4306, R ˆ 0.69),
root (y ˆ 0.00153 3 0.2372, R ˆ 0.78) and calli growth
(y ˆ 0.00098 3 0.163; R ˆ 0.93) and KCl concentration
in the media. These negative correlations gave an ID50
of 40.1 mM KCl for shoots, of 57 mM KCl for roots
and of 72 mM KCl for calli suggesting that calli are
more tolerant to KCl stress than plants.
Nutrient content in plants and calli
Severe KCl stress decreased NO3 levels in the whole
plant (Table 1a). Phosphorus, Ca and Mg decreased only
in stressed shoots. K and Cl , as expected, increased
in the whole plant. With respect to micronutrients, Mn,
Fe and B decreased in stressed roots and leaves. Phosphorus, NO3 , Ca, and Mg decreased in KCl-stressed
calli, while K and Cl levels increased (Table 1b). Only
B and Mn decreased in 100 mM-stressed calli while
other micronutrients were not affected (Table 1b).
Chlorophyll content and fluorescence parameters
Table 2 shows that basal ¯uorescence (F0) was not signi®cantly affected in moderate (25 mM) stressed leaves, but
increased during treatments in 100 and 150 mM-stressed
leaves. Maximal values were obtained in 150 mM-stressed
expanded leaves at the end of treatment. Contrarily, Fm
decreased with KCl stress, in particular in 100 and
150 mM-stressed leaves leading, together with the
increase of F0, to a decrease of (Fm F0)uFm ratio.
In expanded leaves this reduction was signi®cant for
all KCl concentrations at the end of treatment while in
Fig. 1. Effect of KCl on sun¯ower growth: (a) root dry weight, (b) shoot dry weight and (c) calli dry weight (n ˆ 15).
Table 1. Effect of KCl stress on nutrient contents in sun¯ower: (a) plants; (b) calli
Symbol a indicates signi®cantly different means between control and stressed individuals.
(a)
Element
(mg g 1 DW)
Root
Shoot
KCl (0 mM)
KCl (100 mM)
KCl (0 mM)
KCl (100 mM)
NO3
SO24
P
Cl
K
Ca
Mg
Mn
Fe
Cu
Zn
B
3796"387
765"125
1876"355
38"6
8342"725
2343"756
945"156
28"4
96"21
44"15
75"21
56"1
2037"515 a
521"107 a
1956"476
369"87 a
15 845"3 209 a
2098"456
1018"365
20"2 a
62"18 a
26"11 a
60"25
22"7 a
4473"975
946"267
3743"275
41"23
12 753"975
1087"109
1453"234
45"10
87"23
32"7
63"11
98"23
1761"112 a
1021"298
2098"389 a
529"11 a
18 876"1 093 a
818"98 a
826"83 a
28"9 a
41"11 a
38"19
51"16
63"14 a
354
Vieira Santos et al.
Table 1. Continued.
(b)
Element
(mg g 1 DW)
KCl
(0 mM)
KCl
(100 mM)
NO3
SO24
P
Cl
K
Ca
Mg
Mn
Fe
Cu
Zn
B
3786"416
546"127
3872"236
171"54
10 421"857
2578"347
2485"235
42"6
126"23
53"16
76"21
68"18
2046"491 a
638"203
3060"112 a
482"83 a
19 504"2 638 a
1421"672 a
1513"547 a
29"13 a
83"22
65"27
87"16
35"12 a
young leaves this effect was only signi®cant for 100 and
150 mM KCl.
Chlorosis was observed in plants exposed to KCl stress,
and this was con®rmed by a decrease of chlorophyll
content (Fig 2a, b). At the end of treatment chl au
chl b ratio was lower in 25 mM-stressed leaves than in
unstressed, but at the same time this ratio increased in
both 100 and 150 mM stressed leaves (Fig. 2c, d).
Determination of lipid peroxidation and membrane
permeability
Fig. 2. Effect of KCl stress on chlorophyll contents (a, b) and chl auchl b
ratio (c, d) in sun¯ower plants. Symbol a indicates signi®cantly different
means between control and stressed individuals.
Malondialdehyde (MDA) production increased signi®cantly with leaf ageing and this effect was enhanced by
KCl stress. For example, at day 21 unstressed leaves had
a MDA production 1.4 times higher than at day 8, while
leaves exposed to 150 mM produced ®ve times more
MDA than unstressed leaves (Fig. 3b). Also, MDA
production in unstressed plants was always higher in
shoots than in roots (Fig. 3a, b).
MDA production in calli was not signi®cantly affected
by tissue ageing (Fig. 3c). Also, there were no signi®cant
differences of MDA production between unstressed and
25 mM-stressed calli during the whole treatment in
contrast to what happened between unstressed and
25 mM-stressed roots and leaves. MDA production
Table 2. Effect of KCl stress on ¯uorescence parameters in sun¯ower plants
Symbol a indicates signi®cantly different means between control and stressed individuals.
KCl concentration
(mM)
F0
0
25
100
150
Fm
0
25
100
150
(Fm F0)uFm
0
25
100
150
Expanded leaves
Day 15
Young leaves
Day 21
Day 15
Day 21
738"52
777"85
917"66 a
1060"34 a
741"62
744"85
863"44 a
1477"17 a
789"35
738"95
878"48 a
930"23 a
739"56
741"87
842"29 a
1013"32 a
3253"97
3213"88
1122"62 a
1083"47 a
3218"12
2201"95 a
963"43 a
1499"67 a
3386"36
3178"84
2 049"62 a
914"26 a
3241"123
3222"65
1263"108 a
995"87 a
0.773"0.16
0.758"0.23
0.182"0.08 a
0.021"0.01 a
0.769"0.11
0.662"0.18 a
0.104"0.07 a
0.015"0.00 a
0.767"0.15
0.768"0.21
0.571"0.14 a
0.017"0.01 a
0.771"0.12
0.770"0.10
0.333"0.09 a
0.018"0.00 a
Senescence in KCl-stressed cells 355
Fig. 3. Effect of KCl stress on: MDA production in roots (a), shoots (b) and calli (c); solute leakage in roots (d), shoots (e) and calli (f ) and electrolyte
leakage in roots (g), shoots (h) and calli (i). Symbol a indicates signi®cantly different means between control and stressed individuals.
increased however in 100 and 150 mM-stressed calli
which is similar to what happened in plants. At the
end of treatment, 150 mM-stressed calli had a MDA
production 2.4 times higher than unstressed calli.
Similar to MDA formation, the increase of solute
and electrolyte leakage (quanti®ed by LtuLo and RLR)
also increased with plant ageing (Fig. 3d, e, g, h). It can
be seen, by the increases of LtuLo and RLR, that KCl
stress increased membrane damage and this effect was
more evident in leaves than in roots (Fig. 3d, e, g, h). In
150 mM-stressed plants the increments of solute and
electrolyte leakage were more evident during the ®rst
weeks, being higher in leaves. In 100 mM-stressed plants,
solute and electrolyte leakage increases were more
signi®cant during the last week of treatment, being also
more evident in leaves. Calli exposed to 100 and 150 mM
KCl also suffered increases of solute and electrolyte
leakage, but to a lesser extent than stressed plants
(Fig. 3f, i). Solute and electrolyte leakage increases were
evident at the end of the ®rst week in 150 mM-stressed
calli while they were only evident at the second or third
week in 100 mM-stressed calli. Calli exposed to 25 mM
never showed any changes in MDA production or in
membrane permeability (Fig. 3c, f, i).
KCl adapted-calli for 6 months produced only 1.2
times more MDA than unstressed calli, and solute and
356
Vieira Santos et al.
electrolyte leakage was not signi®cantly different from
unstressed calli (Table 3).
Antioxidant enzymes in stressed plants and calli and
in KCl-adapted calli
Catalase activity was higher in unstressed calli than in
shoots while SOD and peroxidase activities were lower
(Fig. 4a, d). All stress conditions decreased catalase,
peroxidase and SOD activities in shoots (Fig. 4a±f ).
Minimum values were obtained in 150 mM-stressed
shoots at day 21 for catalase, peroxidase and SOD.
Signi®cant decreases of SOD and peroxidase activities
were already observed at day 3 in stressed shoots. By
contrast, moderate stress increased catalase, peroxidase
and SOD activities in calli, while higher KCl concentrations decreased peroxidase and SOD activities not
affecting signi®cantly the activity of catalase (Fig. 4d, e, f).
Unstressed shoots had, in average, 17.2"1.2 mg
protein g 1 FW and this content was not signi®cantly
affected by moderate stress. Only 150 mM KCl decreased
the content of protein to 10.3"1.5 mg protein g 1 FW
in shoots. Calli exposed to 100 and 150 mM KCl
also suffered a slight decrease of soluble protein
Table 3. Effect of KCl stress on MDA production, solute leakage, K content and oxidative stress enzyme activities in 100 mM
adapted calli
Symbol a indicates signi®cantly different means between control and stressed individuals.
Unstressed calli
Salt-adapted calli
MDA
formation
(nmol g 1 FW)
Solute
leakage
(LtuLo %)
Solute
leakage
(RLR %)
K content
(mg g 1 DW)
Sol. protein
(mg g 1 FW)
Catalase
(U mg 1 prot)
Peroxidase
(U mg 1 prot)
SOD
(U mg
25.4"6.5
26.7"4.1
31.5"6.6
36.7"10.4
39.3"7.9
39.1"9.4
11 530"2157
18 498"3758 a
13.4"2.8
12.9"1.7
75.3"12.8
176.1"23.0 a
28.4"4.6
95.3"7.1 a
3.8"0.2
11.2"0.7 a
1
prot)
Fig. 4. Effect of KCl on catalase activity in (a) shoots and (d) calli; peroxidase activity in (b) shoots and (e) calli; and SOD activity in (c) plants and
(f ) calli.
Senescence in KCl-stressed cells 357
content: unstressed calli had an average 13.2"0.6 mg
protein g 1 FW, but 100 mM and 150 mM stress decreased protein contents to 11.3"1.1 and 9.5"0.8 mg
protein g 1 FW, respectively. KCl adapted-calli showed
an increase of peroxidase, catalase and SOD activities
respectively to unstressed calli with the same age
(Table 3) while protein content was not signi®cantly
affected.
Discussion
Plant and calli growth in the presence of KCl
In this paper it is demonstrated that KCl stress accelerates the development of senescence characteristics in
sun¯ower cells and that these changes are accompanied
by decreases of antioxidant enzyme activities. Shoots are
more sensitive to KCl than roots having not only a higher
reduced growth but also higher membrane degradation.
Sun¯ower calli are more tolerant to KCl stress than
plants, which is shown by the higher ID50 exhibited by
calli. Also, KCl stress induced higher increases of MDA
production and solute and electrolyte leakage in plants
than in calli. This fact, together with the higher activities
of antioxidant enzymes observed in calli, may justify the
higher tolerance to KCl exhibited by in vitro cells.
It is still a question why, in some genotypes, cells
growing in vitro are more tolerant to salt than intact
plants. When one compares salt tolerance between calli
and plants, several factors must be taken in account:
(1) plants have different stages of differentiation during
their life cycle that may affect their salt tolerance while
calli differentiation can usually be controlled; (2) due to
transpiration mesophyll cells may be exposed to higher
concentrations of Kq than the concentration present in
the medium; (3) culture media used in vitro are usually
richer in salts and sugars than those used for plant growth
and, therefore, in vitro cells may not suffer so easily from
macro and micronutrient de®ciencies. On the other hand,
in vitro cells may be indirectly selected for higher saline
concentrations and reduced osmotic potentials. These
aspects may contribute to the higher salt tolerance
observed in calli.
Nutritional imbalance, lipid peroxidation
and antioxidant metabolism
In several species, the increase of Cl concentration in the
medium leads to a reduction of nitrate levels in tissues
(Grattan and Grieve, 1994). The same effect was found
for KCl-stressed sun¯ower plants and calli. However,
once the pool of free amino acid increased with increasing KCl stress (Santos, 1998), the decrease of nitrate
levels may not only be due to a decrease of N uptake,
but also to an increase of its reutilization by cells mainly
in proline synthesis (Santos, 1998).
KCl stress decreased Ca levels in shoots. This decrease
may, besides other effects, signi®cantly affect membrane
structure and permeability, cell wall composition and
other physiological processes. Cytoplasmic calcium acts
as a second messenger in plant cells, being involved in
signalling pathways mediation and it was reported to be
a primary event in response to ABA, a hormone that was
postulated to play a central role in signalling stress
responses (Ingram and Bartels, 1996; Netting, 2000).
KCl also induced the accumulation of high levels of K
in sun¯ower cells. Potassium uptake seems to be similarly
regulated in 100 mM-stressed and adapted calli because
both systems accumulated similar amounts of K. It was
previously proposed, for other species, that Kq in¯ux
regulation may be due to the allosteric effect of internal
Kq in the cell (Benlloch et al., 1994). However, regulation
of Kq uptake may follow a more complicated pathway than the allosteric response (Benlloch et al., 1994).
Recently, experiments with split roots have shown that
long-distance signals must also play a role (Maathuis and
Amtmann, 1999), and it was postulated that hormones
could be involved in this regulation. For example,
Montero et al. suggested that ABA inhibited Cl and
Naq uptake, and stimulated the uptake of Kq in
NaCl-tolerant bush bean plants (Montero et al., 1997).
Also, stimulation of Kq uptake was also reported to be
promoted by GA3, a hormone that was also associated
with antisenescence in salt-stressed cells (Prakash and
Prathapasenan, 1990). It is therefore important to study
the effect of KCl stress on plant hormones and its
in¯uence on Kq uptake regulation.
On the other hand, there is no de®nitive study on the
effects of KCl on micronutrient accumulation in plants.
Micronutrient accumulation depends on the tissue nature,
the micronutrient concentration in the medium and also
on the type of salt stress (Grattan and Grieve, 1994).
In sun¯ower, micronutrient accumulation with salinity
also depends on the culture system used. The behaviour
of plants and calli was different with respect to the
accumulation of micronutrients with increasing KCl
concentration in the medium: in fact, except for B and
Mn, KCl-stressed calli were not affected in their micronutrient contents. This fact may be due not only to
different cellular organization, but also to the fact that
the MS medium is richer in salts than the culture medium
used for plant growth.
Similarly to what has been reported for this (Santos
and Caldeira, 1999) and other species exposed to NaCl
(Chen et al., 1991; Irigoyen et al., 1992; Lutts et al., 1996),
KCl stress induced changes in sun¯ower cells that
represent a hastening of the natural senescence. On the
other hand, KCl tolerance seemed to correlate with the
stimulation of antioxidant enzymes and the enhanced
ability to remove AOS. In fact, 25 mM-tolerant and
100 mM-adapted calli showed increases of antioxidant
358
Vieira Santos et al.
enzyme activities, while MDA production and membrane
permeability did not differ from unstressed calli. On the
contrary, in all stressed plants the activities of these
enzymes decreased, together with increases of MDA
production and membrane permeability.
Shalata and Tal suggested that, as AOS induce
peroxidation of membrane lipids, resistance to environmental stress may depend on the inhibition of AOS
production or the enhancement of antioxidant levels
(Shalata and Tal, 1998). One of the plant responses to
AOS production is the increase of antioxidant enzyme
activities providing protection from oxidative damage
induced by several environmental stresses. The higher
tolerance of some genotypes to environmental stresses
has been associated with higher activities of antioxidant enzymes. For example, the wild NaCl-tolerant species
Lycopersicon pennellii had lower lipid peroxidation than
the cultivated species L. esculentum, and higher activities
of SOD, peroxidase and catalase (Shalata and Tal, 1998),
suggesting that the wild species is better protected against
oxidative damage. Lopez et al. reported an increase of
peroxidase activity in salt-stressed radish cells (Lopez
et al., 1996). Also, NaCl-sensitive pea cultivars suffered
a decrease of Mn-SOD activity, while in tolerant cultivars
the activity of this isoenzyme increased (Olmos et al.,
1994). The de®ciency of Cu, Zn and Mn also affected
CuuZnSOD and MnSOD activities in lupin leaves (Yu and
Range, 1999) and in bush beans (Mehlhorn and Wenzel,
1996). KCl stress induced de®ciency of Mn in both
100 mM-stressed plants and calli, but Cu decreased only
in plants. On the other hand, SOD and other antioxidant
enzyme activities were more affected in stressed plants.
It must be further clari®ed if there are different mechanisms of removing AOS in stressed calli and plants
that may justify the in¯uence of the cell system to salt
stress response.
fact that the ®rst step in chl b degradation involves its
conversion to chl a (Fang et al., 1998).
The stability of F0, found for moderate KCl stress,
indicates that this low concentration of salt has no
signi®cant changes in the reaction centres. However,
the variations found for higher KCl concentrations
indicate losses of energy transference from pigments to
the reaction centre (probably due to damage of the LHC
centre associated to the PSII). Changes in (Fm F0)uFm
indicate variations in the photochemical ef®ciency of the
PSII (Maxwell and Johnson, 2000). A reduction of this
ratio in KCl-stressed plants is also due to a reduction of
Fm that may re¯ect an increased energy dissipation due
to a destruction of photosynthetic apparatus. Previous
studies showed that salt and osmotic stress induced
changes in the photosynthetic apparatus (Bhorer and
Dorf¯ing, 1993; Lutts et al., 1996), and the membrane
permeability properties of chloroplasts. Dissociation of
the light-harvesting antennae from the PSII core and the
denaturation of the PSII reaction centre have been
postulated for rice NaCl-stressed leaves (Lutts et al.,
1996) and for drought-stressed cotton (Genty et al., 1987)
and sun¯ower (Conroy et al., 1988) plants. This fact
may be the result of chlorophyll degradation anduor
synthesis de®ciency together with a decrease of thylakoid
membrane integrity. Several data sets point to this conclusion: (1) a reduction in Mg and Mn contents together
with a reduction of chlorophyll contents observed in
KCl stressed leaves; (2) an increase of membrane permeability and of lipid peroxidation, probably due to
changes in membrane compositionustructure. It must be
emphasized that KCl stress decreased Ca levels while
increased K, affecting the sites of transport and Ca2q
allocation in membranes and most probably, affecting
plastid membrane structure.
Fluorescence parameters and chlorophyll content
Acquisition of salt tolerance
Some antioxidant enzymes (e.g. MnSOD) are located in
plastids and, as postulated by Osswald et al., compartmentation of the singlet oxygen (formed within thylakoid
membranes) and the detoxifying enzymes, localized in
the stroma, must occur (Osswald et al., 1992). This
compartmentation implies the maintenance of membrane
integrity, and if one of these conditions is destroyed, the
plastid loses integrity affecting photosynthesis.
Photosynthetic parameters such as chlorophyll content
and photosynthetic rate have previously been used as
senescence parameters (Buchanan-Wollaston, 1997). KCl
stress severely affected chlorophyll concentrations. In
leaves exposed to severe stress, not only was the total
chlorophyll content drastically reduced, but the chl auchl b
ratio increased showing that chl b was being degraded at
a higher rate than chl a. This can be explained by the
Calli that survived to 100 mM KCl were subcultured
on the same medium for 6 months. At the end of this
time it was possible to select some calli adapted to this
salinity that had, nevertheless, a retarded growth.
Tolerance to KCl has been reported previously (Rush
and Epstein, 1981) for some tomato genotypes and
by Sumaryati et al. in protoplast-derived colonies of
haploid Nicotiana plumbaginifolia L., that regenerated
into plants (Sumaryati et al., 1992). In all cases, selected
cells accumulated high levels of Kq, similar to what
happens with KCl-tolerant sun¯ower calli. This fact
suggests that KCl-adapted calli developed alternative
mechanisms of tolerating high Kq level that were not
present in KCl-stressed cells and that may contribute to
KCl adaptation. In adapted calli, MDA production and
membrane permeability reached values that were similar
Senescence in KCl-stressed cells 359
to those of unstressed calli while antioxidant enzymes
were much more active.
These data support the idea that salt toleranceuadaptation may be correlated with a stimulation of antioxidant
enzymes and that these may control lipid peroxidation and reduce membrane degradation. In fact, in these
adapted cells, catalase, peroxidase, and SOD activities
were higher than those found for unstressed calli. On the
contrary in 100 mM-stressed calli the activities of these
enzymes decreased during the whole treatment. Further
studies must be done in order to clarify if these changes
in antioxidant enzymes are a direct effect of Kq anduor
Cl in cell, or a response to a primary effect induced by
KCl, such as nutrient imbalance.
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