Plant Physiol. (1996) 110: 321-328
Photosynthesis in Salt-Adapted Heterotrophic Tobacco Cells
and Regenerated Plants'
Locy, Ching-Chun Chang', Brent 1. Nielsen, and Narendra K. Singh*
Department of Botany and Microbiology, Auburn University, Auburn, Alabama 36849
Robert D.
Tobacco (Nicofiana tabacum L.) cells growing heterotrophically
in the light on supplied sucrose ( S O ) have previously been adapted
to grow in 428 mM NaCl (S25). Among the changes occurring in
salinity-adapted cell cultures are (a) elevated levels of chlorophyll
compared to unadapted cells; (b) decreased levels of starch; (c)
alterations in chloroplast ultrastructure, including loss of starch
grains, increased thylakoid membrane structure, and the presence
of plastoglobules; and (d) increased rates of O, evolution, CO,
fixation, and photophosphorylation relative to SO cells. These latter
changes apparently derive from the fact that thylakoid membranes
in S25 cells contain higher levels of photosystem I- and 11-associated
proteins as well as thylakoid ATPase components. S25 chloroplasts
contain immunologically detectable levels of ribulose-1,S-bisphosphate carboxylase/oxygenase, whereas SO completely lack the enzyme. These changes taken together suggest that even in the presente of sucrose, S25 cells have acquired a significant degree of
salt-tolerant photosynthetic competence. This salt-tolerant photoysynthetic capability manifests itself in plants backcrossed with
normal plants for three generations. These plants contain chloroplasts that demonstrate in vitro more salt-tolerant CO, fixation, O,
evolution, and photophosphorylation than do backcross progeny of
plants regenerated from SO cultures.
Numerous cell-suspension culture lines able to grow in
the presence of salt-induced osmotic stress have been established (Dix and Street, 1975; Nabors et al., 1975; Tyagi et
al., 1981; Kochba et al., 1982; Rangan and Vasil, 1983;
Watad et al., 1983; Pandey and Ganapathy, 1984; Binzel et
al., 1985; Rains, 1989; Plaut et al., 1991) for the purpose of
studying cellular adaptation to salinity stress, and these
cell lines have provided much information about salinity
tolerance at the cellular level. The tobacco cell line chosen
for the present study has been widely studied in this regard. Binzel et al. (1985) established a heterotrophically
growing, salt-adapted cell line of tobacco (Nicotiana tabacum L. var Wisconsin 38) by gradually adapting cells to
increasing concentrations of NaC1. The adapted cell line
showed substantially greater growth in salt-containing media than did previously unadapted cells. However,
adapted cells growing in salt showed reduced cell expansion and fresh weight gain but equivalent dry weight gain
'
This is publication No. 6-955101 of the Alabama Agricultura1
Experiment Station.
Present address: Department of Microbiology, Cornell University, Ithaca, NY 14853.
* Corresponding author; e-mail nksingh8ag.auburn.edu; fax
1-334 - 844-1 645.
(Binzel et al., 1985; Bressan et al., 1990) when compared to
unadapted cells growing without salt.
Adaptation of tobacco cells to salt has also been shown to
involve the accumulation of a 26-kD, thaumatin-like protein, called osmotin (Ericson and Alfinito, 1984; Singh et al.,
1985, 1987); alterations in the cell wall and extracellular
polysaccharides (Iraki et al., 1989a, 1989b, 1989~);the accumulation of various osmolytes, including Suc and Pro (Binzel et al., 1987); and the accumulation and partitioning of
inorganic ions such as potassium and sodium (Binzel et al.,
1987, 1988; Watad et al., 1991). These observations support
the concept that in heterotrophically grown cells of the
glycophyte tobacco, the same general principals of osmotic
regulation appear to be at work as are exhibited at the
cellular level by most halophytes, i.e. organic osmolytes
accumulate principally in the cytoplasm, whereas the accumulation of ions occurs principally in the vacuolar compartment (Flowers et al., 1977; Greenway and Muns, 1980;
Binzel et al., 1988).
To date there is little information available regarding the
mechanisms of osmotolerance and/or salinity tolerance of
other organelles within the cell. However, it is clear that, if
osmolytes and other ions accumulate in the cytoplasm of
tobacco cells adapted to salinity as suggested above, there
must be additional mechanisms for organellar adaptation.
Rearrangement of the mitochondrial genome appears to
occur in the adapted cells and in plants regenerated from
them (Rietveld et al., 1988). However, the physiological
significance of this rearrangement is at present unexplained. Furthermore, the initial observation presented
here that S25 cells are substantially greener than SO cells
suggests that chloroplast adaptation to osmotic stress may
be a critica1 component of cellular adaptation to salinity
stress in these cells.
In the present paper we report our initial studies comparing chloroplast activities and photosynthetic capacity in
S25 cells with those in S0 cells.
MATERIALS A N D METHODS
Cell Culture
Tobacco cells (Nicotiana tabacum var Wisconsin 38) were
grown heterotrophically at 27°C with constant shaking at
120 rpm under a photoperiod of 14 h of light (50 pmol m-'
Abbreviations: SO, unadapted tobacco; S25, salt-adapted tobacco; S25-P, plants regenerated from S25 cells; W38, normal tobacco
(Wisconsin 38).
321
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Copyright © 1996 American Society of Plant Biologists. All rights reserved.
322
Locy et al.
s-’) in a Labline gyratory Environmental Shaker (Labline,
Melrose Park, IL 1. Both SO and S25 cells (Binzel et al., 1987)
were obtained from Dr. P.M. Hasegawa (Purdue University, West Lafayette, IN). SO cells were grown in Murashige
and Skoog medium as modified by Hasegawa et al. (19801,
except that casein hydrolysate was omitted. S25 cells were
grown in the same medium containing 428 mM NaC1. The
medium was dispensed into Erlenmeyer flasks (one-fifth of
the flask volume), closed with sponge plugs and aluminum
foil, and autoclaved at 121°C at 15 p.s.i. for 20 min.
Plants
Severa1 plants were regenerated from S25 cells and have
been previously described (Bressan et al., 1985, 1987). In
general, a11 regenerated plants showed thicker leaves and
shortened internodes and were male sterile. The progeny
of one regenerated plant, MJ-14 (used as a female parent),
were used in this study. AI1 experiments presented here
were performed with the progeny of three successive
crosses in which W38 served as the recurrent male parent
and are referred to as S25-P plants. S25-P seeds were grown
in a growth chamber at 26°C using a 14-h light photoperiod
and a light intensity of 200 pmol m-’ spl. After 40 d of
growth, the third leaves from the top of the tobacco seedlings were used in a11 experiments.
Chl Determination
Chl was extracted with 80% acetone, and the concentration of Chl was determined by measuring the A,, and A663
and calculating Chl concentration using the equation of
Arnon (1949).
Chloroplast lsolation
Chloroplasts were isolated using the Percoll gradient
method of Walker et al. (1987) modified as follows. Either
suspension-cultured cells harvested by centrifugation at
1,0009 for 5 min at 4°C or leaf tissues from plants were
suspended in chilled grinding buffer (50 mM Hepes-KOH,
pH 8.3, 350 mM sorbitol, 1 mM MgCl,, 1 mM MnCl,, 2 mM
EDTA, 2 mM EGTA, 0.5% BSA, 4.4 mM ascorbate). For
isolation of chloroplasts from S25 cells, grinding buffer was
osmotically adjusted to 950 mM sorbitol. Cells were homogenized in a Waring Blendor equipped by removing the
normal blades and fastening razor blades in their place.
The blender was run at the top speed three times for 5 s
each. This homogenate was filtered through two layers of
cheesecloth and two layers of Miracloth (Calbiochem), and
the filtrate was centrifuged for 10 min at 10,OOOg. The
supernatants were decanted and discarded, and the pellets
were gently resuspended in grinding buffer using a soft
brush.
The suspension was layered onto a Percoll step gradient
consisting of 40 and 80% Percoll. The gradients were centrifuged at 9000 rpm for 4 min at 4°C in a Sorvall HB-4 rotor
with the brake turned off. The upper band containing
broken membranes was separated from the middle band
containing intact chloroplasts. Resuspension medium (375
mM sorbitol, 35 mM Hepes-KOH, pH 8.3,lO mM Na,HPO,,
Plant Physiol. Vol. 110, 1996
0.96 mM DTT) was added to the material in this band, and
after gentle mixing the mixture was centrifuged at 25008
for 4 min to wash off the Percoll. The supernatant was
discarded, and the pellets were resuspended in resuspension medium to a density of 2 to 3 mg Chl mL-’.
Measurement of O, Evolution
Light-dependent O, evolution of both heterotrophic cells
and leaf discs from regenerated plants was measured using
a Clark-type O, electrode (Yellow Springs Instruments,
Yellow Springs, OH) as described by Robinson et al. (1983).
The basic reaction medium contained 0.1 M sorbitol, 5 mM
MgCI,, 5 mM NaC1, 50 mM Hepes-KOH, pH 7.6, 10 mM
DL-glyceraldehyde, and 10 mM NH,Cl. The Hill reaction
was determined by adding 5 mM potassium ferricyanide as
the electron acceptor before illumination under 200 pmol
m-z s -1 light at 29°C. A11 measurements were performed
in triplicate.
Measurement of 14C0, lncorporation
Two milliliters of cell-suspension culture were transferred into a 50-mL Erlenmeyer flask in which a vial containing 1 pCi of NaH14[C03] (specific activity 58.2 mCi
mmol-I, Amersham Canada Limited) was placed. The
flask was sealed with a serum stopper and illuminated
with 200 pmol m-’ s-’ light intensity at 29°C. The cultures
were allowed to equilibrate for 30 min, and 200 pL of lactic
acid (50%, v/v) were injected through the serum stopper
into the vial to release 14C0,. Dark carbon fixation was
measured in flasks wrapped with aluminum foil. After 30
min, the cells were killed by the addition of 5 mL of 95%
ethanol. Two drops of 1 N HCl were added to acidify the
solution to remove unincorporated 14C0, from solution.
Samples were homogenized for 30 s at top speed using a
Tissuemizer (Tekmar Co., Cleveland, OH), and a fraction of
the homogenate was mixed with 3 volumes of scintillation
cocktail. 14C02incorporation into ethanol-insoluble material was measured in a Sorvall Liquid Scintillation Counter.
A11 measurements were performed in triplicate.
For the measurement of 14C02fixation rates in plants,
three sets of 10 leaf discs each were floated in 3 mL of
distilled water containing different concentrations of NaCl
(O, 25, 50, 75, 100, and 125 mM). The discs were placed in
50-mL Erlenmeyer flasks and treated as described above
except that incubations with 14C0, were carried out for 45
min.
Assay of ATP Formation
Chloroplasts were isolated from S25-P and W38 plants as
described above. Ten microliters of an appropriate intact
chloroplast preparation (equivalent to 20 pg of Chl) were
added to a glass tube (13 X 75 mm) containing 0.19 mL of
reaction medium (0.33 M sorbitol, 2 mM EDTA, 1 mM
MgCl,, 1 mM MnCI,, 50 mM Hepes-KOH, pH 7.6) and
different concentrations of NaCl (O, 25, 50,75, 3 00, and 125
mM). The tubes were illuminated at a light intensity of 200
pmol m-’ spl at 27°C for 1 min. Additional tubes were
wrapped with aluminum foil as dark controls. After illu-
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Copyright © 1996 American Society of Plant Biologists. All rights reserved.
Photosynthesis in Salt-Adapted Tobacco Cells and Plants
mination ATP formation was determined according to the
luciferin-luciferase method as described by Mills (1986).
Luminescence was determined using a Monolight 2001
luminometer (Analytical Luminescence Laboratory, Inc.,
San Diego, CA). Because of the low yield of intact chloroplasts from cultured cells, light-dependent ATP formation
in isolated thylakoid membranes from chloroplasts derived
from salt-adapted S25 and SO was measured as described
above in an assay medium containing 6 mM MgCI,, 25 mM
KC1, 0.1 mM methyl viologen, 1.0 mM ATP, 6 mM DTT, 2.5
~ L diadenosine
M
pentaphosphate, and 2 mM Tricine-KOH,
pH 8.0. Inhibitors in the light reaction of photosynthesis,
such as 50 p~ N,N'-dicyclohexylcarbodiimide or 10 p~
DCMU, were added and incubated for 2 min in the dark
before illumination. For determination of light-dependent
ATP formation, the results were corrected by subtracting
dark ATP formation. A11 experiments were performed in
triplicate.
Starch Determination
Starch was determined by the enzymatic-colorimetric
method described by Rose et al. (1991) with modification.
Cells were harvested on Whatman filter paper by aspiration during the stationary phase of culture growth. Fresh
weight was recorded, and 1 g of cells was placed into a
15-mL centrifuge tube (Corning, Inc., Corning, NY). Five
milliliters of 80% ethanol were added to each tube, and the
cells were homogenized using a Polytron homogenizer
(Brinkmann Instruments) set at No. 7 for 3 X 10 s. After the
cells were homogenized, another 5 mL of 80% ethanol were
added to each tube, and the tubes were placed in a water
bath at 95°C for 10 min. The tubes were cooled on ice and
centrifuged at 900g for 5 min. The supernatants were removed by aspiration. This process was repeated until the
supernatant became clear. The washed precipitates were
dried in a hot water bath, and 5 mL of distilled H,O were
added to each tube. Starch was gelatinized by autoclaving
at 121°C and 15 p.s.i. for 45 min. The samples were cooled,
and 5 mL of 5 mM sodium citrate solution containing 510
units of amyloglucosidase (A-3514, Sigma) were added to
the samples. The samples were mixed well and incubated
in a 30°C water bath overnight. After the volume was
adjusted to 10 mL, the samples were centrifuged as above,
and a 1-mL aliquot of the supernatant was taken to measure the amount of reducing sugar according to the Nelson-Somogyi copper reduction method (Somogyi, 1945).
The amount of starch (in milligrams) was calculated by the
formula of Rose et al. (1991).
Western Blot Analysis
Thylakoid membrane proteins were obtained from the
Perco11 gradient, followed by centrifugation for 2 min at
16,OOOg. Pellets were solubilized in 50 mM Tris-HC1, pH 6.8,
containing 8% SDS, 5% 2-mercaptoethanol, 5 mM PMSF,
and 20% glycerol at room temperature for 2 h. Protein
concentration was determined using the BCA method
(Pierce). Discontinuous gradient SDS-PAGE gels (10-16%)
were prepared, and equal amounts of protein (30 pg) were
323
loaded into each well for electrophoresis according to the
method of Laemmli (1970). For Rubisco determination,
soluble proteins were extracted according to Keys and
Parry (1990). Equal amounts of protein (9 pg) were loaded
into each well of a discontinuous SDS-PAGE (12%)gel for
electrophoresis as above.
After completion of electrophoresis, gels were soaked in
transblotting buffer (48 mM Tris, 39 mM Gly, 20% methanol, pH 9.2) for 1 h and electroblotted onto nitrocellulose
paper (Micron Separations, Westborough, MA) using a
semidry transblotting system (Bio-Rad) at 25 V for 40 min.
After transfer to nitrocellulose, the blots were blocked with
0.2% nonfat milk in buffer (10 mM Tris-HC1, pH 8.0, 150
mM NaC1, 0.5% Triton X-100) for 2 h at room temperature.
Heterologous polyclonal antibody against individual thylakoid membrane proteins and Rubisco were a gift from
Dr. A.K. Mattoo (U.S. Department of Agriculture Plant
Molecular Biology Laboratory, Beltsville, MD) and were
used for detection of respective proteins following the
procedure described by Harlow and Lane (1988).
EM
Cells were collected by centrifugation at 1500g and fixed
in their respective growth media containing 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.0, for 4 h at 4°C.
The samples were then rinsed in chilled cacodylate buffer
for 10 min, postfixed in 1%osmium tetroxide in the above
buffer for 4 h at 4"C, briefly rinsed again in cacodylate
buffer, dehydrated in a graded series of ethanol, soaked in
acetone for 15 min, and embedded in a mixture of Epon
and Araldite (Dylewski et al., 1991).Sections were cut with
a Sorvall-Blum MT-2 ultramicrotome, collected on 100mesh copper grids or single-slot grids, and stained with
uranyl acetate (Watson, 1958) and lead citrate (Venable and
Coggeshall, 1965). The sections were examined with a Zeiss
10-CA transmission electron microscope operated at an
accelerating potential of 60 kV. AI1 measurements are
based on seria1 section analysis of 50 or more examples of
each structure.
RESULTS
Chloroplast Ultrastructure, Chl, and Starch Determination
Typical transmission electron micrographs showing the
ultrastructure of chloroplasts from S25 cells and leukoplasts from SO cells are shown in Figure 1. Leukoplasts
from SO cells were irregular in shape but averaged 3.8 ? 0.2
pm in diameter along their longest axis. They contained
numerous, large starch grains embedded in an electrondense stroma. Heterotrophic cell leukoplasts showed little
organized thylakoid structure. The few thylakoids that
were present averaged 0.05 t- 0.04 pm in diameter and
were composed of two to four thylakoids typically positioned around the starch grains (Fig. 1, A and B).
By comparison, chloroplasts from S25 cells growing in
428 mM NaCl contained fewer and smaller starch grains,
were nearly the same size as leukoplasts from SO cells (3.7
t 0.2 pm), and contained numerous electron-dense plastoglobules (Seeni and Gnanam, 1982). S25 chloroplasts had
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Copyright © 1996 American Society of Plant Biologists. All rights reserved.
324
Locy et al.
Plant Physiol. Vol.
110, 1996
D
Figure 1. Electron micrographs of leukoplasts from tobacco cells growing in the absence of NaCI (SO) and of chloroplasts
from tobacco cells growing in the presence of 428 m,M NaCI (S25). A, Micrograph showing a typical leukoplast from an SO
showing large starch grains (S) and the electron-dense stroma of these plastids. X21,750; bar = 1 /j,m. B, Another view of
an SO leukoplast more clearly showing the thylakoids (indicated by arrows) on the periphery of the starch grains with limited
grana stacking. D, Dictysome. X54,81 0; bar = 0.25 /urn. C, Comparable micrograph of chloroplasts from S25 showing the
absence of starch grains, highly organized thylakoids, and the presence of plastoglobules (indicated by arrows). W, Cell wall.
X21,750; bar = 1 /j,m. D, Higher magnification of a similar chloroplast showing the organized thylakoid membranes with
stacked grana (indicated by arrows). Note the abundance of the thylakoid membranes and that they traverse the entire long
axis of the chloroplast. X54,810; bar = 1 ju,m.
well-organized thylakoid membranes exhibiting stacked
grana. The thylakoids traversed the chloroplast along its
longest axis in most cases (Fig. 1, C and D).
In support of the above morphological observations, S25
cells appeared dark green in contrast to the pale yellow
color of SO cells. S25 cells contained approximately 88.8 jag
Chl g"1 fresh weight (Table I) and demonstrated a Chl a/b
ratio of 2.39. By comparison SO cells contained 2.8 jug Chl
g ' fresh weight with a Chl a/b ratio of 0.97. Furthermore,
the starch content of S25 cells (Table I) growing in salt was
strikingly lower than that of SO cells (150 versus 396 ;u,g
starch mg"1 fresh weight, respectively).
Table I. Chl content, Chl a/b ratio, and average starch content
across the culture cycle of SO and 525 cells
Values are means ± SES.
Cell
Chl
Chl a/b
Line
Content
Ratio
S25
2.8 ± 0.8
88.8 ± 2.2
Because S25 cells contain elevated Chl levels and demonstrate a more typical chloroplast organization, it seemed
appropriate to determine the relative photosynthetic competence of S25 cells by measuring rates of O2 evolution,
CO, fixation, and photophosphorylation. The rate of O2
evolution measured in S25 cells was 10.1 /imol mg Chl"1
h ' (Table II), whereas O2 evolution by SO cells could not
be detected. Similarly, the CO2 fixation rate was 3.54 /umol
mg Chl" 1 h"1 in S25 cells but nearly undetectable in normal tobacco cells (Table II). These results were particularly
Table II. O, evolution, CO, fixation, and light-dependent ATP formation rates in SO and S25 cells
ND, Not detectable.
Cell
Line
lig mg ' fresh wt
fj.g g" ' fresh wt
SO
Starch
Content
Photosynthetic Capacity in SO and S25 Cells
0.97
2.39
396 ± 52.8
150 ± 30.6
SO
Rate
CO2 Fixation
Rate
ATP Formation
Rate
\itno\ O2 mg" '
jamo/ COj mg" '
jj.mol ATP mg" '
Chl h~ '
Chl h '
ND
10.13 ± 0.36
0.0 ± 0.48
3.54 ± 0.69
Chl h- '
ND
21.3 ± 3.27
O2 Evolution
S25
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Copyright © 1996 American Society of Plant Biologists. All rights reserved.
Photosynthesis in Salt-Adapted Tobacco Cells and Plants
surprising, since both S25 and SO cells were grown in 30 g
LT 1 Sue, and cells growing heterotrophically in vitro typically do not demonstrate photosynthetic capabilities.
To determine whether light-dependent ATP formation
occurred in S25 cells, light-dependent ATP formation was
measured using the luciferin-luciferase reaction as described in "Materials and Methods." The rate of lightdependent ATP formation in S25 cells was 21.3 p,mol ATP
mg Chl~' h ~ ' . In contrast, there was no light-dependent
ATP formation detected in SO (Table II). To further confirm
that ATP formation resulted from light-dependent electron
transport in chloroplast thylakoid membranes rather than
electron transport in mitochondria, it was shown that 50
JU.M N,N'-dicyclohexylcarbodiimide or 10 /U.M DCMU completely inhibited light-dependent ATP formation (data not
shown).
Analysis of Thylakoid Membrane Proteins and Rubisco
To demonstrate the presence of membrane proteins that
perform photochemical reactions in S25, western blotting
analysis of thylakoid membrane proteins with heterologous antibodies to the PSII proteins, Dl, D2, 51-kD peptide,
43-kD peptide, and Cyt b55g; to the PSI proteins, PSI-1 and
PSI-2; and to the thylakoid ATPase components, CF0, CF llt ,
and CFlti was carried out. All of these proteins were found
in both S25 and in SO cells. However, the levels of each of
these proteins present in SO cells were substantially reduced in quantity compared to S25 cells (Fig. 2).
Since it was found that S25 cells had light-dependent
CO2 fixation capacity but SO cells did not, immunodetection of Rubisco using western blotting analysis was also
performed. Figure 3 shows that the large subunit of
Rubisco proteins was detected in S25 cells but was not
detectable in SO.
Photosynthetic Capabilities of Regenerated Plants under
High NaCI Conditions
To determine whether plants regenerated from S25 cells
possess the ability to maintain photosynthesis in a highly
Figure 2. Western blot analysis of thylakoid membrane proteins in
50 and S25 cells. Equal amounts (30 jig) of thylakoid membrane
proteins were loaded into each well of a 12 to 16% SDS-PACE gel.
Left lane, SO; right lane, S25. Heterologous antibodies were used to
detect the presence of thylakoid membrane proteins. A, Dl; B, D2;C,
51 kD; D, 43 kD; E, Cyt b 599; F, PSI-I; G, PSI-II; H, CF(); I, CF,,,; J,
SO
325
S25
Figure 3. Western blot analysis of Rubisco in SO and S25. Equal
amounts (9 /xg) of soluble cellular proteins were loaded in each well
of a 12% SDS-PAGE gel, followed by immunoblotting using Rubisco
antibody. Left lane, SO; Right lane, S25.
saline environment, the rate of photosynthetic O2 evolution, CO2 fixation, and ATP formation was determined in
S25-P plants and in normal W38 plants. The rate of O2
evolution was not significantly affected by high concentrations of NaCI in either W38 or S25-P plants (Fig. 4). However, leaf discs of S25-P plants exposed to 125 mM NaCI
showed high CO2 fixation rates, whereas leaf discs from
W38 plants showed decreased CO2 fixation rates as the
NaCI concentration increased to 125 mM (Fig. 5). Similarly,
intact chloroplasts of S25-P exhibited high rates of ATP
formation even at 125 mM of NaCI, whereas the ATP formation rate of chloroplasts from W38 plants was significantly reduced at 50 mM NaCI (Fig. 6). The apparent difference in light-dependent ATP formation rates of
thylakoid membranes from S25 cells versus intact chloroplasts from W38 or S25-P plants (14.8 ju.mol mg CM"1 h^ 1
in S25 thylakoids versus 100-800 pmol mg ChP1 h"1 in
intact chloroplasts) resulted from the utilization of two
different methods for measuring ATP formation. When
osmotically lysed chloroplasts from W38 or S25-P plants
0 mM
25 mM
50 mM
75 mM
100 mM 125 mM
NaCI Concentration
Figure 4. Effect of NaCI on O, evolution in W38 and S25-P. Leaf
discs of W38 and S25-P were incubated in media containing varying
concentrations of NaCI as indicated. Rates of O2 evolution for W38
(black bars) are compared with S25-P (shaded bars). Error bars show
the SE of three measurements.
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Copyright © 1996 American Society of Plant Biologists. All rights reserved.
326
Locy et al.
.......................
-o
T
75 mM
I
100 mM 125 mM
NaCl Concentration
Figure 5. Effect of NaCl on CO, fixation in W38 and S25-P. Leaf
discs of W38 and S25-P were incubated in media containing varying
concentrations of NaCl as indicated. Rates of 14C0, fixation for W38
(black bars) are compared with 525-P (shaded bars). Error bars show
the SE of three measurements.
were used for measurements of light-dependent ATP formation in the absence of NaC1, the rates of ATP formation
measured were comparable to the rates observed with
thylakoid membranes from S25 (not shown). This result is
consistent with the fact that ATP synthesized in chloroplasts is utilized for the carbon fixation reaction.
DISCUSSI ON
The adaptation of heterotrophic tobacco cells to high
levels of NaCl produces a population of cells that are
darker green in appearance and contain substantially more
Chl than cells growing in the absence of NaCl. Adapted
tobacco cells contain chloroplasts that demonstrate morphology typical of functional chloroplasts from leaf tissues,
and by a11 measurements made in this study adapted cells
demonstrate photosynthetic competence, despite the fact
that the cells are growing in medium containing SUCin the
light. It should be noted that even when grown in darkness
S25 cells are darker green in appearance than SO cells
grown in the light (data not shown). By comparison, unadapted cells contain chloroplasts that appear not to be
photosynthetically competent. Thus, it can be concluded
that the adaptation of cultured tobacco cells to NaCl stress
involves an increase in chloroplast functionality or adaptation establishes a physiological state in which chloroplast
functionality is either permitted or favored.
Although experiments that directly distinguish between
these two alternatives remain to be conducted, it is apparent from other studies using these same cell lines that
adapted cell lines of tobacco grow well in darkness on
medium containing 428 mM NaCl (Schnapp et al., 1990).
Thus, it is clear that photosynthetic capability is not an
obligate requirement for salinity adaptation in tobacco.
However, the possibility that photosynthesis contributes to
an accelerated growth rate or to a greater carbon use efficiency in adapted cells still exists.
Plant Physiol. Vol. 110, 1996
Alfalfa cells adapted to grow in only 1%(10 g L-', 171
mM) NaCl showed salt-induced greening similar to that
reported here (Winicov and Button, 1991). In addition,
mRNAs for a number of photosynthetic genes were salt
induced in adapted alfalfa cells (Winicov and Seemann,
1990; Winicov and Button, 1991).The studies reported here
with tobacco expand beyond the alfalfa studies to show
alterations in levels of proteins present in the chloroplast,
in chloroplast morphology, and in chloroplast function.
Alfalfa cells adapted to 1%NaCl require light for optimal
growth, and growth is inhibited by photosynthetic inhibitor herbicides (Winicovand Seeman, 1990).At least relative
to a light requirement for growth, the behavior of S25 cells
used in the present study does not appear to correlate with
the alfalfa studies. It is unclear whether this is related to the
different levels of adaptive pressure applied to the respective cell lines or to species-specific differences.
In many respects S25 cells resemble what are described
in the literature as photomixotrophic cells (Horn and Widholm, 1984). Photomixotrophic cultures are usually obtained by subculturing heterotrophic cells into media that
promote culture greening, especially in media containing
lower carbohydrate content, although photomixotrophic
cells require both a supply of carbohydrate and light for
optimal growth. Photomixotrophic cultures usually do not
demonstrate net CO, fixation or net O, evolution, but they
have well-developed, functional chloroplasts, and they
demonstrate the capacity to accomplish photosynthetic carbon fixation (Neumann and Bender, 1987). The contribution of photosynthesis to the carbon balance of such cells
can be substantial (Nishida et al., 1980; Neumann and
Bender, 1987). In photomixotrophic cultures the level of
Chl and the degree of photosynthetic capability is inversely
correlated with the level of SUCsupplied in the medium
(LaRosa et al., 1984; Neumann and Bender, 1987; Rebeille,
1988). This seems to be a factor that distinguishes tobacco
l,OOO/l
...............................
o "O
mM /25 mM ;O
mM 7'5
mM <O0 mM <25 mM'
NaCl Concentration
Figure 6. Effect of NaCl o n ATP formation in W38 and S25-P.
Chloroplasts from leaves of W38 and S25-P were incubated in media
containing varying concentrations of NaCl as indicated. Rates of
light-dependent ATP formation for W38 (black bars) are compared
with S25-P (shaded bars). Error bars show t h e SE of three measurements.
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Copyright © 1996 American Society of Plant Biologists. All rights reserved.
Photosynthesis in Salt-Adapted Tobacco Cells and Plants
cells adapted to grow in 428 mM NaCl from photomixotrophic cultures, because photosynthetic competence can be
demonstrated in adapted cultures at the same SUCleve1 at
which unadapted cells grow as strictly heterotrophic
cultures.
Three hypotheses have been developed and supported to
explain the effect of Suc on the loss of Chl and photosynthetic capability in photomixotrophic cells. First, Suc may
have a direct effect on the photosynthetic apparatus or its
development, affecting photosynthetic competence and ultimately leading to lowered Chl levels (LaRosa et al., 1984).
Second, SUCmay stimulate growth to a greater extent than
it does Chl synthesis and chloroplast biogenesis, leading to
a decrease in cellular photosynthetic capability when Suc is
supplied to cultures (Edelman and Hanson, 1971; LaRosa et
al., 1984). The third possibility is that Suc has a direct
inhibitory effect on Chl synthesis, which ultimately leads to
reduced photosynthetic capability (Pamplin and Chapman,
1975). In tobacco cells adapted to high salinity, it is known
that cell growth on a fresh weight basis is slowed (Binzel et
al., 1985). Thus, it is reasonable to hypothesize that salinity
adaptation acts to increase Chl content by slowing growth
relative to Chl synthesis and chloroplast biogenesis, putting these processes back in balance to create a situation
favorable to chloroplast function. In addition, salinity adaptation could alter the effect of Suc on Chl biosynthesis or
on chloroplast biogenesis, or salinity adaptation may affect
the uptake of SUCor its metabolites into the chloroplast
compartment.
It is clear from the cell culture studies presented here that
the chloroplast compartment of adapted cells has undergone an adaptation to the elevated levels of salinity and
osmoticum present in the cytoplasm of adapted cells (Binzel et al., 1987). More detailed investigations of the mechanisms associated with this adaptation are currently underway in our laboratories. Although chloroplasts of S25
are fully functional, they cannot provide enough energy to
support normal cell growth without supplemental SUC.
Thus, it does not appear likely that the sole basis of chloroplast salinity adaptation is to provide additional fixed
carbon to support growth. This further suggests that chloroplast function in adapted cells may be related to the
production of energy for cellular processes.
Plants regenerated from 525 cells exhibit severa1 altered
characteristics, such as male sterility, reduced growth rate,
and increased ability to survive under high levels of NaCl
(Bressan et al., 1985). The reduced growth rate and other
halophytic features are passed on to sexual progeny, resulting at least partly from a cytoplasmic pattern of inheritance (Bressan et al., 1987). Based on the studies presented
here with progeny of plants derived from adapted cultures,
whatever the mechanism of chloroplast adaptation to salt
is, this adaptation of the chloroplast is carried forward in
three generations of progeny derived from 525 cultures.
Progeny of regenerated plants, which were crossed with
W38 plants (as male parents), still maintain high salt tolerance in photosynthesis as measured by CO, fixation and
photophosphorylation for as long as six sexual generations
(data not shown). These observations are consistent with
327
the hypothesis that such chloroplast adaptation is maternally inherited, although this has not been verified by
reciproca1 crosses because of the limited male fertility of
the plants and progeny obtained.
In conclusion, we have shown that the process of adapting tobacco cells to grow in 428 mM NaCl produces a
population of cells with decreased starch content, elevated
Chl content, and increased photosynthetic capability.
These phenomena apparently result at least in part from an
increased salinity and/or osmotic stress tolerance of the
chloroplast compartment, which can be stably inherited in
the progeny of plants derived from the adapted cultures.
ACKNOWLEDCMENTS
The authors gratefully acknowledge the late Dr. Danniel P.
Dylewski for his assistance with the preparation of electron micrographs and Drs. R.A. Bressan and P.M. Hasegawa (Purdue
University) for generously supplying the plant materials and original cell lines used in these studies.
Received June 7, 1995; accepted October 9, 1995.
Copyright Clearance Center: 0032-0889/96/110/0321 /OS.
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