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The effects of sodium sulphate and sodium chloride on growth morphology
photosynthesis and water use efficiency of
henopodium rubrum
Plant Physiology Research Group Department of Biological Sciences University of Calgary Calgary Alta.
Canada N IN4
Received June 14, 1989
W A R N E , ., G U Y ,R . D . ,
ROLLINS,
. , and REI D, D. M. 1990. The effects of sodium sulphate and sodium chloride on
growth, morphology, photosynthesis, and water use efficiency of
Chenopodium rubrum.
Can. J Bot. 68: 999-1006.
The effects of sodium sulphate and sodium chloride salinity on the anatomy, water relations, and photosynthesis of Chen-
opodium rubrum L. were compared. Low concentrations of either salt stimulated growth, but higher concentrations resulted
in large decreases in dry weight and leaf area. Leaf succulence and the number of layers of palisade cells were increased,
but these effects were more pronounced with NaCl than with N S0 4. Stomatal density was reduced at low to moderate
salinities, but then increased again at high salinity. Stomatal size was reduced at all salinities. Increasing salinity had no
great effect on photosynthetic rates except with older plants grown at the highest level of Na'SO,. Stomatal cond uctan ce
decreased at all salinities. This reduced transpiration and led to increased intrinsic water use efficiency. Total tissue stable
carbon isotope ratios also indicated that water use efficiency was improved.
Chenopodium rubrum
adjusted osmotically by
accumulating electrolytes from the nutrient solution and by synthesizing glycinebetaine. Plants in NaCl limited osmotic
adjustment more than those growing in Na2 S0,. Despite this, N S0 4 was more dama ging than NaCl and caused earlier leaf
senescence at high concentrations.
W A R N E , ., G U Y ,R . D . ,
ROLLINS,
. , et REID,D. M . 1990. The effects of sodium sulphate and sodium chloride on growth,
morphology, photosynthesis, and water use efficiency of Chetiopodiurn rubrum. a n . . B ot . 6 8 9 99 -1 00 6.
Les effets de la salinitt du sulfate de sodium et du chlorure de sodium sur I'anatomie, les relations hydriques et la
photosynthkse d u
Chenopodium rubrum
sont compares. Les basses concentrations de I'un ou I'autre sel stirnulent la crois-
sance , mais les concentrations plus Clevtes causent de s diminutions importantes de la masse sec he et de la surface des feuilles.
La succulence des feuil les et le nom bre de couches d e cellules palissadiques augm entent, mais ces effets sont plus pr onon cts
avec NaCl q u'avec Na 2S0 4. La den sit t stomatique est r tdu ite aux salinites basses B modtrtes, mais, par la suite, augmente
B forte salinitt. La dimension des stomates est rtdu ite toutes les salinitts. L'augm entation de la salinitt n'a pa s d'effet
important sur les taux de photosynthkse sauf chez les plantes plus igtes au niveau le plus ClevC de NaZSO,. La conductance
stomatique dim inue B toutes les salinitCs. Ceci rtd uit la transpiration et conduit une augmentation d e I'efficacitC de l 'uti-
lisation d e I'eau. Les ratios en isotope de carbone stable des tissus indiquent aussi que I'effic acitt d e I'utilisation de I'eau
est amCliorte.
Cherlopodium rubrum
s'ajuste osmotiquement par I'accumulation des Clectrolytes de la solution nutritive et
par la synthkse de glycinebetaine. Les plantes dans la solution de NaCl limitent I'ajustement osmotique plus que celles en
prtsen ce de Na2 S04. En dtp it de ce fai t , Na,S04 est plus domm ageable que NaCl et cause une stnescenc e foliaire plus
hitive aux concentrations Clevtes.
[Traduit par la revue]
ntroduction
Within the Chen opodiaceae are found many species that are
highly tolerant to salinity. Typically, these species are able to
survive at soil osmotic potentials below . 5 MPa (Sharma
1 9 8 2 ) .
The exposure of halophytes to salinity often induces
alterations in morphology and (or) anatomy (Poljakoff-Mayber
1 9 7 5 ) . Less apparent effects also occur at the physiological
level. Several chenopods accumulate large quantities of inor-
ganic ions in their leaf tissues as soil salt concentrations
increase. This is usually accompanied by the accumulation of
glycinebetaine. Though low or moderate salinity may stimu-
late growth, high salinity results in decreased growth. How-
ever, the photosynthetic rate of many chenopods is only
slightly affected by chronic salinity stress. Many chenopods
also have low er transpiration rates compared w ith other plants
and may therefore use water more efficiently.
Chenopodiurn rubrum is one of several halophytic cheno-
pods found on saline soils in western Canada. Many of these
soils contain a high proportion of sodium sulphate (Dodd et
al. 1 9 6 4 ; Lilley 1 9 8 2 ) . Very little information is available
'Present address: Department of Forest Sciences, The University
of British Columbia, Vancouver, B .C ., Canada V6T 1 W5.
'Author to whom correspondence should be addressed.
regarding the effects of Na2S0, salinity on various aspects o
plant growth, with most studies utilizing only sodium chloride
This paper compares the effects of both Na2S0, and NaCl o
grow th, succulence, stomata1 size and nu mb er, accumulatio
of glycinebetaine, water relations, intrinsic water use effi
ciency, and photosynthetic capacity of C . rubrum.
Methods and materials
Seeds of
C. rubrum
L. (obtained from several plants that in tur
originated from seed collected near Nanton, Alberta) were scarifie
and sown on to 4-inch (40.8-cm ) pots contain ing granite grit No.
(Imasco). Pots were suspended by their rims from Lucite covers i
deep plastic trays (six pots per tray) containing 5 L of nutrient solu
tion. Th e lower cm of each pot was submerged in the solution
which was maintained at constant level by the automatic addition o
deionized water. Solutions were continuously aerated to keep them
well mixed and to prevent anaerobic conditions. Nutrient solution
consisted of half-strength Hoagland's solution (Hoagland and Amo
1950) with salt (either NaCl or anhy drou s Na,S04 ACS certified
added to obtain the solute potentials desired in any particular exper
iment. Media solute potentials (MSP) were confirmed by psychro
metry. Th e solute potential of half-strength Hoa gland 's solution
about -0. 03 MPa. For our purposes, this small contribution is n
included in further references to MSP. Plants were allowed to adju
to increased salinities by incrementally dropping MSP by no mor
Printcd in anada Imprim e au anada
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1000 CAN. J. BOT.
than -0 .2 MPa every 2 days. Solutions were changed weekly after
maximum salt concentrations were reached. Plants were maintained
at the final salinities for at least 2 weeks prior to harvesting.
When an Econaire (Winnipeg) growth chamber was used,
daymight
temperatures were 22:12 C. The light source was Sylvania gro-lux
lamps, which provided an irradiance (PAR) of 313 IJ.Em-' s- at
plant height. Irradiance was increased to 483 IJ.Em-' s- ' for the
photosynthesis experiments. Photoperiod was 16 h, and humidity was
46% during the day and 75% at night. Treatments were represented
by only a single tray of pots each, but experiments were repeated
where warranted and tray positions within the growth chamber were
randomized each time.
In experiments performed in the greenho use, irradiance ranged from
about 220 IJ.Em -Z s- on overcast days to about 1250 IJ.E n-, s-
on sunny days. Relative humidity was variable, ranging from 30 to
40% during the day and up to 70% at night. Temperature varied from
approximately 23C on overcast days to 33OC on sunny days and
dropped to a minimum of 14C at night. All treatments were repre-
sented by on e tray in each of two randomized block s (i.e., 12 plants
per treatment).
Unless otherwise noted, measurements of foliar characteristics refer
to the leaf from the 7th node up from the base of the plant. Succu lence
(water content per unit leaf area) was measured by cutting leaf disks
of equal area with a cork borer and determining the difference between
fresh and dry weights. One disk was cut from near the middle of the
7th leaf from each of six plants per treatment. Major veins were
avoided. Changes in the size and number of layers of leaf cells were
determined by exam ining leaf cross-sections under a microscope. Leaf
samples were taken near the centre of the leaf and fixed in 3% glu-
taraldehyde buffered at pH 6.8 with 0.05 M phosphate buffer. After
dehydration, tissues were embedded in LKB Historesin and sections
cut with a glass knife on an LKB 2218 microtome. Mounted sections
were stained with periodic acid -Schiff's reagent (Feder and O'Brien
1968) and counterstained with toluidine blue
0
Leaf thickness was
estimated from freehand and cross-sections.
Numbers and sizes of stomata were determined from nail polish
leaf impressions (modified from Sampson, 1961) examined under a
Reichert projecting microscope (magnification 800 X . Stomata1 sizes
were measured lengthwise and classified into two categories: large
(>3 2 IJ.M) r small (
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WARNE ET AL. 100
TABLE
. Effect of NaCl and NqSO, salinity on leaf area (one surface), stornatal size, stornatal density, and number of stomata per leaf f
the 7th leaf up from the base on the main axis of C. ru rlitn
Stomata1 density
No. of stomata per leaf
Mean area Stornatal size
Solute potential
(no./mm2)
per leaf ( >32 km
X
10-7
Salt
MPa (cm')
long) Abaxial
Adaxial
Abaxial Adaxial
Control
0 14.97k 1.99 64.7 22 .3 152.9k 3.3
105.8k4.0
226.9k29.6 165.8221.6
NaCl .4 15.38k2.09 43.3?4.8 92. 0k 1.9 6 5. 2k 2. 0 147.0224.0 105.8k 18.0
1.0 10. 112 1.22 14.9 2.7 105.5?2.1 63. 9k 1.4 107.4k 14.2 66.5 k9 .6
-2.1 6.75 0.92 0.7 k0 .7
124.4k2.9 79.72 2.0
86. 8k 12.4 56.1 k8 .5
N S04 .4 19.04k2.41 41.2&7.7 104.7k 2.5 75. 92 2.6 200.3 22 7. 2 155.2225.4
1.0 9.1350.67 8.4 k2. 5 110.62 2.6 75.2 k 1.9 99.7k 6.9 68.2k5.1
-2.1 4.27 0.55 0 209.6 4. 131. 823. 2 86 .5 k 10.7 55 .4 27 .2
NOTE:
Each datum represents the mean + SE of 12 plants or, for effects on stomatal size (abaxial leaf surface only), six plants per treatment. For density determinations, stoma
were counted in five different areas on each leaf.
NaCl
MEDI A SOLUT E POT ENT I AL MPa)
FIG.2. Effects of NaCl or Na,SO, on leaf succulence. Data points
represent means k SE of six plants per treatment.
part to the development of larger cells in the palisade layer an
an increase in the number of cell layers. Leaves of contro
plants averaged 2.3
+
0.1 (SE) layers of palisade cell
whereas at MS P of -0 .8 MP a, this increased to 2.96 .0
layers with NaCl and to 2.89 + 0.1 with N S04. The spong
meso phyll lay er a136 increased in thicknes s in NaCl trea
ments, but very little increase was observed in Na,S0
treatments.
Stomatal numbers atid sizes
In view of the fact that the growth of plants declined as sa
concentrations increased, it seemed reasonable to investigat
the effects of salt on stomatal number and size, since an
changes would presumably influence photosynthetic carbo
gain. Stomatal densities on both abaxial and adaxial leaf sur
faces decreased at moderate salinities, but increased again a
higher salinities (Table 1). This trend was particularly eviden
in the Na,S0 4 treatment where density was actually higher
-2. 1 MPa than in the control treatment. However, there wa
a tendency towards larger leaves at low salinity before lea
area decreased again at higher salinities. T hes e trends appeare
to be at least partially independent, as evidenced by the fa
that their combined action resulted in a reduction in the tota
number of stomata per leaf across all salt treatments (Tab
1). A change in the size stomata was also observed (Table 1
In the absence of salt, 65 of stomata were greater than 3
p,m in length . Aperture length was reduced as M SP decreas ed
and O. l9
with
Na S04 (both
at
M P a )
The ratio
such that there were very
few
stomata of this size seen at .
decreased again
at MSP
.6 MPa'
The
low
MPa, Trends were virtually identical for both salts. Interes
irradiance within the growth cham ber (compared with the nat-
ural environment) might have prevented or precluded a more
ingly, at - 0. 4 MP a, stomatal size was reduced relative
substantial response. Hence, an experiment was conducted in
controls, even though stomatal density was lowe r and leaf are
the greenhouse to investigate whether o r not irradiance in com-
was greater.
bination with salinity would influence the expression of pros-
trate growth. No such effect was found.
smotica accumulation
Like many halophytic chenopods,
C.
rubrum readily accu
Succulence
Increasing salt concentrations had a significant effect on leaf
succulence (Fig. 2). Both salts caused substantial increases
from 0 to - 0. 8 MP a, but NaCl had a much greater effect
beyond this point. Leaf thickness also increased as MSP
decreased and again, NaCl had the greatest effect (data not
shown ). For examp le, leaf thickness at MS P of 1.0 MPa
was increased over controls by 50 in NaC1-treated plants and
by 21 in Na,S04-treated plants. This increase was due in
mulates large quantities of salt within its tissues. Ash conten
increased almost linearly with decreasing MSP, ranging fro
14 of leaf dry weight in controls to 40 at the highest sa
concentrations. Results were the same for both salts (data n
shown). A ccumulation of glycinebetaine was also similar wi
both Na,S04 and NaCl (Fig. 3). Data in Fig. 3 are presente
in terms of organic matter (i.e ., dry weight less the ash weigh
to correct for the bias caused by the high salt content of th
leaves. The glycinebetaine content of plants grown at MSP o
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1002 CAN.
J. BOT
VOL. 68, 1990
M E DI A S O LU TE P O TE N TI AL M P ~ )
FIG.3. Leaf glycinebetaine content of C. rubrum grown in NaCl
and N% S04. Data points represent means SE of four plants per
treatment. Error bars not visible where smaller than the symbol size.
.1 MPa was about 420 pmol per gram of organic matter
(equivalent to 4.9% of the leaf biomass).
Water relations
To determine the extent and pattern of osmotic adjustment
in C. rubrum water and solute potentials of leaf tissue were
measured. Results presented in Fig. 4 show that as salt con-
centration increased, both
*,
and Ts ecreased and were
maintained below the MSP. Estimated leaf
Tp
emained rel-
atively constant in NaCl treatments, but increased with
increasing Na,SO, concentrations. As MSP below .4 MP a,
leaf 9 nd Ts ere lower in N% S0 4 treatments than in NaCl
treatments. For example, at 1.6 MPa Na2S0 4, the leaf Tw
was .84 MP a, whereas plants at .0 MPa NaCl had a
Tw f only .5 MP a. Superficially, then, it would appear
that plants grown in
N% S04 should have had an advantage
over those grown in NaC1. As already described, this was not
the case.
Photosynthetic CO, assimilation and related variables
Results already presented showed changes in stomatal num-
bers and length that, in addition to possible changes in stomatal
opening, could influence gas exchange. Under steady-state
conditions, A was about 22 pm ol CO, m-'s-' for plants
grown without salt (Fig. 5 . This rate decreased slightly at
-0 .4 MPa (for both salts) but increased again at 1 0 and
.0 MP a (NaCl) to a level similar to that of the controls. At
1.6 MPa N%SO,, however, the photosynthetic rate dropped
to approximately 10 pmol CO, m-'s ,perhaps reflecting the
poor condition of plants at this treatment level.
At all salt concentrations there was a significant decrease in
stomatal conductance (Fig. 6A) to a level approximately one-
third that of the con trols. Decreased conductance can limit the
rate of CO, assimilation. In C. rubrum however, was only
modestly reduced at most salt concentrations (the notable
MEDIA SOLUTE POTENTIAL
M P ~
FIG. 4
Wate r relations of C.
rubrum
leaves from 50-day-o ld plan
grown in N %S 04 and NaC1. Each data point represents the mean
S E of three to five samples per treatment. D ashed diagon al lines re
resent the isomotic limit, where leaf water or solute potent
equals media solute potential.
0
Y ;
?
9, ;Ys.
rror bars n
visible where smaller than the symbol size.
exception being the 1.6 MP a Na 2S0 4 (treatment). Cons
quently, CJC, ratios were reduced at all salinities other tha
the .6 MPa Na 2S 04 treatment (Fig. 6B). The fact that
was m uch lower (and yet the C i of plants from this treatme
did not differ from unsalinated controls) implies that photo
synthesis was somehow impaired at the biochemical leve
Trends in WU E (Fig. 6C) closely mirrored CJC,, as would
expected.
The photosynthesis experiments were repeated under ide
tical conditions with the on e modification that the plants we
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T
AL.
100
MED IA SOLU TE POTEN TIAL MPa)
FIG. 5 . Steady-state net p hotosyn thetic rates of leaves of 50-d ay-
old C. rubrum plants grown in NaCl and Ns SO ,. Data points rep-
resent means SE of six plants per treatment.
1 week youn ger at the time of measurem ent. Results were very
similar to those shown in Figs. 5 and 6 except that the effects
of the .6 MP a Na,SO, treatmen t on A conductance,
C,/C,, and water use efficiency we re not significantly differen t
from the
0
MPa NaCl treatment. The effects on CJC, are
presented in Table 2 Measurem ents were repeated 7 days later,
by which time the leaves of .6 MPa N S0 4 plants were
beginning to show visual damage. These plants were again
behaving as shown in Figs. 5 and 6. It thus appears that N S0 4
at high con centrations induces earlier leaf senescence that does
NaCl.
Carbon isotope analysis
Isotopic composition of C. rubrum leaf tissue is presented
in Fig. 7 . Genera lly, less negative 613C values were obta ined
with increasing salt concentrations. Plants grown with NaCl
appeared to have slightly greater changes in 613C than those
grown with N S0 4, but there was no significant difference
(by t-test) between the slopes of regression lines for both sets
of data.
iscussion
At low to moderate salinities, similar effects on growth of
C. rubrum were obtained with both NaCl and N S0 4. For
example, small amou nts of the salt promoted growth and higher
concentrations inhibited growth. Th is pattern is typical of many
halophytes. Morphological changes induced by the two salts
were also similar though different in degree. Hence, stomata1
density responded in a complex fashion, but trends were still
more or less the same for both NaCl and N S0 4. Leaf suc-
culence was increased with either salt, but m ore so with NaCl.
Similar observations were made by Poljakoff-Mayber (1975).
Increased succulence is frequently observed in halophytes
exposed to increasing salinity and various suggestions have
M ED IA S O LU T E P O TE N TI AL M P
FIG.6. Effects of increasing salt concentrations on various g
exchange parameters under steady-state conditions (same 50-day-o
plants as in Fig. 5).
(A)
Stomata1 conductance to diffusion of CO
(B) intercellular to ambient CO, ratio (CjC ,); (C) intrinsic water-u
efficiency (WU E). 0 aCL;
a
Na,SO,.
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N a C l
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M ED IA S O LU TE P O TE NT IA L M P ~ )
FIG.
7
Effect of increasing concentrations of NaCl or N SO, on the isotopic composition (613C value) of leaves of 50-day-old C .
rubrum
plants. Analyses were performed on pooled samples from six plants per treatment.
T A B L E
.
The effect of NaCl and Na,SO, salinity on inter-
cellular to ambient CO, ratio (C,/C,) for leaves from two
ages of C.
rubrurn
Solute potential C,/C, ratio
Salt MPa 43 days 50 days
NaCl 0
.4
1.0
.6
NOTE ns, not significantly different (by I-test) from the NaCl 0 MPa
treatment of the 43-day-old plants; , indicates significance at
P