Change in Uptake, Transport and Accumulation of Ions in Nerium oleander
(Rosebay) as Affected by Different Nitrogen Sources and Salinity
AHMAD ABDOLZADEH
1,3,
*, KAZUTO SHIMA
2
, HANS LAMBERS
3
and KYOZO CHIBA
2
1
Gorgan University of Agricultural Sciences and Natural Resources, POB 49175-386, Gorgan, Iran,
2
Laboratory of
Applied Plant Ecology, Graduate School of Natural Sciences and Technology, Okayama University, Tsushima-naka,
Okayama 700, Japan and
3
School of Plant Biology, The University of Western Australia, 35 Stirling Highway,
Crawley WA 6009, Australia
Received: 17 May 2008 Returned for revision: 23 June 2008 Accepted: 22 July 2008 Published electronically: 4 September 2008
† Background and Aims The source of nitrogen plays an important role in salt tolerance of plants. In this study, the
effects of NaCl on net uptake, accumulation and transport of ions were investigated in Nerium oleander with
ammonium or nitrate as the nitrogen source in order to analyse differences in uptake and cycling of ions within
plants.
† Methods Plants were grown in a greenhouse in hydroponics under different salt treatments (control vs. 100 mM
NaCl) with ammonium or nitrate as the nitrogen source, and changes in ion concentration in plants, xylem sap
exuded from roots and stems, and phloem sap were determined.
† Key Results Plant weight, leaf area and photosynthetic rate showed a higher salt tolerance of nitrate-fed plants com-
pared with that of ammonium-fed plants. The total amount of Na
þ
transported in the xylem in roots, accumulated in
the shoot and retranslocated in the phloem of ammonium-fed plants under salt treatment was 1
.
8, 1
.
9 and 2
.
7 times
more, respectively, than that of nitrate-treated plants. However, the amount of Na
þ
accumulated in roots in nitrate-
fed plants was about 1
.
5 times higher than that in ammonium-fed plants. Similarly, Cl
2
transport via the xylem to
the shoot and its retranslocation via the phloem (Cl
2
cycling) were far greater with ammonium treatment than with
nitrate treatment under conditions of salinity. The uptake and accumulation of K
þ
in shoots decreased more due to
salinity in ammonium-fed plants compared with nitrate-fed plants. In contrast, K
þ
cycling in shoots increased due to
salinity, with higher rates in the ammonium-treated plants.
† Conclusions The faster growth of nitrate-fed plants under conditions of salinity was associated with a lower trans-
port and accumulation of Na
þ
and Cl
2
in the shoot, whereas in ammonium-fed plants accumulation and cycling of
Na
þ
and Cl
2
in shoots probably caused harmful effects and reduced growth of plants.
Key words: Mineral cycling, Nerium oleander, nitrogen source, salinity, xylem and phloem transport.
INTRODUCTION
Nerium oleander is an evergreen sclerophyllous C
3
shrub,
native to arid regions in a broad area from Morocco to
southern China, and planted widely as an ornamental
plant in many warm parts of the world, including hot
desert areas (Huxley, 1992). It is of interest for revegetation
and landscaping, because of its ornamental value and its
capacity to acclimate to adverse environmental conditions.
Several investigators have reported remarkable drought tol-
erance of N. oleander (Bjo¨rkman and Powles, 1984;
Demmig et al., 1988). Salinity can inhibit plant growth
by a range of mechanisms, including low external water
potential, ion toxicity and interference with the uptake of
nutrients, particularly K
þ
(Munns, 1993; Tester and
Davenport, 2003). Previous studies have indicated that the
salt tolerance of N. oleander is achieved by inhibition of
uptake of Na
þ
and Cl
2
and their accumulation in the
shoot, as well as maintenance of a high K
þ
/Na
þ
ratio in
plants (Hajji, 1979; Abdolzadeh et al., 1998).
Plant metabolism of nitrogen is influenced by the inor-
ganic form of nitrogen supplied. Since salinity affects the
uptake and assimilation of nitrogen in various plant
species, the nitrogen source plays an important role in salt
tolerance of plants. In most studied species, including
wheat (Leidi et al., 1991), maize (Lewis et al., 1989),
peanuts (Silberbush and Lips, 1988), pea (Speer et al.,
1994; Frechilla et al., 2001), muskmelon (Adler and
Wilcox, 1995) and sunflower (Ashraf, 1999), plants grown
with ammonium as the nitrogen source show a higher
level of salt sensitivity than those grown with nitrate.
Similarly, a study of salt tolerance of N. oleander with
different nitrogen sources indicates that ammonium-fed
plants show a greater reduction in dry weight under saline
conditions than those grown with nitrate. One of the main
reasons for such a difference might be related to a different
distribution and accumulation of Na
þ
and Cl
2
in roots and
shoots (Abdolzadeh et al., 1998). These different patterns
were suggested to be the result of the relative contribution
of xylem and phloem import and of export via the
phloem. It is generally accepted that an increased K
þ
/Na
þ
ratio and reduced Na
þ
translocation from the root to the
shoot contribute to the overall salt tolerance in glycophytes
(Tester and Davenport, 2003; Parida and Das, 2005). It is of
interest, therefore, to evaluate the rate of phloem and xylem
transport between roots and shoots to quantify the effects of
NaCl on uptake and transport of ions within intact plants
using different nitrogen sources. In Ricinus communis,
* For correspondence E-mail abdolzadeh@gau.ac.ir or ah_ab99@
yahoo.com
# The Author 2008. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
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Annals of Botany 102: 735–746, 2008
doi:10.1093/aob/mcn156, available online at www.aob.oxfordjournals.org

Lupinus albus and Banksia prionotes, small amounts of
phloem exudate can be collected after a small incision is
made in the aerial part of plants. Most of our knowledge
about transport of ions comes from these species
(Marschner, 1995; Jeschke and Pate, 1995). The same prop-
erty was found in N. oleander; it spontaneously exudes
phloem sap that can be collected by making shallow
incisions in the stem (A. Abdolzadeh, pers. obs.).
Armstrong and Kirkby (1979) used a model that assumes
Ca
2þ
is not translocated in the phloem. Such an assumption
may not be completely valid, as illustrated by the finding of
45
Ca in a part of the root system that was not supplied with
labelled Ca
2þ
in a spilt-root experiment (Lambers et al.,
1982); however, the Ca
2þ
concentration in phloem sap in
comparison with other macronutrients is, indeed, negli-
gible. Thus, the xylem flux of Ca
2þ
can be estimated
from the accumulation of Ca
2þ
in shoots. The xylem
fluxes of other ions are then calculated from the Ca
2þ
flux and the ion/Ca
2þ
ratios in the xylem sap. Finally, the
phloem fluxes are calculated from the difference between
accumulation rates and xylem fluxes (Armstrong and
Kirkby, 1979; Touraine et al., 1988; Gouia et al., 1994,
Lu et al., 2005; Niu et al., 2007).
In the present study, growth and changes in ion concen-
trations in whole plants, xylem sap exuded from roots and
stems, and phloem sap were determined in N. oleander
exposed to saline conditions and grown with either
ammonium or nitrate. Based on the above model, the
uptake and flows of major ions in shoots and roots were
estimated in order to elucidate the relationship between
some mineral cycling and salinity tolerance as influenced
by different nitrogen sources.
MATERIALS AND METHODS
Plant material and growth conditions
Plants were grown in a greenhouse from shoot cuttings of a
single tree of N. oleander L. (Rosebay). Plants of a similar
size were transferred to a hydroponics culture with
ammonium or nitrate as the nitrogen source. The nutrient
solution was made with tap water and contained 0
.
5mM
MgSO
4
,0
.
5mM KH
2
PO
4
and micronutrients (Gibson,
1987) in all treatments. Other nutrients included 2
.
5mM
KNO
3
and 1
.
5mM Ca(NO
3
)
2
for the nitrate treatment, and
1
.
5mM CaCl
2
,2
.
75 mM (NH
4
)
2
SO
4
and 2
.
5mM KCl for
the ammonium treatment. The pH of the nutrient solution
was adjusted daily to 6
.
3+ 0
.
2 with 0
.
1 M KOH or
H
2
SO
4
, and the nutrient solution was changed every
week. Salinity treatments (100 mM NaCl) were started 3
weeks after transfer to nutrient solution. Salinity treatments
were started with 25 mM NaCl and increased in three steps
to 100 mM to avoid osmotic shock. The average daily
maximum and minimum temperature in the greenhouse
during the growing period were 32 and 188C, respectively.
The relative humidity was between 42 and 75%. Four plants
in each treatment were harvested at days 1, 3, 7, 15 and 30
after starting the salt treatment for growth assessment and
chemical analyses. The phloem and xylem sap collections
were carried out on the same days (days 1, 3, 7, 15 and
30 after starting the salt treatment). Leaf area was deter-
mined by an automatic leaf area meter (Model AAM-7
Hyashi Denkoh Co. Ltd, Tokyo, Japan).
Gas exchange measurements
CO
2
assimilation (net photosynthesis) and transpiration
rates of attached leaves were measured using a portable gas
exchange system (ADC Infrared Gas Analyzer type LCA4
with PLC4 chamber, Hertfordshire, UK) at various time inter-
vals during the study. Measurements were taken from 1000 h
to 1500 h, at a CO
2
concentration of approx. 360 mmol mol
21
,
vapour pressure deficit of 1
.
2 kPa and a photosynthetuic
photon flux density of 1000 mmol m
22
s
21
. Each measure-
ment took approx. 7 min, and the parameters were recorded
every 30 s. The mean of the values obtained during the
stable part of the experiment (lasting 4 min) was used as
one value. Water-use efficiency (mmol CO
2
fixed per mmol
water lost) was calculated by dividing net photosynthesis by
transpiration rates.
Xylem and phloem sap collection
Phloem exudate was collected from 0900 h to 1100 h by
making shallow incisions in the middle of the stem using a
sharp razor blade. Phloem sap exuded from the incisions
almost immediately in small droplets. The first drop of
sap may contain cell sap and was discarded (Jeschke and
Pate, 1995). Approximately 50–100 mL of phloem sap
was collected by a Hamilton syringe over a 2–3 min
period, and pooled into small plastic vials. Total carbon,
nitrogen and carbohydrate concentrations were measured
in the exuded sap. The total C and N concentration was
determined in freeze-dried phloem sap after the dried
powder was mixed with 4 g of cobalt oxide using a C-N
Corder (Yanaco Co. Type TNC-600, Kyoto, Japan).
Hippuric acid was use as a standard. The freeze-dried
samples were dissolved in 7 mL of distilled water and
passed through an ion exchange column (Amberlite
CG-120 cation exchange resin and Amberlite CG-400
anion exchange resin, Sigma, Tokyo, Japan) before quanti-
fication of soluble sugars by high-performance liquid
chromatography (HPLC; Shimazu Co., Kyoto, Japan).
The mobile phase was water, and separation was carried
out at 808Cataflowrateof0
.
5mL min
21
. Xylem sap
was collected from stems (from 1000 h to 1200 h) and
roots separately. In stems, after collection of phloem sap
the external tissue of the vascular cambium, including the
phloem, was peeled off, and then the stem was cut and
the exuded sap was collected. After approx. 1–2 min,
about 100 mL of xylem sap was collected. In the case of
small volumes for plants under a prolonged period of sal-
inity, the stem segments were gently pressurized with air.
For roots, one or two of the main cut roots were sealed in
a pressure chamber and mildly pressurized with air, and
the exuded sap was collected. For evaluation of the purity
of the xylem sap, the concentration of carbohydrates was
measured in xylem sap as described above. All sap
samples were stored at –208C until inorganic ion analysis.
Abdolzadeh et al. — Ion Uptake and Transport with Salinity and N Source736