CSIRO PUBLISHING

Functional Plant Biology

http://dx.doi.org/10.1071/FP12352

The importance of iron supply during repetitive harvesting

of Aster tripolium

Yvonne Ventura A, Malika Myrzabayeva B, Zerekbay Alikulov B, Shabtai Cohen C,

Zion Shemer C and Moshe Sagi A,D
A
Albert Katz Department of Dryland Biotechnologies, Jacob Blaustein Institutes for Desert Research,
Ben-Gurion University, PO Box 653, Beer Sheva 84105, Israel.
B
L.N Gumilyov Eurasian National University, Department of Biology and Biotechnology,
5 Munaitpasov St., 473021, Astana, Kazakhstan.
C
Ramat Negev Desert Agro-Research Station, Halutza 85515, Israel.
D
Corresponding author. Email: gizi@bgu.ac.il

This paper originates from a presentation at the COST WG2 Meeting ‘Putting halophytes to work – genetics, biochemistry
and physiology’ Hannover, Germany, 28–31 August 2012.

Abstract. Aster tripolium L. is a salt marsh halophyte that has recently gained interest as a cash crop vegetable. Leaf yield
and quality were investigated in plants grown with salinity in experiments with Perlite in pots and in plots on dune sand. Plants
were repetitively harvested in a 14-day cycle. A. tripolium irrigated with 50 mM NaCl exhibited the highest yield when
grown in pots, whereas in the plot experiment no significant differences in biomass accumulation occurred up to 80 mM
NaCl in the irrigation water. Chemical leaf composition changed with salinity, exhibiting higher levels of electrical
conductivity, total soluble solutes and the non-enzymatic antioxidant compounds ascorbic acid and polyphenols compared
with control plants grown without NaCl supplementation. Using the repetitive harvest regime, leaf chlorosis occurred, a
symptom shared by deficiencies in either nitrogen or iron. Comparative applications of five iron chelate formulations in
plants grown with 50 mM NaCl in pots revealed improved leaf colour and chlorophyll content for only two of the applied
Fe-chelates. Concomitantly with leaf colour restoration, the activity of nitrate reductase, the first enzyme during nitrate
assimilation, which requires heme-iron for its proper function, increased 3-fold as a result of the iron treatment in the plot
experiment. Importantly, the enhancement of nitrate reductase activity was associated with a considerable decrease in the
leaf nitrate concentration. Therefore, we concluded that iron deficiency, in addition to leaf chlorosis, reduces A. tripolium
leaf quality as a vegetable by increasing the leaf nitrate content. Furthermore, nitrate reductase (NR) activity levels in
A. tripolium leaves may act as an indicator of iron deficiency that manifests itself as reduced nitrate content owing to the
higher NR activity upon proper iron nutrition. These results demonstrate the importance of salinity level and the
application of an appropriate iron-chelating formulation to generate marketable yields of Aster tripolium leafy
vegetable when grown commercially on dune sand.

Additional keywords: antioxidants, cash crop cultivation.

Received 21 November 2012, accepted 21 February 2013, published online 19 March 2013

Introduction Salicornia persica H. Akhani’ and Sarcocornia fructicosa (L.)

Dwindling fresh irrigation water resources and increasing soil
salinisation are ongoing problems in arid and semiarid areas.
A promising approach to utilise salt-contaminated soils and low
quality saline irrigation water is the commercial cultivation of
naturally salt-tolerant plants as leafy vegetables (Glenn et al.
1999). However, halophytes can be converted into economically
valuable crops only if a high yield potential is realised (Rozema
and Flowers 2008). Halophytes have growth rates comparable
to those of conventional crops and they only show reductions in
yield at salinity levels that are much higher than those of major
agricultural crops (Rozema and Flowers 2008). For example,
Journal compilation  CSIRO 2013
A. J. Scott. were introduced as leafy vegetables irrigable with
water at salinities up to that of seawater: even at such extreme
salinity, not only did plant yields exceed those of other crops, but
also the nutritional value of the vegetable was enhanced over
crops irrigated with non-saline by the induction of secondary
metabolites with antioxidant capacity (Ventura et al. 2011;
Ventura and Sagi 2012).
Aster tripolium L. (sea aster) is a biennial to short-lived
perennial halophyte commonly found in the coastal areas and
sometimes in the inland salt marshes of north-western Europe
(Clapham et al. 1942). As a typical halophyte, A. tripolium can
www.publish.csiro.au/journals/fpb
B Functional Plant Biology
grow at salt levels of up to 300 mM NaCl, which is equivalent
to about two-thirds the strength of seawater (Shennan et al.
1987; Sági and Erdei 2005). Recently, interest in the
commercial cultivation of A. tripolium as a vegetable has
grown substantially (Lieth et al. 2000). Owing to their mild
salty taste and high nutritional value, A. tripolium leaves are
valued as gourmet health food (Wagenvoort et al. 1989).
The salt tolerance of halophytic plants relies for the most
part on controlled ion uptake and compartmentalisation of
Na+, K+, and Cl in the vacuoles together with the synthesis
of organic compatible solutes in the cytoplasm (Flowers and
Colmer 2008). In addition to osmotically active molecules such
as sucrose, an array of small, non-enzymatic compounds with
antioxidant properties – such as ascorbate, carotenoids and
polyphenols – are amassed in the plant cells to compensate for
the oxidative stress caused by salinity (Flowers and Colmer
2008; Geissler et al. 2010), being potentially valuable for
the nutritional quality of the vegetable product (Cieslik et al.
2006).
Before a halophyte can be introduced for commercial
cultivation as a vegetable, new agro-techniques must be
developed (Glenn et al. 1999; Lieth et al. 2000). For example,
a non-selective repetitive harvest system was applied to
Salicornia spp, where fast re-growth after shoot removal
allowed for more than five harvests (Ventura et al. 2010,
2011). Frequent removal of the plant canopy during sequential
harvesting implies the need for a suitable nutrient supply to
avoid reduced biomass accumulation (Ventura et al. 2010) and
the appearance of symptoms of leaf chlorosis that may reduce
produce quality.
Salinisation may result in micronutrient deficiency that can
derive from both, soil alkanisation and ion competition (Grattan
and Grieve 1998). Hence, leaf chlorosis, which occurs in plants
irrigated with saline water, could be attributed either to
microelement (i.e. iron) or nitrate deficiency (Imsande 1998).
The enzyme nitrate reductase (NR) catalyses the first and rate-
limiting step – the reduction of nitrate to nitrite – in plant nitrate
assimilation (Crawford 1995). NR in plants consists of three
functional domains: the N-terminal domain associated with
the molybdenum cofactor (Moco), the central heme-binding
cytochrome b5, and the C-terminal flavine adenine
dinucleotide (FAD)-binding domain. Critical to its activity,
NR requires iron in the form of iron-heme to function.
Additionally, the biosynthesis of Moco, also essential for NR
activity, involves the complex interaction of six proteins in a
four-step process that includes iron indispensably (Mendel
2007). Hence, NR activity levels may be a good indicator of
iron deficiency in plants, as was suggested for young maize plants
where a reduction in NR activity was observed to be a direct
effect of iron deficiency (Nenova and Stoyano 1995). Moreover,
since leaf chlorosis is a symptom shared by nitrate and iron
deficiencies (Imsande 1998), measuring NR activity levels
together with the leaf tissue nitrate content provides a method
for distinguishing the cause of the chlorosis. For example, low
NR activity together with high leaf nitrate content may indicate
that leaf chlorosis is due not to a nitrate deficiency, but rather to
an iron deficiency.
This study investigated the effect of salinity level and
Fe-chelate application on the yield performance and chemical
Y. Ventura et al.
leaf quality of A. tripolium, a halophytic vegetable crop suitable
for cultivation on sand dune soils with saline irrigation.
Materials and methods
Plant material
Seeds of Aster tripolium L. originating from the Atlantic coastal
region of the Netherlands and Belgium were germinated in a
growth room on a double layer of filter paper (Whatman No. 1,
Maidstone, Kent, UK) in 16-cm diameter Petri dishes at 25C
under a 16-h light regime. The seeds germinated within a 3 week
period. Germinated seedlings were transplanted into seedling
trays (cell size 3  3  8 cm) filled with either Perlite (Agrekal
Habonim Industries Ltd, Moshav Habonim, Israel) for pot
experiments or commercial potting mixture HR-1 (composed
of peat, tuff, and synthetic sponge, including slow release
fertiliser; Shacham Givat Ada Ltd, Givat Ada, Israel) for plot
experiments. After an additional month of cultivation in a
temperature-controlled greenhouse (2033C), plants were
transferred either to their final pot size (14 cm diameter, 12 cm
high) or to the plastic-house plots as described below.
Growing conditions
All pot experiments were conducted in Beer Sheva (31140 N,
34470 E), Israel, in a temperature controlled, plastic-covered
greenhouse that includes automatic heating/cooling system to
keep the internal temperature between 20 and 33C. Midday
photosynthetic photon flux density in the greenhouse was
300–500 mmol m–2 s–1. When plants attained sizes of 3–4 true
leaves, they were transferred from seedling trays to 14 cm
diameter plastic pots (12 cm high) filled with Perlite.
The plot experiments were conducted at the Ramat HaNegev
(30520 N 34470 E) experimental station, Israel, in plastic-covered
greenhouses (27  6 m) located on native sand dunes. When
they were ~4 weeks old, A. tripolium plants were transplanted
in sand-dune soil (96% sand, 0.8% silt, 3.1% clay, <0.1% organic
matter, pH 8). Because of the dune soil composition, the soil
pH values did not change after salt treatment applications. Each
plot sized 1  2.25 m and 12 plots were placed in a row resulting
in 27 m length. Salt treatments and irrigation were supplied via
a drip irrigation system.
Salinity levels experiment
In the pot experiments, salinity treatments were applied by
irrigating the plants upon their requirement (see below), every
second day, with the respective salt concentration (0, 50 or
100 mM NaCl) supplemented with commercial N-P-K
fertiliser (20-20-20 + microelements (MEs); Haifa Chemicals
Ltd, Haifa, Israel) at a final concentration of 2.85 mM
nitrogen. Each salinity treatment was replicated three times,
which resulted in a total of nine pots. The experiment was
replicated twice. Five repetitive harvests were conducted
during the 10-week experiment. Leaf fresh biomass in grams
per plant was determined after each harvest.
In the plot experiment, A. tripolium plants were planted at
final density of 50 plants per square meter. Plots were irrigated
with three salt concentrations: control without added salt and
salt treatments of 50 and 80 mM NaCl, supplemented with
commercial N-P-K fertiliser (5-3-8 + MEs; Haifa Chemicals
Fe-chelate formulation in Aster tripolium
Ltd) at a final concentration of 5.7 mM nitrogen + 42 9mM Fe,
218 mM Mn, 92 mM Zn, 14 mM Cu, and 6.6 mM Mo in the
irrigation water. Plants were irrigated three times per day.
Irrigation requirements were determined according to our
experience allowing 30% of the applied water to leach out in
the pot experiment and applying 130% of the evapotranspiration
estimated by the pan evaporation method according Grismer
et al. (2002) in the plastic-house plot experiments.
During the summer months, the plastic-house was covered
by shading net, which reduced the midday photosynthetic
photon flux density to ~500 mmol m–2 s–1. The experiment was
conducted over 30 weeks and a total of 15 harvests were
performed. Determined parameters were: total and marketable
yield (kg m–2 year–1) and leaf constituents, such as pH, electrical
conductivity (EC) (dS m–1), total soluble solids (TSS) (%),
ascorbic acid (mg 100 g–1 FW), polyphenols (mg 100 g–1 FW),
b-carotene (mg g–1 FW), chlorophyll (mg g–1 FW) (for a detailed
description of the methods see below).
After the appearance of chlorosis in all salt treatments, a
single application of Fe chelated as EDDHA (4 g m–2) was
applied manually in three replication within the salinity
treatments.
Fe-chelate experiment
Fe-chelates were given at a salinity level of 50 mM NaCl and
a concentration of 447 mM iron in a half-strength Hoagland
solution in a pot experiment (Hoagland and Arnon 1938).
Plants were grown in Perlite and the pH of the irrigation
solution was adjusted to pH 7 and 8. The following
Fe-chelateswere tested: (i) ethylenediamine-N,N0 -bis (2-
hydroxyphenylacetic acid) (EDDHA); (ii) ethylenediamine
(o-hydroxy-p-methylphenylacetic) acid (EDDHMA);
(iii) ethylenediamine (2-hydroxy-5-sulfophenylacetic) acid
(EDDHSA); (iv) hydroxy acetate; and (v) a mixture of equal
quantities of EDDHMA, Diethylenetriamine pentaacetic acid
(DTPA) and EDTA (EDTA). Experiments were completely
randomised, and each treatment was replicated five times
resulting in a total of 25 pots. The experiment lasted for
8 weeks, resulting in a sum of four harvests. Leaf fresh
biomass in grams per plant was determined after each
harvest. Six weeks after treatment initiation chlorophyll was
extracted from the leaf samples and expressed in mg gram–1 FW.
Micro-elements experiment
The application of specific MEs was tested in a plot experiment
on sand dune soil using fresh tap water supplemented with
50 mM NaCl (salinity level of the local saline water) and with
commercial N-P-K fertiliser (5-3-8 + MEs; Haifa Chemicals
Ltd). Four different MEs combination treatments were given
in addition to the regular nutrient solution at the concentrations
recommended by the manufacturer: (i) Fe chelated as EDDHA
(applied concentration: 400 mM); (ii) a combination of MEs
chelated in EDTA, which include a little Fe (36 mM), but
mainly Mn (20 mM), Zn (8.6 mM), Cu (1.3 mM) and Mo
(5.6 mM); (iii) a mixture of (i) and (ii) representing the
concentrations of (i) and (ii) as a sum; (iv) control, fresh tap-
water, nothing added. MEs treatments were applied manually
after each harvest by evenly distributing 3 L of solution, at the
Functional Plant Biology C
designated concentration, per plot. Each treatment was
replicated four times. A sum of eight harvests was conducted
during the four months of cultivation. The following parameters
were tested: total and marketable yield (kg m–2 year–1),
chlorophyll content (mg g–1 FW), nitrate reductase activity
(nmol nitrite g–1 FW h–1), nitrate content (mg 100 g–1 FW).
Harvest regime and marketability
Plants were harvested for the first time as soon as their leaves
reached commercial size (5–10 cm tall and 2–3 cm wide) by
cutting the complete plant canopy ~3 cm above ground. After
plant re-growth, leaves were harvested in the same manner at a
14 days interval. After the harvest, leaf samples were sorted by
size and rated for their marketability, such that only green, turgid
leaves of commercial size without any physical damage (e.g. leaf
cuts caused by the previous harvest) were counted as marketable
(see Fig. S1, available as Supplementary Material to this paper).
Nitrate reductase activity
The activity of NR, an enzyme that catalyses the reduction of
nitrate to nitrite, was determined in vivo in freshly collected
samples following the procedure described by Ventura et al.
(2010) with minor modifications. Briefly, 6–8 leaf strips ~15 mm
long and 5 mm wide were cut out of the middle part of the first
fully developed leaf and used to determine NR activity. Samples
were vacuum infiltrated for 3 min in a reaction mixture containing
0.1 M KNO3 and 0.1% ethanol in 50 mM potassium phosphate
buffer (pH 7.5) at a 1 : 20 (w/v) ratio. The reaction was allowed to
proceed for 30 min at 30C in the dark and the levels of nitrite in
samples removed at the beginning (time 0) and at the end of the
assay were detected spectrophotometrically after the addition
of 1% sulfanilamide in a 1 : 1 (v/v) mixture of 3 M HCl and
0.02% N-1-naphthyl ethylenediamine at 540 nm. NaCl in the
range of the plants concentration did not interfere with the colour
formation during the assay. NR activity was expressed in
nmol nitrite g–1 FW h–1.
Metabolite determination
Immediately after collection, leaf samples were snap-frozen in
liquid nitrogen and stored at 80C until extraction. Frozen leaf
samples were homogenised on ice with a Polytrone (Kinematica,
Lucerne, Switzerland) at a ratio of 1 : 4 (w/v) with double
distilled water as described previously (Ventura et al. 2011).
The homogenate was centrifuged at 17 500i for 20 min at 4C,
filtered through Whatman filter paper No. 1, and then kept on
ice. The electrical conductivity in the supernatant (EC, dS m–1)
was measured with a conductivity meter (CyberSan 500 Con,
Eutech Instruments, Singapore), pH was detected with a pH
meter (pH211 microprocessor pH meter, Hanna Instruments,
Romania) and total soluble solids (TSS, %) were determined
with a digital refractometer (Atago PR-1, Atago Co. Ltd, Tokyo,
Japan). Ascorbic acid and nitrate were determined with a RQflex
10 instrument (Merck KGaA, Darmstadt, Germany) using the
Reflectoquant strips for the ascorbic acid test (1.16981.0001;
measuring range 25–450 mg L–1) and nitrate (1.16971.0001;
measuring range 5–225 mg L–1) respectively. Importantly, the
Cl– level in the diluted plant extract was below the threshold
limit of Cl– interference for nitrate determination.
D Functional Plant Biology Y. Ventura et al.

Chlorophyll and b-carotene were extracted and determined in Harvest 1 Harvest 2 Harvest 3 Harvest 4 Harvest 5
dark conditions. The 100-mg samples frozen in liquid nitrogen b a b
40
were crushed with glass beads (Sigma-Aldrich, Inc., St Louis,

Accumulated yield (g plant–1)

MO, USA) twice for 14 s in an electrical tissue grinder (Softly 8, 35
de Götzen, S.r.l., Olgiate Olona, Italy). After the addition of 30
80% acetone, the extracts were centrifuged at 15 000g for 10 min 25
and the supernatant was collected. The remaining pellets were
20
mixed again with 80% acetone, centrifuged and the supernatant
was added to the supernatant previously collected. This step 15
was repeated twice resulting in a final dilution of 1 : 10 (w/v). 10
After a further 1 : 4 (v/v) dilution of the extract, chlorophyll 5
and b-carotene were determined with a spectrophotometer
(JASCO, V-530, JASCO Inc., Mary’s Court, Easton, MD, 0
0 mM 50 mM 100 mM
USA) at 652 nm and 480 nm respectively (Arnon 1949; NaCl concentration
Ben-Amotz et al. 1988).
Total polyphenols, non-enzymatic antioxidant compounds, Fig. 1. Effect of salinity on yield accumulation of Aster tripolium. The fresh
were extracted from shoot material according to Singleton and biomass was accumulated in five subsequent harvests, conducted with a
Rossi (1965) in 0.4 M phosphate buffer (pH 7) at a ratio of 1 : 3.75 14 day cropping regime, during 10 weeks of cultivation. Values represent
(w/v). The extract was centrifuged at 22 500g for 20 min means  s.e. (n = 6). Values followed by different letters indicate significant
differences between treatments at P < 0.05.
at 4C. Aliquots of the collected supernatant were incubated
with Folin-Cioclateur’s phenol (Sigma) and 7.5% Na2CO3 for
2 h at 30C in a water bath. Absorbance was determined Table 1. Yield and leaf constituents of Aster tripolium grown under
spectrophotometrically (JASCO, V-530, Easton, MD, USA) at greenhouse plot conditions (control, 50 and 80 mM NaCl)
a wavelength of 765 nm. Values are the means  s.e. (n = 4–6). Values followed by different letters are
significantly different at P < 0.05; n.d., not determined

Statistical analysis
Irrigation water salinity
All datasets were tested for normality and homogeneity of the Control 50 mM NaCl 80 mM NaCl
variance applying the Shapiro-Wilk and Lilliefors tests and the
F-test, respectively, at critical values of P < 0.05 using XLSTAT Total yield 16.5 ± 0.7a 18.3 ± 1.2a 18.6 ± 1.2a
(kg m–2 year–1)
2012 software. Subsequently, significant differences between
Marketable yield 10.0 ± 0.5a 11.5 ± 1.1a 11.7 ± 1.0a
treatments were analysed by single factor analysis of variance (kg m–2 year–1)
(ANOVA) for all pot and the micro-elements experiment using pH 6.11 ± 0.03a 6.10 ± 0.07a 6.22 ± 0.02a
the JumpIn 5.0.1a software package (SAS, Cary, NC, USA). EC (dS m–1) 20.3 ± 0.39c 22.5 ± 0.43b 24.2 ± 0.57a
When ANOVA indicated significance, we compared treatment TSS (%) 3.26 ± 0.07a 3.40 ± 0.27a 3.60 ± 0.11a
means according to the Tukey-Kramer HSD with the same Ascorbic acid 13.7 ± 1.7a n.d. 15.1 ± 1.4a
software. (mg 100 g–1 FW)
Polyphenols 31.6 ± 2.2b 37.0 ± 2.7b 45.9 ± 2.2a
Results (mg 100 g–1 FW)
b-carotene (mg g–1 FW) 36.9 ± 5.8a 32.6 ± 2.9a 30.5 ± 3.1a
Effect of salinity on yield accumulation Chlorophyll (mg g–1 FW) 198.6 ± 36.8a 153.6 ± 25.5a 140.0 ± 19.0a
Greenhouse pot experiment
A non-selective repetitive cropping regime was tested in a between the salt treatments, when growing the plants in native
pot experiment in which plants were irrigated with one of three sandy soil. An average total yield of 17.8 kg m–2 was achieved at
different salt concentrations. Complete canopy removal resulted the salt treatments (Table 1). Importantly, the mean marketable
in the fast re-growth of newly developed young leaves that yield, accounted for 62.1% of the total yield.
reached commercial size within 2 weeks, thus, allowing
additional harvests. After five harvests, a significantly higher
yield was obtained at 50 mM NaCl and similar biomass harvests Effect of salinity on leaf constituents
were found for 100 mM NaCl and in the control treatment
Irrigation with saline water altered some, but not all of the
(0 mM NaCl) (Fig. 1).
chemical quality parameters in the leaves of A. tripolium
(Table 1). In particular, EC values, which are a measure of the
Greenhouse plot experiment total accumulated salts in the leaf tissue and provide an indication
Three irrigation water salinities were tested in a greenhouse about the desired salty taste, increased by 16% as a result of
plot experiment. Plants were irrigated with salinised water primarily Na+ and Cl accumulation (Table 1). An assessment of
comprising fresh tap water supplemented with 50 or 80 mM compatible organic solutes, such as sugars, is given by the TSS
NaCl compared with a control without NaCl addition. No value, which showed the absence of a significant difference
significant differences in total and marketable leaf yield but the increasing tendency when plants were irrigated with
accumulation during a harvest period of 1 year were observed increasing salinity. The total polyphenol content exhibited
Fe-chelate formulation in Aster tripolium
31% enhancement compared with the control levels, but
salinity had no significant effect on the antioxidant ascorbic
acid content in the leaves. The b-carotene and chlorophyll
contents showed no significant changes with salinity.
Chlorosis appearance after multiple harvests
During the experiment, leaf chlorosis appeared irrespective of the
salinity treatments, which was characterised by leaf chlorophyll
concentration. After a single iron application, chelated as Fe-
EDDHA, the chlorosis disappeared and an approximate 2-fold
increase in chlorophyll concentration was observed (Fig. 2).
Since no differences were found between the salt treatments,
the subsequent iron-chelate investigations were conducted on
the 50 mM salt level.
Effect of Fe-chelate formulations on biomass accumulation
and chlorophyll level in A. tripolium leaves
To clarify the effect of Fe-chelate formulations on plant
productivity, we tested in a pot experiment five commercially
available Fe-chelate formulations and the effectiveness of each
on biomass accumulation and chlorophyll concentration in
repetitively harvested A. tripolium plants. Plants treated with
Fe-hydroxyacetate accumulated the lowest biomass, followed
by the EDDHSA treatment (Fig. 3a). Significantly higher
yields were obtained in plants fertilised with iron formulated
in EDDHA and in the EDTA-EDDHMA-DTPA combination.
The EDDHMA treatment did not differ significantly from the
former treatments in terms of biomass accumulation. In contrast,
the best leaf colouring, expressed as high chlorophyll levels, was
observed only in the EDDHA and EDDHMA treatment
formulations (Fig. 3b). No significant differences could be
determined between the pH levels; neither for biomass
accumulation nor chlorophyll content.
Effect of microelement application on marketable leaf
attributes
To exclude the possibility that a deficiency in a microelement
other than iron may be the cause of leaf chlorosis, we tested the
application of Fe-EDDHA chelate with or without a solution
–Fe EDDHA +Fe EDDHA
250
b a b a b a
Chlorophyll (μg g–1 FW)
200
150
100
50
0 post-harvest re-growth phase, thereby compensating for the
Control 50 mM 80 mM
NaCl concentration
Fig. 2. The effect of a single Fe-chelate application (Fe-EDDHA) at
increasing irrigation water salinities on the leaf chlorophyll concentration.
Values represent means  s.e. (n = 3). Values followed by different letters
indicate significant differences between treatments at P < 0.05.
Functional Plant Biology E
containing additional MEs in a field plot experiment at optimal
salinity. Treatment without the addition of MEs or Fe-EDDHA
chelate served as a control. Leaf chlorosis occurred in the control
plants, which had ultimately lower total yields (Table 2; Fig. 4).
The marketable yield of control plants was reduced significantly
and comprised only 40% of the total yield (Table 2). Fertilising
plants with a combination of MEs, including Fe-EDTA, had no
effect compared with the control on either yield accumulation or
marketable yield (Table 2). However, although the application of
iron chelated in an EDDHA complex did not change total biomass
accumulation, the percentage of marketable leaves increased to
87%, more than double the marketable yield of the control
treatment. An additional supplement of the MEs combination
to the EDDHA treatment did not further improve yield (Table 2).
The marketability of A. tripolium leaves was mostly related to
leaf colour, since control plants and plants fertilised with MEs
exhibited leaf chlorosis and low chlorophyll contents. However,
plants to which Fe-EDDHA was added had green leaves
accompanied by enhanced chlorophyll content (Fig. 4).
Iron deficiency results in reduced nitrate reductase activity
and high nitrate content in A. tripolium leaves
NR activity, measured to determine whether low iron availability
was the limiting factor for the NR enzyme, was 3-fold higher in
Fe-EDDHA irrigated plants than in control plants. Fertilising
plants with the added MEs mixture, which includes iron as EDTA,
had no effect on NR activity (Fig. 5a).
The lower rate of NR activity in control plants led to an
accumulation of nitrate in the leaves, thus, further supporting the
notion that a nitrogen shortage was not the cause of the appearance
of yellow leaves (Fig. 5b). Accordingly, higher NR activity
reduced the leaf nitrate content significantly in the EDDHA
treatment. We noted that leaves treated with MEs accumulated
nearly double the amount of nitrate than the control treatment
(Fig. 5b).
Discussion
Growing a halophyte using irrigation water that is much more
saline than that tolerated by conventional agricultural crops
requires novel agrotechnical adaptations to ensure the plant’s
success as a commercial cash crop (Boyko 1966). We
successfully applied a repetitive harvest regime during
cultivation of A. tripolium (Fig. 1) that entailed complete
canopy removal, after which the leaves quickly grew back to
allow additional harvests at ~2-week intervals. Under such a
harvest regime, the highest yields – doubling of the harvested
fresh biomass – were obtained in a pot experiment from plants
irrigated with salinity levels of 50 mM NaCl (Fig. 1), a finding
that demonstrates the plants’ halophytic character (Ramani et al.
2006). For plants grown at higher salinity levels, such as
100 mM, harvest intervals can be extended to provide a longer
slower growth rates typical at the higher salinities. Gallagher
(1985) reported yields equivalent to 2.1 kg m–2 fresh biomass
per harvest of Atriplex triangularis, a potential fresh vegetable
crop for human consumption, when grown at high salinity.
Unfortunately, no information was available about the time
intervals between harvests. Nevertheless, our long-term
F Functional Plant Biology Y. Ventura et al.

(a)
Harvest 1 Harvest 2 Harvest 3 Harvest 4

25
Accumulated FW (g plant–1)

20

15

10

(b)
700 pH 7 pH 8
Chlorophyll (μg g–1 FW)

600

500

400

300

200

100

Fig. 3. The effect of Fe-chelate application on (a) biomass accumulation during four consecutive harvests and
(b) the chlorophyll concentration. Values represent means  s.e. (n = 5). Upper case letters indicate the differences
between Fe-chelates; lower case letters indicate the differences between the two pH levels at each specific Fe-
chelate at P < 0.05.

Table 2. Total and marketable yields as affected by MEs and Fe-EDDHA values are known to be related to the low solubility of
chelate treatments micronutrients to the plant and as a consequence, plants grown
Yields were calculated on the accumulation based on a one year growing cycle. in soils with high pH values often experience deficiencies in these
Values are means  s.e. (n = 4). Table entries identified by different letters are microelements (Lindsay 1984; Grattan and Grieve 1998). In view
significantly different at P < 0.05 of the symptoms of chlorosis that appeared in the leaves after
repetitive harvesting, we anticipated the need of Fe application
Total yield Marketable yield and thus tested several commercially available iron-chelate
(kg m–2 year–1) (kg m–2 year–1) fertilisers at pH 7 and 8. The limit for iron selectivity in the
Control 13.5 ± 2.1a 5.4 ± 1.2b
commonly used EDTA is below pH 6.3, while EDDHA chelates
MEs 14.6 ± 3.4a 6.3 ± 1.3b iron over a pH range of 4–9 (Sekhon 2003). Only the iron
Fe-EDDHA 14.4 ± 3.3a 12.6 ± 1.1a formulation in EDDHMA led to commercially acceptable leaf
Fe-EDDHA + MEs 18.3 ± 1.9a 14.0 ± 1.4a colour and chlorophyll content that were comparable to the results
with EDDHA (Fig. 3). Nevertheless, a combination of three
different chelates EDTA, EDDHMA, and DTPA, the last of
greenhouse plot experiment, during which plants were harvested which according to Sekhon (2003) binds iron up to a pH of
every 14 days for 7.5 months, emphasised the potential of 7.5, led to biomass accumulation similar to that of either of the two
A. tripolium to achieve high yields reaching an annual total of preferred chelates (EDDHA, EDDHMA). Despite the high total
18.6 kg m–2 when it was grown in sand dune soils at salinity yield detected, the chlorophyll concentration was significantly
levels of up to 80 mM NaCl (Table 1), highlighting the lower as compared with the EDDHA and EDDHMA treatments
importance of large-scale investigations. (Fig. 3), thereby limiting the marketability of A. tripolium leaves.
Soils that are available and suitable for saline irrigation in EDDHA is reportedly highly selective for iron over a broad pH
arid and semiarid regions are often alkaline, and high soil pH range from 4 to 9 (Sekhon 2003), and therefore, can make iron
Fe-chelate formulation in Aster tripolium Functional Plant Biology G

600 the current experiments. At pH values higher than 6.8, EDTA

preferably reacts with Ca2+ (Sekhon 2003), thus, making it an
Chlorophyll (μg g–1 FW)

500 ineffective iron chelate for A. tripolium grown at high pH

400
conditions. Therefore, although plants were fertilised with
commercial N-P-K fertiliser, including supplemental iron
300 (formulated as EDTA) and microelements, during cultivation
on sand dune soil at medium saline conditions (50 mM NaCl),
200
leaf chlorosis occurred after several harvests, thus, reducing
b b a a leaf marketability (Fig. 4; Table 2). Conversely, plants
100
receiving equal amounts of iron formulated in different
0 chelates, showed significant differences in leaf colour and
Control ME EDDHA EDDHA + ME
chlorophyll concentration (Fig. 3), hence, not the iron
concentration in the irrigation water, but probably the better
iron availability to the plant using EDDHA and EDDHMA
chelates resulted in the greener leaf colour. In addition, a
single application of iron chelated in EDDHA of 4 g m–2
Fig. 4. Chlorophyll concentration and leaf appearance as affected by the between two successive harvests, was able ‘to paint’ the plants
application of microelements (MEs), Fe-EDDHA chelate, or a combination of green within one week (Figs 2, S2). The observed enhancement
both. A treatment without the addition of MEs or Fe-EDDHA chelate served in leaf colour in the Fe-EDDHA treatment coincided further
as a control. Values represent means  s.e. (n = 12). Means indicated by with a shift in the proportion of leaves exhibiting a larger leaf
different letters are significantly different at P < 0.001. size (data not shown), hence, further improving marketability.
The additional iron chelated as EDDHA not only restored leaf
colour, but it also enhanced the NR activity of A. tripolium leaves
800 (Figs 4, 5a). The nitrate reducing capacity in a plant system
(a)
NRA (nmol g–1 FW hour–1)

700 depends inter alia on the level of functional NR, which is

600 determined by the amount of total NR polypeptide and by the
availabilities of the cofactors and of the metals iron and
500
molybdenum (Campbell 1999). Accordingly, NR activity was
400 found to be 3-fold higher in Fe-EDDHA treated plants than in
300 non-treated plants, which, in turn, probably increased iron
b b a ab
availability. Thus, NR activity may be a reliable tool for
200
determining accessibility to iron.
100 High NR activity, as demonstrated in transgenic Nicotiana
0 plumbaginifolia plants, reduces leaf nitrate levels (Crawford
1995; Foyer et al. 1993). Indeed, the increase in NR activity
200 significantly reduced the nitrate content of A. tripolium leaves
180
(b) treated with EDDHA (Fig. 5) to levels comparable to those
detected in vegetables classified as low nitrate-accumulators
NO3 (mg 100g–1 FW)

160
140 ranging from 200 to 500 mg nitrate per kg fresh vegetable
120
(Santamaria 2006). This result further supports the conclusion
100 b a c bc
that the leaf chlorosis that appeared after the repetitive harvesting
80
is a result of low Fe availability and NR activity rather than low
nitrate level in the plant tissue. We therefore concluded that iron
60
deficiency, in addition to leaf chlorosis, may further reduce
40
A. tripolium leaf quality as a vegetable by increasing the leaf
20
nitrate content.
0
Control ME EDDHA EDDHA + ME Devoid of salt excreting leaf structures, the halophyte
A. tripolium depends on Na+ and Cl compartmentalisation
Fig. 5. Effects of Fe-EDDHA and MEs treatments on (a) NR activity and into the vacuole to survive salt stress (Shennan et al. 1987;
(b) nitrate concentration. Values represent means  s.e. (n = 4–9). Means Ueda et al. 2003). Indeed, the plants’ EC values (Table 1)
indicated by different letters are significantly different, P < 0.01. were positively influenced by salt treatment and produced the
vegetable’s salty taste that is sought after by the consumer
available to A. tripolium plants growing in a broad range of soil (Wagenvoort et al. 1989). A significant increase in TSS due to
pH, including alkaline soils. Compared with the control, no the synthesis of sugars, being osmoprotectants, to adjust the
significant effects on leaf colour, chlorophyll content, or osmotic balance, was not found in A. tripolium leaves
marketable yield were noticeable in the treatment with Fe- (Table 1) (Türkan and Demiral 2009).
EDTA, which was applied together with the microelement The degree of oxidative damage suffered by a plant exposed
mixture (Table 2; Fig. 4). These results may have been caused to abiotic stresses is controlled by the plant’s capacity to protect
by the alkaline pH values of ~8 detected in the dune soil used in itself from oxidative agents (Flowers and Colmer 2008). Salt
H Functional Plant Biology
tolerance was found to promote an increased antioxidant
capacity and higher polyphenol content in the halophyte
Cakile maritima (Ksouri et al. 2007). Ascorbic acid, an
additional non-enzymatic antioxidant, plays an important role
in scavenging reactive oxygen species, whose production is
elevated during both biotic and abiotic stresses, including
salinity (Noctor and Foyer 1998). Moreover, higher ascorbate
levels and antioxidant enzyme activities in the leaf tissue were
associated with higher salt tolerance as a result of the NaCl-
induced alleviation of oxidative damage in one accession of
C. maritima, (Ksouri et al. 2007). Seen in this light, the
significant polyphenol content enhancement in A. tripolium
leaves in response to salinity found in our study not only
increased the plants’ salt tolerance, but also improved the
vegetable’s chemical quality (Table 1). A reduction in
chlorophyll and b-carotene content with increasing salinity, a
commonly observed phenomenon in both glycophytes and
halophytes (Ali et al. 2004; Sai Kachout et al. 2009) was not
observed in A. tripolium leaves (Table 1), supporting its salt
tolerance and the good performance of green marketable
produce also at elevated irrigation salinities.
Conclusions
In view of approaching commercial cultivation we investigated
pot and plastic-house plot experiments applying commonly
utilised agrotechnical practices. We showed that moderate
salinity levels in the irrigation water enhanced yield and
improved quality of the A. tripolium leafy vegetable harvested
using a non-selective repetitive harvest regime of 2–3 weeks
intervals. Additionally, we demonstrated that iron deficiency
reduced NR activity, which was associated with higher leaf
nitrate content. We have also shown that Fe-chelate
formulation is important to prevent the leaf chlorosis that
appears during intensive A. tripolium cultivation on dune sand.
Acknowledgements
The research was supported partly by a grant from the Chief Scientist, Ministry
of Agriculture and Rural Development, Israel grant no. 884–0186–06 and
by research grant No. TA-MOU-02-CA21–026, funded by the USA-Israel
Cooperative Development Research Program, Bureau for Economic Growth,
Agriculture, and Trade, USA Agency for International Development. The
authors gratefully thank Dr Levi Gheber for his support regarding the
statistical analysis and Mrs Michal Amichay for her excellent technical
assistance at the Ramat HaNegev Research Station.
References
Ali Y, Aslam Z, Ashraf MY, Tahir GR (2004) Effect of salinity on
chlorophyll concentration, leaf area, yield and yield components of
rice genotypes grown under saline environment. International Journal
of Environmental Science and Technology 1, 221–225.
Arnon DI (1949) Copper enzymes in isolated chloroplasts.
Polyphenoloxidase in Beta vulgaris. Plant Physiology 24, 1–15.
doi:10.1104/pp.24.1.1
Ben-Amotz A, Lers A, Avron M (1988) Stereoisomers of b-carotene and
phytoene in the alga Dunaliella bardawil. Plant Physiology 86,
1286–1291. doi:10.1104/pp.86.4.1286
Boyko H (Ed.) (1966) ‘Salinity and aridity: new approaches to old problems.
Monographiae Biologicae Vol. XVI.’ (Dr W Junk: The Hague, The
Netherlands)
Y. Ventura et al.
Campbell WH (1999) Nitrate reductase structure, function and regulation.
Bridging the gap between biochemistry and physiology. Annual Review
of Plant Physiology and Plant Molecular Biology 50, 277–303.
doi:10.1146/annurev.arplant.50.1.277
Cieslik E, Greda A, Adamus W (2006) Contents of polyphenols in fruits
and vegetables. Food Chemistry 94, 135–142. doi:10.1016/j.foodchem.
2004.11.015
Clapham AR, Pearsall WH, Richards PW (1942) Biological flora of the
British Isles. Aster tripolium L. Journal of Ecology 30, 385–395.
doi:10.2307/2256580
Crawford NM (1995) Nitrate: nutrient and signal for plant growth. The
Plant Cell 7, 859–868.
Flowers TJ, Colmer TD (2008) Salinity tolerance in halophytes. New
Phytologist 179, 945–963. doi:10.1111/j.1469-8137.2008.02531.x
Foyer CH, Lefebvre C, Provot M, Vincentz M, Vaucheret H (1993)
Modulation of nitrogen and carbon metabolism in transformed
Nicotiana plumbaginifolia mutant E23 lines expressing either
increased or decreased nitrate reductase activity. Aspects of Applied
Biology 34, 137–145.
Gallagher JL (1985) Halophytic crops for cultivation at seawater salinity.
Plant and Soil 89, 323–336. doi:10.1007/BF02182251
Geissler N, Hussin S, Koyro HW (2010) Elevated atmospheric CO2
concentration enhances salinity tolerance in Aster tripolium L. Planta
231, 583–594. doi:10.1007/s00425-009-1064-6
Glenn EP, Brown JJ, Blumwald E (1999) Salt tolerance and crop potential of
halophytes. Critical Reviews in Plant Sciences 18, 227–255. doi:10.1016/
S0735-2689(99)00388-3
Grattan SR, Grieve CM (1998) Salinity-mineral nutrient relations in
horticultural crops. Scientia Horticulturae 78, 127–157. doi:10.1016/
S0304-4238(98)00192-7
Grismer ME, Orang M, Snyder R, Matyac R (2002) Pan evaporation to
reference evapotranspiration conversion methods. Journal of Irrigation
and Drainage Engineering 128, 180–184. doi:10.1061/(ASCE)0733-
9437(2002)128:3(180)
Hoagland DR, Arnon DI (1938) The water culture method for growing
plants without soil. California Agricultural Experiment Station Circle
347, 1–39.
Imsande J (1998) Iron, sulfur, and chlorophyll deficiencies: a need for an
integrative approach in plant physiology. Physiologia Plantarum 103,
139–144. doi:10.1034/j.1399-3054.1998.1030117.x
Ksouri R, Megdiche W, Debez A, Falleh H, Grignon C, Abdelly C (2007)
Salinity effects on polyphenol content and antioxidant activities in
leaves of the halophyte Cakile maritime. Plant Physiology and
Biochemistry 45, 244–249. doi:10.1016/j.plaphy.2007.02.001
Lieth H, Lohmann M, Güth M, Menzel U (2000) ‘Cash crop halophytes
for future halophytes growers.’ (Institute of Environmental System
Research: University of Osnabrück, Osnabrück, Germany)
Lindsay WL (1984) Soil and plant relationships associated with iron
deficiency with emphasis on nutrient interactions. Journal of Plant
Nutrition 7, 489–500. doi:10.1080/01904168409363215
Mendel RR (2007) Biology of the molybdenum cofactor. Journal of
Experimental Botany 58, 2289–2296. doi:10.1093/jxb/erm024
Nenova V, Stoyano I (1995) Physiological and biochemical changes in
young maize plants under iron deficiency: 2. Catalase, peroxidase, and
nitrate reductase activities in leaves. Journal of Plant Nutrition 18,
2081–2091. doi:10.1080/01904169509365046
Noctor G, Foyer C (1998) Ascorbate and glutathione: keeping active
oxygen under control. Annual Review of Plant Physiology and Plant
Molecular Biology 49, 249–279. doi:10.1146/annurev.arplant.49.1.249
Ramani B, Reeck T, Debez A, Stelzer R, Huchzermeyer B, Schmidt A,
Papenbrock J (2006) Aster tripolium L. and Sesuvium portulacastrum L.:
two halophytes, two strategies to survive in saline habitats. Plant
Physiology and Biochemistry 44, 395–408. doi:10.1016/j.plaphy.2006.
06.007
Fe-chelate formulation in Aster tripolium
Rozema J, Flowers T (2008) Crops for a salinized world. Science 322,
1478–1480. doi:10.1126/science.1168572
Sági B, Erdei L (2005) Adaptive response to high salinity of two subspecies
of Aster tripolium on different nitrogen sources. Acta Biologica
Szegediensis 46, 115–116.
Sai Kachout S, Ben Mansoura A, Jaffel K, Leclerc JC, Rejeb MN, Ouerghi Z
(2009) The effect of salinity on the growth of the halophyte Atriplex
hortensis (Chenopodiaceae). Applied Ecology and Environmental
Research 7, 319–332.
Santamaria P (2006) Nitrate in vegetables: toxicity, content, intake and
EC regulation. Journal of the Science of Food and Agriculture 86,
10–17. doi:10.1002/jsfa.2351
Sekhon BS (2003) Chelates for micronutrient nutrition among crops.
Resonance 8, 46–53. doi:10.1007/BF02834402
Shennan C, Hunt R, Macrobbie EAC (1987) Salt tolerance in Aster tripolium
L. II. Ionic regulation. Plant, Cell & Environment 10, 67–74. doi:10.1111/
j.1365-3040.1987.tb02081.x
Singleton VL, Rossi JA (1965) Colorimetry of total phenolics with
phosphomolybdic-phosphotungstic acid reagents. American Journal of
Enology and Viticulture 16, 144–158.
Türkan I, Demiral T (2009) Recent developments in understanding
salinity tolerance. Environmental and Experimental Botany 67, 2–9.
doi:10.1016/j.envexpbot.2009.05.008
www.publish.csiro.au/journals/fpb
Functional Plant Biology I
Ueda A, Kanechi M, Uno Y, Inagaki N (2003) Photosynthetic limitations of
a halophyte sea aster (Aster tripolium L.) under water stress and NaCl
stress. Journal of Plant Research 116, 65–70.
Ventura Y, Sagi M (2012) Halophyte crop cultivation: the case for Salicornia
and Sarcocornia. Environmental and Experimental Botany, in press.
doi:10.1016/j.envexpbot.2012.07.010
Ventura Y, Wuddineh WA, Ephrath Y, Shpigel M, Sagi M (2010)
Molybdenum as an essential element for improving total yield in
seawater-grown Salicornia europaea (L.). Scientia Horticulturae 126,
395–401. doi:10.1016/j.scienta.2010.07.015
Ventura Y, Wuddineh WA, Myrzabayeva M, Khozin-Goldberg I, Shpigel M,
Samocha TM, Sagi M (2011) Effect of seawater concentration on the
productivity and nutritional value of annual Salicornia and perennial
Sarcocornia halophytes as leafy vegetable crops. Scientia Horticulturae
128, 189–196. doi:10.1016/j.scienta.2011.02.001
Wagenvoort WA, van de Vooren JG, Brandenburg WA (1989) Plant
domestication and the development of sea starwort (Aster tripolium L.)
as a new vegetable crop. Acta Horticulturae 242, 115–122.