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The effect of growing spinach (Spinacia oleracea L.) at two light intensities on
the amounts of oxalate, ascorbate and nitrate in their leaves

Article  in  Journal of Horticultural Science and Biotechnology · July 2004

DOI: 10.1080/14620316.2004.11511814

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Journal of Horticultural Science & Biotechnology (2004) 79 (4) 606–609

The effect of growing spinach (Spinacia oleracea L.) at two light

intensities on the amounts of oxalate, ascorbate and nitrate in their
leaves

By S. PROIETTI2, S. MOSCATELLO1, A. LECCESE1, G. COLLA2 and A. BATTISTELLI1*

1
Istituto di Biologia Agroambientale e Forestale, Consiglio Nazionale delle Ricerche, Viale Marconi,
2, 05010 Porano (TR) Italy
2
Dipartimento di Produzione Vegetale, Università degli Studi della Tuscia, Via S. Camillo de Lellis,
01100 Viterbo (VT) Italy
(e-mail: a.battistelli@ibaf.cnr.it) (Accepted 14 March 2004)

SUMMARY
The effect of growing spinach at two light intensities on the content of oxalate, ascorbate and nitrate in their leaves
was determined. Plants were grown for five weeks in a growth cabinet under a 10 h light/14 h dark photoperiod and a
photon flux density of either 800 or 200 µmol quanta m–2 s–1. The content of oxalate, ascorbate and nitrate in the fourth
and fifth fully expanded true leaves was then determined at three times during the photoperiod. Spinach plants grown
at the lower light intensity showed decreased growth, a decreased leaf area per plant and an increased shoot to root
ratio. Leaves from plants grown under low light contained less ascorbate but more oxalate and nitrate. Our data show
that the nutritional quality of spinach is thereby decreased by growth in low light intensities and suggest that the
content of oxalic acid in leaves may depend on the rate of its catabolism.

S pinach (Spinacia oleracea L.) is a widely grown

commercial crop used both fresh and after
processing. In terms of nutritional value, spinach is a
good source of minerals and vitamins, especially ascorbic
acid (vitamin C) (Kawazu et al., 2003), however, it can
contain harmful substances such as nitrate (Gangolli
et al., 1994) and oxalate (Kawazu et al., 2003) amounts of
which are altered by growth conditions. Dietary oxalate
is a health hazard for humans (Holmes and Kennedy,
2000; Stevenson et al., 2003) since it can lead to the
formation of calcium oxalate urinary stones (Siener
et al., 2002; Siener et al., 2003) and it can impair the
uptake of iron and calcium from food in the gut (Kawazu
et al., 2003; Hodgkinson, 1981). These effects of oxalate
can be partially counteracted by the presence of
ascorbate in foodstuffs (Siener et al., 2003).
In plants, oxalic acid is synthesized from ascorbic acid,
which in turn, is made from glucose (Smirnoff, 1996;
Loewus, 1999). Oxalic acid synthesis is also associated
with the reduction of nitrate to ammonium in leaves, to
counteract the alkalinity produced by this process
(Davies, 1986). Rinallo and Modi (2002) found this to
occur in kiwifruit. Oxalic acid can be catabolized in
plants by oxalate oxidase (EC 1.2.3.4.) which oxidizes it
to 2CO2 and H2O2 (Loewus, 1999).
The content of oxalate in vegetables can be affected
by cultivation practices. For example, nitrate-based
fertilizers often lead to an higher oxalate content in crops
than ammonium-based fertilizers (Libert and
Franceschi, 1987; Rinallo and Modi, 2002). In Tetragonia
tetragonioides both plant age and salinity affect oxalate
content (Ahmed and Johnson, 2000). In spinach, both the
time of sowing and variety were found to affect oxalate
*Author for correspondence.
content (Zimmermann, 1966; Kawazu et al., 2003). In
commercial production the light intensity under which
spinach is grown differs because it is cultivated in a wide
range of geographic regions, at different times of the year
and both in the field and greenhouse.
Although the effect of growing plants at different light
intensities on their content of ascorbate and nitrate has
been subject to much study (Gaudreau et al., 1995;
Dapoigny et al., 2000; Toledo et al., 2003, and references
therein), less is known about how this affects oxalate
content (Zimmermann, 1966; Franceschi and Horner,
1980; Libert and Franceschi, 1987).
The aims of this work were, first, to evaluate how
growing spinach at different light intensities alters the
leaf content of oxalate, ascorbate and nitrate. This was
done in growth cabinets to reduce the effect of variables
such as temperature. Secondly, to evaluate whether the
oxalate content of leaves is linked to their nitrate and
ascorbate contents.
MATERIALS AND METHODS
Plant material and growth conditions
Spinach seeds (‘Gigante d’Inverno’) were sown in
perlite in 1 l plastic pots (three seeds per pot). Pots were
placed in a growth cabinet (Fitotron SGD170 Sanyo
Gallenkamp UK) under a 10 h light/14 h dark
photoperiod. Photon flux density was 200 µmol quanta
m–2 s–1 (at a wavelength of 400–700 nm). Temperature
was 25 ± 0.5°C during the light period and 20 ± 0.5°C
during the dark period. Relative humidity was 70% and
CO2 concentration 360 ppm. After expansion of the
cotyledons, pots were placed at either 800 µmol quanta
m–2 s–1 or 200 µmol quanta m–2 s–1 for the high and low
light treatment respectively. Plants were supplied with a
 S. PROIETTI, S. MOSCATELLO, A. LECCESE, G. COLLA and A. BATTISTELLI 607

full nutrient solution (Edward and Walker, 1983) Statistical analysis

containing: 5 mM NH4NO3, 4 mM CaCl2, 1 mM K2SO4,
1.3 mM NaH2PO4.H2O, 1.5 mM MgSO4.H2O and 2 µM
Mn SO4, Zn SO4, Cu SO4, 1 µM NaMoO4, 100 µM H3BO3,
0.4 µM Co(NO3).6H2O, 200 µM NaCl and 1 µM EDTA,
with pH of 6.5 and EC 1.7 mS cm–1. EC was maintained
at 1.7 ± 0.1 mS cm–1 by watering daily each pot with 1 l of
distilled water, which was allowed to drain off, and then
with 1 l of nutrient solution, which was also allowed to
drain away.
Statistical analysis was done by ANOVA using the
STATISTICA software package (StatSoft for Windows,
1998). Growth characteristics were analysed by one-way
analysis of variance (ANOVA). Data on oxalate,
ascorbate and nitrate content were analysed using two-
way ANOVA, with the TIME of sampling and LIGHT
intensity as factors. Differences between averages were
tested by a LSD test for a significance level of P = 0.05.

Sampling and analysis RESULTS

Five weeks after germination, 12 plants from each Growing spinach plants at different light intensities
treatment were used for analysis. Leaf area was measured had a marked effect on their development. At the lower
using an area meter (Delta-T Devices Ltd, UK). Shoots light intensity both dry and fresh weight and leaf area
and roots were separated and fresh weights measured. were reduced (Table I). This is probably because at low
The tissues were then placed in an oven at 80°C until dry light, photosynthesis was reduced (data not shown). The
and the dry weights were then noted. The nitrate, structure of the plants was also different. Under low
ascorbate and oxalate content of the fourth and fifth fully light, leaves were thinner as shown by a decrease in
expanded leaves were determined for freshly harvested specific leaf dry weight (SLDW, Table I). In addition,
material. This was done for leaves collected at the end of plants grown under low light possessed a higher shoot:
the dark period and after 4 h and 8 h of the light period. root ratio (Table I).
To determine nitrate content, five leaves from different The amounts of ascorbate, oxalate and nitrate in
plants were harvested, and after measurement of fresh leaves were also affected by the intensity of light the
weight, were freeze-dried to constant weight. The dry
material was then ground to a fine powder in a mortar
and 10 mg of the powder was extracted by placing in 2 ml
of water at 70°C for 40 min. After centrifugation at 12000
g for 5 min, the supernatant was used for determination
of nitrate content by an enzymatic assay (Beutler and
Wurst, 1986). Ascorbic acid content was determined using
four leaf discs each of area 1.3 cm2. Each disc was taken
from a different plant, immediately frozen and then
stored in liquid nitrogen until required. Discs were
ground using a glass-glass homogenizer containing 1.5 ml
of 10% (w/v) TCA, and then centrifuged at 12000 g for 15
min. Reduced ascorbate (AsA), oxidized ascorbate
(dehydroascorbate, DAsA) and total ascorbate (AsA +
DAsA), in the supernatant were measured using a
colorimetric assay (Kampfenkel et al., 1995). For each
sample, DAsA was calculated as the difference between
total ascorbate (after reduction of DAsA with 2 mM
dithiothreitol) and reduced ascorbate. For the
determination of oxalic acid, four spinach leaves, each
from a different plant, were harvested and reduced to a
fine powder by grinding in liquid nitrogen using a mortar
and pestle. Then, 150 mg of powder was mixed with 1.5 ml
of distilled water and the pH adjusted to 2.8 with 1 N
HCl. The mixture was then placed at 50°C for 15 min.
Further extraction steps, and the determination of oxalic
acid content by enzymatic assay, were as described by
Beutler et al. (1980).

TABLE I
Characteristics of spinach plants grown at high and low light. Each value
is the mean of 12 replicates (for 12 different plants). For all variables the
F test (ANOVA) was statistically significant (P=0.05) FIG. l
Ascorbate (A, B), oxalate (C, D) and nitrate (E, F) contents of spinach
Light intensity leaves. Leaves were sampled three times during the photoperiod and
(µmol quanta m–2 s–1) under two light intensities (see Materials and Methods). The F tests
200 800 (ANOVA) for the interaction TIME of the day
LIGHT intensity and
Plant fresh weight (g plant–1) 3.7 11.2 for the factor TIME were not statistically significant, hence averages of
Plant dry weight (g plant–1) 0.5 1.2 the factor TIME (panel A, C and E) and of the factor LIGHT intensity
Leaf area (LA) (cm2 plant–1) 52 123 (panels B, D and F) are reported separately. The F test (ANOVA) for
Specific leaf dry weight (SLDW)(mg cm–2) 5.3 6.6 the factor LIGHT intensity was statistically significant for all three
Shoot to root ratio 5.6 3.2 compounds. Differences between averages were tested by LSD test
for P = 0.05.
608 Light and oxalate content of spinach
plants were grown under (Figure 1). Leaves of plants
grown at high light contained more ascorbate but less
nitrate than those from plants grown under low light.
These differences were maintained throughout the
photoperiod (Figure 1). In low-light grown plants,
ascorbate content was about half that of leaves from
plants grown under high light (Figure 1B). On the other
hand, the content of oxalate was about 25% lower in
leaves of plants grown under high light (Figure 1D), and
nitrate was about 65% lower (Figure 1F). The average
redox state of the ascorbate pool (AsA/DAsA+AsA) in
all leaves was about 89% and there was no significant
difference between the treatments (data not shown).
Statistical analysis showed no significant interaction
between the two factors TIME and LIGHT intensity
(Figure 1A, 1C, 1E), and no significant effects of the
factor TIME for the content of ascorbate, oxalate and
nitrate in leaves.
DISCUSSION
Other authors have reported the effect of light
intensity on oxalate accumulation in plants (Loewus,
1999). However, studies such as this one, in which light
intensity is carefully controlled throughout growth, and
interactions with other factors such as nutrition or
temperature are reduced, have not been done.
Zimmermann (1966), for example, found a variation of
oxalate content in spinach sown at different times during
the year and fed different amounts and types of
nitrogenous fertilizer. However, he could not separate
clearly the effects of all the factors that changed during
the experiment such as temperature, photoperiod and
water availability. Sugiyama and Okutani (1996) found a
decrease of oxalate in leaves of spinach kept in the light
with respect to leaves kept in the dark. However their
experiments were done with detached leaves over a
short period of 4 h.
The results obtained in this study show that growing
spinach plants at different light intensities alters the
plants in a physiological way. The light intensities were
chosen so as to be in the range not too far from that
likely to be encountered by plants grown commercially,
then our results are applicable to plant produced in the
field or greenhouse. This is confirmed by the fact that
spinach plants acclimated to the low light intensity by
altering their structure as expected in response to light
limited photosynthesis (Poorter and Nagel, 2000).
Contents of ascorbic acid, nitrate and oxalate under
the two light regime suggest a role for the oxalate
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