Vol.2, No.4, 477-486 (2011)
opyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/AS/
Agricultural Scienc es
Reduced nitrogen availability during growth improves
quality in red oak lettuce leaves by minimizing nitrate
content, and increasing antioxidant capacity and leaf
mineral content
Dario Stefanelli*, Sonja Winkler, Rod Jones
Future Farming Systems Research Division, Department of Primary Industries, Knoxfield Centre, Victoria, Australia;
*Corresponding Author: dario.stefanelli@dpi.vic.gov.au
Received 5 September 2011; revised 15 October 2011; accepted 25 October 2011.
Overuse of N in lettuce production can lead to
environmental problems caused by leaching an d
the accumulation of harmful nitrates in edible
tissues. This study investigated the effect of
applied nitrogen (N) concentrations between 40
and 2400 mg·L–1 on growth, nitrate accumula-
tion, mineral leaf content, and antioxidant ca-
pacity in Oak Leaf lettuce cv. “Shiraz” grown
under hydroponic conditions in Australia. Yield
(g FW) increased with nitrogen (N) application
rate up to 1200 mg·L–1, as did leaf N content,
while C:N declined. Nitrogen Utilization Effi-
ciency (NUtE) increased rapidly from 40 to 75
mg·L–1 applied N, leveling at 150 mg·L–1 with no
subsequent effect of N concentrations between
400 and 2400 mg·L–1. Nitrate content rose sig-
nificantly with increased N, particularly at 1200
and 2400 mg·L–1. Leaf total plant phenolic con-
tent (TPP) and antioxidant capacity (measured
by ferric reducing antioxidant power—FRAP)
were both maximal at 75 and 400 mg·L–1 applied
N, while highest oxygen radical absorption ca-
pacity (ORAC) values were found in leaves
supplied with lo w N (40 to 400 mg·L–1). Applied N
as calcium nitrate also significan tly affecte d leaf
mineral content as B, Mg, Mn, and Zn signifi-
cantly decreased with increasing N. These re-
sults indicate that N applications of 1200 mg·L–1
or higher can result in reduced antioxidant ca-
pacity and mineral content in lettuce leaves.
Keywords: Lactuca sativa L.; Hydroponic;
Phenolic Content; Zinc; Manganese; Magne sium
In the past 50 years the use of nitrogen-phosphorus
potassium-based (NPK) fertilizers has increased dra-
matically around the world, particularly in North Amer-
ica and Europe [1]. Overuse of NPK can lead to two
major problems—N leaching and P runoff from agricul-
ture is widely considered the main cause of eutrophica-
tion in fresh and salt water supplies throughout the world
[2]. Secondly, leafy vegetable crops, such as lettuce,
accumulate nitrates when grown with high N availability
and low light [3], and this can be deleterious to human
health as nitrates can be converted to harmful nitrites
post-harvest [4]. Thus the efficient use of N is an impor-
tant environmental and social issue [1], in addition to the
potential cost savings of reduced fertilizer use [5].
Maximizing nitrogen use and nitrogen utilization effi-
ciency (NUtE) of crop production can be achieved by 1)
optimizing the supply of N to meet the requirements of a
crop during growth and development [6]; 2) optimizing
N supply in correlation with the desired final produce
quality [7], or 3) by selecting and growing N-efficient
crop genotypes [5,8].
Lettuce is considered the one of most economically
important leafy vegetable crop in the world [9] and is
widely consumed in Western diets. It is therefore an im-
portant source of dietary antioxidants, primarily in the
form of phenolic compounds such as caffeic acid deriva-
tives and flavonols [10]. There is increasing evidence
that antioxidants contained within fruits and vegetables
may protect against serious diseases, including cardio-
vascular disease and certain cancers, if consumed regu-
larly [11,12]. The major flavonols contributing to anti-
oxidant activity found in lettuce are quercetin and
kaempferol derivatives [13], while isorhamnetin is less
common. Quercetin has been extensively studied in vitro
D. Stefanelli et al. / Agricultural Sciences 2 (2011) 477-486
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and is known to be a potent free-radical scavenger and
anti-oxidant [14,15]. Recently, the closely related com-
pound, kaempferol, has also been shown to possess an-
tioxidant activity in its own right, but lower than que-
rcetin [16]. Quercetin and kaempferol are also known to
act synergistically in the inhibition of cell proliferation
in human gut cancer lines [17].
When grown under high N (200 kg·ha–1) other quality
indices (dry matter, sugar and vitamin C content) de-
clined in Crisphead lettuce [18]. Furthermore, Butter-
head, Romaine and Oak Leaf lettuce quality, as per-
ceived by a sensory panel, was maximized by as little as
80 kg·ha–1, significantly less than the normal recom-
mended N application rate for field-grown lettuce in
Italy [19].
Minimizing N availability in lettuce, while maintain-
ing yield and quality, is a subject of much recent study [7,
20], but little is known of the effect of minimal N on
antioxidant and mineral contents in lettuce. Nitrogen
deficiency has resulted in increased flavonol accumula-
tion in Arabidopsis [21], broccoli [22] and tomato leaves
[23], but had no effect in onion bulbs [24]. Limited N
also resulted in higher total plant phenolic content in
basil leaves [25] while, conversely, increased N via
foliar urea application resulted in an increase in free
radical scavenging activity in lettuce [26].
It is estimated that up to two-thirds of the world’s
population might be at risk of deficiency in one or more
essential mineral nutrients [27], and the concentration of
mineral elements in edible plant tissues is therefore of
fundamental importance to human nutrition [28]. A re-
cent area of research has been termed “ionomics” [29],
which focuses on the quantification and characterization
of the mineral elements of plant tissues [28,30,31]. The
ionome is influenced significantly by developmental,
environmental, and agronomical factors [27,32] which
are fundamental not only to mineral nutrition of the plant,
but also for increasing the concentrations of mineral
elements in edible tissues for human consumption [27].
Fortification of horticultural produce and leafy vegeta-
bles in particular, could be a successful strategy for im-
proving human diets [33], resulting in an increase in the
value and quality of the produce itself [7].
This study investigates the effect of N concentrations
between 40 and 2400 mg·L–1 on yield, nitrate accumula-
tion, mineral leaf content, and antioxidant capacity in
Oak Leaf lettuce cv. Shiraz grown under hydroponic
conditions in Australia.
2.1. Experimental Design
A greenhouse experiment was conducted in Novem-
ber-December 2008 at the Department of Primary Indus-
tries (DPI) Knoxfield, Victoria, Australia. Thirty Lactuca
sativa L. var. “Shiraz” plants were grown hydroponically
in 800 mL square plastic pots with perlite as growing
medium. Plants were germinated on site and transplanted
into pots as plugs after three weeks. Greenhouse tem-
perature was maintained between 18˚C (night) and 24˚C
(day) [34] by an evaporative cooling system. Irrigation
was delivered thrice per day by an automatic system
with two drippers (300 ml·day1·plant1) per pot. Saucers
underneath the pots collected irrigation water and main-
tained medium (perlite) moisture. An especially prepared
commercial lettuce hydroponic fertilizer (Hysol Twin,
Duralite, Melbourne, Australia) in which all traces of N
were removed from the manufacturer, was applied as
base fertilizer to all pots at the industry standard rate of
1.0 to 1.2 g·L1. Six N levels were applied as calcium
nitrate (Ca:NO3—19:15.5) derived N (CaNO3-N) [35].
Both base fertilizers and N levels (40, 75, 150, 400, 1200,
2400 mg·L1 of actual CaNO3-N) were applied manually
on alternate days throughout the experiment for a total of
13 applications of 30 ml each. Plants received a total of
15.6, 29.3, 58.5, 156, 468, and 936 mg of N per plant
during the experiment, equivalent to 1.7, 3.2, 6.4, 17.2,
51.5, and 103.0 kg/ha of N. Water collected in the sau-
cers was checked with an EC meter (NZ Hydroponics
LTD, Tauranga, NZ) to avoid excessive build up of salts
[36]. To avoid influencing elements absorption, EC was
maintained at similar levels in all treatments by periodi-
cally flushing excess salts from the high N concentra-
tions with de-ionized water.
2.2. Measurements
At the completion of the 30 day trial, plants were
weighed for FW and immediately frozen at –20˚C and
freeze-dried. Fresh and dry weight, total N and C were
measured in roots and leaves from all plants. Addition-
ally, mineral (Al, B, Ca, Cu, Fe, Mg, Mn, K, P, S, Na, Zn)
and NO3 content in leaves from all CaNO3-N levels was
also measured. Leaves from the 40 mg·L–1 treatment
were excluded due to lack of material.
2.3. Leaf Anal ysis
Mineral Leaf Content: Approximately 0.5 g (DW) of
freeze-dried sample from each plant was weighed into a
test tube and 5mL of a 1:4 mixture of perchloric and
nitric acids was added. Test tubes were left overnight at
20˚C to “cold digest”. The following day the mixture
was heated to 80˚C on an aluminium heating block for
30 minutes, then the temperature was increased to 150˚C
for 1.5 hours, and then raised again to 185˚C for ap-
proximately 1 to 2 hours or until white perchloric acid
fumes were observed. The resulting digest was made up
D. Stefanelli et al. / Agricultural Sciences 2 (2011) 477-486
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to 25 mL with distilled de-ionized water and the solution
measured by ICP-OES VISTA (VARIAN Inc., Palo Alto,
USA) against external calibrates following the suggested
methodology [37].
Total N and C were measured with a LECO CNS2000
(LECO Corporation, St. Joseph, USA) following the
suggested methodology [38].
Nitrate Content: NO3 content in leaves was assessed
by weighing approximately 0.2 g into a plastic tube
filled with 100 mL of distilled de-ionized water. Tubes
were shaken for 20 minutes and filtered through a 0.45
um filter disk. The collected solution was analyzed fol-
lowing the APHA method 4500-NO3-F [39].
Total plant phenolics: Water soluble TPP, as an as-
sessment of antioxidant activity, were measured follow-
ing the Folin-Cioccalteau (FC) method [40].
Oxygen radical absorbance capacity: antioxidant ca-
pacity, as measurement of peroxyl radical scavenging
activity [41], was measured following the microplate
fluorescence reading method [42] and carried out on a
Varioskan Flash (Thermoscientific Corp., Melbourne,
Ferric reducing antioxidant power: the antioxidant
capacity potential, as measurement of Fe(III) reducing
activity [41] was performed as previously described by
Benzie and Strain [43] and was also carried out on a
Varioskan Flash (Thermoscientific Corp., Melbourne,
Leaf chlorophyll content was measured by SPAD
(Konica-Minolta SPAD-502 Chlorophyll meter, Braeside,
Australia) on two fully expanded leaves before each
CaNO3-N fertilization to monitor chlorophyll content.
2.4. Statistical Method
The experiment was a complete randomized block de-
sign with five replications. Data were analyzed by
Anova (p < 0.005) with Genstat 12.0 (VSN International
Ltd, Hemel Hempstead, UK). Regression curves were
performed with SigmaPlot 10 (Systat Software Inc, Chi-
cago, USA). Plants fertilized with 40, 75, and 150 mg·L–1
CaNO3-N were harvested at 45 days after transplant due
to lack of growth at 30 days. All data were converted to
a per day basis for uniform comparison and then either
reported as such or compared at 30 days.
3.1. Yield
Lettuce leaf yield, expressed in g FW per day, was
significantly affected by N application rate (Figure 1),
with higher N applications resulting in increased yield.
Figure 1 represents a typical rise to maximum growth
N solution concentration (mg L
05001000 1500 2000 2500 3000
Yield - leaves fresh wgt per d ay (g day
Figure 1. Lettuce yield (g·day–1 FW) as affected by N supplied
at 40, 75, 150, 400, 1200 or 2400 mg·L–1. Fitted curve is a Rise
to max, equation = 3.0887(1e(0.0015X)) R2 = 0.90, p < 0.0001.
Each data point represents a single plant.
curve for lettuce (R2 = 0.90; p < 0.0001) with significant
increases in FW as N increased from 40 to approxi-
mately 1200 mg·L–1. Fresh weight continued to increase
between 1200 and 2400 mg·L–1 applied N, but at a
slower rate. Similarly, leaf N uptake, as expressed by N
accumulation within lettuce leaves, also increased with
greater N availability (Figure 2(a)) and increased more
rapidly from 40 to 400 mg·L–1 applied N than from 400
to 2400 mg·L–1. N uptake slowed markedly between 1200
and 2400 mg·L–1 applied N, showing a logarithmic growth
curve overall (R2 = 0.84, p < 0.0001) (Figure 2(a)).
Leaf NUtE, calculated as the amount of leaf FW per g
of N accumulated, showed a rise to maximum growth
curve (R2 = 0.65, p < 0.0001) (Figure 2(b)). NUtE grew
rapidly from 40 to 75 mg·L–1 applied N, leveling at 150
mg·L –1, and with no subsequent effect of N concentra-
tions between 400 and 2400 mg·L–1 (Figure 2(b)). This
pattern was also reflected in the logarithmic decrease in
the carbon to nitrogen ratio (C:N) curve (R2 = 0.92, p <
0.0001) (Figure 3). C:N is inversely proportional to N
leaf accumulation, decreasing with the increase of N
concentration in the fertilizer solution. Total leaf nitrate
increased exponentially (R2 = 0.84; p < 0.0001) with
increased N supply (Figure 4), particularly at application
rates of 400 mg·L–1 N or higher, reaching a maximum at
2400 mg·L–1 applied N.
3.2. Phenolic Content, Antioxidant Capacity
and Chlorophyll
Total plant phenolics and antioxidant capacity, as
measured by both FRAP and ORAC, were significantly
affected by applied N rate (Table 1), with the highest
levels of all three indices recorded between 75 and 400
mg·L –1 applied N. Highest TPP was recorded at 75 and
D. Stefanelli et al. / Agricultural Sciences 2 (2011) 477-486
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400 mg·L–1, which both had significantly higher TPP
than the other N rates (Table 1). It is noteworthy that
TPP and FRAP in leaves supplied with 150 mg·L–1 N
were significantly lower than leaves supplied either 75
or 400 mg·L–1, but the reason for this result is unknown
(Ta b l e 1 ). FRAP peaked at 400 mg·L–1, which was sig-
nificantly higher than 40, 150 and 2400 mg·L–1 applied
N (Ta ble 1). There was no significant difference in TPP
and FRAP values for leaves supplied with lowest (40
mg·L –1) or highest (2400 mg·L–1) N. Antioxidant capac-
ity as measured by ORAC was also significantly lower
in leaves supplied with 1200 or 2400 mg·L–1 N, com-
pared with lower N application rates (Table 1). There
were no statistical differences between chlorophyll lev-
els measured by SPAD. Levels oscillated between 15 (40
mg·L –1 N) and 20 (2400 mg·L–1 N) SPAD units (data not
shown), indicating that N application rate did not poten-
tially affect photosynthesis.
Figure 2. (a) Total N content (mg·kg–1 DW) and (b) Nitrogen Utilization Efficiency (NUtE) in lettuce leaves as affected by N sup-
plied at 40, 75, 150, 400, 1200 or 2400 mg·L–1. Fitted curve equations: A = –2.1973 + 0.8207lnx; R2 = 0.84; p < 0.0001; B = Expo-
nential rise to max = 22.5277(1e(0.0152X)); R2 = 0.65; p < 0.0001. NUtE was calculated by the ratio between total fresh yield and total
nitrogen accumulated in leaves. Each data point represents a single plant.
Figure 3. Total nitrate content (mg·kg–1 DW) in lettuce leaves as
affected by N supplied at 40, 75, 150, 400, 1200 or 2400 mg·L–1.
Fitted exponential curve equation = 719.9728e0.0011X R2 = 0.84; p
< 0.0001. Each data point represents a single plant.
Figure 4. Carbon:Nitrogen ratio in lettuce leaves as affected by
N supplied at 40, 75, 150, 400, 1200 or 2400 mg·L–1. Fitted loga-
rithmic curve equation = 35.5525 – 0.0427lnx – 0.4604lnx2; R2 =
0.92; p < 0.0001. Each data point represents a single plant.
Openly accessible at
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Table 1. Total Plant Phenolics (TPP) and antioxidant capacity, as measured by FRAP (µmol Fe2+ g–1 FW) or ORAC (µmol Trolox
equivalents.100 g–1 DW) in lettuce leaves supplied with N at 40, 75, 150, 400, 1200 or 2400 mg·L–1. Values represent averages of five
N solution concentration (mg·L–1) TPP (mg GAE g–1 DW) FRAP (µmol Fe 2+ g–1 DW)ORAC (µmol Trolox equiv 100 g–1 DW)
40 26.24 b* 259.7 cd 3369 a
75 32.19 a 335.5 ab 3631 a
150 21.07 b 186.7 d 3283 a
400 32.18 a 396.2 a 3384 a
1200 26.43 ab 328.8 abc 2647 b
2400 24.91 b 293.8 bc 2635 b
lsd 5.68 74.8 508
* = Values with different letters in the same column are significantly different at p < 0.05.
Table 2. Lettuce leaf macronutrient composition in dry weight (DW) as affected by N supplied at 75, 150, 400, 1200 or 2400 mg·L–1.
Values represent averages of five plants.
Ca Mg P K Na S
N solution concentration (mg·L–1)
g 100 g–1 DW
75 0.33 c* 0.40 a 0.59 5.8 a 0.10 0.21
150 0.31 c 0.36 a 0.63 5.1 b 0.09 0.24
400 0.34 c 0.24 c 0.58 5.6 ab 0.07 0.21
1200 0.52 b 0.26 bc 0.57 6.0 a 0.08 0.24
0.77 a
0.30 b
5.7 ab
* = Values with different letters in the same column are significantly different at p < 0.05.
Table 3. Lettuce leaf micronutrient composition (DW) as affected by N supplied at 75, 150, 400, 1200 or 2400 mg·L–1. Values repre-
sent averages of five plants.
Al B Cu Fe Mn Zn
N solution concentration (mg·L–1)
mg·kg–1 DW
75 20.3 b* 75.3 a 11.0 a 67.2 b 471 a 125 a
150 27.1 b 76.2 a 10.1 ab 69.1 b 378 b 121 a
400 28.8 ab 47.1 b 9.9 ab 83.6 ab 175 c 57 b
1200 55.7 a 45.4 bc 9.7 ab 108.4 a 121 d 59 b
2400 8.2 b 37.1 c 8.6 b 69.4 b 106 d 42 b
lsd 26.45 7.91 1.75 26.20 32.71 18.83
* = Values with different letters in the same column are significantly different at p < 0.05.
3.3. Leaf Mineral Content
Tables 2 and 3 show lettuce leaf mineral composition
as affected by N solution concentration. Out of the 12
elements measured by ICP only P, Na and S were not
significantly affected by N application rates. Ca accu-
mulated in leaves with increased N rate. The highest
level of Ca accumulation was reached with 2400 mg·L–1
of applied N, followed by 1200 mg·L–1 and with no sig-
nificant difference between 400, 150 and 75 mg·L–1 (Ta-
ble 2). B, Cu, Mg, Mn, and Zn all declined significantly
at the increase of N concentrations. K did not show a
clear trend related to N applications. Metals Fe and Al
content did peak at 1200 mg·L–1 N, which was signifi-
cantly different from 75, 150 and 2400 mg·L–1 N, while
400 was similar to all other treatments (Table 3).
Increased fertilizer utilization efficiency can be achieved
through either improved fertilizer management practices
and/or by cultivating crops and cultivars that genetically
acquire or use elements more efficiently [44,45]. In the
present study, we have illustrated that N application rates
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can be reduced significantly without a significant reduc-
tion in quality as expressed by antioxidant capacity and
mineral content, while yield was marginally reduced.
Increasing N concentration in hydroponic fertilizer solu-
tions is known to stimulate yield, with our data follow-
ing the classic growth N response curve [46] (Figure 1).
However there were no statistical differences in yield
between N rates of 400 mg·L–1 and the commercial
standard rate (Figure 1). These results imply that reduc-
ing N rates in hydroponic lettuce cultivation below the
industry standard rate of 1000 - 1200 mg·L–1 should not
reduce yield significantly, potentially allowing a more
environmentally sustainable production. This finding
was also corroborated by the NUtE data (Figure 2(b))
which reached a plateau between 150 and 400 mg·L–1 N,
while N leaf accumulation continued above 1200 mg·L–1
N (Figure 2(a)). This indicates that the maximal usage
NUtE (from a yield perspective) in lettuce leaves of this
variety was between 150 and 400 mg·L–1. Reduction of
N fertilization rates to increase NUtE is one method
suggested to improve environmentally sustainable agri-
culture [6,28]. Recent research in this area is also at-
tempting to select for higher N-efficient plants with re-
gards to uptake, in combination with improved nitrogen
efficiency [44,47]. Our results indicate that NUtE in let-
tuce leaves could be improved with N application rates
significantly lower than commonly applied in Australia.
In fact chlorophyll levels did not change in the present
study (data not shown), indicating that the lowest ap-
plied N rate (40 mg·L–1) was sufficient to maintain ade-
quate photosynthesis.
Accumulation of N in leaves, especially in the form of
nitrates, is an important negative quality trait for leafy
vegetables [9]. Low N availability generally results in
minimal nitrate accumulation in lettuce [20], while high
N (>150 kg·ha–1) significantly increased nitrate accumu-
lation [9]. Our results (Figure 3) confirm these observa-
tions, with negligible nitrate recorded in lettuce leaves
supplied with 400 mg·L–1 N (Figure 4). The European
Community has set upper nitrate limits in lettuce leaves
of between 3500 and 4500 mg·kg–1 FW [48]. Our data
shows 2400 mg·L–1 N resulted in a mean nitrate content
of 10,000 mg·kg–1 DW, which translates in the order of
700 mg·kg–1 FW, therefore quite within the limits of the
acceptable daily intake [49].
High nitrogen availability is known to inhibit phenolic
production and subsequently antioxidant capacity, in a
range of leafy vegetables [7]. In lettuce, fertilization treat-
ments that resulted in relatively high soil N content caused
a reduction in phenolic content, specifically coumaric
acid and antioxidant capacity [50]. Highest antioxidant
capacity (measured by FRAP and DPPH) and TPP in
Chinese cabbage were recorded in plants grown in soil
under 0 mg/kg applied N, with higher N application rates
resulting in lower TPP and antioxidant capacity [51].
Similarly highest phenolic content in basil grown hydro-
ponically was found after minimal N application (0.1 mM)
[25], with lowest antioxidant capacity in leaves grown
under the highest N availability. Our data partially
agrees with these studies in that highest TPP and anti-
oxidant capacity were recorded in plants supplied with
low N, specifically 400 mg·L–1 (Table 1). However,
plants supplied with the lowest N (40 mg·L–1) were not
significantly different with respect to TPP or FRAP from
those supplied with 2400 mg·L–1 N. TPP and FRAP
peaked at 75 or 400 mg·L–1 applied N (Ta ble 1), while
antioxidant capacity, as measured by ORAC, was maxi-
mal at 40 to 400 mg·L–1 N (Table 1). Thus, there appears
to be a lower effective limit for N supply of approxi-
mately 75 mg·L–1 N, below which TPP and antioxidant
capacity declined. Furthermore, there was no significant
difference in TPP, FRAP and ORAC values in leaves
supplied with 400 mg·L–1 N and 75 mg·L–1 (Table 1),
while yield was significantly greater in plants supplied
with 400 mg·L–1 N (Figure 1). Maximizing antioxidant
capacity in lettuce cv. “Shiraz” can be therefore be
achieved with 400 mg·L–1 N
A variety of stresses are known to result in an increase
in the phenolic synthesis pathway in plants [52]. Wound-
ing, drought, nutrient deficiency and high light intensity
all resulted in increased phenolic compounds in lettuce
leaves due to upregulation of the phenylpropanoid path-
way [53]. Accumulation of the flavonols quercetin and
kaempferol, which would result in both increased TPP
and antioxidant capacity, is known to be induced by N
depletion through enhanced synthesis [54]. Low N
availability specifically stimulated phenolic synthesis in
tomato leaves [21]; and fruit [55]. Nitrogen-depleted
Matricaria rosettes showed a significant increase in PAL
activity and a concomitant increase in phenolic content
[56]; an increase in PAL and TPP was also seen in yar-
row leaves grown with low N (0.1 mM) for 4 months
The phenolic compounds quercetin, kaempferol, isor-
hamnetin and anthocyanins are all commonly found in
leafy vegetables and contribute significantly to antioxi-
dant capacity [58]. Our results indicate that a similar
increase in phenolic compounds (TPP in Table 1) was
seen under low N nutrition, but the largest increase was
not at the lowest N rate (40 mg·L–1) but between 75 and
400 mg·L–1. It is not known why TPP and FRAP values
were significantly lower at 150 mg·L–1 compared with
either 75 or 400 mg·L–1. Despite this anomaly, it is pos-
sible to grow lettuce plants at 400 mg·L–1 N and achieve
high antioxidant capacity with a yield not particularly
depressed when compared with plants grown under 1200
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mg·L –1—currently the industry standard N application
rate for lettuce plants in Australia.
It is not certain, that the increase in phenolic com-
pounds was solely responsible for increased antioxidant
capacity. In lettuce leaves, the major contributor to anti-
oxidant capacity, as measured by DPPH was ascorbic
acid (57% - 63%) [50]. Furthermore, a good correlation
between TPP and antioxidant capacity was found in only
5 out of 10 lettuce varieties studied by Heimler et al. [59]
most likely due to interference by ascorbic acid. Ascor-
bic acid in spinach is also known to increase with low N
availability [60], therefore we can speculate that the high
FRAP and ORAC levels at applied N of 400 mg·L–1 or
less in the present study (Ta bl e 1 ) could be due at least
in part to higher ascorbic acid levels, as well as the ob-
served significant increase in TPP at 400 and 75 mg·L–1
applied N. Ascorbic acid levels were not recorded in our
study. Conversely, Chiesa et al. [9] found high N (150
kg·ha–1) increased ascorbic acid content in lettuce, indi-
cating this area requires further investigation.
The Carbon/Nutrient Balance Theory (CNB) [61]
predicts that production of carbon-based secondary me-
tabolites (e.g. phenolics) decreases with increased N as
C is needed primarily for plant growth and development.
This hypothesis supposes that a decrease in N supply
causes a restriction in photosynthesis and plant growth
and reduces the demand for amino acids for protein syn-
thesis. In lettuce plants, however, it seems that the theory
is only partially accurate, as highest TPP and antioxidant
capacity was found not at the lowest N availability, as
predicted by the model, but at more “intermediate” rates,
vis. 75 or 400 mg·L–1.
In our experiment, it appears that the ionome [27,28,
32] of lettuce cv. “Shiraz” was affected by varying N rate.
Nitrogen was applied as calcium-nitrate, thereby in-
creasing the N concentration in the solution at the same
time as Ca. This resulted in an increased accumulation of
Ca in leaves (Ta b le 2 ) agreeing with Neeser et al. [33]
who reported increasing Ca in the fertilizer solution was
an effective method to biofortify lettuce for this element.
Our results indicate a further influence of increased N
and Ca availability on other essential minerals. Tables 2
and 3 show that B, Mg, Mn, and Zn all decreased sig-
nificantly with increasing N and Ca applications. This is
possibly due to 2+ cation absorption-competition interac-
tions between Ca and Mg, Mn, and Zn or the 1 anion
competition between borate and nitrate [46]. More re-
search is necessary to better identify specific ion interac-
tions and correlations with plant physiological pathways.
In lettuce several factors can influence leaf mineral
content. High nutrient solution electrical conductivity
(EC) has been reported to diminish Fe and Zn and in-
crease Mn concentrations, while N, P, and K were not
affected [62]. Gent [36], however, reported only a small
increase in the accumulation of nitrates with increased
EC. Nitrogen form in the fertilizer solution had no effect
on nitrate accumulation, with organic forms stimulating
K absorption [35]. In our trials EC was maintained as
similar as possible in all treatments by flushing excess
salts from the high N concentrations with deionised wa-
ter periodically, therefore reducing the influence on the
elements absorption to immediately after fertilization.
In conclusion, leaf total plant phenolic content (TPP)
and antioxidant capacity (measured by FRAP) were both
maximal at 75 or 400 mg·L–1 applied N, while highest
ORAC values were found in leaves supplied with low N
(40 to 400 mg·L–1). Applied N also significantly affected
leaf mineral content: Ca rose with increased N, while B,
Mg, Mn, and Zn significantly decreased. These results
indicate that CaNO3-N applications of 1200 mg·L–1 or
higher can result in reduced antioxidant capacity and
mineral content in lettuce leaves. It is possible to reduce
N concentration in the hydroponic solution for lettuce
production in Australia without having a detrimental
effect on yield. This in addition will increase lettuce
quality reducing nitrate content, increasing essential
elements such as Mn, Zn, Mg, and Ca and increasing
antioxidant capacity.
This is a publication from Vital Vegetables, a Trans Tasman research
project jointly funded and supported by Horticulture Australia Ltd.,
New Zealand Institute for Crop and Food Research Ltd., the New
Zealand Foundation for Research Science and Technology, the Austra-
lian Vegetable and Potato Growers Federation Inc, New Zealand Vege-
table and Potato Growers Federation Inc and the Victorian Department
of Primary Industries. Also thanks to Janet Tregenza, Christine Frisina,
and Bret Henderson for their technical assistance, and to Bruce Tom-
kins and Dr Mark Downey for proof reading.
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