Reduced nitrogen availability during growth improves quality in red oak lettuce leaves by minimizing nitrate content, and increasing antioxidant capacity and leaf mineral content

doi:10.4236/as.2011.24061

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Vol.2, No.4, 477-486 (2011)

doi:10.4236/as.2011.24061

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.

ABSTRACT

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

1. INTRODUCTION

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

478

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. MATERIALS AND METHODS

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·day−1·plant−1) 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·L−1. 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·L−1 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

479479

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,

Australia).

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,

Australia).

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. RESULTS

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

-1

)

05001000 1500 2000 2500 3000

Yield - leaves fresh wgt per d ay (g day

-1

)

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(1−e(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

480

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(1−e(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

D. Stefanelli et al. / Agricultural Sciences 2 (2011) 477-486

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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

plants.

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

2400

lsd

0.77 a

0.07

0.30 b

0.05

0.54

0.09

5.7 ab

0.63

0.09

0.02

0.23

0.03

* = 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).

4. DISCUSSION

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

D. Stefanelli et al. / Agricultural Sciences 2 (2011) 477-486

482

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

[57].

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

D. Stefanelli et al. / Agricultural Sciences 2 (2011) 477-486

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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.

5. CONCLUSIONS

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.

6. ACKNOWLEDGEMENTS

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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