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Composition of hydroponic lettuce: Effect of time of day, plant size, and

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Article  in  Journal of the Science of Food and Agriculture · February 2012

DOI: 10.1002/jsfa.4604 · Source: PubMed

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 Research Article
Received: 31 March 2011 Revised: 7 June 2011 Accepted: 10 July 2011 Published online in Wiley Online Library: 14 September 2011

(wileyonlinelibrary.com) DOI 10.1002/jsfa.4604

Composition of hydroponic lettuce: effect

of time of day, plant size, and season
Martin PN Gent∗

Abstract
BACKGROUND: The diurnal variation of nitrate and sugars in leafy green vegetables may vary with plant size or the ability of
plants to buffer the uptake, synthesis, and use of metabolites. Bibb lettuce was grown in hydroponics in a greenhouse and
sampled at 3 h intervals throughout one day in August 2007 and another day in November 2008 to determine fresh weight, dry
matter, and concentration of nitrate and sugars. Plantings differing in size and age were sampled on each date.

RESULTS: The dry/fresh weight ratio increased during the daylight period. This increase was greater for small compared to large
plants. On a fresh weight basis, tissue nitrate of small plants was only half that of larger plants. The variation in concentration
with time was much less for nitrate than for soluble sugars. Soluble sugars were similar for all plant sizes early in the day, but
they increased far more for small compared to large plants in the long days of summer.

CONCLUSION: The greatest yield on a fresh weight basis was obtained by harvesting lettuce at dawn. Although dry matter or
sugar content increased later in the day, there is no commercial benefit to delaying harvest as consumers do not buy lettuce for
these attributes.

c 2011 Society of Chemical Industry

Keywords: diurnal; dry matter; lettuce; nitrate; sugars

INTRODUCTION
The diurnal variation in light has an effect on many metabolites
in leafy green vegetables. The diurnal variation of sugars in field-
grown crisp-head lettuce was greatest in the outer leaf and in the
stem and least in inner leaves.1 In leaf blades of spinach, sugars
increased fivefold through the photoperiod.2 Nitrate in spinach
increased dramatically over a 14 h dark period, and decreased in
light.3 When grown under low light intensity, this increase occurred
in the first part of the night, and then nitrate was constant toward
the end of the night.4 Similarly in hydroponic lettuce, nitrate
increased for 4 h early in the night and then decreased.5 However,
there was no increase if supplemental light was applied in the
night.
Various studies suggest nitrate is used as an osmoticum in
lettuce and spinach. Nitrate concentration often varies inversely
with that of sugars and organic acids, which are alternatives for this
purpose. In spinach grown at low light intensity, the daily changes
in uptake, reduction, and storage of nitrate were opposite those
for amino acids or sugars.4 The total osmoticum was similar for
lettuce grown under different light intensities, but more light
increased organic acids and sucrose and decreased nitrate.6 Using
phosphate depletion to limit growth of lettuce caused sugars
to accumulate, which depressed the level expected for nitrate.7
An examination of uptake of water and nitrate in young tomato
or lettuce seedlings showed that the changes in nitrate content
were due to changes in the size of the water reservoir,8 while the
concentration of nitrate was relatively constant. Solution electrical
conductivity (EC) affected nitrate concentration in hydroponic
542
Composition of plant tissue changes with size and age of the
plant, and these factors may affect diurnal variation. For field-
grown lettuce, nitrate increased and dry matter decreased for
older or larger plants.10 However, when normalized to plant size,
plant growth and nitrate uptake of hydroponic lettuce decreased
as the plants grew larger.11 The nature of sugars in lettuce changes
late in development or in large plants. Whereas only mono- and
disaccharides predominate in young plants, sugars with three
or four hexose subunits appear later.12 This would reduce the
osmotic effect of an equal weight of sugars.
Composition of plant tissue changes with environment also.
There is a concern regarding accumulation of nitrate in lettuce and
spinach grown under low light conditions in winter. Hydroponic
lettuce has much more nitrate in winter than in summer.13 A study
of seasonal effects found that concentrations of nitrate decreased
and sugars increased from winter to summer in greenhouse
lettuce.14 The magnitude of diurnal variation in nitrate and sugars
is also likely to depend on light intensity and temperature. A
study comparing field-grown lettuce and spinach grown at similar
temperatures but different day lengths showed that nitrate varied
more when grown under a longer day length.15
Models have been used to understand the changes in
composition as a function of plant size in lettuce. A dynamic
∗
Correspondence to: Martin PN Gent, Department of Forestry and Horticulture,
Connecticut Agricultural Experiment Station, PO Box 1106, New Haven, CT
06504, USA. E-mail: Martin.Gent@ct.gov
Department of Forestry and Horticulture, Connecticut Agricultural Experiment

lettuce,9 again suggesting a role in osmotic adjustment. Station, New Haven, CT 06504, USA

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c 2011 Society of Chemical Industry
Diurnal variation of nitrate and sugars in lettuce
process model with explicit metabolic and support tissue
compartments predicted lower nitrogen but increased nitrate with
increasing plant size in lettuce.16 This model included an osmotic
balance such that diurnal variation of nitrate was inversely related
to that of sugars. A model of metabolism with simplified enzyme
reactions in plant tissues also predicted this complementary
diurnal variation in nitrate and sugars.17 However, this model
predicted that nitrate would decrease, and sugars would increase,
during daylight hours, due to the effects of light on nitrate
reduction and photosynthesis, irrespective of any osmotic effect.
These trends were predicted to reverse in the dark, and to be more
dramatic in small compared to large plants.
In this study, lettuce plants were grown in hydroponics in
summer and winter under similar conditions of nutrient supply
to determine how plant size and environment affect the diurnal
variation in composition. Plants were harvested at 3 h intervals
over a day. Plants differing in size, due to different planting dates,
were compared in each season.
EXPERIMENTAL
Growth conditions
The experiments were conducted in Hamden, CT, USA (latitude
42 ◦ N, longitude 73 ◦ W, 50 m above sea level) in hoop-houses,
17.1 m long × 4.3 m wide × 2.7 m high, with a double glazing of
0.1 mm clear polyethylene film. Heating and ventilating set points
were 17 and 22 ◦ C. From mid June to September, the sides were
rolled up to a height of 1.5 m to improve ventilation and 30%
shade cloth was applied. Shaded thermocouples and temperature
humidity transmitters (model 500, Campbell Scientific, Logan, UT,
USA) measured air temperature at a height of 1.5 m in the center
of each greenhouse. Quantum sensors (model 190, LiCor, Lincoln,
NE, USA) measured photosynthetic active radiation (PAR) within
the greenhouse above the height of the crop. Another sensor
measured ambient sunlight in an unobstructed location. Readings
from each sensor were taken every minute, averaged hourly and
recorded.
Two hydroponic systems that could support 12 troughs or rows
of lettuce plants were constructed in each of two greenhouses.
These systems were run simultaneously by a single data-logging
computer. Troughs for the two systems were alternated along the
length of the house. The troughs were supported by a wooden
frame to give a 1.5% slope from inlet to outlet of troughs. The
troughs were 3 m long and 4 × 10 cm in cross-section, with 20 cm
between plants in each trough and between troughs (model CHA
9004/9010, Crop King, Lodi, OH, USA). Each system had a nutrient
reservoir: a 200 L removable-top polyethylene drum (model H-
06 950-35, Cole Parmer Inst. Co., Vernon Hills IL, USA) insulated
and buried in the ground and covered with black polyethylene film
to exclude light. Solution from the nutrient reservoir was passed
through a cotton-string core filter. A magnetic drive pump (model
SM1212-26, Liang, Chula Vista, CA, USA) supplied the high end of
each trough at 1 L min−1 . Solution flowed by gravity to the low
end of each trough and into a manifold that returned solution to
the nutrient reservoir for each system. The hydroponic systems
circulated water for 3 min in a 5, 10 or 20 min cycle, depending on
sunlight intensity.
Water and nutrient concentrates were injected automatically to
maintain a constant system volume and EC. A float switch (model
M800B, Madison Controls, Branford, CT, USA) was mounted inside
the reservoir to maintain 100 L of solution within the reservoir.
Whenever the float switch was below the set point, a solenoid
J Sci Food Agric 2012; 92: 542–550

c 2011 Society of Chemical Industry
www.soci.org
valve (model SV4212DB24, Valcor Engineering, Springfield, NJ,
USA) injected water at 0.12 L min−1 through a flow restriction
consisting of a 10 cm length of 0.75 mm i.d. × 1.6 mm o.d.
PEEK tubing (model 1533, Upchurch Scientific, Oak Harbor, WA,
USA). An electrode to measure conductivity and temperature
(model 35 820-62, ThermoFisher Scientific, Pittsburgh, PA, USA)
was mounted inline in the supply manifold. When the conductivity
cell was below the set point of 1.5 dS m−1 , peristaltic pumps (model
FPU104, Omega Engineering, Stamford, CT, USA) injected nutrient
concentrate solutions at 3 mL min−1 . Two nutrient concentrates
were used. One was a complete fertilizer, 4 : 14 : 34 N : P2 O5 : K2 O
(Hydro Gro, CropKing, Lodi, OH, USA) at 25 g L−1 with concentrated
phosphoric acid at 1.25 mL L−1 . The other was greenhouse-grade
calcium nitrate at 20 g L−1 . The dilute solution had 8 mmol L−1
nitrate, 4 mmol L−1 potassium, and adequate concentrations of all
other essential elements.
Plant material
Seeds of butter head lettuce (Lactuca sativa L.) ‘Buttercrunch’
(Johnny’s Seeds, Winslow, ME, USA) were germinated under con-
trolled conditions in an artificial medium (model LC1 Horticubes,
Smithers Oasis, Kent, OH, USA). Two weeks after germination, the
seedlings were transplanted to the greenhouse hydroponic sys-
tems. Several plantings were grown simultaneously in each of the
systems in each house. In the summer experiment, plants were
harvested on 14 August 2007. These plants were germinated on
12 June, 3 July or 13 July 2007, and they were harvested 63, 42, and
32 days after germination. The greenhouses were covered with
30% shade cloth during growth and harvest in summer. In the
winter experiment, plants were harvested on 17 November 2008.
These plants were germinated on 16 September and 1 October,
and were harvested 62 and 47 days after germination. No shade
cloth covered the greenhouses during growth and harvest in win-
ter. Each system had four troughs per planting in summer, and
eight troughs per planting in winter. Each trough had 14 plants
before the harvest commenced.
Plants were harvested at 3 h intervals during the day from 0615
to 2030 h in summer, and from 0630 to 2130 h in winter. Stated
times are EDT in summer and EST in winter. At each harvest, four
plants were selected, each from a different trough and position
along the trough. The whole plant fresh weights were recorded.
Roots were cut off the Oasis cube, washed in tap water, dried by
centrifugal force and weighed. They were placed in a bag on dry
ice. The shoot of each plant was divided into halves or quarters.
One set of subsamples for tissue analysis was weighed, placed in
a bag and put on dry ice. Another set of subsamples was used
to determine biomass and water content. The midrib (petiole)
was dissected from the leaf blade. These parts were weighed. The
area of leaf blade was determined. This harvest and measurement
operation took 5–10 min for one replicate of each planting. The
biomass samples were dried at 75 ◦ C and reweighed to determine
water content. The tissue samples were lyophilized, and separated
into petiole and leaf blade. The root, petiole, and leaf blade tissues
were weighed, and ground to pass a 20-mesh sieve. These were
stored at −20 ◦ C until analysis.
Chemical analysis
Three or four subsamples of the dried tissue for each planting
and harvest time were extracted and analyzed for nitrate and
soluble sugars using liquid chromatography (LC) (model 1100,
543
Agilent Corp, Palo Alto, CA, USA). For nitrate analysis, a 50 mg
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 www.soci.org
subsample of freeze-dried plant tissue was transferred to a 7 mL
homogenizer cooled in ice and ground with 5 mL ice-cold water.
This sample was diluted 2 : 1, centrifuged and stored at −20 ◦ C in
a micro-centrifuge tube until analysis. Just prior to analysis, the
sample was thawed, diluted 12 : 1 and filtered into a Target vial. A
20 µL subsample was injected into a 4.1 × 100 mm ion exchange
column with pre-column (model PRP-X110S, Hamilton, Reno, NV,
USA) and eluted at 1.5 mL min−1 with a buffer of 1.7 mmol L−1
NaHCO3 , 1.8 mmol L−1 Na2 CO3 , and 0.1 mmol L−1 KSCN. Nitrate
and other anions were detected by UV absorbance at 212 nm. This
LC method and dilution were calibrated with external standards
of nitrate.
For sugar analysis, 100 mg subsamples of freeze-dried plant
tissue were extracted twice with 5 mL of a solution of 240 mL
methanol, 100 mL chloroform, 20 mL water, and 10 mL formic
acid. Six milliliters of water was added to the extract and mixed to
separate liquid phases. The chloroform layer containing pigments
was discarded. The water phase was dried at 50 ◦ C under a stream
of air. Just prior to analysis, the dried residue was resuspended
in 10 mL water and filtered into a Target vial. A 50 µL volume
was injected into a 7.8 × 300 mm LC column with a calcium form
of exchange resin (model HPX-87C, Bio-Rad, Hercules, CA, USA)
with both anion and cation pre-columns to trap all except neutral
compounds. Deionized water at 0.6 mL min−1 flowed through the
pre-columns at room temperature, and through the column at
70 ◦ C. Sugars were detected by refractive index and calibrated
with external standards of sucrose, glucose, and fructose. Values
for polysaccharides, glucose and fructose were combined and
reported as hexoses.
Total nitrogen and elemental composition were determined in
250 mg subsamples digested by boiling for 10 min in a reagent
of 4 mL H2 SO4 then adding dropwise 10 mL H2 O2 . The sample
was cooled and diluted to 100 mL. Most elemental concentrations
were determined by inductively coupled plasma atomic emission
spectroscopy (Atom Scan 16, Thermo-Jarrell Ash, Franklin, MA,
USA). Reduced nitrogen was determined by reacting 0.4 mL
of the digest with 1.0 mL Nessler reagent diluted to 25 mL.
Optical absorbance was measured at 460 nm using a spectrometer
(Evolution 60, ThermoFisher Scientific). The method was calibrated
with mixtures of starch and bovine serum albumin.
Experimental design
The summer and winter experiments were analyzed separately.
There were four hydroponic systems used as replicate plots in
analysis of variance. In the summer experiment, only three systems
were included in the analysis, because a nutrient supply failed one
week before harvest. Plant size or planting was included as a fixed
factor in analysis of variance. First- and second-order polynomials
of time of day were used to examine effects of time. The analysis
included interactions between time and size.
RESULTS
Environment
Day length in the summer experiment was 15 h and maximum
radiation inside the greenhouses was 700 µmol m−2 s−1 PAR,
whereas in the winter these values were 8 h and 500 µmol m−2 s−1
(Fig. 1). In the summer, there is a large variation in air temperature,
with a maximum of 30 ◦ C and a minimum of about 15 ◦ C. The
low temperature occurred at 0600 h and the high temperature
544
persisted from about noon until 1700 h. The solution temperature
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c 2011 Society of Chemical Industry
MPN Gent
40 1600
Summer PAR Winter
Air temp.
Solution temp.
30 1200
Sunlight, µmol m–2 s–1 PAR
Temperature, °C
20 800
10 400
0 0
0 6 12 18 24 6 12 18 24
Hours
Figure 1. The diurnal variation of light and temperature on the harvest
date of summer and winter experiments.
followed a similar pattern, with a lag of several hours between
the maximum/minimum of air and solution temperature. In the
winter, air and solution temperature varied little and was within a
degree of the mean temperature of 17 ◦ C.
Dry matter
The plantings differed greatly in biomass and leaf area in both
summer and winter (Table 1). The small and medium plants
harvested in summer were similar in size to those harvested
in winter. Dry matter increased over the light period for all plant
sizes, and this increase was greater for smaller than for larger plants
(Fig. 2). The dry matter fraction of shoot biomass varied far more
in summer than winter, and the increase in dry matter continued
later in the day. The minimum dry matter fraction was measured
at dawn. This minimum was the same for plants of similar size in
summer and winter. The composition of lettuce tissue was actually
determined in dried tissue. The dry matter fraction was used to
express the composition of the plants on a fresh weight basis.
I report the information on a fresh weight basis, as it is more
relevant to human nutrition.
Nitrate
Averaged over all times, larger plants had a higher concentration of
nitrate than smaller plants (Table 2). On a fresh weight basis, small
plants had one half the nitrate concentration of medium plants in
summer. In winter, this increased to 75% of the value in medium
plants. There was little diurnal change in nitrate concentration
(Fig. 3). In summer, nitrate decreased from the first to second
harvest, dawn to 0900 h, and then increased at the end of the
day. These diurnal changes were not significant. When comparing
plants of similar size, the nitrate concentration was greater at all
times of day in winter than summer (Fig. 3). The increase from
summer to winter, averaged over all times, was more than 100%
for small plants, but only 25% for medium plants.
The change in nitrate concentration over the daylight hours
in summer was significant when expressed on a dry matter basis
(Table 2). Plants of all sizes showed a marked decrease in nitrate
concentration from the first to second harvest (Fig. 4). Thereafter,
nitrate rose towards the end of the day in large and medium plants.
J Sci Food Agric 2012; 92: 542–550
Diurnal variation of nitrate and sugars in lettuce www.soci.org

Table 1. Age, size, and nitrogen content of shoot tissue (leaf blade and petiole) of hydroponic lettuce plantings. Values are means per plant and
standard deviations over all diurnal harvests

Size Days after germination Leaf area (cm2 ) Fresh weight (g) Leaf : shoot dry weight ratio Total N (mol kg−1 )

Summer 2007
Large 63 3451 ± 281 295 ± 49 0.55 ± 0.07 2.77 ± 0.07
Medium 42 1170 ± 140 55 ± 7 0.66 ± 0.06 3.07 ± 0.19
Small 32 123 ± 75 3.6 ± 0.6 0.78 ± 0.08 3.41 ± 0.39
Winter 2008
Medium 62 938 ± 138 54 ± 8 0.65 ± 0.03 3.38 ± 0.29
Small 47 38 ± 8 1.5 ± 0.5 0.70 ± 0.05 3.68 ± 0.35

120 they had 20% less on a dry weight basis. On a dry weight basis,
Summer Winter the primary difference between plant sizes was the concentration
at dawn (Fig. 6). Small plants had less sugar than other plant
100
sizes when harvested in the morning, and sugars increased with
time, but only to a concentration similar to that in medium and
Dry matter, g dw kg–1 fw

80 large plants. The diurnal variation in soluble sugars did not differ
with plant size. The variation during daylight hours was linear in
summer and quadratic in winter.
60

Petiole and leaf blade composition

40
20 Large
Shoot Medium
Small
0
0 6 12 18 24 6 12
Hours
Figure 2. Dry matter content of lettuce shoot tissue as a function of plant
size and time of day in summer and winter. Symbols are means of three
samples in summer and four in winter, error bars indicate standard error,
and lines connect values found at subsequent harvest times.
The effect of plant size was more marked when expressed on a dry
weight than a fresh weight basis. Small plants had only 20% of the
nitrate concentration of large plants in summer, and 60% of that
of medium plants in winter. There was no interaction of the effects
of plant size and time of day on nitrate concentration, whether
expressed on a fresh or dry basis.
Sugars
On a fresh weight basis, the concentration of soluble sugars,
expressed as hexoses, was similar for the three plant sizes early in
the day (Fig. 5). In contrast to nitrate, the concentration of hexoses
on a fresh weight basis changed dramatically over the daylight
hours. Sugars increased linearly with time through most of the
daylight hours in summer (Fig. 5). This increase was far greater in
small plants than in medium and large plants. The diurnal variation
of sugars in small plants was less in winter than in summer, and
more similar to that of medium plants in both summer and winter.
The diurnal patterns were similar for medium and small plants in
winter, except medium plants had more hexoses at dawn.
In summer, the differences in soluble sugar content between
plant sizes were less on a dry weight than on a fresh weight basis
(Table 2). Averaged over all times, small plants had an average of
Much of the difference due to plant size in the composition of the
shoot can be attributed to the fact that the shoot consists of two
tissues, namely leaf blade and petiole. Petiole samples referred to
here include the midrib of each leaf as well as any stem above the
germination cube. The fraction of weight of the shoot in each of
these tissues changes with plant size. The leaf blade accounted for
18 24 about 55%, 65%, and 70–78% of total shoot dry weight, in large,
medium, and small plants, respectively (Table 1). Thus shoot tissue
composition was a weighted average of the concentrations in
leaf blade and petiole. These two tissues differed in composition,
as well as in the diurnal variation over the day. The dry matter
content of leaf blades was 50% greater than petiole at dawn in
the summer, and this difference increased over the daylight hours
(data not shown). The difference in dry matter between leaf blade
and petiole was smaller in winter than in summer. There was less
diurnal variation in petiole than leaf blade tissue, for all plant sizes
in both seasons. These data are not shown, but they can be inferred
from the size of the standard deviations over all harvests (Tables 3
and 4). The standard deviation was smaller for dry matter content
in petiole than leaf tissue, for all plant sizes in both seasons. For
plants of similar size, petiole tissue had greater dry matter in winter
than in summer (Table 3), while the opposite trend was observed
for leaf blades (Table 4).
The nitrate concentration, on either a fresh or dry weight basis,
was greater in petiole than in leaf blade tissue, and concentration
varied less with plant size (Tables 3 and 4). On a fresh weight
basis, the nitrate concentration in leaf blades of large plants was
threefold that of small plants in summer, whereas the difference
in petioles was only 40%. There was a greater difference in nitrate
concentration between petiole and leaf blade for small compared
to large plants. In medium plants, the nitrate concentration in
petioles was twice that in leaf blades in summer, and 50% more in
winter. Small plants had threefold more nitrate in petioles than leaf
blades in summer, and 80% more in winter. The diurnal variation
in nitrate was more significant in leaf blades than petioles on a
fresh weight basis, but the opposite was true on a dry weight
basis. There was no significant diurnal variation in winter (data not
545

55% more hexoses than large plants on a fresh weight basis, but shown).

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 www.soci.org MPN Gent

Table 2. Composition means and standard deviation over time of lettuce shoot tissue (leaf blade and petiole), and analysis of variance due to plant
size and time of harvest. Plant size was a fixed effect; time was included as first- and second-order polynomials

Fresh weight Dry weight

Factor Dry matter (g kg−1 fw) Nitrate (mmol kg−1 ) Hexoses (mmol kg−1 ) Nitrate (mmol kg−1 ) Hexoses (mmol kg−1 )

Summer 2007
Large 51 ± 3 36.4 ± 9.0 41.4 ± 12.1 714 ± 172 801 ± 110
Medium 64 ± 8 31.7 ± 7.9 47.4 ± 15.8 502 ± 130 725 ± 174
Small 92 ± 18 15.7 ± 4.8 64.0 ± 31.2 179 ± 76 654 ± 225
Plant size (S) ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗

Time of day (T) ∗∗∗ NS L∗∗ ∗∗ L∗

S×T L∗∗ NS NS NS NS
Winter 2008
Medium 57 ± 2 51.6 ± 12.0 38.2 ± 12.6 905 ± 216 662 ± 199
Small 66 ± 4 39.2 ± 6.4 36.5 ± 17.5 529 ± 92 481 ± 214
∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗
Plant size (S) NS
Time of day (T) ∗∗∗ NS ∗∗∗ NS ∗∗∗

S×T ∗∗∗ NS NS NS NS

Asterisks indicate effect significance at ∗ P< 0.05; ∗∗ P< 0.01; ∗∗∗ P < 0.001; NS, not significant; L, only first-order effect of time is significant.

120 1200
Summer Winter Summer Winter

100 1000
Large
Medium
Small
Nitrate, mmol kg–1 dw

800
Nitrate, mmol kg–1 fw

80

60 600

40 400

20 200 Large
Medium
Small
0 0
0 6 12 18 24 6 12 18 24 0 6 12 18 24 6 12 18 24
Hours
Hours

Figure 3. Nitrate concentration of lettuce shoot tissue on a fresh weight
basis as a function of plant size and time of day in summer and winter.
Symbols are means of three samples in summer and four in winter, error
bars indicate standard error, and lines connect values found at subsequent
harvest times.
Figure 4. Nitrate concentration of lettuce shoot tissue on a dry weight
basis as a function of plant size and time of day in summer and winter.
Symbols are means of three samples in summer and four in winter, error
bars indicate standard error, and lines connect values found at subsequent
harvest times.

The sugar concentrations and diurnal variation differed in DISCUSSION

petiole and leaf blade tissue. Leaf blades had less sugar than
petioles at dawn (data not shown). Although the sugars in leaf
blades increased rapidly to about 1500 h, they often decreased
at the end of the day. In contrast, petioles showed a more linear
increase in sugars throughout the daylight hours. Consequently,
averaged over the day, the hexose concentration in petioles was
greater than in leaf blades, for all except the small plants (Tables 3
and 4). In summer, small plants had much more sugar in leaf
blades than medium or large plants, although the concentration
in petioles was similar. In winter, there were similar concentrations
of sugars in leaf blades of small and medium plants, but petioles of
medium plants had 30% more hexoses than those of small plants
546
Although these results were presented according to the size of
the plants at harvest, the plants also differed in age or days after
germination (Table 1). Within each experiment, plants of one size
were all the same age. The large plants in summer were similar
in age to the medium plants in winter, and the medium plants
in summer were actually 5 days younger than the small plants
in winter. All of these plants were in a purely vegetative stage
of development at harvest, except for slight stem elongation of
some large plants in summer. In comparisons between summer
and winter, differences were less if plants were compared on the
basis of similar age rather than similar size.
Both plant size and season affected the diurnal variation in

(Tables 3 and 4). concentration of nitrate and sugar in lettuce. Diurnal variation was

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c 2011 Society of Chemical Industry J Sci Food Agric 2012; 92: 542–550
Diurnal variation of nitrate and sugars in lettuce www.soci.org

120 more slowly, but for a longer duration of the daylight hours, than
Summer Winter in leaf blades. The greater contribution of petiole tissue to shoot
biomass would explain the relatively smaller diurnal variation in
100 sugars in large compared to small plants, as well as the relatively
Large
Medium high concentration of sugars and nitrate in large plants.
Small The changes in dry matter content with time of day and plant
Hexoses, mmol kg–1 fw

80
size have been observed under other conditions. Burrage and
Varley18 noted a dip in the relative water content at noon for
60 hydroponic lettuce grown in a greenhouse in May. The relative
water content was less at high EC compared to low EC of the
nutrient solution, 2.9 compared to 1.1 dS m−1 . In the present
40 study, plants grew under an EC = 1.5 dS m−1 . After a day under a
light intensity of 200 µmol m−2 s−1 PAR, dry matter in two cultivars
of lettuce grown in sand culture decreased in the dark by 10 g kg−1
20
fresh weight (fw).8 This was similar to the variation we observed in
medium plants in winter. This and other studies noted a change
0 in dry matter with plant size. For butterhead lettuce grown in
0 6 12 18 24 6 12 18 24 hydroponics, dry matter decreased with size from 70 g kg−1 fw to
Hours 60 and 50 g kg−1 fw, in two cultivars.19 A time study comparing
two lettuce cultivars only noted a difference between cultivars in
Figure 5. Sugar concentration of lettuce shoot tissue on a fresh weight
basis as a function of plant size and time of day in summer and winter. water content after they attained 100 g fw.20 However, for head
Symbols are means of three samples in summer and four in winter, error lettuce grown in a poly-house in winter, water content decreased
bars indicate standard error, and lines connect values found at subsequent as plants grew, and outer leaves had more water content than
harvest times. inner leaves.21
Environmental factors, particularly light intensity, have an
1200 effect on dry matter. For small seedlings, dry matter increased
linearly from 60 to 110 g kg−1 fw as light increased from 100 to
Summer Winter 300 µmol m−2 s−1 , with a greater increase under 24 h compared to
1000 16 h photoperiod, and high compared to low CO2 .22 However, for
hydroponic lettuce in the Netherlands, dry matter of large plants
decreased from 55 to 35 g kg−1 fw from winter to summer,13
Hexoses, mmol kg–1 dw

800
600
400
200 Large
Medium
Small
0 in soil – a range of 63–110 g kg−1 fw23 – which may in part be
0 6 12 18 24
Hours
Figure 6. Sugar concentration of lettuce shoot tissue on a dry weight
basis as a function of plant size and time of day in summer and winter.
Symbols are means of three samples in summer and four in winter, error
bars indicate standard error, and lines connect values found at subsequent
harvest times.
much greater when plants were grown under high light intensity
in summer than when plants were grown under low light intensity
and relatively constant temperature in winter. In summer, the
diurnal changes were greater for small plants than for large plants.
The shoot of lettuce is made up of different tissues, namely leaf
blade and petiole, which differ in composition. As a fraction of
shoot fresh weight, the petioles increased from 25% to 50% or more
of the total, as lettuce grew from a seedling to a large plant. The
petiole appeared to act, in part, as a reservoir to accumulate sugars
and/or nitrate that were not metabolized for growth. There was
more nitrate in petiole than leaf blade tissue at all times. Petioles
perhaps because plant size increased, and this increase had more
of an effect than increasing light. This may be due to a greater
contribution of petiole tissue to dry matter in large plants. We
noted a decrease in petiole dry matter from winter to summer.
This is the first report to document that the changes in diurnal
variation of dry matter vary with plant size in lettuce. Although
there was a change in dry matter in both leaf blade and petiole
with plant size and environment, the diurnal variation decreased
more markedly with increased size in the leaf blade alone. There
is considerable genotypic variation in dry matter of lettuce grown
6 12 18 24 due to genotypic differences in the leaf blade : petiole weight
ratio. When a range of lettuce ecotypes was observed, there was
a linear relationship between nitrate on a dry matter basis and
water content, more water, and higher nitrate across cultivars.20
This relation was stronger in summer than winter. This observation
is consistent with a hypothesis that petioles have a higher nitrate
and water content than leaf blades, and therefore genotypes with
a higher fraction of shoot weight in petiole tissue would also have
a higher nitrate and water content.
Nitrate varied less with plant size or time of day than dry matter
or sugars. The time of day variation was not significant in winter.
This lack of diurnal variation in nitrate on a fresh weight basis
has been noted for root and petiole of tomato.8 A variation was
only seen in leaf blade tissue. We saw a more significant diurnal
variation in nitrate on a fresh weight basis in leaf blades compared
to petioles. The variation across plant sizes in nitrate concentration
we observed in summer – 16, 32, and 36 mmol kg−1 fw, for small,
medium, and large plants, respectively – can be compared to a
range of 23–33 mmol kg−1 fw reported14 for lettuce grown at a
547

had more sugar than leaf blades at dawn, and sugars increased similar time of year.

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 www.soci.org MPN Gent

Table 3. Lettuce petiole tissue composition means and standard deviation over time, and analysis of variance due to plant size and time of harvest.
Plant size was a fixed effect; time was included as first- and second-order polynomials

Fresh weight Dry weight

Factor Dry matter (g kg−1 fw) Nitrate (mmol kg−1 ) Hexoses (mmol kg−1 ) Nitrate (mmol kg−1 ) Hexoses (mmol kg−1 )

Summer 2007
Large 38 ± 2 38.6 ± 12.2 44.0 ± 11.6 1009 ± 302 1146 ± 264
Medium 43 ± 4 38.3 ± 10.8 48.3 ± 13.9 903 ± 245 1119 ± 249
Small 56 ± 8 24.2 ± 9.7 45.7 ± 16.8 435 ± 194 789 ± 201
Plant size (S) ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗

Time of day (T) ∗ ∗ L∗∗ ∗∗ NS

S×T ∗∗ NS NS NS NS
Winter 2008
Medium 50 ± 1 66.5 ± 21.2 47.4 ± 13.8 1320 ± 491 935 ± 254
Small 67 ± 3 53.4 ± 12.3 36.7 ± 15.2 800 ± 184 543 ± 213
∗∗∗ ∗ ∗∗ ∗∗∗ ∗∗∗
Plant size (S)
Time of day (T) ∗∗∗ NS ∗∗∗ NS ∗∗∗

S×T ∗∗∗ NS NS NS NS

Asterisks indicate effect significance at ∗ P< 0.05; ∗∗ P< 0.01; ∗∗∗ P < 0.001; NS, not significant; L, only first-order effect of time is significant.

Table 4. Lettuce leaf blade tissue composition means and standard deviation over time, and analysis of variance due to plant size and time of
harvest. Plant size was a fixed effect; time was included as first- and second-order polynomials

Fresh weight Dry weight

Factor Dry matter (g kg−1 fw) Nitrate (mmol kg−1 ) Hexoses (mmol kg−1 ) Nitrate (mmol kg−1 ) Hexoses (mmol kg−1 )

Summer 2007
Large 61 ± 4 29.0 ± 7.8 32.4 ± 11.4 480 ± 138 523 ± 163
Medium 75 ± 10 21.0 ± 6.5 40.2 ± 15.5 286 ± 102 521 ± 151
Small 102 ± 21 10.4 ± 3.2 67.4 ± 35.4 110 ± 52 618 ± 243
∗∗∗ ∗∗∗ ∗ ∗∗∗
Plant size (S) NS
∗∗∗ ∗∗
Time of day (T) L∗∗ ∗
L∗∗∗
∗∗
S×T NS NS NS NS
Winter 2008
Medium 61 ± 3 41.3 ± 10.0 31.6 ± 13.1 682 ± 174 514 ± 199
Small 79 ± 5 29.5 ± 5.8 36.0 ± 18.9 377 ± 86 446 ± 220
Plant size (S) ∗∗∗ ∗∗∗ NS ∗∗∗ NS
∗∗∗ ∗∗∗ ∗∗∗
Time of day (T) NS NS
∗
S×T NS NS NS NS
∗ ∗∗ ∗∗∗
Asterisks indicate effect significance at P < 0.05; P < 0.01; P < 0.001; NS, not significant; L, only first-order effect of time is significant.

Some studies found a diurnal variation in concentration of
nitrate in lettuce and others did not. Nitrate increased in the
dark from 40 to 50 and from 55 to 65 mmol kg−1 fw in two
cultivars of lettuce grown under controlled conditions.8 For
hydroponic lettuce in a greenhouse, leaf nitrate increased from
61 to 77 mmol kg−1 fw early in the dark, before falling to a
lower value early in the light.5 For hydroponic lettuce grown for
20 days under supplemental light of 16 mol m−2 d−1 , average
shoot nitrate was about 40 mmol kg−1 fw. This increased by 5, 15,
and 5 mmol kg−1 fw on three nights, and decreased through the
day.24 For field lettuce of 65 or 73 g fw grown at 46◦ and 64◦ N
latitude under 8 or 5 h of night, average nitrate concentration was
20 and 43 mmol kg−1 fw, respectively.15 Under an 8 h night, nitrate
increased linearly during two of three nights and decreased during
one day, whereas under a 5 h night there was a linear decrease
548
increase in nitrate in the dark was greater under the 5 h compared
to 8 h night condition: about 14 and 4 mmol kg−1 fw, respectively.
However, diurnal variation was seen in only one of four cultivars
of hydroponic lettuce grown in a greenhouse under low light and
temperature.25
Environment also affects tissue nitrate. Hydroponic lettuce
grown in a greenhouse in Connecticut had two- to threefold
greater nitrate in winter than in summer: a range of 32–54
mmol kg−1 fw in winter and 18–19 mmol kg−1 fw in summer.9
Nitrate concentration decreased from 78 to 45 mmol kg−1 fw from
winter to summer in a greenhouse in the Netherlands.13 Time of
day had little effect on nitrate concentration in winter.
Studies of spinach noted more nitrate in petioles than leaves,
and a greater diurnal variation in leaves. The diurnal variation of
nitrate in the leaf blade was from 42 to 23 mmol kg−1 fw under

only during one day. Although the authors do not state it, the 145 µmol m−2 s−1 PAR light and from 27 to 14 mmol kg−1 fw

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c 2011 Society of Chemical Industry J Sci Food Agric 2012; 92: 542–550
Diurnal variation of nitrate and sugars in lettuce
under 225 µmol m−2 s−1 PAR.4 The nitrate in petioles varied less
than in leaf blades, and averaged 200 and 50 mmol kg−1 fw under
low and high light. For spinach under a 14 h photoperiod, nitrate
in the shoot decreased in the 10 h light period, from 22 to 20, 10,
and 7 mmol kg−1 fw (assuming 100 g kg−1 fw), under 100, 320,
and 510 µmol m−2 s−1 PAR,3 respectively. The variation in the root
and petiole was less than in the leaf. The present study of lettuce
also noted a greater diurnal variation under high than low light,
and higher nitrate in petiole than leaf blade.
In contrast to nitrate, we observed a significant diurnal variation
in sugars during the day in plants of all sizes and in both summer
and winter. Such behavior has been observed in tomato26,27 and
many other annual crop plants. However, there have been few
studies of lettuce. For field-grown crisphead lettuce, only the outer
leaf showed a pattern similar to that which we observed. The outer
leaf had 2 and 12 mmol kg−1 fw of hexoses at 0700 and 1400 h.1
Glucose and fructose in leaf blades decreased during the day
from 17 to 8, and from 25 to 14 mmol kg−1 fw, respectively,
in that study. There was little sucrose, but higher amounts
of glucose and fructose in the petiole. These concentrations
in the petiole and in inner leaves decreased during the day.
A range of 36–44 mmol kg−1 fw of reducing sugar has been
reported for greenhouse lettuce varying in size.14 This compares
to concentrations of 61, 47, and 41 mmol kg−1 fw that we observed
in summer for small, medium, and large plants, respectively.
The inverse pattern of diurnal variation in sugars and nitrate,
increasing for sugars and decreasing for nitrate in the light, has
been hypothesized to be due to a requirement for a stable osmotic
potential in plant tissue.6,28 However, the diurnal changes in sugar
and nitrate observed here are of different magnitudes and patterns.
Most of the decrease in nitrate occurred early in the day, whereas
sugars showed a continuous increase through the day. These
patterns are not consistent with a constant osmotic potential. In
particular, the higher values of both hexoses and nitrate in petiole
compared to leaf blade at dawn, and the remarkable increase in
hexoses with no substantial change in nitrate in leaf blades, are
not consistent with the hypothesis that sugars and nitrate are
interchangeable as an osmoticum. In fact, the remarkable diurnal
variation in dry matter content suggests that the osmotic potential
of plant tissue changes considerably over the day.
One factor that varies considerably between plants of different
sizes is the leaf area index and light interception per unit leaf area
or weight. Leaves of small plants are widely spaced and they are
exposed to the full intensity of sunlight falling on the canopy.
Larger plants have more leaf area and considerable overlap of
leaves, so on average the light intensity per unit leaf area is less.
Per unit weight, small plants would be expected to have higher
rates of photosynthesis and nitrate reduction, and thus greater
changes in sugars and nitrate, due to the greater light intensity per
unit leaf area. We observed that plant size did affect the magnitude
of diurnal changes of sugars but not that of nitrate. Small plants
must also have a faster rate of metabolism in the dark, as the
concentration of sugar at the end of the dark period was less for
small compared to large plants.
CONCLUSION
Time of day affected the dry matter content and concentration
of sugars more than that of nitrate in lettuce. The effects were
most pronounced in small plants grown in summer, equivalent to
micro-greens. For lettuce harvested at a normal commercial size
of 100–200 g fw, the diurnal variation of dry matter and sugar
J Sci Food Agric 2012; 92: 542–550

c 2011 Society of Chemical Industry
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was of the order of 20% of the mean. The greatest yield would be
obtained by harvesting early in the day. As consumers do not buy
lettuce for dry matter or sugar content, there is little commercial
benefit to delaying harvest to later in the day.
ACKNOWLEDGEMENTS
I thank Dr Ido Seginer of the Technion, Haifa, Israel, for suggesting
this study of diurnal variation as a function of plant size, and
Mr Michael Short for assistance with cultivation, harvesting, and
extraction and chemical analysis of lettuce tissues.
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