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Photochemistry and Photobiology, 2014, 90: 574–584
Photobiological Interactions of Blue Light and Photosynthetic Photon
Flux: Effects of Monochromatic and Broad-Spectrum Light Sources
Kevin R. Cope*, M. Chase Snowden and Bruce Bugbee
Crop Physiology Laboratory, Department of Plants Soils and Climate, Utah State University, Logan, Utah
Received 30 July 2013, accepted 10 December 2013, DOI: 10.1111/php.12233
ABSTRACT
Photosynthesis (Pn) and photomorphogenesis (Pm) are
affected by light quality, light intensity and photoperiod.
Although blue light (BL) is necessary for normal develop-
ment, it is less efficient in driving Pn than other wavelengths
of photosynthetically active radiation. The effects of BL on
Pm are highly species dependent. Here we report the inter-
acting effects of BL and photosynthetic photon flux (PPF) on
growth and development of lettuce, radish and pepper. We
used light-emitting diode (LED) arrays to provide BL frac-
tions from 11% to 28% under broad-spectrum white LEDs,
and from 0.3% to 92% under monochromatic LEDs. All
treatments were replicated three times at each of two PPFs
(200 and 500 lmol m
2
s
1
). Other than light quality, envi-
ronmental conditions were uniformly maintained across
chambers. Regardless of PPF, BL was necessary to prevent
shade-avoidance responses in radish and lettuce. For lettuce
and radish, increasing BL reduced stem length, and for both
species, there were significant interactions of BL with PPF
for leaf expansion. Increasing BL reduced petiole length in
radish and flower number in pepper. BL minimally affected
pepper growth and other developmental parameters. Pepper
seedlings were more photobiologically sensitive than older
plants. Surprisingly, there were few interactions between
monochromatic and broad-spectrum light sources.
INTRODUCTION
Photosynthesis (Pn) and photomorphogenesis (Pm) are both
affected by light quality, light intensity (defined as photosyn-
thetic photon flux, PPF) and photoperiod.
Photosynthesis is best defined as quantum yield (moles of
CO
2
fixed per mole of photons absorbed) and is driven by the
absorption of light by chlorophyll and other photosynthetic
accessory pigments, including carotenoids (1). The wavelengths
of light that drive Pn are referred to as photosynthetically active
radiation (PAR), and range from 400 to 700 nm. McCree (2)
concluded that PPF (lmol m
2
s
1
), rather than energy flux
(W m
2
), is a more accurate method for measuring PAR. This
means that although blue photons have more energy than red
photons, both red and blue photons will still drive the same
amount of Pn. PPF assumes that all photons between 400 and
700 nm have equal weight. However, specific wavelengths of
light are absorbed differently, so not all wavelengths are equally
efficient in driving Pn. More specifically, not all absorbed blue
photons (400–500 nm) are used for Pn because approximately
20% of these photons are absorbed by inactive pigments (such
as anthocyanins), and as a result, their energy is not transferred
to photosynthetic reaction centers but is lost as heat and/or fluo-
rescence (3). Furthermore, some blue photons are absorbed by
carotenoids, which have absorption maxima for blue wave-
lengths. Depending on the type of carotenoid and its position
within the photosynthetic apparatus, the efficiency of excitation
energy transfer from carotenoids to chlorophylls ranges from
35% to 90%. This is significantly less than the energy transfer
efficiency in the antenna complexes from chlorophyll to chloro-
phyll (up to 100%) (4). As a result, there is a significant loss of
blue light (BL) energy due to radiation capture by non-photosyn-
thetic pigments and inefficient energy transfer by accessory
pigments. This loss is indicated by the relative quantum efficiency
(RQE) curve (5,6). The RQE of red light (RL, 600–700 nm) is
25–35% more efficient than blue light (BL, 400–500 nm) and
5–30% greater than green light (GL, 500–600 nm). For this rea-
son, a theoretically more accurate measurement of PAR is the
yield photon flux (YPF), which is derived from weighting each
photon’s wavelength using its RQE (7).
Photomorphogenesis is defined as light-mediated develop-
ment, and is regulated by the light perception network in plants,
which is composed of at least three classes of photoreceptors:
phytochromes, cryptochromes and phototropin (8). Phytochromes
are sensitive primarily to red and far-red (FR) radiation
(600–750 nm), cryptochromes to ultraviolet-A (UV-A) and blue
(320–500 nm) and phototropins to UV-A, blue and green
(320–520 nm) (9). In the model plant Arabidopsis thaliana,five
phytochromes (Phy A through Phy E), two cryptochromes (Cry
1 and Cry 2) and two phototropins (Phot 1 and Phot 2) have
been identified (10). The activity of each photoreceptor varies
with developmental stage, and each has been shown to regulate
certain developmental responses in A. thaliana (11). Phyto-
chrome regulates seed germination, de-etiolation, shade-avoid-
ance, circadian entrainment and flowering; cryptochrome also
regulates de-etiolation, circadian entrainment and flowering;
phototropin, however, is unique and regulates phototropism,
chloroplast movement and stomatal opening (11). Interactions
between photoreceptors can be synergistic or antagonistic,
depending on the light signals received by the plant (9). Most if
not all of the photoreceptors in A. thaliana are likely conserved
in other plant species (12). The activity of these photoreceptors
can be manipulated by altering the light spectra (13). For example,
*Corresponding author email: kevinrcope@gmail.com (Kevin R. Cope)
©2013 The American Society of Photobiology
574
phytochrome photoequilibrium (PPE) is a parameter used for pre-
dicting the effect of a given light source on phytochrome. PPE is
calculated as described by Sager et al. (7). Stutte (14) demon-
strated that manipulating PPE with LEDs led to a direct control
of some plant developmental parameters.
Several studies have utilized LEDs to test specific combina-
tions of wavelengths of light and to elucidate their effects on Pn
and Pm. BL has a relatively small effect on Pn, but profound
effects on Pm. Cope and Bugbee (15) reviewed 10 studies that
collectively indicate that some BL is essential for normal devel-
opment, including prevention of shade-avoidance responses.
However, BL effects appear to be species dependent and interact
with light quality and PPF. Because the RQE for BL is low, the
goal of many studies has been to determine the minimum
amount of BL needed to achieve normal development (16).
Wheeler et al. (17) reported that an absolute amount of
30 lmol m
2
s
1
of blue photons were adequate for normal
development of soybeans. Dougher and Bugbee (18) found that
species (wheat, soybean and lettuce) have different BL require-
ments, and that developmental responses (stem elongation and
leaf expansion) can be best predicted by either the relative (% of
total PPF) or the absolute (lmol photons m
2
s
1
) amount of
BL. These results were confirmed and extended by Cope and
Bugbee (15) who studied three types of white LEDs, including
warm, neutral and cool, which contained 11%, 19% and 28%
BL respectively. They analyzed their effect on the growth (dry
mass [DM] gain) and development (plant morphology) of radish,
soybean and wheat and found that variation in BL from 11% to
28% minimally affected growth but significantly altered develop-
ment in radish and soybean. Overall, the low BL from warm
white LEDs increased stem length (SL) and leaf area (LA) while
the high BL from cool white LEDs resulted in more compact
plants. For radish and soybean, absolute BL was a better predic-
tor of SL than relative BL, but relative BL better predicted LA.
Absolute BL better predicted the percent leaf DM in radish and
soybean and percent tiller DM in wheat (15).
Overall, Cope and Bugbee (15) concluded that roughly
50 lmol m
2
s
1
or 15% BL (whichever is greater depending
on the PPF) is likely required to promote normal development in
most plant species. However, their range of BL levels was lim-
ited (11–28%) and they suggested that the optimal amount of BL
likely changes with age as plant communities balance the need
for rapid leaf expansion (which is necessary to maximize radia-
tion capture) with prevention of excessive stem elongation.
We sought to extend the results of previous research by
increasing the range of BL to 0.3–92% using both monochro-
matic and broad-spectrum LEDs to grow three diverse plant spe-
cies. This BL range permitted a better evaluation of the putative
role of phytochrome (a predominately RL photoreceptor) and
cryptochrome (a BL photoreceptor) in shade-avoidance responses
in horticultural crop plants. Furthermore, the use of monochro-
matic and broad-spectrum light sources allowed us to observe
the effects of wavelength interactions. We also examined interac-
tions of BL with developmental stage in pepper.
MATERIALS AND METHODS
Plant material and cultural conditions. The LED growth chamber hard-
ware was identical for all studies, but the cultural conditions for lettuce
(Lactuca sativa, cv. “Waldmann’s Green”) and radish (Raphanus sativus,
cv. “Cherry Belle”) were different than for pepper (Capsicum annum, cv.
“California Wonder”).
Lettuce and radish seeds were pre-germinated until radicle emergence
(48 and 36 h respectively). Subsequently, eight uniform seeds were trans-
planted to root modules measuring 15 918 99cm (L9W9H;
2430 cm
3
). The root modules were filled with horticultural grade soilless
media (one peat: one vermiculite by volume) and 5 g of slow-release
fertilizer (16N-2.6P-11.2K; Polyon
â
1–2 month release, 16-6-13), which
was uniformly mixed into the media. After planting, the root modules
were watered to excess with a complete, dilute fertilizer solution (0.01N-
0.001P-0.008K; Scotts
â
Peat-Lite, 21-5-20; EC =100 mS m
1
), and
allowed to passively drain.
Pepper seeds were pre-germinated for 7 days, and transplanted after
radicle emergence into pots (8 9897 cm; 448 cm
3
)filled with soilless
media identical to that used for lettuce and radish. One gram of slow-
release fertilizer was incorporated into each pot. The pots were then
watered to excess with the same dilute fertilizer solution and allowed
to passively drain. After planting, the pots were placed in a growth
chamber (130 956 9108 cm; 0.79 m
3
) with an average PPF of
300 lmol m
2
s
1
provided by cool white fluorescent (CWF) lamps (see
Table 1 and Fig. 1 for spectral characteristics) and day/night temperature
set-points of 25/20°C. Seedlings emerged within 2 days and the cotyle-
dons were allowed to fully expand. Plants remained in the growth cham-
ber for 33 days after emergence (DAE). For all three replicates, 56 of the
most uniform plants were randomly assigned to 14 groups of four, and
each group of four was transplanted into root modules with the same
dimensions as those used for lettuce and radish. To facilitate studies on
developmental stage in pepper, we also conducted a supplemental study
with pepper seedlings by germinating 12 seeds directly in the LED cham-
bers following the same methods as lettuce and radish.
After planting the pre-germinated lettuce and radish seeds and trans-
ferring the pepper plants, each root module was randomly placed
into one of 14 LED growth chambers. Chambers measured
20 923 930 cm (13800 cm
3
; Fig. 2) and the internal walls were lined
with high-reflectance Mylar
â
. Shortly after lettuce and radish seedling
Table 1. Spectral characteristics of the seven LED treatments. Yield photon flux (YPF) and phytochrome photoequilibrium (PPE) were determined
according to Sager et al. (7). Since the pepper plants were started in a growth chamber with cool white fluorescent (CWF) lamps, the CWF spectral data
are included as a reference.
Light quality LED treatment
Parameter CWF Cool Neutral Warm Blue Red RB RGB
PPF 500 500 500 500 500 500 500 500
YPF 445 429 441 455 351 486 469 447
YPF/PPF 0.89 0.86 0.88 0.91 0.70 0.97 0.94 0.89
% UV-A 1.57 0.17 0.13 0.09 0.18 0.03 0.01 0.11
% Blue 20.6 27.5 19.4 10.8 92.0 0.27 12.0 13.7
% Green 52.3 48.0 45.6 40.9 7.8 1.7 1.7 22.9
% Red 23.8 24.5 35.0 48.3 0.22 98.1 86.3 63.4
PPE 0.84 0.83 0.84 0.84 0.58 0.89 0.89 0.89
R:FR 14.1 6.44 5.67 5.02 0.1/0 >999 >999 170.0/0
Photochemistry and Photobiology, 2014, 90 575
emergence, the four least uniform seedlings were removed and the other
four seedlings continued to grow and develop in their respective light
environment; peppers were not thinned after transplanting into the LED
treatments. Lettuce and radish plants were grown for 21 DAE and pepper
plants were grown for 21 days after transferring them into their assigned
LED treatment. In total, peppers were grown for 54 DAE. Pepper seed-
lings in the single supplemental study were harvested at 12 DAE after
cotyledons were fully expanded.
Ventilation was ample in all growth chambers and controlled to main-
tain the same temperature, CO
2
concentration and relative humidity
(RH). Type-E thermocouples connected to a data-logger (model CR10T;
Campbell Scientific, Logan, UT) were used to continuously monitor tem-
perature at the top of the plant canopy. To avoid partial shading of the
plants, the thermocouples were not shielded; had they been shielded, our
measurements indicate that recorded temperatures would have been
reduced by about 0.5°C in each treatment. Average day/night tempera-
tures were 24.7 0.6/24.1 0.4°C for lettuce, 23.4 0.3/
21.8 0.3°C for radish and 23 0.4/21.7 0.3°C for pepper. Temper-
ature differences among chambers for each replicate were less than
0.5°C. CO
2
concentration was measured using a CO
2
probe (model
GMP222; Vaisala Inc., Finland) and the average daytime CO
2
concentra-
tion for lettuce, radish and pepper was approximately 430 50,
490 70 and 480 70 lmol mol
1
(ppm) respectively. RH was mea-
sured using a RH probe (model HMP110; Vaisala Inc.) and the average
RH for lettuce, radish and pepper was 30 6, 20 4, and 19 3%
respectively. Throughout the study, the same dilute fertilizer solution was
applied as needed to maintain ample root-zone moisture. The slow-
release fertilizer and nutrient solution helped maintain leachate electrical
conductivity measurements between 100 and 150 mS m
1
(1.0 and
1.5 mmhos cm
1
).
Light treatments. The experimental system included 14 chambers
(seven arrays of LEDs at two PPFs [200 and 500 lmol m
2
s
1
]). Each
chamber contained one of three broad-spectrum LED arrays (warm, neu-
tral and cool white [Multicomp; Newark, Gaffney, SC]), or one of five
monochromatic LED arrays (pure red, pure blue and combinations of red
and blue [RB] and red, green and blue [RGB] [(Luxeon Rebel Tri-Star
LEDs; Quadica Developments Inc., Ontario, Canada]). Measurements of
PPF, YPF, PPE and relative (percent of total PPF) amounts of blue
(400–500 nm), green (500–600 nm) and red (600–700 nm) light for all
LED treatments in each growth chamber were made using a spectroradi-
ometer (model PS-200; Apogee Instruments, Logan, UT; Table 1). The
spectral output of each LED array is shown in Fig. 1. The red:far-red (R:
FR) ratio was measured using a R:FR sensor (SKR110; Skye Instru-
ments, UK) (Table 1). During the experiment, PPF was measured using a
Figure 1. Spectral distributions of all seven LED treatments, including: the three types of broad-spectrum white LEDs, the red +blue (RB) and
red +green +blue (RGB) LEDs and the red and blue monochromatic LEDs. Since the pepper plants were started in a growth chamber with cool white
fluorescent (CWF) lamps, the CWF spectrum is included as a reference.
Figure 2. The seven LED treatments that were used at both 200 and 500 lmol m
2
s
1
; each treatment is labeled with the amount of blue light (BL)
present in their respective spectra. Treatments are, left to right: red (0.3%), red +blue (RB; 12%), red +green +blue (RGB; 14%), warm white (11%),
neutral white (19%), cool white (28%) and blue (92%).
576 Kevin R. Cope et al.
quantum sensor (LI-188B; LI-COR, Lincoln, NE) calibrated for each
treatment against the spectroradiometer. PPF was maintained constant
relative to the top of the plant canopy by adjusting the electrical current
sourced to each LED array. Variability in PPF within each growth cham-
ber was less than 5%. Root modules in each chamber were rotated 180°
at each watering event (every 2 or 3 days). The photoperiod was main-
tained across species at 16-h day/8-h night.
Plant measurements. Measurements were made on all four plants in
each treatment. Prior to harvest, relative leaf chlorophyll concentration
(measured as chlorophyll content index, CCI) was measured with a chlo-
rophyll meter (CCM-200; Opti-Sciences Inc., Hudson, NH). CCI measure-
ments were taken from the third set of true leaves in lettuce and radish,
and from the topmost, fully expanded leaves in pepper. Immediately
following harvest, stem and leaf fresh mass were measured. The number
of leaves per plant was counted and total leaf area (LA) was measured
using a portable LA meter (model LI-3000; LI-COR, Lincoln, NE). Total
SL or hypocotyl length (HL) was measured for all three species, but peti-
ole length (PL) was measured in radish and pepper only. For pepper, both
the number of branches >1 cm and the number of flowers per plant were
determined. For all species, stems and leaves were separated and dried for
48 h at 80°C to determine DM. Root mass was not measured. We also
calculated specific leaf area (LA divided by DM) and leaf mass fraction
(LMF; leaf DM divided by total DM) for all three species.
Statistical analysis. There were three replicate studies for each species.
Statistical analysis within a species was conducted using a two-way
analysis of variance (ANOVA) with the PROC-GLM package in SAS
(version 9.3; Cary, NC). Each developmental parameter was averaged
over the four plants in each chamber with a significance level of
P=0.05.
RESULTS
Lettuce and radish were generally more photobiologically sensi-
tive to BL than pepper (Fig. 3).
Stem length
Lettuce SL rapidly decreased with an increase in BL from 0% to
11%; the decrease was much greater at 200 lmol m
2
s
1
(Fig. 4). SL for the 0% BL treatment was significantly higher
than all other treatments. Differences in SL were not significant
above 11% BL.
Radish SL gradually decreased as BL levels increased. Like
lettuce, radish SL was significantly higher for the 0.3% BL treat-
ment compared to the treatments with higher BL; however,
unlike lettuce, radish SL in the 92% BL treatment was signifi-
cantly lower than the 11–28% BL treatments. The 11% and 28%
BL treatments were not significantly different from one another.
The response was similar at both PPFs, and there was no signifi-
cant effect of monochromatic and broad-spectrum light (Fig. 4).
The pepper plants that were transferred into the LED cham-
bers were not only less photobiologically sensitive than lettuce
and radish but surprisingly, they responded in the opposite direc-
tion. SL for pepper under the 0.3% BL treatment was signifi-
cantly lower than the 92% BL treatment (Figs. 4 and 5).
However, HL for the pepper seedlings in the supplemental study
was greater at 0.3% BL than higher BL levels (Fig. 6), which
followed the response of SL in lettuce and radish (Fig. 4). This
Figure 3. Top: Overhead view of lettuce from each treatment at both
PPFs at 21 DAE. Photographs are arranged in increasing order of BL,
except the broad-spectrum light sources (warm, neutral and cool), which
are grouped together. Note the elongated growth in the red LED treat-
ment and the dwarfed plants in the blue LED treatment. Bottom: Side
view of one representative radish plant from each of the LED treatments
at 500 lmol m
2
s
1
taken at 21 DAE. Photographs are arranged in
increasing order of blue light (BL), except the broad-spectrum light
sources (warm, neutral and cool), which are grouped together. Each treat-
ment is labeled with its corresponding percent BL. Note the decrease in
elongated storage roots as percent BL increases.
Figure 4. Effect of blue light (BL;% of total PPF) on total stem length
in lettuce, radish and pepper. Each data point represents the mean of
three replicates of each species (n=3). Error bars represent the standard
error of the mean for each treatment. For some data points, the error bars
are not visible due to the size of the symbol. The solid symbols (light,
medium and dark gray) indicate the three broad-spectrum light sources:
warm, neutral and cool white LEDs respectively. Measurements were
made at harvest, 21 DAE for lettuce and radish and 54 DAE for pepper
(or 21 days in the LED treatments). Note the significant variation in the
response of each species to BL.
Photochemistry and Photobiology, 2014, 90 577
response in pepper was similar at both PPFs. For lettuce and rad-
ish there were no significant interactions between broad-spectrum
and monochromatic light sources with the same percent BL;
however, for pepper, SL was decreased under the monochromatic
sources (Fig. 4).
Petiole length
The response of PL in radish and pepper to BL was similar to
the response of SL (Fig. 7), except that in radish PL was signifi-
cantly longer in the 0.3% BL treatment than the other treatments.
For both species, PL was shorter at 500 lmol m
2
s
1
. PL for
radish was similar between monochromatic and broad-spectrum
treatments with the same percent BL; however, there was a sig-
nificant interaction in pepper, where PL slightly decreased with
increasing BL under the broad-spectrum light sources.
Leaf area
At 500 lmol m
2
s
1
, LA decreased as BL levels increased, but
the magnitude of the response varied by species (Fig. 8). For
lettuce and radish, there was an interaction of the BL response
with PPF. LA decreased for both species at 500 lmol m
2
s
1
as BL increased; however, for lettuce, at 200 lmol m
2
s
1
,LA
initially increased followed by a decrease between 19% and 92%
BL. In radish at 200 lmol m
2
s
1
, BL had no significant effect
on LA (Fig. 8).
There was no interaction of BL with PPF in pepper. At both
PPFs, LA gradually decreased with increasing BL. LA in the
0.3% BL treatment was significantly higher than in the 92% BL
treatment. All other treatments were not significantly different.
There was no significant interaction of monochromatic and
broad-spectrum light sources with the same percent BL.
Relative chlorophyll concentration
For all three species, CCI increased as BL increased from 0%
to about 30% and then decreased as BL further increased to
Figure 5. Side view of pepper plants from both the 0.3% and 92% BL
treatments at both PPFs taken at 54 DAE (or 21 days in the LED treat-
ments). Note that plants from the 92% BL treatment are more elongated
than plants in the 0.3% BL treatment.
Figure 6. Effect of blue light (BL;% of total PPF) on hypocotyl length
(HL) in pepper plants from the supplemental study. Each data point rep-
resents the average of 12 plants per treatment. No error bars are
displayed because this supplemental study was not replicated in time.
The solid symbols (light, medium and dark gray) indicate the three broad
spectrum light sources: warm, neutral and cool white LEDs respectively.
Measurements were made at 12 DAE. Surprisingly, the elongation
response of emerging pepper seedlings to BL was the opposite of older
pepper plants (see Fig. 4).
Figure 7. Effect of blue light (BL;% of total PPF) on petiole length
(PL) in radish and pepper. Each data point represents the mean of three
replicates (n=3). Error bars represent the standard error of the mean for
each treatment. For some data points, the error bars are not visible due to
the size of the symbol. The solid symbols (light, medium and dark gray)
indicate the three broad-spectrum light sources: warm, neutral and cool
white LEDs respectively. Measurements were made at harvest 21 DAE
for radish and 54 DAE for pepper. Note the difference in the response of
PL to varying BL levels compared to SL (see Fig. 4).
578 Kevin R. Cope et al.
92% BL (Fig. 9). The effect of BL was most dramatic in let-
tuce, but CCI was consistently higher at 500 than at
200 lmol m
2
s
1
for all species. Between 11% and 14% BL,
the monochromatic sources tended to have higher chlorophyll
than the broad-spectrum sources with the same percent BL
(Fig. 9).
Dry mass
Growth (DM gain) was significantly reduced at 0.3% and 92%
BL for lettuce and radish, but not pepper; however, growth dif-
ferences between 11% and 28% were not statistically different
(Fig. 10). As expected, DM increased with PPF. For lettuce and
radish, DM was nearly two and half times greater at 500 than
200 lmol m
2
s
1
; however, for pepper, DM was only 40%
greater at 500 than 200 lmol m
2
s
1
. There were no significant
interactions between monochromatic and broad-spectrum light
sources with the same percent BL (Fig. 10).
Specific leaf area and LMF
Increasing BL significantly decreased specific leaf area (SLA;
m
2
g
1
) in all three species at both PPFs except radish at
200 lmol m
2
s
1
. There were no significant interactions
between monochromatic and broad-spectrum sources with the
same percent BL (Fig. 11).
There was a statistically significant (16%) decrease in leaf mass
fraction (LMF; ratio of leaf DM to total DM [g g
-1
]) in radish as
BL increased from 0% to 11%, but it remained constant from 11%
to 92% BL (data not shown). Surprisingly, there was no significant
effect of BL on LMF for lettuce or pepper (data not shown).
Figure 8. Effect of blue light (BL;% of total PPF) on total leaf area
(LA) in lettuce, radish and pepper. Each data point represents the mean
of three replicates (n=3). Error bars represent the standard error of the
mean for each treatment. The solid symbols (light, medium and dark
gray) indicate the three broad-spectrum light sources: warm, neutral and
cool white LEDs respectively. Measurements were made at harvest 21
DAE for lettuce and radish and 54 DAE for pepper (or 21 days in the
LED treatments). Note the significant difference in the response of each
species to varying BL levels.
Figure 9. Effect of blue light (BL;% of total PPF) on relative chloro-
phyll concentration (measured as chlorophyll content index, CCI) in let-
tuce, radish and pepper. Each data point represents the mean of three
replicates (n=3). Error bars represent the standard error of the mean for
each treatment. For some data points, the error bars are not visible due to
the size of the symbol. The solid symbols (light, medium and dark gray)
indicate the three broad-spectrum light sources: warm, neutral and cool
white LEDs respectively. Measurements were made at harvest 21 DAE
for lettuce and radish and 54 DAE for pepper (21 days in the LED treat-
ments). Note the significant species differences.
Photochemistry and Photobiology, 2014, 90 579
Branching and flowering
There was no significant effect of BL on branch number, but
there was a small yet statistically significant reduction in flower
number at 92% BL (data not shown). At 500 lmol m
2
s
1
,
there were 8.1 flowers in the 92% BL treatment compared to
10.3 1.5 in all other treatments; at 200 lmol m
2
s
1
there
were 4.4 flowers in the 92% BL treatment compared to
7.5 1.4 in all other treatments.
DISCUSSION
Smith and Whitelam (19) reported that shade-avoidance is asso-
ciated with significant stem elongation, reduced leaf expansion,
decreased chlorophyll production and early flowering. In the
absence of BL, some plants exhibit a profound shade-avoidance
response which is likely mediated by the combined activity of
phytochrome and cryptochrome. The BL effects in this study
reveal many similarities but some differences to other studies.
Stem elongation
Hoenecke et al. (20) grew lettuce seedlings for 6 days under
multiple BL treatments ranging from 0% to 40% BL at two PPFs
(150 and 300 lmol m
2
s
1
). Similar to our results for lettuce
SL, they reported that HL was significantly and rapidly reduced
as BL levels increased up to 28%. Their results agree with those
of Dougher and Bugbee (18), although Dougher and Bugbee
reported on total SL, not HL. The results from both of these
studies are similar to those of ours. Also similar to our results,
Brown et al. (21) reported that pepper SL decreased as BL
Figure 10. Effect of blue light (BL;% of total PPF) on growth (dry mass
gain) in lettuce, radish and pepper. Each data point represents the mean
of three replicates (n=3). Error bars represent the standard error of the
mean for each treatment. For some data points, the error bars are not visi-
ble due to the size of the symbol. The solid symbols (light, medium and
dark gray) indicate the three broad-spectrum light sources: warm, neutral
and cool white LEDs respectively. Measurements were made at harvest
21 DAE for lettuce and radish and 54 DAE for pepper (or 21 days in the
LED treatments). Note the reduced response of pepper compared to that
of lettuce and radish.
Figure 11. Effect of blue light (BL;% of total PPF) on specific leaf area
(SLA) in lettuce, radish and pepper. Each data point represents the mean
of three replicates (n=3). Error bars represent the standard error of the
mean for each treatment. For some data points, the error bars are not visi-
ble due to the size of the symbol. The solid symbols (light, medium and
dark gray) indicate the three broad-spectrum light sources: warm, neutral
and cool white LEDs respectively. Measurements were made at harvest
21 DAE for lettuce and radish and 54 DAE for pepper (or 21 days in the
LED treatments).
580 Kevin R. Cope et al.
increased up to 21% BL, but they did not use treatments with
higher percentages of BL, so no direct comparison can be made
for treatments with more than 21% BL.
Both phytochrome (22) and cryptochrome (23) may play a
role in the response of SL to BL. Clearly, the activation of phy-
tochrome by 0.3% BL (red LEDs, PPE =0.89) was insufficient
to prevent excessive stem elongation in lettuce and radish
(Figs. 4 and 5). But, with the addition of as little as 11% BL
(warm white LEDs, PPE =0.84), stem elongation was signifi-
cantly reduced, most likely as a direct response to cryptochrome
activation by BL. Under 92% BL (blue LEDs, PPE =0.58), SL
was the lowest, indicating that activation of cryptochrome alone
is sufficient to prevent stem elongation in lettuce and radish.
Interaction of BL and developmental stage in pepper
Our results indicated a surprisingly small response of pepper to
BL; this response tended to be in the opposite direction of the
response in lettuce and radish (Figs. 4 and 5). To our knowledge,
increased BL has not caused an increase in SL in any species in
any other study. This response may be associated with the
unusually high leaf chlorophyll concentration in peppers (Fig. 9),
which typically reduces light penetration and reflectance from
leaf surfaces. In addition, there is some evidence that meristems
may be uniquely sensitive to light quality (24). The apical meri-
stem of peppers is at the top of the stem as opposed to being
enclosed by the rosette, as in lettuce and radish. This difference
in meristem position may be associated with a difference in per-
ception of light quality. Similar to pepper, soybean meristems
are located at the top of the plant, yet Dougher and Bugbee (18)
did not observe an increase in soybean SL as BL increased.
However, they also did not grow soybean under pure red and
nearly pure BL.
Interestingly, our results indicate that there was a significant
interaction between BL and developmental stage in pepper. We
were surprised to find that SL increased with increasing BL in
older pepper plants (Figs. 4 and 5), but in our supplemental
study, HL decreased with increasing BL in emerging pepper
seedlings (Fig. 6). This difference in response to BL at two
developmental stages may be explained by the activity of photo-
receptors whose roles vary throughout the life cycle of the plant
(11). Our results indicate that pepper has a critical period imme-
diately following emergence as it transitions from skotomorpho-
genesis (development below ground) to Pm (development above
ground) (25). It is during this early developmental stage that
plants begin to sense and respond to their light environment
using all three classes of photoreceptors, including phyto-
chromes, cryptochromes and phototropins (9,11,26). Our results
suggest that the response of plants to their light environment at
this early developmental stage may also partially predetermine
how they grow and develop for the rest of their life cycle.
Petiole length
The petiole plays a critical role in orienting the leaf to maximize
radiation capture. Therefore, PL is an important component of
radiation capture and appears to be similarly sensitive to BL as
SL. Few studies have reported the effect of BL on PL, but a sim-
ilar response to BL was observed in petioles of strawberry (13)
and A. thaliana (27). As with SL, the response of PL is likely
regulated by interactions of phytochrome and cryptochrome.
Leaf expansion
Our results for LA are similar to the results of other studies.
Dougher and Bugbee (18) reported a dramatic increase in LA as
BL increased from 0% to 2% and a small response to BL from
6% to 26%. We only had treatments at 0.3% and 11% BL, so no
direct comparison between studies can be made within this
range. However, at 200 lmol m
2
s
1
, lettuce LA increased
from 0.3% to 11% BL. Surprisingly, this increase did not occur
at 500 lmol m
2
s
1
. Similar to Dougher and Bugbee (18) dif-
ferences in LA for both PPFs between 11% and 28% BL were
not significant. Differences between studies may have been
caused by cultivar type (Waldmann’s Green vs Grand Rapids) as
well as complex spectral interactions due to different lamp types.
Dougher and Bugbee (28) observed that the effect of 6% BL
provided by high-pressure sodium and metal halide (MH) lamps
on LA was significantly different regardless of identical BL
levels. Through statistical analysis they were able to associate
these differences with interactions of yellow light (580–600 nm).
These results warrant further investigation into the interactions of
other wavelengths of light with BL.
Our results for LA in radish are comparable to Cope and Bug-
bee (15). Other studies with radishes have not reported BL
effects on LA.
Brown et al. (21) grew peppers and found that addition of
only 1% BL in a red light (RL) source significantly increased
LA; we did not find any significant difference in pepper LA
between 0.3% and 11% BL. The cause for differences between
studies remains unknown, but potential sources of variation
include different cultivars and variation in peak wavelengths of
red LEDs (650 nm vs 630 nm).
Since reduced leaf expansion is a common shade-avoidance
response (19), LA would be expected to decrease when
plants are undergoing a shade-avoidance response at 0.3% BL.
This response occurred with lettuce at 200, but not at
500 lmol m
2
s
1
. For radish and pepper, LA typically
decreased as BL increased at both PPFs, indicating that BL
inhibits leaf expansion. This suggests that the role of BL in leaf
expansion varies by species, and possibly by PPF. Furthermore,
the association of leaf expansion with BL suggests a crypto-
chrome-mediated response.
Relative chlorophyll concentration
Chlorophyll (Chl) synthesis is associated with phytochrome
activity (25,29,30), which is primarily affected by the ratio of
red and far RL (22). Phytochrome activity under a given light
source can be predicted by either measuring the R:FR ratio or by
calculating the PPE using the entire spectrum (7). The only light
source with a significantly different PPE in this study was the
92% BL treatment (0.58 compared to 0.84–0.89 for the other
treatments; Table 1). Thus, phytochrome activity should be sup-
pressed in the 92% BL treatment, and as a result, Chl synthesis
would also be reduced; however, surprisingly, for all three spe-
cies and at both PPFs, CCI was equal to or higher under the
92% BL treatment compared to the 0.3% BL treatment, which
had the highest PPE (0.89). Furthermore, CCI doubled between
0.3% and 11% and remained constant from 11% to 28% BL,
even while PPE remained nearly constant. Similar to this study,
Fan et al. (31) studied the effects of light quality on Chl concen-
tration and Chl biosynthesis precursors in non-heading Chinese
Photochemistry and Photobiology, 2014, 90 581
cabbage. They reported that, although Chl was highest under
white light and RB LEDs, under 100% BL Chl concentration
and Chl biosynthesis precursors were significantly higher than
under 0% BL. Both their results and ours suggest that Chl syn-
thesis is not exclusively regulated by phytochrome, but likely a
complex interaction of phytochrome and cryptochrome since BL
had such a powerful effect on Chl synthesis (Fig. 8). The rela-
tionships between BL, photoreceptors and Chl synthesis remain
poorly understood.
Dry mass gain
Growth (DM gain) is the combined result of light interception
and quantum yield. Light interception is primarily affected by
LA because light capture increases with increasing LA. Since
LA typically decreases with increased BL, growth is thus par-
tially dependent on development. The remaining component of
growth is affected by quantum yield, which is dependent on
wavelength. As reviewed previously, BL is less efficient in driv-
ing Pn than other wavelengths of PAR. Because BL decreases
LA and has low quantum efficiency, minimizing BL in a light
source and replacing it with more photosynthetically efficient
wavelengths (e.g. red) increases the YPF of that light source.
However, BL is essential to prevent shade-avoidance responses.
Thus, the ideal BL level would promote normal development
while minimally reducing Pn.
For all three species, we repeatedly observed maximum
growth under the RB LED treatment at both PPFs. Although this
result was not statistically significant at P=0.05 level, it was a
consistent trend. Reduced growth for lettuce and radish, but not
pepper, was consistently observed in the monochromatic 0.3%
and 92% BL treatments. This was statistically significant in
lettuce and radish, and nearly significant in pepper (Fig. 10).
Reduced growth in the 0.3% BL treatment may be caused by
inactivity of cryptochrome (23). At 92%, BL plants did not exhi-
bit shade-avoidance responses since cryptochrome was likely
activated (12); however, cryptochrome may have been overstimu-
lated which caused a significant reduction in LA and, therefore,
radiation capture. The reduced photosynthetic efficiency of the
blue photons likely reduced the amount of light energy actually
absorbed by photosynthetic pigments. Both factors resulted in
reduced growth.
Similar to this study, Brown et al. (21) grew pepper plants for
21 days in broad-spectrum light prior to introducing them into
light treatments including red LEDs, red +far-red LEDs, red
LEDs +blue fluorescent lamps and MH lamps. Contrary to our
results, there was a significant spectral effect on growth. The
cause for these differences remains unknown, although the fol-
lowing parameters varied: plant age at exposure (21 vs 33 days),
cultivar (Hungarian Wax verses California Wonder), plant den-
sity, light source and spectrum and the root-zone environment
(hydroponic verses soilless media).
Similar to our results for lettuce and radish, Yorio et al. (32)
observed that RB treatments had significantly increased growth
compared to 0% BL treatments, but contrary to our results, RB
had significantly lower growth compared to CWF for radish.
Lettuce growth was similar between the two light sources. The
CWF treatment had double the BL (16%) and nearly 47% more
GL compared to the RB treatment, so differences in growth can-
not be entirely attributed to BL. Since GL was also a variable
factor, it may have played a role in increasing growth.
Specific leaf area and leaf mass fraction
Both SLA and LMF can be sensitive indicators of the shade-
avoidance response. Plants in the shade typically have thin leaves
and elongated stems because less DM is partitioned to the leaves.
This results in a dramatic reduction in both SLA and LMF.
Although both stem elongation (Fig. 4) and leaf expansion
(Fig. 8) were altered by BL, our data indicate that carbon parti-
tioning to stems and leaves stays relatively constant over a wide
range of BL levels. Our results are comparable to Dougher and
Bugbee (18).
Branching
Decreased branching is thought to be a shade-avoidance
response. Dougher and Bugbee (18) reported that wheat tiller
number (branching) tended to decrease with increasing BL (dif-
ferences were not significant) but soybean branch number
increased with increasing BL (differences were significant). In
contrast, Cope and Bugbee (15) saw a slight increase in tiller
number in wheat as BL increased, but observed no change in
branch number in soybean among treatments (11–28% BL). We
saw no significant effect of BL on branching in pepper, which
suggests that the response of branching to BL is species depen-
dent and interacts with other wavelengths of light and PPF.
Branching may therefore be regulated by complex interactions of
phytochrome and cryptochrome.
Flower number
Both Eskins (27) and Cerd
an and Chory (33) reported that flow-
ering time in A. thaliana is affected by light quality. Eskins (27)
found that bolting occurs earlier as BL increases. Cerd
an and
Chory (33) reported that this may be a shade-avoidance response
that occurs as R:FR ratios decrease. They also suggested that this
response is mediated by interactions of specific types of phyto-
chromes and cryptochromes (33). Although we did not measure
days to flowering (or bolting), we found that flowering at 92%
BL was significantly lower than all other treatments. It is possi-
ble that flower initiation occurred earlier in this treatment when
the pepper plant was less capable of supporting flowers. This
may have reduced flower number at harvest.
Green light interactions
GL levels for light sources with RL and BL varied from >1% to
52%, with the highest GL levels in the three broad-spectrum
white LEDs (Table 1). Yorio et al. (32) found that increased GL
significantly increased the growth of radish, but not lettuce. Kim
et al. (34) reported that including GL in a RB light source dra-
matically increased lettuce growth compared to RB lights alone.
We did not find that growth under light sources with high GL
was increased over the RB treatment, which had 0% GL. These
studies warrant further investigation into the importance of
supplemental GL.
Monochromatic BL
Five studies have evaluated the effect of monochromatic BL on
plant growth and development (Arabidopsis (27), potato (35),
strawberry (13,36) and Chinese cabbage (31)). In all studies,
582 Kevin R. Cope et al.
plants grown under the BL treatment were significantly shorter
and had significantly less growth than broad-spectrum treatments.
These results are consistent with this study for lettuce and radish,
but not pepper. The use of increasing BL regardless of the light
source always results in a lower YPF and usually a lower PPE
than light sources with predominately longer wavelengths of
PAR. The lower YPF and lower PPE should individually, and in
combination, result in a shade-avoidance response. Interestingly,
we observed the opposite in the 92% BL treatment (decreased
SL, HL and LA) suggesting that factors other than YPF and PPE
were responsible for the biological response. Furthermore,
growth was decreased in the 92% BL treatment. This response
was likely caused by reduced LA and thus reduced radiation cap-
ture rather than a direct effect of a lower YPF on Pn. These
results suggest that YPF is not a better indicator of Pn than PPF.
Effects of BL on anatomical features
Schuerger et al. (37) evaluated the effect of the same light
sources used by Brown et al. (21) on multiple anatomical fea-
tures in pepper and found that the thickness of leaf cross sections
decreased as BL levels increased. Dougher and Bugbee (38)
studied the histology of leaves and stems in lettuce and soybean
and found that the increase in SL in soybean from 6% to 26%
BL was due to an increase in both the number and size of cells
in the stem. Lettuce and soybean LA was greater under 0% than
26% BL, which was associated with an increase in the size and
number of leaf epidermal cells. Although we did not measure
leaf or stem anatomical features in this study, the results of
Schuerger et al. (37) and Dougher and Bugbee (38) provide
insight into the underlying effects of BL on SL, PL and LA.
CONCLUSIONS
There was an interaction of BL with PPF for LA in lettuce and
radish, but no interaction with PPF in pepper. There were also
significant differences in the BL response among species, which
are likely dependent on the action of at least two photoreceptors,
phytochrome and cryptochrome. Our data confirm that BL is
necessary in most species to prevent shade-avoidance responses;
yet increasing BL above 11% continued to reduce HL, SL, PL
and LA, which in turn reduced radiation capture and growth.
Although there are potential interactions among blue, green,
yellow and red wavelengths, our data indicate few interactions
between broad-spectrum and monochromatic light sources for the
three diverse species we studied.
Acknowledgements—This research was supported by the Utah
Agricultural Experiment Station, Utah State University (approved as
journal paper number 8581). We thank the Space Dynamics Lab (Logan,
UT) for donating microLADA growth chambers. We also thank Ty
Weaver and Alec Hay for their technical support, Saundra G. Rhoades
for her help in obtaining precise measurements and Spencer Tibbitts for
his help in statistically analyzing our data. Mention of trade names is for
information only and does not constitute an endorsement by the authors.
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