Environmental and Experimental Botany 155 (2018) 345–359

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Environmental and Experimental Botany

journal homepage: www.elsevier.com/locate/envexpbot

Blue light associated with low phytochrome activity can promote elongation T
growth as shade-avoidance response: A comparison with red light in four
bedding plant species
⁎
Yun Kong, Michael Stasiak, Michael A. Dixon, Youbin Zheng
School of Environmental Sciences, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada

A R T I C LE I N FO A B S T R A C T

Keywords: To explore the action mode of blue light on elongation growth of bedding plants, the plant growth and mor-
Light-emitting diode phology traits of petunia (Petunia × hybrida, ‘Duvet Red’), calibrachoa (Calibrachoa × hybrida, ‘Kabloom Deep
Phytochrome photostationary state Blue’), geranium (Pelargonium × hortorum, ‘Pinto Premium Salmon’), and marigold (Tagetes erecta, ‘Antigua
Light quality Orange’) were compared under four light quality treatments: (1) R, “pure” red light (660 nm); (2) B, “pure” blue
Light intensity
light (450 nm); (3) BR, “unpure” blue light created by mixing B with a low level of R to provide B/R ≈ 9; (4)
Plant species
BRF, “unpure” blue light created by adding a low level of far red light to BR with red/far red ≈ 1. Continuous
(24-h) light-emitting diode lighting with either 100 or 50 μmol m−2 s−1 photosynthetic photon flux density at ≈
23℃ was used with the above treatments. After 14–20 day of lighting treatment, B promoted elongation growth
compared to R, as demonstrated by a greater canopy height, main stem length, internode length, and daily main
stem extension rate. However, BR showed similar or inhibitory effects on these traits relative to R, while BRF
exhibited similar promotion effects as B. The calculated phytochrome photoequilibrium, an indication of phy-
tochrome activity, was higher for R (0.89) and BR (0.74) than for B (0.49) and BRF (0.63). Adding red (or far
red) light reversed the effects of B (or BR) on elongation growth and the phytochrome photoequilibrium, sug-
gesting that blue light promotion of elongation growth is related to the lower phytochrome activity. Also, B and
BRF, when compared to R or BR, promoted elongation growth to a greater degree at 50 than 100 μmol m−2 s−1
for petunia and calibrachoa. In addition to the promoted elongation growth, B and BRF reduced side branch
number, biomass allocation to side branches, leaf epinasty, leaf angle, and/or leaf chlorophyll content relative to
R or BR, but increased individual leaf area, petiole length, and/or biomass allocation to main stem, which varied
with different species. It suggests that the promoted elongation growth by blue light associated with lower
phytochrome activity is one of shade-avoidance responses with varying sensitivity among species.

1. Introduction elongation (Laskowski and Briggs, 1989; Hoenecke et al., 1992; Huche-
Thelier et al., 2016), many previous studies have shown that blue light
Compact growth is one of the ideal marketable morphological was more effective than red light in suppressing shoot/leaf elongation
characteristics of ornamental plants, especially bedding plants in a range of plant species (Cosgrove, 1981; Appelgren, 1991; Wheeler
(Wollaeger and Runkle, 2015; Mah et al., 2018). Light adjustment et al., 1991; Hoenecke et al., 1992; Cosgrove, 1994; Brown et al., 1995;
technology has been adopted as one of the environmentally friendly Kong et al., 2012). However, in these studies, blue light sources may not
ways to modify crop morphology in greenhouse production (Demotes- have provided “pure”, but rather “contaminated” blue light, i.e., blue
Mainard et al., 2016). Plants require light not only for photosynthesis, light mixed with small amount of other spectral bands (Bergstrand
but also for regulation of their growth and development (Folta and et al., 2014).
Childers, 2008). Previous studies have clearly indicated that at least Studies using light-emitting diode (LED) in recent decades have
two photoreceptor systems, i.e., phytochromes, activated by red light reported contradictory morphological responses to blue vs. red light in
and deactivated by far red light, and blue light receptors, crypto- different species, and even in the same species; and, the effect of blue
chromes, are involved in mediation of elongation growth by light vs. red LED light on stem elongation is species- and even variety-de-
(Cosgrove, 1981). Although both red and blue light can mediate stem pendent (Huche-Thelier et al., 2016). The emission of narrow-

⁎
Corresponding author.
E-mail address: [email protected] (Y. Zheng).

https://doi.org/10.1016/j.envexpbot.2018.07.021
Received 27 June 2018; Received in revised form 24 July 2018; Accepted 24 July 2018
Available online 25 July 2018
0098-8472/ © 2018 Elsevier B.V. All rights reserved.
Y. Kong et al. Environmental and Experimental Botany 155 (2018) 345–359

waveband light by LEDs provides the opportunity to re-evaluate the growth when compared to monochromatic red light, and whether this
effects of “pure” blue light on plant growth and development and its response can be reversed by adding a small amount of far red light (e.g.,
relationship with other wavelengths such as red and far red (Tarakanov red/far red ≈ 1) to de-active phytochrome in these bedding plants.
et al., 2012; Wollaeger and Runkle, 2013). Further study on blue light PPFD level of around 100 μmol m−2 s−1 has been commonly used
using LEDs will certainly contribute to a more in-depth understanding for LED lighting studies in the past decades, and at this light level in-
of the mode of action of blue light, and a more precise control of plant consistent effects of “pure” blue vs. red light on elongation growth have
morphology using LED technologies in the near future (Tarakanov been reported in different species, and even in the same species (Huche-
et al., 2012; Huche-Thelier et al., 2016). Thelier et al., 2016). Surprisingly, under light levels < 100 μmol m−2
Petunia, geranium, calibrachoa, and marigold are popular and s−1 and even as low as 30 μmol m−2 s−1, many LED studies on tissue
economically important bedding plant species. However, limited in- cultured plantlets have consistently reported elongation growth in-
formation is available on the effects of blue vs. red light on these spe- hibition by “pure” blue when compared to red light in a wide range of
cies, especially the effect of narrow-band LED light on plant mor- species such as chrysanthemum (Kim et al., 2004), strawberry (Nhut
phology including elongation growth, even though a number of LED et al., 2003), grape (Poudel et al., 2008; Li et al., 2017), banana (Nhut
studies have been carried out on agronomic crops over the past decades et al., 2002), Cymbidium (Tanaka et al., 1998), and Doritaenopsis (Shin
(Massa et al., 2008). Also, the previous results of blue vs. red light ef- et al., 2008). Nevertheless, it is difficult to compare these previous re-
fects on stem elongation of these bedding plants are not consistent and sults about blue light effects under different light levels due to different
are often contradictory. Some studies have shown blue light to increase environment conditions and plant materials. Different from tissue cul-
shoot elongation compared with red light. For example, in calibrachoa, tured plantlets, seedlings from seeds do not have rooting and/or dif-
propagated cuttings showed greater shoot elongation under mono- ferential induction stages. So, for the seedlings of these four bedding
chromatic blue vs. red LED light with photosynthetic photon flux plant species, further study is required to determine how plant growth
density (PPFD) of 40 or 80 μmol m−2 s−1 and 16-h photoperiod and morphology responds to blue vs. red light under decreased light
(Olschowski et al., 2016). For marigold, monochromatic blue vs. red levels, e.g., PPFD of 50 vs. 100 μmol m−2 s−1.
LED light (90 ± 10 μmol m−2 s−1 PPFD and 16-h photoperiod) in- For different ornamental plant species, blue light has been suggested
creased stem length (Heo et al., 2002). For petunia (‘Baccarat Blue’), to play an equal or greater role than red light in mediating inhibition of
monochromatic blue vs. red LED light promoted stem elongation under stem extension in long-day plants, such as Campanula carpatica ‘Blue
100 μmol m−2 s−1 PPFD and 14-h photoperiod (Fukuda et al., 2011), as Clips’, Coreopsis × grandiflora ‘Early Sunrise’, Lobelia × speciosa
well as under 70 or 150 μmol m−2 s−1 PPFD and 12-h photoperiod ‘Sompliment Scarlet’, Pisum sativum ‘Utrillo’, and Viola × wittrockiana
(Fukuda et al., 2016). Similar results were also observed in petunia ‘Crystal Bowl Yellow’ (Runkle and Heins, 2001; Kim et al., 2002;
(‘Dwarf varieties mix’) during early seedling stage, where a 12-h pho- Shimizu et al., 2005). Petunia and calibrachoa are long-day plants, but
toperiod was used but PPFD information was not mentioned (Akbarian marigold and geranium are short-day and day-neutral plants, respec-
et al., 2016). By contrast, for petunia (‘Wave Pink’), monochromatic tively. In the commercial production of bedding plants, marigolds ap-
blue vs. red LED light (160 μmol m−2 s−1 PPFD and 18-h photoperiod) pear to be less susceptible to stretching than the other three bedding
inhibited leaf and stem expansion (Wollaeger and Runkle, 2014, 2015). plant species based on the information provided by local greenhouse
In these two studies, higher light intensity, longer photoperiod, and growers. It is not known if there are some differences among the four
different petunia varieties were used compared to the previously species in their morphological responses to blue vs. red LED light under
mentioned studies. For geranium, strong inhibitory effects on stem the same environment conditions.
elongation were observed in shoot cultures under blue vs. red light Previous studies have indicated that blue light inhibits plant elon-
(30 μmol m−2 s−1 PPFD and 18-h photoperiod), but fluorescent light gation within seconds, while the inhibition by red light begins
sources rather than LEDs were used for this study (Appelgren, 1991). It 15–90 min after the onset of irradiation, and in some species, blue light
appears that the inconsistent results that have been reported may be inhibition persisted only during the period of irradiation, after which
due to the differences in lighting source, light intensity, genotype, and/ elongation growth quickly returned to the high rate during the dark
or photoperiod. period (Gaba and Black, 1979; Cosgrove, 1981; Kigel and Cosgrove,
Taking into account the increasing evidence of elongation growth 1991). The dark period in the previous studies using periodic lighting
promotion by blue light from LED lighting rather than broad-band may have reduced the difference in blue vs. red light effects on elon-
lighting sources, it may indicate that “pure” blue light needs to act gation growth to a varied degree. Continuous lighting (no dark period)
together with other wavelength(s) to inhibit elongation growth, at least may be able to remove the effects of different photoperiod on elonga-
in some species under a certain range of light levels. Previous study tion growth response to blue vs. red light. However, no studies using
using non-LED lighting on de-etiolated seedlings of Arabidopsis in- continuous (i.e., 24-h) blue or red LED lighting have so far been re-
dicated that blue light mediated inhibition of hypocotyl elongation was ported on bedding plants, and to our best knowledge, just one study has
enhanced by its co-action with a low level of red light, but this effect been performed on a non-bedding plant species, sesame (Hata et al.,
was reversed by an equally low-level far red light, suggesting the in- 2013).
volvement of phytochrome in this process (Ahmad and Cashmore, Based on the above information, the following three hypotheses
1997). However, it is not clear whether blue light action on other plant were formed for the four bedding plant species under continuous
species would have the same effect as with Arabidopsis if using LED lighting using LEDs: (1) co-action with a low level of red light is re-
lighting. In a previous LED study on petunia, the combination of 50% quired for “pure” (or monochromatic) blue light to inhibit elongation
red and 50% blue light inhibited stem elongation when compared to growth at least in some species under a certain range of light levels
100% blue light, but was not different from 100% red light (Fukuda (e.g., ≤100 μmol m−2 s−1 PPFD), but this inhibition effect can be re-
et al., 2016). Similarly, the combination of red and blue LED light versed by addition of far red with an amount equal to red light; in other
(34–85% red light) induced more compact plants when compared to words, blue light effect on elongation growth is related to phytochrome
100% blue LED light in petunia and geranium (Davis et al., 2015). activity at least in some cases; (2) the effects of blue vs. red light on
However, in the above two studies, the effects of adding far red light plant elongation as well as other morphological traits differ under lower
were not tested, and a higher proportion (more than 30%) of red light (e.g., ≈50 μmol m−2 s−1 PPFD) and higher (e.g., ≈100 μmol m−2 s−1
was used for the red and blue LED combination treatment. The question PPFD) light intensity; alternatively, there are interaction effects be-
that remains to be answered is whether “unpure” blue light, created by tween light intensity and light quality; (3) blue vs. red light effects on
mixing “pure” blue light with a small amount (e.g., 10%) of mono- plant growth and morphology vary with different species, especially
chromatic red light to activate phytochrome, can inhibit elongation those with different photoperiod flowering responses.

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Y. Kong et al. Environmental and Experimental Botany 155 (2018) 345–359

The objectives of this study were to explore the mode of action of

Harvesting date

Rep.1: July 19

Rep.1: July 20

Rep.1: July 24
Rep.2: Aug 29

Rep.2: Aug 23

Rep.2: Aug 24

Rep.2: Aug 28
Rep.3: Sep 29

Rep.3: Sep 30
Rep.1:July 25

Rep.3: Oct. 5

Rep.3: Oct. 4
blue light on plant growth and morphology in the four bedding plant
species, by testing the above three hypotheses.

2. Materials and methods

2.1. Plant materials and growing conditions

Transplanting date

Sep.15

Sep.15

Sep.15

Sep.15
The experiment with three replicates over time was conducted on
July 5

July 5

July 5

July 5
Aug 9

Aug 9

Aug 9

Aug 9
the four bedding plant species (Table 1) at the University of Guelph,
Rep.1:
Rep.2:
Rep.3:
Rep.1:
Rep.2:
Rep.3:
Rep.1:
Rep.2:
Rep.3:
Rep.1:
Rep.2:
Rep.3:
Guelph, ON, Canada during the period from Jun. to Oct. of 2017.
Taking into account the varied germination time among the tested plant
species, seed sowing was scheduled at different dates (Table 1) before
transplanting for the tested four species, based on our previous ex-
June 23

June 14

June 14

June 20
July 28

July 19

July 25

perience. Seeds were germinated using 288-cell trays containing Sun-

Aug 25

Aug 25

Aug 31
July19
Sep. 3
Seeding date

shine Mix #5 substrate (Sun Gro Horticulture, MA, USA) covered with a
layer of vermiculite, and were placed inside a growth chamber main-
Rep.1:
Rep.2:
Rep.3:
Rep.1:
Rep.2:
Rep.3:
Rep.1:
Rep.2:
Rep.3:
Rep.1:
Rep.2:
Rep.3:

tained at ≈ 23 °C with a photoperiod of 14-h fluorescent lighting

(Sylvania FP54/841/HO/ECO, Pentron 4100 K 54 W 20906 Hg; Wil-
mington, MA, USA) at a PPFD of 210 μmol m−2 s−1. The seedlings had
two true leaves for geranium and marigold or four true leaves for pet-
PanAmerican Seed Co., West Chicago, IL, USA
Ball Horticultural Co., West Chicago, IL, USA

unia and calibrachoa at the time of transplanting. For each replicate, 72

Express Seed Company, Oberlin, OH, USA

seedlings from each species were transplanted into 8.5 cm × 8.5 cm ×

9.5 cm pots containing Sunshine® Mix #1 substrate (Sun Gro Horti-
Syngenta Flowers, Gilroy, CA, USA

culture), and placed inside a walk-in growth chamber to start the

lighting treatment. The plants were sub-irrigated with nutrient solution
once every 1–5 d with increasing frequency dependent on stage of plant
growth. The nutrient solution was made from 20–8–20 water soluble
fertilizer (Master Plant-Prod Inc., Brampton, Ontario, Canada) with a
fertilizer/water mass ratio of 1:800. The nutrient solution (EC ≈
Seed source

2.6 dS m−1) contained the following nutrients (mg·L–1): 250.0 N,

42.5 P, 207.5 K, 184.0 Ca, 1.9 Mg, 0.6 Cu, 0.3 B, 0.6 Mn, 0.6 Zn, 0.2 Mo,
and 1.3 Fe. The temperature set point in the growth chamber was 23 °C.

2.2. Experimental design and treatments

Photoperiod requirement

The experiment was conducted as a 4 × 2 × 4 factorial (light

quality × light intensity × plant species) in a split-split-plot design
with three replicates over time (Table1). The light quality, light in-
Day-neutral

tensity and plant species treatments were allocated to the main plots,
Short-day
Information of the four bedding plant species and key time points for the experiment.

Long-day

Long-day

subplots and sub-subplots, respectively. Light quality treatments in-

cluded: (1) R, “pure” (monochromatic) red light with peak wavelength
at 660 nm; (2) B, “pure” (monochromatic) blue light with peak wave-
length at 450 nm; (3) BR, “unpure” blue light created by mixing B with
a small amount (10% total PPFD) of R; (4) BRF, “unpure” blue light
Pinto Premium Salmon
Kabloom Deep Blue

created by adding a small amount of far red light to BR with red/far red
≈ 1. Lighting was provided by five-channel water cooled LED arrays
Antigua Orange
Cultivar name

custom designed by Intravision Light Systems Canada (Toronto, ON,

Duvet Red

Canada). Lighting array LEDs were produced by Philips (Amsterdam,

Netherlands) for Deep Red, 5500 K White and Royal Blue (LUXEON
Rebel) and LedEngin (Marblehead, MA, USA) for UV (LZ1-00U600) and
Far Red (LZ1-00R300). The IP 67 electronics with dimming and pulse
width modulation (PWM) control were supplied by MOSO Power
Pelargonium × hortorum
Calibrachoa × hybrida

Supply Technology Co., Ltd (Shenzhen, China). The aluminum LED

supports with integrated liquid cooling were custom manufactured by
Petunia × hybrida

Sapa AS (Oslo, Norway).

Tagetes erecta
Latin name

For each replicate, four light quality treatments were randomly al-
Note: Rep. indicates replicate.

located to four shelves separated by horticultural black/white plastic to

prevent neighbour effects. The light quality treatments were replicated
three times by switching light spectral output of LEDs in each com-
partment within the walk-in chamber (i.e. changing the position of light
Common name

quality treatments within the four compartments) during the three re-
Calibrachoa

plications of the experiment. For each light quality treatment, two light
Geranium
Marigold

intensity treatments of 100 and 50 μmol m−2 s−1 at the plant canopy
Petunia
Table 1

were achieved by changing the distance between lighting fixtures and

the plant canopy. Light spectra and intensities were set up and verified

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Y. Kong et al. Environmental and Experimental Botany 155 (2018) 345–359

Fig. 1. The spectral distribution of four light quality treatments, R

(A), B (B), BR (C), and BRF (D) delivered by different combina-
tions of light-emitting diodes (LEDs). HL: higher light intensity,
100 μmol m−2 s−1; LL: lower light intensity, 50 μmol m−2 s−1
PPFD. R: monochromatic red light; B: monochromatic blue light;
BR: “unpure” blue light created by mixing B with a small amount
(10% total PPFD) of R; BRF: “unpure” blue light created by mixing
BR with a small amount of far red light with red/far red ≈ 1. The
wavelength range, red light (600–700 nm) and far red light
(700–800 nm) was used for calculation of red/far red ratio. (For
interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article).

Table 2
Spectral and environmental data for different light quality and intensity treatments.
Treatment PPFD %B %R Far red Red/far red ratio PPS Temperature (℃) RH
(μmol m–2 s–1) (μmol m–2 s–1) (%)

HL
R 101.7 ± 0.8 – – – – 0.89 ± 0.00 23.6 ± 0.2 61.4 ± 1.4
B 102.5 ± 1.1 – – – – 0.49 ± 0.00 23.3 ± 0.1 59.7 ± 0.8
BR 102.4 ± 0.9 90.1 ± 0.3 9.5 ± 0.3 – – 0.74 ± 0.01 23.4 ± 0.3 60.9 ± 3.9
BRF 103.6 ± 0.8 89.5 ± 0.2 10.1 ± 0.2 10.3 ± 0.4 1.0 ± 0.0 0.63 ± 0.00 23.1 ± 0.3 61.3 ± 0.8

LL
R 51.1 ± 1.7 – – – – 0.89 ± 0.00 23.3 ± 0.2 60.7 ± 1.4
B 52.0 ± 0.1 – – – – 0.49 ± 0.00 23.6 ± 0.4 58.9 ± 0.7
BR 52.0 ± 1.6 90.9 ± 0.7 8.7 ± 0.7 – – 0.73 ± 0.00 23.5 ± 0.1 58.9 ± 2.0
BRF 51.9 ± 0.7 90.1 ± 0.5 9.5 ± 0.5 5.1 ± 0.3 1.0 ± 0.0 0.63 ± 0.00 23.1 ± 0.3 61.1 ± 1.3

Data are means ± SE (n = 3). PPS: phytochrome photostationary state, which is the estimated phytochrome photoequilibrium according to the method by Sager
et al. (1988). HL: higher light intensity, 100 μmol m−2 s−1 PPFD; LL: lower light intensity, 50 μmol m−2 s−1 PPFD. The wavelength ranges for red (600–700 nm) and
far red (700–800 nm) were used for calculation of red/far red ratio.

using a USB2000 + UV–vis spectrometer (Ocean Optics, Inc., Dunedin, LNf − LNi
LER =
FL, USA). The spectral distribution and data of different light treat- t (2)
ments are presented in Fig. 1 and Table 2.
where LSf and LSi are the final and initial MS lengths during the period
of lighting treatment, respectively; LNf and LNi are the final and initial
number of fully expanded leaves during the period of lighting treat-
2.3. Growth and morphology measurements
ment, respectively. The denominator, t, represents the number of days
between the initial and final measurements for each plant species.
For each of the 3 replicates of the experiment, at the beginning of
At 14, 15, 19, and 20 d after the start of lighting treatments in each
the lighting treatments and just before harvest, three plants of each
of the 3 replicates of the experiment, three plants were randomly se-
species were randomly selected from each light treatment combination
lected from each light treatment combination (4 light quality treat-
(4 light quality treatments × 2 light intensity treatments) to measure
ments × 2 light intensity treatments) for final harvest from petunia,
main stem (MS) length and leaf number on the MS. Stem extension rate
calibrachoa, geranium, and marigold, respectively. For each harvested
(SER; cm·d−1) and leaf expansion rate (LER; no.·d−1) during the period
plant, side-view pictures were taken for subsequent measurement of
of lighting treatment were calculated using Eqs. (1) and (2), respec-
leaf angle (defined as angle between main stem and upper side of fully
tively:
expanded leaf) using ImageJ 1.42 software (National Institute of
LSf − LSi Health, USA). Additional measurements included canopy height and
SER = width, petiole length and blade length and width of the 3rd to 5th fully
t (1)
expanded leaf counted downward from top. The leaf SPAD index was

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Y. Kong et al. Environmental and Experimental Botany 155 (2018) 345–359

determined in three fully expanded leaves using a chlorophyll meter were similar or shorter. Canopy width of petunia at both HL and LL and
(SPAD 502; Spectrum Technologies, Inc., Aurora, IL, USA). Leaf epi- geranium at HL showed a similar response to the variation of light
nasty index was determined using the method described by Fukuda quality treatments as above (Fig. 2B). Generally, the whole plant ca-
et al., (2008). Flowering stage, based on visibility or size of flower buds, nopy appeared less compact under B and BRF than under R and BR,
was recorded, and then the plant was cut at the base above the sub- especially in petunia, calibrachoa, and geranium (Fig. 2D–G). This ob-
strate, and after weighing the fresh weight (FW), the aerial part was servation was supported by the results of compact index (Fig. 2C).
separated into MS, leaves on the MS, and side branches (SB). After Light intensity did not change the aforementioned pattern of re-
weighing their FWs, MS length and maximum length of SB were mea- sponse to light quality variation for canopy height of the four species or
sured. Stem diameter was measured at the middle inter-node of the MS for canopy width of petunia (Fig. 2A and B). However, light intensity
using a digital caliper. The number of true leaves, nodes, and SB changed the magnitude of response variation with light quality in ca-
(> 1 cm in length) on the MS per plant were counted. The total area of nopy height for petunia, indicating an interaction effect between light
true leaves on the MS for each plant was measured using a leaf area quality and light intensity (Fig. 2A and B). Also, there were interaction
meter (LI-3100; LI-COR, Lincoln, NE, USA). The separated aerial plant effects on the canopy morphology between plant species and light
parts were transferred into a drying oven at 65 °C until a constant quality (Fig. 2A–C).
weight was achieved to determine the dry weight (DW) of each com-
ponent. Roots were then washed to remove the substrate and dried at 3.2. Stem morphology
the same conditions as aerial plant parts to determine DW. Internode
length of MS, individual leaf area, leaf mass unit area (LMA), Aerial/ With the variation of light quality treatments, MS length and in-
Total DW, MS/Aerial DW, SB/Aerial DW, and plant compact index were ternode length showed similar response pattern as canopy height for
calculated using Eqs. (3)–(9), respectively. petunia, calibrachoa, and geranium at both HL and LL, and marigold at
HL (Fig. 3A and D). This response pattern was also found in MS dia-
MS length(cm)
Internode length(cm) = meter at HL and in node number at both HL and LL for petunia (Fig. 3B
Node number on MS (3)
and C). However, SB number in petunia at LL showed a reverse re-
Total leaf area on MS(cm2) sponse pattern: B and BRF resulted in fewer branches than R and BR,
Individual leaf area(cm2) = and there was no difference between B and BRF or between R and BR
Leaf number on MS (4)
(Fig. 3E). Generally, light quality treatments did not affect maximum SB
DW of leaves on MS(g) length (data not show).
LMA(mg cm-2) = × 1000
Total leaf area on MS(cm2) (5) Light intensity did not change the patterns of response to light
quality variation in MS length and internode length for the tested
DW of aerial part(g)
Aerial/Total DW(%) = × 100 species except marigold (Fig. 3A and D). However, light intensity
DW of whole plant(g) (6) changed the magnitude of variation with light quality in the two stem
morphology traits for petunia, which was supported by the interaction
DW of main stem(g)
MS/Aerial DW(%) = × 100 effects on these traits between light quality and light intensity (Fig. 3A
DW of aerial part(g) (7)
and D). Also, there were interaction effects on the stem morphology
DW of side branches(g) traits, with the exception of node number and maximum SB length,
SB/Aerial DW(%) = × 100 between plant species and light quality (Fig. 3A and B, D and E). These
DW of aerial part(g) (8)
observations were supported by different response patterns and/or
DW of aerial part(g) variation magnitude in these traits among the four species under dif-
Plant compact index = × 100
Canopy height(cm) (9) ferent light quality treatments.

3.3. Leaf morphology

2.4. Statistical analysis
Regardless of plant species and light levels, leaf epinasty index and
Data were subjected to analysis of variance using the Data leaf angle were greater under R than under B, BR, and BRF (Fig. 4C and
Processing System Software (DPS, version 7.05; Refine Information D). With the variation of light quality treatments, individual leaf area
Tech. Co., Hangzhou, China) and were presented as means ± SE showed similar response pattern as canopy height for petunia, cali-
(standard error). Separation of means was performed using Duncan's brachoa, and marigold at HL, and petiole length for petunia and ger-
new multiple range test at α = 0.05 level. Correlation analysis was used anium at both HL and LL (Fig. 4A and B). A reverse response pattern
to determine the relationship between the growth and morphological was obtained in Chlorophyll content for petunia and calibrachoa at
traits and phytochrome photostationary state (PPS) values or blue light both HL and LL and for geranium at LL, and in LMA for calibrachoa at
intensity in the four bedding plant species. The coefficient of variation HL, where B and BRF resulted in a lower value than R and BR, and there
(CV) of each growth and morphological trait among the four light was no difference between B and BRF, but with a higher or similar value
quality treatments was calculated to compare variation magnitude of under BR vs. R (Fig. 4E and F).
light quality responses between the two light intensity treatments, and There were interaction effects between light intensity and light
among the four plant species. Based on the CV data, cluster analysis by quality on the leaf morphology traits except with leaf angle and LMA
Euclidean distance was used to group the tested four plant species. (Fig. 4). Also, there were interaction effects between plant species and
light quality on the leaf morphology traits except for leaf angle, which
3. Results was supported by different response pattern and/or variation magni-
tude in these traits among the four species under different light quality
3.1. Canopy morphology treatments (Fig. 4).

Regardless of plant species and light levels, B vs. R increased canopy 3.4. Plant growth
height, but BR vs. R showed similar or inhibitory effects on this trait,
and BRF vs. R exhibited promotion effects similar to B vs. R (Fig. 2A). In For plant growth, with the variation of light quality treatments, SER
other words, plants under B and BRF were taller than those under R and and MS/aerial DW showed similar response pattern as canopy height
BR, but plants under B and BRF were similar, and those under BR vs. R for petunia, calibrachoa, and geranium at both HL and LL, and marigold

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Y. Kong et al. Environmental and Experimental Botany 155 (2018) 345–359

Fig. 2. Plant canopy morphology under

different light treatments in four bed-
ding plant species. Data are
means ± SE (n = 3). The pictures
(D–G) were taken at 23 d from the start
of lighting treatments. The length of
the blue reference bar in these pictures
is 6.3 cm. HL: higher light intensity,
100 μmol m−2 s−1 PPFD; LL: lower
light intensity, 50 μmol m−2 s−1 PPFD.
R: monochromatic red light; B: mono-
chromatic blue light; BR: “unpure” blue
light created by mixing B with a small
amount (10% total PPFD) of R; BRF:
“unpure” blue light created by mixing
BR with a small amount of far red light
with red/far red ≈ 1. For the same
plant trait, light quality (Q), light in-
tensity (L), plant species (S), or the in-
teraction of light quality and light in-
tensity (Q × L), light quality and plant
species (Q × S), light intensity and
plant species (L × S), or light quality,
light intensity and plant species
(Q × L × S) followed by NS, *, **, or
*** denote treatment effects are not
significant or significant at α = 0.05,
0.01, or 0.001, respectively. For the
same species, the eight treatment
combinations of light quality × light
intensity bearing the same letters are
not significantly different at α = 0.05
according to Duncan’s new multiple
range test. (For interpretation of the
references to colour in this figure le-
gend, the reader is referred to the web
version of this article).

at HL (Fig. 5A and D). Similar response pattern was obtained in aerial and variation magnitude in these traits among the four species under
DW for petunia at HL (Fig. 5C). However, the SB/aerial DW showed a different light quality treatments (Fig. 5).
reverse pattern of response in petunia at both HL and LL and cali-
brachoa at LL where B and BRF resulted in a lower value than R and BR,
4. Discussion
and there was no difference between B and BRF, but with a lower or
similar value under BR vs. R (Fig. 5E). There was no difference in re-
4.1. Blue light effects on elongation growth are related to phytochrome
sponse of aerial/total DW to light quality treatments (data not shown).
activity
Light intensity did not change the pattern of response to light
quality variation in SER and MS/aerial DW for the tested species except
Previous studies have indicated that blue light responses in higher
marigold (Fig. 5A and D). However, light intensity changed the varia-
plants can be mediated not only by specific blue light receptors such as
tion magnitude of the two growth traits in petunia with light quality
cryptochromes, but also by the red/far red photoreversible phyto-
change, which was supported by the interaction effects between light
chrome system (Poppe et al., 1998), suggesting phytochrome, or the
quality and light intensity in this species (Fig. 5A and D). Also, there
phytochrome signal transduction pathway, was somehow implicated in
were interaction effects between plant species and light quality on the
blue-light responses (Ahmad and Cashmore, 1997). Accumulated evi-
two growth traits, which were reflected by different response pattern
dence in Arabidopsis has revealed that cryptochromes and

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Fig. 3. Stem morphology under dif-

ferent light treatments in four bedding
plant species. Data are means ± SE
(n = 3). HL: higher light intensity,
100 μmol m−2 s−1 PPFD; LL: lower
light intensity, 50 μmol m−2 s−1 PPFD.
R: monochromatic red light; B: mono-
chromatic blue light; BR: “unpure” blue
light created by mixing B with a small
amount (10% total PPFD) of R; BRF:
“unpure” blue light created by mixing
BR with a small amount of far red light
with red/far red ≈ 1. MS: main stem;
SB: side branch. For the same plant
trait, light quality (Q), light intensity
(L), plant species (S), or the interaction
of light quality and light intensity
(Q × L), light quality and plant species
(Q × S), light intensity and plant spe-
cies (L × S), or light quality, light in-
tensity and plant species (Q × L × S)
followed by NS, *, **, or *** denote
treatment effects are not significant or
significant at α = 0.05, 0.01, or 0.001,
respectively. For the same species, the
eight treatment combinations of light
quality × light intensity bearing the
same letters are not significantly dif-
ferent at α = 0.05 according to
Duncan’s new multiple range test. (For
interpretation of the references to
colour in this figure legend, the reader
is referred to the web version of this
article).

phytochromes share some common signaling pathways to coordinately same intensity as red light (Ahmad and Cashmore, 1997). Similar re-
regulate transcriptional changes in response to blue light (Liu et al., sults were found on the co-action of blue light with red light and far red
2016; Pedmale et al., 2016; Mishra and Khurana, 2017; Su et al., 2017; light in our present study despite using a different light source, higher
Yang et al., 2017). For example, in de-etiolated seedlings of Arabidopsis, light intensity and more mature plants. For the four bedding plant
blue-light mediated inhibition of hypocotyl elongation can be enhanced species in this study, although the “pure” blue vs. red light promoted
if 10 min blue-light pulses (30 μmol m−2 s−1) are followed by 10 min elongation growth with increased canopy height, main stem length,
pulses of red light (3 μmol m−2 s−1), but can be partially reversed if the internode length, or main stem extension rate, the “unpure” blue light
blue-light pulses are followed by 10 min pulses of far red light with the by mixing with a small amount of red light had a similar or even

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Fig. 4. Leaf morphology under different light treat-

ments in four bedding plant species. The length of
reference bar in pictures is 6 cm. Data are
means ± SE (n = 3). HL: higher light intensity,
100 μmol m−2 s−1 PPFD; LL: lower light intensity,
50 μmol m−2 s−1 PPFD. R: monochromatic red light;
B: monochromatic blue light; BR: “unpure” blue
light created by mixing B with a small amount (10%
total PPFD) of R; BRF: “unpure” blue light created by
mixing BR with a small amount of far red light with
red/far red ≈ 1. LMA: leaf dry mass unit area. For
the same plant trait, light quality (Q), light intensity
(L), plant species (S), or the interaction of light
quality and light intensity (Q × L), light quality and
plant species (Q × S), light intensity and plant spe-
cies (L × S), or light quality, light intensity and plant
species (Q × L × S) followed by NS, *, **, or ***
denote treatment effects are not significant or sig-
nificant at α = 0.05, 0.01, or 0.001, respectively. For
the same species, the eight treatment combinations
of light quality × light intensity bearing the same
letters are not significantly different at α = 0.05 ac-
cording to Duncan’s new multiple range test. (For
interpretation of the references to colour in this
figure legend, the reader is referred to the web ver-
sion of this article).

stronger inhibition effect on these traits compared to “pure” red light. to “pure” red light, with a similar promotion effect to “pure” blue light.
This inhibition effect was reversed by again adding a small amount of Phytochromes are synthesized as Pr (the inactive form) which is con-
far red light. In other words, “unpure” blue light with an equal small verted by red light to Pfr (the active form), and far red light can induce
amount of red and far red light promoted elongation growth compared the reverse reaction by converting Pfr back to Pr, so red/far red

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Fig. 5. Plant growth rate, biomass ac-

cumulation and allocation under dif-
ferent light treatments in four bedding
plant species. Data are means ± SE
(n = 3). HL: higher light intensity,
100 μmol m−2 s−1 PPFD; LL: lower
light intensity, 50 μmol m−2 s−1 PPFD.
R: monochromatic red light; B: mono-
chromatic blue light; BR: “unpure” blue
light created by mixing B with a small
amount (10% total PPFD) of R; BRF:
“unpure” blue light created by mixing
BR with a small amount of far red light
with red/far red ≈ 1. SER: main stem
extension rate; LER: leaf expansion
rate; DW: dry weight; MS: main stem;
SB: side branch. For the same plant
trait, light quality (Q), light intensity
(L), plant species (S), or the interaction
of light quality and light intensity
(Q × L), light quality and plant species
(Q × S), light intensity and plant spe-
cies (L × S), or light quality, light in-
tensity and plant species (Q × L × S)
followed by NS, *, **, or *** denote
treatment effects are not significant or
significant at α = 0.05, 0.01, or 0.001,
respectively. For the same species, the
eight treatment combinations of light
quality × light intensity bearing the
same letters are not significantly dif-
ferent at α = 0.05 according to
Duncan’s new multiple range test. (For
interpretation of the references to
colour in this figure legend, the reader
is referred to the web version of this
article).

reversibility is the classic signature of phytochrome activity (Strasser 2009). It appears that involvement of active phytochrome is necessary
et al., 2010). Also, phytochrome activity can be indicated by the cal- for blue light to inhibit elongation growth of the four bedding plant
culated PPS, which in the present study was the lowest under “pure” species under the tested light levels, which was also supported by a
blue light (0.49), and increased to 0.74 under “unpure” blue light by tight negative correlation of elongation growth to PPS rather than to
adding a small amount of red light, but decreased to 0.63 by again “pure” blue light level in most cases (Table 3). The results from the
adding a small amount of far red (Table 2). The threshold value of PPS present study confirmed the first hypothesis that blue light effect on
to induce active-phytochrome response is controversial, but a general elongation growth is related to phytochrome activity at least in some
consensus is that a PPS > 0.6 may induce the active response (Stutte, cases.

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Table 3
Correlations of phytochrome photostationary state (PPS) or “pure” blue light intensity (BL) with plant growth and morphological traits in four bedding plants.
Plant traits Petunia Calibrachoa Geranium Marigold

PPS BL PPS BL PPS BL PPS BL

Canopy morphology Canopy height −0.80** 0.35 −0.76* 0.28 −0.75* 0.39 −0.44 0.51
Canopy width −0.64 0.64 −0.25 0.56 −0.55 0.60 −0.08 0.47
Compactness 0.54 0.04 0.48 0.09 0.43 0.15 −0.04 0.58

Stem morphology MS length −0.80** 0.35 −0.77* 0.30 −0.75* 0.41 −0.47 0.55
MS diameter −0.38 0.66 −0.31 0.53 −0.15 0.34 −0.23 0.64
Node no. −0.65 0.62 −0.55 0.30 0.14 0.50 −0.27 0.60
Internode length −0.80** 0.38 −0.82** 0.40 −0.54 0.30 −0.70* 0.62
SB number 0.46 0.15 0.37 0.11 −0.21 0.74* 0.06 0.45
Max. SB length 0.20 0.38 −0.30 0.58 −0.04 0.53 0.06 0.38

Leaf morphology Leaf area −0.68* 0.65 -0.82** 0.57 0.18 −0.14 −0.31 0.64
Petiole length −0.79* 0.46 0.32 −0.62 −0.72* 0.25 0.27 −0.48
Leaf epinasty 0.86** −0.78* 0.85** −0.87** 0.78* −0.82** 0.76* −0.77*
Leaf angle 0.79* −0.75* 0.88** −0.83** 0.84** −0.80** 0.83** −0.48
Chl content 0.53 0.13 0.64 −0.02 0.37 0.28 −0.13 0.73*
LMA 0.39 0.16 0.59 −0.08 −0.33 0.72* −0.05 0.59

Growth rate SER −0.79* 0.35 −0.78* 0.32 −0.73* 0.32 −0.60 0.70*
LER −0.44 0.44 −0.44 0.44 0.50 0.08 −0.53 0.54

Biomass accumulation and allocation Aerial DW −0.32 0.62 −0.25 0.49 −0.13 0.46 −0.18 0.60
Aerial/ total DW −0.13 −0.39 −0.77* 0.27 −0.13 0.02 0.17 −0.59
MS/ Aerial DW −0.82** 0.38 −0.74* 0.19 −0.64 0.43 −0.62 0.35
SB/ Aerial DW 0.66 −0.08 0.55 0.03 0.09 0.38 0.28 0.16

Data are correlation coefficient (r) values. * or ** next to the data indicate that correlations are significant at α = 0.05 or 0.01, respectively. PPS is the estimated
phytochrome photoequilibrium according to the method by Sager et al. (1988). MS: main stem; SB: side branch; Chl: chlorophyll; LMA: leaf dry mass unit area; SER:
main stem extension rate; LER: leaf expansion rate; DW: dry weight.
The bold values were used to indicate the significant correlations.

It is possible that in non-LED blue lighting sources, the other light showed a higher PPS than B light (≈0.63 vs. 0.49), the two light
components with a high ratio of red/far red may activate phyto- treatments had similar effects on elongation growth, suggesting that
chromes, which make the above “contaminated” blue light appear more phytochrome only related responses cannot entirely explain this phe-
suppressive on elongation growth than red light. For example, the blue nomenon, because increasing PPS mostly leads to decreased elongation
fluorescent lamp, one of the previously most used blue light sources, (Rajapakse et al., 1999; Dueck et al., 2016). It appears that in either
was reported to have a red/far red ratio of 1.87 (Appelgren, 1991), and “unpure” (BR, or BRF), or “pure” (B) blue light treatment, the important
cool-white fluorescent lamp filtered through blue acetates, another role played by blue light as well as its specific photoreceptors (e.g.,
previously-popular blue light source, did not contain > 700-nm light cryptochromes) in mediating plant elongation cannot be ruled out.
due to the filters employed (Kigel and Cosgrove, 1991). However, in our A recent study found that even for Arabidopsis mutants which lack
present study, when using narrow-waveband LEDs as the lighting all known phytochrome species, the de-etiolated seedlings still showed
source, “pure” blue light showed the lowest PPS (0.49), indicating that shorter hypocotyl length under blue vs. red light (fluorescent lamps in
phytochromes were deactivated, and did not inhibit, but promoted combination with red and blue filters), indicating cryptochromes re-
elongation growth in the four bedding plant species compared to “pure” mained functional in inhibiting stem elongation in this case (Strasser
red light. Also, a recent supplemental LED study on petunia found that et al., 2010). If these phytochrome deficient mutants did not retain
elongation growth was promoted under supplemental “pure” blue vs. considerable residual phytochrome responses (Ahmad and Cashmore,
red lighting, and the promotion effect was stronger in the presence of 1997), it suggests that cryptochrome function and signaling is only
far red light (Gautam et al., 2015). Therefore, in the future applications modified by phytochrome rather than being determined by it (Liu et al.,
of blue LEDs to reduce stretching of the four bedding plant species, a 2016). This was supported by the observed inhibitory effects of “pure”
combination with red LEDs or far-red-filtering covering materials is blue vs. red light on elongation growth of petunia (‘Wave Pink’), as well
necessary to create a background light environment with a higher red/ as some vegetable and agronomic crops even in recent LEDs studies,
far red ratio (e.g., PPS > 0.73). where 160–500 μmol m−2 s−1 PPFD were used (Wollaeger and Runkle,
Due to the fact that the phytochrome itself directly absorbs blue 2014; Snowden, 2015; Wollaeger and Runkle, 2015; Snowden et al.,
light, it is difficult to unequivocally distinguish between pure phyto- 2016). However, in our present study, “pure” blue did not inhibit, but
chrome responses and an influence of phytochrome on the activity of promoted elongation growth in the four bedding plant species com-
cryptochrome (Ahmad and Cashmore, 1997). Similarly, in the present pared to “pure” red light. Similar promotion effects on stem elongation
study, when “pure” blue light was mixed with a small amount of red by “pure” blue light have been reported on petunia (different varieties
light, it showed an inhibitory effect similar to “pure” red light on from ‘Wave Pink’) and marigold seedlings, and calibrachoa cuttings in
elongation growth in some cases (e.g. petunia and calibrachoa). It is recent monochromatic LED light research where 40–150 μmol m−2 s−1
difficult to tell if the response to this “unpure” blue light resulted from PPFD were used (Heo et al., 2002; Fukuda et al., 2011, 2016;
the direct or indirect influence of the small amount of red light added. Olschowski et al., 2016). Possibly, the inconsistent effects of mono-
However, despite the lower PPS (≈0.73 vs. 0.89), BR light had stronger chromatic blue vs. red LED lighting on elongation growth may be due to
inhibitory effects than R light on a number of elongation growth traits, different light intensity, and/or different plant species or even varieties
including canopy height of geranium and marigold, and internode within the same species, which may affect the degree of cryptochrome
length, petiole length and SER of geranium. Also, although BRF light function modification by phytochrome.

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4.2. Blue light affects plant growth and morphology by interaction with light from 25 to 75 μmol m−2 s−1. Possibly, despite the contribution of red
intensity light to red/far red ratio, blue light rather than red light is an important
signal for plants (at least for some species) to sense light intensity
There were interaction effects between light intensity and light changes for the control of elongation growth.
quality on plant growth and morphology, especially elongation growth Based on the interaction effects between light intensity and light
traits, in our present study (Figs. 2–5). This confirmed the second hy- quality, the promotion of stem elongation under B and BRF vs. R or BR,
pothesis that differences in plant response can occur between higher in the present study, appears to be an expression of shade avoidance
and lower light intensity. syndrome (SAS) response mediated by blue light under light levels of
On one hand, light intensity changed the magnitude of variation of 50–100 μmol m−2 s−1. Studies in the past decade have suggested that
elongation growth in response to the change of light quality in some besides low red/far red ratios, reduced blue light levels can also induce
cases, although it generally did not change the response patterns. This shade-avoidance responses including elongation growth
was especially obvious in petunia and calibrachoa where B and BRF vs. (Vandenbussche et al., 2005; Keller et al., 2011). A reduction in blue
R and BR promoted elongation growth such as canopy height, MS light intensity is an indicator of actual shading, whereas the decrease in
length, internode length, or SER to a larger degree under lower than red/far red ratio is an early warning signal of light competition in the
higher light level. It appeared that lower vs. higher light intensity en- future (Keuskamp et al., 2012). A recent study indicated that low blue
hanced the promotion effects on elongation growth induced by blue light perception enhanced plant response to low red/far red ratio (de
light associated with lower PPS (i.e., B or BRF). In other words, higher Wit et al., 2016). In this way, low blue light signals enable plants to tell
intensity of blue light associated with lower PPS (i.e., B or BRF) reduced between true shade and threat of shade (Franklin, 2016). As the major
the promotion of elongation growth to some degree compared to lower mediators in blue light regulated SAS (Keuskamp et al., 2012), cryp-
intensity of blue light. This suggested the possibility that increasing the tochromes are required to sense decreased blue light intensity under
intensity of blue light associated with lower PPS (i.e., B or BRF) beyond canopy shade, and may have evolved a cross talk with other photo-
100 μmol m−2 s−1 up to a threshold value could result in an elongation receptors such as phytochromes and phototropins (another category of
inhibition effect similar to red light, although the threshold intensity blue light receptor) to coordinate SAS response in natural conditions
would seem to be much higher for blue light associated with lower vs. (Nozue et al., 2015; Fraser et al., 2016). Under B and BRF vs. R or BR
higher PPS (i.e., B and BRF vs. BR). This supports the opinion that has light, the light intensity of around 50–100 μmol m−2 s−1 PPFD used in
long been proposed where enhanced intensity of blue light can inhibit the present study also induced obvious SAS responses as described by
elongation growth by activating cryptochrome, although responses Smith and Whitelam (1997). In addition to promotion of elongation
vary with plant species (Cope and Bugbee, 2013). Recent studies on growth, these responses included decreased branching, chlorophyll
Arabidopsis have also confirmed that the physiological activities of content, and/or leaf mass per unit area (i.e., leaf thickness), and/or
cryptochromes are well correlated with blue light-dependent phos- increased individual leaf area, petiole length, biomass allocation to
phorylation, which increases under higher irradiances of blue light (Liu supporting structure (e.g., main stem), and/or advanced flowering time
et al., 2016). In addition to elongation growth, if comparing the mag- (data not shown), which varied with different species. However, under
nitude of variation, represented by the mean value of CV of all the plant B, BR, and BRF vs. R light, for all the four species, plants exhibited
growth and morphology responses to different light quality treatments, reduced leaf epinasty (i.e., increased leaf hyponasty) and leaf angle. It
the plants showed greater mean CV value under the lower light level in appears that although most blue light mediated SAS responses in-
the four species (Fig. 6). Similarly, a previous study on leaf lettuce using cluding elongation growth require the co-action of blue light with red/
monochromatic LEDs indicated that the difference in stem elongation far red signals, some SAS responses such as leaf position are specifically
between plants exposed to red and blue lights was increased when mediated by blue light where phototropins are important controllers
PPFD was decreased from 150 to 50 μmol m−2 s−1, due to the dis- (Hangarter, 1997; Inoue et al., 2008; Van Zanten et al., 2010). Re-
proportional promotional effect of the two light spectra under lower garding geraniums, despite having typical SAS responses such as petiole
intensity, and the promotion of elongation growth was assumed to be a elongation, the plants under B vs. R light also showed some contra-
shade-avoidance response (Hirai et al., 2006). dictory SAS responses such as reduced individual leaf area and in-
On the other hand, different light quality also resulted in different creased leaf mass per unit area under the light intensity of 100 μmol
plant responses to the decrease in light intensity. For example, in pet- m−2 s−1. Such diverse responses are likely to be brought about by
unia, when light intensity was reduced from 100 to 50 μmol m–2 s–1, tissue- or even cell-specific expression of blue light receptors
elongation growth was promoted under B and BRF light, but without (Shimazaki and Tokutomi, 2013), and thus the same cryptochrome
any change under R and BR light. Obviously, at least for this species, signaling mechanism may trigger opposite cellular responses in dif-
elongation growth response was more sensitive to decreased intensity ferent cells in the same plant (Yu et al., 2010). This was possibly due to
of “pure” blue light (with the lowest PPS) than that of “pure” red light different threshold levels of blue light to trigger the SAS response in
(with the highest PPS), and was less sensitive to intensity decrease in different cells (Mishra and Khurana, 2017).
“unpure” blue light associated with higher vs. lower PPS (i.e., BR vs. Most LEDs research on the comparison of blue and red light used
BRF). Similarly, a previous LED study on eggplant also reported that PPFD levels of less than 200 μmol m−2 s−1. These light levels for some
stem elongation showed a higher sensitivity to a light intensity decrease species may also induce SAS responses including elongation growth
of monochromatic blue light (likely also with a much lower PPS) (Snowden, 2015). This may explain previous reports on the promotion
compared with monochromatic red light within a PPFD range of of elongation growth of bedding plants under monochromatic blue vs.
100–150 μmol m–2 s–1 (Hirai et al., 2006). It appears that at least under red LED light (Heo et al., 2002; Fukuda et al., 2011, 2016; Olschowski
light conditions which result in lower phytochrome activity (i.e., lower et al., 2016). Today, supplemental lighting using a PPFD level of
PPS), elongation growth of these plant species is more sensitive to the 100–200 μmol m−2 s−1 is quite often used for successful year round
reduced intensity of blue light than to that of red light. This is also greenhouse crop production (Moe et al., 2006). When using lower-in-
supported by a previous study on tomato seedlings under a combination tensity supplemental lighting for production of the four bedding plant
of blue and red LEDs with likely higher PPS due to no addition of far red species, one should be cautious in the sole use of blue LEDs, especially
light (Nanya et al., 2012). In the above study, when red light intensity under a background light environment with low red/far red ratios (e.g.,
was constant (75 μmol m−2 s−1), stem elongation was promoted with PPS ≤ 0.63). However, it is worthwhile to note that under lower light
the decrease in blue light intensity from 75 to 25 μmol m−2 s−1, but levels (e.g., around 30–100 μmol m−2 s-1) similar to that of our study,
when blue light intensity was constant (75 μmol m−2 s−1), stem elon- inhibited elongation growth was also observed in tissue-cultured
gation was not different under different red light intensities ranging plantlets from “pure” blue vs. red light in a number of previous LEDs

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Y. Kong et al. Environmental and Experimental Botany 155 (2018) 345–359

Fig. 6. CV (coefficient of variation) of

plant traits among different light
quality treatments under different light
levels in four bedding plant species:
petunia (A), calibrachoa (B), geranium
(C), and marigold (D). HL: higher light
intensity, 100 μmol m−2 s−1 PPFD; LL:
lower light intensity, 50 μmol m−2 s−1
PPFD. MS: main stem; SB: side branch;
Chl: chlorophyll; LMA: leaf dry mass
unit area; SER: main stem extension
rate; LER: leaf expansion rate; DW: dry
weight. CV of each growth and mor-
phology traits among the four light
quality treatments was calculated by
standard deviation/mean. The number
beside the legends is the mean CV of all
the tested plant traits. The dash lines
indicate CV = 0.5.

studies (Tanaka et al., 1998; Nhut et al., 2002, 2003; Kim et al., 2004; of tomato, radish, soybean, cucumber, and lettuce when compared to
Poudel et al., 2008; Shin et al., 2008; Li et al., 2017). This may be due to red light, but blue and red light showed similar effects on wheat and
the cross effects of the addition of hormone in the medium, relative air- pepper seedlings (Snowden, 2015; Snowden et al., 2016). Apparently,
tight environments and/or involvement of the differentiation-induction even with the higher light intensity, elongation growth response to
stage on the growth of plantlets rather than seedlings (Hahn et al., “pure” blue vs. red light varies with different species. Future studies
2000; Chang et al., 2016). Nevertheless, recent studies using LEDs with need to be carried out under higher light levels to investigate blue light
higher light intensities of up to 200 and 500 μmol m−2 s−1 demon- responses of the bedding plant species used in our present study.
strated that “pure” blue light inhibited elongation growth in seedlings

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4.3. Plant growth and morphology response to blue light vary with different growth (Mishra and Khurana, 2017). This is sometimes further com-
plant species plicated by inconsistent light intensities among light quality treatments
that introduce confounding factors and possibly inaccurate conclusions
In the present study, the four bedding plant species studied showed about light quality effects (Lefsrud et al., 2008; Snowden, 2015). An-
different magnitudes of variation in plant elongation response to dif- other possibility is that nearly all the previous LEDs studies except one
ferent light quality treatments, which was supported by strong inter- used periodic rather than continuous lighting. In these cases, during
action effects between plant species and light quality on canopy height, dark vs. light periods, plant elongation is normally faster, and may be
MS length, internode length, and SER. Based on the morphological affected to a larger degree by small temperature differences among light
appearances, B and BRF vs. R and BR promoted elongation growth to a treatments, and/or by trace light pollution (such as red or far red light),
larger degree in petunia and calibrachoa than in geranium and mar- which varies with different dark period lengths (Dowson-Day and
igold (Fig. 2D–G). This was also supported by the higher CVs (> 0.5) of Millar, 1999; Kurepin et al., 2011). However, in the present study,
elongation traits under different light quality treatments for the former continuous lighting might provide a better platform to present and even
two species, but lower ones (< 0.5) for the latter two species (Fig. 6). amplify blue vs. red light effects on plant elongation, due to the dif-
As discussed before, the tested bedding plants showed typical SAS re- ference in action of red and blue light on elongation growth during light
sponses under B and BRF vs. R or BR. So, the above different sensitivity and dark periods (Gaba and Black, 1979; Cosgrove, 1981; Kigel and
in elongation growth responses may be due to different shade-avoid- Cosgrove, 1991). Under the continuous lighting conditions used in the
ance strategies for the different plant species, since elongation growth is present study, some calibrachoa plants from the blue light treatments
just one of the most striking SAS responses (Smith and Whitelam, 1997; showed interveinal necrosis in some leaves varying to different degrees,
Franklin, 2008). which has also been reported on sesame plants in a previous study using
In addition to the promoted elongation growth with different continuous lighting (Hata et al., 2013). This leaf injury did not occur in
magnitudes, the four plant species under B and BRF vs. R or BR showed the other three plant species in the present study. The species-specific
either one, two or three species-specific SAS responses (Supplemental response differences will provide first-hand information for bedding
Fig. S1). Among all the tested growth and morphological traits, the total plant production using continuous lighting.
number of SAS response traits induced by B and BRF was 12 for petunia In summary, at PPFD of 50 or 100 μmol m−2 s-1, B (“pure” blue
and calibrachoa, but only 9 for geranium and marigold. Furthermore, light) vs. R (“pure” red light) promoted elongation growth of the four
for a certain plant trait, blue light could induce a SAS response in one bedding plant species, but the promotion effects of B were reversed by
species, but sometimes could repress the response in another plant adding a low level of R (i.e., BR, “unpure” blue light), and then re-
species (Van Zanten et al., 2010). For example, in the present study, covered by again adding a low level of far red (i.e., BRF, “unpure” blue
under B or BRF vs. R, leaf chlorophyll content decreased in petunia, light). The PPS values, indicating phytochrome activity, were higher for
calibrachoa, and geranium, but increased in marigold. The adaptive/ R (0.89) and BR (0.74), compared to B (0.49) and BRF (0.63). This
acclimation implications of such species-specific differences deserve suggests that effects of blue light on elongation growth are related to
further attention. Nevertheless, it suggests that blue light mediated SAS phytochrome activity, and if associated with a lower phytochrome ac-
responses vary strongly among different species. When taking all the tivity (e.g., PPS ≤ 0.63), blue light, compared to red light, can promote,
plant traits into consideration, the mean magnitude of variation (i.e., rather than inhibit, plant elongation. The promotion magnitudes were
CV) under the different light quality treatments in the four plant species greater at PPFD of 50 vs. 100 μmol m−2 s-1 for petunia and calibrachoa.
showed the following order: petunia > calibrachoa > geranium > In addition to elongation promotion, some shade avoidance syndromes
marigold (Fig. 6). Cluster analysis based on the CVs of all the plant varying with species were also induced by B or BRF, relative to R or BR.
traits indicated that the four plant species could be generally classified It suggests that plant elongation promoted by blue light with a lower
into two groups (Supplemental Fig. S2). One group included two long- PPS (i.e., B or BRF) is one of shade-avoidance responses varying sen-
day plants species, petunia and calibrachoa, and the other one included sitivity with plant species and light intensities.
short-day and day-neutral plants, marigold and geranium, respectively.
Although further confirmation is needed in more plant species, it ap- Author contributions
pears that long-day plants are more sensitive to light quality changes
than short-day and day-neutral plants, which confirmed the third hy- YK designed and performed the experiments, and collected and
pothesis that response to blue vs. red light varies in plant species with analyzed the data, and wrote and revised the manuscript. MS helped in
different photoperiod flowering responses. Similarly, previous study designing the experiments, setting up the lighting treatments and par-
using photo-selective coverings reported that all the five tested long- ticipated in manuscript preparation and editing. MD provided the
day plants were sensitive to blue light, showing promoted elongation growth chamber and critically revised the manuscript. YZ was the
growth under blue light-deficient covering to different degrees varying principle investigator who participated in experimental design, data
with species (Runkle and Heins, 2001; Shimizu et al., 2005). collection and analyzing, manuscript preparation and editing. All au-
It is worthwhile to note that in the present study elongation growth thors approved the final manuscript.
was promoted by B or BRF relative to R or BR in all the four bedding
plant species, despite the different magnitudes. Similar results were
found in the de-etiolated seedlings of tobacco and sunflower, tested Conflict of interest
with the same light treatments (unpublished). Thus, under a certain
range of light intensities (e.g., 50–100 μmol m−2 s-1), stem elongation The authors declare that the research was conducted in the absence
promotion by blue light associated with lower PPS (i.e., B or BRF) re- of any commercial or financial relationships that could be construed as
lative to red light might be a common phenomenon among various a potential conflict of interest.
shade-avoidance plant species. If this speculation is true, there are at
least two possible explanations for the contradictory results related to Acknowledgments
“pure” blue vs. red effects on elongation growth under similar light
intensity in previous and our present LED studies. One possibility is that Thanks for the financial support awarded to Dr. Youbin Zheng by
different plant species (e.g., shade-avoidance or -tolerance) have dif- the Natural Sciences and Engineering Research Council of Canada and
ferent ways to deal with shade (Franklin, 2008), and even the shade- Ontario Ministry of Agricultural, Food and Rural Affairs. We thank Sean
avoidance plant species may have a different critical value of light in- Ratcliffe and Jasmine Mah for their technical support, and Drs. Michelle
tensity to induce blue light-mediated SAS response including elongation Edwards and Yuhong Wei for their help with statistical analysis.

357
Y. Kong et al. Environmental and Experimental Botany 155 (2018) 345–359

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30, 1–8.
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online version, at doi:https://doi.org/10.1016/j.envexpbot.2018.07. bedding plants as affected by monochromic or mixture radiation provided by a light-
021. emitting diode (LED). Plant Growth Regul. 38, 225–230.
Hirai, T., Amaki, W., Watanabe, H., 2006. Action of blue or red monochromatic light on
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