International Journal of

Molecular Sciences

Article
Effects of Light-Emitting Diode Irradiation on
Growth Characteristics and Regulation of Porphyrin
Biosynthesis in Rice Seedlings
Lien Hong Tran and Sunyo Jung *
School of Life Sciences and Biotechnology, BK21 Plus KNU Creative Bioresearch Group,
Kyungpook National University, Daegu 41566, Korea; thlien91@yahoo.com
* Correspondence: sjung@knu.ac.kr; Tel.: +82-53-950-7364

Academic Editor: Jianhua Zhu

Received: 17 January 2017; Accepted: 13 March 2017; Published: 16 March 2017

Abstract: We examined the effects of light quality on growth characteristics and porphyrin
biosynthesis of rice seedlings grown under different wavelengths from light emitting diodes
(LEDs). After 10 days of exposure to various wavelengths of LEDs, leaf area and shoot biomass
were greater in seedlings grown under white and blue LEDs than those of green and red LEDs.
Both green and red LED treatments drastically decreased levels of protoporphyrin IX (Proto IX)
and Mg-porphyrins compared to those of white LED, while levels of Mg-Proto IX monomethyl
ester and protochlorophyllide under blue LED were decreased by 21% and 49%, respectively.
Transcript levels of PPO1 were greatly upregulated in seedlings grown under red LED compared
to white LED, whereas transcript levels of HO2 and CHLD were upregulated under blue LED.
Overall, most porphyrin biosynthetic genes in the Fe-porphyrin branch remained almost constant or
upregulated, while most genes in the Mg-porphyrin branch were downregulated. Expression levels
of nuclear-encoded photosynthetic genes Lhcb and RbcS noticeably decreased after exposure to blue
and red LEDs, compared to white LED. Our study suggests that specific wavelengths of LED greatly
influence characteristics of growth in plants partly through altering the metabolic regulation of the
porphyrin biosynthetic pathway, and possibly contribute to affect retrograde signaling.

Keywords: growth; LED (light emitting diode); nuclear-encoded photosynthetic gene; photosynthetic
pigment; porphyrin biosynthesis

1. Introduction
Tetrapyrroles play an essential role in photosynthesis, cellular respiration, catalytic activities
of enzymes, and signal transduction [1,2]. In higher plants, 5-aminolevulinic acid (ALA) is formed
from glutamic acid and subsequently converted to protoporphyrin IX (Proto IX) through a variety
of reactions [1] (Figure 1). Proto IX is the point at which the chlorophyll (Mg-porphyrin) and the
heme (Fe-porphyrin) pathways diverge. Metabolites in the porphyrin biosynthetic pathway are highly
photoreactive and can easily be excited and transfer the energy to O2 . Proto IX and protochlorophyllide
(Pchlide) are especially reactive with O2 to form reactive oxygen species (ROS), which is harmful to
cells and causes the peroxidation of membrane lipids [3,4].
Porphyrin-containing compounds are cofactors of apoproteins involved in photosynthesis and
electron transfer reactions [5]. Plants have a remarkable ability to adapt their photosynthetic machinery
to environmental condition such as different light qualities. Acclimatory responses of plants to
light quantity and light spectral distribution often involve changes in the relative amounts of
pigment-binding protein complexes, such as light-harvesting chlorophyll proteins (Lhcs), accompanied
by corresponding changes in chlorophyll content in chloroplasts [6,7]. This ensures the coordinated

Int. J. Mol. Sci. 2017, 18, 641; doi:10.3390/ijms18030641 www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2017, 18, 641 2 of 11

Int. J. Mol. Sci. 2017, 18, 641 2 of 11

assembly of the photosynthetic apparatus and avoids cellular photosensitization by free photoreactive
photoreactive tetrapyrroles [8]. In addition, nuclear gene expression including Lhc is regulated by the
tetrapyrroles [8]. In addition, nuclear gene expression including Lhc is regulated by the functional and
functional and developmental states of chloroplasts via retrograde signaling [9].
developmental states of chloroplasts via retrograde signaling [9].

Figure 1.1. TheThe

Figure porphyrin
porphyrin pathway
pathway in plants
in plants showing
showing metabolites
metabolites and genesand genes in
analyzed analyzed in
this study.
this study. Metabolites quantified in this study are highlighted. Proto IX, protoporphyrin
Metabolites quantified in this study are highlighted. Proto IX, protoporphyrin IX; Mg-Proto IX, Mg-
IX; Mg-Proto IX, IX;
protoporphyrin Mg-protoporphyrin
Mg-Proto IX ME, IX; Mg-Proto IX ME, Mg-protoporphyrin
Mg-protoporphyrin IX monomethyl IX monomethyl
ester; Pchlide,
ester; Pchlide, protochlorophyllide. Genes and enzymes that correspond to the gene names:
protochlorophyllide. Genes and enzymes that correspond to the gene names: HEMA, glutamyl-tRNA
HEMA, glutamyl-tRNA
reductase; reductase;
GSA, glutamate GSA, glutamate
1-semialdehyde 1-semialdehyde
aminotransferase; aminotransferase;
ALAD, ALAD, PPO,
ALA dehydratase; ALA
dehydratase; PPO, protoporphyrinogen oxidase; FC2, Fe-chelatase 2; HO, heme oxygenase;
protoporphyrinogen oxidase; FC2, Fe-chelatase 2; HO, heme oxygenase; CHLD, D-subunit of CHLD,
D -subunit of Mg-chelatase; CHLH, H-subunit of Mg-chelatase; CHLI, I-subunit of Mg-chelatase; PORB,
Mg-chelatase; CHLH, H-subunit of Mg-chelatase; CHLI, I-subunit of Mg-chelatase; PORB,
protochlorophyllide oxidoreductase B. Solid and dashed arrows indicate a single step and multiple
protochlorophyllide oxidoreductase B. Solid and dashed arrows indicate a single step and multiple
steps, respectively.
steps, respectively.

Light isisanan
important
importantenvironmental
environmental factorfactor
affecting plant development
affecting and growth
plant development andbygrowth
regulating
by
morphological changes [10,11]
regulating morphological changesas well as as
[10,11] possible
well asmetabolic pathways.
possible metabolic In photobiological
pathways. studies,
In photobiological
light-emitting diodes (LEDs)
studies, light-emitting diodesare an efficient
(LEDs) are ansystem
efficientof system
narrow-band light output
of narrow-band thatoutput
light can change the
that can
wavelength of light in enclosed
change the wavelength of lightenvironment. Although a number
in enclosed environment. of studies
Although usingof
a number LED lightsusing
studies have been
LED
performed
lights haveon the effect
been of theon
performed light
thespectral quality
effect of on the
the light plant growth
spectral qualityandon morphogenesis
the plant growth [11,12],
and
the underlying mechanisms
morphogenesis of the effectmechanisms
[11,12], the underlying of different light
of thespectra
effectonof porphyrin metabolism
different light spectraandon
retrograde signaling have not yet been elucidated.
porphyrin metabolism and retrograde signaling have not yet been elucidated.
In this study,
study, wewe examined
examined the effect of light light quality on the regulation of plant growth and
morphology in rice seedlings grown under different wavelengths of light. We attempted to control
light quality for plant growth by using various wavelengths wavelengths fromfrom LEDs
LEDs (Supplemental
(Supplemental TableTable S1).
S1).
Moreover, to shed light on plant growth driven by a specific wavelength of light, we monitored the
regulatory mechanism
mechanismof ofporphyrin
porphyrinmetabolites
metabolites and
and expression
expression levels
levels of their
of their biosynthetic
biosynthetic genes.
genes. We
We
alsoalso investigated
investigated howhowthethe expression
expression ofofnuclear-encoded
nuclear-encodedphotosynthetic
photosyntheticgenesgenesisis regulated
regulated by
different wavelength of LEDs. This This study demonstrates
demonstrates that light qualities influence not only the
growth properties of rice seedlings following different LED treatments treatments through altering porphyrin
porphyrin
metabolism, but also possibly retrograde
retrograde signaling.
signaling.
Int. J. Mol. Sci. 2017, 18, 641 3 of 11
Int. J. Mol. Sci. 2017, 18, 641 3 of 11

2. Results
2. Results andand Discussion
Discussion

2.1. Effects
2.1. Effects of Different
of Different Wavelengths
Wavelengths of of LEDsononthe
LEDs theGrowth
Growthand
and Morphological
Morphological Appearances
Appearancesof of Rice Seedlings
Rice Seedlings
The effects of light quality on growth characteristics in rice seedlings were examined using
The effects of light quality on growth characteristics in rice seedlings were examined using
different light wavelengths from LEDs. The five-day-old etiolated rice seedlings were exposed to
different light wavelengths from LEDs. The five-day-old etiolated rice seedlings were exposed to
white, blue, green, or red LED light in cycles of 14 h light and 10 h dark or maintained in continuous
white, blue, green, or red LED light in cycles of 14 h light and 10 h dark or maintained in continuous
darkness for either
darkness 5 or
for either 10 10
5 or days.
days.Exposure
Exposureof of etiolated seedlingstotodifferent
etiolated seedlings different wavelengths
wavelengths not only
not only
greatly enhanced plant growth, but also altered morphological characteristics (Figure 2).
greatly enhanced plant growth, but also altered morphological characteristics (Figure 2). In response In response
to five
to days of LED
five days illumination,
of LED shoot
illumination, shootheight
heightofof seedling treatedwith
seedling treated with white
white or or blue
blue LEDLED
werewere shorter
shorter
thanthan
those of green
those LED,
of green redred
LED, LED,
LED,orordark
darkconditions (Table1).
conditions (Table 1).Leaf
Leafarea
area
of of blue
blue LED,
LED, as indicated
as indicated
by length
by leaf leaf length
andand width,
width, was
was thegreatest
the greatestamong
among all LED
LEDtreatments,
treatments, whereas
whereas drydry
biomass
biomassdid not
did not
prominently
prominently differ. differ.

Figure 2. Effects of different wavelengths of LEDs on the growth parameters of rice seedlings.
Figure 2. Effects of different wavelengths of LEDs on the growth parameters of rice seedlings. The
The five-day-old
five-day-oldetiolated
etiolated
ricerice seedlings
seedlings were exposed
were exposed to light-emitting
to different different light-emitting
diodes (LEDs)diodes (LEDs)
with a 14-
withh-light/10-h-dark
a 14-h-light/10-h-dark photoperiod
photoperiod or underdark
or under constant constant darkforcondition
condition for dark;
10 days. D, 10 days. D, dark;
W, broad
W, broad spectrum-white
spectrum-white LED (420
LED (420680 nm)−as
680a nm) as aB,control;
control; blue LED blue LEDnm);
B, (460490 −490
(460G, greennm);
LEDG, green LED
(520550
(520− 550R,
nm); nm);
red R,
LEDred(620650
LED (620 −650 nm).
nm).

TableTable 1. Effect
1. Effect of LED
of LED lights
lights ononthe
themorphological
morphological characteristics
characteristicsofofrice seedlings
rice after
seedlings different
after different
wavelengths of LED illumination.
wavelengths of LED illumination.
Treatment Shoot Height (cm) Leaf Length (cm) Leaf Width (cm) Dry Weight (mg·plant−1)
Treatment
5 days Shoot Height (cm) Leaf Length (cm) Leaf Width (cm) Dry Weight (mg·plant−1 )
Dark 17.9 ± 1.6 a 6.1 ± 1.0 b 0.19 ± 0.01 d 11.7 ± 1.3 ab
5 days
White 14.3 ± 1.1 c 5.4 ± 1.0 c 0.24 ± 0.03 b 11.6 ± 1.5 abc
Dark 17.9 ± 1.6 a 6.1 ± 1.0 b 0.19 ± 0.01 d 11.7 ± 1.3 ab
Blue 13.6 ± 0.7 d 7.1 ± 0.9 a 0.33 ± 0.05 a 12.0 ± 1.4 a
White Green 14.3 ± 1.1± c1.3 b
16.9 5.8±± 1.0
5.4 1.0 cab 0.24
0.22 ± 0.03
± 0.02 c b 1.9 c± 1.5 abc
10.8 ±11.6
Blue Red 13.6 ±
17.7 ±d1.3 a
0.7 7.16.3±±0.9
1.2 ab ±
0.20 ± 0.02 d a
0.33 0.05 11.1 ±12.0 ± 1.4 a
1.7 bc
Green10 days 16.9 ± 1.3 b 5.8 ± 1.0 ab 0.22 ± 0.02 c 10.8 ± 1.9 c
Red Dark 17.7 ± 1.3±a3.3 c
22.5 ±±1.2
6.37.4 2.1bd 0.20
0.26 ± 0.02
± 0.03 d d 1.6 c± 1.7 bc
10.6 ±11.1
White 27.3 ± 3.2 a 17.0 ± 2.5 a 0.33 ± 0.03 b 15.7 ± 2.2 a
10 days
Blue 22.4 ± 2.3 c 13.8 ± 1.5 b 0.37 ± 0.02 a 14.9 ± 2.2 a
Dark 22.5 ± 3.3 c 7.4 ± 2.1 d 0.26 ± 0.03 d 10.6 ± 1.6 c
Green 24.4 ± 1.5 b 11.8 ± 3.0 c 0.32 ± 0.03 b 12.7 ± 2.2 b
White 27.3 ± 3.2 a 17.0 ± 2.5 a 0.33 ± 0.03 b 15.7 ± 2.2 a
Red 27.5 ± 1.7 a 14.1 ± 2.2 b 0.28 ± 0.03 c 12.6 ± 1.3 b
Blue 22.4 ± 2.3 c 13.8 ± 1.5 b 0.37 ± 0.02 a 14.9 ± 2.2 a
The five-day-old rice seedlings were exposed to different LEDs with a 14-h-light/10-h-dark
Green 24.4 ± 1.5 b 11.8 ± 3.0 c 0.32 ± 0.03 b 12.7 ± 2.2 b
Redphotoperiod27.5
or under
± 1.7constant
a dark 14.1
condition
± 2.2forb either 5 or0.28
10 days.
± 0.03The
c data represent12.6
the± mean
1.3 b±
SD of at least 30 plants from two independent experiments. Within each column, means denoted by
The five-day-old rice seedlings were exposed to different LEDs with a 14-h-light/10-h-dark photoperiod or under
the same
constant letter did for
dark condition noteither
differ 5significantly at p <data
or 10 days. The 0.05represent
according tomean
the Duncan’s
± SDmultiple range
of at least test. from two
30 plants
independent experiments. Within each column, means denoted by the same letter did not differ significantly at
Following
p < 0.05 accordingprolonged
to Duncan’sexposure to 10 test.
multiple range days of various LED light, the shoot height of rice seedlings
treated with white LED or red LED was taller than that of blue LED, green LED or dark condition
(Table 1). Rice
Following seedlingsexposure
prolonged grown intodarkness
10 daysinitially showed
of various LEDalight,
fasterthe
growth,
shootbut their of
height growth was
rice seedlings
subdued in the subsequent phase, which was probably due to depletion of nutrition in the
treated with white LED or red LED was taller than that of blue LED, green LED or dark condition
endosperms. By contrast, seedlings grown under white LED had a faster developmental pace in the
(Table 1). Rice seedlings grown in darkness initially showed a faster growth, but their growth was
later phase of the light exposure, implying that photosynthetic production is crucial for robust growth
subdued in the subsequent phase, which was probably due to depletion of nutrition in the endosperms.
By contrast, seedlings grown under white LED had a faster developmental pace in the later phase
of the light exposure, implying that photosynthetic production is crucial for robust growth in this
Int. J. Mol. Sci. 2017, 18, 641 4 of 11

phase.
Int.The
J. Mol.blue light
Sci. 2017, 18,signal
641 inhibits elongation of leaf sheaths through repression of gibberellin 4 of 11(GA)
biosynthetic-related genes and induction of GA inactivation-related genes [13]. Short shoot height
in this
of rice phase. The
seedlings under blueblue
lightLED
signalmight
inhibits
beelongation
connected ofwith
leaf sheaths through
the report. repression
Leaf length ofof gibberellin
rice seedlings
(GA) biosynthetic-related genes and induction of GA inactivation-related
treated with green LED was significantly shorter than seedlings treated with other LEDs, genes [13]. Short shoot
with the
height
longest leafoflength
rice seedlings under
in seedlings blue
with LED LED.
white mightNoticeably,
be connected bluewith the report. Leafseedlings
LED-illuminated length of showed
rice
seedlings treated with green LED was significantly shorter than seedlings treated with other LEDs,
a significant increase in leaf width compared to white LED. Unrolling (leaf width) of leaves was also
with the longest leaf length in seedlings with white LED. Noticeably, blue LED-illuminated seedlings
promoted by blue light in etiolated rice seedlings, but not by red light [14]. Leaf width of orchid
showed a significant increase in leaf width compared to white LED. Unrolling (leaf width) of leaves
seedlings
was also waspromoted
expandedbyunder whiteinand
blue light blue LEDs
etiolated and wasbut
rice seedlings, narrower
not by under
red lightgreen
[14].and
Leafred LEDs
width of [15].
The seedlings
orchid seedlings was expanded under white and blue LEDs and was narrower under green and redother
treated with white or blue LEDs produced significantly greater dry biomass than
LEDsLEDs
(Table [15].1).
TheWheat grown
seedlings under
treated with red
whiteLED + 10%
or blue LEDs blue fluorescent
produced light greater
significantly had greater shoot dry
dry biomass
matter
than accumulation
other LEDs (Table relative to wheat
1). Wheat grown
grown underunder red +LED
red LED 10% [16]. In higher plants,
blue fluorescent light hadblue light is
greater
mainly
shootperceived
dry matter by accumulation
cryptochromes and phototropins,
relative to wheat grown which
undersubsequently
red LED [16]. mediate
In higheraplants,
varietyblueof blue
light is mainly
light-dependent perceivedincluding
responses by cryptochromes and phototropins,
phototropism, which subsequently
photomorphogenesis, stomatalmediate
opening,a variety
and rapid
of blueoflight-dependent
inhibition responses[12,17].
hypocotyl elongation includingIn phototropism,
our study, leafphotomorphogenesis,
growth and shoot biomassstomatal were
opening,greatly
and rapid inhibition of hypocotyl elongation [12,17]. In our study, leaf
promoted by white and blue light, demonstrating that morphology and growth of rice seedlings growth and shoot biomasswere
were greatly promoted by white and blue light, demonstrating that morphology and growth of rice
controlled by the light quality of LEDs.
seedlings were controlled by the light quality of LEDs.
2.2. Effects of Different Wavelengths of LEDs on the Levels of Photosynthetic Pigments and Porphyrin Metabolites
2.2. Effects of Different Wavelengths of LEDs on the Levels of Photosynthetic Pigments and
To assessMetabolites
Porphyrin the effect of different light qualities on photosynthetic pigments, we quantitated the
amount ofTototal chlorophylls
assess the effect ofand carotenoids
different in rice seedlings
light qualities grown under
on photosynthetic various
pigments, LED lights the
we quantitated having
different
amount of total chlorophylls and carotenoids in rice seedlings grown under various LED lights of
wavelengths. The seedlings grown in constant dark conditions showed a negligible level
chlorophylls, but rapidly
having different accumulated
wavelengths. chlorophylls
The seedlings grownin in response to LED
constant dark illumination
conditions showed (Figure 3). There
a negligible
werelevel
considerable differences
of chlorophylls, in the
but rapidly content of chlorophylls
accumulated chlorophyll,inwhichresponseis the endillumination
to LED product of (Figure
porphyrin
3). Therepathway,
biosynthetic were considerable
when rice differences
seedlingsinwerethe content
grown of chlorophyll,
under different which
LEDissources.
the end In product of
comparison
porphyrin biosynthetic pathway, when rice seedlings were grown under
to broad-spectrum white LED, chlorophyll contents of green and red LEDs were decreased by different LED sources. In
comparison35%
approximately to broad-spectrum white LED,
and 21%, respectively, chlorophyll
whereas contents
chlorophyll of green
contents and LED
in blue red were
LEDs similar
were to
decreased by approximately 35% and 21%, respectively, whereas chlorophyll
those of white LED (Figure 3A). In etiolated pea seedlings, illumination with blue light caused contents in blue LEDa large
were similar to those of white LED (Figure 3A). In etiolated pea seedlings, illumination with blue
increment of chlorophyll within 48 h, while white light induced a small increment [18]. On the other
light caused a large increment of chlorophyll within 48 h, while white light induced a small increment
hand, the chlorophyll a/b ratio increased in blue, red, and green LEDs compared to that of white LED,
[18]. On the other hand, the chlorophyll a/b ratio increased in blue, red, and green LEDs compared to
with that
the greatest increase
of white LED, withinthe
blue LED increase
greatest (Figure in 3B). Elevated
blue chlorophyll
LED (Figure a/b ratio,
3B). Elevated particularly
chlorophyll in blue
a/b ratio,
LED,particularly
could reflect a reduced
in blue light-harvesting
LED, could antenna
reflect a reduced size of photosystem
light-harvesting antenna size II (PSII). Levels ofIItotal
of photosystem
carotenoids also exhibited declines in blue, green, and red LEDs, compared to
(PSII). Levels of total carotenoids also exhibited declines in blue, green, and red LEDs, compared white LED (Figureto 3C).
These results
white LEDindicate thatThese
(Figure 3C). a narrow
resultsspectrum
indicate thatLED influenced
a narrow theLED
spectrum influenceda/b
chlorophyll theratio as well as
chlorophyll
a/b ratio
content as well as content
of chlorophylls of chlorophylls
and carotenoids. and carotenoids.
Particularly, Particularly,
rice seedlings grown riceunder
seedlings
blue grown
LED under
sustained
blue
higher LEDof
levels sustained higherand
chlorophylls levels of chlorophylls
carotenoids and carotenoids
than those of green andthanred
those
LEDs.of green
Blue and
lightred
is LEDs.
important
Blue light isdevelopment
for chloroplast important for chloroplast
and chlorophylldevelopment
formationand [18,19].
chlorophyll formation [18,19].

Figure 3. Effects of different wavelengths of LEDs on the content of total chlorophyll (A); chlorophyll
Figure 3. Effects of different wavelengths of LEDs on the content of total chlorophyll (A); chlorophyll
a/b ratio (B);(B);
a/b ratio andandcarotenoid
carotenoid(C)
(C)ininrice
riceseedlings. Theplants
seedlings. The plantswere
weresubjected
subjected to the
to the same
same treatments
treatments as as
in Figure 2, and
in Figure treatment
2, and notations
treatment notationsare arethe
thesame
same as
as in
in Figure
Figure 2.2.The
Thedata
data represent
represent thethe
mean ± SD± SD
mean
(n = 6).
(n =Means denoted
6). Means denoted byby
thethe
same
sameletter
letterdid
did not
not differ significantlyatatp <p 0.05
differ significantly < 0.05 according
according to Duncan’s
to Duncan’s
multiple range
multiple test.test.
range
Int. J. Mol. Sci. 2017, 18, 641 5 of 11
Int. J. Mol. Sci. 2017, 18, 641 5 of 11

Toexplore
To explorethe therelationship
relationship between
between porphyrin
porphyrin biosynthesis
biosynthesis andand light
light quality,
quality, wewe examined
examined the the
effect of
effect of different
different LEDs
LEDs on on porphyrin
porphyrin intermediates
intermediates of the the biosynthetic
biosynthetic pathway in rice seedlings.
Althoughnegligible
Although negligibleamount
amount of Mg-Proto
of Mg-Proto IX monomethyl
IX monomethyl ester ester (ME)great
(ME) and andaccumulation
great accumulation
of Pchlide of
Pchlide
were were observed
observed under dark under dark conditions,
conditions, Proto IX andProto IX and Mg-Proto
Mg-Proto IX were not IXdetected
were not(Figure
detected4). (Figure
Levels
4).Proto
of LevelsIXof Proto
and IX andIX
Mg-Proto Mg-Proto IX intreated
in seedlings seedlings
withtreated withwere
blue LED bluesimilar
LED were similar
to those to those
of white LED,of
white LED,
whereas levelswhereas
of Mg-ProtolevelsIX of
MEMg-Proto
and Pchlide IX were
ME decreased
and Pchlide wereand
by 49% decreased by 49% (Figure
44%, respectively and 44%, 4).
respectively
Both green and (Figure
red LED 4). treatments
Both greengreatly
and red LED treatments
decreased levels of greatly
Proto IXdecreased levels of Proto
and Mg-tetrapyrroles IX and
including
Mg-tetrapyrroles
Mg-Proto including
IX, Mg-Proto IX ME, Mg-Proto IX, Mg-Proto
and Pchlide, with lowerIXlevels
ME, inand Pchlide,
green LED. with lower
Our data levelsevidence
provide in green
LED.a Our
that narrowdataspectrum
provide evidence
of each LEDthattreatment
a narrow hasspectrum of each
a striking LED
effect on treatment
the porphyrinhas ametabolism
striking effectin
on the
rice porphyrin
seedlings. metabolism
Particularly, bluein riceappears
light seedlings.
to beParticularly,
essential forblue lightbiosynthesis
efficient appears to be essential
of Proto for
IX and
efficient biosynthesis
Mg-porphyrins. of Proto
Metabolic IX and
control in Mg-porphyrins. Metabolic control
the porphyrin biosynthetic pathway in the porphyrin
is critical biosynthetic
for the fitness of
pathway
plants is Therefore,
[20]. critical for plants
the fitness of plants
can trigger [20].photodynamic
severe Therefore, plants can trigger
damage severe photodynamic
if this control mechanism is
damage if by
perturbed thisaltered
control mechanism
growth is perturbed
conditions [21,22]. by altered growth conditions [21,22].

Figure
Figure 4. Effectsof
4. Effects ofdifferent
differentwavelengths
wavelengthsofofLEDs
LEDsononthethe distribution
distribution of porphyrin
of porphyrin intermediates.
intermediates. (A)
(A)
Proto IX; (B) Mg-Proto IX; (C) Mg-Proto IX ME; and (D) Pchlide. The plants were subjected to
Proto IX; (B) Mg-Proto IX; (C) Mg-Proto IX ME; and (D) Pchlide. The plants were subjected to the
the
same
same treatments
treatmentsas asin
in Figure
Figure 2,
2, and
and treatment
treatment notations
notations are
are the
the same
same as
as in
in Figure
Figure 2. N.D., not
2. N.D., not detected.
detected.
The
The data
data represent
represent the mean ±± SD
the mean SD (n
(n == 6–8). Means denoted
6–8). Means denoted by by the
the same
same letter
letter did
did not
not differ
differ
significantly at p < 0.05 according to Duncan’s multiple range
significantly at p < 0.05 according to Duncan’s multiple range test. test.

2.3.
2.3. Different
Different Wavelengths
Wavelengths of
of LEDs
LEDs Influence
Influence Porphyrin
PorphyrinBiosynthesis
Biosynthesisand
andRetrograde
RetrogradeSignaling
Signaling
Photo-excitation
Photo-excitationofofporphyrins
porphyrinswith with blue
bluelight, particularly
light, in the
particularly wavelength
in the wavelength range of 405–470
range nm,
of 405–470
results in the production of ROS, such as 1 O , H 1 O , and O2– [23].2–Therefore, we focused on the effects
nm, results in the production of ROS, such2 as 2O22, H2O2, and O [23]. Therefore, we focused on the
of blue and
effects red and
of blue light,red
both of which
light, both ofarewhich
sensedare by sensed
specificby
photoreceptors in plants [24,25].
specific photoreceptors Expression
in plants [24,25].
levels of biosynthetic genes in the porphyrin pathway were examined using qRT-PCR
Expression levels of biosynthetic genes in the porphyrin pathway were examined using qRT-PCR in in rice seedlings
grown under broad
rice seedlings grownspectrum-white
under broadLED as well as blueLED
spectrum-white and red
as LEDs.
well asDuring prolonged
blue and red and
red LEDs. blue
During
LED treatments, transcript levels of HEMA1 involving ALA-synthesizing activity
prolonged red and blue LED treatments, transcript levels of HEMA1 involving ALA-synthesizing and ALAD—which
encodes ALAALAD—which
activity and dehydratase—were encodeslower
ALA than those of white LED
dehydratase—were (Figure
lower than5A), implying
those of whitethat
LEDa specific
(Figure
wavelength
5A), implying of light
that affects the wavelength
a specific early step of ofporphyrin biosynthesis.
light affects the early step of porphyrin biosynthesis.
Transcript
Transcript levels Glutamate-1-Semialdehyde
levels ofofGlutamate-1-Semialdehyde Aminotransferase
Aminotransferase (GSA),
(GSA), whichwhich also involves
also involves ALA-
ALA-synthesizing activity, were similar among all three LEDs. Transcript
synthesizing activity, were similar among all three LEDs. Transcript levels of PPO1 encoding levels of PPO1 encodingthe
the protoporphyrinogen
protoporphyrinogen oxidase
oxidase (PPO),
(PPO), which which produces
produces ProtoProto
IX in IX
theinlast
thestep
lastbefore
step before the branch
the branch point,
were greatly upregulated in seedlings treated with red LED, compared to those of white and blue
Int. J. Mol. Sci. 2017, 18, 641 6 of 11
Int. J. Mol. Sci. 2017, 18, 641 6 of 11

point, However,
LEDs. were greatly
thisupregulated in of
upregulation seedlings
PPO1 intreated
red LED with
didred
notLED,
resultcompared to those ofincrease
in a corresponding white and
in
blue LEDs.
Proto IX. However, this upregulation of PPO1 in red LED did not result in a corresponding increase
in Proto IX.

Figure 5.
Figure Effects of
5. Effects of different
different wavelengths
wavelengths of
of LEDs
LEDs on
on the
the expression
expression of
of genes
genes encoding
encoding the
the porphyrin
porphyrin
pathway enzymes. (A) Common branch; (B) Fe-porphyrin branch; and (C) Mg-porphyrin
pathway enzymes. (A) Common branch; (B) Fe-porphyrin branch; and (C) Mg-porphyrin branch. branch.
The
The plants were subjected to the same treatments as in Figure 2. Treatment notations are the
plants were subjected to the same treatments as in Figure 2. Treatment notations are the same as same as in
in
Figure 2. Total RNAs were purified from plants and reverse transcribed. The resultant cDNAs were
Figure 2. Total RNAs were purified from plants and reverse transcribed. The resultant cDNAs were
used as templates for qRT-PCR using Actin as an internal control. The white LED (control) was used
used as templates for qRT-PCR using Actin as an internal control. The white LED (control) was used
for normalization, with the expression level of the sample set to 1. Error bars indicate SEM from three
for normalization, with the expression level of the sample set to 1. Error bars indicate SEM from three
independent experiments (n = 3); each experiment has three biological replicates. Means denoted by
independent experiments (n = 3); each experiment has three biological replicates. Means denoted by
the same letter did not differ significantly at p < 0.05 according to Duncan’s multiple range test.
the same letter did not differ significantly at p < 0.05 according to Duncan’s multiple range test.

As different
As different wavelengths
wavelengths of of LEDs
LEDs affected
affected the levels of Mg-porphyrin intermediates
intermediates and and
chlorophylls, we investigated whether these changes can be explained
chlorophylls, explained by aa regulatory
regulatory switch
switch inin Proto
Proto
IX distribution
IX distribution through
through regulating
regulating the
the genes
genes encoding
encoding enzymes
enzymes in in Fe-
Fe- and
and Mg-porphyrin
Mg-porphyrinbranches.
branches.
The transcript
transcript level
level of FC2—which encodes
of FC2—which encodes the
the plastidic
plastidic isoform
isoform of
ofFe-chelatase—in
Fe-chelatase—in seedlings
seedlings treated
treated
similar to that
with blue LED was similar that of
of white
white LED,
LED, but
but decreased
decreased inin red
red LED
LED (Figure
(Figure 5B).
5B). The
The transcript
transcript
levels of HO1 encoding heme oxygenase (HO)—which catalyzes the oxygen-dependent
levels of HO1 encoding heme oxygenase (HO)—which catalyzes the oxygen-dependent degradation degradation
of heme
of heme to biliverdin IXα [26,27]—were the same in all all LED
LED treatments. Interestingly, seedlings
treatments. Interestingly, seedlings
treated with
treated with blue
blue LED
LED exhibited
exhibited aa 2.6-fold
2.6-fold increase
increase in
in transcript
transcript level
level of HO2, compared
of HO2, compared to to seedlings
seedlings
treated with white
treated with white LED, implying
implying more conversion of heme to biliverdin IXα. The principal
The principal HO HO
reaction product,
reaction product, biliverdin-IXα,
biliverdin-IXα, prevents oxidative cell damage as part part ofof the
the antioxidant
antioxidant machinery
machinery
in animal
in animal cells
cells [28].
[28]. Its
Its role
role in
in protection
protection against
against oxidative
oxidative stress
stress in
in plant
plant systems
systems is also suggested
suggested
by other reports [29–31]. In addition, HO2 shows strong binding of the heme precursor Proto IX in
vitro, implicating a possible function of HO2 in the regulation of tetrapyrrole metabolism [32]. The
Int. J. Mol. Sci. 2017, 18, 641 7 of 11

Int. J. Mol. Sci. 2017, 18, 641 7 of 11

by other reports [29–31]. In addition, HO2 shows strong binding of the heme precursor Proto IX
in vitro,
high implicating
transcript level aofpossible
HO2 in function
seedlingsof HO2 in
treated theblue
with regulation
LED might of tetrapyrrole
contribute to metabolism
scavenging[32]. the
The high transcript level of
photosensitizing effect of Proto IX. HO2 in seedlings treated with blue LED might contribute to scavenging
the photosensitizing
Among the genes effect of Protothe
encoding IX.three Mg-chelatase subunits CHLD, CHLH, and CHLI in the
Among the genes encoding
Mg-porphyrin branch, transcript levels the three Mg-chelatase
of CHLH and CHLI subunits
decreasedCHLD, CHLH, and
in seedlings treatedCHLIwith inblue
the
Mg-porphyrin
or red LED compared branch, transcript
to white LED, of CHLHtranscript
levelswhereas and CHLIlevel decreased
of CHLD in seedlings
in blue LED treated with blue
exhibited a two-or
red LED compared to white LED, whereas transcript level of CHLD in
fold increase (Figure 5C). CHLH is thought to have an additional function in mediating plastid-to- blue LED exhibited a two-fold
increase
nucleus (Figure
retrograde 5C).signaling
CHLH is by thought to havethe
controlling an metabolism
additional function in mediating
of Mg-Proto IX, which plastid-to-nucleus
is an important
retrograde signaling by controlling the metabolism of Mg-Proto IX,
molecule for retrograde signaling [33,34]. Both blue and red LEDs resulted in lower transcript which is an important molecule
levels
for retrograde signaling [33,34]. Both blue and red LEDs resulted
of Protochlorophyllide Oxidoreductase (PORB), encoding the enzyme that generates chlorophyllide in lower transcript levels fromof
Protochlorophyllide
Pchlide, than whiteOxidoreductase (PORB), encoding
LED, while chlorophyll contents didthe enzyme that generates
not significantly differ inchlorophyllide
samples takenfrom from
Pchlide,
plants exposed to white and blue LEDs (Figure 3). This discrepancy is referred to asindifferential
than white LED, while chlorophyll contents did not significantly differ samples taken post-
from plants exposed
transcriptional controls to white
underand blue LEDs
different LEDs. (Figure 3). This
Notably, discrepancy
upregulations is referred
of HO2 and CHLDto as differential
under blue
post-transcriptional controls under different LEDs. Notably,
LED as well as PPO1 under red LED suggest their important roles as key regulatory genesupregulations of HO2 and CHLD under in
blue LED as well as PPO1 under red LED suggest their important
porphyrin metabolism under altered light environment. Our results demonstrate that light spectralroles as key regulatory genes in
porphyrin
quality, asmetabolism
indicated by under altered
different light environment.
wavelengths, controlsOur results
levels demonstrate
of porphyrin that light spectral
metabolites through
quality, as indicated by different wavelengths, controls
regulating expression of genes involved in porphyrin biosynthesis pathway. levels of porphyrin metabolites through
regulating expression
Expression of Lhcofgenes
genesdepends
involved onintheporphyrin biosynthesis
developmental pathway. state of chloroplasts [35].
and functional
Expression
Transcript levelsofofLhc genes
Lhcb, the depends
genes foron theof
Lhcs developmental
PSII, and the RbcS and functional
gene encoding state the
of chloroplasts
small subunit [35].
of
Transcript levels of Lhcb, the genes for Lhcs of PSII, and the
Rubisco were determined as markers of photosynthetic gene expression patterns in rice seedlings RbcS gene encoding the small
subunit
grown underof Rubisco were LEDs.
different determined as markerslevels
The expression of photosynthetic gene expression
of nuclear-encoded patternsgenes—
photosynthetic in rice
seedlings grown under different LEDs. The expression levels of nuclear-encoded
including Lhcb1, Lhcb6, and RbcS—decreased after exposure to blue and red LEDs, compared to those photosynthetic
genes—including
of white LED (Figure Lhcb1,6). Lhcb6, and RbcS—decreased
The greater declines in transcript after exposure to blue
levels of Lhcb andand RbcSred LEDs,
than compared
in chlorophyll
to those can
content of white LED (Figure
be explained 6). reduced
by the The greater declines in transcript
light-harvesting antenna sizelevelsof of Lhcbasand
PSII, RbcS than
indicated by thein
chlorophyll content can be explained by the reduced light-harvesting antenna
increased chlorophyll a/b ratio in blue and red LEDs (Figures 3 and 6). It might be expected that the size of PSII, as indicated
by the increased
regulatory patternschlorophyll
of CHLH, a/bCHLI,
ratio and
in bluePORBandin red LEDs (Figuresbranch
Mg-porphyrin 3 and 6). It might
as well be expected
as Lhcb and RbcS that
by
the regulatory
white patterns
light would of CHLH, CHLI,
correspond to theand PORB in effects
combined Mg-porphyrin
of red branch
and blue as well Lhcb andfrom
light.as Signals RbcS theby
white light would
chloroplast regulatecorrespond to the combined
the expression effects of red and blue
of the nuclear-encoded genes,light.
LhcbSignals fromHEMA1,
[34] and the chloroplast
which
regulate
encodes the expression
glutamyl tRNA of the nuclear-encoded
reductase, genes, Lhcbstep
the first committed [34] and HEMA1, which
of tetrapyrrole encodes glutamyl
biosynthesis in plants
tRNA reductase,
[2,36,37]. Particularthe first committed
porphyrin step of may
molecules tetrapyrrole
absorb biosynthesis in plants [2,36,37].
a specific wavelength of light, Particular
thereby
porphyrin
involving retrograde signaling which sends signals to the nucleus to control expression ofsignaling
molecules may absorb a specific wavelength of light, thereby involving retrograde nuclear-
which
encoded sends signals to the
photosynthetic nucleus
genes encodingto control expression of nuclear-encoded
the pigment-binding protein complexphotosynthetic
of the photosynthetic genes
encoding
machinery. the pigment-binding protein complex of the photosynthetic machinery.

Figure6.6.Effects
Figure of different
Effects wavelengths
of different of LEDs of
wavelengths on the
LEDs expression
on theofexpression
nuclear-encoded photosynthetic
of nuclear-encoded
genes encoding the chlorophyll-binding proteins. (A) Lhcb1; (B) Lhcb2; and
photosynthetic genes encoding the chlorophyll-binding proteins. (A) Lhcb1; (B) Lhcb2; and (C) RbcS. The plants
(C) were
RbcS.
subjected to the same treatments as in Figure 2. Treatment notations are the same
The plants were subjected to the same treatments as in Figure 2. Treatment notations are the same as as in Figure 2.
Total RNAs
in Figure were RNAs
2. Total purified from
were plantsfrom
purified and plants
reverseandtranscribed. The resultant
reverse transcribed. cDNAs were
The resultant cDNAs used as
were
templates for qRT-PCR using Actin as an internal control. The white LED (control)
used as templates for qRT-PCR using Actin as an internal control. The white LED (control) was used was used for
normalization, withwith
for normalization, the the
expression level
expression of the
level sample
of the sample setset
toto1. 1.Error
Errorbars
barsindicate
indicateSEM
SEMfromfromthree
three
independent
independent experiments (n = 3); each experiment has three biological replicates. Means denoted by
experiments (n = 3); each experiment has three biological replicates. Means denoted by
the
thesame
sameletter
letterdid
didnotnotdiffer significantlyatatpp<<0.05
differsignificantly 0.05according
accordingto toDuncan’s
Duncan’smultiple
multiplerange
rangetest.
test.
Int. J. Mol. Sci. 2017, 18, 641 8 of 11

3. Materials and Methods

3.1. Plant Growth and Light Treatment Conditions

Rice (Oryza sativa cv. Dongjin) seeds were germinated at 25 ◦ C under dark condition for five days
before irradiation. Then, the rice seedlings were grown under various types of light-emitting diodes
(LEDs; Union LED Electronic Co., Siheung-si, Korea) for 10 days. In each cycle, about 100 seedlings
were used for each LED treatment. Each LED treatment was conducted in separately controlled
chambers to be free from spectral interference among treatments. The LED array chambers were
programmed to provide a 14-h-light/10-h-dark photoperiod at photosynthetic photon flux maintained
of approximately 150 µmol·m−2 ·s−1 . The rice seedlings were grown under four different light
sources (Supplemental Table S1) with broad spectrum-white LED (420−680 nm) as a control, blue
LED (460−490 nm), green LED (520−550 nm), and red LED (620−650 nm), or under constant dark
conditions. For pigment and RNA samples, shoot parts of rice seedlings were sampled at 4 h after
illumination (11 AM) on the last day of LED treatments.

3.2. Determination of Morphological Characteristics

For measurement of shoot height, the length from seed (endosperm) to the top of the leaf in
rice seedlings was measured by a Vernier Caliper. The length and the width of the youngest, fully
developed leaf in each seedling were measured for the estimation of leaf area. To measure dry biomass,
the shoot part of rice seedlings were dried at 80 ◦ C for 48 h and weighed.

3.3. Extraction and Analysis of Porphyrin Intermediates and Photosynthetic Pigments

For measurement of porphyrin content, porphyrins were extracted and analyzed following
the method of Lermontova and Grimm [38] with some modifications. Leaf tissue (0.1 g) from rice
seedlings after 10 days of light exposures or darkness were macerated with liquid N2 and extracted
in 1.5 mL of extraction buffer (methanol:acetone: 0.1 N NaOH, 9:10:1, v/v/v) [31]. The homogenate
was centrifuged at 10,000× g for 10 min and the supernatant was taken for HPLC analysis. Porphyrin
intermediates were separated by HPLC (1525 Binary HPLC Pump, Waters, Milford, MA, USA) using
a Novapak C18 column (4 µm particle size, 4.6 mm × 250 mm, Waters). Porphyrin intermediates were
eluted with a solvent system of 0.1 M ammonium phosphate (pH 5.8) and methanol at a flow rate of
1 mL·min–1 . The elution profile of porphyrins from column was monitored with a fluorescence detector
(2474, Waters) at excitation and emission wavelengths of 400 and 630 nm for Proto IX, 440 and 630 nm
for Pchlide, and 415 and 595 nm for Mg-Proto IX and Mg-Proto IX ME, respectively. All porphyrins
were identified and quantified using authentic standards. The contents of total chlorophylls and
carotenoids were spectrophotometrically determined according to the method of Lichtenthaler [39].

3.4. RNA Extraction and qRT-PCR Analysis

Total RNA was isolated from leaf tissue (0.1 g) after 10 days of light exposure using TRIZOL
Reagent (Invitrogen, Carlsbad, CA, USA) in accordance with manufacturer’s instructions. The 5 µg
of purified total RNA from each sample was used for the first strand cDNA synthesis by the reverse
transcription reaction (SuperScript III First-Strand Synthesis System, Invitrogen) in the total volume of
20 µL following manufacturer’s instructions. Subsequently, 50 ng of cDNA and specific primers for
genes (Supplemental Table S2) were used for qRT-PCR analysis. The qRT-PCR analysis was performed
with the 7300 Real-Time PCR system (Applied Biosystems, Waltham, MA, USA) using Power SYBR
Green PCR Master Mix (Applied Biosystems). The thermal cycling of qRT-PCR program consisted
of an initial step at 50 ◦ C for 2 min, at 95 ◦ C for 10 min, and 40 cycles of 15 s at 95 ◦ C, and 1 min at
60 ◦ C [21]. A melting curve analysis was performed after every PCR reaction to confirm the accuracy
of each amplified product. All reactions were set up in triplicate. The sample of white LED treatment
was used as the calibrator, with the expression level of the sample set to 1. Actin was used as the
internal control.
Int. J. Mol. Sci. 2017, 18, 641 9 of 11

3.5. Data Analysis

Data were expressed as means ± SE. Differences were analyzed by the Duncan’s multiple range
test. p-values < 0.05 were considered to be significant. The analyses were performed using the SPSS
software (SPSS Inc., Chicago, IL, USA).

4. Conclusions
In this study, levels of porphyrin metabolites and photosynthetic pigments as well as growth
properties were greatly influenced by different wavelengths of LEDs. The greater leaf width and dry
biomass in rice seedlings grown under blue LED and broad spectrum-white LED may be related to the
fact that porphyrin compounds absorb blue wavelength intensely. Our data also show that different
wavelengths of LEDs have marked effects on the metabolic regulation of the porphyrin biosynthetic
pathway. In comparison to white LED, most porphyrin genes in the Fe-porphyrin branch remained
constant or upregulated under blue and red LEDs, while most genes in the Mg-porphyrin branch were
downregulated. Particularly, the accumulation of HO under blue LED may be due to a higher energy
level of blue light, however, the relevant mechanism is unknown. Expression levels of Lhcb and RbcS
noticeably decreased after exposure to blue and red LEDs, compared to white LED, implying that
particular wavelengths of light from different LEDs may possibly alter retrograde signaling which
sends chloroplast signals to the nucleus to control expression of nuclear-encoded photosynthetic genes.
Further studies are needed to elucidate the signaling pathway involved in the light
wavelength-dependent physiological modifications in plants. This may act through altering porphyrin
biosynthesis, although it remains necessary to explain how a specific LED wavelength leads to the
altered porphyrin biosynthesis.

Supplementary Materials: Supplementary materials can be found at www.mdpi.com/1422-0067/18/3/641/s1.

Acknowledgments: This research was supported by Basic Science Research Program through the National
Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2015R1D1A3A01020365).
Author Contributions: Sunyo Jung designed the experiment; Lien Hong Tran performed the experiments;
Lien Hong Tran and Sunyo Jung analyzed the results; Sunyo Jung wrote the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.

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