R E S E A R C H A R T I C L E Open Access
Full moonlight-induced circadian clock
entrainment in Coffea arabica
, D. Djerrab
, S. Leran
, L. Toniutti
, C. Guittin
, D. Severac
, M. Pratlong
, A. Dereeper
and B. Bertrand
Background: It is now well documented that moonlight affects the life cycle of invertebrates, birds, reptiles, and
mammals. The lunisolar tide is also well-known to alter plant growth and development. However, although plants
are known to be very photosensitive, few studies have been undertaken to explore the effect of moonlight on
Results: Here for the first time we report a massive transcriptional modification in Coffea arabica genes under full
moonlight conditions, particularly at full moon zenith and 3 h later. Among the 3387 deregulated genes found in
our study, the main core clock genes were affected.
Conclusions: Moonlight also negatively influenced many genes involved in photosynthesis, chlorophyll biosynthesis
and chloroplast machinery at the end of the night, suggesting that the full moon has a negative effect on primary
photosynthetic machinery at dawn. Moreover, full moonlight promotes the transcription of major rhythmic redox
genes and many heat shock proteins, suggesting that moonlight is perceived as stress. We confirmed this huge impact
of weak light (less than 6 lx) on the transcription of circadian clock genes in controlled conditions mimicking full
Beyond tales and legends, there is no longer a doubt that
solar radiation reflected by the moon can be perceived by
many organisms on Earth, and the informational role of
moonlight as an environmental cue is not questioned .
Moonlight and the lunar cycle can affect reproduction,
communication, foraging and predation in invertebrates,
birds, reptiles, and mammals [1,2].
Peter W. Barlow’s work clearly demonstrated the impact
of local gravimetric oscillations on plant growth and de-
velopment. These gravimetric variations, i.e. the lunisolar
gravity cycle or lunisolar tide, occur daily as result of the
impact of the sun and moon on the earth’s surface gravity.
Lunisolar tide influences plant phenomena such as leaf
movement, stem elongation, fluctuations in tree stem
diameter, root growth, biophoton emission by seedlings,
and chlorophyll fluorescence . Recently, Gallep and co-
workers demonstrated co-variation between ultra-weak
light emission, coffee seedling growth patterns and luni-
solar gravity cycles . These authors corroborated results
previously found in seedlings of other species . The
moon’s influence on plant growth and development is well
documented with regard to its action on local gravity, but
it could also have an effect through the sunlight it reflects.
Light is crucial for plant life, and perception of the
light environment dictates plant growth, morphology,
and developmental changes. Although plants are highly
photosensitive, very few studies have explored the effect
of moonlight on plant physiology, and most of the re-
sults have generally been conflicting. Between 1926 and
1935, Kolisko showed that the particular phase of the
moon at sowing time influences the period and percent-
age of germination as well as the subsequent plant
growth [5–7]. Charles Darwin studied the nyctinastic
movement of leaves during the night and concluded that
this phenomenon was caused by radiation from the sky
. Thanks to the work of Peter W. Barlow, we now
know that in most of these studies the influence of the
moon was due to its local effect on gravimetry, not to
moonlight. But the hypothesis of an influence of
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* Correspondence: firstname.lastname@example.org
CIRAD, UMR IPME, F-34398 Montpellier, France
UMR IPME, Univ. Montpellier, CIRAD, IRD, F-34394 Montpellier, France
Full list of author information is available at the end of the article
Breitler et al. BMC Plant Biology (2020) 20:24
moonlight on plants does not seem as foolish when we
consider that coral can perceive blue light from the
moon, which in turn induces gametogenesis and spawn-
ing . Bünning and Mose in 1969 hypothesized that a
light intensity as low as 0.1 lx (equivalent to the light
from a very small candle) can influence photoperiodism
in plants . They suggested that nyctinastic leaf fold-
ing in legumes could be a means of preventing moon-
light from activating the red form of the pigment
phytochrome in the upper leaf epidermis. Following this
pioneering study, several recent studies have highlighted
the effects that artificial light can have on plants at night.
Artificial lighting (also sometimes referred to as light
pollution) alters natural light regimes (spatially, tempor-
ally, and spectrally), when light is perceived as an infor-
mation source and not as a resource [11,12]. Kadman-
Zahavi and Peiper (1987) reported that, in their experi-
mental conditions, plants exposed to moonlight flowered
2–3 days late. They suggested that, while full moonlight
may be perceived in the photoperiodic reaction, in the
natural environment it would only have a very slight ef-
fect on the time of flower induction at the most .
These studies showed that plants can perceive even very
low moonlight but they provided no information about
how moonlight is perceived at the molecular level and
can affect plant physiology, particularly transcriptional
activation. But maybe the findings of these studies need
to be reinterpreted in the light of the recent work of P.
Plants use the circadian clock to synchronize their
physiology and development with daily and yearly
changes in the environment . The aim of the present
study was to investigate whether coffee photoreceptors
can perceive moonlight and deregulate circadian clock
mechanisms. One key aspect of clock-driven physio-
logical patterns in plants is that they match environmen-
tal patterns while relying on accurate prediction of day
and night lengths. Genes orthologous to circadian light
perception in Arabidopsis and genes involved in photo-
synthesis pathways and regulation are present in the cof-
fee genome. The expression pattern of core clock genes
in coffee trees is similar to that in Arabidopsis, suggest-
ing a high level of conservation. While studying the cir-
cadian cycle of young Arabica coffee seedlings in an
artificial environment (phytotron, 12/12 h photoperiod),
we decided to also check our results by analyzing older
plants in the greenhouse. We conducted sampling at
three hourly intervals at spring solstice (12 h day, 12 h
night). When we analyzed the key core clock gene LHY
using qRT-PCR, we observed a surprising phenomenon.
The gene expression profile showed a completely unex-
pected peak in the middle of the night. By chance, on
the night of our study, the moon was full “at the exquis-
ite hour when a vast and tender peacefulness seems to
descend from the firmament”(freely adapted from a
poem by Paul Verlaine entitled “The good song”, 1871).
To further investigate this discovery, we analyzed our
samples using RNAseq and confirmed our results at
spring solstice in plants grown under the same environ-
mental conditions, but also in plants grown in a
Particularities of moonlight
Different communities worldwide traditionally use lunar
rhythms as a tool to pinpoint the best germination and
harvest times. The moon can act in two ways on plants,
via its gravitational effect or via the sunlight it reflects.
The gravitational effect is now well known, but the effect
of full moon (FM) light is completely unknown. Com-
pared with sunlight, the wavelength of full moonlight is
generally centered around 400 nm (580 nm for the sun)
with a very low energy level (0.2 lx or 0.0024 μmol m
). The red:far red (R:FR) ratio of sunlight during the
day is more than 1.2, while that of moonlight is between
0.18 and 0.22 (Fig. 1).
Massive transcriptional up and down regulation induced
by full moonlight
Full moonlight was reported to induce transcriptional up
or down regulation of many coffee leaf genes compared
with new moon (NM) light (Fig. 2a). Under our experi-
mental conditions, we monitored transcript accumula-
tion by RNAseq every 3 h over a 24 h period in March
2016 (Additional file 1: Tables 0, 1, 2, 3, 4, 5, 6, 7, 8 and
9). Taking the whole transcriptome (25,574 genes) into
account, we observed only slight differences between the
FM and NM at ZT6, ZT9, ZT18, with only 0.3 to 1.2%
of genes being differentially regulated. We observed two
maxima at ZT15 (4.8%) that corresponded to the FM
zenith and 3 h later at ZT21, with more than 6.8% of the
genes being differentially regulated (Fig. 2b-c). Overall,
we found that 3387 genes were differentially regulated.
These results clearly demonstrate that moonlight was
perceived by the coffee leaves.
Effect of FM on photoreceptor transcription
Phytochromes (PHY), cryptochromes (CRY), ZEITLUPE
(ZTL) family proteins and phototropins (PHOT) are
known to be major red/far-red and blue light photore-
ceptors. It is likely that several of these photoreceptors
could be involved in moonlight perception, but most
them are unaffected at the transcription level. Only
phototropins were highly expressed at the FM zenith
(ZT15) (Fig. 3). We observed that PHOT1 gene expres-
sion was highly correlated with several genes involved in
chlorophyll biosynthesis. For example, the correlation
with the magnesium chelatase gene was r = 0.91 (Fig. 3).
Breitler et al. BMC Plant Biology (2020) 20:24 Page 2 of 11
Not surprisingly, the PHOT2 gene, which is known to
react to strong blue light, was less differentially
expressed than PHOT1 (log2FoldChange 0.69 and 1.40,
respectively). Zeaxanthin epoxidase (ZEP), beta-carotene
3-hydroxylase (CRTZ) and phytoene synthase (PSY1)gene
expressions were also highly correlated with PHOT1.We
observed higher gene expression at ZT15, indicating that
the carotenoid biosynthesis pathway was activated by full
Key core clock genes are affected by full moonlight
The accumulation of coffee putative clock gene transcripts
(LHY, Gigantea, Elf3, Elf4, Lux, PRR 5/7/9, PIF1, PIF4,
Constans-like 2/4/9/16) was affected by full moonlight at
ZT3, ZT12, ZT15, ZT18, ZT21, ZT24 (Additional file 1:
Table 2, 5, 6, 7, 8, 9). In a parallel study using Arabica
plantlets and RNA sequencing time-course data, we deter-
mined the cycling transcripts by running JTK_CYCLE for
two cycles (48 h). Out of the 25,574 genes of the whole
transcriptome, we found 4126 (16%) rhythmic genes at
their level of transcription, including 83% that were
similar to Arabidopsis rhythmic genes (Additional file 1:
Figure S10). Of the 3387 genes differentially expressed be-
tween FM and NM, 40% were rhythmic, which is a signifi-
cantly larger proportion than 18% of the total number of
genes (p< 0.0001), thus showing that the core clock alter-
ation caused by the FM influenced many genes, with most
of them being rhythmic genes.
We found that the accumulation of coffee putative clock
gene transcripts (LATE ELONGATED HYPOCOTYL
(LHY), TIMING OF CAB EXPRESSION 1 (TOC1),
GIGANTEA (GI), EARLY FLOWERING 3 and 4 (Elf3,
Elf4), LUX ARHYTHMO (LUX), PSEUDO-RESPONSE
REGULATOR (PRR 5, 7, and 9), PHYTOCHROME IN-
TEGRATING FACTOR (PIF1, PIF3, PIF4, PIF7),
CONSTANTS-like 2, 4, 9, and 16 (CO)) were affected by
full moonlight. Pairwise phase plots (Additional file 1:
Figure S11) showed similar relationships between FM and
Fig. 1 Spectrometer natural and simulated full moonlight and natural sunlight measurements
Breitler et al. BMC Plant Biology (2020) 20:24 Page 3 of 11
NM, but with unusual full-moon loops, thus illustrating
the influence of the FM while changing the relationships
between key circadian rhythm genes in a very punctual
but marked manner. Taken together, our data suggest
that core clock genes are altered in amplitude by the
FM (Fig. 2candAdditionalfile1:Tables0,1,2,3,4,
5, 6, 7, 8, 9 and 10 and Fig. S11). However, the FM
also changed the phase of several rhythmic genes
(Additional file 1: Figure S12) and led to phase delays
(at least 6 h in our study).
Full moonlight affects the expression of many regulator
More than 490 putative pentatricopeptides (PPR) have
been predicted in the coffee genome (http://coffee-gen-
ome.org/advanced). Here we showed (Fig. 2c) that 130
genes of this family were upregulated at ZT21 while only
four were down-regulated. Of the 130 up-regulated PPR
genes, 97 were rythmics and 127 were negatively corre-
lated with LHY gene expression (r ranging from 0.5 to
0.88, P< 0.01). At ZT15, 29 PPR genes were up-regulated
and 8 were down-regulated. We also observed high dis-
equilibrium in ribosomal activity at ZT21, where 69 ribo-
somal genes were up-regulated and only 4 were down-
regulated (data not shown).
Transcription of photosynthesis-related genes, heat shock
and lipid biosynthesis genes is drastically affected by full
Regarding photosynthesis-related genes, we observed
(Fig. 2c), that 50 genes of this pathway were strongly up
or down-regulated during the night. Light-harvesting a-b
binding proteins (CAB 1C-4/8/21/36) were highly up-
regulated at FM at ZT15 and ZT18. On the other hand,
a many photosynthesis-related genes were found to be
down-regulated before dawn at ZT21, but mostly at
ZT24 (Fig. 2c). Logically, several photosynthesis-related
genes were highly correlated with major redox genes
((Additional file 1: Table S13) for which they are the
main target of redox regulation. Indeed, we observed
(Fig. 2c) up-regulation of genes belonging to the heat
shock protein family HSFs). Thirteen genes were up-
Fig. 2 How the full moon clock and the new moon clock differ. aNormalization of the data, example for ZT 15: scatterplot of the log fold-
change for the FullMoon vs NewMoon comparison against the log-counts-per-million logs in reads for each gene. The log fold-change of the
data was centered on 0, showing that the libraries were correctly normalized. In the figure, differentially expressed genes are shown in red (p<
0.05) between the two conditions. bIn response to the FM, many coffee leaf genes were transcriptionally down or up regulated compared with
the the response to the NM at the different Zeitgeber times –(ZT0 = dawn, ZT12 = dusk), (color pink = down; color green = up). cExamples of
these responses (from inside to outside) include histone gene expression, heat shock genes, pentatricopeptide family genes, photosynthesis
related genes (photosystem, Calvin cycle, chlorophyll metabolism, carotenoid). Numbers associated with the up or down arrows indicate the
number of genes up or down-regulated, respectively, at each ZT. We provide both numbers for ZT24 and ZT0 (ZT24 in italics) (coffee plant photo
credit, Breitler jean-christophe)
Breitler et al. BMC Plant Biology (2020) 20:24 Page 4 of 11
regulated at ZT15, six at ZT18, eight at ZT21, and
seven at ZT24, while only one gene was down-
regulated at ZT24. The majority of these genes were
classified as rhythmic. Many genes of the lipid biosynthesis
pathway peaked differentially at ZT15 (Additional file 1:
Table S14), showing that the lipid biosynthesis pathway was
also altered by full moonlight.
Coffee trees perceive the moonlight that deregulates
their gene expression
At spring solstice in 2016, using RT-QPCR, we assessed
the expression of clock genes (LHY, GI, LUX ARRY
THMO, TOC1), chlorophyll biosynthesis genes (Proto-
chlorophyllide Oxidoreductases a (POR1A)), and starch
metabolism gene (Alpha-glucan water dikinase 1
(GWD1)), during the FM and NM. We repeated the ex-
periment during the FM in March 2017, with the same
plants in the same greenhouse. In this new experiment,
we also placed half of the plants in a phytotron (12/12 h
photoperiod) where the plants did not receive any light
at night throughout the month of March. We found no
difference in gene expression between plants exposed to
the NM in 2016 and 2017 and plants placed in the phy-
totron (Additional file 1: Figure S16). This lack of differ-
ence is illustrated in Fig. 4for LHY. In addition, these
curves can be compared to that showing the LHY ex-
pression pattern in Fig. 3b obtained with RNASeq data
during the NM.
When RT-QPCR was used to compare the expression
of LHY, GI, LUX ARRYTHMO, POR1A, POR1B,
GWD1 and ISA3 genes between the FM of 2016, 2017
and the NM of 2016 or 2017, we observed the same un-
expected peak when the plants were exposed to full
moonlight (Fig. 4, Additional file 1: Figure S17). The ex-
pression very clearly peaked in 2016, quite similar to the
peak observed by RNA-seq (Fig. 3) for all genes under
study. However, in 2017 the variations displayed a lower
amplitude and the atypical expression peak of LHY had
shifted to ZT18 and was of lower amplitude than in
2016. The difference between the 2 years was likely due
to the partial cloud cover that prevailed during the
nights preceding the FM in March 2017.
Artificial full moonlight deregulates gene expression
In order to confirm the huge impact of weak light on gene
transcription, we designed a combination of LEDs to re-
produce full moonlight in a growth chamber. We set up
four different types of LED lighting to reproduce the
bright spectrum of the FM as well as possible (Fig. 1). The
ratio between the blue light intensity and green light
Fig. 3 aRNAseq expression profile of PHOT1,zeaxanthin epoxidase,bLHY and Reveille 3. Both genes showed a transcription peak at ZT15, 3 h
after the moon zenith. cZeaxanthin epoxidase had an expression pattern similar to that of PHOT1,dwhile the pattern obtained for Reveille 3 was
similar to that of LHY. RNAseq data were standardized by DESeq2. Full moon (solid line); new moon (dotted); grey: subjective night
Breitler et al. BMC Plant Biology (2020) 20:24 Page 5 of 11
intensity at the FM zenith was about 1.30, which is
quite similar to the 1.41 ratio reproduced in our growth
chamber. We regulated the overall intensity at less than
6 lx (0.073 μmol m
ceived by the plant was less than 1 photosynthetically
active radiation unit (PAR). Technically, we were un-
able to increase the light intensity to mimic that emit-
ted at moonrise and at the moon zenith. We switched
on the light at full intensity at 10:00 pm. Despite the
difficulty in reproducing the light of the FM, after 7
days of treatment, plants exposed to this artificial
‘moonlight’showed atypical transcription at ZT21 of
LHY,PHOT1 and PHOT2 genes in the RT-qPCR ana-
lysis (Fig. 5). The night peak was produced at ZT21 in-
stead of ZT15 under natural FM conditions.
Plants are exposed to repeated changes in light quantity
and quality and they use a set of photoreceptors to
recognize the surrounding light environments [16,17].
Are these photoreceptors able to perceive full moon-
light? The full moonlight PAR is clearly inadequate for
photosynthetically supported growth, but from a qualita-
tive viewpoint moonlight mainly consists of blue and
far-red light, i.e. two wavelengths perceived by plants
and known to affect both their physiology and develop-
ment . On the other hand, full moonlight can be
perceived by plant photoreceptors as it mainly consists
of blue light with a very low R:FR ratio. We are almost
certain that this is only a moonlight effect, but we can-
not completely rule out gravity effects. Plants placed in
Fig. 4 Atypical accumulation of LHY transcripts under moonlight exposure. March 2016 NM (solid dark line), March 2016 FM (solid orange line),
March 2017 phytotron (dashed dark line) and March 2017 FM (dashed orange line)
Fig. 5 Spectrometer measurements on a NM day, a full sun day and in a growth chamber. We simulated the luminous intensity of the moon in a
growth chamber using four types of LED programmed at the main wavelengths 450 nm (blue), 660 nm (red), 730 nm (red) and in white light to
emit a light intensity of 6 lx (< 2 PAR). Light intensity spectra (cd) and histograms of the color quality scale (CQS) and light values measured by a
Rainbow-Light Portable Spectrometer MR-16 PPF under a full moon, a full sun day and in a growth chamber are shown. LHY and PHOT1 gene
expression were analyzed by RT-qPCR. Plants exposed to this artificial ‘moonlight’showed atypical transcription at ZT21 (dashed line)
Breitler et al. BMC Plant Biology (2020) 20:24 Page 6 of 11
the phytotron during the FM of 2017 showed the same
gene expression profiles as those obtained for the NM of
2017 and 2016. This control shows that it is indeed
moonlight that is perceived and not a variation of gravity
between the beginning and middle of the month. As the
qRT-PCR results were similar for the NM in 2016 and
in the culture chamber during the FM of 2017, we could
conclude that moonlight was responsible for the gene
expression modification, not gravitiational forces.
Phytochromes (PHY), cryptochromes (CRY), ZEI-
TLUPE (ZTL) family proteins and phototropins (PHOT)
are known to be major red/far-red and blue light photo-
receptors [19,20]. The PHOT protein acts as a blue light
photoreceptor . Zeaxanthin epoxidase (ZEP) is
known to respond to red light . It is likely that sev-
eral of these photoreceptors are involved in moonlight
perception. At the transcription level, most of them were
unaffected, except phototropins, which were highly
expressed at the FM zenith. Phototropins are blue-light
receptors controlling a range of responses that serve to
optimize the photosynthetic efficiency of plants. These
include phototropism, light-induced stomatal opening,
and chloroplast movements in response to changes in
light intensity . We observed that PHOT1 gene ex-
pression was highly correlated with several genes in-
volved in chlorophyll biosynthesis or within the
chloroplast, and also with some genes involved in the ca-
rotenoid biosynthesis pathway. Over-expression of ZEP,
which is known to respond to red light , CRTZ and
PSY1 indicated that the carotenoid biosynthesis pathway
was activated by full moonlight.
The circadian clock produces rhythmic variations in a
suite of biochemical and physiological processes that
help to optimize plant growth in daily cycles. Regular
environmental changes, especially the sunrise and sun-
set, coordinate these rhythmic behaviours. Photorecep-
tors and metabolites produced during photosynthesis,
operate to synchronize the internal timing clock with
lighting cues. In our study, we hypothesized that massive
transcriptional activation would be a good way to dem-
onstrate the effect of moonlight on plants. Moreover,
transcript abundance is useful to assess the effects of ex-
ternal clues on circadian oscillations. Light-regulated
changes in the morphology of a dicot or monocot seed-
ling are accompanied by an alteration in the expression
of up to 20% genes in Arabidopsis and rice . The cir-
cadian clock provides a mechanism for plants to antici-
pate events such as sunrise and to adjust their
transcriptional programs to coordinate environmental
signals and endogenous pathways. Clock activity can be
reset by environmental cues such as temperature, photo-
period and metabolic state . A change in ambient
light signals induces changes in a molecular pacemaker
called the circadian clock , which is a biological
network of interconnected feedback loops . Here we
demonstrated that weak full moonlight had a profound
impact on numerous genes, particularly at FM zenith
and 3 h later. The main core clock genes were deregu-
lated among the 3387 affected genes.
We observed atypical expression of the main core
clock genes during FM when the findings were corre-
lated with those of many other genes like REVEILLE3
(REV3). Several genes showed expression patterns iden-
tical to those of core clock genes. REV3 expression was
correlated with LHY (r= 0.98), suggesting that these two
genes were probably co-regulated (Fig. 2). REV3 plays a
photoperiod role in growth regulation . In fact, many
genes with patterns similar to LHY behave as if a day
phase takes place at night. Of the 3387 genes differen-
tially expressed between FM and NM, 40% were rhyth-
mic, indicating that the core clock alteration caused by
the FM exerted impacted a large number of genes, in-
cluding a majority of rhythmic genes.
Among the 3387 deregulated genes, we also observed
many genes involved in transcriptional and post-
transcriptional processes including ribosomal genes and
PRR proteins, respectively. PPR proteins are RNA bind-
ing proteins involved in post-transcriptional processes
(RNA processing and translation) in mitochondria and
chloroplasts, where they can affect gene expression in
various ways . Here we hypothesize that, once a
plant has perceived moonlight, ribosomal genes and PPR
proteins serve as regulatory factors and reprogram nu-
clear and organellar gene expression earlier.
Regarding photosynthesis-related genes, 50 genes of this
pathway were deregulated by moonlight. Many of these
genes were found to be down-regulated just before and at
dawn, suggesting that full moonlight has a negative effect
on the primary photosynthetic machinery at dawn.
We demonstrated that the weak intensity of the FM
was able to alter the transcription of many important
genes. However, it is still unclear how this transcription
alteration is translated phenotypically. Components of
the photosynthetic apparatus vary over the course of the
day to maximize energy absorption while limiting dam-
age caused by excessive light harvesting. Lai and co-
workers showed that the circadian clock coordinates
ROS homeostasis and the transcriptional response .
Here we found that several redox genes which regulate
the photosynthetic machinery were remarkably highly
correlated with LHY (Additional file 1: Table S13). The
modification of the transcription of major rhythmic
redox genes, many heat shock proteins and carotenoids
genes seemed to be prove that the moonlight was per-
ceived as stress by the plant. Activation of stress-
responsive pathways is energetically demanding, which
raises the question as to what is the plant protecting it-
Breitler et al. BMC Plant Biology (2020) 20:24 Page 7 of 11
Could moonlight be an environmental cue perceived by
the plant to channel some of its plant resources towards
reproduction or defense? These early results pave the
way for future studies on the impact of moonlight on
plant physiology. FM nights in natural conditions are
not easy to study because the sunrise and moonset times
vary and weather conditions are not always favorable.
Moreover the moon’s trajectory resembles a complex
ballet around the earth. Artificial culture conditions can
facilitate studies on the effect of moonlight on model
plants but also the effects of light pollution on plants.
We think that the start of the artificial lunar light was
too late in our experiment, which shifted the expression
of the genes concerned. However, in artificial conditions,
our results confirmed that very low luminous intensities
could be perceived by plants and that they had the cap-
acity to modify the transcription of one photoreceptor
and one core clock gene.
Plant material and growth conditions
The C. arabica var. Caturra seeds came from the La Cum-
plida Research Center (Matagalpa, Nicaragua). To deter-
mine the effects of moonlight, the plants were cultivated
in a glasshouse under natural daylight (65–75% humidity,
25 °C temperature, 12/12 h photoperiod) at IRD (Montpel-
lier, France) in 3 L pots containing a GO M2 (Jiffygroup)
potting soil mixture with watering as necessary. Leaf sam-
ples were collected from 1 year old plants for RNA extrac-
tion at Zeitgeber time (ZT) point ZT0 (sunrise), ZT3,
ZT6, ZT9, ZT12 (sunset), ZT15, ZT18, ZT21, and ZT24
in March 2016, and from the same plants in March 2017.
Sampling was done at the spring solstice FM and the fol-
lowing NM (4 biological replicates). During the FM, sam-
ples were also taken from control plants cultivated in a
phytotron under artificial light (CRYONEXT, model RTH
1200 L, with the following parameters: 12/12 h light/dark
photoperiod, 80% humidity, 25 °C temperature and 600
We performed an experiment using the same phyto-
tron and conditions to identify the set of genes with
rhythmic expression. We generated a 48 h transcrip-
tomic time-course dataset. Leaves were snap frozen in li-
quid nitrogen and stored at −80 °C until RNA analysis.
During sampling, three biological replicates were per-
formed using three plants for all RNAseq experiments
and four biological replicates with the plants exposed to
simulated moonlight. To classify the time points at
which the sampling was carried out, we used Zeitgeber
time (ZT), which is defined as the time in hours from
the start of a normal 12/12 h photoperiod cycle (photo-
period 12 h/12 h). For this purpose, we collected leaf
samples at 3 h resolution from ZT0 to ZT24.
Solar and lunar light was analyzed in 2016 and 2017 at
spring solstice using an MR-16v4 Rainbow-Light Port-
able Light Measuring Instrument. This spectrometer
uses micro-electromechanical systems (MEMS) and dy-
namic thermal equilibrium (DTE) technologies, with
high accuracy (spectral deviation in +/−0.1 nm, measur-
ing difference < 0.3%) and high stability (repeated meas-
urement error < 0.04).
Simulation of moonlight in a growth chamber using LEDs
In order to simulate the luminous intensity of the moon
in a growth chamber, we measured the real luminous in-
tensity emitted by the moon under a FM and NM. We
then programmed four types of LED in the growth cham-
ber to emit a light intensity of 6 lx at the main wave-
lengths: 450 nm (blue), 660 nm (red), 730 nm (red) and in
white light. We measured the light intensities to obtain
the real intensity value in the growth chamber. We used
three devices: a Rainbow-Light Portable Spectrometer
(version MR-16 PPF) to generate a light intensity
spectrum, a TopSafe light meter to obtain illuminance
(lx) and a photometric PAR probe to obtain the photo-
synthetic photon flux density (PPFD) expressed in
/s. No background noise was detectable with
the light meter or the photometric PAR probe, but the
spectrometer showed a background noise spectrum
(Fig. 1). The experiment was conducted in a growth
chamber at 25 °C and 60% humidity. We placed the
LEDs on a shelf and programmed them to emit a light
intensity of 350 PAR between ZT0 and ZT12, corre-
sponding to NM conditions. We programmed LEDs on
another shelf to emit a light intensity of 350 PAR be-
tween ZT0 and ZT12 and of 6 lx between ZT15 and
ZT20, corresponding to FM conditions. We exposed 10
Coffea arabica plants to NM conditions for 10 days to
acclimatize them to the growth chamber. We then ex-
posed 5 plants among the 10 acclimated plants in the
FM conditions for 7 days. At the end of the 7 day
period, the first sample was taken at ZT0, and then
every 3 h for 24 h. Samples (4 biological replicates) were
taken from 5 plants per condition. The samples were
taken from the 3rd and 4th leaves of the coffee plants.
Total RNA was extracted from leaves prefrozen in liquid
nitrogen that were subsequently ground and processed
as described previously . RNA quantification was
performed using a NanoDropTM 1000 Spectrophotom-
eter (Thermo Fisher Scientific, Waltham, MA, USA) and
the quality was assessed using the Agilent 2100 Bioana-
lyzer system with the RNA 6000 Nano™kit.
Breitler et al. BMC Plant Biology (2020) 20:24 Page 8 of 11
Real-time RT-qPCR assays
PCR experiments were performed as previously described
. Primers were designed using Primer3Plus web-based
plus/primer3plus.cgi). Based on published data, we targeted
three key genes of the circadian clock CcLHY (Cc02_
g39990), CcGIGANTEA (Cc10_g15270) and CcLUX-ARRY
THMO (Cc06_g20160). The specificity of the PCR products
generated for each primer set was checked by analyzing the
Tm (dissociation) of the amplified products. PCR efficiency
(E) was estimated using absolute fluorescence data captured
during the exponential phase of amplification of each reac-
tion with the eq. (1 + E)=10
(Ramakers et al. 2003)
(Additional file 1: Table S15). Expression levels were calcu-
lated by applying the formula (1 + E)
reference sample, with the T
sample used as reference for
each construct. Expression levels were normalized with the
expression of the CaGAPDH gene (GB accession number
GW445811 using primer pair GAPDH-F/R) serving as en-
dogenous control .
RNA sequencing and bioinformatics analysis
RNA sequencing (RNAseq) was carried out by the MGX
platform (Montpellier GenomiX, Institut de Génomique
Fonctionnelle, Montpellier, France; www.mgx.cnrs.fr/).
RNAseq libraries were constructed with the TruSeq
Stranded mRNA Sample Preparation kit from Illumina.
One microgram of total RNA was used for the library
construction. SuperScript IV reverse transcriptase and
random primers were used to produce first strand cDNA
from cleaved RNA fragments. This was followed by
second-strand cDNA synthesis. The cDNA fragments
were repaired, before the addition of a single ‘A’base
and the subsequent ligature of the adapter. The final
cDNA libraries were validated with a Bioanalyzer kit
(Standard Sensitivity NGS) and quantified by qPCR
(ROCHE Light Cycler 480). Libraries were pooled in
equal proportions, before denaturation with NaOH and
dilution to 17 pM, and before clustering on two lanes in
a flow cell. Clustering and 100 nt single read sequencing
were performed with a Hiseq 2500 according to the
manufacturer’s instructions. Image analysis and base
calling were performed using HiSeq Control Software
(HCS) and the Real-Time Analysis component (Illu-
mina). The data quality was assessed using FastQC from
the Babraham Institute (http://www.bioinformatics.bab-
raham.ac.uk/projects/fastqc/) and Illumina Sequence
Analysis Viewer (SAV) software. We obtained an average
of 21 million single end reads per sample.
Differential expression analysis
Before differential expression (DE) analysis, genes whose
sum of counts (by summing the counts per repetition
(3)) was below 45 were discarded. Reads were then stan-
dardized across libraries using the normalization proced-
ure in DESeq2 . FM/NM comparisons were
performed at ZT0, ZT3, ZT6, ZT9, ZT12, ZT15, ZT18,
ZT21 and ZT24. Differential expression was considered
statistically significant at p< 0.05. All genes of interest
were analyzed and compared using the TopHat2 2.1.1
(with Bowtie 2.2.9) algorithm against the Coffea cane-
phora genome (Coffee Genome Hub) (splice junction
mapping) and BWA-backtrack 0.7.15 algorithm against
the Coffea arabica transcriptome  (mapping and
Differential expression (DE) analysis was performed using
R 3.4.2 software and the DESeq2 1.18.1 package. Rhythmic
gene expression, period and phase parameters were mea-
sured using JTK_CYCLE implemented in MetaCycle
v1.1.0 .. To identify the rhythmic transcripts, we ana-
lyzed the DESeq2 normalized data. JTK_CYCLE uses a
non-parametric test to detect cycling transcripts 
considered transcripts with Benjamini-Hochberg q values
(BH.Q) < 0.05 as rhythmic transcripts. JTK-CYCLE was
run with a 21–27 h range of periods. A χ
test (P < 0.05)
was used to determine if the rhythmic genes in the differ-
ential expressed gene set were present in greater numbers
than expected by chance. Graphs were plotted using Excel,
or R. The R codes are available from the corresponding
Supplementary information accompanies this paper at https://doi.org/10.
Additional file 1: Table S0. REPLICATES. Table S1. Genes differentially
expressed between Full moon and new Moon at ZT0. Table S2. Genes
differentially expressed between Full moon and new Moon at ZT3. Table S3.
Genes differentially expressed between Full moon and new Moon at ZT6.
Table S4. Genes differentially expressed between Full moon and new Moon
at ZT9. Table S5. Genes differentially expressed between Full moon and new
Moon at ZT12. Table S6. Genes differentially expressed between Full moon
and new Moon at ZT15. Table S7. Genes differentially expressed between
Full moon and new Moon at ZT18. Table S8. Genes differentially expressed
between Full moon and new Moon at ZT21. Table S9. Genes differentially
expressed between Full moon and new Moon at ZT24. Figure S10.
Rhythmic genes in common between Coffee and Arabidopsis thaliana.
Figure S11. Pairwise phase plots of the core clock genes. Figure S12.
Number of genes per phase under new moon and the full moon. Table
S13. Correlation matrix between Redox homeostasis genes and LHY. Table
S14. Correlation matrix between genes involved in lipids pathways and LHY.
Table S14. Primers used for genes studied by qRT-PCR. Figure S16. RT-QPCR
of LHY gene during new moon. Figure S17. RT-QPCR time-course of core
clock genes, POR1A and GWD1.
DTE: Dynamic thermal equilibrium; FM: Full moon; HSP: Heat shock protein;
NM: New moon; PAR: Photosynthetically active radiation unit;
PPFD: Photosynthetic photon flux density; PPR: Putative pentatricopeptides;
ZT: Zeitgeber time
Breitler et al. BMC Plant Biology (2020) 20:24 Page 9 of 11
We thank the MGX platform (CNRS, Montpellier, France; https://www.mgx.
cnrs.fr) for conducting the RNAseq analyses. We are also highly grateful to
Jean-Louis Cuquemelle (Alpheus SARL, Montgeron, France, https://www.al
pheus-led.com/) for setting up the LED lighting and computer programs to
reproduce full moon conditions. We also thank David Manley for English
revision of the manuscript.
J-CB, HE and BB designed the study and drew up the experimental design. J-CB,
SL, DD, HE, LT, CG, AD, DS and BB implemented the research experiments. J-CB,
DD, BB and MP analyzed the data. J-CB and BB wrote the paper. All authors read
and approved the final manuscript.
RNAseq and artificial moon light development were funded by the European
Union’s Horizon2020 research and innovation programme under grant
agreement n. 727934 (BREEDCAFS project, http://www.breedcafs.eu). There is
no role of the funding body in the design of the study and collection,
analysis, and interpretation of data and in writing the manuscript.
Availability of data and materials
All data generated or analysed during this study are included in this
published article and its additional files.
Ethics approval and consent to participate
The research project and this study have been approved by an ethics
committee which found them conform to all national and international
guidelines for conservation of endangered species. The plant material
(variety Caturra) used is a common variety which is not endangered.
Consent for publication
The authors declare that they have no competing interests.
CIRAD, UMR IPME, F-34398 Montpellier, France.
UMR IPME, Univ.
Montpellier, CIRAD, IRD, F-34394 Montpellier, France.
BioMimic, 34394 Xalapa Enríquez, Ver, Mexico.
CNRS, Montpellier GenomiX,
c/o Institut de Génomique Fonctionnelle, 141 rue de la Cardonille, Cedex 34
Received: 5 June 2019 Accepted: 3 January 2020
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