1. Introduction
Light is among the most important environmental factors modifying plant
growth and development. Higher plants have evolved sophisticated
mechanisms to perceive light signals through specialized photoreceptors
that respond to various light properties, such as light intensity, light
spectral quality, and photoperiod (Briggs & Olney, 2001; Zoratti et
al., 2014a). Different visible light photoreceptors and UV-B receptors
can sense light signals from a broad range of solar spectrum between 280
nm to 750 nm (Möglich et al., 2010). Most of the transducible
wavelengths absorbed by plants fall within 400-700 nm (from blue to
red), which is also commonly referred to as photosynthetically active
radiation (PAR; Chen et al., 2004). Within this range, phytochrome B has
a specialized function towards red light while cryptochrome sense blue
light to promote photomorphogenesis (Li & Yang 2007; Lu et al., 2015).
After light perception, phytochrome and cryptochrome interact with the
E3 ubiquitin ligase constitutive photomorphogenesis protein 1 (COP1),
which is the key light signaling regulator (Lau et al., 2012). In the
dark, COP1 directly interacts and represses the action of elongated
hypocotyl (HY5) gene inhibiting light signal transmittance and circadian
clock genes, also the flowering regulators, such as CONSTANS (CO) via
the proteasomal degradation complex (Zoratti et al., 2014a; Bhatnagar et
al., 2020). Under light, COP1 activity is repressed allowing the
expression of HY5 and positive transcriptional regulators in a
number of developmental processes and metabolic pathways, including
anthocyanin biosynthesis (Wu et al., 2019).
Light quality has a significant influence on plant secondary metabolism
(Ouzounis et al., 2015). For example, biosynthesis of polyphenols,
anthocyanins, glucosinolates, terpenes, and carotenoids in plant tissues
are responsive to light quality and they have important roles for
example in photoprotection (Ballaré, 2014; Holopainen et al., 2018).
Light quality also influences the metabolite accumulation in fruits and
berries, as shown by several pre- and postharvest light treatments in
different fruit crops (Koyama et al., 2012; Tao et al., 2018; Kokalj et
al., 2019). High light intensity has generally been reported to increase
anthocyanin accumulation in fruits, but it is also affected by light
quality (Jaakola et al., 2013; Ma et al., 2019). For instance, red light
has been reported to increase anthocyanin content in strawberry
(Fragaria x ananassa) , and blue light radiation increased
anthocyanin levels after selective bagging treatment in pear
(Pyrus communis L.) fruit (Miao et al., 2016; Tao et al., 2018).
In bilberry, Zoratti et al., (2014b) showed that short term treatment
with supplemental monochromatic light affected the anthocyanin profile
during bilberry fruit development. However, the regulatory mechanisms
behind the effect of red and blue light wavelengths on anthocyanin
biosynthesis are not well understood.
Anthocyanins are prominent phenolic compounds in plants that are
biosynthesized from the well-studied flavonoid pathway, which branches
from phenylpropanoid biosynthesis (Tohge et al., 2017). The major early
biosynthetic enzymes involved in flavonoid biosynthesis are
phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), and chalcone
isomerase (CHI). At the branchpoint of flavonoid biosynthesis, flavonoid
3’ hydroxylase (F3ʹH) and flavonoid 3ʹ5ʹ hydroxylase (F3ʹ5ʹH) direct
biosynthesis to cyanidin and delphinidin compounds, respectively
(Jaakola et al., 2002). The six major anthocyanin aglycone end-products,
namely cyanidin, delphinidin, pelargonidin, petunidin, malvidin, and
peonidin, are biosynthesized by the late biosynthetic enzymes
dihydroflavonol 4-reductase (DFR) and anthocyanin synthase (ANS) and
further glycosylated by UDP-glucose:
flavonoid-O -glycosyltransferase (UFGT) as the last step in
anthocyanin biosynthesis (Wu et al., 2017). Anthocyanins are transported
to the vacuole after their biosynthesis. The mechanisms of anthocyanin
transport are not fully understood but common transporter proteins, such
as ATP-binding cassettes (ABCs), multidrug and toxic extrusion (MATEs)
and glutathione S-transferases (GSTs), are commonly believed to be
responsible for transportation to vacuolar membrane and lumen (Behrens
et al., 2019). Another proposed model has been vesicular trafficking by
phagosomes involving engulfment of anthocyanin bodies by endosomes
before reaching the vacuole (Chanoca et al., 2015). The vesicular
transportation is mediated by SNARE (solubleN -ethylmaleimide-sensitive fusion protein attachment protein
receptors) protein complexes, which are proposed to have a role in
cellular transport in higher plants under stress responses (Pečenková et
al., 2017).
The biosynthesis of flavonoids is directly controlled by the
transcriptional regulatory MBW complex, consisting of MYB
(myeloblastosis), bHLH (basic helix-loop-helix) transcription factors
and WD-40 repeat proteins (Feller et al., 2011; Xu et al., 2015). R2R3
MYB transcription factors (TFs) are known as the key regulators of
anthocyanin biosynthesis and are responsive to shifts in light spectral
quality (Zoratti et al., 2014a). In grapes, two R2R3 MYB TFs,VvMYBA1 and VvMYBA2, controlling anthocyanin biosynthesis
specifically regulate UFGT (Walker et al., 2007). In apple and
peach, R2R3 MYBA-type TFs activate anthocyanin biosynthesis by
interacting with both the UFGT and DFR promoters during
fruit ripening (Takos et al., 2006; Ravaglia et al., 2013).
In contrast to climacteric fruits, the ripening process of
non-climacteric fruits is independent of respiratory burst and ethylene
accumulation (Cherian et al., 2014). Instead, abscisic acid (ABA), which
is synthesized by the key cleavage enzyme 9-cis-epoxycarotenoid
dioxygenase (NCED) in apocarotenoid pathway, has been shown to be a
major regulator of ripening in non-climacteric fruits, such as
strawberry, grapes and bilberry (Jia et al., 2011; Ferrera et al., 2015;
Karppinen et al., 2018). The ABA signal transduction is known to be
mediated by pyrabactin resistance/like receptors (PYR/PYL) and ABA
responsive element binding factors (ABFs) through SQUAMOSA-MADS box
(TDR-type) transcription factors leading to regulation of the MBW
complex proteins (Chung et al., 2019). Another model has been proposed
illustrating that ABA interacts directly with PYR by inhibiting type2C
protein phosphatases subsequently binding with ABFs and transduces the
ABA signaling pathway (Park et al., 2009).
Bilberry (Vaccinium myrtillus L.), also known as European
blueberry, is one of the most important wild perennial berry species ofVaccinium genus and predominantly found in Northern Europe (Chu
et al., 2011; Karppinen et al. 2016a; Zoratti, et al., 2016). The
species has gained global interest due to its abundant health-beneficial
bioactive compounds, including phenolic compounds, carotenoids, vitamins
but especially anthocyanins, which constitutes 90% of total phenolics
in these berries and give distinct deep blue color to both skin and
flesh (Karppinen et al., 2016a). Several studies have reported
consumption of bilberries to reduce risk of metabolic syndrome and
various microbial and degenerative diseases (Chu et al., 2011; Nile &
Park, 2014; Bujor et al., 2016). In bilberry, delphinidin and cyanidin
glycosides are the major anthocyanins followed by malvidin and petunidin
glycosides (Müller et al., 2012; Zoratti et al., 2016; Thornthwaite et
al., 2020). In particular, delphinidins, which are abundant in northern
clones alongside malvidins, have been recently linked to many biological
and health-beneficial activities (Nakaoga et al., 2019; Heysieattalab &
Sadeghi, 2020).
In the present study, we utilized the Illumina-based RNA-seq approach to
produce transcriptome libraries from ripening bilberry fruit grown under
supplemental red and blue light conditions. We specifically focused on
the differences in red and blue light signal transduction in regulation
of anthocyanin biosynthesis. The red and blue light emitting diodes
(LEDs) used in our study give an opportunity to provide high intensity
spectral wavelengths to plants as source of light for studying the
effect of light quality to biosynthesis of phytochemicals.