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.