Differences in carotenoid biosynthesis and ABA metabolism suggest red light regulation through ABA
A significant increase in the expression of the carotenoid early biosynthetic genes and late cleavage genes has earlier been found in bilberry fruit ripening under red light (Karppinen et al., 2016b). Our results are in agreement with this study but also showed that not only the red light but also blue light treatment up-regulated expression levels of many of the carotenoid biosynthetic genes. However, the expression of ZDS andCYP450 -BCH genes branching towards α-carotene and further to lutein biosynthesis (LUT1 ) varied between the red and blue light treatments (Figure 6). Earlier it has been shown that the up-regulated expression levels of carotenoid biosynthetic genes in red light conditions led to low levels of carotenoids indicating elevated cleavage reactions (Karppinen et al., 2016b).
The cleavage of carotenoids leads to the production of plant hormone ABA through action of the key enzyme NCED (Karppinen et al., 2013). Many studies have demonstrated the exogenous application of ABA increasing the anthocyanin levels of non-climacteric fruits when applied at the time of fruit ripening (Wheeler et al., 2009; Ferrero et al., 2018; Karppinen et al., 2018). In strawberry, the effect of endogenous ABA levels in response to light-regulated anthocyanin biosynthesis has been reported through the activation of FvMYB10 , the key gene in strawberry anthocyanin biosynthesis (Kadomura-Ishikawa et al., 2015). In our study, the red light treatment induced higher expression ofNCED compared to control and blue light treatment. Also, we showed that expression levels of ABA-8’ hydroxylase gene, responsible for degradation of ABA, increased 2-fold higher in red light treatment and was down-regulated in blue light treatment. It has been shown that the endogenous levels of ABA in plant cells are maintained only by the inhibition of this enzyme (Kondo et al., 2012). A similar trend of increase in expression of NCED and ABA-8’ hydroxylase genes alongside higher anthocyanin accumulation in response to red light irradiation was observed earlier in grapevine (Kondo et al., 2014). Rodyoung et al. (2016) have shown that in grapes, the expression of both of these genes was higher at veraison in response to red and blue light irradiation. In many fruits, the ABA catabolism might not directly coincide with anthocyanin biosynthesis but the upstream ABA biosynthesis and endogenous levels of ABA may be directly involved in interaction with the flavonoid pathway. Here we show that in ripening bilberry fruit the red light activates both the biosynthetic and catabolic ABA pathways and correlates with anthocyanin accumulation. Furthermore, the changes in delphinidin levels showing a strong correlation with anthocyanin biosynthesis and direct regulatory genes, ABA signaling and metabolism genes (Figure 9) suggests a well-orchestrated regulatory network of ABA-regulated anthocyanin biosynthesis occurring under supplemental red light.
The signal transduction from ABA to the regulation of anthocyanin biosynthesis has been reported earlier in other fruit species, such asLycium fruits, where ABA was found to interact directly with the MBW complex and other key flavonoid genes (Li et al., 2019). In our study, we showed that light treatments during ripening process activated signal transduction cascade via ABA signaling lead to anthocyanin accumulation. It has been shown in one of the recent studies that red light increased the expression level of AGAMOUS -like (AGL) regulators in tomato (Solanum lycopersicum L.) during fruit ripening (Zhang et al., 2020). These AGL-like MADS box TFs are also orthologs to VmTDR4 in bilberry, a key player in bilberry anthocyanin accumulation (Jaakola et al., 2010). This provides us with an understanding that red light might mediate ABA-regulated anthocyanin biosynthesis through SQUAMOSA-MADS box type TF’s, abscisic acid binding receptors such as pyrabactin resistance like (PYR /PYL ), ABA insensitive (ABI5 ) gene and MBW complex TFs (Chung et al., 2019). It should also be noted that the SPL type of TFs (SQUAMOSA promoter binding like) that were up-regulated in response to blue light can be associated with the increase in expression of FLOWERING LOCUS-T (FT) gene involved in flowering and circadian clock related mechanisms as the TF acts downstream to FT expression (Wang et al., 2009).