Introduction
Whether epidermal anthocyanins offer effective protection to plants facing excessive light is still a debated issue (for review articles, see Hughes, 2011; Manetas, 2006; Landi, Tattini, & Gould, 2015; Steyn, Wand, Holcroft, & Jacobs, 2002). There are several reasons responsible for this ‘apparently irrelevant’ discussion, since anthocyanins effectively absorb photons over a wide portion of the solar spectrum (Gould, Jay-Allemand, Logan, Baissac, & Bidel, 2018; Tattini et al., 2017). First, juvenile red leaves or leaves that became transiently red during the winter season (so-called ‘winter reddening’) have been compared to mature green leaves in many instances (Hughes, Neufeld, & Burkey, 2005; Kytridis, Karageorgou, Levizou, & Manetas, 2008; Ranjan, Singh, Singh, Pathre, & Shirke, 2014; Zeliou, Manetas, & Petropoulou, 2009; Zhang et al., 2018a). We note that many traits other than the biosynthesis of anthocyanins may largely vary because of leaf age or season, between cyanic and acyanic leaves (Rasulov, Bichele, Laisk, & Niinemets, 2014; Tattini et al., 2014). Second, light irradiance at which plants have been grown strikingly differs among studies. In some instances, plants acclimated to relatively low irradiance (greenhouse/growth chamber studies) have been suddenly and transiently exposed to excessive light (Gould et al., 2018; Landi, Guidi, Pardossi, Tattini, & Gould, 2014; Logan, Stafstrom, Walsh, Reblin, & Gould, 2015). Other studies have instead compared cyanic and acyanic individual exposed for long periods to high light irradiance in the field (Liakopoulos et al., 2006; Tattini et al., 2017; Zhang, Zhong, Wang, Sui, & Xu, 2016). Third, there is the general, inexact belief that anthocyanins are effective in absorbing photons over the green region (over the 500-550 nm waveband), but quite ineffective in absorbing photons over other portions of the visible solar spectrum (Hughes, 2011; Kyparissis, Grammatikopoulos, & Manetas, 2007; Steyn et al., 2002). It has been argued, therefore, that anthocyanins may play only a marginal role in photoprotection (Liakopoulos et al., 2006; Neill & Gould, 1999; Nikoforou, Nikopoulos, & Manetas, 2011), given that chlorophylls mostly absorb over the blue (400-500 nm) and the red (600-700 nm), but poorly over the green portion of the solar spectrum. Nonetheless, light-induced depression in both maximal (Fv/Fm) and operational (ΦPSII) photosystem II (PSII) quantum yield is lower in cyanic compared to acyanic leaves over a broad range of species (Gould et al., 2018; Hughes & Smith, 2007; Landi et al., 2014; Tattini et al., 2017). There is also evidence that photoinhibition (Long, Humphries, & Falkowski, 1994), estimated from morning-to-midday depression in photosynthesis is lower in ‘constitutively’ cyanic leaves (leaves that remain red throughout their entire life cycle) compared to the green counterparts (Tattini et al., 2014; 2017). This is consistent with the observation that, while having molar extinction coefficient (Ɛ) maxima in the 510-540 nm waveband, anthocyanins also substantially absorb blue photons (and red photons to a lesser degree, Fig. 1), depending on decoration and tissue molar concentration (Jordheim et al., 2016; Merzlyak, Chivkunova, Solovchenko, & Naqvi, 2008; Tattini et al., 2014; Gould et al., 2018).
It is a matter of fact, that the capacity of anthocyanins to absorb maximally over the green portion of the solar spectrum does not fit to the long-reported ‘shade syndrome’ displayed by cyanic leaves (Manetas, Petropoulou, Psara, & Drinia, 2003; Tattini et al., 2014, see end of section for details). High green light availability actually induces shade avoidance responses in leaves and individuals (Dhingra, Dies, Lehner, & Folta, 2006; Wang & Folta, 2013; Smith, McAuslan, & Murchie, 2017), as is the case of leaves growing in the understorey (true shade leaves), which perceive light strongly enriched in green and far-red (FR) wavelengths. Green light stimulates early stem elongation indeed, and opposes responses to blue- and red light-activated signaling pathways (e.g. blue/red light-induced stomata opening, Folta & Mahrunic, 2007). Transcripts encoding proteins of PSI, PSII and the stroma (psaA, psbD, and rbcL), which are long known to accumulate in response to high light, are largely downregulated upon a pulse of green light (Wang & Folta, 2013). We also argue that the shade nature of cyanic leaves is unlikely the result of the UV-screening ability of anthocyanins, which may be substantial for anthocyanins acylated with hydroxycinnamic acid derivatives (Jordheim et al., 2016; Tattini et al., 2014). Effective UV-absorbing compounds, such as the colorless flavonols, accumulate more in high light-exposed green leaves compared to the corresponding red counterparts (Tattini et al., 2014; 2017), consistent with the strong competition between flavonol and anthocyanin biosynthetic pathways (Yuan, Rebocho, Sagawa, Stanley, & Bradshaw, 2016). The lower UV-absorbing potential of red compared to green leaves should oppose indeed the shade avoidance response (Hayes, Velanis, Jenkins, & Franklin, 2014; Mazza & Ballarè, 2015). On the other hand, the ability of anthocyanins in absorbing over the red waveband, thus reducing the red (R) to far-red (FR) ratio (R/FR), as occurs when leaves grow under a dense canopy (Franklin, 2008), may be responsible for the shade syndrome displayed by cyanic leaves. The absorption spectra of anthocyanins, especially when conjugated with ‘colorless’ flavonoids (so-called co-pigmentation, Trouilas et al., 2016) may have an appreciable tail over the 600-630 nm waveband (Gould et al., 2018; Jordheim et al., 2016; Fig. 1). Since the epidermal concentration of colorless flavonoids is high (in the low mM range, Agati & Tattini, 2010) in both green and red leaves growing in sunlight (Tattini et al., 2014, 2017), co-pigmentation is strongly favored. However, the extent to which anthocyanin-induced decline in R/FR may contribute to the shade avoidance response in cyanic leaves needs deeper investigation: true shade leaves may experience R/FR ratios even an order of magnitude lower than that perceived by sun-exposed leaves (Fankhauser & Batschauer, 2016).
The functional significance of blue-light absorption by epidermal anthocyanins has been early-emphasized (Chalker-Scott, 1999; Drumm-Herrell & Mohr, 1985), but largely ignored thereafter. Nonetheless, red stems of Cornus stolonifera transmitted just 25% of blue light compared to green stems (Cooney, Schafer, Logan, Cox, & Gould, 2015; Gould, Dudle, & Neufeld, 2010), and blue light absorption by the epidermal peel of activation-tagged pap1-D(production of anthocyanin pigment 1- Dominant ) mutant ofArabidopsis was as much as 70% of the absorbance over the green-yellow waveband (Gould et al., 2018). The decline in blue photons reaching the photosynthetic apparatus limits the efficient use of incident radiant energy for photosynthesis and imposes to cyanic leaves a profound adjustment in the light harvesting system (Horton, 2012; Ruban, 2018). Consistently, cyanic leaves have much greater concentration of chlorophylls (Chl), a significantly lower Chlato Chlb ratio (Chla /Chlb ) and, usually display lower photosynthetic rates than the green counterparts (Gould, Vogelmann, Han, & Clearwater, 2002; Menzies et al., 2015; Zhang et al., 2018a), unless when long exposed to high solar irradiance (Liakopoulos et al., 2006; Tattini et al., 2014; 2017). These observations well explain the shade syndrome displayed by cyanic leaves, even when growing in full sunlight (Manetas et al., 2003; Hughes et al., 2005; Tattini et al., 2017; Zeliou et al., 2009). In fact, red leaves are thinner, with less compact mesophyll tissues and lower proportion of palisade to spongy parenchyma with respect to green leaves (Boardmann, 1977; Franklin, 2008; Manetas et al., 2003; Kyparissis et al., 2007; Tattini et al., 2014). The shade nature of cyanic leaves is also manifested through a lower concentration of de-epoxided xanthophylls, and consequently, of a lower potential (or of a lower need, Tattini et al., 2017) to dissipate excess energy via nonphotochemical quenching (NPQ) compared to green leaves, irrespective of light availability (Landi et al., 2015; Tattini et al., 2014).
Here we discuss about the suite of molecular events, which operate at very different levels of scale (from cellular to organism, up to whole-plant levels), that follow blue light absorption by epidermal anthocyanins. We offer clear evidence that the blue light-absorbing properties of anthocyanins are responsible for the shade nature of cyanic leaves/individuals and, as a corollary, this strongly supports the view of an effective photoprotective role of anthocyanins, consistent with the notion that blue light contributes substantially to the action spectrum for photodamage (Takahashi et al., 2010).