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).