Dissecting molecular events at the base of the shade nature of cyanic leaves
Low blue light availability evokes shade avoidance responses similar to those induced by low R/FR (Sellaro et al., 2010; Pedmale et al., 2016), and the cellular reprogramming because of blue light depletion may be even larger than in response to a decrease in R/FR (Ballarè & Pierik, 2017; Ballarè, Scopel, & Sanchez, 1990). Plants are very sensitive to changes in blue light irradiance, as up to 26% of gene expression varies in response to blue light, even on a very short, time-scale level (Jiao et al., 2003). This conforms to the notions that stems perceive reductions in blue light well before leaves are shaded, and that low blue light is an indicator of actual shading, whereas plants use the reduction in R/FR as an early warning signal of future competition (Ballaré et al., 1990; Keuskamp, Keller, Ballarè, & Pierik, 2012). As a consequence, the blue light absorbing properties of anthocyanins cannot be ignored when exploring response mechanisms of green vs red individuals to excessive light as well as to conclusively assess the photoprotective functions of anthocyanins.
While functional analysis of genes involved in secondary metabolite biosynthesis has been investigated in some instances(Jin et al., 2018; Kim et al., 2018; Torre et al., 2016), there is very limited information about molecular events that govern morpho-anatomical and physiological traits in red compared to green individuals (Tattini et al., 2017). Here we have extended the analysis of differentially expressed genes reported in Tattini et al. (2017) for purple and green basil, with special emphasis on a suite of blue light-responsive genes (Fig. 2), which regulate developmental processes at the base of the shade nature of cyanic leaves/individuals. Overall, low blue light-regulated genes aimed at maximizing light harvesting are over-expressed in purple leaves (Fig. 2). These include a gene coding for auxilin-like J-domain protein required for chloroplast accumulation response 1 (JAC1 ) under low blue light (Suetsugu, Kagawa, & Wada, 2005), thus re-locating chloroplasts perpendicular to the light flux (chloroplasts move to the periclinal cell wall). The expressions of genes coding for Chlorophylla-b binding protein CP2410A (CAP10A), and the nitrogen fixing (niFU-like3) protein, all involved in light harvesting in PSI (Ganadeg, Klimmek, & Jansson, 2004; Yabe, Morimoto, Nishio, Terashima, & Nakai 2004) are also higher in cyanic leaves. This is also the case of CURVATURE THYLAKOID 1A (CURT1A), which is effective in optimizing PSII photochemistry under low light conditions (Pribil et al., 2018). The need of ‘maximizing’ light harvesting in purple leaves is also well documented by the large expression of genes, such as Far Red Impaired Response 1 (FAR1 ) and Protochlorophyllide-a oxygenase (PTC52 ), which promote Chl biosynthesis (Bartsch et al., 2008; Reinbothe et al., 2006; Tanaka, Tanaka, Tanaka, Yoshida, & Okada, 1998). As expected, a range of high light-responsive genes is downregulated in red basil. This includes the Filamenting Temperature-Sensitive Z1 (FtsZ1 ) that promotes chloroplast division and the photo-relocation of chloroplasts toward the anticlinal wall of palisade cells (so-called chloroplast avoidance response, Dutta et al., 2017; Kong & Wada, 2011; Koniger, Delamaide, Marlow, & Harris, 2008). The transcript abundance of genes that encode for proteins that either reduce the synthesis (early light-induced protein2 ELIP2, Tzvetkova-Chevolleau et al., 2007) or sustain the catabolism of Chl (Chlorophyllase1, CLH1, Banaś, Labuz, Sztatelman, Gabrys, & Fiedor, 2011), and of Chlb (Non Yellow Coloring1, NYC1, Horie, Ito, Kusaba, Tanaka, & Tanaka, 2009), is also low in purple basil. Notably, red leaves have lower expression levels of bothEXECUTER1 (EX1 ) and Plastid-Lipid-Associated 6 , (PAP6 ), which are genes involved in singlet oxygen-induced retrograde signal and in the transport of lipophilic antioxidants, respectively, under light stress (Langenkamper et al., 2011; Lee, Kim, Landgraf, & Apel, 2007). This increase in oxidative stress signaling (sensu Foyer, Ruban, & Noctor, 2017) displayed by green leaves adds further evidence to previous suggestions that anthocyanins are effective photoprotective pigments (Gould, 2004; Gould et al., 2010; Hughes et al., 2005, Hughes & Smith, 2007).
The expression of genes involved in the shade avoidance responses at leaf and whole plant levels is also higher in cyanic leaves. These include three members of the Phytochrome Kinase Substrate gene family (PKS1, PKS3, PKS4 ), which operate downstream of Phototropin1 (phot1) under low blue light irradiance, and act as negative regulators of phytochrome (PHY) signaling. Overexpression ofPKS s promotes hypocotyl elongation and leaf flattening as well, early events in shade avoidance responses (de Carbonell et al., 2010; Lariguet et al., 2003). We also observe that the expressions of bothDWARF27 (β-carotene isomerase ), a gene involved in the first committed step of strigolactone biosynthesis and two genes, coding for members of the ABC (ATP BINDING Cassette) superfamily transport proteins (ABCG5 and ABCG11, also known as pleiotropic drug resistance (PDR) proteins) are low in purple basil. DWARF27 and ABCGs regulate canopy architecture, by promoting axillary branching indeed (Bienert et al., 2012; Kretzschmar et al., 2012; Yasuno et al., 2009), a common plant response to high light irradiance. Other relevant genes involved in shade avoidance responses such as LONGIFOLIA 1/2 and a set ofEXPANSINs (1,4,8,10) (Christie, 2007; Sasidharan, Chinnappa, Voesenek, & Pierik, 2008) are also overexpressed in red compared to green basil leaves. LONGIFOLIA and EXPANSIN both promote leaf and stem elongation, by enhancing polar cell elongation at the expense of cell proliferation (Lee et al., 2018), and disrupting noncovalent bonds between cellulose microfibrils and matrix polysaccharides, respectively (Choi, Lee, Cho, & Kende, 2003; Marowa, Ding, & Kong, 2016).
Consistent with their shade nature, red individuals invest less carbon to leaf construction, as also occurs when green leaves grow in low light. This is because R2R3MYB genes, such as the MYB6 ,MYB75 , and MYB308 genes detected in our study, while promoting anthocyanin biosynthesis, repress the synthesis of early products of the general phenylpropanoid metabolism, including lignin (Bhargava, Mansfield, Hall, Douglas, & Ellis, 2010; Tamagnone et al., 1998). This is in line with the notion that blue light strongly promotes secondary cell wall thickening (Zhang et al. 2018b). It has been recently shown that a range of transcription factors that are responsive to low blue light irradiance, and involved in the regulation of leaf cell fate, are largely overexpressed in high light-grown red compared to corresponding green basil leaves (Tattini et al., 2017). This includes relevant members of the HD-ZIP family, such as ATHB1, ATHB2 andATHB12 genes, which repress cell proliferation and the proper development of palisade cells, thereby sustaining shade avoidance responses (Ciolfi et al., 2013; Hur et al., 2015). Our findings are therefore consistent with and may conclusively explain why cyanic leaves are thinner, have much less compact mesophyll and lower leaf mass per area (LMA) with respect to acyanic leaves (Manetas et al., 2003; Kyparissis et al., 2007; Wang, Zhou, Jiang, & Liu, 2016), especially when growing in high light (Tattini et al., 2014).