• anthocyanins
• carbohydrate
• leaf senescence
• photoprotection
• photosynthesis
• sugar-buffering

Red or green leaves might differ in photoprotective mechanisms and, also in the photosynthetic rates to different environmental stress conditions. The debate on the ecological role of foliar anthocyanins is controversial in the scientific community and their photoprotective versus other potential roles still remains a point to be solved. Moreover, the high cost in carbon skeletons required for the anthocyanin biosynthesis raises questions about their possible role as sugar-buffers.
For this purpose, four experiments were conducted using two Prunus cerasifera morphs. One morph is characterised by green leaves (P. cerasifera clone 29C; GLP) and the other is characterised by red leaves (P. cerasifera var. pissardii; RLP) due to the presence of anthocyanins. The research aim was understanding the possible physiological and metabolic influences of anthocyanin presence in leaves.
Firstly, leaves of P. cerasifera morphs, at three leaf developmental stages, young, mature and senescent (1, 7 and 13-week-old, respectively), were analysed in terms of photosynthetic rate, carbon metabolism and photoprotective mechanisms. The goal was to test whether anthocyanin-equipped leaves perform better than anthocyanin-less leaves and whether photoprotection is the primary role of epidermally-located anthocyanins, using for the first time a recently developed parameter of chlorophyll fluorescence (qPd). Anthocyanins contributed to leaf photoprotection throughout the leaf development but were tightly coordinated with carotenoids. Besides their photoprotecting role, we propose that epidermal anthocyanins may be principally synthetised to maintain an efficient carbon-sink strength in young and senescent leaves, thus extending the RLP leaf lifespan.
In the second experiment, a transcriptomic approach has been exploited on green (GLP) and red (RLP) leaves of P. cerasifera from mature to senescent (7 and 13-week-old, respectively). Our analysis revealed 3,070 differentially expressed genes (DEGs), between mature and senescent stage in GLP, whereas red morph showed a slightly higher gene regulation (4,925 DEGs). Significant differences were detected in cellular metabolism of red leaves compared to green: genes involved in photosynthetic processes (e.g. transcripts coding for chlorophyll binding proteins) were highly down-regulated in green genotype, whereas transcripts involved in anthocyanin biosynthesis (e.g. flavonoid 3-O-glucosyltransferase) were up-regulated exclusively in red leaves. In conclusion, RNA-seq analysis suggests that in P. cerasifera red genotype, there is a gene regulation and molecular pathways to cope with the senescence processes in order to promote a delayed leaf ontogenesis compared to the green one.
In the third experiment, to elucidate the possible role exerted by anthocyanins against an abiotic stress, the physiological biochemical responses of mature leaves to a severe PEG6000-induced water stress prolonged for 20 days in the two morphs of P. cerasifera were evaluated. After 10 days of the treatment, the reduction in photosynthetic rate was 83.4 and 76.5% in GLP and RLP, respectively, as compared to their relative controls. In both the morphs, the decrease was attributable mostly to stomatal closure but also to a reduction of PSII photochemical efficiency. Analysis of leaf sugar pattern revealed that stress promoted biosynthesis of anthocyanins paralleled to slight increments of sucrose in red-morph leaves at an early stage of the stress whereas anthocyanin-less green-morph leaves exhibited a severe accumulation of soluble sugars. It is possible that a part of carbon assimilated by red-morph leaves was invested into anthocyanin synthesis and this, in turn, lowered accumulation of soluble sugars and limited sink-induced photoinhibition. In conclusion, a positive effect given by anthocyanin presence, in terms of light abatement and photosystem II (PSII) protection, was observed, and also the evidence that the sugar-buffering hypothesis proposed for anthocyanins might make the difference in terms of drought sensitivity between red versus green leaves is provided.
In the 4th experiment, shoots from the two P. cerasifera morphs were girdled to understand the role of leaf anthocyanin on photosynthesis, photoprotection and sugar metabolism. Shoot girdling was operated in order to evaluate the anthocyanin influence on the sugar accumulation promoted by girdling. Leaf gas exchange, carbohydrate and anthocyanin concentration were measured daily during the three days after girdling. On the first day at 12:00, net photosynthesis and stomatal conductance were only reduced in girdled green-morph (29 and 33 %, respectively) which also showed higher soluble sugar concentrations than controls. Girdled red morph showed the first reduction of A390 only at 18:00 with no significant differences in sucrose, glucose and fructose concentration. However, girdling highlighted the constitutive ability of girdled RLP to produce more sorbitol than starch if compared to respective GLP. The increase in anthocyanin concentration detected in girdled RLP at the second and third day had contributed in lowering the glucose and fructose accumulation that, together to the constitutive ability of RLP to produce more sorbitol than starch, influenced the circadian leaf starch metabolism and also attenuated the feedback down-regulation of photosynthesis. The effectiveness of non-photochemical quenching (pNPQ) also revealed the ability of anthocyanins to photoprotect PSII from supernumerary photons reaching the chloroplast. Overall, a sugar-buffering role exerted by anthocyanins might have positively influenced the feedback regulation of photosynthesis and, on another hand, anthocyanin accumulation also improved the PSII photoprotection from excitation energy excess. This study represents a starting point to understand the probable link between photosynthesis regulation through sugar-signalling and anthocyanin upregulation.
In summary, besides the classical role proposed for anthocyanins as sunscreen compounds, as confirmed in these experiments, a possible modulation of sugar metabolism was proposed, that might further give a wider meaning of “leaf protectants” to this versatile class of flavonoids. This study opens a wider perspective in which anthocyanin synthesis limits the increase in leaf sugars during stress conditions, and at the same time ‘sunscreening’ the leaf, all of this to limit the excitation pressure on PSII reaction centers.