Dissection of carotenoids and apocarotenoids biosynthesis in crop plants

Carotenoids and apocarotenoids are one of the largest and most important classes of natural compounds. They play an important role in human nutrition because of their pro-vitamin A activity and their roles as nutritional antioxidants. In plants, they play essential functions in several mechanisms ranging from photoprotection to attraction of pollinators, to antagonism against phytophages, to hormone and volatile precursors. In the present study, biosynthesis of these metabolites has been investigated in two important plant systems, tomato fruit and saffron stigma, which are known to accumulate, respectively, valuable carotenoids and apocarotenoids. n tomato, the aim of the project has been to elucidate the molecular-biochemical mechanisms underlying carotenoid accumulation in fruit, a model system for studying carotenoid pathway, due to the distinctive color changes during berry development. A collection of single mutants in 5 enzymatic steps in the pathway (apricot, yellow flesh, tangerine, Beta and Delta) has been analyzed at three different ripening stages of ripening, i.e. mature green, breaker and full ripe by a combined approach of Real-time PCR and LC-PDA-APCI-MS. Interestingly, large and unscheduled alterations have been found both in the expression profiles of genes involved in both carotenoid and other isoprenoid pathways, as well as in the corresponding metabolite accumulation. Overall, a dramatic shift in the "local" metabolism, as well as in several more distant areas of the metabolism has been observed. For instance, in the Beta mutant, an overexpression oboth early and late genes has been observed, in particular ZISO, ZDS, CHY2 and LUT5, lead to accumulation of zeaxanthin, a rare metabolite in tomato fruit. Correlation analyses of the gene-metabolite interactions have been employed to visualize and predict multi-level alterations in order to obtain a better understanding of their regulation during the fruit ripening, to be taken into account for future classic breeding or genetic engineering manipulation. Since the availability of stable mutants is limited to relatively few carotenoid genes, a virus-induced gene silencing (VIGS) approach has been used to study the biosynthetic pathway, using a visual reporter system. My contribution to this work has been the characterization of fruits of the yellow flesh and tangerine mutants and their comparison with fruits silenced in the corresponding genes (PSY1 and CrtISO). A second project has been focused on a “pathway discovery” approach in saffron stigma. Saffron stigmas have been used as a spice since the Greek-Minoan civilization, due to the presence of several valuable metabolites and, particularly, of the apocarotenoids crocin, picrocrocin and safranal, contributing respectively to the red color, the bitter taste and the aroma. Apocarotenoid biosynthesis occurs in the frame of carotenoid catabolism, through a cleavage reaction catalyzed by a class of enzymes called CCDs (Carotenoid Cleavage Dioxygenases). Conflicting data had been previously reported about the candidate enzyme catalyzing the cleavage reaction in saffron (Zeaxanthin Cleavage Dioxygenase or ZCD), and two different enzymes acting on alternative substrates (!-carotene and zeaxanthin) have been proposed to perform this enzymatic step. We took an RNA-Seq approach to identify all CCDs expressed during 6 stigma developmental stages: globally, 9 CCDs have been found and cloned into an expression vector optimized to yield large amounts of protein. I have accomplished the expression of all the CCDs in E. coli cells engineered to accumulate several carotenoid substrates. Then I have undertaken a detailed enzymatic characterization of two CCDs, CCD2 and the previously discovered ZCD, accumulated during early and late stigma development, respectively. These analyses strongly suggest that the newly identified CCD2 catalyzes the cleavage step in saffron apocarotenoid biosynthesis from zeaxanthin. In the same assays, ZCD did not show any cleavage activity, in contrast to previous literature.