Investigating molecular mechanisms controlling phenylpropanoid production in potato and tomato

Plant phenylpropanoids draw an outstanding interest due to their effect in both human nutrition and plant defence. In fact, compounds produced through the phenylpropanoid biochemical pathway represent indispensable elements for plants to face environmental stresses. Moreover, thanks to their strong antioxidant activity, these compounds are important components of functional food. In this work we focused on phenylpropanoid pathway, with a particular interest for the braches of flavonoids. In particular, the major aim of our research was to investigate the genetic mechanism controlling the production of flavonoids in order to clarify either their role in plant stress tolerance or to enhance the production of useful metabolites in plant derived foods. As regard the role of flavonoids in plant environmental tolerance, we focused on the ability of anthocyanin pigments to induce cold stress tolerance in potato. Since no molecular information was available on anthocyanin regulation in potato leaves, the first part of our research focused onto the determination of MYB/bHLH complex that influences anthocyanin production in vegetative tissues of the cultivated potato Solanum tuberosum. In particular, we found that StAN1, a gene codifying a MYB factor, displays intraspecific sequence variability in both coding/non-coding regions and in the promoter. In addition, expression analysis suggested that leaf pigmentation is associated with StAN1 expression and that a bHLH, named StJAF13, acts as putative StAN1 co-regulator for anthocyanin gene expression in leaves of red-leaf varieties. Functional analysis through protein/protein interaction and ectopic transgenic expression, further confirmed AN1/StJAF13 interaction complex to induce anthocyanin accumulation. Once characterized this anthocyanin complex, we compared the anthocyanin genes of the wild cold tolerant species Solanum commersonii with those of cultivated varieties under cold stress condition. As suggested by functional and metabolic analysis in Nicotiana benthamiana, ScAN2, a paralog gene of ScAN1, evolved differently between cultivated and wild species. In S. commersonii, ScAN2 seemed to keep a pleiotropic and ancestral function with respect to ScAN1, inducing a multiple activation of several phenylpropanoid branches to respond to cold injury. Consistently, we found that ScAN2 was up regulated after cold treatment only in the cold tolerant S. commersonii. In addition, metabolic and microscopy analyses suggested that ScAN2 is connected to the production of phenolic compounds located on plasma membrane and cell wall of transformed tobacco cells. The third part of our work was mainly concentrated on the group of flavonols which are flavonoids with a strong anti-inflammatory activity as well as protective role against cardiovascular diseases. In this study, we tried to understand which were the genes that normally contribute to flavonol accumulation in tomato flesh. We choose an eQTLs approach to move in the intricate gene regulation architecture that may influence flavonol accumulation in tomato flesh. The material used was the introgression population developed from crosses between S. pennellii and the cultivated tomato S. lycopersicum, cultivar “M82”. The most significant outcome from this research was the identification of two potential negative regulators we named SlMYB4 and SlELVIRA. The function of these two genes was studied using VIGS (virus induced gene silencing) approach. The transient silencing of the SlMYB4 and SlELVIRA resulted in an increase of flavonols as well as of chlorogenic acid in tomato flesh. This suggested a negative action of these two genes in flavonoid regulation. Ultimately, we believe that these studies may provide a new framework to explore how phenylpropanoid genes regulate the different branches of phenylpropanoid pathway to either increase plant tolerance to external stresses or to enhance the accumulation of human beneficial metabolites in important crops.