Identification of direct transcriptional targets of PIF (Phytochrome-Interacting factors)In tocopherol biosynthetic pathway

Abstract

Tomato fruits are source of health-promoting antioxidant substances, such as ascorbic acid, carotenoids and tocopherols (Vitamin E, VTE). The accumulation of these plastid-derived compounds is affected by light, as this signal has a strong role in regulating chloroplast maintenance and activity. For example, silencing of DE-ETIOLATED 1 gene, a negative effector in light signal transduction pathway, results in higher chloroplast number and Chl accumulation in the green fruit and higher carotenoid and VTE content at ripe stages. On the other hand, silencing of HYPOCOTYL ELONGATED 5 (HY5) transcription factor (TF), an inducer of light response, results in green fruits with deficient chloroplasts and ripe fruits with lower carotenoid levels.In Arabidopsis, plastid differentiation and activity are governed by a complex signaling network controlled by light. Phytochrome-Interacting Factors (PIFs) are central components of this cellular hub, controlling plant development since seed germination towards plant senescence. PIFs are TFs responsible for repression of photomorphogenesis in the dark. Although PIF genes have been extensively studied in Arabidopsis, there are but few studies in other species. My PhD project funded by FAPESP aims the identification and functional characterization of PIF genes in tomato. In this context, we have identified eight PIF-encoding genes in tomato genome and described their expression profiles in different tissues in response to light/dark treatments. Based on the transcription profiles and the mRNA abundance, we have chosen four PIF-encoding loci for further functional characterization. We also observed that PIF1a gene is up-regulated along fruit ripening. The group of Prof. Manuel Rodriguez-Concepción, has shown by chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) that PIF1a regulates fruit carotenoid accumulation by direct interaction with the promoter region of PHYTOENE SYNTHASE 1 gene, repressing its transcription and impairing the first step of carotenogenesis.For the past few years, our group has been studying the biosynthesis of VTE. Tocopherols are non-enzymatic lipid-soluble antioxidants which protective effect against reactive oxygen species generated during photosynthesis, especially under high-light conditions. Our group has demonstrated that VTE biosynthesis is tightly related to chlorophyll and carotenoid metabolism. The expression of the genes involved in these pathways is spatial and temporally regulated in a coordinated manner, and many of the co-regulated genes share cis-regulatory motifs in their promoter regions, suggesting regulation by common TFs.Aiming to gain further knowledge about the interplay between light and VTE accumulation, we decided to investigate whether light regulates VTE biosynthesis. We found higher levels of VTE in fruits ripened under constant light, indicating that this stimulus regulates VTE accumulation. We also observed differential expression of genes involved in VTE biosynthesis in response to light and dark treatments, suggesting that VTE biosynthesis is regulated at the transcriptional level. By performing a de novo search, we found both exclusive and shared cis-regulatory motifs recognized by PIFs and HY5 on the promoter regions of the differentially expressed genes. These results allowed us to hypothesize that VTE biosynthesis might be controlled by light at the transcriptional level by PIFs and HY5 TFs.This internship proposal aims to investigate whether genes of VTE biosynthesis are direct targets of PIFs. This goal will be achieved using a ChIP-qPCR technique developed by Prof. Manuel Rodriguez-Concepción in Barcelona, Spain, to analyse promoter-transcription factor interaction based on transient expression in tomato fruits. This methodology will be very useful both for my thesis and for the research line developed at our lab. (AU)