Functional Characterization Of The Gentiana Lutea Carotenoid Biosynthetic Gene Promoters

Carotenoids are naturally tetraterpenoids. They are important in nutuition for human. Inaddition carotenoids function as attractants to pollinators and seed dispersal agents. Duringthe last two decades, significant advances have been made in cloning most of thecarotenogenic genes and the genetic engineering of plants with the intention of manipulationof carotenoid content. However, a major limitation is our poor knowledge of how endogenouscarotenogenic genes are regulated in higher plants. Poor knowledge of the regulation ofcarotenogenic gene expression has thus far limited the predictive carotenoid engineering ofplants for enhancing carotenoid content and composition.The investigation of promoter was one of means for attempts to overcome theseroadblocks in plants by breaking through them or going around them. So the promoter andtheir cis-elements were researched. During the development of Gentiana lutea petals, atemporal correlation among the accumulation of carotenoids, the formation of chromoplasts,and the coordinated up-regulation of carotenogenic gene expression is evident. In this report,in order to elucidate the molecular control mechanisms of the coordinated up-regulation ofcarotenogenic gene expression, six promoters (GlZEP, GlPDS, GlZDS, GlLYCB, GlBCH andGlLYCE) were isolated by inverse PCR from G. lutea. All of these promoters were able todrive developmentally regulated expression of the gusA reporter gene in transgenic tomato(Solanum lycopersicum cv. Micro-Tom).We transformed tomato plants with the gusA gene encoding the reporter enzymeβ-glucuronidase (GUS) under the control of the GlZEP promoter, and investigated thereporter expression profile. We detected high levels of gusA expression and GUS activity inchromoplast containing flowers and fruits, but minimal levels in immature fruits containinggreen chloroplasts, in sepals, leaves, stems and roots. GlZEP-gusA expression was strictlyassociated with fruit development and chromoplast differentiation, suggesting anevolutionarily-conserved link between ZEP and the differentiation of organelles that storecarotenoid pigments.We transformed tomato plants with the gusA gene encoding the reporter enzymeβ-glucuronidase (GUS) under the control of the five genes(GlPDS, GlZDS, GlLYCB, GlBCHand GlLYCE) promoters respectively. High levels of gusA expression driven by thesepromoters were found in chromoplast-containing mature green (MG) fruits, but very lowlevels of expression, if any, were seen in chloroplast-containing immature green (IM) fruitsfor both GlLYCB and GlBCH promoters. The expression of these promoters is strictlyassociated with tomato fruit development and chromoplast differentiation. In addition to corepromoter elements (TATA box and CAAT box), three common cis-regulatory motifs involvedin methyl jasmonate (MeJA) responsiveness (CGTCA-motif), and binding an ethyleneresponse transcription factor (ATCTA-motif)，required for endosperm expression Skn-1motif(GTCAT) were found in all the carotenogenic gene (GlPDS, GlZDS, GlLYCB, GlBCH, GlZEP and GlLYCE) promoters of G. lutea. Our present investigation provides new insights into theregulatory architecture of the coordinated up-regulation of carotenogenic gene expression inpetals of G. lutea. The impact of our results on current models for the regulation ofcarotenogenesis in plants is discussed.