IDENTIFICATION AND FUNCTIONAL STUDIES OF THE REQUIREMENTS FOR EMBRYONIC NADPMALIC ENZYME GENE TRANSCRIPTION

GISMONDI, MAURO; SOUZA, DANIEL; PERMINGEAT, HUGO; BORRÁS, LUCAS; ANDREO, CARLOS S.; DRINCOVICH, MARÍA FABIANA; GROTEWOLD, ERICH; SAIGO, MARIANA

Congreso; SAIB LX - XIV PABMB; 2019

physiological functions of its substrates and products (malate, and pyruvate and NADPH, respectively). In plants, this gene family containsseveral members although the biological roles of each of them remain still unknown. Phylogenetic analysis of plant NADP-ME proteinsequences revealed the existence of different clades, being embryo-specific ME1lineage the only one that contains monocot and dicot members.Although the members of each clade have common subcellular localization, biochemical and structural features, and abundance in particularorgans and during development, little is known about the mechanisms that govern their gene expression. Here, we present the in silico analysisof ME1 lineage-specific features and performed experimental approaches on ZmME1 promoter, to discover new players that could control ME1particular abundance in plants. We verified the functionality of promZmME1::GFP constructs in transient expression experiments consisting ofisolated maize embryo gene bombardments. Moreover, we performed a Yeast One Hybrid (Y1H) screen with a TFome library (containing onlytranscription regulators) and using a histidine auxotrophy-based selection of putative positive interactions. Different members of particular TFfamilies (GNAT, MADS, bZIP, MYB, bHLH, HB) and also different combinations of TFs were identified as capable of binding to atranscriptional start proximal region of ZmME1 gene, suggesting their importance of these protein-DNA interactions in its transcriptionregulation. Validations were carried out by direct transformation of identified TFs in yeast bait strain cells. Finally, the integration of obtainedresults with reported gene expression data in maize and other monocot species increased the confidence about the participation of the identifiedTFs in ME1 clade-specific transcription. These findings provide new insights and trigger new hypothesis to better understand ME1 lineagefunction and the importance of their substrates and products for embryo development in species of agronomical interest.