Identification and functional studies of the requirements for embryonic NADP malic enzyme gene transcription

PERMINGEAT H; DRINCOVICH MF; GASMUNDI M; BORRAS L; GROTEWOLD E; SOUZA D; ANDREO CS; SAIGO M

Salta

Congreso; LV Annual SAIB-XIV PAMBM 2019; 2019

SAIB

NADP-dependent malic enzyme (NADP-ME) is widespread in nature and involved in different metabolic pathways due to the relevant physiological functions of its substrates and products (malate, and pyruvate and NADPH, respectively). In plants, this gene family contains several members although the biological roles of each of them remain still unknown. Phylogenetic analysis of plant NADP-ME protein sequences 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 particular organs and during development, little is known about the mechanisms that govern their gene expression. Here, we present the in silico analysis of ME1 lineage-specific features and performed experimental approaches on ZmME1 promoter, to discover new players that could control ME1 particular abundance in plants. We verified the functionality of promZmME1::GFP constructs in transient expression experiments consisting of isolated maize embryo gene bombardments. Moreover, we performed a Yeast One Hybrid (Y1H) screen with a TFome library (containing only transcription regulators) and using a histidine auxotrophy-based selection of putative positive interactions. Different members of particular TF families (GNAT, MADS, bZIP, MYB, bHLH, HB) and also different combinations of TFs were identified as capable of binding to a transcriptional start proximal region of ZmME1 gene, suggesting their importance of these protein-DNA interactions in its transcription regulation. Validations were carried out by direct transformation of identified TFs in yeast bait strain cells. Finally, the integration of obtained results with reported gene expression data in maize and other monocot species increased the confidence about the participation of the identified TFs in ME1 clade-specific transcription. These findings provide new insights and trigger new hypothesis to better understand ME1 lineage function and the importance of their substrates and products for embryo development in species of agronomical interest.