ROLE OF A NOVEL KNOX1 TRANSCRIPTION FACTOR OF MEDICAGO TRUNCATULA IN THE DETERMINATION OF LEAF MORPHOLOGY

In higher plants, KNOX1 homeobox transcription factors play a critical role in compound leaf development. The presence of KNOX1 in developing leaves characterizes compound-leaved versus simple-leaved species, where KNOX1 genes are expressed only in the shoot apical meristem (SAM) and permanently down-regulated during leaf development. It has been recently shown that modifications of KNOX1 gene expression, through changes of cis-regulatory properties, represent the main evolutionary step of leaf morphology complexity. In simple-leaved species, KNOX1 are essential for proper formation and morphogenetic activity of the SAM, whereas in most of compound-leaved species they exert their role in leaf dissection through the maintenance of a transient indeterminacy and morphogenetic activity at the marginal blastozone. In the inverted repeat-lacking clade (IRLC) of legumes, an alternative pathway, involving the Arabidopsis LEAFY (LFY) orthologs - UNIFOLIATA (UNI) of pea and SINGLE LEAFLET1 (SGL1) of M. truncatula - was proposed to act in the formation of compound leaves. In order to explore the role of KNOX1 transcription factors in the development of trifoliate compound leaves in Medicago truncatula, a model species for forage legumes, six MtKNOXs have been identified and characterized in our lab. Here we present the molecular and functional characterization of MtKNOX6, a homologue of Arabidopsis SHOOTMERISTEMLESS (STM) that is located in a separate branch of the phylogenetic tree with respect to the strictly STM-like proteins. MtKNOX6 expresses in developing leaves, in the SAM and during inflorescence development, is regulated through alternative splicing and the translated proteins retain the ability to functionally interact with the BEL protein PNY/BLH9 to enter the nucleus. Transgenic lines that overexpressed MtKNOX6 in the heterologous system Arabidopsis thaliana did not phenocopy Arabidopsis plants that overexpressed STM, that suggesting different biochemical and molecular properties of MtKNOX6 with respect to STM. Also, overexpression of either STM or MtKNOX6 in M. truncatula did not trigger the same developmental alterations, that confirming the data obtained in Arabidopsis. Two mtknox6 knock-out lines were isolated in collaboration with The Samuel Roberts Noble Foundation (US) and their characterization is in progress.