Characterization of KNOX Homeobox Genes in Medicago truncatula as a Resource for Genetic Improvement of Legume Species

Forage legumes are the main source of plant proteins. Among legumes, alfalfa has also a considerable potential as a feedstock for production of ethanol. Crucial quality traits of forage legumes are nitrogen fixation ability, lignin content and leafiness. Alterations in function of KNOTTED1-like homeobox (KNOX) genes have the potential to affect these traits. Therefore,weused the model species Medicago truncatula, a close relative of alfalfa, for developmental biology studies to identify and characterize novel KNOX genes as a resource for genetic improvement in legumes. In this study, quantitative RT-PCR and in situ hybridization techniques were used to analyze: the expression of MtKNOX6, a class 1 STM-like homeobox gene, in the shoot apical meristem, leaf development and flower formation - the expression of MtKNOX3, a cytokinin-inducible homolog of the Arabidopsis KNAT3, during the formation of root nodules in response to rhizobium infection. MtKNOX6 was temporarily down-regulated at the site of leaf primordia initiation but restored during trifoliate compound leaf formation and development, differently from what previously shown for other legume species. Interestingly, the transcript accumulated at the sites of leaflets initiation and, later, in the parenchyma tissues that assist leaf secondary vein formation. MtKNOX6 was post-transcriptionally regulated. Four alternatively spliced transcripts were identified and presented distinct tissuespecific pattern of regulation and response to hormonal treatments. These data suggest a possible role of MtKNOX6 in compound leaf development and point towards the importance of alternative splicing in the processes regulated by KNOX transcription factors. In collaboration with the LIPM and the ISV–CNRS in France, we found the expression of three MtKNOXs modulated in response to rhizobia infection. MtKNOX3 induction was confirmed by qRTPCR. MtKNOX3 transcript localization in nodule meristem since early stages of nodule induction let us hypothesize a role of KNOX transcription factors in the cytokinin-mediated nodule formation process.