Factors involved in root formation in Medicago truncatula

The fact that auxin induces root formation has been known for more than half a century. However, despite the recent progress in this field, neither the molecular processes in which the auxin-responsive genes leading to root formation nor the interactions between phytohormones and other bioactive molecules during the commitment phase of root formation are well understood. Here the effect of biomolecules such as cytokinin, glutathione, and flavonoids, as well as the expression of several transcription factors in in vitro root formation in model legume Medicago truncatula are presented. It was demonstrated that auxin NAA (1-naphthaleneacetic acid) pretreatment for 7 d can irreversibly interrupt somatic embryo formation, whilst both reduced and oxidized forms of glutathione enhance root formation via a mechanism independent of ethylene perception, as determined by analysis of the ethylene-insensitive skl mutant. It was also shown that quercetin and the well-known auxin transport inhibitor NPA (N-1-naphthylphthalamic acid), which has a similar structure to quercetin, and isoflavonoids formononetin and genistein caused severe reduction in root formation. Also, the relative expression of several transcription factors was analysed in 1-week-old NAA-treated explants (stem cell niche formation stage), in NAA- and BAP-treated explants (no root formation), and in the roots of germinated seeds. The results showed, for the first time in a legume, that the transcription factors homeodomain WOX5 and the AP2-domain containing PLETHORA1 and 2, BABY BOOM1 were strongly induced by auxin addition, while cytokinin addition dramatically reduced their expression, indicating a role for these genes in the formation of root stem cell niches.