Cytokinin oxidase/dehydrogenases (CKXs) play a key role in the irreversible degradation of phytohormone cytokinin that is necessary for various plant growth and development processes. However, thus far, detailed investigations of the CKX gene family in the model legume Medicago truncatula are limited. In this study, we identified 9 putative CKX homologues with conserved FAD- and cytokinin-binding domains in the M. truncatula genome. We analyzed their phylogenetic relationship, gene structure, conserved domain, expression pattern, protein subcellular locations and other properties. The tissue-specific expression profiles of the MtCKX genes are different among different members and these MtCKXs also displayed different patterns in response to synthetic cytokinin 6-benzylaminopurine (6-BA) and indole-3-acetic acid (IAA), suggesting their diverse roles in M. truncatula development. To further understand the biological function of MtCKXs, we identified and characterized mutants of each MtCKX by taking advantage of the Tnt1 mutant population in M. truncatula. Results indicated that M. truncatula plants harboring Tnt1 insertions in each single MtCKX genes showed no morphological changes in aerial parts, suggesting functional redundancy of MtCKXs in M. truncatula shoot development. However, disruption of Medtr4g126160, which is predominantly expressed in mots, leads to an obvious reduced primary mot length and increased lateral root number, indicating the specific roles of cytokinin in regulating mot architecture. We systematically analyzed the MtCKX gene family at the genome-wide level and revealed their possible roles in M. truncatula shoot and mot development, which shed lights on understanding the biological function of CKX family genes in related legume plants.
