HEADLESS represses a CYP735 monooxygenase gene and promotes iP-type cytokinin accumulation in the Medicago truncatula shoot apex.

Cytokinins comprise a class of phytohormones that promote cell division and participate in diverse developmental processes. Cytokinin activity and steady-state levels in tissues are maintained through biosynthesis, signaling, degradation, side-chain modification, transport, and storage. Mutating HEADLESS (HDL), a Medicago truncatula homolog of Arabidopsis WUSCHEL (WUS), terminates the shoot apical meristem (SAM); thus, hdl mutants continuously generate leaves. In this study, we isolated a direct target of HDL [(HDL Target 1, HDT1)] based on RNA-seq transcriptome analyses using inducible HDL overexpression lines and the hdl mutant. HDT1 encodes a putative cytokinin hydroxylase homologous to Arabidopsis Cytochrome P450 mono-oxygenase CYP735A1 and CYP735A2. HDT1 trans-hydroxylates the prenyl side chain of cytokinins, converting the isopentenyl-adenine (iP)-type into trans-Zeatin (tZ)-type cytokinins. We show that HDL directly binds to the HDT1 promoter and represses its transcription, promoting the preferential accumulation of iP-type cytokinins. Overexpressing HDT1 slowed plant growth, leading to shorter plants caused by inhibited internode elongation, which is reminiscent of the hdl mutant phenotype albeit less severe. Both the hdl mutant and HDT1 overexpression lines accumulated lower levels of the iP-type but higher levels of the tZ-type cytokinins in shoots, indicating that HDT1 promotes tZ-type cytokinin biosynthesis and that HDL represses this process. Overall, this study uncovers the role of HDL in cytokinin biosynthesis and suggests that iP is preferred over tZ for SAM function in M. truncatula, opening avenues of research to better understand HDL function and the molecular mechanisms that regulate cytokinin biosynthesis.