Cytokinin histidine kinase receptors regulate multiple aspects of rice growth and development

The phytohormone cytokinin has diverse functions in plants, most of which have been primarily elucidated in the eudicotyledonous plant Arabidopsis (Arabidopsis thaliana). To further characterize the role of cytokinin in a monocot, we used CRISPR-Cas9 to generate rice (Oryza sativa) lines with frameshift alleles in genes encoding the 4 putative histidine kinase (HK) cytokinin receptors, resulting in all single and multiple mutant combinations. The hk3,4,5,6 quadruple mutant transcriptome was completely insensitive to cytokinin, suggesting that all transcription regulated through cytokinin signaling acts through these 4 HKs, despite the presence of a putative cytokinin-binding CHASE-containing Ser/Thr kinase in rice. Rice HKs functioned partially redundantly in diverse aspects of growth and development, with different HKs playing more prominent roles in various processes. This functional specialization was most striking in panicle architecture, where HK4 promoted fertility, HK3 and HK5 drove secondary branch formation, and HK6 drove primary branch formation. Furthermore, unlike Arabidopsis, cytokinin signaling was essential for shoot formation in rice, as the hk3,4,5,6 quadruple mutant was shootless, likely due to a failure to develop a shoot apical meristem. The necessity for shoot formation is consistent with one of the first roles of cytokinin uncovered by Skoog and Miller in the 1950s. Overall, this work elucidates the many roles of cytokinin perception throughout the lifespan of rice, some of which diverge from those known in Arabidopsis, highlighting the need to study alternative model systems.