Cytokinins regulate spatially specific ethylene production to control root growth in Arabidopsis

Two principal growth regulators, cytokinins and ethylene, are known to interact in the regulation of plant growth. However, information about the underlying molecular mechanism and positional specificity of cytokinin/ethylene crosstalk in the control of root growth is scarce. We have identified the spatial specificity of cytokinin-regulated root elongation and root apical meristem (RAM) size, both of which we demonstrate to be dependent on ethylene biosynthesis. Upregulation of the cytokinin biosynthetic gene ISOPENTENYLTRANSFERASE (IPT) in proximal and peripheral tissues leads to both root and RAM shortening. By contrast, IPT activation in distal and inner tissues reduces RAM size while leaving the root length comparable to that of mock-treated controls. We show that cytokinins regulate two steps specific to ethylene biosynthesis: production of the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC) by ACC SYNTHASEs (ACSs) and its conversion to ethylene by ACC OXIDASEs (ACOs). We describe cytokinin- and ethylene-specific regulation controlling the activity of ACSs and ACOs that are spatially discrete along both proximo/distal and radial root axes. Using direct ethylene measurements, we identify ACO2, ACO3, and ACO4 as being responsible for ethylene biosynthesis and ethylene-regulated root and RAM shortening in cytokinin-treated Arabidopsis. Direct interaction between ARABIDOPSIS RESPONSE REGULATOR 2 (ARR2), a member of the multistep phosphorelay cascade, and the C-terminal portion of ETHYLENE INSENSITIVE 2 (EIN2-C), a key regulator of canonical ethylene signaling, is involved in the cytokinin-induced, ethylene-mediated control of ACO4. We propose tight cooperation between cytokinin and ethylene signaling in the spatially specific regulation of ethylene biosynthesis as a key aspect of the hormonal control of root growth.