Plant postembryonic development takes place in the meristems, where stem cells self-renew and produce daughter cells that differentiate and give rise to different organ structures. For the maintenance of meristems, the rate of differentiation of daughter cells must equal the generation of new cells: How this is achieved is a central question in plant development. In the Arabidopsis root meristem, stem cells surround a small group of organizing cells, the quiescent center. Together they form a stem cell niche [1, 2], whose position and activity depends on the combinatorial action of two sets of genes - PLETHORA1 (PLT1) and PLETHORA2 (PLT2)[3, 4] and SCARECROW (SCR) and SHORTROOT (SHR)[2] - as well as on polar auxin transport. In contrast, the mechanisms controlling meristematic cell differentiation remain unclear. Here, we report that cytokinins control the rate of meristematic cell differentiation and thus determine root-meristem size via a two-component receptor histidine kinase-transcription factor signaling pathway. Analysis of the root meristems of cytokinin mutants, spatial cytokinin depletion, and exogenous cytokinin application indicates that cytokinins act in a restricted region of the root meristem, where they antagonize a non-cell-autonomous cell-division signal, and we provide evidence that this signal is auxin.

Highlights d scRNA-seq of Arabidopsis root cells captures precise spatiotemporal information d Defining expression features for cell types identify new developmental regulators d Cluster arrangement reflects developmental time with a centrally localized niche d Intricate waves of gene expression finely resolve developmental trajectories

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In Arabidopsis, interplay between nuclear auxin perception and trans-cellular polar auxin transport determines the transcriptional auxin response. In brevis radix (brx) mutants, this response is impaired, probably indirectly because of disturbed crosstalk between the auxin and brassinosteroid pathways. Here we provide evidence that BRX protein is plasma membrane-associated, but translocates to the nucleus upon auxin treatment to modulate cellular growth, possibly in conjunction with NGATHA class B3 domain-type transcription factors. Application of the polar auxin transport inhibitor naphthalene phthalamic acid (NPA) resulted in increased BRX abundance at the plasma membrane. Thus, nuclear translocation of BRX could depend on cellular auxin concentration or on auxin flux. Supporting this idea, NPA treatment of wild-type roots phenocopied the brx root meristem phenotype. Moreover, BRX is constitutively turned over by the proteasome pathway in the nucleus. However, a stabilized C-terminal BRX fragment significantly rescued the brx root growth phenotype and triggered a hypocotyl gain-of-function phenotype, similar to strong overexpressors of full length BRX. Therefore, although BRX activity is required in the nucleus, excess activity interferes with normal development. Finally, similar to the PIN-FORMED 1 (PIN1) auxin efflux carrier, BRX is polarly localized in vascular cells and subject to endocytic recycling. Expression of BRX under control of the PIN1 promoter fully rescued the brx short root phenotype, suggesting that the two genes act in the same tissues. Collectively, our results suggest that BRX might provide a contextual readout to synchronize cellular growth with the auxin concentration gradient across the root tip.

A central question in developmental biology is how multicellular organisms coordinate cell division and differentiation to determine organ size. In Arabidopsis roots, this balance is controlled by cytokinin-induced expression of SHORT HYPOCOTYL 2 (SHY2) in the so-called transition zone of the meristem, where SHY2 negatively regulates auxin response factors (ARFs) by protein-protein interaction. The resulting down-regulation of PIN-FORMED (PIN) auxin efflux carriers is considered the key event in promoting differentiation of meristematic cells. Here we show that this regulation involves additional, intermediary factors and is spatio-temporally constrained. We found that the described cytokinin-auxin crosstalk antagonizes BREVIS RADIX (BRX) activity in the developing protophloem. BRX is an auxin-responsive target of the prototypical ARF MONOPTEROS (MP), a key promoter of vascular development, and transiently enhances PIN3 expression to promote meristem growth in young roots. At later stages, cytokinin induction of SHY2 in the vascular transition zone restricts BRX expression to down-regulate PIN3 and thus limit meristem growth. Interestingly, proper SHY2 expression requires BRX, which could reflect feedback on the auxin responsiveness of SHY2 because BRX protein can directly interact with MP, likely acting as a cofactor. Thus, cross-regulatory antagonism between BRX and SHY2 could determine ARF activity in the protophloem. Our data suggest a model in which the regulatory interactions favor BRX expression in the early proximal meristem and SHY2 prevails because of supplementary cytokinin induction in the later distal meristem. The complex equilibrium of this regulatory module might represent a universal switch in the transition toward differentiation in various developmental contexts.