Visualization of hormonal signaling input and output is of key importance for understanding regulation of multicellular development. The plant signaling molecule auxin triggers many growth and developmental responses, but current tools lack sensitivity or precision to visualize these. We developed a set of novel fluorescent reporters that allow sensitive and semi-quantitative readout of auxin responses at cellular resolution in Arabidopsis. These generic tools are suitable for any transformable plant species.

While apical growth in plants initiates upon seed germination, radial growth is only primed during early ontogenesis in procambium cells and activated later by the vascular cambium 1 . Although it is not known how radial growth is organized and regulated in plants, this system resembles the developmental competence observed in some animal systems, in which pre-existing patterns of developmental potential are established early on 2,3 . Here we show that the initiation of radial growth occurs around early protophloem sieve element (PSE) cell files of the root procambial tissue in Arabidopsis. In this domain cytokinin signalling promotes expression of a pair of novel mobile transcription factors, PHLOEM EARLY DOF (PEAR1, PEAR2) and their four homologs (DOF6, TMO6, OBP2 and HCA2), collectively called PEAR proteins. The PEAR proteins form a short-range concentration gradient peaking at PSE and activating gene expression that promotes radial growth. The expression and function of PEAR proteins are antagonized by well-established polarity transcription factors, HD-ZIP III 4 , whose expression is concentrated in the more internal domain of radially non-dividing procambial cells by the function of auxin and mobile miR165/166. The PEAR proteins locally promote transcription of their inhibitory HD-ZIP III genes, thereby establishing a negative feedback loop that forms a robust boundary demarking the zone of cell divisions. Taken together, we have established a network, in which the PEAR -HD-ZIP III module integrates spatial information of the hormonal domains and miRNA gradients during root procambial development, to provide adjacent zones of dividing and more quiescent cells as a foundation for further radial growth. Cambial growth in plants is initiated within the procambial tissues of the apical meristems through periclinal (i.e. longitudinal) divisions associated with formation of the vascular tissues xylem and phloem 1 (Extended Data Fig. 1a). It has been established that during procambial development in Arabidopsis roots there are distinct domains for high auxin and cytokinin signalling, which mark the regions for further development of xylem and phloem/procambium, respectively 5-8 . To accurately map the spatial distribution of the periclinal divisions, we established a new nomenclature for the root procambial cells, including PSE-lateral neighbours (PSE-LN) as cells directly contacting both PSE and the pericycle, the outer procambial cells (OPC) as procambial cells adjacent to the pericycle but not contacting PSE, and SE-internal neighbours (PSE-IN) as cells located internal to and directly contacting PSE (Fig. 1a). Both the PSE cell and PSE-LN showed higher activity of periclinal cell division than the OPC and PSE-IN (Fig. 1b, Extended Data Fig. 1b-d and Supplementary Information).We observed virtually no periclinal divisions in metaxylem (MX) and internal procambial cells (IPC) (Fig. 1b). Furthermore, blocking symplastic transport genetically 9 between the PSE and the surrounding cells results in a dramatic reduct...

Optimal plant growth is hampered by deficiency of the essential macronutrient phosphate in most soils. Plant roots can, however, increase their root hair density to efficiently forage the soil for this immobile nutrient. By generating and exploiting a high-resolution single-cell gene expression atlas of Arabidopsis roots, we show an enrichment of TARGET OF MONOPTEROS 5 / LONESOME HIGHWAY (TMO5/LHW) target gene responses in root hair cells. The TMO5/LHW heterodimer triggers biosynthesis of mobile cytokinin in vascular cells and increases root hair density during low phosphate conditions by modifying both the length and cell fate of epidermal cells. Moreover, root hair responses in phosphate deprived conditions are TMO5 and cytokinin dependent. In conclusion, cytokinin signaling links root hair responses in the epidermis to perception of phosphate depletion in vascular cells.

Growth regulation tailors plant development to its environment. A showcase is response to gravity, where shoots bend up and roots down 1 . This paradox is based on opposite effects of the phytohormone auxin, which promotes cell expansion in shoots, while inhibiting it in roots via a yet unknown cellular mechanism 2 . Here, by combining micro uidics, live imaging, genetic engineering and phosphoproteomics in Arabidopsis thaliana, we advance our understanding how auxin inhibits root growth. We show that auxin activates two distinct, antagonistically acting signalling pathways that converge on the rapid regulation of the apoplastic pH, a causative growth determinant. Cell surface-based TRANSMEMBRANE KINASE1 (TMK1) interacts with and mediates phosphorylation and activation of plasma membrane H + -ATPases for apoplast acidi cation, while intracellular canonical auxin signalling promotes net cellular H + -in ux, causing apoplast alkalinisation. The simultaneous activation of these two counteracting mechanisms poises the root for a rapid, ne-tuned growth modulation while navigating complex soil environment. MainAuxin, a major growth regulator in plants, acts oppositely in shoots and roots. In shoots, canonical/intracellular auxin TRANSPORT INHIBITOR RESPONSE1 (TIR1)/AUXIN-SIGNALING F-BOX (AFB) receptors by downstream transcriptional regulation activate H + -pumps to acidify the apoplast a promote cell elongation 3,4 , in accordance with the Acid Growth Theory, which postulates that low apoplastic pH promotes growth 5 . In roots of many species including Arabidopsis, auxin inhibits growth. These contrasting responses are the basis for positive versus negative bending of roots and shoots in response to gravity and light 1 . The inhibitory auxin effect in roots also involves TIR1/AFB receptors but its rapid timing points towards an unknown non-transcriptional signalling branch 6 . Besides, a cell surface-based pathway involving TMK1 regulates development 7 , including differential growth in the apical hook 8 , while its role in auxin-regulated root growth remains unclear. Hence, the auxin signalling mechanism and the downstream processes for regulating root growth remain elusive.In this study, we revealed antagonistic action of intracellular TIR1/AFB and cell surface TMK1 auxin signalling converging on regulation of apoplastic pH, which we con rm as the key cellular mechanism allowing immediate and sensitive root growth regulation. Growth inhibition correlates with H + -in uxAuxin rapidly inhibits root growth through a non-transcriptional branch of TIR1/AFB signalling 6 . Although several cellular processes, including cortical microtubule (CMT) reorientation 9,10 , vacuolar fragmentation 11 and apoplastic pH changes [12][13][14] have been implicated, the causal mechanism remains unidenti ed.

Plant cells cannot rearrange their positions; therefore, sharp tissue boundaries must be accurately programmed. Movement of the cell fate regulator SHORT-ROOT from the stele to the ground tissue has been associated with transferring positional information across tissue boundaries. The zinc finger BIRD protein JACKDAW has been shown to constrain SHORT-ROOT movement to a single layer, and other BIRD family proteins were postulated to counteract JACKDAW's role in restricting SHORT-ROOT action range. Here, we report that regulation of SHORT-ROOT movement requires additional BIRD proteins whose action is critical for the establishment and maintenance of the boundary between stele and ground tissue. We show that BIRD proteins act in concert and not in opposition. The exploitation of asymmetric redundancies allows the separation of two BIRD functions: constraining SHORT-ROOT spread through nuclear retention and transcriptional regulation of key downstream SHORT-ROOT targets, including SCARECROW and CYCLIND6. Our data indicate that BIRD proteins promote formative divisions and tissue specification in the Arabidopsis thaliana root meristem ground tissue by tethering and regulating transcriptional competence of SHORT-ROOT complexes. As a result, a tissue boundary is not "locked in" after initial patterning like in many animal systems, but possesses considerable developmental plasticity due to continuous reliance on mobile transcription factors.

SummaryThe root endodermis surrounds the central vasculature as a protective sheath, analogous to an animal polarised epithelium, and restricts diffusion by its ring-shaped Casparian strips (CS)1. Following a lag phase, individual endodermal cells suberise in an apparently random fashion, leading to a “patchy” suberisation that eventually gives rise to a zone of continuous deposition2. Casparian strips and suberin lamellae affect paracellular and transcellular transport, respectively. Interestingly, most angiosperms maintain some isolated cells in an unsuberised state3. These so-called “passage cells” are speculated to allow uptake across an otherwise impermeable endodermal barrier. Here, we demonstrate that these passage cells are late emanations of a meristematic patterning process that reads-out the underlying non-radial symmetry of the vasculature. This process is mediated by non-cell autonomous cytokinin repression in the root meristem, leading to distinct phloem and xylem pole-associated endodermal cells. The latter can resist ABA-dependent suberisation and give rise to passage cell formation. Our data further demonstrate that during meristematic patterning, xylem pole-associated endodermal cells can dynamically adapt passage cell numbers in response to nutrient status and that passage cells express transporters and locally impact their expression in adjacent cortical cells.

In Arabidopsis, more than 1000 putative small signalling peptides have been predicted, but very few have been functionally characterized. One class of small post-translationally modified signalling peptides is the C-TERMINALLY ENCODED PEPTIDE (CEP) family, of which one member has been shown to be involved in regulating root architecture. This work applied a bioinformatics approach to identify more members of the CEP family. It identified 10 additional members and revealed that this family only emerged in flowering plants and was absent from extant members of more primitive plants. The data suggest that the CEP proteins form two subgroups according to the CEP domain. This study further provides an overview of specific CEP expression patterns that offers a comprehensive framework to study the role of the CEP signalling peptides in plant development. For example, expression patterns point to a role in aboveground tissues which was corroborated by the analysis of transgenic lines with perturbed CEP levels. These results form the basis for further exploration of the mechanisms underlying this family of peptides and suggest their putative roles in distinct developmental events of higher plants.

Summary To create a three-dimensional structure, plants rely on oriented cell divisions and cell elongation. Oriented cell divisions are specifically important in procambium cells of the root to establish the different vascular cell types [ 1 , 2 ]. These divisions are in part controlled by the auxin-controlled TARGET OF MONOPTEROS5 (TMO5) and LONESOME HIGHWAY (LHW) transcription factor complex [ 3 , 4 , 5 , 6 , 7 ]. Loss-of-function of tmo5 or lhw clade members results in strongly reduced vascular cell file numbers, whereas ectopic expression of both TMO5 and LHW can ubiquitously induce periclinal and radial cell divisions in all cell types of the root meristem. TMO5 and LHW interact only in young xylem cells, where they promote expression of two direct target genes involved in the final step of cytokinin (CK) biosynthesis, LONELY GUY3 ( LOG3 ) and LOG4 [ 8 , 9 ] Therefore, CK was hypothesized to act as a mobile signal from the xylem to trigger divisions in the neighboring procambium cells [ 3 , 6 ]. To unravel how TMO5/LHW-dependent cytokinin regulates cell proliferation, we analyzed the transcriptional responses upon simultaneous induction of both transcription factors. Using inferred network analysis, we identified AT2G28510/DOF2.1 as a cytokinin-dependent downstream target gene. We further showed that DOF2.1 controls specific procambium cell divisions without inducing other cytokinin-dependent effects such as the inhibition of vascular differentiation. In summary, our results suggest that DOF2.1 and its closest homologs control vascular cell proliferation, thus leading to radial expansion of the root.