Vascular transcription factors guide plant epidermal responses to limiting phosphate conditions

Roots primed for better phosphate uptake Phosphate is a key resource for plants, and remediating phosphate deficiency drives considerable fertilizer use. In low-phosphate conditions, roots make more root hairs, which makes them better able to take up what little phosphate can be found. Wendrich et al. performed single-cell transcriptomics on the developing Arabidopsis root and queried the resulting gene-expression atlas for responses related to vascular development. The authors found that signals regulating root hair development began in the inner vasculature of the root with transcription factors that drove the production of the hormone cytokinin. Response cascades identified through the transcriptome database pointed to genes in epidermal cells that regulate root hair development. Science, this issue p. eaay4970 Single-cell transcriptomics uncovers a hormone signaling pathway that drives development of root hairs in search of phosphate. INTRODUCTION In plants, vascular tissues serve a dual function by providing both structural support and transport of water, nutrients, hormones, and other signaling molecules throughout the plant body. Proliferation of vascular tissues is, in part, controlled by the TARGET OF MONOPTEROS 5/LONESOME HIGHWAY (TMO5/LHW) transcription factor complex and involves intricate cell communication through mobile factors. The activity of this heterodimer complex is limited to young xylem cells and is required and sufficient to control vascular cell proliferation by inducing expression of rate-limiting enzymes in the cytokinin biosynthetic pathway. Because cytokinin is mobile, the molecular and developmental responses to this hormone signaling cascade are likely to have an effect in various cell types surrounding the xylem and perhaps even outside the vascular bundle. To what extent this is the case and how this controls developmental processes other than vascular proliferation remain unknown. RATIONALE By intersecting a high-resolution single-cell gene expression atlas with TMO5/LHW target genes, we probed the tissue-specific distribution of transcriptional responses upon induction of this transcription factor complex in the Arabidopsis root meristem. We next used a combination of genetic and molecular tools to validate the existence and importance of a cytokinin-dependent signaling mechanism bridging the inner vascular tissues and the outer epidermis cell layers. RESULTS We generated a single-cell gene expression atlas that represents all known cell types and sub–cell types of the Arabidopsis root apical meristem. Cell-type annotations and developmental trajectories were validated through expression of known marker genes, newly generated promoter reporter lines, and analysis of the endoreduplication status in each cell cluster. We next intersected this expression atlas with a set of known target genes of the vascular TMO5/LHW transcription factor complex and found that a large portion of the target genes are expressed in, and are even restricted to, the outermost root hair cells. This suggests a possible involvement of the TMO5/LHW complex in root hair development. In line with this hypothesis, misexpression of TMO5 and LHW in the entire root meristem was found to increase root hair density, a phenotype resembling wild-type roots grown under phosphate-limiting conditions. Moreover, the response of epidermal cells to phosphate deficit was shown to be TMO5-dependent. Phosphate-limiting conditions were found to induce TMO5 expression, and genetically increasing TMO5 expression only in the vascular bundle is sufficient to trigger epidermal responses. These results indicate that the TMO5-dependent increase in root hair density under low-phosphate conditions occurs through a cell nonautonomous effect. TMO5/LHW-dependent vascular cytokinin biosynthesis was found to be sufficient to increase epidermal root hair density because mutants with reduced cytokinin levels are less sensitive to the effects of phosphate-limiting conditions on root hair density. Furthermore, phosphate-limiting conditions increase cytokinin signaling in epidermal cells of wild-type roots, and treatment with exogenous cytokinin can restore root hair densities back to wild-type levels in the absence of TMO5 activity. This suggests that cytokinin is the mobile signal linking TMO5/LHW activity in vascular cells to root hair density changes in response to phosphate deficit. The TMO5 and cytokinin-dependent root hair density increase under low-phosphate conditions was found to originate not only from changes in epidermal cell length but also from changes in cell identity because both phosphate-limiting conditions and exogenous cytokinin treatment were shown to scramble identities of the otherwise highly organized epidermal cells. CONCLUSION Through its effect on cytokinin biosynthesis, the vascular TMO5/LHW heterodimer complex controls epidermal root hair density by modifying cell length and cell identity. We hypothesize that phosphate-limiting conditions may trigger increased auxin signaling in xylem cells, inducing activity of the TMO5/LHW pathway and downstream local cytokinin biosynthesis. Cytokinin may then diffuse outward from the vasculature to direct both length and fate of the outer trichoblast cells. This hormone signaling cascade spans multiple tissue layers, allowing roots to efficiently forage the soil for phosphate. Vascular transcription factors guide epidermal responses. A validated high-resolution single-cell gene expression atlas of the Arabidopsis root was intersected with TMO5/LHW target genes, uncovering an enrichment of epidermal-restricted expression patterns. By activating local cytokinin biosynthesis, the vascular TMO5/LHW complex was shown to regulate epidermal root hair density in response to the availability of phosphate. scRNA-seq, single-cell RNA-sequencing; mock, untreated condition; DEX, dexamethasone; Pi, inorganic phosphate. 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. Cytokinin signaling links root hair responses in the epidermis to perception of phosphate depletion in vascular cells.