Vascular transcription factors guide plant epidermal responses to limiting phosphate conditions

Abstract

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.

Figure 1. Identification of Arabidopsis root meristem cell types using scRNA-seq A.

Color-coded tSNE plot showing the classification of 5,145 high quality (UMI count > 17,290) cells into distinct cell identities corresponding to the schematic representation of the root meristem on the left. Grey dots represent predicted doublet cells. All inferred and validated developmental trajectories are projected onto the tSNE plot as black lines. Cells within dotted line are initials. QC: quiescent center, ppc: phloem procambium, se: sieve element, cc: companion cell, px: protoxylem, mx: metaxylem. B. Dot plot showing the expression of known tissue specific reporter genes in the scRNA-seq dataset, validating the annotation of tissue specific clusters. Size of the circles represents the percentage of cells with expression (pct.exp.), while the color indicates the scaled average expression (avg exp. scale). Dotted boxes represent major tissue types and cellular stages present in the root. C. Projection of predicted ploidy levels of each cell onto the tSNE plot.

Figure 2. TMO5 activity is required for root hair responses to low phosphate conditions

A. Number of TMO5/LHW target genes expressed in each of the tissue types of the Arabidopsis root meristem. Note the high number of trichoblast expressed genes. B. Predicted (left) and validated (right) expression of root hair specific target genes in the root hair. Arrowheads indicate nuclear expression. C. Root hair phenotype of dGR (induced or non-induced with dex) and wild type, tmo5 single mutant and tmo5 tmo5like1 double mutants grown on control conditions (high phosphate) or phosphate limiting conditions (low phosphate). D-E. Quantification of the root hair density (D) and epidermal cell length (E) of the lines depicted in C. Lower case letters on top of boxplots indicate significantly different groups as determined by one-way ANOVA with post-hoc Tukey HSD testing (p<0.001); the number of individuals is shown at the bottom of the plot and biological repeats are indicated using different symbols.

Figure 3. Vascular TMO5/LHW expression increases root hair density

A-B. Expression (A) and quantification (B) of the pTMO5::n3GFP reporter line in the root meristem under high (HP) and low (LP) phosphate conditions by confocal microscopy. C-E. Root hair phenotype and quantification of wild type, pTMO5::TMO5:GR, pSHR::TMO5:GR and pWOL::XVE>>TMO5:YFP roots grown on high phosphate conditions or induced by dexamethasone or estradiol (see Fig 2 for wild type control). Lower case letters on top of boxplots indicate significantly different groups as determined by one-way ANOVA with posthoc Tukey HSD testing (p<0.001 in C and E, p<0.01 in D); the number of individuals is shown at the bottom of the plot and biological repeats are indicated using different symbols.

Figure 4. TMO5/LHW dependent cytokinin triggers root hair responses

A. Expression and quantification of pTCSn::ntdTomato in the root meristem under high (HP) and low (LP) phosphate conditions by confocal microscopy. Asterisks indicates epidermal cell layer. B. Root hair phenotype and quantification of wild type and tmo5 tmo5like1 roots grown on high phosphate conditions or induced by cytokinin (BAP). C. Root hair phenotype and quantification of wild type and log347 and pRPS5A::CKX3 roots grown on high or low phosphate conditions. D. Root hair phenotype and quantification of pWOL::XVE>>LOG4:YFP roots grown high phosphate conditions or induced by estradiol (see Fig 3 for wild type control). Lower case letters on top of boxplots indicate significantly different groups as determined by one-way ANOVA with post-hoc Tukey HSD testing (p<0.001); the number of individuals is shown at the bottom of the plot and biological repeats are indicated using different symbols.

Figure 5. Cytokinin scrambles epidermal cell identities

A. Expression of pCOBL9::GFP and pGL2::GFP in roots grown in high phosphate (HP), cytokinin (BAP), or low phosphate (LP). B-C. Quantification of number of cells per mm of root in non-hair (NH) or hair (H) positions with GFP expression. D-E. Quantification of the number of cells in non-hair position that form root hairs, along one mm of root from dGR, wild type, tmo5, tmo5 tmo5l1, log347 or pRPS5A::CKX3 grown under indicated conditions. Lower case letters on top of the boxplots indicate significantly different groups as determined by one-way ANOVA with post-hoc Tukey HSD testing (p<0.001); the number of individuals is shown at the bottom of the plot and biological repeats are indicated using different symbols.