Cell signalling by microRNA165/6 directs gene dose-dependent root cell fate

Abstract

A key question in developmental biology is how cells exchange positional information for proper patterning during organ development. In plant roots the radial tissue organization is highly conserved with a central vascular cylinder in which two water conducting cell types, protoxylem and metaxylem, are patterned centripetally. We show that this patterning occurs through crosstalk between the vascular cylinder and the surrounding endodermis mediated by cell-to-cell movement of a transcription factor in one direction and microRNAs in the other. SHORT ROOT, produced in the vascular cylinder, moves into the endodermis to activate SCARECROW. Together these transcription factors activate MIR165a and MIR166b. Endodermally produced microRNA165/6 then acts to degrade its target mRNAs encoding class III homeodomain-leucine zipper transcription factors in the endodermis and stele periphery. The resulting differential distribution of target mRNA in the vascular cylinder determines xylem cell types in a dosage-dependent manner.

Figure 1. Endodermal SHR and SCR control xylem patterning via PHB

a, Schematic representation of the Arabidopsis root meristem and stele. Cells in the ground tissue layer adjacent to the stele are marked with asterisks. b, CRE1::SHRΔNLELDV:nlsGFP in shr-2. c, UAS::SHR:YFP in shr-2 harbouring J0571. d, UAS::SCR:YFP in shr-2, J0571. e–i, Toluidine blue-stained cross-sections and confocal laser scanning micrographs of basic fuchsin-stained xylem of wild type (WT) (e), shr-2 (f), scr-4 (g), phb-7d (h), and phb-6 shr-2 (i). Filled arrowhead indicates metaxylem, and unfilled indicates protoxylem. Scale bars, 10 μm.

Figure 2. SHR post-transcriptionally represses PHB

a, b, In situ hybridization with a PHB mRNA specific probe on cross- and longitudinal sections of WT (a) and phb-7d (b) roots. c–e, In situ hybridization with CNA (c), ATHB8 (d) and REV (e) specific probes on cross-sections of WT roots. f, Confocal laser scanning micrographs of transcriptional fusion of PHB to GFP in WT and shr-2. g, PHB mRNA in situ hybridization to cross-section of shr-2. Inset is a section of shr-2 hybridized with a PHB sense probe. h, Expression of translational fusion of PHB to GFP in WT and shr-2. i, Expression of translational fusion of PHB with the phb-7d mutation to GFP. Asterisks, endodermis position; arrowheads, protoxylem position; scale bars, 10 μm.

Figure 3. miR165/6 activated by SHR inthe endodermis is active in the stele

a, Expression of pMIR165a::GFP in WT and scr-1 meristems and in maturation zone. b, Expression of pMIR166b::GFP in WT, scr-1 and shr-2 meristems. c, Real-time PCR of ChIP on the upstream regulatory regions of MIR165a and MIR166b using anti-GFP antibodies and a transgenic plant expressing pSHR::SHR:GFP. The binding to the MAGPIE (MGP) promoter confirmed previously was used as positive control. Asterisks, endodermis position.

Figure 4. Non-cell-autonomous action of MIR165a.

a, miR165/6 GFP sensor under the U2 promoter in WT and shr-2. b, miR166-specific LNA probe hybridization to sections proximal to the quiescent centre of WT, athb8-11 cna-2 phb-13 phv-11 rev-6 and shr-2. c, Protoxylem forms in shr and scr backgrounds when UAS::MIR165a is introduced into shr-2 and scr-4 harbouring J0571. d, Real-time RT–PCR of pri-MIR165/6 and HD-ZIP III in WT and a line with UAS::MIR165a in shr-2, J0571. n 54. Error bars indicate ±s.d. Asterisks, endodermis/ground tissue position; arrowheads, protoxylem position; scale bars, 10 μm.

Figure 5. HD-ZIP III levels determine xylem type

a, Basic fuchsin-stained xylem and cross-section of cna-2 phb-13 phv-11 rev-6. b, Cleared root and cross-section of athb8-11 cna-2 phb-13 phv-11 rev-6. c, d, Stained xylem of roots in which MIR165a is expressed from the CRE1 promoter in WT and shr-2. Asterisks, endodermis position; arrowheads, protoxylem position; scale bars, 10 μm.