Regulation of MIR165/166 by class II and class III homeodomain leucine zipper proteins establishes leaf polarity

Abstract

A defining feature of plant leaves is their flattened shape. This shape depends on an antagonism between the genes that specify adaxial (top) and abaxial (bottom) tissue identity; however, the molecular nature of this antagonism remains poorly understood. Class III homeodomain leucine zipper (HD-ZIP) transcription factors are key mediators in the regulation of adaxial-abaxial patterning. Their expression is restricted adaxially during early development by the abaxially expressed microRNA (MIR)165/166, yet the mechanism that restricts MIR165/166 expression to abaxial leaf tissues remains unknown. Here, we show that class III and class II HD-ZIP proteins act together to repress MIR165/166 via a conserved cis-element in their promoters. Organ morphology and tissue patterning in plants, therefore, depend on a bidirectional repressive circuit involving a set of miRNAs and its targets.

Keywords: MIR165/166; class II HD-ZIP; class III HD-ZIP; leaf morphogenesis; organ patterning.

Fig. 1.

HAT3 and ATHB4 are required for repressing MIR165/166 expression. (A–D) Expression of pREV::REV-2xYPET (red) in combination with the auxin efflux carrier PIN-FORMED1 (30, 31) fused to GFP (pPIN::PIN1-GFP) (blue) in the shoot apex of 4-d-old control (A and C) and hat3 athb4 plants (B and D). pPIN::PIN1-GFP is used here to outline the tissue. (C and D) Cross-sections of the same leaf primordia shown in A and B, respectively. (E–H) Expression of pMIR165a::mTagBFP-ER (green) in the shoot apex of 4-d-old control (E and G) and hat3 athb4 plants (F and H). (G and H) Cross-sections of the same leaf primordia shown in E and F, respectively. (I–L) Expression of a miR165/166 sensitive biosensor (white) containing the miRNA target sequence from REV fused to the UV-photoconvertible fluorescent protein mEos2FP (pUBQ10::REV-mEos2FP-ER) in the shoot apex of 4-d-old control (I and K) and hat3 athb4 plants (J and L). The sensitive biosensor is inactivated in cells where miR165/166 are active. (K and L) Cross-sections of the same leaf primordia shown in I and J, respectively. Chlorophyll autofluorescence: red (E–L). (Scale bars, 50 μm.) Ad, adaxial side; Ab, abaxial side. (M) Small RNA Northern blot showing expression levels of miR165/166 and U6 snRNA in Col-0 WT (lane 1), p35S::miR165a (lane 2), hat1 hat2 (lane 3), and hat3 athb4 plants (lane 4).

Fig. 2.

pREV::REVr-2xVENUS rescues the hat3 athb4 and hat3 athb4 athb2 leaf phenotype. (A–C) Expression pattern of pREV::REVr-2xVENUS (green) in the shoot apex of 4-d-old control (A), hat3 athb4 (B), and hat3 athb4 athb2 plants (C). (D) Expression of a control REV translational reporter (pREV::REV-2xVENUS) (green) in the shoot apex of 4-d-old hat3 athb4 athb2 plants (control). (E–H) Phenotype of 15-d-old control (E), hat3 athb4 (F), and hat3 athb4 athb2 plants (G) transformed with pREV::REVr-2xVENUS. (H) Phenotype of 15-d-old hat3 athb4 athb2 plants transformed with pREV::REV-2xVENUS. Chlorophyll autofluorescence: red (A–D). [Scale bars, 50 μm (A–D) and 5 mm (E–H).]

Fig. 3.

HAT3 and ATHB4 physically interact with REV. (A) Levels of mature miR165/166 in pML1>>VENUS-HAT3 plants. Fold changes relative to ACTIN2 (ACT2; AT3G18780) in response to DEX treatment (+DEX; green bars) and in control conditions (0.1% ethanol; −DEX; gray bars) are shown. Data are represented as mean ± SD of three biological replicates. *P ≤ 0.05. Phenotype of 10-d-old pML1>>VENUS-HAT3 plants grown on DEX-GM and control medium is shown. [Scale bars, 1 mm (A).] (B–E) Expression of pHAT3::VENUS-HAT3 (red) (B and D) and pATHB4::VENUS-ATHB4 (green) (C and E) combined with pMIR165a::GFP-ER (blue) in the shoot apex of 3-d-old Col-0 plants. (D and E) Cross-sections of the same leaf primordia shown in B and C, respectively. Colocalization of pHAT3::VENUS-HAT3/pATHB4::VENUS-ATHB4 and pMIR165a::GFP-ER is indicated by arrowheads in D and E, respectively. [Scale bars, 50 μm (B–E).] (F) REV and HAT3 interaction in a yeast two-hybrid assay. Growth of yeast on selective medium (−Trp, −Leu, −His, and +3-AT) for REV–HAT3 combination indicates protein–protein interaction. Five transformed colonies per prey/bait combination were analyzed for their growth on −Trp, −Leu, −His, and +3-AT plates as well as on −Trp, −Leu, +His, and +3-AT plates using dilution series (1:1, 1:5, 1:10, and 1:50). AD, activation domain; BD, binding domain. (G) REV–HAT3 interaction in nuclei of tobacco leaf epidermal cells detected through a FRET–FLIM assay. GFP–REV (green) works as a donor and RFP–HAT3 (red) as an acceptor. GFP–REV (green) x RFP (red) and GFP–REV (green) x RFP–ZPR3 (red) combinations were used as negative and positive controls, respectively. GFP fluorescence lifetime (in nanoseconds, ns) quantification is shown. Error bars show mean ± SE of 10 nuclei.

Fig. 4.

REV requires HAT3 and ATHB4 to repress MIR165/166 expression. (A and C) Expression of pMIR166a::GFP-ER (blue) after MIR165a expression driven by the UBQ10 promoter in the shoot apex of Col-0 plants 4 d after germination on DEX-GM medium. (C) Cross-section of the same leaf primordia shown in A. (B and D) Phenotype of 7-d-old plants expressing MIR165a under the UBQ10 promoter. (E–H) Expression of pHAT3::VENUS-HAT3 (red) (E and G) or pATHB4::VENUS-ATHB4 (yellow) (F and H) combined with pMIR165a::GFP-ER (purple) after MIR165a expression driven by the UBQ10 promoter in the shoot apex of Col-0 plants 4 d after germination on DEX-GM medium. (G and H) Cross-sections of the same leaf primordia shown in E and F, respectively. Colocalization of pHAT3::VENUS-HAT3/pATHB4::VENUS-ATHB4 and pMIR165a::GFP-ER is indicated by arrowheads in G and H, respectively. (I–L) Expression of REVr-2xVENUS, which is miR165/166 resistant, driven by the ML1 promoter (green) and pMIR165a::BFP-ER (purple) in the shoot apex of control (I and K) and hat3 athb4 plants (J and L) 4 d after germination on DEX-GM medium. (K and L) Cross-sections of the same leaf primordia shown in I and J, respectively. Colocalization of pML1>>REVr-2xVENUS and pMIR165a::BFP-ER is indicated by an arrowhead in L. Chlorophyll autofluorescence: red (A and C). [Scale bars, 50 μm (A, C, and E–L) and 1 mm (B).]

Fig. 5.

REV and HAT3 bind a conserved cis-element required to restrict MIR165a expression. (A–C) Expression of pML1>>REVr-2xVENUS (green), pMIR165a::GFP-ER (purple), and pMIR165a(−cis)::BFP-ER (blue) in the shoot apex of 7-d-old Col-0 plants 2 d after transferring to 0.1% ethanol (mock) (A and C) or DEX-GM medium (B). Longitudinal sections of the second pair of leaves are shown (A–C). Colocalization of pML1>>REVr-2xVENUS and pMIR165a(−cis)::BFP-ER is indicated by an arrowhead in B. (D) Phenotype of 10-d-old transgenic pML1>>REVr-2xVENUS_ pMIR165a::GFP-ER_pMIR165a(−cis)::BFP-ER plants grown on DEX-GM medium. [Scale bars, 50 μm (A–C) and 1 mm (D).] Ad, adaxial side; Ab, abaxial side (A). (E) Levels of mature miR165/166 in p35S::GR-REVr plants (14). Fold changes relative to ACT2 in response to DEX treatment (+DEX; green and red bars) and in control conditions (0.1% ethanol; −DEX; gray bars) are plotted. Green bars show expression changes in the absence of the protein biosynthesis inhibitor CHX; red bars show expression changes in the presence of CHX (+CHX). HAT3, ATHB4, ZPR3, and AT1G20823 were tested as known direct or indirect REV targets. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. (F) ChIP-qPCR on genomic regions surrounding MIR165a using anti-GFP antibody and the DEX-inducible transgenic lines pML1>>VENUS-HAT3 and pML1>>REVr-2xVENUS. A diagram of the MIR165a genomic region is shown. The black lines, red line, and green box represent the regions amplified by ChIP-qPCR, the cis-element (25), and the MIR165a locus, respectively. ChIP-qPCR data were normalized to the percent of preimmunoprecipitation (pre-IP) input for each sample. Error bars show means ± SD of three biological replicates treated with 10 μM DEX (+DEX; yellow and blue bars) or mock (0.1% ethanol; −DEX; gray bars). ACT2 and ZPR3 were also tested as negative and positive controls, respectively (26, 27). *P ≤ 0.05; **P ≤ 0.01. (G) Interaction of REV and HAT3/ATHB4 with the MIR165a cis-element in a yeast one-hybrid assay. Growth of yeast on selective medium (−Ura, −Trp, −Leu, −His, and +3-AT) for REV–HAT3 and REV–ATHB4 combinations indicates protein–DNA interaction. A mutated version of the cis-element, previously referred to as M1 (25), was used as a negative control. Three transformed colonies per prey/bait combination were analyzed for their growth on −Ura, −Trp, −Leu, −His, and +3-AT selection plates using dilution series (1:5, 1:10, 1:20, 1:50). AD, activation domain.

Fig. 6.

A regulatory network involving MIR165/166, class II and class III HD-ZIP genes controls adaxial–abaxial patterning of the leaf. P1 and P2, leaf primordia; SAM, shoot apical meristem.