Regulation of stomatal development by stomatal lineage miRNAs

Abstract

Stomata in the plant epidermis play a critical role in growth and survival by controlling gas exchange, transpiration, and immunity to pathogens. Plants modulate stomatal cell fate and patterning through key transcriptional factors and signaling pathways. MicroRNAs (miRNAs) are known to contribute to developmental plasticity in multicellular organisms; however, no miRNAs appear to target the known regulators of stomatal development. It remains unclear as to whether miRNAs are involved in stomatal development. Here, we report highly dynamic, developmentally stage-specific miRNA expression profiles from stomatal lineage cells. We demonstrate that stomatal lineage miRNAs positively and negatively regulate stomatal formation and patterning to avoid clustered stomata. Target prediction of stomatal lineage miRNAs implicates potential cellular processes in stomatal development. We show that miR399-mediated PHO2 regulation, involved in phosphate homeostasis, contributes to the control of stomatal development. Our study demonstrates that miRNAs constitute a critical component in the regulatory mechanisms controlling stomatal development.

Keywords: PHO2; stomatal development; stomatal lineage miRNA.

Fig. 1.

Profiling of stomatal lineage miRNAs. (A) A diagram showing an expression window of each developmental stage-specific marker in the stomatal lineage. (B) Confocal images of transgenic plants expressing GFP-AGO1DAH under the control of the promoters of the marker genes. AGO1DAH from epidermal and GCs was immunoprecipitated to isolate stomatal lineage miRNAs associated with AGO1. Promoters used: ML1 (epidermal cells), SPCH (MMC, Meristemoid), EPF2 (Meristemoid), MUTE (Meristemoid, GMC), EPF1 (GMC, young GC), and FAMA (GMC, young GC). Cell outlines are visualized using FM4-64. (Scale bars, 20 μm.) (C) The heatmap of MIR gene sequences shows the position of AGO1-associated 21-nt small RNAs isolated from stomatal lineage cells, at each developmental stage, harboring AGO1DAH driven by the promoter of the marker genes. MIR genomic sequences are presented in the 120-bp window with mature miRNA sequences placed in the center (arrowhead). The abundance of aligned reads was normalized to the total mapped reads in the individual samples and further normalized to the maximum abundance among 120-bp window across the six stages. (D) Principal-component analysis (PCA) of 266 AGO1-associated miRNAs in stomatal lineage cells indicates divergence in the miRNA populations of the SPCH–MUTE–FAMA (red arrows) and EPF2–EPF1 (blue arrows) paths.

Fig. 2.

miRNA expression profiles in the SPCH–MUTE–FAMA and EPF2–EPF1 paths during stomatal development. (A) The heatmaps show clusters of DE miRNAs expressed in the stomatal lineage cells. DE miRNAs of the SPCH–MUTE–FAMA (Left) and EPF2–EPF2 (Right) paths were grouped into three developmental stages (stomatal entry, differentiation, and commitment) and two developmental stages (stomatal entry and differentiation), respectively. The color bars of the heatmaps represent the gradient scale of relative log₂-RPM values for each DE miRNA, which was normalized to the minimum and maximum log₂-RPMs across SPCH, MUTE, and FAMA stages (A, Left) as well as EPF2 and EPF1 stages (A, Right). The numbers in parentheses indicate number of miRNAs at each stage. (B) Venn diagrams show overlapping and distinct DE miRNAs between the SPCH–MUTE–FAMA and EPF2–EPF1 paths at the stomatal entry or differentiation stage. (C–E) The heatmaps show the expression levels of miR165a-3p (C), miR157d (D), and miR167c-3p (E) in each of the stomatal lineage cells. Representative confocal images of the epidermis of at least three independent proMIR165a::GFP-GUS (C), proMIR157d::GFP (D), and proMIR167c::GFP-GUS (E) transgenic plants. The arrows indicate cells at each stage of stomatal lineage progression. GC, guard cell; GMC, guard mother cell. Cell outlines are visualized by FM4-64. (Scale bars, 20 μm.)

Fig. 3.

Stomatal lineage miRNAs modulate stomatal formation and patterning. (A–C) Stomatal development phenotypes of transgenic plants in which stage-specific miRNAs were overexpressed or down-regulated. The heatmaps show the expression levels of miR829-5p (A), miR3932 (B), and miR861 (C) in the stomatal lineage cells. Representative confocal images of stomata of at least three independent Col-0 (WT), pro35S::MIR829 (A), pro35S::MIR3932a (B), and STTM-miR861 (C) transgenic plants. Mature GCs are highlighted in blue for elucidation, and the brackets indicate stomatal pairs. Cell outlines are visualized by FM4-64. (Scale bar, 50 μm.) (D) Stomatal density in pro35S:MIR829 transgenic plants compared to WT. The number of GCs per unit area (780 × 780 μm²) was scored from at least 10 seedlings for each line. Error bars represent mean ± SD. Two-sided Student’s t test P values; **P < 0.01. (E and F) Numbers of stomatal pairs in pro35S:MIR2932 and STTM-MIR861 plants compared to WT. Percentage of plants having stomatal pairs per area (780 × 780 μm²) in cotyledons of 10-d-old seedlings. Error bars represent mean ± SD calculated from at least 10 seedlings. Two-sided Student’s t test P values; ****P < 0.0001.

Fig. 4.

Predicted cellular pathways modulated by stomatal lineage miRNAs and anticorrelated DEGs. (A) Anticorrelation between stomatal lineage miRNAs and their predicted target DEGs. GO enrichment-based cellular activities at SPCH, MUTE, and FAMA stages are shown. The numbers in parentheses indicate number of miRNAs at each stage. (B) Regulatory cellular networks consisted of hub DE miRNAs and their predicted target mRNAs at the stomatal entry stage. The gray and light gray lines indicate the predicted miRNA-target gene and known protein–protein interactions, respectively.

Fig. 5.

miR399-mediated regulation of E3 ubiquitin ligase PHO2 guides stomatal development. (A) The heatmap shows the expression levels of miR399b and miR399c-3p in the stomatal lineage cells. (B) Anticorrelation in expression levels of miR399b and PHO2 during stomatal development. (C) Representative confocal images show stomatal development phenotypes of at least three independent WT, pro35S:MIR399, and pho2 plants. Mature GCs are highlighted in blue for elucidation, and the brackets indicate stomatal pairs. Cell outlines are visualized by FM4-64. (Scale bar, 50 μm.) (D) Stomatal density is increased in pro35S:MIR399 and pho2 plants compared to WT plants. The number of GCs per unit area (780 × 780 μm²) in cotyledons of 10-d-old seedlings. Error bars represent mean ± SD calculated from at least 10 plants. Two-sided Student’s t test P values: ***P < 0.001. (E) Numbers of stomatal pairs in pro35S:MIR399 and pho2 plants compared to WT. Percentage of plants having stomatal pairs per unit area (780 × 780 μm²) in cotyledons of 10-d-old seedlings. Error bars represent mean ± SD calculated from at least 10 seedlings. Two-sided Student’s t test P values; **P < 0.01; ***P < 0.001. (F) Expression levels of the key regulators of stomatal development in 4-d-old WT and pro35S:MIR399 seedlings. The expression levels were normalized to ACTIN2. Error bars represent mean ± SD calculated from three independent biological repeats. Two-sided Student’s t test P values; *P < 0.05. (G) miRNA399-guided 3′ cleavage products of PHO2 mRNA were detected in pro35S:MIR399 plants. The arrowheads indicate the miRNA-guided cleavage products. ARF10 and UBQ5 were used as internal controls. The arrow above the sequences indicates the cleavage site verified from 10 out of 10 clones sequenced.