Direct roles of SPEECHLESS in the specification of stomatal self-renewing cells

Abstract

Lineage-specific stem cells are critical for the production and maintenance of specific cell types and tissues in multicellular organisms. In Arabidopsis, the initiation and proliferation of stomatal lineage cells is controlled by the basic helix-loop-helix transcription factor SPEECHLESS (SPCH). SPCH-driven asymmetric and self-renewing divisions allow flexibility in stomatal production and overall organ growth. How SPCH directs stomatal lineage cell behaviors, however, is unclear. Here, we improved the chromatin immunoprecipitation (ChIP) assay and profiled the genome-wide targets of Arabidopsis SPCH in vivo. We found that SPCH controls key regulators of cell fate and asymmetric cell divisions and modulates responsiveness to peptide and phytohormone-mediated intercellular communication. Our results delineate the molecular pathways that regulate an essential adult stem cell lineage in plants.

Fig. 1. Chromatin immunoprecipitation (ChIP) optimized for cell-type-specific studies in vivo

(A) Arabidopsis stomatal development scheme. SPCH controls the initiation and proliferation of the stem cell-like stomatal lineage precursors (pink and red cells). (B) Model for improving target enrichment in ChIPs through increasing experimental scale. (C and D) ChIPs at larger scales improve target enrichments. ChIP-qPCR assays of a SPCH variant on the TMM promoter performed at the indicated conditions (C). SPCH ChIP-Seq profiles at TMM (D) generated from ChIPs at 4× and 16× (blue and red box in C, respectively). The y-axis represents the enrichment values; note scales. Dashed box marks the SPCH-binding region.

Fig. 2. Genome-wide analysis of SPCH-binding targets reveals direct roles in lineage specification and asymmetric cell divisions

(A) Distribution of SPCH-binding peaks relative to gene structure. (B) Top-scoring motif (E-value: 7.5×10⁻³⁶⁵) and the position of its three variants in SPCH-binding peaks. (C and D) Percentage of SPCH targets among differentially-expressed genes in RNA-Seq analysis of inducible SPCH1-4A (C), and microarray analysis of meristemoid–enriched (scrmD mute) or –depleted (spch) mutants (13) (D). P-values are calculated by Fisher’s exact test. Dashed line indicates percentage by chance. (E) Select enriched GO terms of SPCH target genes. (F and G) SPCH binds and activates key stomatal regulators. ChIP-Seq profiles of select stomatal genes (F). The y-axis represents peak score (CSAR) and arrows indicate gene orientation and transcriptional start sites. Gene expression changes upon induction of SPCH in RNA-Seq analysis (G). (H) Importance of SPCH-binding motif (red) on ICE1 expression. Mutation of two motifs (purple; mICE1pro) within the SPCH-binding peak (blue shading) abrogates ICE1 expression (yellow). Confocal images of 4-dpg abaxial cotyledons have ML1pro:mCherry-RCI2A-marked cell outlines (purple). Scale bar, 40 μm.

Fig. 3. SPCH regulates asymmetric cell division (ACD) through a preprophase band-localized kinesin

(A) SPCH ChIP-Seq profile of ARK3/KINUa. (B to E) Expression of ARK3pro:ARK3-YFP (yellow) and its co-expression with SPCHpro:SPCH-CFP (blue) (C to E only) before (C), during (B and D) and after (E) a stomatal ACD. Arrowheads indicate the preprophase band. (F to I) ACD defects in SPCHpro:amiR-ark3 (G and I), compared to Col (F and H). Brackets mark clusters of small cells (G) or guard cells (I). Confocal images are of 3- (B to E), 4- (F and G) and 11-day (H and I) abaxial cotyledons with ML1pro:mCherry-RCI2A-marked cell outlines. Scale bars, 10 μm (C to E), 50 μm (F to I).

Fig. 4. Feedback regulation of brassinosteroid biosynthesis and signaling by SPCH

(A) ChIP-Seq profiles of select brassinosteroid (BR) pathway genes (labeled as in Fig. 2F). (B) RT-qPCR analysis of BR genes in 4-dpg SPCHpro:SPCH2-4A-YFP and Col seedlings. Values are means+/−SEM. (C & D) Confocal images of 3-dpg adaxial cotyledons with propidium iodide-stained cell outlines (purple). Stomatal lineage-specific expression of hyperactive BIN2 (yellow) induces lineage proliferation (bracket) (C). Stomatal lineage expression pattern of BIM2pro:YFP-YFP (yellow) (D). (E) Alteration of stomatal density in gain-of-function BES1pro:bes1-D and bes1-RNAi knockdown. *: p<0.05, ***: p<0.001 (Wilcoxon ranksum test). (F) Model of SPCH-BR pathway interactions. SPCH, a target of BR signaling, feeds back (positively, red arrows or negatively, red T-bars) upon transcription of multiple pathway members.