Volume 8, Issue 5, Pages 783-795 (May 2015) Phosphorylation of Serine 186 of bHLH Transcription Factor SPEECHLESS Promotes Stomatal Development in Arabidopsis  Ke-Zhen Yang, Min Jiang, Ming Wang, Shan Xue, Ling-Ling Zhu, Hong-Zhe Wang, Jun-Jie Zou, Eun-Kyoung Lee, Fred Sack, Jie Le  Molecular Plant  Volume 8, Issue 5, Pages 783-795 (May 2015) DOI: 10.1016/j.molp.2014.12.014 Copyright © 2015 The Author Terms and Conditions

Figure 1 SPCH Function Depends on the Expression of CDKA;1. (A–D) Cotyledon growth is strongly inhibited in a severe cdka;1 mutant seedling from line 14 (A) and slightly less so in the weak line 11 (C). Stomata are absent from the cotyledon epidermis in line 14 (B). A few stomata can be found in the cotyledon epidermis in the weak transgenic line 11 (D). Arrows indicate stomata. (E and F) TMMpro:TMM-GFP is expressed in stomatal lineage cells in the wild-type (E) but not in the cdka;1 (F) cotyledon epidermis. (G and H) BASLpro:GFP-BASL expression in cotyledons is present in the wild-type (G) but not in the cdka;1 (H) cotyledon epidermis. (I–K) Differential Interference Contrast (DIC) images of the cotyledon epidermis in wild-type and transgenic plants harboring the dominant-negative CDKA;1.N146 construct driven by either the SPCH or TMM promoters. Compared with the wild-type (I), SPCHpro:CDKA;1.N146 (J) and TMMpro:CDKA;1.N146 (K) display reduced stomatal production. (L) Real-time PCR shows a slight reduction (about a 20%–30%) in SPCH transcription levels in cdka;1 transgenic lines 14 and 11. (M) A wild-type cotyledon showing fluorescence from a SPCHpro:nucGFP transcriptional fusion construct in the stomatal lineage as well as in adjacent young cells. (N) In cdka;1, SPCHpro:nucGFP fluorescence persists in most epidermal cells. (O) Fluorescence from a SPCHpro:SPCH-GFP translational fusion construct in the wild-type epidermis is present mostly in meristemoids (arrowheads). (P) cdka;1 epidermis showing the absence of SPCHpro:SPCH-GFP fluorescence. Scale bars represent 100 μm in A and C; 20 μm in B, D, E–H, I–K, and M–P. Molecular Plant 2015 8, 783-795DOI: (10.1016/j.molp.2014.12.014) Copyright © 2015 The Author Terms and Conditions

Figure 2 CDKA;1 Directly Interacts with SPCH. (A) SPCH-GFP protein was not detected in Western blots from cdka;1 seedlings harboring SPCHpro:SPCH-GFP that were grown on BR-supplemented medium (1 μM eBL). The bottom box shows the loading controls after Coomassie brilliant blue (CBB) gel staining. (B and C) The CDKA;1.N146 protein binds SPCH in yeast two-hybrid (B) and BiFC assays (C). Scale bars represent 20 μm. Molecular Plant 2015 8, 783-795DOI: (10.1016/j.molp.2014.12.014) Copyright © 2015 The Author Terms and Conditions

Figure 3 SPCH Is Phosphorylated by the CDKA;1 Complex. (A) Schematic diagram of phosphorylation sites in the SPCH protein. Red letters indicate three predicted phosphorylation sites by CDKA;1 in the SPCH protein. Red squares mark BIN2 phosphorylation target sites. Green triangles indicate MPK phosphorylation target sites. Deletions are shown as solid underline for SPCH Δ93, long-dashed underline for SPCH Δ49, and a short-dashed underline for SPCH Δ31 (according to Lampard et al., 2008). (B) Autoradiographs show that SPCH was phosphorylated by active CDKA;1 complexes (CDKA;1-CYCD3;1-CDKF;1). By contrast, the MUTE and FAMA proteins were not phosphorylated by CDKA;1. (C and D) The extent of phosphorylation of the mutated SPCH proteins, SPCHSPKR(65-68)A/Δ186-222 (C) and SPCHS186A (D), was obviously reduced. The bottom boxes in (B–D) show the loading controls after Coomassie brilliant blue gel staining. Molecular Plant 2015 8, 783-795DOI: (10.1016/j.molp.2014.12.014) Copyright © 2015 The Author Terms and Conditions

Figure 4 Expression of Different SPCH Variants Induces Distinct Epidermal Phenotypes. (A–F) DIC images from mature leaves of an spch-4 mutant transformed with SPCH variants. Stomatal defects in the spch-4 allele are rescued by SPCHpro:SPCHSPKR(65-68)A (A) and the SPCHpro:SPCHSPYR(219-222)A (B), but not by SPCHpro:SPCHSPRK(186-189)A (C), SPCHpro:SPCHSPKR(65-68)A/SPRK(186-189)A (D), SPCHpro:SPCHSPKR(65-68)A/Δ186-222 (E), or SPCHpro:SPCHS186A (F). (G and H) DIC images of the epidermis of mature leaves of wild-type plants transformed with SPCH variants driven by the SPCH native promoter. Transformation with SPCHpro:SPCHSPRK(186-189)A (H) and SPCHpro:SPCHSPKR(65-68)A/Δ186-222 (I) induces excess cell divisions but does not induce the formation of extra stomata. Scale bars represent 20 μm. (I) Schematic diagram shows the SPCH variants. Molecular Plant 2015 8, 783-795DOI: (10.1016/j.molp.2014.12.014) Copyright © 2015 The Author Terms and Conditions

Figure 5 Phosphorylation at Serine 186 Promotes Stomatal Production. (A) Extensive clusters arise in 35pro:SPCHΔ93. (B) Expression of 35Spro:SPCHS/T38-44A induces ectopic symmetric divisions in pavement cells, indicated by green arrows. (C) Stomatal clusters in 35pro:SPCHS/T38-44A/Δ93. (D) Stomatal clusters in 35Spro:SPCHS186D line 1, indicated by red arrows. (E) No stomatal clusters found in 35Spro:SPCHS186D line 4. (F) Expression of 35Spro:SPCHS186D rescued the stomatal phenotypes in SPCHpro:CDKA;1.N146. See the quantitative analysis in Table 2. Scale bars represent 20 μm. Molecular Plant 2015 8, 783-795DOI: (10.1016/j.molp.2014.12.014) Copyright © 2015 The Author Terms and Conditions

Figure 6 Expression of Different SPCH Variants Induces Distinct Epidermal Phenotypes. (A–C) DIC images from mature leaves of wild-type plants transformed with 35Spro:SPCH (A), 35Spro:SPCHSPKR(65-68)A (B), and 35Spro:SPCHSPYR(219-222)A (C). Green arrows indicate ectopic symmetric divisions in pavement cells. (D and E) Besides symmetric divisions in pavement cells, expression of 35Spro:SPCHSPRK(186-189)A (D) and 35Spro:SPCHS186A (E) also induces excess physically asymmetric divisions. Red stars mark precursor-like cells. (F and G) Most precursor-like cells in a 35Spro:SPCHS186A epidermis fail to express the stomatal lineage marker TMMpro:TMM-GFP (F) as well as MUTEpro:MUTE-GFP (G). (H–J) DIC images from mature leaves of 35Spro:SPCHSPKR(65-68)A/SPRK(186-189)A (H), 35Spro:SPCHSPKR(65-68)A/SPRK(186-189)A/SPYR(219-222)A (I), and 35Spro:SPCHSPKR(65-68)A/Δ186-222 (J). Red stars mark precursor-like cells. (K and L) Excessive divisions produce extra small cells in the 35Spro:SPCHSPKR(65-68)A/Δ186-222 epidermis. However, most of the precursor-like cells fail to express TMMpro:TMM-GFP (K) and MUTEpro:MUTE-GFP (L). Brackets in (F) and (K) indicate patchy expression of TMMpro:TMM-GFP. Arrowheads in (G) and (L) indicate occasional MUTEpro:MUTE-GFP fluorescence in GMCs. Arrows point to GFP-negative precursor-like cells. Scale bars represent 20 μm. Molecular Plant 2015 8, 783-795DOI: (10.1016/j.molp.2014.12.014) Copyright © 2015 The Author Terms and Conditions

Figure 7 Schematic Diagram of Stomatal Development and Its Regulatory Pathway. The bHLH transcriptional factor SPEECHLESS (SPCH) controls entry into the stomatal cell lineage by first forming a Meristemoid Mother Cell (MMC). The MMC divides asymmetrically, yielding a Meristemoid (M) and a larger sister cell. SPCH is a phosphorylation target of a signal transduction cascade that includes MAPKKK (YODA), MKK4/5, and MPK3/6. Brassinosteroid (BR) regulates stomatal initiation via BIN2 kinase, which directly or indirectly (via MAPK cascade) phosphorylates SPCH. CDKA;1 promotes stomatal initiation by enhancing SPCH activity. Levels of SPCH transcription are likely regulated by RBR, which is targeted by CDKA;1. MUTE is a key bHLH transcription factor required for the fate transition from M to Guard Mother Cell (GMC). FAMA, which is also a bHLH transcription factor, as well as the R2R3 MYB transcription factors FOUR LIPS (FLP) and MYB88, restrict GMC divisions to one, thus ensuring that stomata consist of only two GCs. Loss of CDKA;1 or CDKB1 activity blocks GMC division, resulting in single guard cells. CDKB1 and CDKA;1 transcription is negatively regulated by FLP/MYB88 and FAMA. The heterodimerization of ICE1 and SCRM2 with SPCH, MUTE, and FAMA is required respectively for stomatal initiation, the meristemoid to GMC transition, and GC differentiation. Molecular Plant 2015 8, 783-795DOI: (10.1016/j.molp.2014.12.014) Copyright © 2015 The Author Terms and Conditions