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A spatiotemporal molecular switch governs plant asymmetric cell division

Nature Plants volume 7, pages 667–680 (2021)Cite this article

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Abstract

Asymmetric cell division (ACD) requires protein polarization in the mother cell to produce daughter cells with distinct identities (cell-fate asymmetry). Here, we define a previously undocumented mechanism for establishing cell-fate asymmetry in Arabidopsis stomatal stem cells. In particular, we show that polarization of the protein phosphatase BSL1 promotes stomatal ACD by establishing kinase-based signalling asymmetry in the two daughter cells. BSL1 polarization in the stomatal ACD mother cell is triggered at the onset of mitosis. Polarized BSL1 is inherited by the differentiating daughter cell, where it suppresses cell division and promotes cell-fate determination. Plants lacking BSL proteins exhibit stomatal overproliferation, which demonstrates that the BSL family plays an essential role in stomatal development. Our findings establish that BSL1 polarization provides a spatiotemporal molecular switch that enables cell-fate asymmetry in stomatal ACD daughter cells. We propose that BSL1 polarization is triggered by an ACD checkpoint in the mother cell that monitors the establishment of division-plane asymmetry.

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Fig. 1: Identification of BSL proteins as putative physical partners of the polarity protein BASL.
Fig. 2: Polarization of BSL1 requires BASL in Arabidopsis.
Fig. 3: The association of BSL1 with the polarity complex occurs in MMCs at the entry of mitosis.
Fig. 4: The association of BSL1 with the polarity complex promotes BIN2 partitioning to the nucleus.
Fig. 5: The association of BSL1 with the polarity complex activates YDA and MAPK signalling.
Fig. 6: BSL requires BIN2 and YDA to regulate stomatal development in Arabidopsis.

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All generated and analysed data from this study are included in the published article and its Supplementary Information. Source data are provided with this paper.

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Acknowledgements

We thank the ABRC Stock Center for providing T-DNA insertional seeds, the Biological Mass Spectrometry Facility of Robert Wood Johnson Medical School for performing the MS analyses, and J. Winkelman for assistance with the data analyses. This research was supported by grants NIH GM109080/GM131827 and NSF 825885/1952823 to J.D., NIH GM066258 to Z.-Y.W., and NIH GM118059 to B.E.N.

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Authors and Affiliations

  1. Waksman Institute of Microbiology, Rutgers, The State University of New Jersey, Piscataway, NJ, USA

    Xiaoyu Guo, Bryce E. Nickels & Juan Dong

  2. Department of Plant Biology, Carnegie Institution for Science, Stanford, CA, USA

    Chan Ho Park & Zhi-Yong Wang

  3. Department of Genetics, Rutgers, The State University of New Jersey, Piscataway, NJ, USA

    Bryce E. Nickels

  4. Department of Plant Biology, Rutgers, The State University of New Jersey, New Brunswick, NJ, USA

    Juan Dong

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Contributions

X.G. and J.D. designed the research. X.G. performed most of the experiments. C.H.P. assisted with the mutant analysis. X.G., C.H.P., Z.-Y.W., B.E.N. and J.D. wrote the manuscript.

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Correspondence to Juan Dong.

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Peer review information Nature Plants thanks Laurie Smith and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Genetic Pathways Involving BSL Proteins in Arabidopsis.

a, Compositional and functional changes of the polarity complex before and after a stomatal ACD. In a young leaf, the initiation of the stomatal lineage cell (MMCs), is driven by the expression of the transcription factor SPEECHLESS (SPCH)11. The plant-specific protein BASL is polarized before, during and after a stomatal ACD13. Multiple components in the BASL polarity complex have been identified to regulate cellular events in stomatal ACD. The BRX proteins interact with BASL to attach the polarity complex to the plasma membrane15. The POLAR proteins associate with the polarity complex and require BASL to become polarized14. It is known that POLAR recruits the BIN2 GSK3-like kinases to the polarity complex in the MMC, where BIN2 phosphorylates and inhibits the MAPKKK YDA, leading to alleviated MAPK-mediated suppression of SPCH, thereby the MMC undergoes cell division19. It is also known that, BASL interacts with YDA to locally concentrate MAPK signaling, thus SPCH is suppressed in the large daughter cell SLGC that inherits the polarity complex, thereby the SLGC after an ACD undergoes differentiation to become a pavement cell18,35. Thus, a successful stomatal ACD necessitates the changes of the key regulators, that is BIN2 to be preferentially membrane-localized before ACD but nucleus-partitioned after ACD, whereas YDA to be suppressed before ACD but activated after ACD. Light blue, stomatal fate; dark blue, stomatal guard cells; pink, non-stomatal fate; light pink, pavement cell. Fading cells, PrCs not converted to MMC become non-stomatal pavement cells. Dotted rectangle, regions enlarged in bottom, containing protein components of the polarity complex. Bottom, enlarged view of polarity protein complexes required for the progenitor cell (MMC, light blue) and the daughter cell (SLGC, pink), respectively, in stomatal ACD. ?, unidentified regulator/s for POLAR to associate with the polarity complex. Fragmented molecules (SPCH and POLAR) indicate proteins undergo degradation. b, Schematics depicting simplified signal transduction pathways in stomatal development (left) and Brassinosteroid (BR) signaling (right). The models are mainly based on Arabidopsis research. In stomatal development, the receptor-like kinases/protein (TMM, TOO MANY MOUTHS; ERf, ERECTA family; SERKf, SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE family)27,30,31 transduce signals from the EPF peptide ligands (not specified here) to activate the YODA (YDA) MAPK signaling that suppresses the transcription factors (SPCH, SPEECHLESS; ICE1/SCRM, SCREAM) and stomatal development17,20,56. The BIN2 GSK-3 like kinases regulate stomatal development by inhibiting YDA and SPCH21,22. The polarity protein BASL interacts with YDA and forms a positive feedback regulation with the MAPK cascade to regulate stomatal ACD18. In this study, we identified the BSL protein phosphatases as BASL partners in the polarity complex. In BR signaling, the hormone (BR) is perceived by the receptor-like kinase (BRI1, BRASSINOSTEROID INSENSITIVE 1)57 that triggers cytoplasmic signal transduction mediated by the BSL protein phosphatases23 and the BIN2 GSK3-like kinases58 to ultimately active the expression of the transcription factors (BZR1, BES1)59,60 for plant responses. Both pathways involve BSL Ser/Thr protein phosphatases and the GSK3-like BIN2 kinase. Block lines indicate negative regulation and arrows indicate positive regulation. PM, plasma membrane; NE, nuclear envelope.

Extended Data Fig. 2 BSLf Physically Interact with BASL in Plants.

a-b, Co-IP assays using purified fusion proteins that are transiently overexpressed (driven by the ubiquitous 35S promoter) in N. benthamiana leaf cells. a, Results show physical association of FLAG-tagged BSL1/2/3 proteins with GFP-BASL. GFP-BASL (right) or GFP (left, control) was used as bait to bind to the GFP-Trap agarose. Immunoprecipitated proteins were detected by anti-FLAG. Data represent results of three biological repeats. b, Results show physical association of FLAG-tagged BSL1D584N (catalytically inactive BSL1 variant) proteins with GFP-BASL and BIN2-YFP. GFP alone (left, control), GFP-BASL (middle) or BIN2-YFP (right) was used as bait to bind to the GFP-Trap agarose. Immunoprecipitated proteins were detected by anti-FLAG. Data represent results of three biological repeats.

Source data

Extended Data Fig. 3 BSL1, BSL2 and BSL3 Co-localize with BASL in Arabidopsis.

a, qPCR data show relative expression levels of BSL1 (driven by the native promoter) in bsl1 (left) or basl-2 (right). Three independent transgenic lines were used. Total RNAs were extracted from 3-day-old seedlings. Gene expression levels were normalized by ACTIN2 and relative expression levels of BSL1 were compared with the values in the wild type. Data are presented as mean ± SD. n = three independent experiments. b, Co-expression of GFP-BASL (green) with BSL1-mRFP (magenta), both driven by the BASL promoter, in a true leaf of 5-dpg seedling. c, Representative images of co-localization of BSL1-mRFP, BSL2-mRFP and BSL3-mRFP (magenta) with GFP-BASL (green), all driven by the BASL promoter. Arrows mark protein polarization at the cell cortex. d, Expression patterns of endogenous promoter driven GFP-BASL (green) and BSL1-YFP (magenta) in progressive cell types during stomatal development. PrC (protodermal cells), early MMC (Meristemoid Mother Cell, small and rectangular), late MMC (asymmetrically expanded and triangle), M (Meristemoid), and SLGC (Stomatal Lineage Ground Cell). Protein polarizations were indicated by arrows. Scale bars in (b-d), 5 μm. e, qPCR data show relative expression levels of BASL and BSL in indicated genetic backgrounds. Total RNAs were extracted from 3-day-old seedlings. Gene expression levels were relative to ACTIN2. Data are presented as mean ± SD. n = three independent experiments. Primers used were listed in Supplementary Table 2.

Extended Data Fig. 4 Association of BSL1 with the Polarity Complex Promotes BIN2 Partitioning to the Nucleus.

a, qPCR data show relative expression levels of BIN2 (left) and BSL1/BSL1D584N (right) in the indicated genetic backgrounds. Total RNAs were extracted from 3-day-old seedlings. Gene expression levels were normalized by ACTIN2 and relative expression levels of BIN2 or BSL1 were compared with the values in the wild-type background. Data are presented as mean ± SD. n = three independent experiments. Primers used were listed in Supplementary Table 2. b, Representative confocal images show differential membrane/nuclear partitioning of BIN2 (yellow) in stomatal lineage cells from the wild type vs. in bsl-quad mutants vs. in BSL1 overexpression plants (driven by the TMM promoter). The expression of BIN2-YFP is driven by the native promoter. Right, florescence intensity profiling of the dotted lines drawn across the stomatal lineage cells shown on the left. Confocal images were taken with same settings and absolute fluorescence intensity values were obtained and profiled by Fiji. M, cell membrane; N, nucleus. Scale, 5 μm c, Co-expression of the TMM promoter driven BSL1-mRFP (red) with the endogenous promoter driven BIN2-YFP (yellow). The nuclear enrichment of BIN2 was verified by positive DAPI staining (cyan). Scale, 10 µm.

Extended Data Fig. 5 BSL1 Interferes the Interaction of BIN2-BASL in vitro.

a, In vitro pull-down assays using recombinant proteins to test the BIN2-BASL interaction in the presence of an increasing amount of MBP-BSL1. His-BASL was used as bait and the amount of GST-BIN2 being pulled down reflects the interaction strength of BIN2-BASL. MBP was used as negative control. Histograms (below) show quantification of relative protein levels of BIN2 in the assay above. Results suggest the addition of MBP-BSL1 reduced the amount of BIN2 that interacted with BASL. Data are presented as mean ± SD. n = three independent experiments. Two-tailed Student’s t-tests. ** P < 0.005. b, Co-IP assays using purified fusion proteins produced by N. benthamiana leaves show physical association of BIN2-YFP with Myc-POLAR was not influenced by the presence of FLAG-tagged BSL1 or BSL1D584N. BIN2-YFP was used as bait. Immunoprecipitated proteins were detected by anti-Myc. Data represent results of experiments repeated three times. c, Results of yeast two-hybrid assays show no interactions between BSL1 and POLAR were identified. BD indicates Gal4 DNA-binding domain. AD indicates Gal4 activation domain. ‘Interaction tests’, assays performed using synthetic dropout medium (-Leu-Trp-His; 1 mM 3-AT added to suppress auto-activation); ‘growth controls’, assays performed using rich media (-Leu-Trp).

Source data

Extended Data Fig. 6 BSL1 Interferes the Interaction of BIN2-BASL in Planta.

a, BiFC assays show interactions between BIN2 and BASL (yellow) in N. benthamiana leaves. Half YFPs (nYFP and cYFP) were used as negative controls. Arrows indicate protein polarization in epidermal cells. b, BiFC interaction tests for BIN2-BASL (yellow) in the presence of BSL1-CFP (cyan, top) or BSL1D584N-CFP (cyan, bottom). Note, in the presence of BSL1, YFP signals were found diminished along the cell periphery but increased in the nucleus (arrowheads), suggesting BIN2-BASL interactions at the cell periphery were disturbed by the expression of wild-type BSL1 but not the phosphatase-dead BSL1D584N. Scale bars in (a-b), 50 μm. c, Quantification of YFP polarization at the cell periphery in the BIN2-BASL and BIN2-POLAR BiFC assays. The method for quantification of polarization in the BiFC assays was described in Fig. 1. *** P < 0.0001. n.s., not significant. d, BiFC assays show protein-protein interaction between BIN2 and POLAR (yellow). Half YFPs (nYFP or cYFP) were used as negative controls. e, BiFC assays show the interaction of BIN2-POLAR (yellow) was not changed by the expression of BSL1-CFP (cyan, top) or BSL1D584N-CFP (cyan, bottom). Representative individual cells were chosen from three independent experiments. Scale bars in (d-e), 50 μm. f, Quantification of membrane/nuclear (M/N) partition in the BIN2-BASL and BIN2-POLAR BiFC assays. Average fluorescence intensity values were taken from the cell periphery and from nucleus area for calculation. To generate c and f, confocal images were captured with same settings and absolute fluorescence intensity from z-stacked images were measured by Fiji. Box plot shows first and third quartiles, median (line) and mean (cross). n, number of cells. One-way ANOVA followed by Tukey’s post hoc test were used to compare with their respective control. *** P < 0.0001. n.s., not significant. g, Representative confocal images show differential membrane/nuclear partitioning of BIN2-YFP (driven by the endogenous promoter) in the Arabidopsis root elongation zone of the wild-type vs. in bsl-quad mutants. Scale bars, 10 μm. (z), images are z-stacked. Right, quantification of membrane/nuclear (M/N) partition of BIN2–YFP. Box plot shows first and third quartiles, median (line) and mean (cross). n, number of cells counted. Two-tailed Student’s t-tests were used to compare with in the wild type. *** P < 0.0001.

Extended Data Fig. 7 BSL1 Physically Interacts with YDA in vitro and in vivo.

a, In vitro pull-down assays using purified recombinant proteins show GST-BSL1 interaction with MBP-YDA. MBP-YDA used as bait. GST (negative control) or GST-BSL1 interaction were detected by anti-GST. b, Co-IP data show in vivo interaction between BSL1 and YDA. BSL1-FLAG was used as bait to detect the binding of YDAKI-Myc in 5-dpg Arabidopsis seedlings. The expression of BSL1-FLAG is driven by the TMM promoter and the expression of YDA is driven by the BASL promoter. YDAKI (Kinase Inactive YDA) was used to avoid the activity of YDA strongly suppressing plant growth. Data represent results of experiments repeated three times in a and b. c, BiFC assays show the interaction between BSL1 and YDAKI (kinase inactive YDA variant used to suppress active YDA-triggered cell death) occurs at the cell membrane in N. benthamiana leaf epidermis. Positive protein-protein interactions were visualized by YFP signal (yellow). Half YFPs (nYFP or cYFP) were uses as negative controls. Scale bars, 50 μm.

Source data

Extended Data Fig. 8 Characterization of the Loss-of-Function bsl Mutants.

a, Semi-quantitative RT–PCR analysis of single bsl T-DNA insertion lines using total RNAs isolated from Arabidopsis seedlings at 3-dpg. gDNA, genomic DNA from the wild type used as template. Primers used for RT–PCR were listed in Supplementary Table 2. Red color indicates the mutant lines used for generation of high order mutants in this study. b, Stomatal phenotype in 5-dpg adaxial cotyledon epidermis of wild type, single bsl T-DNA insertional mutants (top), and double mutants of bsl1;bsl2, bsl2;bsl3 and bsl1;bsl3 (bottom, cells are traced with blue for stomatal guard cells, dark pink for stomatal lineage cells, light pink for pavement cells). Note, mildly clustered guard cells appear in the double mutants. Scale bars, 40 μm. c, Quantification of Stomatal Index (# stomata relative to # total cells) of the designated genotypes in b. Box plot shows first and third quartiles, median (line) and mean (cross). n, number of cotyledons used for quantification of each genetic background. One-way ANOVA with Tukey’s post hoc test were used to compare with the wild type. n.s., not significant. * P < 0.05, ** P < 0.005. d, Elevated number of cells expressing the stomatal lineage identity marker SPCH in bsl-quad mutants. Left: Confocal images (z-projections) show the expression of SPCH-CFP (cyan) in the wild-type (top) and bsl-quad mutants (bottom) at different time point (after gemination) during stomatal development. Cell outlines were stained with PI (magenta). Each representative image was selected from 5 comparable cotyledons for each stage. Scale bars, 50 μm. Right, quantification of SPCH-CFP expression in wild-type and in bsl-quad mutant. Ratios of CFP-positive cells relative to the total cell numbers in a given areas were calculated. Box plot shows first and third quartiles, median (line) and mean (cross). n, total number of cells collected from > 20 cotyledons at 36- and 54-hpg. Data were analysed by two-tailed Student’s t-test. * P = 0.0169.

Extended Data Fig. 9 BSL Requires BIN2 and YDA to Regulate Stomatal Development in Arabidopsis.

a, Overexpression phenotypes of the TMM promoter driven BSL1 (green) in 5-day adaxial cotyledon epidermis. Right, qPCR data show relative expression levels of BSL1 in two independent overexpression lines used in this study. Total RNAs were extracted from 3-day-old seedlings. Gene expression levels were normalized by ACTIN2 and relative expression levels of BSL1 were compared with the values in the wild type. Data are presented as mean ± SD. n = three independent experiments. Primers used were listed in Supplementary Table 2. b, Stomatal phenotypes in 5-day adaxial cotyledon epidermis overexpressing phosphatase-dead BSL1D584N (green) in wild-type (left) and overexpressing wild-type BSL1 (red, right) in the basl-2 mutant (right), both driven by the TMM promoter. Note the stomatal phenotype generated by BSL1D584N resembled that observed for bsl-quad mutants, suggesting a dominant negative effect of BSL1D584N. c, Overexpression of BSL1 (green) driven by the TMM promoter in the loss of function yda-3 mutant did not alter the stomatal phenotypes in yda-3. d, Overexpression of constitutively active YDA (YDACA, green) driven by the SPCH promoter in the wild-type (left) and in bsl-quad mutants (right). The activities of YDACA suppressed the loss-of-function phenotypes in bsl-quad. e, Overexpression of BSL1 (green) driven by the TMM promoter (BSL1 ++) in the loss-of-function mutants bin2-3;bil1;bil2 (bin). bin mutations appeared to be epistatic. Cell outlines were stained with Propidium Iodide (magenta). f, Stomatal phenotype in 5-dpg adaxial cotyledon epidermis of Ws-2 (background of bin2). Right, quantification of Stomatal Index (# stomata relative to # total cells) of the designated genotypes. Box plot shows first and third quartiles, median (line) and mean (cross). n, number of cotyledons used for quantification. One-way ANOVA with Tukey’s post hoc test were used to compare with the wild-type Col-0. n.s., not significant. *** P < 0.0001. Scale bars in (a-e), 50 μm.

Extended Data Fig. 10 The Membrane Receptors ERECTA, SERK, and TMM are not required for BSL to Regulate Stomatal Development in Arabidopsis.

a-c, Confocal images show stomatal development (black/white) and protein expression (green) in 5-day adaxial cotyledon epidermis of the designated genotypes. Overexpression of BSL1 (BSL1 ++, green) was driven by the TMM promoter in loss-of-function receptor mutants, that is er;erl1;erl2 (a), serk1;2;3;4 (b), and tmm (c). Cell outlines were highlighted by PI-staining (magenta). Representative images were selected from at least 10 individual cotyledons. Scale bars, 50 μm. d, Quantification of Stomatal Lineage Index (SLI) of the genotypes shown in (a-c). Box plot shows first and third quartiles, median (line) and mean (cross). More than five cotyledons were scored for each genotype. Two-tailed Student’s t-test were used to compare with their respective genetic backgrounds. ** P < 0.005, *** P < 0.0001.

Supplementary information

Supplementary Information

Supplementary Figs. 1 and 2 and Supplementary Table 2.

Reporting Summary

Supplementary Table 1

Co-IP and MS identification of BSL proteins as BASL-interacting proteins in Arabidopsis.

Source data

Source Data Fig. 4

Unprocessed western blots.

Source Data Fig. 5

Unprocessed western blots.

Source Data Extended Data Fig. 2

Unprocessed western blots.

Source Data Extended Data Fig. 5

Unprocessed western blots.

Source Data Extended Data Fig. 7

Unprocessed western blots.

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Guo, X., Park, C.H., Wang, ZY. et al. A spatiotemporal molecular switch governs plant asymmetric cell division. Nat. Plants 7, 667–680 (2021). https://doi.org/10.1038/s41477-021-00906-0

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