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IQ67 DOMAIN proteins facilitate preprophase band formation and division-plane orientation

Nature Plants volume 7, pages 739–747 (2021)Cite this article

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Abstract

Spatiotemporal control of cell division is essential for the growth and development of multicellular organisms. In plant cells, proper cell plate insertion during cytokinesis relies on the premitotic establishment of the division plane at the cell cortex. Two plant-specific cytoskeleton arrays, the preprophase band (PPB) and the phragmoplast, play important roles in division-plane orientation and cell plate formation, respectively1. Microtubule organization and dynamics and their communication with membranes at the cortex and cell plate are coordinated by multiple, mostly distinct microtubule-associated proteins2. How division-plane selection and establishment are linked, however, is still unknown. Here, we report members of the Arabidopsis IQ67 DOMAIN (IQD) family3 as microtubule-targeted proteins that localize to the PPB and phragmoplast and additionally reside at the cell plate and a polarized cortical region including the cortical division zone (CDZ). IQDs physically interact with PHRAGMOPLAST ORIENTING KINESIN (POK) proteins4,5 and PLECKSTRIN HOMOLOGY GTPase ACTIVATING (PHGAP) proteins6, which are core components of the CDZ1. The loss of IQD function impairs PPB formation and affects CDZ recruitment of POKs and PHGAPs, resulting in division-plane positioning defects. We propose that IQDs act as cellular scaffolds that facilitate PPB formation and CDZ set-up during symmetric cell division.

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Fig. 1: IQD8 functions in the spatial control of cytokinesis.
Fig. 2: Loss of IQD8 and related proteins affects PPB formation and division-plane orientation.
Fig. 3: IQD8 and related proteins interact with key players in division-plane maintenance.
Fig. 4: IQD8 contributes to efficient POK and PHGAP localization to the CDZ.

Data availability

All sequence information was obtained from the Arabidopsis Information Resource.

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Acknowledgements

This research was funded by the Deutsche Forschungsgemeinschaft (DFG, grant nos. BU 2955/1-1 and BU 2955/2-1; K.B.), the Erasmus Mundus programme (P.K.) and core funding of the Leibniz Association (K.B. and S.A.). The laboratory of S.M. is hosted by the Center of Plant Molecular Biology at the University of Tübingen and funded by DFG, grant nos. MU3133/3-2, MU3133/6-1 and MU3133/8-1 (Heisenberg Fellowship). Y.P. acknowledges the support of iDiv funded by the German Research Foundation (DFG–FZT 118, grant no. 202548816). We thank H. Buschmann for providing materials. We thank R. Plötner, D. Mitra and S. Bourbon for assistance with the establishment of mutant and transgenic lines.

Author information

Author notes

  1. Arvid Hermann

    Present address: Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA

  2. These authors contributed equally: Pratibha Kumari, Pradeep Dahiya.

Authors and Affiliations

  1. Department of Molecular Signal Processing, Leibniz Institute of Plant Biochemistry, Halle (Saale), Germany

    Pratibha Kumari, Pradeep Dahiya, Luise Zergiebel, Malte Kölling, Gina Stamm, Steffen Abel & Katharina Bürstenbinder

  2. Department of Developmental Genetics, Center for Plant Molecular Biology (ZMBP), Tübingen, Germany

    Pantelis Livanos, Arvid Hermann & Sabine Müller

  3. German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig, Leipzig, Germany

    Yvonne Poeschl

  4. Institute of Biodiversity, Friedrich Schiller University Jena, Jena, Germany

    Yvonne Poeschl

  5. Institute of Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany

    Steffen Abel

  6. Department of Plant Sciences, University of California Davis, Davis, CA, USA

    Steffen Abel

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Contributions

K.B. conceptualized the study. P.K., P.D., S.M. and K.B. designed the experiments. P.K., P.D., P.L., M.K., L.Z., G.S. and A.H. performed the experiments. Y.P. performed the statistical analysis. All authors analysed the data. K.B. wrote the manuscript draft. All authors discussed the work and edited the manuscript.

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Correspondence to Katharina Bürstenbinder.

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Peer review information Nature Plants thanks Zhaosheng Kong 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 Histochemical GUS staining and GFP fluorescence in transgenic pIQD6:GFP-GUS (pIQD6), pIQD7:GFP-GUS (pIQD7), and pIQD8:GFP-GUS (pIQD8) lines.

a-i, GUS staining in seeds with embryos at globular (a), heart (b), and torpedo stage (c), and mature embryos (d). e, Root tips of 4-day-old seedlings; f, Shoots of 10-day-old seedlings. g-i, Flower buds (g), mature flowers (h) and siliques (i) of 6–7-week-old plants. Bars, 100 µm (a-d); 50 µm (e); 1 mm (f-i). j-l, GFP fluorescence in root tips of 4-day-old seedlings (green). Cell outlines are visualized by propidium iodide staining (magenta). Images are single optical sections. Bars, 20 µm. See Supplementary Table 2 for information on statistics and reproducibility.

Extended Data Fig. 2 Growth phenotypes of iqd mutants throughout development.

a–c, Morphology of iqd6, iqd7 and iqd8 single, double and triple mutants and the pIQD8:IQD8-GFP/iqd678 line (comp/iqd678) compared to wild type (Col-0) in 7-day-old seedlings grown under long day conditions (a), in shoots of 18-day-old seedlings (b), and in mature plants 49 days after germination (c). d, Representative images of siliques from 49-day-old plants of wild type, the iqd678 mutant and the comp/iqd678 line. Bars; 1 cm, (a, b); 4.5 cm (c); 0.5 cm (d). e-g, Quantitative analysis of root length in 7-day-old seedlings (e), of rosette leaf area in 18-day-old seedlings (f) and of silique length (g). Center lines show medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. Siliques were collected from more than 5 independent plants. p values of statistically significant differences calculated by one-way ANOVA and post hoc Tukey HSD test are indicated on top. See Supplementary Table 2 for information on statistics and reproducibility.

Extended Data Fig. 3 Phenotypes of iqd6, iqd7, and iqd8 single, double and triple mutants.

Representative images of PI stained root tips from 7 day-old seedlings of iqd6, iqd7, and iqd8 single mutants, iqd6iqd7 (iqd67), iqd6iqd8 (iqd68), and iqd7iqd8 (iqd78) double mutants, the iqd6iqd7iqd8 triple mutant (iqd678) and the pIQD8:IQD8-GFP/iqd678 complementation line (comp/iqd678) grown under long-day conditions. Red arrowheads point to oblique cell walls. Bars, 50 μm. See Supplementary Table 2 for information on statistics and reproducibility.

Extended Data Fig. 4 Cell division patterns in root tissue layers of wild type, iqd678 mutants and the comp/iqd678 line and expression domains of IQD8-GFP.

a–c, Z-stack images of propidium iodide (PI)-stained root meristems of 7-day-old wild type (a), iqd678 seedlings (b), and of PI and GFP signals in the comp/iqd678 line (c). Longitudinal sections (top, XY view) show layers of root cap (left); epidermis (middle) and cortex (right). The bottom images show corresponding cross sections of maximum projections (XZ) extracted at positions indicated by the cross (yellow lines). Bars, 20 µm. See Supplementary Table 2 for information on statistics and reproducibility.

Extended Data Fig. 5 Subcellular localization of IQD6-GFP, IQD7-GFP, and IQD8-GFP in transgenic Arabidopsis lines.

a,b, Analysis of GFP fluorescence in pIQD8:GFP-GUS lines (a) and in pIQD8:gIQD8-GFP/iqd678 lines (b). Cell outlines were visualized with propidium iodide. Left, GFP fluorescence; right, merged image. c,d, Analysis of subcellular IQD8-GFP localization during prophase (c) and relative to RFP-MBD labeled microtubules in interphase cells of pIQD8:IQD8-GFP/RFP-MBD seedlings (d). e–g, Expression domain and subcellular localization of IQD6-GFP in root tips of pIQD6:IQD6-GFP/iqd6 lines. Overview images (e,f), and magnified view of cells during prophase (upper row) and during cytokinesis (bottom row) (g). Orange arrowheads indicate PPB and CDZ during prophase and cytokinesis, respectively. White arrowheads indicate phragmoplasts, and white asterisks indicate cell plates. h–j, Expression domain and subcellular localization of IQD7-GFP in pIQD7:IQD7-GFP lines. Overview (h), magnification of stem cell niche (i) and close up of individual cells (j). Cell walls or membranes were visualized with propidium iodide (e,f,h-i) and FM4-64 (g), respectively. Bars, 50 µm (a,b,e,f,h), 5 µm (c,g) and 10 µm (d,i,j). See Supplementary Table 2 for information on statistics and reproducibility.

Extended Data Fig. 6 Bimolecular fluorescence complementation (BiFC) assays between IQD8 and POKs and PHGAPs.

a–k, BiFC analysis of IQD8 fused to the N-terminal half of YFP (YN) with C-terminal half of YFP (YC)-fused POK1C (b), POK2C (c), PHGAP1 (d) and PHGAP2 (e). YC -CaM2 (a) and YC -TRM1 (f) were included as positive and negative controls, respectively. BiFC analyses of YN-TRM1 with YC-PHGAP1 (g), YC-PHGAP2 (h), YC-POK1C (i) and YC-POK2C (j) were included as negative controls for POKs and PHGAPs. All combinations were analyzed with identical laser settings (a-j). As positive control for expression of YN-TRM1, YC-TON1a was included in an independent experiment (k). Single optical section of YFP signal (left), the corresponding bright-field (right) and magnified view (inset). Bars, 20 µm (left) and 5 µm (inset). See Supplementary Table 2 for information on statistics and reproducibility.

Extended Data Fig. 7 Subcellular localization of YFP-POK1 in iqd678 mutants.

a,b, Subcellular localization and expression of YFP-POK1 expressed under control of the native promoter in root tips of 7 day-old seedlings of pPOK1:YFP-POK1 (a), and pPOK1:YFP-POK1/iqd678 lines (b) in an RFP-MBD background. White arrows point to YFP-POK1 signals at the PPB and CDZ during prophase and metaphase/cytokinesis, respectively (a), which frequently are absent in iqd678 mutants. Images are single optical sections of YFP (left), RFP-MBD (center) and merged signals (right). Bars, 10 µm. See Supplementary Table 2 for information on statistics and reproducibility.

Extended Data Fig. 8 Subcellular localization of GFP-PHGAP2 in iqd678 mutants.

a–d, Subcellular localization of GFP-PHGAP2 expressed under control of the native promoter in pPHGAP2:GFP-PHGAP2 (a,c) and in pPHGAP2:GFP-PHGAP2/iqd678 lines (b,d) in an RFP-MBD background. Overview images of root tips (a,b) and close ups of cells in prophase (top), meta-/anaphase (middle) and during cytokinesis (bottom) (c,d). White arrows point to cytokinetic cells lacking cortical GFP-PHGAP2 accumulation in iqd678 mutants (b). Images are single optical sections (c-d) of GFP (left), RFP-MBD (center) and merged signals (right) and maximum projections of merged signals from z stacks (Z-projection) Bars, 50 µm (a,b) and 5 µm (c,d). See Supplementary Table 2 for information on statistics and reproducibility.

Extended Data Fig. 9 Genetic interaction of IQD6, IQD7, and IQD8 with PHGAPs and POKs.

a, b, Morphology of wild type (Col-0), iqd678, phgap12, pok12, iqd678pok12, and iqd678phgap12 mutants in aerial parts of 28 day-old plants (a) and in 8 day-old seedling grown on soil under long day conditions (b) Bars, 2.5 cm (a) and 1 cm (b). c, Quantification of root growth in seedlings shown in (b). Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots; n = 30 – 37. Different letters denote statistically significant differences (p ≤ 0.01) calculated by one-way ANOVA and Tukey post-hoc test. d, Propidium iodide stained root tips of seedlings shown in (b). Images are single optical sections. Bars, 50 µm. e, Quantitative analysis of division angles in wild type, iqd678, phgap12, and iqd678phgap12 mutants. Violin plots show the distribution of division angles (e). The shape of the violins outlines densities; center lines, medians; white dots, means. See Supplementary Tables 2 and 11 for information on statistics and reproducibility.

Extended Data Fig. 10 Proposed model of IQD8 function during cell division.

a, During G2 to M transition, microtubules reorganize into the PPB, which is the first visible marker of the future division plane. A plant-specific TTP complex, consisting of TRMs, TON1, and a heterotrimeric PP2A phosphatase is required for PPB formation. After PPB disassembly, a microtubule-depleted zone, termed CDZ remains at the cell cortex. The CDZ is occupied by several proteins, including POKs and PHGAPs, and maintains the positional information of the future division plane throughout cell division. IQD8-GFP localizes to a polarized cortical domain (PCD) of unknown composition that includes the CDZ/CDS. In addition, IQD8 is associated with mitotic microtubules at the PPB and phragmoplast and labels the cell plate. b, In iqd678 mutants, PPB formation is abolished in ~50% of cells, accompanied by an enrichment of perinuclear microtubules. In addition, YFP-POK1 and GFP-PHGAP2 recruitment to the CDZ is delayed, which correlates with increased frequencies of phragmoplast mispositioning and aberrant cross wall placement. Model modified from4. c, We propose that IQD8 and related proteins act as cellular scaffolds during PPB formation and CDZ set up that regulate microtubule organization and macromolecular complex assembly. PPB formation requires a functional TTP complex and IQD proteins. How or if TTP and IQDs function together is unknown. During CDZ set up, IQD8 physically interacts with POKs and PHGAPs, which are core constituents of the CDZ essential for division plane determination. POKs physically interact with PHGAPs and are required for PHGAP recruitment. Whether tripartite interactions between IQDs, POKs and PHGAPs are required for proper CDZ set up remains to be investigated.

Supplementary information

Supplementary Information

Supplementary Figs. 1–12, source files for Supplementary Fig. 4b,c and Tables 1–11.

Reporting Summary

Supplementary Data 1

Manually edited sequence alignment of the 33 Arabidopsis IQD proteins.

Supplementary Data 2

List of genes in the coexpression network of IQD8.

Source Data Fig 3

Unprocessed western blots for Figure 3i,j.

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Kumari, P., Dahiya, P., Livanos, P. et al. IQ67 DOMAIN proteins facilitate preprophase band formation and division-plane orientation. Nat. Plants 7, 739–747 (2021). https://doi.org/10.1038/s41477-021-00923-z

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