Abstract
Stomata are pivotal for gas exchange during photosynthesis and transpiration and are therefore critical in plant growth and global water cycles. However, the mechanistic role of cell wall architecture in grass stomatal function remains elusive. Here immunolabelling and mechanical mapping revealed local distribution of methylesterified pectin at the stiffer polar ends of maize stomata. Expression-knockdown maize with reduced pectin labelling showed decreased polar stiffness and increased stomatal aperture. Finite element modelling corroborated these findings, suggesting that in contrast to non-grass stomata, the size and modulus of the polar materials limit maize stomatal opening. Surveys from various plant species suggest that polar-enriched methylesterified pectin is a unique feature of grass stomata. Xylanase pretreatment diminished pectin labelling at the polar ends, implying associations between pectin and xylan. Our multi-scale research uncovers a pectin–xylan–cellulose composite mediating polar fixation during maize stomatal movement, unveiling new targets for stomata engineering and crop breeding.
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Data availability
All analyses were conducted using standard software. The settings of software used for analyses are described in Methods. The original .cae file for FEM is provided as source data. The previously published single-cell RNA-seq data and bulk RNA-seq data have been deposited in the GenBank SRA database as BioProject PRJNA740934 (ref. 28). All primers were designed with SnapGene (https://www.snapgene.com) and are listed in Supplementary Table 2. The plant materials used in this study are available upon request. Source data are provided with this paper.
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Acknowledgements
We thank Y. Zhou, L. Guo, S. Wang, J. Huang and J. Ma for helpful discussions and constructive suggestions. C. T. Anderson helped with improving the paper. D. Mohnen, A. Voxeur and S. Vernhettes suggested methods of detecting the activity of EPG and PME. M. Cheng provided technical assistance and maintenance of AFM. L. Wei from Bruker Inc. provided the nano-IR measurements. Vectors for maize VIGS experiments were provided by Y. Liu from Tsinghua University and T. Zhou from China Agricultural University. L. Qiao helped with the use of the two-photon microscope. J. Zheng helped with the use of the laser microdissection system. X. Xu and C. Huang provided rice plant materials. X. Zhang offered Arabidopsis plants. This work was supported by the National Natural Science Foundation of China (grant nos. U21A20206 to C.-P.S. and 32100292 to T.Z.), the Natural Science Foundation of Henan Province (grant nos. 242300421026 to S.G. and 222301420103 to Z.L.), the Key Scientific Research Project in Colleges and Universities of Henan Province of China (grant no. 23A180003 to Z.L.), the Zhongzhou Laboratory for Integrative Biology (grant no. CXK2024GG0106 to S.G.) and the Key Research Project of the Shennong Laboratory (grant no. SN02-2024-02 to C.-P.S.).
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C.-P.S. and T.Z. conceptualized the project. T.Z., L.Y., Yueyuan Wang, P.L., X.F., G.J., F.Z., Z.Y., X.L., X.S., Yongqi Wang, L.M., W.S., Z.L. and C.Z. developed the methodology. T.Z., L.Y., Yueyuan Wang, P.L., X.F., G.J., F.Z., Z.Y., X.L., X.S., Yongqi Wang, L.M. and T.W. conducted the investigation. T.Z., Yueyuan Wang, P.L., L.Y. and X.L. wrote the original draft of the paper. C.-P.S. and T.Z. reviewed and edited the paper. C.-P.S., S.G., T.Z. and Z.L. acquired the funding. C.-P.S., T.Z. and Z.L. supervised the project. X.F. and G.J. contributed equally.
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Extended data
Extended Data Fig. 1 Immunofluorescence labeling in cross sections of maize stomata.
a, M154 labeling of arabinoxylan was confirmed by xylanase pretreatment. At least ten stomata from each group were imaged. The experiment was repeated twice independently. buffer: n = 20 from 10 GCs; xylanase pretreatment: n = 23 from 12 GCs, **P = 0.0048. Two-sided student t-test. b-f, Immunofluorescence labeling for esterified HG, cellulose and arabinoxylan in stomatal cross sections. For each group, at least ten stomata from two maize plants were imaged each time. The experiment was repeated three times independently.
Extended Data Fig. 2 Immunofluorescence labeling of MLG, AGP and de-esterified HGs in both paradermal and cross sections.
a–c, For each group, at least ten stomata from two maize plants were imaged each time. The experiments were repeated three times independently.
Extended Data Fig. 3 Expression of ZmGOLS2 in maize stomata.
a–c, Analysis of previously published single-cell RNA-seq (scRNA-seq) data suggests that ZmGOLS2 was expressed in maize stomata. The cluster annotation was based on Satterlee et al. (2020). Cluster 11 was annotated as developing stomata. For expression in epidermal peels, SCs: subsidiary cells; GCs: guard cells; BSCs: bundle sheath-related cells; MCs: mesophyll cells; PCs-A: pavement cells with active anthocyanin biosynthesis; PCs-N: pavement cells lacking active anthocyanin biosynthesis.
Extended Data Fig. 4 ZmGOLS2-VIGS and ZmGOLS2-OE maize plants are similar in stomatal density, halftime of light-induced stomatal response and assimilation rate compared to the wild type.
a, Construction of ZmGOLS2-VIGS lines and ZmGOLS2-OE lines. b, Positive control for ZmGOLS2-VIGS showing leaf bleaching by suppression of isopentenyl/dimethylallyl diphosphate synthase (ZmIspH) expression. c, Confirmation for transcription level of ZmGOLS2 by qRT-PCR. Values represent mean ± standard deviation, n = 3. **P =0.0064, 0.0062 and 0.0099 for VIGS-2, VIGS-3 and VIGS-5 versus B73; ***P = 1.69E-4, ****P =1.70E-5 and *P =0.0237 for OE-130, OE-132 and OE-134 versus B104. Two-sided student t-test. The experiments were repeated three times independently. d, Stomatal density of ZmGOLS2-VIGS and ZmGOLS2-OE maize plants. Values represent mean ± standard deviation, n = 15. P = 0.6194, P = 0.6391 and P >1.0000 for VIGS-2, VIGS-3 and VIGS-5 versus B73; P = 0.9821, 0.5870 and 0.0587 versus OE-130, OE-132 and OE-134 versus B104. Two-sided student t-test. The experiment was repeated twice independently. e, Halftime of light-induced stomatal response. n = 9. P = 0.4709 for ZmGOLS2-VIGS versus B73 for stomata close to open and P = 1.0000 for ZmGOLS2-VIGS versus B73 for stomata open to close. P = 0.6183, 0.5659 and 0.7676 for OE-130, OE-132 and OE-134 versus B104 for stomata close to open and P = 0.4216, 0.8617 and 0.1611 for OE-130, OE-132 and OE-134 versus B104 for stomata open to close. Two-sided student t-test. The experiment was repeated three times. f, Assimilation of ZmGOLS2-VIGS and ZmGOLS2-OE maize plants. Values represent mean ± standard deviation, n = 9. The experiment was repeated three times.
Extended Data Fig. 5 Immunofluorescence labeling of arabinoxylan and cellulose in ZmGOLS2-VIGS and ZmGOLS2-OE maize stomata.
a, Arabinoxylan labeling was weaker at the polar ends of ZmGOLS2-VIGS maize GCs. B73: n = 18 from 9 GCs; ZmGOLS2-VIGS: n = 43 from 22 GCs, **P = 0.0020. Student t-test. The experiment was repeated twice independently. b, Cellulose labeling was weaker at the polar ends of ZmGOLS2-VIGS maize GCs. B73: n = 20 from 10 GCs; ZmGOLS2-VIGS: n = 62 from 31 GCs, ****P = 7.43E-9. Student t-test. The experiment was repeated twice independently. c, Arabinoxylan labeling was stronger at the polar ends of ZmGOLS2-OE maize GCs. B104: n = 44 from 22 GCs; ZmGOLS2-OE: n = 50 from 25 GCs, **P = 0.0064. Student t-test. The experiment was repeated three times independently. d, Cellulose labeling was stronger at the polar ends of ZmGOLS2-OE maize stomata. B104: n = 34 from 17 GCs; ZmGOLS2-OE: n = 36 from 18 GCs, ****P = 1.17E-8. Student t-test. The experiment was repeated three times independently. e, De-methylesterfieid pectin was barely labelled in ZmGOLS2-VIGS, ZmGOLS2-OE and the wild-type maize plants. For each experiment mentioned above, three ZmGOLS2-VIGS plants and three plants of three ZmGOLS2-OE lines were used for collecting samples.
Extended Data Fig. 6 Laser ablation results indicate the compression of GCs by SCs.
a, Micrographs before and after laser ablation of SCs and measurement for stomatal pore width, pore length and GC length. Values represent mean ± standard deviation, n = 34. ***P = 2.83E-13 of pore width, P = 0.9687 of pore length, and P = 0.1422. Student t-test. The experiment was repeated three times independently. b, Micrographs before and after laser ablation of neighboring epidermal cells and measurement for stomatal pore width, pore length and GC length. Values represent mean ± standard deviation, n = 30. P = 0.8901 of pore width, P = 0.8700 of pore length, and **P = 0.0075. Student t-test. The experiment was repeated three times independently.
Extended Data Fig. 7 With different sets of turgor pressure, stiffer polar materials still result in smaller stomatal pore width.
a, Stomatal pore width with different turgor pressures of GCs. Ratio of turgor pressure between GCs and SCs was maintained at 2/0.35. b, Stomatal pore width with different turgor pressures of SCs. Turgor pressure of GCs was maintained at 2 MPa.
Extended Data Fig. 8 Quantitative analysis of LM19 labeling suggest that there is no polar distribution of de-esterified HG in the stomata of fern and fava bean.
a, Quantitative analysis of LM19 labeling in fern GCs. n = 120 from 6 fern GCs, P < 1.00E-15. b, Quantitative analysis of LM19 labeling in fava bean. n = 100 from 5 fava beans, P < 1.00E-15. Statistical differences were determined by ANOVA with a post hoc Tukey test and indicated by different letters. The experiment was repeated twice independently.
Extended Data Fig. 9 AFM-IR imaging shows different spectra at different regions of embedded sections.
a–d, Spectra of different colors correspond to locations marked by colored lines or dots in the height map. Lines of blue indicate stomatal cell walls and black dots point to resin.
Extended Data Fig. 10 Ruthenium red staining of low-methylesterified pectin gel and high-methylesterified pectin gel, indicating activity of EPG and PME respectively.
a–d, The white or lighter circles in low methyl ester pectin gel suggest EPG activity whereas the darker purple circles in high methyl ester pectin gel indicate PME activity. The experiment was repeated three times independently.
Supplementary information
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3D two-photon image of a closed maize stomata.
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3D two-photon image of an open maize stomata.
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Statistical source data. The previously published single-cell RNA-seq data and bulk RNA-seq data from epidermal peels have been deposited in the GenBank SRA database as BioProject PRJNA740934.
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Zhang, T., Yu, L., Wang, Y. et al. Esterified-pectin-coupled polar stiffening controls grass stomatal opening. Nat. Plants 12, 191–204 (2026). https://doi.org/10.1038/s41477-025-02194-4
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DOI: https://doi.org/10.1038/s41477-025-02194-4
