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A new family of proteins is required for tethering of Casparian strip membrane domain and nutrient homoeostasis in rice

Nature Plants volume 9, pages 1749–1759 (2023)Cite this article

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Abstract

Cell–cell junctions are essential for multicellular organisms to maintain nutrient homoeostasis. A plant-type tight junction, the Casparian strip (CS)–Casparian strip membrane domain (CSD) that seals the paracellular space between adjacent endodermal cells, has been known for more than one hundred years. However, the molecular basis of this structure remains unknown. Here we report that a new family of proteins containing a glycine/alanine/proline-rich domain, a lectin domain and a secretory signal peptide (GAPLESS) mediates tethering of the plasma membrane to the CS in rice. The GAPLESS proteins are specifically localized in the CS of root endodermal cells, and loss of their functions results in a disabled cell–cell junction and disrupted nutrient homoeostasis. The GAPLESS protein forms a tight complex with OsCASP1 in the plasma membrane, thereby mediating the CS–CSD junction. This study provides valuable insights into the junctional complex of plant endodermal cells, shedding light on our understanding of nutrient homoeostasis in crops and the cell junctions of eukaryotes.

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Fig. 1: GAPLESS is a family of endodermis-specific genes encoding proteins enriched with glycine, alanine and proline at the C termini.
Fig. 2: GAPLESS proteins are required for rice growth, yield and nutrient homoeostasis.
Fig. 3: The family of GAPLESS proteins is required for apoplastic diffusion.
Fig. 4: The family of GAPLESS proteins is required for tethering of CSD to CS in endodermis.
Fig. 5: GAPLESS proteins mediate adhesion of CS by forming a complex with OsCASP1 in CSD.

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Data availability

Data and databases used in this study: Alphafold models of AtCASP1 (uniprot ID:Q9SIH4) and OsCASP1 (uniprot ID:Q7XPU9). Domain prediction: InterPro (v.92.0, IPR036426). RNA-seq database: http://ipf.sustech.edu.cn/pub/ricerna/. Single-cell database of rice root was obtained from http://www.elabcaas.cn/rcar/index.html. The signal peptides of the identified sequences were verified using SignalP (https://services.healthtech.dtu.dk/service.php?SignalP). The sequence BLAST was searched against the PLAZA database: https://bioinformatics.psb.ugent.be/plaza. Code used in generating co-expression network is available on GitHub (https://github.com/Yorks0n/Os_endo_network). Any additional information is available from the corresponding author upon request. Source data are provided with this paper.

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Acknowledgements

We thank N. Geldner for suggestions and editing on this manuscript; W.-J. Cai, S.-N. Yin, Z.-P. Zhang, X.-Y. Gao, J.-Q. Li, L. Liu, M.-L. Ma (CAS Centre for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, CAS), J.-S. Xue (Shanghai Normal University), X.-X. Li, Y. Feng, C. Peng (Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Science) and Zhenjiang Lehua Technology for technical support.

This work was supported by the National Natural Science Foundation of China (31930024, 32061130209), the Chinese Academy of Sciences (XDB27010103) and the Newton Fund (NAF\R1\201264, NIF\R1\191915).

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Authors and Affiliations

  1. National Key Laboratory of Plant Molecular Genetics, Shanghai Center for Plant Stress Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China

    Tao Song, Ying-Qi Tian, Chu-Bin Liu, Ya-Ling Wang, Jing Zhang, Yu Su, Li-Na Xu, Mei-Ling Han & Dai-Yin Chao

  2. University of Chinese Academy of Sciences, Beijing, China

    Tao Song, Ying-Qi Tian, Chu-Bin Liu, Jing Zhang & Yu Su

  3. Future Food Beacon of Excellence and School of Biosciences, University of Nottingham, Nottingham, UK

    Yi-Qun Gao & David E. Salt

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  1. Tao Song
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Contributions

D.-Y.C. directed the research. T.S. performed most of the experiments. Y.-Q.T., C.-B.L., Y.-Q.G., Y.-L.W., J.Z. and L.-N.X. performed some of the experiments. T.S., Y.-Q.T., C.-B.L., Y.S. and M.-L.H. contributed to the analytical work. D.E.S. oversaw the entire study. D.-Y.C. and T.S. wrote the manuscript.

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Correspondence to Dai-Yin Chao.

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Extended data

Extended Data Fig. 1 Expression of GAPLESS genes in rice and phylogenetic analysis of GAPLESS orthologs.

a, Single cell transcriptome data of GAPLESS1, GAPLESS2 and GAPLESS3 in rice roots. The expression data were collected from the Root Cell Atlas in Rice (RCAR) (http://www.elabcaas.cn/rcar/index.html). b, Representative images after GUS staining. Longitudinal section images after GUS staining of pGAPLESS1:GUS, pGAPLESS2:GUS and pGAPLESS3:GUS. Scale bars: 400 μm. c, Phylogenetic analysis of GAPLESS proteins in plant kindom. The tree was built using the full-length amino acid sequences for GAPLESS proteins. Different colors represent different species: Monocots, Gymnosperms, ANA grade, Magnoliales and Eudicots. d, Amino acid composition of GAP domain of GAPLESS proteins and their orthologs in different plant species. G, Glycine. A, Alanine, P, Proline.

Extended Data Fig. 2 Mutated sequences of three GAPLESS genes in the gapless single and double mutants.

a-c, The genotypes of GAPLESS1 (a), GAPLESS2 (b) and GAPLESS3 (c) in different single mutants and double mutants as indicated. Red lines represent targeted sites of GAPLESS. Insert bases were marked with red colors and deletions were replaced by blue lines.

Extended Data Fig. 3 The growth phenotypes of gapless mutants.

a-d, The growth phenotypes of independent gapless1 mutant lines. e-h, The growth phenotypes of independent gapless2 mutant lines. i-l, The growth phenotypes of independent gapless3 mutant lines. m-p, The growth phenotypes of independent gapless1/2 mutant lines. Representative images are shown for ZH11 and different mutant lines of gapless1 (a), gapless2 (e), gapless3 (i) and gapless1/2 (m) grown in paddy field for 75 days. Quantification of plant height (b, f, j and n), tiller numbers (c, g, k and o) and grain numbers per plant (d, h, l and p) of the independent lines of single mutants and wild-type ZH11, respectively. Scale bars, 10 cm in (a, e, i, m). The values in (b-d, f-h, j-l, n-p) are shown as the mean ± s.d. Data points are independent samples. In (b), n = 19; in (c), n = 14 for ZH11, n = 10 for gapless1-1, n = 9 for gapless1-2, n = 11 for gapless1-3; in (d), n = 7; in (f), n = 7 for ZH11 and gapless2-1, n = 6 for gapless2-2 and gapless2-3; in (g), n = 8 for ZH11, n = 6 for gapless2-1, gapless2-2 and gapless2-3; in (h), n = 6; in (j), n = 8 for ZH11 and gapless3-1, n = 6 for gapless3-2, n = 7 for gapless3-3; in (k), n = 10 for ZH11, n = 9 for gapless3-1, n = 7 for gapless3-2 and gapless3-3. The different letters indicate significant differences at P < 0.05 (one-way ANOVA with Tukey’s post hoc test). Exact P values are listed in Source data.

Source data

Extended Data Fig. 4 The growth and inomic phenotypes of the gapless2/3 mutant.

a, Representative images of ZH11, the double mutant gapless2/3 and the single mutant gapless1 grown in paddy field for 75 days. b,c, Quantification of plant height (b) and tiller numbers (c) of the gapless2/3, gapless1 mutants and wild-type ZH11, respectively. Data points are independent samples. In b, n = 12 for ZH11, n = 7 for gapless2/3, n = 10 for gapless1; in c, n = 10 for ZH11, n = 7 for gapless2/3, n = 8 for gapless1. d, Representative images of grains from ZH11 and gapless mutants as indicated. Statistics of grain weight per plant are shown on the pictures. e, Representative images of old leaves from ZH11, the gapless2/3 and the gapless1 mutant grown in paddy fields. f, Representative images of XRF showing distribution of potassium (K) and calcium (Ca) in leaves of ZH11 and the gapless mutants as indicated. g,h, Potasium (g) and calcium (h) concentrations in old leaves, as revealed by ICP-MS. Data points are independent samples in figures. In (g) and (h), n = 12 for ZH11, n = 6 for gapless2/3, n = 7 for gapless1.Scale bars, 10 cm in (a), 5 cm in (d), 2 cm in (e,) and 5 mm in (f). The values in (b, c, d, g and h) are shown as the mean ± s.d. The different letters indicate significant differences at P < 0.05 (one-way ANOVA with Tukey’s post hoc test). Exact P values are listed in Source data.

Source data

Extended Data Fig. 5 Phenotypes of young seedlings in gapless mutants and the gapless mutants are sensitive to potassium defective treatment.

a, Representative images of ZH11, single mutants of gapless1, gapless2, gapless3 and double mutant gapless1/2 grown in full nutrient conditions for 7 days. b,c, Quantification of adventitious lateral root length (b) and shoot length (c) of ZH11, single mutants of gapless1, gapless2, gapless3 and double mutant gapless1/2, respectively. d, 7-day-old seedlings of the mutant and ZH11 were grown in a nutritive solution with (+K) or without (-K) KNO3 for another 10 days. Representative pictures are presented. e,f, Quantification of shoot length of ZH11 and the mutants without (e) or with (f) K deficiency treatment, respectively. g,h, Quantification of root length of ZH11 and the mutants without (g) or with (h) K deficiency treatment, respectively. The values in (b, c, e, f, g, h) are shown as the mean ± s.d. (n ≥ 6). The different letters indicate significant differences at P < 0.05 (one-way ANOVA with Tukey’s post hoc test). Numbers of independent samples were indicated in the columns respectively. Data points are independent samples in figures. Exact P values are listed in Source data.

Source data

Extended Data Fig. 6 Heatmap representing the ionomic profiles (Z-scores) in old leaves.

Plants of wild type ZH11, single mutants gapless1, gapless2, gapless3 and double mutant gapless1/2 were grown in paddy field for 75 days. Elements contents were determined by ICP-MS. Color-code indicates changes of elements accumulation in mutants compared to ZH11 (Red up, blue down).

Source data

Extended Data Fig. 7 Suberin deposition of gapless mutant with TEM.

Representative TEM images showing suberin lamellae in CS at 30 mm from the root tips. Suberin lamellae were marked with yellow color on the left. The TEM images without coloring are on the right. CS, Casparian strip. Scale bars: 200 nm.

Extended Data Fig. 8 Subcellular localization of GAPLESS1 and OsCASP1.

a, A specific antibody of GAPLESS1 was used to perform immunofluorescence assay. Calcofluor white showed cell wall signal. The secondary antibody is goat anti-rabbit IgG conjugated with Alexa Fluor 555. An assay with gapless1/2 was used as a negative control. b, Representative images show OsCASP1 signals of pOsCASP1:RFP-OsCASP1 at two different positions from the wild-type root tips: 2-5.5 mm (left) and >5.5 mm (right). OsCASP1 is localized around the plasma membrane (AP) at 2-5.5 mm and is localized exclusively at the Casparian strip domain (CSD) at the position >5.5 mm. c, Representative images show GAPLESS1 signals of the wild type NIP and oscasp1 at the position of 8 mm from the root tips. d, Transient expression of GFP-tagged GAPLESS1 and GFP-tagged GAPLESS1 without the secretory signal peptide in tabacco leaves. Representative images show the location of GAPLESS1 and GAPLESS1 lacking the secretory signal peptide (ΔSS) in tobacco leaves. To observe the cell wall localization of GAPLESS1, a plasmolysis assay was performed by treating the leaves with 0.8 M Mannitol. PM, plasma membrane. CW, cell wall. Scale bars: 10 μm in (a, c), 25 μm in (b), 50 μm in (d).

Extended Data Fig. 9 TEM at Casparian strips and ion concentrations in oscasp1 and Nipponbare.

a, Transmission electron micrographs of CS in oscasp1 and Nipponbare (NIP) after plasmolysis at 8 mm from the root tips. CS, Casparian strip; PM, plasma membrane. Scale bars: 200 nm. b, Length of anchored plasma membrane (PM) in oscasp1 (n = 25) and NIP (n = 16) were presented in violin plot. Significant differences were determined in comparison with NIP using a two-sided Student’s t test (P < 0.01). Data points are independent samples in figures. c,d, Plants were grown in paddy field for 75 days and old leaves were picked for by ICP-MS assay to detect the elemental concentrations. The concentrations of potassium (K) and calcium (Ca) in NIP (n = 6) and oscasp1 (n = 6) are listed. Data represent as mean ± s.d. Data points are independent samples in figures. The different letters in (c) and (d) indicate significant differences at P < 0.05 (one-way ANOVA with Tukey’s post hoc test). Exact P values in (c) and (d) are listed in Source data.

Source data

Extended Data Fig. 10 N terminus and C-terminus of OsCASP1 are exposed to apoplast and cytoplasm.

a,b, Predicted 3D structure of AtCASP1 (a) and OsCASP1 (b) were obtained from the Alphafold Protein Structure Database (https://www.alphafold.ebi.ac.uk/). c,d, Tandem RFP-GFP proteins were fused to C-terminus and the N-terminus of OsCASP1 respectively, and were expressed in tobacco leaves. Ratio of GFP/RFP reflects the pH of the environment where the tags are located. e, Representative images of tobacco leaves expressing RFP-GFP-OsCASP1, with deletion of the long N-terminus. Ratio of GFP/RFP reflects the pH of the environment where the tags are located. f, Surface immunolabelling of rice protoplasts expressing OsCASP1 or AtCASP1 fused with a GFP tag at the N-terminus. The immunostaning of GFP was performed using a GFP antibody together with an Alexa Fluor 647-conjugated sencondary antidbody. Scale bars: 100 μm in (c, d and e), 10 μm in (f).

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Song, T., Tian, YQ., Liu, CB. et al. A new family of proteins is required for tethering of Casparian strip membrane domain and nutrient homoeostasis in rice. Nat. Plants 9, 1749–1759 (2023). https://doi.org/10.1038/s41477-023-01503-z

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