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A distinct giant coat protein complex II vesicle population in Arabidopsis thaliana

Nature Plants volume 7, pages 1335–1346 (2021)Cite this article

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Abstract

Plants live as sessile organisms with large-scale gene duplication events and subsequent paralogue divergence during evolution. Notably, plant paralogues are expressed tissue-specifically and fine-tuned by phytohormones during various developmental processes. The coat protein complex II (COPII) is a highly conserved vesiculation machinery mediating protein transport from the endoplasmic reticulum to the Golgi apparatus in eukaryotes1. Intriguingly, Arabidopsis COPII paralogues greatly outnumber those in yeast and mammals2,3,4,5,6. However, the functional diversity and underlying mechanism of distinct COPII paralogues in regulating protein endoplasmic reticulum export and coping with various adverse environmental stresses are poorly understood. Here we characterize a novel population of COPII vesicles produced in response to abscisic acid, a key phytohormone regulating abiotic stress responses in plants. These hormone-induced giant COPII vesicles are regulated by an Arabidopsis-specific COPII paralogue and carry stress-related channels/transporters for alleviating stresses. This study thus provides a new mechanism underlying abscisic acid-induced stress responses via the giant COPII vesicles and answers a long-standing question on the evolutionary significance of gene duplications in Arabidopsis.

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Fig. 1: In vitro reconstitution of Arabidopsis COPII vesicles.
Fig. 2: ABA-sensitive giant COPII vesicles are AtSar1a-dependent.
Fig. 3: AtSar1a-dependent COPII vesicles are involved in abiotic stress responses in plants.
Fig. 4: ABA-triggered AtSar1a-dependent giant COPII vesicles mediate the transportation of stress-related large transporters.

Data availability

The Arabidopsis lines and plasmids generated in this study are available from the corresponding authors upon request. Dataset GSE49418 is publicly available. Source data are provided with this paper.

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Acknowledgements

We thank R. Schekman and D. G. Robinson for critical suggestions; D. Inzé for the PSBD suspension cultures; C. Gao for sharing ABF4 and ABI5 constructs; P. Rodriguez for sharing the Arabidopsis pyr/pyl112458 and hab1-1abi1-2abi2-2 mutants; the Hong Kong Polytechnic University Research Facility in Chemical and Environmental Analysis (UCEA) for the MS platform; and L. Lo for the MS technical assistance. This work was supported by grants from the National Natural Science Foundation of China (31670179 and 91854201), the Research Grants Council of Hong Kong (CUHK14130716, 14102417, 14100818, 14101219, C4012-16E, C4033-19E, C4002-17G, C4002-20W, R4005-18 and AoE/M-05/12) and the CUHK Research Committee to L.J.; Y.G. was supported by Hong Kong Research Grants Council Grants (26100315, 16101116, 16102218, AoE/M-05/12 and C4002-17G) and by the National Natural Science Foundation of China (31871421). Z.Y. was supported by the Research Grants Council Collaborative Research Fund (C5031-14E). W.C.Y.L. was supported by the Research Grants Council General Research Fund (14105517), CUHK Seed Fund Research Support and CUHK Direct Grant for Research (4053182).

Author information

Author notes

  1. These authors contributed equally: Baiying Li, Yonglun Zeng.

Authors and Affiliations

  1. School of Life Sciences, Centre for Cell & Developmental Biology and State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Hong Kong, China

    Baiying Li, Yonglun Zeng, Wenhan Cao, Wenxin Zhang, Lixin Cheng, Xiangfeng Wang, Wilson Chun Yu Lau & Liwen Jiang

  2. Department of Critical Care Medicine, Shenzhen People’s Hospital, The Second Clinical Medicine College of Ji’nan University, Shenzhen, China

    Lixin Cheng

  3. State Key Laboratory of Chemical Biology and Drug Discovery and Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hong Kong, China

    Haidi Yin, Qian Wu & Zhong-Ping Yao

  4. State Key Laboratory of Plant Physiology and Biochemistry, Department of Plant Sciences, College of Biological Sciences, China Agricultural University, Beijing, China

    Xiangfeng Wang

  5. Division of Life Science, Hong Kong University of Science and Technology, Hong Kong, China

    Yan Huang & Yusong Guo

  6. CUHK Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, China

    Liwen Jiang

  7. Institute of Plant Molecular Biology and Agricultural Biotechnology, The Chinese University of Hong Kong, Hong Kong, China

    Liwen Jiang

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Contributions

B.L., Y.Z. and L.J. conceived the project. B.L., Y.Z., Y.G. and L.J. designed the research. B.L., Y.Z., W.C., W.Z., X.W., H.Y., Q.W., Y.H. and W.C.Y.L. performed experiments. B.L., Y.Z., L.C and Z.-P.Y analysed the data. B.L. and Y.Z. wrote the manuscript.

Corresponding authors

Correspondence to Yusong Guo or Liwen Jiang.

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The authors declare no competing interests.

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Peer review information Nature Plants thanks the anonymous reviewers for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Membrane and cytosol preparation for in vitro reconstitution assay.

a, Immunoblot of membrane fractions extracted from 4-d Arabidopsis suspension PSBD cell culture and separated in 25–50% continuous sucrose gradient. b, Diagram describing the workflow of microsomes and cytosol purification. 4-d PSBD cell culture is digested, lysed and homogenized before ultracentrifugation for gradient separation. Microsomes were purified in discontinuous sucrose gradients (HS, high speed; 86,000 × g). Homogenized protoplasts (C1) were medium speed (MS, 10,000 x g) centrifuged with supernatant collected (C2) followed by three rounds of ultracentrifugation (HS, high speed; 259,000 × g) and supernatant collection (C3). c, Immunoblots of membrane fractions collected for microsomes. The 1.0–1.5 M heavy fraction was enriched with ER markers. The 0.6–1.0 M light fraction containing a mixture membrane sources was used as control. H, heavy fraction. L, light fraction. d, Immunoblot of the purification profile in cytosol extraction. The final supernatant C3 contains mainly cytosolic proteins including essential COPII coat proteins with little membrane protein contamination. The experiments in a, c and d were repeated independently three times with similar results.

Source data

Extended Data Fig. 2 Temperature dependency of plant COPII vesicle formation and in vitro reconstitution of mammalian COPII vesicles.

a, The in vitro vesicle formation at different temperatures. In the presence of the energy regeneration system, vesicles form actively at 27 °C. NT, nucleotides, GTP and ATP energy regeneration system (rATP). b, Quantification of a. Means ± SD with all individual data points plotted, n = 3 independent budding reactions derived from a batch of prepared cytosol, microsomes and energy sources, one-way ANOVA analysis followed by Šídák’s multiple comparisons test. ***p < 0.001, Exact p values are ***p < 0.0001 for all ***p values. n.s, not significant, for ManI blots, p = 0.1863 in 20°C -NT/ +NT group, p = 0.5666 in 37°C -NT/ +NT group; for Sec22 blot, p = 0.3572 in 20°C -NT/ +NT group. c, Fractions were collected from the top to the bottom in the Optiprep gradient. When nucleotides supply (+NT), intact vesicles show up on the top fraction showing cargo signals ManI and Sec22. d, ER resident protein Sec61 was excluded from the in vitro reconstituted nascent COPII vesicles. e, TEM of negatively stained reconstituted COPII vesicles from plants under different conditions. The in vitro formed COPII vesicle number decreases in the absence of nucleotides or addition of GTPγS or Sar1-antibody. Scale bar, 100 nm. f, TEM observation of negatively stained reconstituted vesicles from semi-intact HEK293 cells. The addition of purified Sar1ADN protein or GTPγS inhibits the vesicle formation. Scale bar, 100 nm. g, Quantification of e,f. Means ± SD with all individual data points plotted, n = 3 independent budding reactions derived from a batch of prepared cytosol, microsomes and energy sources, TEM images then taken at x 50,000 enlargement were used for analysis. One-way ANOVA analysis followed by Dunnett’s multiple test; ***p < 0.001. Exact p values are ***p < 0.0001 for all comparing groups. The experiments in a, c, d, e and f were repeated independently for at least three times with similar results.

Source data

Extended Data Fig. 3 The formation of the giant vesicles was arrested in ABA-insensitive mutants but activated in ABA-sensitive mutants.

a, 3D Electron Tomography displaying in vivo ER-derived vesicles in root tips of 4 DAG Arabidopsis ABA insensitive mutant pyr/pyl112458 or ABA hypersensitive mutant hab1-1abi1-2abi2-2. Scale bars, 50 nm. Arrows indicate the ER-derived vesicles. b, Quantification of vesicle size in a. Means ± SD with all individual data points plotted, n = 9, 8, 16, 11 vesicles for pyr/pyl112458, pyr/pyl112458 + ABA, hab1-1abi1-2abi2-2, hab1-1abi1-2abi2-2 + ABA group respectively. Two-way ANOVA analysis followed by Šídák’s multiple comparisons test; ***p < 0.001. Exact p values are ***p < 0.0001 for pyr/pyl112458 comparing with hab1-1abi1-2abi2-2 and for pyr/pyl112458 + ABA comparing with hab1-1abi1-2abi2-2 + ABA. n.s, not significant, p = 0.9989 for pyr/pyl112458 comparing with pyr/pyl112458 + ABA, p > 0.9999 for hab1-1abi1-2abi2-2 comparing with hab1-1abi1-2abi2-2 + ABA. The experiments in a were repeated independently for at least three times with similar results.

Source data

Extended Data Fig. 4 eBL or MeJA treatment does not induce the formation of AtSar1a-dependent giant vesicles in vitro and in vivo.

a, Confocal microscopy analysis of AtSar1a-GFP in AtSar1a::AtSar1a-GFP transgenic plants upon eBL or MeJA treatments. Scale bars, 10 μm. b, Quantification of puncta intensity of AtSar1a-GFP upon mock, eBL or MeJA treatments in a. c, 3D Electron Tomography displaying in vivo COPII vesicles in 4 DAG WT Arabidopsis root tips under mock condition or upon eBL or MeJA treatments. Scale bars, 50 nm. Arrows indicate ER-derived vesicles. d, Quantification of vesicle size in c. Two-tailed unpaired t test. Means ± SD with all individual data points plotted, n = 11, 12 vesicles from eBL or MeJA treated seedlings respectively. n.s, not significant, p = 0.8669 e, TEM of negatively stained plant COPII vesicles reconstituted under mock condition or upon eBL or MeJA treatments with (+NT) or without (-NT) energy supplies. Scale bars, 100 nm. f-g, Quantification of vesicle number in e. Means ± SD with all individual data points plotted, n = 4 independent budding reactions derived from a batch of prepared cytosol, microsomes and energy sources for + NT groups in f, or n = 3 for all other groups; TEM images taken at x 50,000 enlargement were used for analysis. One way ANOVA followed by Šídák’s multiple comparisons test. ***p < 0.001, exact p values are: ***p < 0.0001. n.s, not significant, p = 0.9932 for + eBL group, p = 0.7394 for + MeJA group. The experiments in a, c and e were repeated independently for at least three times with similar results.

Source data

Extended Data Fig. 5 AtSar1a exhibit specific effect on in vitro giant vesicle formation upon ABA treatment.

a, Recombinant protein purification of the four dominant negative AtSar1s (AtSar1DNs). b, TEM of negatively stained reconstituted COPII vesicles upon ABA treatments in the presence of AtSar1a/b/c/dDN. Scale bars, 100 nm. c, Quantification of b. Means ± SD with all individual data points plotted, n = 3 independent budding reactions derived from a batch of prepared cytosol, microsomes and energy sources, TEM images taken at x 50,000 enlargement were used for analysis. One-way ANOVA followed by Dunnett’s multiple test. *p < 0.05, exact p value is *p = 0.0421. n.s, not significant, p > 0.9999 for + NT/ +AtSar1bDN, p = 0.9998 for + NT/ +AtSar1cDN, p = 0.9999 for + NT/ +AtSar1dDN. The experiment in b was repeated independently for at least three times with similar results.

Source data

Extended Data Fig. 6 In vivo analysis of AtSar1 puncta upon ABA treatment.

a, Confocal imaging analysis displaying the unchanged AtSar1c-RFP pattern in native promoter driven AtSar1c::AtSar1c-RFP transgenic plants upon ABA treatment. Scale bars, 10 μm. b-c, Comparison of the intensity or diameter between native AtSar1a-GFP and native AtSar1c-RFP positive puncta under mock condition or in response to ABA treatment. Means ± SD with all individual data points plotted, n = 10 puncta per group. Two way ANOVA followed by Tukey’s multiple comparisons test. **p < 0.01, ***p < 0.001, exact p values are **p = 0.0010 in b, ***p < 0.0001 in c. n.s, not significant, p = 0.2712 in b, p = 0.9929 in c. d, AtSar1a-positive giant COPII vesicles formed alongside the ER network upon ABA treatment but vanished upon H89 treatment. e, Quantification of d. Means ± SD with all individual data points plotted, n = 6 root cell cross-sections per group, one-way ANOVA followed by Dunnett’s multiple test. ***p < 0.001, exact p value is ***p < 0.0001. n.s, not significant, p = 0.2283. The experiments in a and d were repeated independently for at least three times with similar results.

Source data

Extended Data Fig. 7 Osmotic stress triggered by PEG or mannitol treatment induced the formation of AtSar1a-dependent giant vesicles in vitro and in vivo.

a, Confocal microscopy analysis of AtSar1a-GFP in AtSar1a::AtSar1a-GFP transgenic plants upon mannitol or PEG treatments. Scale bars, 10 μm. b, Quantification of puncta intensity of AtSar1a-GFP under mock condition or upon mannitol or PEG treatments in a. c, 3D Electron tomography displaying in vivo COPII vesicles in root tips of 4 DAG WT Arabidopsis under mock condition or upon mannitol or PEG treatments. Scale bars, 50 nm. Arrows indicate the ER-derived vesicles. d, Quantification of vesicle size in c. Means ± SD with all individual data points plotted, n = 10, 15, 29 vesicles from Mock, +PEG or +Mannitol groups respectively, one way ANOVA followed by Tukey’s multiple comparisons test. **p < 0.01, ***p < 0.001, exact p values are **p = 0.0072, ***p < 0.0001. n.s, not significant, p = 0.4019. e, TEM of negatively stained vesicles reconstituted under mock condition or upon mannitol or PEG treatments with (+NT) or without (-NT) energy supplies. Scale bars, 100 nm. f-g, Quantification of vesicle numbers in e. Means ± SD with all individual data points plotted, n = 3 independent budding reactions derived from a batch of prepared cytosol, microsomes and energy sources, TEM images taken at x 50,000 enlargement were used for analysis. One way ANOVA followed by Šídák’s multiple comparisons test. ***p < 0.001, exact p values are: ***p = 0.0001 for + Mannitol group, ***p = 0.0002 for + PEG group. n.s, not significant, p = 0.6619 for + Mannitol group, p = 0.9189 for + PEG group. The experiments in a, c and e were repeated independently for at least three times with similar results.

Source data

Extended Data Fig. 8 iTRAQ analysis of in vitro budding cargoes under normal condition or upon ABA treatment and verification of cargoes identified from iTRAQ.

a, Volcano plot depicting the log2 fold change of proteins identified in AtSar1cDN groups versus the + NT group. Orange dots indicate potential cargo proteins significantly down-regulated for 1.2 folds or more in the presence of AtSar1cDN with *p < 0.05. b, Volcano plot depicting the log2 fold change of proteins identified in AtSar1aDN groups versus the + NT group. Orange dots indicate potential cargo proteins significantly down-regulated for 1.2 folds or more in the presence of AtSar1aDN with *p < 0.05. c, Scatter plot depicting AtSar1aDN/+NT cargo protein profile under ABA treatment from two biological repeats. Coloured dots indicate unique proteins down-regulated for 1.2 folds or more in at least one repeat. Orange dots specify proteins down-regulated for 1.2–1.5 folds in both repeats. Dark orange dots are proteins down-regulated for 1.5 folds or more in at least one repeat. Pink proteins are highly confident cargoes down-regulated for 1.5 folds or more in both repeats. Cargoes of special interests are annotated as red (> 1.5 folds in both repeats) or green (> 1.2 folds in at least one repeat) dots. Canonical COPII cargoes and coat proteins are highlighted in red text. d, Verification of the sucrose transporter AtSUC5 and proline transporter AtPROT2 from the quantitative MS list as AtSar1a-specific cargo proteins upon ABA treatment. Cargoes were co-transiently expressed in Arabidopsis protoplasts with AtSar1a or AtSar1aDN with or without ABA. Scale bars, 10 μm. e, Quantification of the localization pattern in d, means ± SD with all individual data points plotted, n = 3 biologically independent experiments with at least 50 protoplasts per group used for analysis, one way ANOVA followed by Šídák’s multiple comparisons test. ***p < 0.001. Exact p values are: ***p < 0.0001 for all comparing groups. The experiments in a, b and d were repeated independently for at least three times with similar results.

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Extended Data Fig. 9 Giant COPII vesicles package the ABCG36 transporter upon ABA treatment.

a, Confocal analysis of the ABCG36 transporter and AtSar1a-positive vesicles upon ABA treatment. ABCG36 transporter was transiently co-expressed in Arabidopsis protoplasts with AtSar1a-GFP and treated with ABA before confocal observation in early (8 h) and late (16 h) time points. Scale bars, 10 μm. b, Budding efficiency of ABCG36 transporter in plant COPII vesicles was assessed under ABA treatment with/without AtSar1aDN. NT, nucleotides, GTP and ATP energy regeneration system (rATP). c, Quantification of b. Means ± SD with all individual data points plotted, n = 3 independent budding reactions derived from a batch of prepared cytosol, microsomes and energy sources, two-tailed unpaired t test, ***p < 0.001, exact p value is ***p < 0.0001. d, TEM images of negatively stained ABA-induced reconstituted vesicles after IEM using 6 nm gold particles and ABCG36 antibody. Scale bars, 100 nm. Arrows indicate gold particle labelling. Right panels showing 2X enlarged images from the overview (indicated by white dotted line boxes). The experiments in a, b and d were repeated independently for at least three times with similar results.

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Extended Data Fig. 10 Working model of giant COPII vesicle formation under stress conditions in Arabidopsis.

Giant COPII vesicle formation is mediated by AtSar1a upon ABA treatment or under stress conditions for plants to accommodate to the increased demanding of large transporters or ion channels trafficking in responses to stresses.

Supplementary information

Supplementary Information

Supplementary Figs. 1–9, unprocessed gels or blots for supplementary Figs. 1, 2, 6 and 9, and Table of primers.

Reporting Summary

Supplementary Table 1

Analysed iTRAQ quantitative mass spectrometry results for volcano plots in Fig. 4 and Extended Data Fig. 8.

Supplementary Video 1

3D electron tomography movie of in vivo COPII vesicle in the 4 DAG WT Arabidopsis root tip cell.

Supplementary Video 2

3D electron tomography movie of in vivo COPII vesicle in the 4 DAG WT Arabidopsis root tip cell upon ABA treatment.

Supplementary Video 3

3D electron tomography movie of in vivo COPII vesicles in the 4 DAG sar1a-1 Arabidopsis root tip cell upon ABA treatment.

Supplementary Video 4

3D electron tomography movie of in vivo COPII vesicles in the 4 DAG pyr/pyl112458 Arabidopsis root tip cells under mock condition or upon ABA treatment.

Supplementary Video 5

3D electron tomography movie of in vivo COPII vesicles in the 4 DAG hab1-1abi1-2abi2-2 Arabidopsis root tip cells under mock condition or upon ABA treatment.

Supplementary Video 6

3D electron tomography movie of in vivo COPII vesicles in the 4 DAG WT Arabidopsis root tip cell under mock condition.

Supplementary Video 7

3D electron tomography movie of in vivo COPII vesicles in the 4 DAG WT Arabidopsis root tip cell upon eBL treatment.

Supplementary Video 8

3D electron tomography movie of in vivo COPII vesicle in the 4 DAG WT Arabidopsis root tip cell upon MeJA treatment.

Supplementary Video 9

3D electron tomography movie of in vivo COPII vesicles in the 4 DAG WT Arabidopsis root tip cells upon PEG or mannitol treatment.

Supplementary Data 1

RNAseq analysis of total gene expression in mock and ABA-treated Arabidopsis seedlings.

Supplementary Data 2

Raw iTRAQ quantitative mass spectrometry search results.

Supplementary Data 3

Statistical source data for supplementary figures.

Source data

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Li, B., Zeng, Y., Cao, W. et al. A distinct giant coat protein complex II vesicle population in Arabidopsis thaliana. Nat. Plants 7, 1335–1346 (2021). https://doi.org/10.1038/s41477-021-00997-9

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