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A non-canonical role of ATG8 in Golgi recovery from heat stress in plants

Nature Plants volume 9, pages 749–765 (2023)Cite this article

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Abstract

Above-optimal growth temperatures, usually referred to as heat stress (HS), pose a challenge to organisms’ survival as they interfere with essential physiological functions and disrupt cellular organization. Previous studies have elucidated the complex transcriptional regulatory networks involved in plant HS responses, but the mechanisms of organellar remodelling and homeostasis during plant HS adaptations remain elusive. Here we report a non-canonical function of ATG8 in regulating the restoration of plant Golgi damaged by HS. Short-term acute HS causes vacuolation of the Golgi apparatus and translocation of ATG8 to the dilated Golgi membrane. The inactivation of the ATG conjugation system, but not of the upstream autophagic initiators, abolishes the targeting of ATG8 to the swollen Golgi, causing a delay in Golgi recovery after HS. Using TurboID-based proximity labelling, we identified CLATHRIN LIGHT CHAIN 2 (CLC2) as an interacting partner of ATG8 via the AIM–LDS interface. CLC2 is recruited to the cisternal membrane by ATG8 to facilitate Golgi reassembly. Collectively, our study reveals a hitherto unanticipated process of Golgi stack recovery from HS in plant cells and uncovers a previously unknown mechanism of organelle resilience involving ATG8.

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Fig. 1: Induction of ATG8 puncta by HS relies on the ATG conjugation system.
Fig. 2: ATG8 is recruited to the vesicular Golgi upon HS.
Fig. 3: Mutation of ATG5 delays Golgi reconstruction during recovery from HS.
Fig. 4: Identification of the neighbour proteome of ATG8 on the vesicular Golgi.
Fig. 5: HS increases the interaction between CLC2 and ATG8.
Fig. 6: Recruitment of CLC2 by ATG8 facilitates Golgi remodelling.
Fig. 7: The dynamics of ATG8-positive vesicles.

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Data availability

The data that support the results in this study are available in the Article and its Supplementary Information files. The raw mass spectrometry data were searched against the TAIR10 database (https://www.arabidopsis.org/). The proteomics data have been deposited at the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the iProX partner repository83 (identifier, PXD039608). Source data are provided with this paper.

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Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (grant nos. 32061160467, 32270291 and 31870171) and Fok Ying-Tong Education Foundation for Young Teachers in the Higher Education Institutions of China (grant no. 171014) to C.G., the National Science Foundation of China (grant no. 31600288) and the Basic Research Program of Guangzhou (grant no. 202201010508) to J.Z., and Hong Kong Research Grant Council (grant nos. GRF14113921, GRF14109222, N_CUHK462/22 and C4002-20W) to B.-H.K. We acknowledge the support of the 2022 Guangdong–Hong Kong–Macao Greater Bay Area Exchange Programs of SCNU.

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Author notes

  1. These authors contributed equally: Jun Zhou, Juncai Ma, Chao Yang.

Authors and Affiliations

  1. MOE Key Laboratory & Guangdong Provincial Key Laboratory of Laser Life Science, Guangzhou Key Laboratory of Spectral Analysis and Functional Probes, College of Biophotonics, Guangdong Provincial Key Laboratory of Biotechnology for Plant Development, School of Life Sciences, South China Normal University, Guangzhou, China

    Jun Zhou, Chao Yang, Xiu Zhu, Xuanang Zheng, Xibao Li, Siyu Chen, Lei Feng, Jun Liao & Caiji Gao

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

    Juncai Ma, Jing Li, Lei Feng, Pengfei Wang, Man Ip Ho, Wenlong Ma, Xiaohong Zhuang, Liwen Jiang & Byung-Ho Kang

  3. Guangdong Provincial Key Laboratory of Applied Botany & Key Laboratory of South China Agricultural Plant Molecular Analysis and Genetic Improvement, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, China

    Chao Yang

  4. College of Life Sciences, South China Agricultural University, Guangzhou, China

    Faqiang Li

  5. College of Life Sciences, Shaoxing University, Shaoxing, China

    Chao Wang

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Contributions

J.Z., J.M., B.-H.K. and C.G. designed the experiments. J.Z., J.M., C.Y., X. Zhu, J. Li, X. Zheng, X.L., S.C. and J. Liao performed the experiments. J.Z., J.M., J. Li, L.F., P.W., M.I.H., W.M., X. Zhuang, L.J., B.-H.K. and C.G. analysed the data. J.M., J. Li, P.W., M.I.H., W.M. and B.-H.K. contributed to the EM analysis. J.Z., J.M., C.Y., F.L., C.W., X. Zhuang, L.J., B.-H.K. and C.G. wrote and edited the paper. All authors reviewed the paper.

Corresponding authors

Correspondence to Jun Zhou, Byung-Ho Kang or Caiji Gao.

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Nature Plants thanks Yohann Boutte, Tamar Avin-Wittenberg 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 Acute heat stress induces the formation of ATG8 puncta.

a, Analysis of the patterns of GFP-ATG8a puncta at different temperatures. Five-day-old GFP-ATG8a transgenic seedlings were exposed to various temperatures for 5 mins, then recovered for 3 hrs at 22 °C before confocal imaging. The intensity profiles were calculated along the dotted line indicated in 5x Enlargement. Scale bar, 20 µm. b, Confocal images of HS-induced GFP-ATG8a puncta in different tissues. Five-day-old GFP-ATG8a seedlings were exposed to 45 °C for 5 mins, then recovered for 3 hrs at 22 °C before confocal imaging. c, Analyzing the response of various ATG8 isoforms (ATG8a-i) to HS. Five-day-old GFP-ATG8a, YFP-ATG8b/c/d/e/g/h/i and mCherry-ATG8f transgenic seedlings were exposed to 45 °C for 5 mins, then recovered for 3 hrs at 22 °C before confocal imaging. Scale bars, 20 µm. Similar confocal imaging results in (a–c) were obtained in at least 10 individual roots/leaves with three times of replicates.

Extended Data Fig. 2 Characterize the dynamics of ATG8 puncta.

a, A schematic workflow of heat stress regimes applied to probe YFP-ATG8e response. b, The effect of heat duration on YFP-ATG8e puncta formation. Five-day-old YFP-ATG8e seedlings were exposed to 45 °C for the indicated time points, then recovered for 3 hrs at 22 °C before confocal imaging. Scale bar, 20 µm. c, Time series analysis of the formation of YFP-ATG8e puncta after HS treatment (45 °C, 5 mins). Scale bar, 25 µm. d, The specific response of the mCherry-ATG8f to HS treatment. Five-day-old GFP x mCherry-ATG8f double transgenic seedlings were incubated in liquid 1/2 MS medium without/with 45 °C HS treatment for 5 mins, then visualized under the confocal microscopy after 3 hrs recovery at 22 °C. Scale bar, 20 µm. e, Immunolabeling with ATG8 antibodies showing the punctate structures of native ATG8 in wild-type Col-0 root meristematic cells upon HS treatment. Scale bars, 20 µm. The experiments (b–e) were repeated independently three times with consistent results.

Extended Data Fig. 3 ATG8 puncta induced by HS differ from conventional autophagosomes.

a, Colocalization analysis of mCherry-ATG8f and NBR1-GFP under normal condition or upon HS treatment. Arrowheads indicated colocalized structures, while arrows indicated those that did not colocalize. Scale bars, 20 µm. b, Quantification of colocalization ratio showing in (a). The PSC coefficient was calculated by Image J with the PSC Colocalization plugin. Data represent means ± SD. n = 5 confocal microscopic images (112.5 µm x 112.5 µm) from individual roots. P values were determined by two-tailed unpaired Student’s t-test. c, GFP-ATG8a cleavage assay showing the autophagic activity during the HS recovery phase. The ratio of free GFP to GFP-ATG8a was quantified by Image J. Ponceau S staining indicated the protein loading. Asterisk indicated an unknown band. d, HS-induced GFP-ATG8a puncta were sensitive to ConcA. A schematic diagram illustrating the HS treatment and the addition of ConcA (1 μM) was given. Scale bar, 20 µm. e, Time-course analysis the disappearance of GFP-ATG8a puncta following ConcA application. A schematic diagram illustrating the HS treatment and the addition of ConcA was given on the left. White arrowheads indicated the representative disappearing GFP-ATG8a puncta. Scale bar, 50 µm. f, HS reduced autophagic body formation. The schematic diagram showed the time points of HS treatment and ConcA addition. Scale bar, 20 µm. g, Quantify the ATG8 positive bodies shown in (f). Data represent means ± SD. n = 10 confocal microscopic images (112.5 µm x 112.5 µm) from individual roots. P values were determined by two-tailed unpaired Student’s t-test. The confocal imaging (a, d–f) and western blot (c) were repeated independently three times with consistent results.

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Extended Data Fig. 4 Possible role of ATG16 in HS-induced ATG8 puncta formation.

a, Interaction analysis of ATG16 and V-ATPase subunits by Y2H assay. The TGN-localized V0 subunits VHA-a1 and VHA-e1 as well as V1 subunits VHA-A and VHA-C were test for their interaction with full-length ATG16 and the WD40 domain of ATG16. SD-2, synthetic dropout medium lacking Leu and Trp. SD-4, synthetic dropout medium lacking Leu, Trp, His and Ade. b, Response of YFP-ATG16 to HS treatment in Col-0, atg5-1 and atg7-2 root cells. Five-day-old YFP-ATG16/Col-0, YFP-ATG16/atg5-1 and YFP-ATG16/atg7-2 seedlings were incubated in liquid 1/2 MS medium with 45 °C HS treatment for 5 mins, then visualized under the confocal microscopy after recovery at 22 °C for 3 hrs. Scale bar, 20 µm. c, Colocalization analysis of YFP-ATG16 and mCherry-ATG8f or VHA-a1-RFP under normal condition or upon HS treatment. Five-day-old YFP-ATG16 x mCherry-ATG8f and YFP-ATG16 x VHA-a1-RFP double transgenic plants were incubated in liquid 1/2 MS medium at 45 °C for 5 mins, then visualized under the confocal microscopy after recovery for 3 hrs. Scale bar, 20 µm. d, Colocalization analysis of RFP-SYP32 and ATG5-GFP or ATG12A-GFP under normal condition or upon HS treatment. Five-day-old ATG5-GFP x RFP-SYP32 and ATG12A-GFP x RFP-SYP32 double transgenic plants were incubated in liquid 1/2 MS medium at 45 °C for 5 mins, then visualized under the confocal microscopy after recovery for 3 hrs. Scale bar, 20 µm. The experiments were repeated at least three times with similar results.

Extended Data Fig. 5 Restacking of Golgi cisternae is delayed in atg5-1 mutant.

a, Representative TEM images of Golgi apparatus in Col-0 and atg5-1 root cells at different recovery time points (3, 6 and 12 hrs). Arrowheads indicate restacking of Golgi cisternae. Similar TEM results were obtained in four individual EM blocks with three times of replicates. Scale bars, 500 nm. b, Quantification of the numbers of Golgi with stacks per cell showing in (a). Data represent means ± SD. n = 20 cells from four individual root blocks were used for quantification analysis. P values were determined by two-tailed unpaired Student’s t-test. c, Positional relationship between VHA-a1-YFP and RFP-SYP32 at different stages of recovery. The intensity profiles were calculated along the dotted arrow indicated in 5x Enlargement. Similar confocal imaging results were obtained in at least 10 individual roots with three times of replicates. Scale bar, 10 µm. d, Quantification of association between VHA-a1-YFP and RFP-SYP32 in (c). The center-to-center distance between VHA-a1-YFP and RFP-SYP32 was calculated with Image J plugin DiAna. n = 1634 closest objects from ten confocal microscopic images (112.5 µm × 112.5 µm). The median (solid line) and quartiles (dotted line) were given in violin plot. P values correspond to two-tailed unpaired Student’s t-test.

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Extended Data Fig. 6 HS-induced Golgi swelling inhibits the post-Golgi trafficking.

a, A cell wall polysaccharide, xyloglucan, accumulated in the swollen Golgi lumen after HS treatment. Two xyloglucan-specific antibodies (anti-CCRCM1 and anti-XG) were used for immuno-EM analysis. CW, cell wall; G, Golgi apparatus; M, mitochondria; P, plastid; V, Vacuole. Red arrows indicate gold particles (10 nm). Scale bars, 500 nm. Similar immuno-EM results were obtained in at least four individual root blocks. b, HS-induced punctate signals of SEC-GFP accumulated inside the cells. Five-day-old SEC-GFP seedlings were incubated in liquid 1/2 MS medium with 45 °C HS treatment for 5 mins, then visualized under confocal microscopy after recovery at 22 °C for 3 hrs. Scale bar, 10 µm. c, HS inhibited the internalization of BOR1-GFP in response to high-boron treatment. The BOR1-GFP transgenic plants were grown on low-boron medium (1 µM boric acid) for 5 days and then transferred to high-boron (150 µM boric acid) liquid medium with/without 50 µM BFA for 1 hr before confocal imaging. Scale bar, 20 µm. d, Quantitative analysis of the ratio of BOR1-GFP signal intensity. The fluorescence intensity was calculated with Image J from thirteen root meristematic regions (n = 13). Data represent means ± SD. P values were determined by two-tailed unpaired Student’s t-test. e-f, FRAP analysis of BOR1-GFP dynamics in response to HS. The 5-day-old BOR1-GFP seedlings were treated with CHX (50 μM) for 30 mins prior to FRAP experiments (e), followed by relative fluorescent intensity analysis of BOR1-GFP dynamics (f). Scale bars, 10 μm. Data represent means ± SD from three individual roots (n = 3). The experiments were repeated three times with similar results.

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Extended Data Fig. 7 HS abolishes GFP-SYP32 and VHA-a1-RFP response to BFA.

a-b, Effect of HS and BFA on the localization of mCherry-ATG8f and GFP-SYP32 (a), as well as GFP-ATG8a and VHA-a1-RFP (b). The schematic diagrams illustrating the HS treatment and the addition of BFA were given on the left. Similar confocal imaging results were obtained in at least 10 individual roots with three times of replicates. Scale bars, 20 µm.

Extended Data Fig. 8 CLC2 interacts with all nine ATG8 isoforms (ATG8a-i).

a, Y2H examining the interaction between CLC2 with nine ATG8 isoforms (ATG8a-i). The Y2H assays were repeated three times with similar results. b, Multiple sequence alignment of the CLC proteins (CLC1 (ABF85787.1), CLC2 (AEC09770.1) and CLC3 (AEE78859.1)) using DNAMAN software. The AIM site in CLC2 was indicated with a red dashed box. Shaded regions show identical amino acid residues among CLC proteins. c, A working model for the in vivo recruitment assay. ATG8 was fused to ER-located calnexin (CNX) to generate a fusion protein CNX-RFP-ATG8. When co-expression of the potential interactors/cargoes of ATG8, they could be recognized by CNX-RFP-ATG8 and then recruited to ER, displaying a linear ER pattern. d, The ER-anchored ATG8e could recruit CLC2-YFP but not CLC2(ΔAIM)-YFP. The different plasmid combinations as indicated were transient expressed in mesophyll protoplasts. After incubated in the dark for 16 hrs, the protoplasts were heat treated at 45 °C for 5 mins, then recovered for 3 hrs at 22 °C before confocal imaging. Scale bar, 10 µm. The in vivo recruitment assays were repeated twice with similar results.

Extended Data Fig. 9 Characterize the dynamics of ATG8 puncta during long-term HS recovery.

a, Representative confocal images showing the patterns of GFP-ATG8a after recovery for 3 to 24 hrs. Five-day-old GFP-ATG8a transgenic seedlings were incubated in liquid 1/2 MS medium with 45 °C HS treatment for 5 mins, then visualized under the confocal microscopy after recovery at 22 °C for the indicated time points. Scale bar, 20 µm. b-c, Colocalization analysis of GFP-ATG8a and VHA-a1-RFP (b) as well as mCherry-ATG8f and NBR1-GFP (c) in root cells after recovery for 3, 6 and 12 hrs. Arrows indicate the separated dots. Scale bars, 20 µm. The colocalization ratio between VHA-a1-RFP and GFP-ATG8a was calculated with Image J plugin JACoP based on distance between centres of mass. Data represent means ± SD. n = 10 confocal microscopic images (112.5 µm × 112.5 µm) from individual roots. P values were analyzed with two-tailed unpaired Student’s t-test. Similar confocal imaging results (a–c) were obtained in at least 10 individual roots with three times of replicates.

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Supplementary information

Supplementary Information

Supplementary Figs. 1 and 2.

Reporting Summary

Supplementary Tables

Supplementary Tables 1–5.

Supplementary Video 1

Accumulation of GFP–ATG8a-labelled puncta in root cells after exposure to 45 °C for 5 minutes.

Supplementary Video 2

Accumulation of GFP–ATG8a-labelled puncta in mesophyll cells after exposure to 45 °C for 5 minutes.

Supplementary Video 3

Time-lapse imaging of YFP–ATG8e puncta formation after HS treatment.

Supplementary Video 4

ConcA dissociates ATG8 from the HS-induced punctate structures.

Supplementary Video 5

3D electron tomography analysis of the buds generated from the dilated Golgi cisternae after 4 h of recovery from HS.

Supplementary Video 6

Time-lapse imaging of a small dot released from mCherry–ATG8f puncta at 4 h after recovery from HS.

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Zhou, J., Ma, J., Yang, C. et al. A non-canonical role of ATG8 in Golgi recovery from heat stress in plants. Nat. Plants 9, 749–765 (2023). https://doi.org/10.1038/s41477-023-01398-w

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