Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

ARP2/3 complex associates with peroxisomes to participate in pexophagy in plants

Nature Plants volume 9, pages 1874–1889 (2023)Cite this article

Subjects

Abstract

Actin-related protein (ARP2/3) complex is a heteroheptameric protein complex, evolutionary conserved in all eukaryotic organisms. Its conserved role is based on the induction of actin polymerization at the interface between membranes and the cytoplasm. Plant ARP2/3 has been reported to participate in actin reorganization at the plasma membrane during polarized growth of trichomes and at the plasma membrane–endoplasmic reticulum contact sites. Here we demonstrate that individual plant subunits of ARP2/3 fused to fluorescent proteins form motile spot-like structures in the cytoplasm that are associated with peroxisomes in Arabidopsis and tobacco. ARP2/3 is found at the peroxisome periphery and contains the assembled ARP2/3 complex and the WAVE/SCAR complex subunit NAP1. This ARP2/3-positive peroxisomal domain colocalizes with the autophagosome and, under conditions that affect the autophagy, colocalization between ARP2/3 and the autophagosome increases. ARP2/3 subunits co-immunoprecipitate with ATG8f and peroxisome-associated ARP2/3 interact in vivo with the ATG8f marker. Since mutants lacking functional ARP2/3 complex have more peroxisomes than wild type, we suggest that ARP2/3 has a novel role in the process of peroxisome degradation by autophagy, called pexophagy.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: ARP2/3 complex subunits GFP–ARPC2 and GFP–ARPC5 and NAP1–GFP, a subunit of an ARP2/3-activating complex, colocalize with peroxisomes.
Fig. 2: Colocalization of ARP2/3 with actin.
Fig. 3: GFP–ARPC5 localization pattern in peroxisomes is dependent on the complex assembly and activation status.
Fig. 4: ARP2/3 mutant peroxisomes are larger, more abundant and functional, ARP2/3 colocalizes with peroxisomes and ATG8f.
Fig. 5: The number of ARP2/3 spots and its colocalization with autophagosomes is sensitive to autophagy induction and inhibition.

Similar content being viewed by others

Data availability

All the main data supporting the findings of this study are available within the article and its supplementary information files. A complete list of proteins identified in proteomic analysis is available as source data. Materials used in this study are available on request from the corresponding author. Source images for Fig. 3e and Supplementary Fig. 5 were uploaded to the Figshare repository (https://doi.org/10.6084/m9.figshare.24064845). All the supporting information and source data can be found in the Zenodo repository (https://doi.org/10.5281/zenodo.8276211). Source data are provided with this paper.

Code availability

Script used for automated analysis of colocalization can be found in the Zenodo repository https://zenodo.org/record/7709848. Parameters used in the script are listed in Supplementary Table 3. Code for all statistical tests is available in the GitHub repository https://github.com/vosolsob/arposomes.

References

  1. Kollmar, M., Lbik, D. & Enge, S. Evolution of the eukaryotic ARP2/3 activators of the WASP family: WASP, WAVE, WASH, and WHAMM, and the proposed new family members WAWH and WAML. BMC Res. Notes 5, 88 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Welch, M. D., DePace, A. H., Verma, S., Iwamatsu, A. & Mitchison, T. J. The human Arp2/3 complex is composed of evolutionarily conserved subunits and is localized to cellular regions of dynamic actin filament assembly. J. Cell Biol. 138, 375–384 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Rotty, J. D., Wu, C. & Bear, J. E. New insights into the regulation and cellular functions of the ARP2/3 complex. Nat. Rev. Mol. Cell Biol. 14, 7–12 (2013).

    CAS  PubMed  Google Scholar 

  4. Welch, M. D., Rosenblatt, J., Skoble, J., Portnoy, D. A. & Mitchison, T. J. Interaction of human Arp2/3 complex and the Listeria monocytogenes ActA protein in actin filament nucleation. Science 281, 105–108 (1998).

    CAS  PubMed  Google Scholar 

  5. Yanagisawa, M., Zhang, C. & Szymanski, D. B. ARP2/3-dependent growth in the plant kingdom: SCARs for life. Front. Plant Sci. 4, 166 (2013).

    PubMed  PubMed Central  Google Scholar 

  6. Pollard, T. D. & Borisy, G. G. Cellular motility driven by assembly and disassembly of actin filaments. Cell 112, 453–465 (2003).

    CAS  PubMed  Google Scholar 

  7. Sawa, M. et al. Essential role of the C. elegans Arp2/3 complex in cell migration during ventral enclosure. J. Cell Sci. 116, 1505–1518 (2003).

  8. Korobova, F. & Svitkina, T. Arp2/3 complex is important for filopodia formation, growth cone motility, and neuritogenesis in neuronal cells. Mol. Biol. Cell 19, 1561–1574 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Kalil, K. & Dent, E. W. Branch management: mechanisms of axon branching in the developing vertebrate CNS. Nat. Rev. Neurosci. 15, 7–18 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Zicha, D. et al. Chemotaxis of macrophages is abolished in the Wiskott–Aldrich syndrome. Br. J. Haematol. 101, 659–665 (1998).

    CAS  PubMed  Google Scholar 

  11. Young, M. E., Cooper, J. A. & Bridgman, P. C. Yeast actin patches are networks of branched actin filaments. J. Cell Biol. 166, 629–635 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Kotchoni, S. O. et al. The association of the Arabidopsis actin-related protein2/3 complex with cell membranes is linked to its assembly status but not its activation. Plant Physiol. 151, 2095–2109 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Ivakov, A. & Persson, S. Plant cell shape: modulators and measurements. Front. Plant Sci. 4, 439 (2013).

  14. Mathur, J., Mathur, N., Kernebeck, B. & Hülskamp, M. Mutations in actin-related proteins 2 and 3 affect cell shape development in Arabidopsis. Plant Cell 15, 1632–1645 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Mathur, J. et al. Arabidopsis CROOKED encodes for the smallest subunit of the ARP2/3 complex and controls cell shape by region specific fine F-actin formation. Development 130, 3137–3146 (2003).

    CAS  PubMed  Google Scholar 

  16. El‐Din El‐Assal, S., Le, J., Basu, D. & Mallery, E. L. DISTORTED2 encodes an ARPC2 subunit of the putative Arabidopsis ARP2/3 complex. Plant J. 38, 526–538 (2004).

  17. Saedler, R. et al. Actin control over microtubules suggested by DISTORTED2 encoding the Arabidopsis ARPC2 subunit homolog. Plant Cell Physiol. 45, 813–822 (2004).

    CAS  PubMed  Google Scholar 

  18. Hossain, M. S. et al. Lotus japonicus ARPC1 is required for rhizobial infection. Plant Physiol. 160, 917–928 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. García-González, J. et al. Arp2/3 complex is required for auxin-driven cell expansion through regulation of auxin transporter homeostasis. Front. Plant Sci. 11, 486 (2020).

    PubMed  PubMed Central  Google Scholar 

  20. Wang, P. & Hussey, P. J. Interactions between plant endomembrane systems and the actin cytoskeleton. Front. Plant Sci. 6, 422 (2015).

    PubMed  PubMed Central  Google Scholar 

  21. Harries, P. A., Pan, A. & Quatrano, R. S. Actin-related protein2/3 complex component ARPC1 is required for proper cell morphogenesis and polarized cell growth in Physcomitrella patens. Plant Cell 17, 2327–2339 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Finka, A. et al. The knock-out of ARP3a gene affects F-actin cytoskeleton organization altering cellular tip growth, morphology and development in moss Physcomitrella patens. Cell Motil. Cytoskeleton 65, 769–784 (2008).

    CAS  PubMed  Google Scholar 

  23. Perroud, P.-F. & Quatrano, R. S. BRICK1 is required for apical cell growth in filaments of the moss Physcomitrella patens but not for gametophore morphology. Plant Cell 20, 411–422 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Menand, B., Calder, G. & Dolan, L. Both chloronemal and caulonemal cells expand by tip growth in the moss Physcomitrella patens. J. Exp. Bot. 58, 1843–1849 (2007).

    CAS  PubMed  Google Scholar 

  25. Li, S., Blanchoin, L., Yang, Z. & Lord, E. M. The putative Arabidopsis arp2/3 complex controls leaf cell morphogenesis. Plant Physiol. 132, 2034–2044 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Chin, S. et al. Spatial and temporal localization of SPIRRIG and WAVE/SCAR reveal roles for these proteins in actin-mediated root hair development. Plant Cell 33, 2131–2148 (2021).

    PubMed  PubMed Central  Google Scholar 

  27. Van Gestel, K. et al. Immunological evidence for the presence of plant homologues of the actin-related protein Arp3 in tobacco and maize: subcellular localization to actin-enriched pit fields and emerging root hairs. Protoplasma 222, 45–52 (2003).

    PubMed  Google Scholar 

  28. Denninger, P. et al. Distinct RopGEFs successively drive polarization and outgrowth of root hairs. Curr. Biol. 29, 1854–1865.e5 (2019).

    CAS  PubMed  Google Scholar 

  29. Yokota, K. et al. Rearrangement of actin cytoskeleton mediates invasion of Lotus japonicus roots by Mesorhizobium loti. Plant Cell 21, 267–284 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Qiu, L. et al. SCARN a novel class of SCAR protein that is required for root-hair infection during legume nodulation. PLoS Genet. 11, e1005623 (2015).

    PubMed  PubMed Central  Google Scholar 

  31. Miyahara, A. et al. Conservation in function of a SCAR/WAVE component during infection thread and root hair growth in Medicago truncatula. Mol. Plant. Microbe Interact. 23, 1553–1562 (2010).

    CAS  PubMed  Google Scholar 

  32. Gavrin, A., Jansen, V., Ivanov, S., Bisseling, T. & Fedorova, E. ARP2/3-mediated actin nucleation associated with symbiosome membrane is essential for the development of symbiosomes in infected cells of Medicago truncatula root nodules. Mol. Plant. Microbe Interact. 28, 605–614 (2015).

    CAS  PubMed  Google Scholar 

  33. Isner, J.-C. et al. Actin filament reorganisation controlled by the SCAR/WAVE complex mediates stomatal response to darkness. N. Phytol. 215, 1059–1067 (2017).

    CAS  Google Scholar 

  34. Jiang, K. et al. The ARP2/3 complex mediates guard cell actin reorganization and stomatal movement in Arabidopsis. Plant Cell 24, 2031–2040 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Fišerová, J., Schwarzerová, K., Petrášek, J. & Opatrný, Z. ARP2 and ARP3 are localized to sites of actin filament nucleation in tobacco BY-2 cells. Protoplasma 227, 119–128 (2006).

    PubMed  Google Scholar 

  36. Maisch, J., Fiserová, J., Fischer, L. & Nick, P. Tobacco Arp3 is localized to actin-nucleating sites in vivo. J. Exp. Bot. 60, 603–614 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Yanagisawa, M. et al. Patterning mechanisms of cytoskeletal and cell wall systems during leaf trichome morphogenesis. Nat. Plants 1, 15014 (2015).

    CAS  PubMed  Google Scholar 

  38. Zhang, C., Mallery, E. L. & Szymanski, D. B. ARP2/3 localization in Arabidopsis leaf pavement cells: a diversity of intracellular pools and cytoskeletal interactions. Front. Plant Sci. 4, 238 (2013).

    PubMed  PubMed Central  Google Scholar 

  39. Havelková, L. et al. Arp2/3 complex subunit ARPC2 binds to microtubules. Plant Sci. 241, 96–108 (2015).

    PubMed  Google Scholar 

  40. Zhang, C. et al. The endoplasmic reticulum is a reservoir for WAVE/SCAR regulatory complex signaling in the Arabidopsis leaf. Plant Physiol. 162, 689–706 (2013).

  41. Yanagisawa, M., Alonso, J. M. & Szymanski, D. B. Microtubule-dependent confinement of a cell signaling and actin polymerization control module regulates polarized cell growth. Curr. Biol. 28, 2459–2466.e4 (2018).

    CAS  PubMed  Google Scholar 

  42. Wang, P., Richardson, C., Hawes, C. & Hussey, P. J. Arabidopsis NAP1 regulates the formation of autophagosomes. Curr. Biol. 26, 2060–2069 (2016).

    CAS  PubMed  Google Scholar 

  43. Wang, P. et al. Plant AtEH/Pan1 proteins drive autophagosome formation at ER–PM contact sites with actin and endocytic machinery. Nat. Commun. 10, 5132 (2019).

    PubMed  PubMed Central  Google Scholar 

  44. Monastyrska, I. et al. Arp2 links autophagic machinery with the actin cytoskeleton. Mol. Biol. Cell 19, 1962–1975 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Kast, D. J., Zajac, A. L., Holzbaur, E. L. F., Ostap, E. M. & Dominguez, R. WHAMM directs the Arp2/3 complex to the ER for autophagosome biogenesis through an actin comet tail mechanism. Curr. Biol. 25, 1791–1797 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Coutts, A. S. & La Thangue, N. B. Regulation of actin nucleation and autophagosome formation. Cell. Mol. Life Sci. 73, 3249–3263 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Kast, D. J. & Dominguez, R. WHAMM links actin assembly via the Arp2/3 complex to autophagy. Autophagy 11, 1702–1704 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Robinson, R. C. et al. Crystal structure of Arp2/3 complex. Science 294, 1679–1684 (2001).

    CAS  PubMed  Google Scholar 

  49. Fahy, D. et al. Impact of salt stress, cell death, and autophagy on peroxisomes: quantitative and morphological analyses using small fluorescent probe N-BODIPY. Sci. Rep. 7, 39069 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Adham, A. R., Zolman, B. K., Millius, A. & Bartel, B. Mutations in Arabidopsis acyl-CoA oxidase genes reveal distinct and overlapping roles in β-oxidation. Plant J. 41, 859–874 (2005).

    CAS  PubMed  Google Scholar 

  51. Graham, I. A. Seed storage oil mobilization. Annu. Rev. Plant Biol. 59, 115–142 (2008).

    CAS  PubMed  Google Scholar 

  52. Farmer, L. M. et al. Disrupting autophagy restores peroxisome function to an Arabidopsis lon2 mutant and reveals a role for the LON2 protease in peroxisomal matrix protein degradation. Plant Cell 25, 4085–4100 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Rodriguez, E. et al. Autophagy mediates temporary reprogramming and dedifferentiation in plant somatic cells. EMBO J. 39, e103315 (2020).

  54. Takatsuka, C., Inoue, Y., Matsuoka, K. & Moriyasu, Y. 3-Methyladenine inhibits autophagy in tobacco culture cells under sucrose starvation conditions. Plant Cell Physiol. 45, 265–274 (2004).

    CAS  PubMed  Google Scholar 

  55. Voitsekhovskaja, O. V., Schiermeyer, A. & Reumann, S. Plant peroxisomes are degraded by starvation-induced and constitutive autophagy in tobacco BY-2 suspension-cultured cells. Front. Plant Sci. 5, 629 (2014).

    PubMed  PubMed Central  Google Scholar 

  56. Chung, T., Phillips, A. R. & Vierstra, R. D. ATG8 lipidation and ATG8-mediated autophagy in Arabidopsis require ATG12 expressed from the differentially controlled ATG12A and ATG12B loci. Plant J. 62, 483–493 (2010).

    CAS  PubMed  Google Scholar 

  57. Bassham, D. C. Methods for analysis of autophagy in plants. Methods 75, 181–188 (2015).

    CAS  PubMed  Google Scholar 

  58. Albrecht, V. et al. The cytoskeleton and the peroxisomal-targeted snowy cotyledon3 protein are required for chloroplast development in Arabidopsis. Plant Cell 22, 3423–3438 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Reumann, S. et al. In-depth proteome analysis of Arabidopsis leaf peroxisomes combined with in vivo subcellular targeting verification indicates novel metabolic and regulatory functions of peroxisomes. Plant Physiol. 150, 125–143 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. Robin, G. P. et al. Subcellular localization screening of colletotrichum higginsianum effector candidates identifies fungal proteins targeted to plant peroxisomes, golgi bodies, and microtubules. Front. Plant Sci. 9, 562 (2018).

    PubMed  PubMed Central  Google Scholar 

  61. Zimmermann, I., Saedler, R., Mutondo, M. & Hulskamp, M. The Arabidopsis GNARLED gene encodes the NAP125 homolog and controls several actin-based cell shape changes. Mol. Genet. Genomics 272, 290–296 (2004).

    CAS  PubMed  Google Scholar 

  62. Mano, S. et al. Distribution and characterization of peroxisomes in Arabidopsis by visualization with GFP: dynamic morphology and actin-dependent movement. Plant Cell Physiol. 43, 331–341 (2002).

    CAS  PubMed  Google Scholar 

  63. Mathur, J., Mathur, N. & Hülskamp, M. Simultaneous visualization of peroxisomes and cytoskeletal elements reveals actin and not microtubule-based peroxisome motility in plants. Plant Physiol. 128, 1031–1045 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  64. Blancaflor, E. B. Cortical actin filaments potentially interact with corticalmicrotubules in regulating polarity of cell expansion in primary roots of maize (Zea mays L.). J. Plant Growth Regul. 19, 406–414 (2000).

    CAS  PubMed  Google Scholar 

  65. Ketelaar, T. et al. The actin-interacting protein AIP1 is essential for actin organization and plant development. Curr. Biol. 14, 145–149 (2004).

    CAS  PubMed  Google Scholar 

  66. Riedl, J. et al. Lifeact: a versatile marker to visualize F-actin. Nat. Methods 5, 605–607 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  67. Kaur, N., Reumann, S. & Hu, J. Peroxisome biogenesis and function. Arabidopsis Book 7, e0123 (2009).

    PubMed  PubMed Central  Google Scholar 

  68. Petriv, O. I., Tang, L., Titorenko, V. I. & Rachubinski, R. A. A new definition for the consensus sequence of the peroxisome targeting signal type 2. J. Mol. Biol. 341, 119–134 (2004).

    CAS  PubMed  Google Scholar 

  69. Lee, H. N., Kim, J. & Chung, T. Degradation of plant peroxisomes by autophagy. Front. Plant Sci. 5, 139 (2014).

    PubMed  PubMed Central  Google Scholar 

  70. Kast, D. J. & Dominguez, R. The cytoskeleton–autophagy connection. Curr. Biol. 27, R318–R326 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  71. Xia, P. et al. WASH inhibits autophagy through suppression of beclin 1 ubiquitination. EMBO J. 32, 2685–2696 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  72. Zavodszky, E. et al. Mutation in VPS35 associated with Parkinson’s disease impairs WASH complex association and inhibits autophagy. Nat. Commun. 5, 3828 (2014).

  73. Coutts, A. S. & La Thangue, N. B. Actin nucleation by WH2 domains at the autophagosome. Nat. Commun. 6, 7888 (2015).

  74. Mi, N. et al. CapZ regulates autophagosomal membrane shaping by promoting actin assembly inside the isolation membrane. Nat. Cell Biol. 17, 1112–1123 (2015).

    CAS  PubMed  Google Scholar 

  75. Mathiowetz, A. J. et al. An Amish founder mutation disrupts a PI(3)P-WHAMM-Arp2/3 complex-driven autophagosomal remodeling pathway. Mol. Biol. Cell 28, 2492–2507 (2017).

  76. Rivers, E. et al. Wiskott Aldrich syndrome protein regulates non-selective autophagy and mitochondrial homeostasis in human myeloid cells. eLife 9, e55547 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  77. Sarkar, S., Olsen, A. L., Sygnecka, K., Lohr, K. M. & Feany, M. B. α-Synuclein impairs autophagosome maturation through abnormal actin stabilization. PLoS Genet. 17, e1009359 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  78. Galiani, S. et al. Super-resolution microscopy reveals compartmentalization of peroxisomal membrane proteins. J. Biol. Chem. 291, 16948–16962 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  79. Quan, S. et al. Proteome analysis of peroxisomes from etiolated Arabidopsis seedlings identifies a peroxisomal protease involved in β-oxidation and development. Plant Physiol. 163, 1518–1538 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  80. Pan, R. et al. Proteome analysis of peroxisomes from dark-treated senescent Arabidopsis leaves. J. Integr. Plant Biol. 60, 1028–1050 (2018).

    CAS  PubMed  Google Scholar 

  81. Wright, Z. J. & Bartel, B. Peroxisomes form intralumenal vesicles with roles in fatty acid catabolism and protein compartmentalization in Arabidopsis. Nat. Commun. 11, 6221 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  82. Kulich, I. et al. Arabidopsis exocyst subcomplex containing subunit EXO70B1 is involved in autophagy-related transport to the vacuole. Traffic 14, 1155–1165 (2013).

    CAS  PubMed  Google Scholar 

  83. Sahi, V. P. et al. Arabidopsis thaliana plants lacking the ARP2/3 complex show defects in cell wall assembly and auxin distribution. Ann. Bot. 122, 777–789 (2018).

    Google Scholar 

  84. Nagata, T., Nemoto, Y. & Hasezawa, S. in International Review of Cytology (eds Jeon, K. W. & Friedlander, M.) 132, 1–30 (Academic Press, 1992).

  85. Zuo, J., Niu, Q.-W. & Chua, N.-H. An estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plants. Plant J. 24, 265–273 (2000).

  86. Nelson, B. K., Cai, X. & Nebenführ, A. A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. Plant J. 51, 1126–1136 (2007).

    CAS  PubMed  Google Scholar 

  87. Honig, A., Avin-Wittenberg, T., Ufaz, S. & Galili, G. A new type of compartment, defined by plant-specific Atg8-interacting proteins, is induced upon exposure of Arabidopsis plants to carbon starvation. Plant Cell 24, 288–303 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  88. Voigt, B. et al. GFP–FABD2 fusion construct allows in vivo visualization of the dynamic actin cytoskeleton in all cells of Arabidopsis seedlings. Eur. J. Cell Biol. 84, 595–608 (2005).

    CAS  PubMed  Google Scholar 

  89. Fendrych, M. et al. Visualization of the exocyst complex dynamics at the plasma membrane of Arabidopsis thaliana. Mol. Biol. Cell 24, 510–520 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  90. Zhang, X., Henriques, R., Lin, S.-S., Niu, Q.-W. & Chua, N.-H. Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nat. Protoc. 1, 641–646 (2006).

    CAS  PubMed  Google Scholar 

  91. Li, J.-F., Park, E., von Arnim, A. G. & Nebenführ, A. The FAST technique: a simplified Agrobacterium-based transformation method for transient gene expression analysis in seedlings of Arabidopsis and other plant species. Plant Methods 5, 6 (2009).

  92. Klíma, P. et al. Plant cell lines in cell morphogenesis research: from phenotyping to -omics. Methods Mol. Biol. 1992, 367–376 (2019).

  93. Konopka, C. A. & Bednarek, S. Y. Variable-angle epifluorescence microscopy: a new way to look at protein dynamics in the plant cell cortex. Plant J. 53, 186–196 (2008).

    CAS  PubMed  Google Scholar 

  94. Tinevez, J.-Y. et al. TrackMate: an open and extensible platform for single-particle tracking. Methods 115, 80–90 (2017).

    CAS  PubMed  Google Scholar 

  95. Goedhart, J. SuperPlotsOfData—a web app for the transparent display and quantitative comparison of continuous data from different conditions. Mol. Biol. Cell 32, 470–474 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  96. Ripley, B. D. The R project in statistical computing. MSOR Connect. 1, 23–25 (2001).

    Google Scholar 

  97. Cribari-Neto, F. & Zeileis, A. Beta regression in R. J. Stat. Softw. 34, 1–24 (2010).

    Google Scholar 

  98. Searle, S. R., Speed, F. M. & Milliken, G. A. Population marginal means in the linear model: an alternative to least squares means. Am. Stat. 34, 216–221 (1980).

    Google Scholar 

  99. Vosolsobě, S. “arposomes” GitHub, 2023, https://github.com/vosolsob/arposomes

Download references

Acknowledgements

Czech Science Foundation project no. 19-10845S (K.S.). Grant Agency of the Charles University nos. 816217 (J.M. and K.S.) and 374522 (B.J. and K.S.). Leverhulme Trust funding RPG-2015-106 (I.S.). Microscopy was performed in the Viničná Microscopy Core Facility co-financed by the Czech-BioImaging large RI project LM2023050. Computational resources were supplied by the project ‘e-Infrastruktura CZ’ (e-INFRA LM2018140) provided within the programme Projects of Large Research, Development, and Innovations Infrastructures. Super-resolution microscopy was performed in the Imaging Facility of the Institute of Experimental Botany ASCR, which was supported by the MEYS CR (Large RI Project LM2023050 Czech-BioImaging). Thanks to K. Harant and P. Talacko (Laboratory of Mass Spectrometry, Charles University, Faculty of Science) for proteomic and mass spectrometric analysis. We thank R. J. O’Connell (INRAE) for ChEC51 and ChEC96 vectors. We also thank M. Fendrych and J. Petrášek for many useful comments and suggestions to the manuscript.

Author information

Authors and Affiliations

  1. Department of Experimental Plant Biology, Faculty of Science, Charles University, Prague, Czech Republic

    Jan Martinek, Petra Cifrová, Stanislav Vosolsobě, Judith García-González, Zdeňka Mauerová, Barbora Jelínková, Jana Krtková, Lenka Sikorová & Kateřina Schwarzerová

  2. Imaging Facility of Institute of Experimental Botany AS CR, Prague, Czech Republic

    Kateřina Malínská

  3. Biosciences, CLES, Exeter University, Exeter, UK

    Ian Leaves

  4. School of Biological Sciences, University of Bristol, Bristol, UK

    Imogen Sparkes

Authors
  1. Jan Martinek
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Petra Cifrová
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Stanislav Vosolsobě
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Judith García-González
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kateřina Malínská
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Zdeňka Mauerová
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Barbora Jelínková
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jana Krtková
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Lenka Sikorová
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Ian Leaves
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Imogen Sparkes
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Kateřina Schwarzerová
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.M., P.C., S.V., J.K. and K.S. prepared the experimental design and executed most of the experiments. J.M., P.C., S.V. J.G.-G., K.M., B.J. and K.S. contributed to microscopy and image analysis. J.M., P.C., S.V. and K.S. contributed to the data analysis and statistics. J.M., J.K., L.S., I.S., I.L. and K.S. contributed to cloning and transformation of the plant and cell lines. J.M., P.C., Z.M. and K.S. contributed to western blotting and proteomic analysis, J.M., P.C. and K.S. contributed to the writing of the manuscript.

Corresponding author

Correspondence to Kateřina Schwarzerová.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Plants thanks Tomokazu Kawashima, Luisa Sandalio and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figs. 1–8, full blots, Tables 1–3 and Methods 1 and 2.

Reporting Summary

Supplementary Video 1

Motility of peroxisomes (mCherry–PTS1, magenta) with the ARP2/3 domain (GFP–NtARPC2, green) in hypocotyl cells of transgenic Arabidopsis thaliana. Confocal microscopy.

Supplementary Video 2

VAEM microscopy of GFP–ARPC2 and GFP–ARPC5 in hypocotyls of Arabidopsis thaliana, as shown in Supplementary Fig. 2l–o.

Supplementary Video 3

Motility of peroxisomes (mCherry–PTS1, magenta) with the ARP2/3 domain (GFP–NtARPC2, green) along actin filaments (FABD–GFP, green) in pavement cells of transgenic Arabidopsis thaliana. Confocal microscopy.

Supplementary Video 4

In vivo localization of GFP–ARPC5 in arpc2 mutants. Super-resolution Airyscan microscopy.

Supplementary Video 5

VAEM microscopy of GFP–ARPC5 in the mutant background, as shown in Fig. 3f–h.

Supplementary Video 6

VAEM microscopy of colocalization of GFP–ARPC2 and RFP–ATG8f, as shown in Supplementary Fig. 6a,b.

Source data

Supplementary Source Data Table 1

. Source data for Supplementary Fig. 4. Supplementary Source Data Table 2. Source data for Supplementary Fig. 5. Supplementary Source Data Table 3. Source data for Supplementary Table 1. Supplementary Source Data Table 4. Source data for Supplementary Table 1. Supplementary Source Data 1. Source data for Supplementary Fig. 5.

Supplementary Source Data Figs. 2 and 4

Uncropped blots for Figs. 2 and 4.

Supplementary Source Data Fig. 2

Source data for Fig. 2 graphs.

Supplementary Source Data Fig. 3

Source data for Fig. 3 graphs.

Supplementary Source Data Fig. 4

Source data for Fig. 4 graphs.

Supplementary Source Data Fig. 5

Source data for Fig. 5 graphs.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Martinek, J., Cifrová, P., Vosolsobě, S. et al. ARP2/3 complex associates with peroxisomes to participate in pexophagy in plants. Nat. Plants 9, 1874–1889 (2023). https://doi.org/10.1038/s41477-023-01542-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41477-023-01542-6

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing