Protoplasma

, Volume 255, Issue 3, pp 829–839 | Cite as

Novel fluorochromes label tonoplast in living plant cells and reveal changes in vacuolar organization after treatment with protein phosphatase inhibitors

  • Miklós Nagy
  • Sándor Kéki
  • Dávid Rácz
  • Jaideep Mathur
  • György Vereb
  • Tamás Garda
  • Márta M-Hamvas
  • François Chaumont
  • Károly Bóka
  • Béla Böddi
  • Csongor Freytag
  • Gábor Vasas
  • Csaba Máthé
Original Article

Abstract

The recently synthesized isocyanonaphtalene derivatives ACAIN and CACAIN are fluorochromes excitable at wavelengths of around 366 nm and bind cysteine-rich proteins with hydrophobic motifs. We show that these compounds preferentially label tonoplasts in living Arabidopsis and tobacco (Nicotiana tabacum SR1) cells. ACAIN-labeled membranes co-localized with the GFP signal in plants expressing GFP-δ-TIP (TIP2;1) (a tonoplast aquaporin) fusion protein. ACAIN preserved the dynamics of vacuolar structures. tip2;1 and triple tip1;1-tip1;2-tip2;1 knockout mutants showed weaker ACAIN signal in tonoplasts. The fluorochrome is also suitable for the labeling and detection of specific (cysteine-rich, hydrophobic) proteins from crude cell protein extracts following SDS-PAGE and TIP mutants show altered labeling patterns; however, it appears that ACAIN labels a large variety of tonoplast proteins. ACAIN/CACAIN could be used for the detection of altered vacuolar organization induced by the heptapeptide natural toxin microcystin-LR (MCY-LR), a potent inhibitor of both type 1 and 2A protein phosphatases and a ROS inducer. As revealed both in plants with GFP-TIP2;1 fusions and in wild-type (Columbia) plants labeled with ACAIN/CACAIN, MCY-LR induces the formation of small vesicles, concomitantly with the absence of the large vegetative vacuoles characteristic for differentiated cells. TEM studies of MCY-LR-treated Arabidopsis cells proved the presence of multimembrane vesicles, with characteristics of lytic vacuoles or autophagosomes. Moreover, MCY-LR is a stronger inducer of small vesicle formation than okadaic acid (which inhibits preferentially PP2A) and tautomycin (which inhibits preferentially PP1). ACAIN and CACAIN emerge as useful novel tools to study plant vacuole biogenesis and programmed cell death.

Keywords

ACAIN/CACAIN Arabidopsis Tonoplast Hypocotyl Microcystin-LR Vacuolar organization 

Notes

Acknowledgements

This work was financially supported by the grants K-116465 and K-120638 given by NKFIH (National Research, Development and Innovation Office, Hungary) and GINOP-2.3.2-15-2016-00041 and GINOP-2.3.3.-15-2016-00030 project. The project is co-financed by the European Union and the European Regional Development Fund. CM was supported by the Balassi Institute/Campus Hungary Mobility Support No. B2/2H/7717 for a mobility to the University of Guelph, ON, Canada in 2014. MN was supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences. JM acknowledges a Discovery Grant from NSERC, Canada. We would like to thank Prof. Ferenc Erdődi for providing us with okadaic acid and tautomycin and Sean Cutler for the GFP-TIP2;1 transgenic Arabidopsis line.

Compliance with ethical standards

Conflict of interest

The authors declare there is no conflict of interest regarding the contents of this manuscript.

Supplementary material

709_2017_1190_MOESM1_ESM.avi (3.8 mb)
Supplementary Movie 1 Normal vacuolar dynamics in a control GFP-TIP2;1 plant labeled with ACAIN: composite ACAIN labeling and GFP signal are seen (AVI 3890 kb)
709_2017_1190_MOESM2_ESM.avi (3.3 mb)
Supplementary Movie 2 Vacuolar dynamics in a GFP-TIP2;1 plant treated for 4 h with 1 μM MCY-LR, GFP signal is seen. The absence of large vacuoles and impaired dynamics of tonoplast-coated vesicles are seen (AVI 3381 kb)
709_2017_1190_MOESM3_ESM.jpg (12.7 mb)
Supplementary Fig. 1 ac ACAIN does not label ER and the Golgi apparatus as shown by the different distribution patterns of fluorescence labeling for these two membrane compartments in Arabidopsis hypocotyls. a EYFP-ER plants (Nelson et al. 2007) showing the ER network and bright yellow ER bodies; b GFP-ERD2 plants (Boevink et al. 1998; Mathur et al. 2003) showing labeling of Golgi (green) and chlorophyll autofluorescence in plastids (blue); c ACAIN labeling of tonoplasts. LSM excitation and emission settings were the characteristic GFP and YFP settings of the Leica TCS-SP5 confocal microscope with the use of the HCX APO L U-V-I 40.0 × 0.80 water immersion objective for (a, b) and the Zeiss LSM 880 confocal microscope setting for ACAIN labeling (c); they were as specified in the “Materials and methods” section. d, e ACAIN labeling of plasmolyzed hypocotyl cells from YFP-PIP2a plants (Cutler et al. 2000). Plasmolysis was done with 5% (w/v) NaCl for 5 min. These images show that ACAIN labels preferentially the tonoplast and not the plasma membrane. Merged images show both the ACAIN and YFP label as well as the brightfield background; the latter is for showing cell contours. e Detail of the merged image of (d). H, Hechtian strands showing only the YFP label; v, shrunk vacuoles showing only the ACAIN label. GFP and YFP settings were those of the Zeiss LSM 880. Scale bars: 30 μm (JPEG 13046 kb)
709_2017_1190_MOESM4_ESM.jpg (1.6 mb)
Supplementary Fig. 2 Preparation of Col-0 protein extracts in the standard Laemmli buffer without boiling gives sufficient protection against proteolytic degradation as shown by both ACAIN labeling and Coomassie Blue staining. A1—extract prepared in the presence of 0.5% (v/v) protease inhibitor cocktail without boiling; A2—extract prepared without protease inhibition and boiling; A3—extract prepared without protease inhibition, but boiled; M—position of the bands of molecular weight marker (JPEG 1686 kb)
709_2017_1190_MOESM5_ESM.jpg (2.7 mb)
Supplementary Fig. 3 a TEM image of a hypocotyl cell treated for 4 days with 1 μM MCY-LR showing a multimembrane vesicle (arrow) after its incorporation into the central vacuole (CV). b TEM image of autophagosome-like structures (a) inside a large vacuole of a hypocotyl cortex cell treated with 1 μM MCY-LR for 3 days. Scale bars: 1 μm (JPEG 2731 kb)

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Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2017

Authors and Affiliations

  • Miklós Nagy
    • 1
  • Sándor Kéki
    • 1
  • Dávid Rácz
    • 1
  • Jaideep Mathur
    • 2
  • György Vereb
    • 3
  • Tamás Garda
    • 4
  • Márta M-Hamvas
    • 4
  • François Chaumont
    • 5
  • Károly Bóka
    • 6
  • Béla Böddi
    • 6
  • Csongor Freytag
    • 4
  • Gábor Vasas
    • 4
  • Csaba Máthé
    • 4
  1. 1.Faculty of Science and Technology, Department of Applied ChemistryUniversity of DebrecenDebrecenHungary
  2. 2.Department of Molecular and Cellular Biology, Laboratory of Plant Development and InteractionsUniversity of GuelphGuelphCanada
  3. 3.Faculty of Medicine, Department of Biophysics and Cell BiologyUniversity of DebrecenDebrecenHungary
  4. 4.Faculty of Science and Technology, Department of BotanyUniversity of DebrecenDebrecenHungary
  5. 5.Institut des Sciences de la VieUniversité catholique de LouvainLouvain-la-NeuveBelgium
  6. 6.ELTE Eötvös Loránd University, Department of Plant Anatomy, Institute of Biology, Faculty of ScienceBudapestHungary

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