Novel fluorochromes label tonoplast in living plant cells and reveal changes in vacuolar organization after treatment with protein phosphatase inhibitors
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.
KeywordsACAIN/CACAIN Arabidopsis Tonoplast Hypocotyl Microcystin-LR Vacuolar organization
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.
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Conflict of interest
The authors declare there is no conflict of interest regarding the contents of this manuscript.
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