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
Microcystin-LR (MCY-LR) is a natural cyclic heptapeptide toxin produced by several cyanobacterial genera. It is known to be a potent inhibitor of the serine-threonine protein phosphatases, mainly of type 1 and 2A (PP1 and PP2A), and of PP3, PP4, PP5, and PP6 (Swingle et al. 2007). It is also a strong inducer of reactive oxygen species (ROS) (Bouaïcha and Maatouk 2004; Campos and Vasconcelos 2010). Due to these two properties, it affects key cellular events: metabolic regulation, signal transduction events, and cell-cycle regulation. Thus, it affects eukaryotic subcellular structures: chromatin, cytoskeletal, and endomembrane (mainly ER) organization (Chen and Xie 2016; Máthé et al. 2013, 2016). For this reason, it is ideal for the study of alterations of subcellular organization under stress. If ACAIN and CACAIN can label specific subcellular structures, MCY-LR could be ideal to be used as a tool for the detection of changes highlighted by these fluorochromes.
In the light of the above arguments, our main goals were (1) to detect the subcellular structures labeled by ACAIN and CACAIN and to assess whether they can label proteins related to those subcellular structures, and (2) to test these fluorochromes for their suitability to detect stress-induced subcellular alterations induced by MCY-LR. We show that ACAIN/CACAIN are suitable as alternative labels for specific cellular structures—tonoplasts—and for detecting changes in the organization of tonoplast-coated endomembranes.
Materials and methods
ACAIN/CACAIN were synthesized as we have previously described (Nagy et al. 2016). Briefly, the synthesis started from 1,5-diaminonaphthalene (Sigma-Aldrich, St. Louis, MO, USA) by Hofmann isonitrile synthesis using chloroform and potassium hydroxide, followed by the addition of epichlorohydrin on the amine group. Secondary hydroxyl group formed was then acylated with acryloyl chloride (ACAIN) or 2-choloroacrylic acid (CACAIN). MCY-LR was purified from Microcystis aeruginosa BGSD243 according to the procedure of Kós et al. (1995) modified by Vasas et al. (2004). Briefly, methanolic extracts of M. aeruginosa cells were vacuum evaporated, re-extracted with 10 mM Tris–HCl, pH 7.5, and subjected to ion-exchange chromatography on DEAE-cellulose (DE-52; Whatman, Maidstone, UK) columns. This was followed by desalting and semi-preparative HPLC to obtain MCY-LR of purity ≥ 95%.
Plant material and treatments
Arabidopsis thaliana genotypes used were Col-0 (wild-type), plants bearing GFP-TIP2;1 (δ-TIP) fusion protein described by Cutler et al. (2000), and four knockout mutants in Col-0 background of tonoplast intrinsic proteins (TIPs). These were tip1;1, tip1;2, tip2;1, and their triple mutant obtained as described previously (Reinhardt et al. 2016). The tobacco genotype used was Nicotiana tabacum SR1. Seeds were surface sterilized by rinsing two times for 10 min with 10% of a sodium-hypochlorite-containing solution, then washed four to five times for 5 min with sterile water. Seeds were transferred to Murashige–Skoog (MS) basal medium supplemented with Gamborg’s vitamins, 2% (w/v) sucrose (Molar, Budapest, Hungary), and 0.8% (w/v) Bacto-agar (Difco, Lawrence, KS, USA) (Gamborg et al. 1968; Murashige and Skoog 1962). After a 48-h cold treatment (used for Arabidopsis), plates were placed in a tissue culture room (14:10 h photoperiod, 22 ± 2 °C, 60 μmol m−2 s−1 photon flux density in the light period). After 1 week of culture, part of the seedlings was directly subjected to ACAIN/CACAIN cytological labeling or ACAIN staining of root and shoot extracts after SDS-PAGE (see sections below). For detecting ACAIN-labeled proteins on SDS-PAGE, Lemna minor shoots axenically subcultured for 1 week on liquid Allen medium (Allen 1968) were used as well. Part of Arabidopsis (Col-0 and GFP-TIP2;1) seedlings were further treated with protein phosphatase inhibitors. Col-0 and GFP-TIP2;1 seedlings were treated with 1–5 μM MCY-LR for 4–72 h. Col-0 plants were further stained with ACAIN/CACAIN. GFP-TIP2;1 seedlings were also treated with 1 μM okadaic acid (OA) for 24 h and 1 μM tautomycin (Tm) for 4–24 h.
ACAIN/CACAIN staining and microscopy
Control and protein phosphatase-treated whole seedlings were placed in four-well tissue culture chambers for microscopy (Sarstedt, Nümbrecht, Germany) and washed with phosphate buffered saline (PBS). ACAIN/CACAIN stocks dissolved in DMSO were diluted in PBS with or without 0.05% (v/v) Triton X-100 (Reanal, Budapest, Hungary) to obtain final concentrations of 20 μg mL−1, used for labeling of seedlings. Duration of labeling was 30 min, followed by three consecutive 5-min washes with PBS. Samples were then first visualized with a conventional Olympus Provis AX-70 (Olympus, Tokyo, Japan) fluorescence microscope at excitation wavelength of 320–360 nm. This was followed by CLSM analysis. ACAIN/CACAIN stained Col-0 and GFP-TIP2;1 seedlings were analyzed with a Zeiss LSM 880 confocal microscope by using a × 40 C-Apochromat water immersion objective. Excitation and emission wavelengths were 405/408–473 nm (ACAIN/CACAIN), 488/492–540 nm (GFP), and 543/547–614 nm (chlorophyll autofluorescence). For treatment of GFP::TIP2;1 seedlings with protein phosphatase inhibitors (without ACAIN/CACAIN labeling), a Leica TCS-SP5 confocal microscope/HCX APO L U-V-I 40.0 × 0.80 water immersion objective was used. GFP was excited with a 488 nm Ar laser, and emission was at 503–515 nm. For chlorophyll autofluorescence detection, the 543 HeNe laser was used. Emission was at 650–710 nm, and chlorophyll autofluorescence was pseudocolored in blue on the GFP-chlorophyll composite images.
All cytological experiments were performed at least three times, and representative results are presented (“Results” section).
Col-0 seedlings were treated with 1–2 μM MCY-LR for 72 h (see above). TEM preparations were made as follows: segments of control and microcystin treated Arabidopsis thaliana hypocotyls were fixed in 2% glutaraldehyde for 3 h and in 1% osmium tetroxide for 2 h (both fixatives were dissolved in 0.1 M K–Na–phosphate buffer, pH 7.2) rinsed in the buffer and embedded in Durcupan resin after dehydration in an ethanol series ended with propylene oxide. Ultra-thin sections (70 nm) were cut with a Reichert Ultracut E ultramicrotome (Leica Microsystems, Vienna, Austria), mounted on uncoated Cu/Pd grids (Polysciences, Warrington, PA, USA), and stained with 2% uranyl acetate dissolved in methanol for 4 min and lead citrate for 6 min. Sections were studied with a Hitachi 7100 transmission electron microscope (Hitachi, Tokyo, Japan) at 75 kV.
ACAIN labeling of proteins following SDS-PAGE
Protein extracts from shoots and roots of Arabidopsis and tobacco seedlings as well as Lemna minor shoots were prepared in the SDS (Sigma-Aldrich) containing standard Laemmli buffer (Laemmli 1970), but without boiling to allow further renaturation of proteins (see 2-propanol treatments below). To check whether this extraction does not allow protein degradation, alternatively extracts were prepared in the presence of a protease inhibitor cocktail (0.5% v/v; Roche Applied Science, Indianapolis, USA) as well as without protease inhibitors and with standard protein boiling. Protein contents of samples were assayed by the Bradford (1976) method. Extracts containing 40 μg protein per well were loaded along with molecular weight markers (Sigma-Aldrich, BLUeye Prestained Protein Ladder). SDS-containing gels were prepared essentially by the Laemmli (1970) method except that 7.5–18% polyacrylamide gradient (running) gels proved to be the best for our purposes and used accordingly. After SDS-PAGE, gels were rinsed for 10 min in sterile water, then twice for 10 min in 20% (v/v) 2-propanol (VWR, Radnor, USA) and twice for 20 min in PBS. Afterwards, labeling was performed for 5 h with 35 μg mL−1 ACAIN in PBS. Gels were then washed for 10 min with 20% 2-propanol, followed by two consecutive washes with PBS for 5 min each. ACAIN-labeled proteins were detected by illumination at 365 nm and photographed with an UVITEC transilluminator/gel documentation equipment (UVITEC, Cambridge, UK). MW of proteins was determined with the UVITEC® software. Band intensities and intensity curves were determined with the aid of the GelAnalyzer 2010 software. Besides ACAIN staining, gel slices containing the same protein samples were stained with 0.2% Coomassie Brilliant Blue (Serva, Heidelberg, Germany) for 4 h.
All SDS-PAGE work was performed in three independent experiments and images of representative gels are shown in the “Results” section.
ACAIN labels tonoplasts in different plant species and TIPs contribute to this labeling pattern
ACAIN labels proteins on SDS-polyacrylamide gels
ACAIN/CACAIN labeling reveals changes in vacuolar organization induced by protein phosphatase inhibitors
GFP signal in hypocotyls from GFP-TIP2;1 seedlings treated with protein phosphatase inhibitors showed the following: controls showed normal vacuolar system (Fig. 6d). Four hours of MCY-LR treatment revealed the absence of large vacuoles and the presence of small tonoplast-coated vesicles similarly to the pattern revealed by CACAIN/ACAIN staining (Fig. 6e). Moreover, the movement/dynamics of these vesicles was very slow (Supplementary Movie 2). OA treatments did not alter vacuolar organization (Fig. 6f). In contrast, in the presence of Tm, the proportion of large vacuoles decreased in many cells (while for MCY-LR, large vacuoles were completely lacking in several cells), and this effect increased over time (9 vs. 24 h) (Fig. 6g–i). Thus, Tm did not have such dramatic effects on vacuolar organization as MCY-LR.
TEM studies showed normal organization of endomembranes in control Col-0 plants, with large vacuoles and smaller, abundant vesicles (Fig. 6j). After 72 h of treatment with 1–2 μM MCY-LR, an increase in the abundance of multimembrane vesicles as well as autophagosome-like structures incorporated into the large vacuoles were observed (Fig. 6k; Supplementary Fig. 3). Apparently, multimembrane vesicles formed in the cytosol (Fig. 6k) were later incorporated in the large vacuoles (Supplementary Fig. 3a).
ACAIN and CACAIN label preferentially tonoplasts in living hypocotyl cells of Arabidopsis and tobacco, in a similar manner (Figs. 2f, g and 6a, b). Tonoplast labeling is proven by the co-localization of fluorochrome signals with GFP signals from Arabidopsis plants expressing GFP-TIP2;1 fusion protein (Fig. 2e) reported to highlight all endomembranes delimited by a tonoplast (Cutler et al. 2000). The second evidence for preferential tonoplast labeling is that Hechtian strands known to consist mainly of plasma membrane fractions that tend to attach to the cell wall are showing almost exclusively the YFP label when plasmolyzed hypocotyl cells of YFP-PIP2a plants (Cutler et al. 2000) are labeled with ACAIN (Supplementary Fig. 1d, e). Meanwhile, shrunk vacuoles of plasmolyzed cells probably delimited only by tonoplasts are labeled exclusively by ACAIN (Supplementary Fig. 1d, e). The surface of shrunken protoplasm is apparently showing both the ACAIN and YFP label (Supplementary Fig. 1e). However, one should note that during plasmolysis, tonoplast or segments of tonoplast may be tightly adhered to the plasma membrane (Oparka 1994). While the resolution of light microscopy/CLSM might not allow us to distinguish plasma membrane and tonoplast under these conditions, the possible tight adherence of the two membrane types may lead one to false co-localization. The third evidence is the different fluorescence signal pattern (as compared to ACAIN/CACAIN) for other endomembranes (ER, Golgi) in Arabidopsis plants with GFP/YFP-fusion proteins labeling those cell compartments (Supplementary Fig. 1a–c). The fourth evidence for the peculiar ACAIN/CACAIN labeling pattern is that the use of known surfactants (Triton X-100) neither increased the intensity of labeling nor caused labeling of other subcellular structures than tonoplasts (Fig. 2e–g). Data presented in the “Results” section suggest that the isocyanonaphthalene derivatives used in this study can be of broad use for tonoplast labeling in living plant cells. Since it works well in another model plant too (N. tabacum SR1), ACAIN/CACAIN labeling could be a good alternative to plants expressing GFP-fusion proteins for cytological studies on the tonoplast. It can be used for species where transgenic lines with fluorescent protein labels of tonoplasts are not available.
What could be the nature of proteins labeled by our isocyanonaphthalene derivatives? Theoretically, a large variety of proteins containing hydrophobic motifs must be labeled by ACAIN and its derivatives (Nagy et al. 2016). Among plant vacuolar membrane proteins, tonoplast intrinsic proteins (TIPs)—since they are aquaporins—are fitting, although not exclusively into this category. Indeed, two Arabidopsis knockout mutants (tip2;1 and tip1;1-tip1;2-tip2;1 triple mutant) showed much weaker signal than the wild-type (Col-0) plants as revealed by CLSM (Fig. 2). Thus, at least some of the TIPs are labeled by ACAIN (and CACAIN), but not exclusively TIPs are labeled. Tonoplasts are characterized by a large variety of channel proteins characterized by hydrophobic transmembrane domains (Batistič 2012; Maeshima 2001). Indeed, ACAIN staining of protein gels revealed a large number of labeled proteins. Among them, the 22–26-kDa TIPs (see Maeshima 2001 and Schüssler et al. 2008 for molecular weights) were presumably labeled, but several other proteins were also well detectable (Fig. 4). Interestingly, the intensity of ACAIN labeling decreased for the 22–26-kDa proteins in the tip1;1 and tip2;1 mutants, but increased in the triple knockout mutant (Figs. 3 and 4), predicting that for the latter mutant, the absence of three TIP proteins is compensated by increased expression of other proteins. It should be noted, however, that in roots, in general the absence of specific TIPs in the mutants is not compensated by other TIP isoforms (Reinhardt et al. 2016). Thus, our prediction might be valid only for shoot extracts, where ACAIN labeling of proteins following SDS-PAGE was clearly visible (Fig. 4) or for other proteins than TIPs. It should be noted that to date, 10 proteins of the TIP family were detected in Arabidopsis (the expression of several TIPs is organ and tissue specific) and many other proteins with hydrophobic motifs are present in the tonoplast (Beebo et al. 2009). One of the most characteristic changes revealed by SDS-PAGE of the proteins from wild-type plants and mutants were the 18–19-kDa proteins (according to their MW, probably not TIPs) that were almost completely absent in the triple mutant (Figs. 4 and 5, “Results” section). At this stage, we can assume that our fluorochromes are suitable for the detection of specific proteins, among them tonoplast proteins in a crude cellular extract.
What is the uniqueness of ACAIN in the labeling of tonoplasts in live cells? There are several, relatively low molecular weight fluorochromes used for the labeling of plant vacuolar membranes. Different probes including pH markers, Lysosensor yellow/blue, glutathione/sulfhydryls, etc. were used for the labeling of vacuoles (mainly lytic vacuoles) in barley aleurone protoplasts (Swanson et al. 1998). Dyes of the FM family like FM4-64 can label tonoplasts, but other membrane types including those in animal cells (Golgi membranes, vesicles in the phragmoplast for plant cells during cytokinesis, or even the plasma membrane) are also labeled, thus their specificity is lower (Bolte et al. 2004). For plant cells, labeling of other membranes than tonoplasts by FM4-64 occurs after shorter labeling time (Bolte et al. 2004). In contrast, shorter ACAIN labeling times than 30 min (as specified in the “Materials and methods” section) will label tonoplasts preferentially, although labeling intensity is weaker (data not shown). This is probably due to the stable/covalent binding of the fluorochrome to certain tonoplast proteins, as it is known to establish stable bonds with hydrophobic proteins (Nagy et al. 2016) in contrast to FM4-64 known to be a partitioning dye that is preferentially binding to hydrophobic supramolecular structures like biological membranes. FM1–43, excitable at 488 nm, was described to be targeted to tonoplast over time and proven to be a good endocytosis marker in tobacco BY-2 cells (Emans et al. 2002). This dye is first targeted to the plasma membrane (Emans et al. 2002; Zhao and Dixon 2009), meaning that a short-term incubation does not label the tonoplast. Moreover, FM1–43 was proven to be cytotoxic for Vicia faba cells where it was used for plasma membrane and tonoplast labeling (Meckel et al. 2004). This is in contrast to our finding: ACAIN preserves cell viability at least for short term. In the light of the above data, we can assume that ACAIN and CACAIN are attractive alternatives for labeling of tonoplasts in living cells for the above reasons: (1) they label all tonoplast-coated compartments including large vacuoles and small vesicles, (2) they are preferentially labeling the tonoplast, and (3) they preserve cell viability at least for 1 h (see Supplementary Movie 1). In addition, they are excitable at 405 nm, so they can be easily used when multiple labeling procedures involving blue (488 nm), green (543 nm), and/or red (633 nm) wavelength excitation are used simultaneously.
In the next step, we were looking for the possible use of ACAIN/CACAIN for the study of stress-induced changes of vacuolar organization. We used microcystin-LR (MCY-LR) for this purpose (see “Introduction” section for reasons). This toxin is widely used as a tool for the regulation of structural organization and dynamics of subcellular compartments (Máthé et al. 2016). Vacuolar organization including the formation of lytic vacuoles and autophagosomes is sensitive to biotic and abiotic stresses including oxidative stress (Liu and Bassham 2012). ACAIN/CACAIN labeling revealed that MCY-LR, known to be a potent inhibitor of serine-threonine protein phosphatases (mainly PP1 and PP2A) and a ROS inducer, induced the formation of small tonoplast-coated vesicles, several of which engulfed chloroplasts (Fig. 6c). At this stage, we cannot state unequivocally the nature of these vesicles. Our TEM studies revealed that MCY-LR induced the formation of multimembrane vesicles as well as vesicles incorporated into the large vacuoles resembling plant autophagosome morphology (Fig. 6k, Supplementary Fig. 3) as described by Gao et al. (2015) and Liu et al. (2012). Autophagosomes can be not only double membrane but multimembrane vesicles as well. The multimembrane structures (Fig. 6k) were similar to autophagosomes in pathogen-infected Nicotiana benthamiana cells, as described by Liu et al. (2005). Autophagosomes are known to fuse with vacuoles, where their contents are degraded (Liu and Bassham 2012). The formation of multimembrane vesicles detected by our TEM studies could be the result of altered lytic vacuole formation in Arabidopsis as well (Feraru et al. 2010).
If the multimembrane vesicles detected are autophagosomes, they are possibly not identical to the small vesicles detected in GFP-TIP2;1 plants or by ACAIN labeling. The presence of those small vesicles may indicate two distinct phenomena. The first one is the alteration of vacuole biogenesis similar to that shown in Arabidopsis pat mutants (Zwiewka et al. 2011). Tonoplast proteins may be present in the membrane systems of prevacuolar compartments (PVCs), when membrane traffic between PVC and vacuolar compartments is inhibited (Foresti et al. 2006). The second possibility is the enhanced lytic vacuole formation. To our knowledge, TIP3;1 and not TIP2;1 is associated to membranes of autophagosomes (Moriyasu et al. 2003). On the other hand, vesicles observed in this work are clearly delimited by tonoplasts. TIP2;1 used in our study as one of the tonoplast markers is known to be localized in both lytic and storage vacuoles (Martinoia et al. 2000).
GFP-TIP2;1 signals showed MCY-LR-induced alterations of vacuolar organization, similar to those shown by ACAIN/CACAIN labeling (Fig. 6a–e). MCY-LR (an inhibitor of both PP1 and PP2A) induced more pronounced vacuolar alterations than OA (inhibits preferentially PP2A) and Tm (PP1) (Fig. 6f–i). Therefore, probably both PP1 and PP2A play an important role in the differentiation of a normal vacuolar system. It is worth mentioning that to date, there is practically no information on how protein phosphatase inhibitors influence plant endomembrane integrity. For vertebrate cells, it is known that MCY-LR induces ER stress, mitochondrial damage, and autophagy (Chen and Xie 2016). Thus, our new fluorochromes ACAIN and CACAIN are presumably suitable for the detection of multiple vacuole types and stress-induced vacuolar changes. Taken together, the ACAIN/CACAIN labeling and TEM studies, the protein phosphatase inhibitor MCY-LR induces probably two types of changes: (1) alteration of vacuolar organization and (2) the formation of autophagosome-like structures.
In conclusion, ACAIN and CACAIN can be of universal use for the preferential labeling of tonoplasts in living plant cells and the study of tonoplast dynamics. Therefore, they may be good alternatives to the use of tonoplast-specific GFP-fusion proteins widely used for this purpose. These fluorochromes are suitable for the detection of stress-related changes of vacuolar organization as revealed by treatments with protein phosphatase inhibitors. In addition, even though ACAIN labeling is not restricted to a single protein with hydrophobic motifs, it is suitable for the detection and possible further identification of proteins with cysteine-rich hydrophobic motifs, among them tonoplast-specific proteins following SDS-PAGE.
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.
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