Download Architectural remodeling of the tonoplast during fluid

research paper
Research paper
Plant Signaling & Behavior 8:7, e24793; July 2013; © 2013 Landes Bioscience
Architectural remodeling of the tonoplast during
fluid-phase endocytosis
Ed Etxeberria,1,* Pedro Gonzalez1 and Javier Pozueta-Romero2
Department of Horticultural Sciences; University of Florida; Institute of Food and Agricultural Sciences; Citrus Research and Education Center; Lake Alfred, FL USA; 2Instituto
de Agrobiotecnologia; Universidad Publica de Navarra/Consejo de Investigaciones Cientificas/Gobierno de Navarra; Nafarroa, Spain
1
Keywords: exocytosis, retrograde vesicles, sucrose transport, tonoplast
Abbreviations: ADP, adenosine-5'-diphosphate; ATP, adenosine-5'-triphosphate; BSA, bovine serum albumin; BTP, bis-trispropane; DTT, dithiothreitol; FPE, fluid phase endocitosis; MES, 2-(N-morpholino)ethanesulfonic acid; NAD, nicotinamide
adenine dinucleotide; NADH, nicotinamide adenine dinucleotide-H; pCMBS, p-Chloromercuribenzene sulfonate; Tris,
tris(hydroxymethyl)aminomethane; V-ATPase, vacuolar-type H+ -ATPase
During fluid phase endocytosis (FPE) in plant storage cells, the vacuole receives a considerable amount of membrane
and fluid contents. If allowed to accumulate over a period of time, the enlarging tonoplast and increase in fluids would
invariably disrupt the structural equilibrium of the mature cells. Therefore, a membrane retrieval process must exist that
will guarantee membrane homeostasis in light of tonoplast expansion by membrane addition during FPE. We examined
the morphological changes to the vacuolar structure during endocytosis in red beet hypocotyl tissue using scanning
laser confocal microscopy and immunohistochemistry. The heavily pigmented storage vacuole allowed us to visualize all
architectural transformations during treatment. When red beet tissue was incubated in 200 mM sucrose, a portion of the
sucrose accumulated entered the cell by means of FPE. The accumulation process was accompanied by the development
of vacuole-derived vesicles which transiently counterbalanced the addition of surplus endocytic membrane during rapid
rates of endocytosis. Topographic fluorescent confocal micrographs showed an ensuing reduction in the size of the
vacuole-derived vesicles and further suggest their reincorporation into the vacuole to maintain vacuolar unity and solute
concentration.
Introduction
Plant storage cells accumulate a portion of their photoassimilates
in the vacuole via fluid phase endocytosis (FPE),1-6 a process characterized by the nonspecific bulk uptake of extracellular solutes
in plasma membrane-bound vesicles.7 The portion of assimilates
taken up by FPE varies according to the apoplastic solute concentration,8 the remaining difference being transported by membrane-bound carriers. Although firmly established that all types
of endocytic systems (i.e., clathrin-mediated, caveolin-mediated,
lipid raft-dependent, flotillin-dependent and macropinocytosis9
carry a fluid-phase component,10,11 FPE is distinguished by their
relatively larger vesicle size11,12 and cargo capacity. Solutes trapped
by FPE eventually reach the vacuole; however,13,14 some systematic cargo distribution within the endosome has been observed
using a combination of soluble membrane-impermeable endocytic markers5,15 and fluorescent nano-particles.16
In plants, FPE has been documented primarily for storage
cells.3-6,8,17-19 During accumulation of photoassimilates at low
external concentrations, membrane-bound solute transporters
support the cellular energy and metabolic demands, in addition
to transporting surplus solutes to the vacuole.20-22 This constitutes the hyperbolic, high-affinity phase of the characteristic
curvilinear photoassimilate uptake mechanism.23 At high extracellular sucrose, mannitol or glucose concentrations, FPE is triggered and functions largely to carry surplus photoassimilates
into the vacuole bypassing the cytosol,5,8 constituting the linear
phase of the uptake curve.23 The simultaneous action of these
multiple transport mechanisms was demonstrated using fluorescent deoxy-glucose, evacuolated cytoplasts and inhibitors of
both transport systems24 and their coordinated action detailed
by Pozueta-Romero et al.8 Similar conclusions were obtained
by Bandmann and Homann5 with tobacco BY-2 cells. The
results provided evidence that FPE and membrane transporters
can work in parallel and are under strict control of the external
photoassimilate supply. A number of studies have revealed the
formation of endocytic vesicles of different sizes ranging from
0.5–6 μm,6,25 a size more in accordance with macropinocytosis.26
However, membrane capacitance measurements by Bandmann
and Homann5 and Gall et al.27 suggested an endocytic vesicle
diameter of 80–220 nm. Whether or not the larger vesicles carrying endocytic markers observed during FPE result from the
*Correspondence to: Ed Etxeberria; Email: [email protected]
Submitted: 04/05/13; Revised: 04/23/13; Accepted: 04/24/13
Citation: Etexeberria E, Gonzalez P, Pozueta-Romero J. Architectural remodeling of the tonoplast during fluid-phase endocytosis;. Plant Signal Behav 2013; 8:
e24793; http://dx.doi.org/10.4161/psb.24793
www.landesbioscience.com
Plant Signaling & Behavior
e24793-1
Figure 1. Red beet protoplasts isolated from tissue incubated in the presence and absence of the membrane-impermeable, endocytic-marker bluefluorescent Alexa-350. (A) Fluorescent micrograph of protoplasts isolated from tissue incubated in 200 mM sucrose and 100 μM Alexa-350. (B) Light
micrograph of (A). (C) High magnification fluorescent micrograph of a protoplast demonstrating Alexa-350 blue fluorescence in the vacuole and
vacuole derived vesicles. (D) Fluorescent micrograph of protoplasts prepared from control tissue incubated in betaine.
fusion of smaller vesicles28,29 prior to merging with the vacuole
has not been determined.
During the vesicle delivery process, the vacuole receives a
considerable amount of membrane and fluid content which,
if allowed to accumulate over a period of time, would invariably disrupt the structural equilibrium of the mature cells.
Furthermore, given the disproportional membrane surfacearea/volume added by endocytic vesicles, membrane influx in
the absence of a compensatory efflux process would result in
increased dilution of the vacuolar contents. Therefore a retrieval
process must exist that would guarantee membrane homeostasis
in light of tonoplast expansion by membrane addition during
FPE.30 Despite the volumes of literature on endocytosis in both
animal and plant cells, the structural integrity of the tonoplast/
lysosome during endocytosis remains unexplained, largely overlooked by the difficulty in visualizing, measuring and quantifying such changes. In an attempt to explain the lack of observed
e24793-2
variations in the lysosome volume during macropinocytosis
in macrophages, Swanson 26 suggested a system of retrograde
vesicles of much reduced in size compared with the endocytic
vesicles, a way to minimize solute loss while disproportionally
removing excessive membrane surface area. If the diameter of
the efflux vesicles were considerably smaller than that of the
macropinosome (endocytic vesicle) the cell could balance incoming and outgoing membrane, and at the same time, restrict the
retro-efflux of fluid. Nevertheless, regardless of the size of the
exocytic vesicles, some portion of the vacuolar fluids would be
returned to the exterior, creating an ancillary futile cycle. The
remaining water and other permeable solutes would cross the
lysosome membrane leaving membrane impermeable solutes to
concentrate in the lysosome. Such a system could explain how
solutes are concentrated in the storage lysosome/vacuole thereby
maintaining the membrane surface area without significant loss
of trapped solutes.31 In support of this proposed system, clathrin
Plant Signaling & Behavior
Volume 8 Issue 7
Table 1. Changes of plasma membrane marker vanadate-sensitive ATPase activity in red beet cells incubated in sucrose or betaine for 24 h
24 h
Vanadate sensitive P-ATPase
(*Units/ total ATPase in tonoplast fraction)
Time 0
Betaine
Sucrose
% increase
0.17 ± 0.03
0.23 ± 0.01
0.31 ± 0.02
20%
*One unit defined as 1 nkat. Purified tonoplast fractions were isolated from fresh tissue and from tissue incubated in sucrose or betaine for 24 h. VATPase activity was measured in the presence and absence of vanadate.
coated retrieval vesicles have been observed at the surface of lysosomes,32 whereas retrograde membrane traffic systems from the
vacuole have been identified as part of the membrane recycling
process in Saccharomyces cerevisiae,33,34 in tobacco BY-2 cells35
and from the pre-vacuolar compartment in Arabidopsis.36
Throughout our investigations into FPE in plant cells with
a variety of membrane-impermeable probes, 2,15,16,25 we consistently observed their eventual deposition in the vacuole. For
such mature cells to endocytose constitutively, a stable tonoplast
surface area/volume ratio must be preserved by an unknown
accompanying process of membrane turnover. Based on reports
of retrograde vesicle budding,32 the exceptional plasticity of the
tonoplast 37 and our unpublished observations, we hypothesize
the existence of one or several cellular, morphological and/
or metabolic events that will assist in vacuolar stability during endocytosis. As part of our continued investigation on FPE
in plant cells, we examined the morphological changes to the
vacuolar structure during rapid rates of endocytosis. To follow
these essential modifications, we used red beet hypocotyl tissue. The heavily pigmented vacuole of the storage parenchyma
cells allowed us to microscopically visualize all architectural
transformations during treatment and distinguish these from
any possible plasma membrane-derived osmocytotic structures
as those reported by Goodwin et al.38 and Oparka et al.39 The
present study revealed that, under conditions of rapid FPE
proliferation, the cell transiently counterbalances the addition
of surplus membrane by vacuole fragmentation with a system
of vacuole-derived vesicles. Topographic fluorescent confocal
micrographs showed an ensuing reduction in the size of the
vacuole-derived vesicles and further suggested their reincorporation into the vacuole.
Results
Endoytic sucrose uptake. Protoplasts prepared from red beet
storage tissue after incubation in 200 mM sucrose and the bluefluorescent membrane-impermeable endocytic marker Alexa-350
for 24 h exhibited a pronounced blue fluorescence in the vacuole
(Fig. 1A–C). The blue fluorescence was not due to autofluorescence by the vacuolar pigment betacyanin. Although betacyanin has a wide fluorescent spectrum (560–750 nm) 40 it does
not overlap with that of Alexa-350 (400–550 nm). Furthermore,
and most notably, protoplasts prepared from control tissue incubated in Alexa-350 and betaine instead of sucrose showed no
fluorescence (Fig. 1D). Sucrose induced FPE has been reported
for other tissues including turnip storage parenchyma,8 citrus
juice cells3 and sycamore cultured cells.2,16
www.landesbioscience.com
Consistent with the participation of FPE in the process of
sucrose uptake, determinations of membrane flux indicated
a significant movement of plasma membrane elements to the
tonoplast. A 20% increase in vanadate-sensitive ATPase activity
(plasma membrane H+ -ATPase marker) was measured in purified
tonoplast fractions from tissue incubated in sucrose when compared with control samples incubated in equal concentrations of
betaine (Table 1). The above observations are in agreement with
other storage tissues1-6 and collectively indicate that transport of
Alexa-350 to the vacuole (Fig. 1) was mediated in part by sucroseinduced FPE.
Vacuole restructuring during endocytosis. During dormant
or resting stage, red beet hypocotyl storage cells contained predominantly one large central vacuole (Fig. 2A and B). However,
after tissue incubation in 200 mM sucrose for 24 h, a series of
vacuole-derived organelles become evident (Fig. 2C and D), a
condition scarcely observed when betaine was used as osmotic
control. These vesicles are unquestionably of vacuolar origin
given their red betacyanin content (Fig. 2C and D). The vacuolederived vesicles not only varied in size and number between cells
from different beet hypocotyl preparations, but also between
cells from the same hypocotyl. Lack of synchrony is not unusual
since not all cells participate equally in the endocytic process.26,30
The absence of vacuole-derived vesicles in protoplasts obtained
from control tissue cultured in betaine is in clear contrast to
the prominent tonoplast vesiculation observed by Diekmann
et al.41 using guard cells protoplasts subjected to osmotic excursions. However, it is noteworthy that guard cells are highly differentiated cells primarily specialized to undergo rapid volume
adjustments based on vacuole fragmentation and reformation in
response to osmotic changes42 as opposed to sucrose accumulating beet storage parenchyma cells.
A Z-stack 3D scanning laser confocal analysis revealed the
abundance of vesicles and their large variation in size (Fig. 3A
and B). Whether the larger vacuole-derived vesicles resulted from
the fusion of smaller ones cannot be determined from the static
data generated by these images. Nevertheless, merger of vesicles
would not offer the cell any organizational advantage during
photoassimilate accumulation since fusion of two spherical compartments would generate a larger surface area/volume structure
resulting in solute dilution.
Fluorescence comparison of vacuole and vacuole-derived vesicles revealed comparable fluorescence intensity (Fig. 4). Since
signal strength is a function of the concentration of fluorescent
molecules,27 the concentration of betacyanin (and hence all
solutes) within the vacuole-derived vesicles must, therefore, be
reasonably similar in all compartments (Fig. 4B). This apparent
Plant Signaling & Behavior
e24793-3
Figure 2. Light Nomarski (A and C) and fluorescent laser scanning confocal micrographs (B and D) of protoplasts from red beet storage tissue. Protoplasts were prepared from hypocotyls at dormant state (A and B) and after 24 h incubation in 200 mM sucrose (C and D).
uniformity in fluorescence remained despite some expected pigment dilution generated by incoming endocytic fluids.
Apparent volume contraction in vacuole-derived vesicles.
The vacuole-derived vesicles formed during incubation in sucrose
underwent visible changes when cells were further incubated for
an additional 24 h in the absence of sucrose. At the end of the
additional incubation period, there were no observable vesicles, or
when present, their numbers were substantially reduced suggesting a re-fusion process with the large central vacuole. The likelihood that the pigmented vacuole-derived vesicles merged with the
plasma membrane in an exocytic manner was disregarded since
there was no betacyanin bleeding into the media during incubation. During a more rigorous examination, the enduring vesicles
appeared darker in color under light microscopy, although not so
apparent in photomicrographs (Fig. 5A and B). However, the perceived increase in color intensity was confirmed by a landscape
e24793-4
view of a topographic fluorescence analysis (Fig. 5C). The vesicle fluorescence peaks were an average 20–30% taller than
those emanating from the vacuole indicating higher betacyanin
concentration.
In a distinct type of image analysis performed on Z-stacks
obtained from separate cells, topographic surface reconstruction
of maximum fluorescence intensity analysis of protoplasts only
revealed the outlining tonoplast (Fig. 6A and B). At this setting,
fluorescence of the central vacuole was eliminated whereas the
vacuole-derived vesicle(s) remained intensely fluorescent indicating that the concentration of betacyanin (and thus all membrane
impermeable solutes) within the vacuole-derived vesicles was considerably higher than in the large central vacuole (arrows). The
increase in fluorescence intensity strongly suggests that vacuolederived vesicles underwent a process of size reduction and their
disappearance denotes their eventual fusion with the vacuole.
Plant Signaling & Behavior
Volume 8 Issue 7
Figure 3. 3D reconstruction of Z-stack fluorescent images of red beet protoplasts isolated from tissue incubated 24 h in 200 mM sucrose. Given the
wide fluorescence spectrum of betacyanin, samples were examined under an emission range of 620–650 nm. (A) Entire protoplast showing vacuole
and vacuole-derived vesicles of a wide range of sizes. (B) Close up of a section of a protoplast. The dark region represents the cytosol, whereas the red
structures are the vacuole, vacuole-derived vesicles and external medium still fluorescing from residual dye.
Discussion
In eukaryotic cells, the dynamics of membrane maintenance
requires a constant turnover of endosome-intrinsic constituents
mediated by vesicle transport and the processes of exocytosis
and endocytosis.43 In each of these processes, membrane vesicles
composed of lipid bilayers and various membrane proteins are
pinched off from one organelle and delivered to other subcellular
or extracellular compartments that are undergoing expansion or
reconstruction.43 During cell growth, much of this trafficking is
directed outwards toward the plasma membrane and, in plants,
toward the enlargement and assembling of the vacuole. However,
in non-expanding cells, membrane degrading and transport pathways must be kept in balance to maintain the proper functioning size of cellular constituents.44 As part of internal trafficking
between different organelles, several inter-endosomal retrograde
systems have been hypothesized that undoubtedly contribute to
membrane homeostasis.32,33,45
In mature plant storage cells, sugar accumulation takes place
in the confined space of a non-expanding vacuole. Aside from
sugar transport by tonoplast-bound transporters,20-22 the partial
delivery of sugars in cargo-filed endocytic vesicles to the vacuole
imposes a significant structural and spatial constraint difficult to
overcome by the reported clathrin-dependent vacuolar retrograde
systems.32-35 Merging of the relatively large FPE vesicles with the
vacuole generates a dual imbalance of membrane and vacuolar
fluids. The data we present in this communication confirm the
endocytic uptake of extracellular fluids induced by sucrose (Fig.
1) and reveal the transitional re-arrangement of the tonoplast/
vacuole architecture during this process (Figs. 2–6). Vacuole
www.landesbioscience.com
fragmentation allows for the temporary adjustment of membrane
surplus and, at the same time, assuring the eventual concentration of solutes. This conclusion is supported by three interrelated pieces of evidence. First, incorporation of the fluorescent
endocytic marker Alexa-350 into the vacuole in the presence of
sucrose indicated the involvement of FPE. Second, a substantial
and consistent fragmentation of the vacuole was observed upon
sucrose accumulation by FPE (Figs. 2–4). Third, the increase
intensity of pigment fluorescence denoted volume shrinkage of
the vacuole-derived vesicles (Figs. 5 and 6). Although speculative at this moment, disappearance of the vacuole-derived vesicles
suggests their merging with the vacuole. A similar conclusion was
reached for Saccharomyces at times of membrane expansion where
vesicle trafficking and nuclear membrane deformations maintain
nuclear/cell volume ratio.46 In plant cells, fragmentation of the
vacuole is commonly observed in glandular cells of carnivorous
plants during endocytosis.6 Another type of tonoplast deformation (termed “bulbs”) has been implicated in the rearrangement
of the vacuole.47 Bulbs are tonoplast-delimited spherical protrusions of 1–3 μm and formed from cytoplasmic folds protruding
into the vacuole. These are believed to serve as reservoir of tonoplast to prepare for quick volume changes.48
The process of vacuole fragmentation described here is fundamentally similar in concept to that conjectured by Traub et
al.32 and Swanson26 in that excess membrane from the vacuole/
lysosome is removed in a retrograde manner. However, our findings differ in that the “retrograde” vesicles generated during
FPE are unequally sized and are not recycled within the endosome as would be those using a clathrin-assisted vesicle system.
Fundamental to the reduction in size of the vacuole-derived
Plant Signaling & Behavior
e24793-5
Figure 4. Topographic fluorescence analysis of red beet protoplasts isolated from tissue incubated 24 h in 200 mM sucrose at two different stages. (A)
Protoplast from dormant beet hypocotyls with a large central vacuole. (B) Protoplast from tissue incubated in 200 mM sucrose for 24 h. Fluorescence
intensity of vacuole-derived vesicles is similar to that emanating from the vacuole.
vesicles (Figs. 5 and 6) is the participation of an additional, yet
unknown, ancillary mechanism(s) involved in the removal of
membrane components and volume regulation. Much of this permanent reduction can be performed by vacuolar lipases, which
have been shown to be essential for membrane digestion during
the macroautophagocytic process in yeast.49 In fact, mutation of
cvt17, which encodes for a putative lipase in yeast, disrupted the
proper turnover of endocytic membrane.49 In our experiments,
it is possible that tonoplast digestion also took place in the large
vacuole; however, since its volume was considerably larger than
that of vacuole-derived vesicles, any reduction in size would have
been proportionally smaller than that of vesicles. This disproportional reduction in size would allow the vacuole-derived vesicles
to outshine the vacuole (Figs. 5 and 6).
Involvement of other proposed mechanisms that could conceivably participate in reducing or preventing expansion of the
tonoplast during FPE are not supported by our data. For example, macrophagocytosis, the engulfing of protoplasm into the
vacuole by tonoplast foldings,50 if operative, would have appeared
as discolored areas within the vacuole, a situation never observed
in our 3-D reconstructions of Z-stacks. The same absence of
membrane inclusions makes microphagocytosis of vacuolar
tubules an unlikely event. Finally, “kiss-and-run,” the fast release
of contents by the endocytic vesicle into the targeted organelle
and its immediate retrieval,51 can be disregarded by the observed
increase presence of plasma membrane P-ATPase in vacuolar
fractions (Table 1).
The tonoplast’s inherent plasticity is often overlooked as an
essential factor in vacuolar maintenance.36,52,53 Capable of withstanding substantial deformations, the tonoplast can absorb
e24793-6
extensive changes in surface area without the need for permanent removal. In fact this plasticity is likely the initial buffer
mechanism during FPE before the formation of vacuole-derived
vesicles.
Studies of membrane rearrangement during endocytosis
have been precluded by the absence of an indisputable recycling
cargo,44,54 a limitation overcame in this study by the use of red
beet hypocotyl cells. These data indicate that tonoplast homeostasis during FPE is a coordinated event that may involve one or
many processes. Here, we present evidence for the architectural
remodeling of the tonoplast during rapid rates of FPE and suggests the participation of an axillary membrane recycling mechanism where vacuole-derived vesicles diminish their size before
re-incorporation to the vacuole and maintaining vacuolar unity.
Materials and Methods
Plant material. Fresh red beet hypocotyls were purchased at a
local grocery store and maintained refrigerated until use. Beets
were brought to room temperature 24 h before experimentation.
Tissue incubation. Tissue disks of approximately 0.5 mm
in thickness were obtained from cylinders excised using a #5
cork borer. The disks were thoroughly rinsed in deionized water
until bleeding stopped. After blotting dry, 1 g of tissue was incubated in a 25 mL Erlenmeyer flask containing 5 mL of a solution
of 10 mM MES (pH 5.6), 200 mM sucrose, 1 mM CaCl 2, 1
mM MgCl2 and 2 mM DTT, incubated for 24 h at 30°C while
being agitated at 80 rpm. Treatment solutions were changed
every 24 h as required by individual experiments. When indicated, inhibitors of endocytosis (10 μM Latrunculin-B) and
Plant Signaling & Behavior
Volume 8 Issue 7
Figure 5. Red beet protoplast isolated from tissue incubated 24 h in 200 mM sucrose followed by a 24 h period of sucrose starvation. (A and B) represent fluorescent and light laser scanning confocal micrographs, respectively. (C and D) are topographic analyses of (A and B), respectively. Fluorescent
peaks on (C) correspond to the vacuole-derived vesicles in (A).
membrane-bound sucrose carriers (1 μM pCMBS) were added
to pre-incubation and incubation solutions. The pre-incubation
solution was similar to the incubation solution with 200 mM
betaine added instead of sucrose.
At the end of the incubation time, tissue was rinsed three
times with 5 mL of pre-incubation media for 10 min each.
Samples were immediately frozen in 5 mL of 25 mM Tris/MES
(pH 5.6) until further analysis. Sucrose was determined following the procedure of Van Handel55 after thawing the tissue in a
rotary shaker for 1 h at 40 rpm and 30°C.
www.landesbioscience.com
Protoplast preparation. Protoplasts were prepared from tissue incubated according to individual experiments by replacing the incubation medium with 10 mL of 100 mM MES (pH
5.6), 2% Cellulase from Trichoderma viride (Onozuka-RS;
SERVA Cat no. 16420), 2% Pectinase from Aspergillus niger
(CalBiochem Cat no. 515883), 1% Macerozyme R-10 from
Rhizopus sp (SERVA cat no. 28302), 1% BSA, 125 mM CaCl 2,
125 mM KCl and incubated overnight in a rotary shaker at 30°C
and 40 rpm. Prior to microscopic observation, the protoplasts were
washed with a similar solution without hydrolyzing enzymes.
Plant Signaling & Behavior
e24793-7
Figure 6. Topographic surface reconstruction of maximum fluorescence intensity of two red beet protoplasts prepared from tissue after a 24 h incubation in 200 mM sucrose followed by a 24 h period of sucrose starvation. Vacuole-derived vesicles (arrows) remain highly fluorescent.
Tonoplast and plasma membrane vesicle isolation. Highly
purified tonoplast and plasma membrane samples were isolated
using a discontinuous sucrose gradient as described by Bennett et
al.56 and Etxeberria and Gonzalez.57 The purities of the membrane
fractions were corroborated measuring the total ATPase activities with and without vanadate and nitrate inhibitors. Individual
membrane fractions were brought to 1.0 mL with storage buffer
containing 250 mM sucrose, 10 mM MES pH 5.6 and 2 mM
DTT and stored at −80°C.
ATPase activity. V-ATPase activity in tonoplast fractions was
determined by coupling the production of ADP to the oxidation
of NADH at 340 nm using a pyruvic kinase/lactate dehydrogenase system (PK-LDH). ATPase reaction solution contained
50 mM BTP/MES (pH 7.5), 4 mM dithiothreitol (DTT), 0.4
mgmL−1 bovine serum albumin (BSA), 250 mM sorbitol, 50
mM KCl, 10 mM gramicidin, 4 mM ATP, 4 mM MgSO4, 5
mL PK:LDH enzymes (Sigma P-0294), 0.25 mgmL−1 NADH,
1 mM phosphoenolpyruvate and approximately 100 mg vesicle
protein in a total volume of 1.0 mL.58 ADP generated was calculated from the extinction coefficient of NAD (6.22) at 340 nm.
References
1. Baluška F, Baroja-Fernandez E, Pozueta-Romero J,
Hlavacka J, Etxeberria E, Samaj J. Endocytic uptake
of nutrients, cell wall molecules, and fluidized cell wall
portions into heterotrophic plant cells. In: Šamaj J,
Baluška F, Menzel D, ed(s). Plant Endocytosis, Plant Cell
Monograph, Springer Verlag, Berlin. 2005:1-17.
2. Etxeberria E, Baroja-Fernandez E, Muñoz FJ, PozuetaRomero J. Sucrose-inducible endocytosis as a mechanism for nutrient uptake in heterotrophic plant cells.
Plant Cell Physiol 2005a; 46:474-81; PMID:15695454;
http://dx.doi.org/10.1093/pcp/pci044
3. Etxeberria E, Gonzalez P, Pozueta-Romero J. Sucrose
transport into Citrus juice cells: evidence for an endocytic transport system. J Am Soc Hortic Sci 2005b;
130:269-74
e24793-8
4.
5.
6.
All reactions were carried with and without 50 mM vandate at
30°C in a Shimadzu UV-160.
Microscopy. Light microscopy was performed using an
Olympus BH-2 microscope equipped with a Canon Power Shot
S3-15 digital camera connected to a Dell monitor. Fluorescent
images were obtained using a Leica TCS-SL confocal laser scanning microscopy.
Given the wide range of fluorescence emission by the vacuolar pigment betacyanin, for experiments containing the red
fluorescent membrane marker FM 4-64 FX, we used ex/em =
488/490-510. We captured FM 4-64 fluorescence at ex/em =
543/650-750. For all other experiments not containing FM 4-64,
betacyanin was observed at ex/em = 543/590-650. Images were
analyzed and created using Leica imaging system. To observe
protoplast loaded with Alexa 350 we used a Zeiss Scope A-1 fluorescent microscope equipped with Zeiss Filter No. 49 (DAPI; Ex:
G365, Em:BP445/50) and Zeiss Axio Cam ICc1.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Etxeberria E, Gonzalez P, Pozueta-Romero J. Mannitolenhanced, fluid-phase endocytosis in storage parenchyma cells of celery (Apium graveolens; Apiaceae) petioles.
Am J Bot 2007a; 94:1041-5; PMID:21636473; http://
dx.doi.org/10.3732/ajb.94.6.1041
Bandmann V, Homann U. Clathrin-independent endocytosis contributes to uptake of glucose into BY-2
protoplasts. Plant J 2012; 70:578-84; PMID:22211449;
http://dx.doi.org/10.1111/j.1365-313X.2011.04892.x
Adlassnig W, Koller-Peroutka M, Bauer S, Koshkin E,
Lendl T, Lichtscheidl IK. Endocytotic uptake of nutrients in carnivorous plants. Plant J 2012; 71:303-13;
PMID:22417315; http://dx.doi.org/10.1111/j.1365313X.2012.04997.x
Plant Signaling & Behavior
7.
Khalil IA, Kogure K, Akita H, Harashima H. Uptake
pathways and subsequent intracellular trafficking in
nonviral gene delivery. Pharmacol Rev 2006; 58:32-45;
PMID:16507881; http://dx.doi.org/10.1124/pr.58.1.8
8. Pozueta-Romero D, Gonzalez P, Pozueta-Romero J,
Etxeberria E. The hyperbolic and linear phases of the
sucrose accumulation curve in turnip (Brassica campestris) storage cells denote carrier-mediated and fluid-phase
endocytic transport, respectively. J Am Soc Hortic Sci
2008; 133:612-8
9. Lin AEJ, Guttman JA. Hijacking the endocytic machinery by microbial pathogens. Protoplasma 2010; 244:7590; PMID:20574860; http://dx.doi.org/10.1007/
s00709-010-0164-2
Volume 8 Issue 7
10. Holstein SE. Clathrin and plant endocytosis. Traffic
2002; 3:614-20; PMID:12191013; http://dx.doi.
org/10.1034/j.1600-0854.2002.30903.x
11. Kruth HS, Jones NL, Huang W, Zhao B, Ishii I, Chang
J, et al. Macropinocytosis is the endocytic pathway that
mediates macrophage foam cell formation with native
low density lipoprotein. J Biol Chem 2004; 280:235260; PMID:15533943; http://dx.doi.org/10.1074/jbc.
M407167200
12. Cao H, Chen J, Awoniyi M, Henley JR, McNiven
MA. Dynamin 2 mediates fluid-phase micropinocytosis in epithelial cells. J Cell Sci 2007; 120:416777; PMID:18003703; http://dx.doi.org/10.1242/
jcs.010686
13. Lazzaro MD, Thompson WW. Endocytosis of lanthanum nitrate in organic acid-secreting trichomes of
chickpea (Cicer arietinum). Am J Bot 1992; 79:1113-8;
http://dx.doi.org/10.2307/2445210
14. Šamaj J, Šamajova O, Peters M, Baluška F, Lichtscheidl
I, Knox JP, et al. Immunolocalization of LM2 arabinogalactan protein epitope associated with endomembranes
of plant cells. Protoplasma 2000; 212:186-96; http://
dx.doi.org/10.1007/BF01282919
15. Etxeberria E, Gonzalez P, Pozueta-Romero J. Evidence
for two endocytic transport pathways in plant cells.
Plant Sci 2009; 177:341-8; http://dx.doi.org/10.1016/j.
plantsci.2009.06.014
16. Etxeberria E, Gonzalez P, Baroja-Fernandez E, PozuetaRomero J. Fluid phase uptake of artificial nano-spheres
and fluorescent quantum-dots by sycamore cultured cells.
Plant Signal Behav 2006; 1:196-200; PMID:19521485;
http://dx.doi.org/10.4161/psb.1.4.3142
17. Paramonova NV. Structural bases of interrelationships
between the symplast and apoplast in the root of Beta
vulgaris during the period of assimilate influx from the
leaves. ?????? ???? 1974; 21:578-88
18. Baroja-Fernandez E, Etxeberria E, Muñoz FJ, MoránZorzano MT, Alonso-Casajús N, Gonzalez P, et al. An
important pool of sucrose linked to starch biosynthesis
is taken up by endocytosis in heterotrophic cells. Plant
Cell Physiol 2006; 47:447-56; PMID:16434435; http://
dx.doi.org/10.1093/pcp/pcj011
19. Onelli E, Prescianotto-Baschong C, Caccianiga M,
Moscatelli A. Clathrin-dependent and independent
endocytic pathways in tobacco protoplasts revealed
by labelling with charged nanogold. J Exp Bot
2008; 59:3051-68; PMID:18603619; http://dx.doi.
org/10.1093/jxb/ern154
20. Briskin DP, Thornley WR, Wyse RE. Membrane transport in isolated vesicles from sugarbeet taproot. Plant
Physiol 1985; 78:871-5; PMID:16664343; http://
dx.doi.org/10.1104/pp.78.4.871
21. Getz HP. Sucrose transport in tonoplast vesicles of beet
roots is linked to ATP hydrolysis. Planta 1991; 185:2618; http://dx.doi.org/10.1007/BF00194069
22. Schulz A, Beyhl D, Marten I, Wormit A, Neuhaus
E, Poschet G, et al. Proton-driven sucrose symport
and antiport are provided by the vacuolar transporters SUC4 and TMT1/2. Plant J 2011; 68:129-36;
PMID:21668536; http://dx.doi.org/10.1111/j.1365313X.2011.04672.x
23. Ayre BG. Membrane-transport systems for sucrose
in relation to whole-plant carbon partitioning. Mol
Plant 2011; 4:377-94; PMID:21502663; http://dx.doi.
org/10.1093/mp/ssr014
24. Etxeberria E, González PC, Tomlinson P, PozuetaRomero J. Existence of two parallel mechanisms for
glucose uptake in heterotrophic plant cells. J Exp Bot
2005c; 56:1905-12; PMID:15911561; http://dx.doi.
org/10.1093/jxb/eri185
25. Etxeberria E, Gonzalez P, Pozueta-Romero J. Fluid phase
endocytosis in Citrus juice cells is independent from
vacuolar pH and inhibited by chlorpromazine, a PI-3
kinase and clathrin-mediated endocytosis inhibitor. J
Hortic Sci Biotechnol 2007b; 82:900-7
26. Swanson JA. Phorbol esters stimulate macropinocytosis
and solute flow through macrophages. J Cell Sci 1989;
94:135-42; PMID:2613767
www.landesbioscience.com
27. Gall L, Stan RC, Kress A, Hertel B, Thiel G, Meckel
T. Fluorescent detection of GFP allows for the in
vivo estimation of endocytic vesicle sizes in plant cells
with sub-diffraction accuracy. Traffic 2010; 11:548-59;
PMID:20136778; http://dx.doi.org/10.1111/j.16000854.2010.01037.x
28. Murphy RF. Analysis and isolation of endocytic vesicles by flow cytometry and sorting: demonstration of
three kinetically distinct compartments involved in
fluid-phase endocytosis. Proc Natl Acad Sci USA 1985;
82:8523-6; PMID:2867544; http://dx.doi.org/10.1073/
pnas.82.24.8523
29. Cupers P, Veithen A, Kiss A, Baudhuin P, Courtoy
PJ. Clathrin polymerization is not required for bulkphase endocytosis in rat fetal fibroblasts. J Cell Biol
1994; 127:725-35; PMID:7962055; http://dx.doi.
org/10.1083/jcb.127.3.725
30. Müller O, Sattler T, Flötenmeyer M, Schwarz H,
Plattner H, Mayer A. Autophagic tubes: vacuolar invaginations involved in lateral membrane sorting and
inverse vesicle budding. J Cell Biol 2000; 151:51928; PMID:11062254; http://dx.doi.org/10.1083/
jcb.151.3.519
31. Swanson J, Yirinec B, Burke E, Bushnell A, Silverstein
SC. Effect of alterations in the size of the vacuolar compartment on pinocytosis in J774.2 macrophages. J Cell
Physiol 1986; 128:195-201; PMID:3733886; http://
dx.doi.org/10.1002/jcp.1041280209
32. Traub LM, Bannykh SI, Rodel JE, Aridor M, Balch WE,
Kornfeld S. AP-2-containing clathrin coats assemble
on mature lysosomes. J Cell Biol 1996; 135:180114; PMID:8991092; http://dx.doi.org/10.1083/
jcb.135.6.1801
33. Bryant NJ, Piper RC, Weisman LS, Stevens TH.
Retrograde traffic out of the yeast vacuole to the TGN
occurs via the prevacuolar/endosomal compartment. J
Cell Biol 1998; 142:651-63; PMID:9700156; http://
dx.doi.org/10.1083/jcb.142.3.651
34. Brown CR, Hung GC, Dunton D, Chiang HL. The
TOR complex 1 is distributed in endosomes and in
retrograde vesicles that form from the vacuole membrane
and plays an important role in the vacuole import and
degradation pathway. J Biol Chem 2010; 285:2335970; PMID:20457600; http://dx.doi.org/10.1074/jbc.
M109.075143
35. Yano K, Matsui S, Tsuchiya T, Maeshima M, Kutsuna
N, Hasezawa S, et al. Contribution of the plasma membrane and central vacuole in the formation of autolysosomes in cultured tobacco cells. Plant Cell Physiol 2004;
45:951-7; PMID:15295079; http://dx.doi.org/10.1093/
pcp/pch105
36. Otegui MS, Spitzer C. Endosomal functions in plants.
Traffic 2008; 9:1589-98; PMID:18627577; http://
dx.doi.org/10.1111/j.1600-0854.2008.00787.x
37.Makarenko SP, Konenkina TA, Salyaev RK.
Characteristics of fatty acid composition of lipids in
higher plant vacuolar membranes. Membr Cell Biol
2000; 13:687-95; PMID:10987391
38.Goodwin PB, Shepherd V, Erwee MG.
Compartmentation of florescent tracers injected into
the epidermal cells of Egeria densa leaves. Planta 1990;
181:129-36; http://dx.doi.org/10.1007/BF00202335
39. Oparka KJ, Prior DAM, Harris N. Osmotic induction
of fluid-phase endocytosis in onion epidermal cells.
Planta 1990; 180:555-61; http://dx.doi.org/10.1007/
BF02411454
40. Rabasovic MS, Sevic D, Terzic M, Savic-Sevic S, Muric
B, Pantelic D, et al. Measurement of beet root extract
flurescence using TR-LIF technique. Acta Physiol Pol
2009; 116:570-2
41. Diekman W, Hedrich R, Raschke K, Robinson DG.
Osmocytosis and vacuolar fragmentation in guard cell
protoplasts: their relevance to osmotically-induced volume changes in guard cells. J Exp Bot 1993; 44:1569-77;
http://dx.doi.org/10.1093/jxb/44.10.1569
Plant Signaling & Behavior
42. Gao XQ, Li CG, Wei PC, Zhang XY, Chen J, Wang
XC. The dynamic changes of tonoplasts in guard cells are
important for stomatal movement in Vicia faba. Plant
Physiol 2005; 139:1207-16; PMID:16244153; http://
dx.doi.org/10.1104/pp.105.067520
43. Verma DPS, Hong Z. The ins and outs in membrane
dynamics: tabulation and vesiculation. Trends Pharmacol
Sci 2005; 10:159-65; http://dx.doi.org/10.1016/j.
tplants.2005.02.004
44. Efe JA, Botelho RJ, Emr SD. The Fab1 phosphatidylinositol kinase pathway in the regulation of vacuole morphology. Curr Opin Cell Biol 2005; 17:4028; PMID:15975782; http://dx.doi.org/10.1016/j.
ceb.2005.06.002
45. Hawes C. The ER/Golgi interface – is there anything
in-between? Frontiers in Plant Sci. 2012; http://dx.doi.
org/10.3389/fpls.2012.00073
46. Webster MT, McCaffery JM, Cohen-Fix O. Vesicle
trafficking maintains nuclear shape in Saccharomyces
cerevisiae during membrane proliferation. J Cell Biol
2010; 191:1079-88; PMID:21135138; http://dx.doi.
org/10.1083/jcb.201006083
47. Saito C, Uemura T, Awai C, Tominaga M, Ebine
K, Ito J, et al. The occurrence of ‘bulbs’, a complex
configuration of the vacuolar membrane, is affected by
mutations of vacuolar SNARE and phospholipase in
Arabidopsis. Plant J 2011; 68:64-73; PMID:21645145;
http://dx.doi.org/10.1111/j.1365-313X.2011.04665.x
48. Saito C, Ueda T, Abe H, Wada Y, Kuroiwa T, Hisada
A, et al. A complex and mobile structure forms a
distinct subregion within the continuous vacuolar
membrane in young cotyledons of Arabidopsis. Plant
J 2002; 29:245-55; PMID:11844103; http://dx.doi.
org/10.1046/j.0960-7412.2001.01189.x
49. Teter SA, Eggerton KP, Scott SV, Kim J, Fischer AM,
Klionsky DJ. Degradation of lipid vesicles in the yeast
vacuole requires function of Cvt17, a putative lipase. J
Biol Chem 2001; 276:2083-7; PMID:11085977
50. Toyooka K, Moriyasu Y, Goto Y, Takeuchi M, Fukuda
H, Matsuoka K. Protein aggregates are transported to
vacuoles by a macroautophagic mechanism in nutrient-starved plant cells. Autophagy 2006; 2:96-106;
PMID:16874101
51. Taraska JW, Almers W. Bilayers merge even when
exocytosis is transient. Proc Natl Acad Sci USA
2004; 101:8780-5; PMID:15173592; http://dx.doi.
org/10.1073/pnas.0401316101
52. Makarenko SP, Salyaev RK. The structure of plant
vacuolar membranes according to infrared spectroscopy.
Membr Cell Biol 1998; 12:385-400; PMID:10024971
53. Reisen D, Marty F, Leborgne-Castel N. New insights
into the tonoplast architecture of plant vacuoles and
vacuolar dynamics during osmotic stress. BMC Plant
Biol 2005; 5:13; PMID:16080795; http://dx.doi.
org/10.1186/1471-2229-5-13
54. Floyd FE. 1942. The carnivorous plants. Ronald Press,
New York
55. Van Handel E. Direct microdetermination of sucrose.
Anal Biochem 1968; 22:280-3; PMID:5641848; http://
dx.doi.org/10.1016/0003-2697(68)90317-5
56. Bennett AB, O’neill SD, Spanswick RM. H+-ATPase
activity from storage tissue of Beta vulgaris. I.
Identification and characterization of an anion sensitive H+-ATPase. Plant Physiol 1984; 74:53844; PMID:16663457; http://dx.doi.org/10.1104/
pp.74.3.538
57. Etxeberria E, Gonzalez P. Simultaneous isolation of
tonoplast and plasma membrane from Citrus juice cells:
combination of sucrose gradient and two phase partitioning. HortScience 2004; 39:174-6
58. Brune A, Muller M, Taiz L, Gonzalez PC, Etxeberria
E. Vacuolar acidification in citrus fruit: Comparison
between acid lime (Citrus aurantifolia) and sweet lime
(Citrus limettioides) juice cells. J Am Soc Hortic Sci 2002;
127:171-7
e24793-9