The Plant Journal (2003) 35, 545±555
doi: 10.1046/j.1365-313X.2003.01822.x
TECHNICAL ADVANCE
Why green ¯uorescent fusion proteins have not been
observed in the vacuoles of higher plants
Kentaro Tamura1, Tomoo Shimada1, Eiichiro Ono2, Yoshikazu Tanaka2, Akira Nagatani1, Sho-ich Higashi3,
Masakatsu Watanabe3,4, Mikio Nishimura3,4 and Ikuko Hara-Nishimura1,
1
Department of Botany, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan,
2
Institute for Advanced Technology, Suntory Ltd, Osaka 618-8503, Japan,
3
National Institute for Basic Biology, Okazaki 444-8585, Japan, and
4
School of Advanced Sciences, The Graduate University for Advanced Studies, Hayama 240-0193, Japan
Received 20 March 2003; revised 18 May 2003; accepted 28 May 2003.
For correspondence (fax ‡81 75 753 4142; e-mail [email protected]).
Summary
Green ¯uorescent protein (GFP) makes it possible for organelles and protein transport pathways to be
visualized in living cells. However, GFP ¯uorescence has not yet been observed in the vacuoles of any
organs of higher plants. We found that the ¯uorescence of a vacuole-targeted GFP was stably observed
in the vacuoles of transgenic Arabidopsis plants under dark conditions, and that the ¯uorescence rapidly
disappeared under light conditions. The vacuolar GFP was rapidly degraded within 1 h in the light, especially blue light. An inhibitor of vacuolar type H‡-ATPase, concanamycin A, and an inhibitor of papain-type
cysteine proteinase, E-64d, abolished both the light-dependent disappearance of GFP ¯uorescence and GFP
degradation in the vacuoles. An in vitro assay showed that bacterially expressed GFP was degraded by
extracts of Arabidopsis cultured-cell protoplasts at an acidic pH in the light. These results suggest that blue
light induced a conformational change in GFP, and the resulting GFP in the vacuole was easily degraded by
vacuolar papain-type cysteine proteinase(s) under the acidic pH. The light-dependent degradation accounts
for the failure to observe GFP ¯uorescence in the vacuoles of plant organs. Our results show that stable
GFP-¯uoresced vacuoles are achieved by transferring the plants from the light into the dark before inspection with a ¯uorescent microscope. This might eliminate a large hurdle in studies of the vacuolar-targeting
machinery and the organ- and stage-speci®c differentiation of endomembrane systems in plants.
Keywords: vacuole, green ¯uorescent protein, Arabidopsis, light, degradation.
Introduction
Green ¯uorescent protein (GFP) from luminescent jelly®sh
Aequorea victoria has been widely used as a reporter in the
determination of gene expression and protein localization in living cells and in real time (Chal®e et al., 1994;
Lippincott-Schwartz et al., 2001; Tsien, 1998). This approach has been highly successful in animal and yeast cells
(Cubitt et al., 1995). In higher plants, also, GFP should be a
tool to investigate the retention or sorting signals of proteins delivered through the secretory pathway. Modi®ed
GFPs have been reported to be localized in various plant
endomembranes: endoplasmic reticulum (Hayashi et al.,
2001; Matsushima et al., 2002; Saito et al., 1999); Golgi
ß 2003 Blackwell Publishing Ltd
complex (Nebenfuhr et al., 1999; Saint-Jore et al., 2002);
plasma membrane (Brandizzi et al., 2002); and vacuolar
membrane (Saito et al., 2002; Uemura et al., 2002) in plant
organs. On the contrary, GFP ¯uorescence has not been
observed in the acidic vacuoles of organs of higher plants,
in spite of extensive studies of GFP ¯uorescence in vacuoles
of protoplasts (Jin et al., 2001; Sansebastiano et al., 1998,
2001; Takeuchi et al., 2000) or cultured cells (Mitsuhashi
et al., 2000). This raises a question of whether the failure to
detect GFP ¯uorescence in vacuoles is caused by degradation and/or protonation of GFP under the acidic conditions
that are found in vacuoles of higher plants.
545
546 Kentaro Tamura et al.
Vacuoles are the largest organelles in plant cells and are
transformed morphologically or functionally during development (Bethke and Jones, 2000; Marty, 1999). In vegetative organs, for example, vacuoles contain various lytic
enzymes and function as lytic organelles, as do yeast
vacuoles and animal lysosomes. Vacuoles maintain an
acidic condition that is generated by the action of vacuolar
H‡-ATPase and H‡-pyrophosphatase using the energy
derived from hydrolysis of their substrates (ATP and pyrophosphate, respectively) (Maeshima, 2000; Sze et al., 1999).
In seed cells, the vacuoles contain reserve proteins and
soluble carbohydrates and function as storage organelles.
Such morphologically or functionally different vacuoles are
known to co-exist in the same cells of maturing pea cotyledons (Hoh et al., 1995), barley roots (Paris et al., 1996),
and barley aleurone cells (Swanson et al., 1998). Vacuolar
proteins are synthesized on the rough endoplasmic reticulum and are sorted and delivered to the respective vacuoles
in these cells. Therefore, different mechanisms might be
involved in targeting proteins to the different vacuoles.
Targeting of vacuolar proteins requires a positive targeting
signal in their polypeptides. A number of signals mediating
the vacuolar transport of soluble proteins have been characterized in higher plants (Neuhaus and Rogers, 1998;
Vitale and Raikhel, 1999). The signals are separated into
three classes: N-terminal propeptides (NTPPs), internal
peptides, and C-terminal propeptides (CTPPs). Analysis
of these targeting signals at the molecular level is essential
to elucidate the complex vacuolar-targeting machinery in
various plant organs.
If stable GFP ¯uorescence could be achieved in acidic
vacuoles, it could be a powerful tool for studying the
vacuolar-targeting machinery and to investigate organand stage-speci®c differentiation of endomembrane systems in plants. To understand the behavior of GFP in
vacuoles, we expressed a GFP fusion protein with a vacuolar-targeting signal in transgenic plants and cultured cells of
Arabidopsis. We found that light caused the rapid disappearance of GFP ¯uorescence in the vacuoles and the rapid
degradation of GFP by the proteinase activity that is present
in vacuoles. We succeeded in stabilizing GFP ¯uorescence
within acidic vacuoles in a variety of organs of Arabidopsis
plants.
Results
Vacuolar GFP fluorescence disappeared under the light
condition
GFP ¯uorescence has not been observed in the acidic and
lytic vacuoles in higher plants. To unveil the in planta
behavior of GFP in vacuoles of Arabidopsis, we generated
transgenic Arabidopsis plants that stably expressed a
vacuole-targeted GFP, SP-GFP-2SC, composed of a signal
peptide and GFP followed by a vacuolar-targeting signal
(the C-terminal 18-amino-acid sequence) of pumpkin 2S
albumin (Mitsuhashi et al., 2000; see Figure 2a). GFP ¯uorescence was not observed in the vacuoles of the transgenic
plants that were grown under the continuous light condition. However, we found that GFP ¯uorescence was detectable in the vacuolar lumen only when the transgenic plants
were placed in the dark for 2 days, as shown in Figure 1.
The vacuoles of root cells (Figure 1a), root hairs (Figure 1b),
and guard cells (Figure 1c) of transgenic seedlings exhibited a bright ¯uorescence, although the vacuoles of leaf
epidermal cells exhibited faint ¯uorescence (Figure 1c).
When the plants were placed in the light for 1 day, the
¯uorescence in the vacuoles completely disappeared, but
the ¯uorescence on the endoplasmic reticulum and the
small granular structures of Golgi complex and/or prevacuolar compartments did not (Figure 1d±f).
To determine whether GFP ¯uorescence in vacuoles of
protoplasts also disappears in the light, we transformed
Arabidopsis suspension-cultured cells (T87) with three
chimeric genes encoding the GFP fusion proteins (SPGFP-2SC, SP-GFP-CTPP, and SP-NTPP-GFP) with each
vacuolar-targeting signal (Figure 2a). CTPP was derived
Figure 1. Light-dependent disappearance of GFP ¯uorescence in vacuoles
of transgenic Arabidopsis plants.
Transgenic Arabidopsis seedlings expressing a vacuole-targeted GFP, SPGFP-2SC (see Figure 2a), were incubated under the dark and light conditions
for 2 days and were inspected with a confocal laser scanning microscope.
GFP ¯uorescence was detected in the vacuolar lumens in the roots (a), the
root hairs (b) and the guard cells of the cotyledons (c) under the dark
condition. Fluorescence was not detected in the vacuolar lumens of any
of these tissues, but it was detected on the networks of the endoplasmic
reticulum and the small granular structures under the light condition (d±f).
Bars ˆ 20 mm.
ß Blackwell Publishing Ltd, The Plant Journal, (2003), 35, 545±555
Stable accumulation of GFP in plant vacuoles
547
condition for 1 day and then were transferred to the light
condition. Figure 3(a, lower panel) shows the rapid disappearance of the GFP ¯uorescence in the vacuoles. The halflife of the vacuolar GFP ¯uorescence was calculated to be
approximately 19 min (Figure 3b, open circles). On the
contrary, the intensity of ¯uorescence in the vacuoles
remained at a high level until 60 min under the dark condition (Figure 3a, upper panel; 3b, closed circles). The
results obtained with the transgenic plants (Figure 1) and
the transformed protoplasts (Figures 2 and 3) show that the
light-induced disappearance of the GFP ¯uorescence was
strictly limited to the vacuolar lumen.
Why does the GFP ¯uorescence in the vacuoles disappear? One possibility is that GFP is degraded in the
vacuoles. Another possibility is that GFP ¯uorescence is
Figure 2. Light-dependent disappearance of GFP ¯uorescence in vacuoles
of transformed Arabidopsis suspension-cultured cells expressing the GFP
fusion proteins with various vacuolar-targeting signals.
(a) Constructs of three modi®ed GFP fusion proteins with various vacuolartargeting signals for expression in Arabidopsis cells. SP, the signal peptide
of pumpkin 2S albumin. Vacuolar-targeting signals used were as follows:
2SC, the C-terminal propeptide of pumpkin 2S albumin; CTPP, the C-terminal propeptide of tobacco chitinase A; NTPP, the N-terminal propeptide of
Arabidopsis aleurain homolog.
(b±g) Protoplasts prepared from Arabidopsis cultured cells were transformed with a chimeric gene for each modi®ed GFP fusion protein. The
protoplasts expressing these GFP fusion proteins were incubated under the
dark (b±d) or light (e±g) condition for 2 days and were inspected with a
confocal laser scanning microscope (FL) or a differential-interferencecontrast microscope (DIC). V, vacuoles.
Bar ˆ 20 mm.
from the C-terminal 6-amino-acid propeptide of tobacco
chitinase A (Sansebastiano et al., 1998), and NTPP was
derived from the N-terminal 140-amino-acid propeptide
of an Arabidopsis aleurain homolog (Ahmed et al., 2000).
Each transformed protoplast exhibited strong ¯uorescence
in the vacuoles when placed in the dark, as can be seen on
the endoplasmic reticulum and the small granular structures within the cytosol (Figure 2b±d). However, once the
protoplasts were transferred to the light condition, the
¯uorescence in the vacuoles disappeared within 1 day
(Figure 2e±g). The ¯uorescence on the network and small
granular structures did not disappear at all. These results
indicated that light induced the disappearance of the
vacuolar GFP ¯uorescence.
Rapid degradation of vacuolar GFP fusion protein in
the light
We investigated the light-induced disappearance of GFP
¯uorescence in vacuoles in greater detail. The protoplasts
expressing SP-GFP-2SC were incubated under the dark
ß Blackwell Publishing Ltd, The Plant Journal, (2003), 35, 545±555
Figure 3. Time course for the degradation of GFP fusion protein in vacuoles
under the light condition.
(a) A time-lapse analysis of the disappearance of GFP ¯uorescence in
vacuoles of protoplasts from Arabidopsis suspension-cultured cells expressing SP-GFP-2SC. The protoplasts were incubated under the dark condition
for 1 day and then were transferred into either the light condition or the dark
condition. Fluorescent images of GFP in the vacuoles were obtained at 10min intervals. Bar ˆ 20 mm.
(b) The levels of the vacuolar GFP ¯uorescence shown in (a) were densitometrically determined with protoplasts incubated under the light (open
circles) or dark (closed circles) condition (SE, n ˆ 6). Relative intensity is
the percentage of the initial values.
(c) The protein pro®les of GFP in transgenic Arabidopsis plants expressing
SP-GFP-2SC. Seven-day-light-grown seedlings (lane 1) were incubated
under the dark condition (D) for 6 h (lane 2), 24 h (lane 3), and 48 h (lane
4), and then were incubated under the light condition (L) for 5 min (lane 5),
10 min (lane 6), 20 min (lane 7), 30 min (lane 8), 1 h (lane 9), and 4 h (lane 10).
These seedlings were subjected to SDS±PAGE followed by immunoblot with
anti-GFP antibodies. Molecular masses are given on the right in kDa.
548 Kentaro Tamura et al.
attenuated by protonation of the ¯uorophore of the GFP
molecule. We further investigated the protein pro®le of the
GFP fusion protein in the transgenic Arabidopsis plants
after transferring the plants to the light condition. Each
extract from the seedlings harvested at the indicated period
was subjected to an immunoblot analysis with anti-GFP
antibodies (Figure 3c). A 30-kDa protein was accumulated
in the 7-day-light-grown seedlings (Figure 3c, lane 1),
which exhibited GFP ¯uorescence on the network and small
granular structures but not in the vacuolar lumen (data not
shown). The molecular mass of 30 kDa was consistent with
30 162 Da of the calculated mass of GFP-2SC. The seedlings
accumulated another protein with a molecular mass of
27-kDa during the time that they were in the dark (Figure 3c,
lanes 2±4). The value of 27-kDa was consistent with the
molecular mass of the GFP itself, suggesting that the Cterminal peptide (2SC) was removed from the GFP-2SC
molecule. The 27-kDa GFP was rapidly degraded by irradiation with light (Figure 3c, lanes 5±10). The reduction of the
27-kDa GFP level in the light was consistent with the lightinduced disappearance of the ¯uorescence found in the
vacuoles (Figure 3a). On the other hand, the 30-kDa GFP2SC was not degraded, but slightly increased in the level by
irradiation (Figure 3c, discussed below). These results
implied that the 30-kDa GFP-2SC was not localized in the
vacuoles, although the 27-kDa GFP was localized in the
vacuoles.
To clarify the subcellular localization of the 30-kDa GFP2SC and 27-kDa GFP, the homogenate from the transgenic
Arabidopsis seedlings was subjected to differential centrifugation. Figure 4 shows that the 30-kDa GFP-2SC was
detected speci®cally in the P100 fraction (microsome-rich
fraction), while the 27-kDa GFP was detected speci®cally in
the S100 fraction (vacuole-rich fraction). This result indicates that the 27-kDa GFP was localized in the vacuoles, and
that the 30-kDa GFP-2SC was localized in the endoplasmic
reticulum, Golgi complex, and/or the pre-vacuolar compartments. Thus, the 30-kDa GFP-2SC synthesized was transported from the endoplasmic reticulum to the vacuoles and
Figure 4. Differential subcellular localization of two forms of GFP fusion
proteins in transgenic Arabidopsis plants.
Total homogenate from 10-day-old seedlings of transgenic plants expressing SP-GFP-2SC was subjected to differential centrifugation to obtain the
1000 g supernatant (total), 100 000 g pellet (P100, microsomal fraction), and
100 000 g supernatant (S100, vacuolar fraction). Each subcellular fraction
was subjected to an immunoblot with antibodies against each of GFP, a
putative vacuolar-sorting receptor (AtELP), and an Arabidopsis aleurain
homolog (AtALEU). Molecular masses are given on the right in kDa.
then proteolytically processed to produce the 27-kDa GFP
within the vacuoles. The slight increase in the amount of the
30-kDa GFP-2SC by irradiation (Figure 3c) might be caused
by more active synthesis of the protein in the light than in
the dark. It is possible that disappearance of GFP in
vacuoles under the light condition was caused by degradation of the 27-kDa GFP but not by protonation of GFP
molecule in the vacuoles.
Involvement of pre-existing cysteine proteinase(s) in the
light-dependent degradation of vacuolar GFP fusion
protein
To elucidate the mechanism for the light-dependent degradation of GFP fusion protein in the vacuoles, we examined
the effect of E-64d, a membrane-permeable inhibitor of
cysteine proteinases of the papain family, on the degradation process. The protoplasts transiently expressing SPGFP-2SC were incubated under the dark condition for
1 day and then treated with 50 mM E-64d before incubation
under the light condition for 1 day. The light-dependent
disappearance of the GFP ¯uorescence in the vacuoles was
observed in the untreated protoplasts (Figure 5a, upper
panel). We found that the treatment with E-64d prevented
the ¯uorescence in the vacuoles from disappearing even in
the light condition (Figure 5a, lower panel). A similar inhibitory effect of E-64d was observed with the protoplasts
that were incubated under the light condition for 1 day
before treating with E-64d for 1 day (Figure 5b).
We also veri®ed an effect of E-64d on the degradation of
the GFP fusion protein in the transgenic Arabidopsis plants.
The 7-day-light-grown transgenic Arabidopsis seedlings
were incubated for 2 days in the dark and then incubated
for 1 day in the light. The 27-kDa vacuolar form of GFP was
not detected in the seedlings, while the 30-kDa microsomal
form was detected (Figure 5c, lane 1). As expected from the
above observation (Figure 5a), the 27-kDa GFP was detectable in the seedlings that had been treated with E-64d
before incubation in the light for 1 day (Figure 5c, lane
2). Similarly, the 27-kDa GFP was detectable in the seedlings that had been treated with E-64d even under the
continuous light conditions (Figure 5d, lanes 1 and 2).
These results indicate that cysteine proteinase(s) of the
papain family are involved in the light-dependent degradation of the GFP fusion proteins within vacuoles.
To clarify whether the light-dependent degradation of
the GFP fusion protein in the vacuoles needs de novo
protein synthesis, we treated the transgenic Arabidopsis
plants with cycloheximide, an inhibitor of protein synthesis. Two-day-dark-adapted seedlings were treated with
900 mM cycloheximide for 4 h in the dark and then were
irradiated with the light for 1 h to monitor the light-dependent degradation of GFP. Before the irradiation, both the
30-kDa microsomal form and the 27-kDa vacuolar form of
ß Blackwell Publishing Ltd, The Plant Journal, (2003), 35, 545±555
Stable accumulation of GFP in plant vacuoles
549
proteinase(s) in the vacuoles may be responsible for the
GFP degradation.
Vacuolar H‡-ATPase activity is required for lightdependent degradation of GFP fusion protein
Figure 5. Involvement of pre-existing cysteine proteinase(s) in the lightdependent degradation of the vacuolar form of GFP and light-dependent
disappearance of GFP ¯uorescence in vacuoles.
(a, b) Protoplasts transformed with the gene for SP-GFP-2SC were incubated
for 1 day under (a) the dark or (b) the light condition. The protoplasts were
treated with or without E-64d before incubation under the light condition for
1 day, as indicated by arrows. Fluorescent images of the vacuoles in the
transformed protoplasts were obtained with a confocal laser scanning
microscope. V, vacuoles. Bar ˆ 20 mm.
(c, d) The 7-day-light-grown transgenic Arabidopsis seedlings were incubated for 2 days under (c) the dark or (d) the light condition. They were
treated with or without E-64d before incubation under the light condition for
1 day, as indicated by arrows. These seedlings were subjected to an immunoblot with anti-GFP antibodies. Molecular masses are in kDa.
(e) Dark-adapted transgenic Arabidopsis seedlings expressing SP-GFP-2SC
were treated under the dark condition with (lanes 3 and 4) or without (lanes 1
and 2) 900 mM cycloheximide for 4 h. These seedlings were irradiated with
the light for 0 h (lanes 1 and 3) and 1 h (lanes 2 and 4), and then were
subjected to an immunoblot with anti-GFP antibodies. Molecular masses
are in kDa.
GFP were detected on the immunoblot of untreated seedlings (Figure 5e, lane 1). The 30-kDa microsomal form in the
seedlings disappeared with the cycloheximide treatment
(Figure 5e, lane 3), indicating that the treatment actually
inhibited de novo synthesis of the GFP fusion protein. On
the contrary, the 27-kDa vacuolar form did not disappear as
a result of the treatment (Figure 5e, lane 3). After the
irradiation, the vacuolar form of GFP was degraded in both
untreated (Figure 5e, lane 2) and treated (Figure 5e, lane 4)
seedlings. This result indicates that the light-dependent
degradation of the GFP fusion protein in the vacuoles does
not need de novo protein synthesis. Pre-existing cysteine
ß Blackwell Publishing Ltd, The Plant Journal, (2003), 35, 545±555
It has been shown that vacuolar enzymes have an acidic
optimum pH. The interior acidic pH of vacuoles is maintained by H‡-ATPase and H‡-pyrophosphatase on vacuolar
membranes. We examined the effect of pH on the degradation of the GFP fusion protein in the vacuoles by using
concanamycin A, a speci®c inhibitor of vacuolar H‡-ATPase
(Drose et al., 1993; Matsuoka et al., 1997). The 1-day-darkadapted transgenic seedlings were treated with 0.5 mM
concanamycin A for 1 day and then irradiated for 1 h.
The light-dependent degradation of the 27-kDa vacuolar
form of GFP was monitored on the immunoblot of the
seedlings. Figure 6(a) shows that the light-dependent
degradation of the vacuolar form was completely inhibited
by the concanamycin A treatment. As expected from the
result, the transgenic Arabidopsis seedlings with the concanamycin A treatment exhibited GFP ¯uorescence in the
vacuoles of the root cells in the light (Figure 6b, right panel)
in contrast to the seedlings without concanamycin A treatment (left panel). These results indicated that an acidic pH is
necessary for the light-dependent degradation of the
GFP fusion proteins in the vacuoles. Sodium pamidronate
(Gordon-Weeks et al., 1999), a speci®c inhibitor of vacuolar
Figure 6. Concanamycin A, an inhibitor of vacuolar H‡-ATPase inhibited the
light-dependent degradation of the vacuolar form of GFP.
(a) The 1-day-dark-adapted transgenic Arabidopsis seedlings expressing
SP-GFP-2SC were treated with (lane 2) or without (lane 1) 0.5 mM concanamycin A for 1 day under the dark condition and then irradiated for 1 h. These
seedlings were subjected to an immunoblot with anti-GFP antibodies.
Molecular masses are given on the right in kDa.
(b) Fluorescent image of roots of transgenic Arabidopsis seedlings expressing SP-GFP-2SC that were treated with or without 0.5 mM concanamycin A
for 2 days under the light condition. Bar ˆ 20 mm.
550 Kentaro Tamura et al.
H‡-pyrophosphatase, which is another pump that maintains the acid conditions in the vacuoles, did not inhibit the
degradation of vacuolar GFP under the light condition
(data not shown). This result suggests that vacuolar
H‡-pyrophosphatase is not the main pump for the acidi®cation of vacuoles.
The Arabidopsis det3 mutant is the only vacuolar H‡ATPase-de®cient mutant known in plants (Schumacher
et al., 1999). In det3, a low-level expression of subunit C
of vacuolar H‡-ATPase causes the reduction of vacuolar
H‡-ATPase activity to only about 40% of the values in the
wild type. To determine whether the degradation of the GFP
fusion protein is affected in the det3 mutant, we made an
Arabidopsis det3 mutant expressing SP-GFP-2SC and analyzed the GFP fusion protein by an immunoblot. After 1-h
irradiation, the 27-kDa vacuolar form of GFP was not
degraded in the det3 mutant seedlings (Figure 7a, lane
4), while it was completely degraded in the transgenic
seedlings (Figure 3c, lane 9). Longer irradiation (>4 h)
degraded the vacuolar form of GFP (Figure 7a, lanes 5
and 6). The half-life of the vacuolar form of GFP in the
det3 mutant was longer than that in the transgenic Arabidopsis seedlings (Figure 7b). The slow degradation of GFP
in the det3 mutant may re¯ect the increase in vacuolar pH
caused by the reduced expression of DET3 that would affect
Figure 8. In vitro light-dependent degradation of recombinant GFP with
extracts from protoplasts of Arabidopsis suspension-cultured cells.
The recombinant His-tagged GFP was incubated with extracts of protoplasts
from wild-type cells to degrade it at pH 5.5 or pH 7.5 with (b) or without (a)
50 mM E-64d under the light or dark condition, respectively. The reactions
were subjected to an immunoblot with anti-GFP antibodies. Closed triangles, intact recombinant GFP; open triangles, truncated form of GFP.
activities of proteinases. This result indicates that the lightdependent degradation of the vacuolar GFP involves
vacuolar H‡-ATPase activity that maintains the acidic
condition in the vacuolar lumen (Sze et al., 1999).
In vitro degradation of GFP with the protoplast extract
from Arabidopsis cultured cells in a light-dependent
manner
Figure 7. Time course for the light-dependent degradation of GFP fusion
proteins in Arabidopsis det3 mutant expressing SP-GFP-2SC.
(a) The 7-day-light-grown seedlings of the det3 mutant with a low activity of
vacuolar H‡-ATPase were incubated under the dark condition for 0 h (lane 1)
and 48 h (lane 2), and then were incubated under the light condition for 0.5 h
(lane 3), 1 h (lane 4), 4 h (lane 5), and 24 h (lane 6). These seedlings were
subjected to an immunoblot with anti-GFP antibodies. Molecular masses
are given on the right in kDa.
(b) Amounts of the 27-kDa vacuolar type of GFP shown in (a) were densitometrically determined with the mutant plants. The change in the level of
the 27-kDa GFP in det3 mutants (open circles) is compared with that of the
transgenic plants (closed circles, Figure 3c). Relative amounts are the percentages of the initial values.
We examined the effect of light on the degradation of GFP
in vitro. The recombinant His-tagged 31-kDa GFP (rGFP)
that had a hexa His-tag and additional amino acids at the
N-terminus was incubated under light and dark conditions
with the protoplast extracts from the cultured cells. rGFP
was limitedly degraded to produce a 27-kDa GFP at pH 5.5
within 1 h both in the light and in the dark (Figure 8a, lanes
3 and 9). An immunoblot with anti-hexa His antibody
revealed that the 27-kDa GFP lacked the His-tag domain
(data not shown). Further 16-h incubation in the light at
pH 5.5 resulted in the complete degradation of the 27-kDa
GFP (Figure 8a, lane 6). On the contrary, the 27-kDa GFP
was not degraded in the dark (Figure 8a, lane 12). The
in vitro light-dependent degradation was consistent
with the in vivo light-dependent degradation shown in
Figure 3(c). On the other hand, the 27-kDa GFP was stable
until 16 h even in the light at pH 7.5 (Figure 8a, lane 18). A
cysteine proteinase inhibitor E-64d partially inhibited the
degradation of the 27-kDa GFP in the light at pH 5.5
(Figure 8b, lane 6). These results indicate that the degradation of GFP needs both an acidic environment, which will
activate vacuolar proteinases, and light.
ß Blackwell Publishing Ltd, The Plant Journal, (2003), 35, 545±555
Stable accumulation of GFP in plant vacuoles
551
489 nm, which is an absorption lmax of GFP at the neutral
condition (Elsliger et al., 1999).
Discussion
Visualization of vacuoles in living cells of higher plants
Figure 9. Wavelength dependency of degradation of the vacuolar form of
GFP.
(a) The 7-day-light- plus 2-day-dark-grown transgenic Arabidopsis seedlings
were irradiated with blue light (B, peak at 450 nm), red light (R, at 660 nm)
and far-red light (FR, at 730 nm) for 0±4 h. These seedlings were subjected to
an immunoblot with anti-GFP antibodies. Molecular masses are given on
the right in kDa.
(b) An equal-quantum action spectrum for the degradation of 27-kDa GFP.
The 7-day-light- plus 2-day-dark-grown transgenic Arabidopsis seedlings
were irradiated with monochromatic-light (wavelength from 400 to 700 nm).
These seedlings were subjected to an immunoblot with anti-GFP antibodies.
Amounts of the 27-kDa vacuolar type of GFP were densitometrically determined. Relative reduced amounts are the percentages of the maximum
values. Vertical bar is SE.
Blue light specifically induces degradation of the GFP
fusion protein in the vacuoles
To determine which wavelengths of light are most effective
at inducing the degradation of GFP fusion protein in the
vacuoles, 2-day-dark-adapted transgenic Arabidopsis seedlings were irradiated with blue, red, and far-red light (with
peaks at 450, 660, and 730 nm). After 0-, 1-, and 4-h irradiation, the seedlings were subjected to an immunoblot to
determine the degradation degree of the vacuolar form of
GFP. Figure 9(a) shows that only blue light was effective for
the degradation. To obtain the equal-quantum action spectrum for the degradation of the vacuolar form of GFP, we
used the Okazaki Large Spectrograph. The 2-day-darkadapted transgenic Arabidopsis seedlings were irradiated
with the monochromatic light at 20-nm interval from 400 to
700 nm and at ¯uence rate of 30 mmol m 2 sec 1 for 16 h.
Figure 9(b) shows that the action spectrum was consistent
with the above result. The spectrum showed that the degradation of the vacuolar form of GFP was enhanced as the
wavelength was shorter than 440 nm. The spectrum was in
agreement with the fact that the GFP (S65T) has an absorption lmax of 394 nm in the acidic condition but no peak at
ß Blackwell Publishing Ltd, The Plant Journal, (2003), 35, 545±555
Several studies have detected GFP ¯uorescence in the
vacuoles of cultured cells or protoplasts, including Arabidopsis protoplasts (Jin et al., 2001; Takeuchi et al., 2000),
tobacco mesophyll protoplasts (Sansebastiano et al.,
2001), and tobacco cultured cells (Mitsuhashi et al.,
2000). Sansebastiano et al. (2001) detected GFP ¯uorescence in the vacuoles of tobacco protoplasts, but they
did not detect it in the tobacco plants themselves. Thus,
to our knowledge, no one has succeeded in obtaining GFP
¯uorescence in the vacuoles of higher plants. We have
succeeded in accumulating GFP ¯uorescence in vacuoles
of transgenic Arabidopsis plants by growing them in the
dark. In the previous studies, the cultured cells and protoplasts of transformants were incubated under the dark
condition. This may be the reason why stable GFP ¯uorescence was observed in the vacuoles. On the other hand,
plants are usually grown under the light condition. Because
of this, GFP ¯uorescence would not have been detected in
the vacuoles of plant bodies.
The light condition abolished the GFP ¯uorescence in the
vacuoles (Figures 1±3). However, even under the light condition, a stable accumulation of the GFP fusion protein was
observed in the vacuoles of the cells treated with either E64d or concanamycin A (Figures 5 and 6, discussed below).
These results indicate that the light condition does not
in¯uence the transport of the GFP fusion protein to the
vacuoles.
The vacuole-targeted GFP, SP-GFP-2SC, was synthesized
on the endoplasmic reticulum to be transported to the
vacuoles via the secretory pathway by which proteins are
transported to the vacuoles in plant cells. Transient expression of the GFP fusion protein in Arabidopsis roots under
the dark condition allowed the visualization of the secretory
pathway. GFP ¯uorescence was ®rst detected in the endoplasmic reticulum and then in the central vacuoles via the
small granules, which are possibly Golgi complexes and/or
pre-vacuolar compartments (unpublished data). Similarly,
the endoplasmic reticulum, the small granules, and the
vacuoles also ¯uoresced in the cells in the stable transgenic
Arabidopsis plant organs expressing SP-GFP-2SC
(Figure 1). Transgenic Arabidopsis plants expressing SPGFP-2SC are a powerful tool to study organ- and stagespeci®c differentiation of the endomembrane system
including vacuoles.
A variety of GFP-related ¯uorescent proteins including
¯uorescence resonance energy transfer (FRET)-based
552 Kentaro Tamura et al.
probes have been used to determine Ca2‡ concentration,
pH, various activities of signaling molecules, and protein±
protein interactions in living cells of animals (see for a
review, Zhang et al., 2002). These ¯uorescent proteins
should be valuable and versatile tools to investigate vacuolar functions in various organs of plants. For such experiments, transgenic plants are grown in the light that causes
disappearance of GFP ¯uorescence in the vacuoles. We
discovered that stable GFP-¯uoresced vacuoles are
achieved by transferring the plants from the light into the
dark a half day before inspection with a ¯uorescent microscope.
Instability of GFP in acidic vacuoles under the light
condition
We showed that GFP is unstable in the acidic vacuoles of
plants under the light condition. Crystallographic studies
indicate that GFP is tightly folded. The GFP tertiary
structure resembles a barrel consisting of 11 antiparallel
b-sheets and a single central a-helix surrounded by
the b-sheets (Ormo et al., 1996; Yang et al., 1996). Most
of the b-sheets are connected by small loops of one to four
amino acids. The ¯uorophore is buried in the center of the
barrel, completely shielded from the external environment.
Unfolded GFP does not ¯uoresce because the ¯uorophore
becomes accessible to the solvent and is no longer
restricted to the center of the barrel. GFP retains its ¯uorescence upon extensive exposure to various proteinases
(Bokman and Ward, 1981), even though the GFP molecule
has several putative recognition sites for proteinases, such
as trypsin, chymotrypsin, and subtilisin, in the regions of
the loops in the tertiary structure (Yang et al., 1996). Other
b-barrel proteins such as OmpA and a-hemolysin are also
highly resistant to proteolysis (Cheley et al., 1997; Surrey
and Jahnig, 1992). For this reason, GFP has been thought to
be highly resistant to proteolysis.
Chiang et al. (2001) constructed GFP variants containing
sequences recognized by various proteinases that attack
the loops that link b-sheets. They found that the ¯uorescence of these GFP variants was not signi®cantly affected
by the proteinases, indicating that GFP has a remarkably
stable structure. Our immunoblot analysis revealed that
30-kDa GFP-2SC was truncated to produce 27-kDa GFP
in vivo (Figure 3c), and that the 31-kDa recombinant GFP
was truncated to produce the 27-kDa GFP in vitro (Figure 8).
This result suggests that an additional peptide at the N- or
C-terminus, unlike the core of GFP, is quite susceptible to
attack by proteinases. The resulting 27-kDa GFP is tightly
folded and ¯uoresces.
However, we clearly demonstrated that light caused the
rapid disappearance of GFP ¯uorescence in the vacuoles of
Arabidopsis plants (Figure 3a), and that light also caused
the rapid degradation of the 27-kDa GFP by the pre-existing
proteinase in vacuoles (Figure 5). The 27-kDa GFP was also
effectively degraded at pH 5.5 in the light by the protoplast
extract (Figure 8a). We found that the degradation of the
27-kDa GFP was induced by blue light (Figure 9), which is
near the absorption peak wavelength (at 394 nm) of sGFP
(S65T) under the acidic condition (Elsliger et al., 1999). Our
results suggest that absorption of the blue light at the lower
pH made the GFP susceptible to proteinase attack, which
was able to lead to its complete degradation. This is the
reason why ¯uorescent vacuoles have not been observed in
the transgenic plant bodies.
GFP ¯uorescence in the vacuoles re¯ects the degradation
activity of vacuoles. We observed different GFP ¯uorescent
intensities in vacuoles of various organs of the transgenic
Arabidopsis seedlings (Figure 1). For example, leaf epidermal cells showed a lower ¯uorescent intensity than root
cells, suggesting that the vacuoles of the leaf cells have
a higher degradation activity than those of the roots
(Figure 1). Therefore, transgenic Arabidopsis plants
expressing SP-GFP-2SC can be a useful tool for investigating degradation activity of vacuoles in various organs and
various developmental stages of living cells of higher plants.
Experimental procedures
Plant materials and growth conditions
Arabidopsis thaliana (ecotype Columbia) and the Arabidopsis
mutant of det3-1 (Schumacher et al., 1999) were used. Seeds
were sown onto 0.5% Gellan Gum (Wako, Tokyo, Japan) with
Murashige±Skoog medium (Murashige and Skoog, 1962) supplemented with B5 vitamins (Gamborg et al., 1968) and 3% sucrose,
and were grown at 228C under continuous light condition
(150 mmol m 2 sec 1). Suspension-cultured cells of Arabidopsis
(T87) were kindly provided by the Bio Resource Center (BRC) of
RIKEN (Tsukuba, Ibaraki, Japan). The cells were subcultured as
described previously by Axelos et al. (1992). The cells were transformed with each of the following GFP-chimeric genes.
Plasmid construction
GFP was kindly provided by Dr Y. Niwa of University of Shizuoka
(Chiu et al., 1996). The chimeric genes encoding SP-GFP and SPGFP-2SC were constructed previously by Mitsuhashi et al. (2000).
SP-GFP consists of a signal peptide of pumpkin 2S albumin
followed by sGFP (S65T), and SP-GFP-2SC consists of SP-GFP
and the vacuolar-targeting signal of the C-terminal 18-amino-acid
sequence of pumpkin 2S albumin.
For the chimeric gene encoding SP-GFP-CTPP, two complementary oligonucleotides (50 -GATCTCCTCGTGGATACCATGTGAG-30
as the sense strand and 50 -AATTCCCTGGTATCCACGAGGA-30 as
the antisense strand) were synthesized and annealed to produce a
double-strand DNA with restriction ends to be ligated to the Bgl II±
EcoRI sites of pSP-GFP (Mitsuhashi et al., 2000). This chimeric
gene encodes the signal peptide followed by GFP and the vacuolar-targeting signal of the C-terminal 5-amino-acid sequence of
tobacco chitinase A, Leu-Asp-Thr-Met (LDVTM) (Sansebastiano
et al., 1998).
ß Blackwell Publishing Ltd, The Plant Journal, (2003), 35, 545±555
Stable accumulation of GFP in plant vacuoles
For the chimeric gene encoding SP-NTPP-GFP, the DNA fragment was produced by PCR ampli®cation using an expressed
sequence tag (EST) clone for Arabidopsis aleurain homolog
(Accession number 153D20T7) as a template and a set of the
oligonucleotide primers, 50 -CCACATGTCTGCGAAAACAAT-30 and
50 -GTTTCCATGGTAGCTGCTTCTGTGACCT-30 . This produced fragment was inserted into the A¯III±NcoI sites of pSP-GFP. This chimeric
gene encodes the signal peptide, the N-terminal 140-amino-acid
sequence including the vacuolar-targeting signal, Asn-Pro-Ile-Arg
(NPIR), of Arabidopsis aleurain homolog, and GFP, in that order.
553
An immunoblot analysis was performed essentially as
described previously by Matsushima et al. (2002), except that
dilutions of the antibodies were as follows: anti-GFP (1 : 5000)
(Clontech, Palo Alto, CA, USA), anti-PV72 (AtELP) 1 : 1000
(Shimada et al., 1997), and anti-AtALEU (1 : 10 000) (Rogers et al.,
1997).
Confocal laser scanning microscopy
All chimeric genes were inserted into pBI221 for transient expression. Protoplasts prepared from Arabidopsis suspension-cultured
cells (T87) were transformed with each of the chimeric genes using
polyethylene-glycol as described previously by Shimada et al.
(2002). The chimeric gene encoding SP-GFP-2SC was also inserted
into pBI121 for stable expression in Arabidopsis plants according
to the in planta method (Bechtold and Pelletier, 1998).
Green ¯uorescent protein ¯uorescent images of the transgenic
plants and the transformants of cultured cells were inspected with
a ¯uorescence microscope (Axioplan 2 Imaging, Carl Zeiss, Jena,
Germany) equipped with a confocal laser scanning unit (CSU 10,
Yokogawa Electric, Tokyo, Japan) and the laser unit (Sapphire 488
Coherent, CA, USA). We used the ®lter set for GFP ¯uorescence
(510AF23 Omega Optical, VT, USA). Images were acquired by a
cooled color charge-coupled device camera (ORCA-ER, Hamamatsu Photonics, Japan) and processed by the IPLab software
(Scanalytics, Fairfax, VA, USA) and Adobe Photoshop 5.5 (Adobe
Systems, Tokyo, Japan).
Treatments with inhibitors
In vitro degradation of the recombinant GFP
Stock solutions of inhibitors used were 300 mM cycloheximide
dissolved in c. 100% ethanol, 10 mM (L-3-trans-ethoxcycarbonyloxirane-2-carbonyl)-L-leucine (3-methylbutyl) amide (E-64d) in
c. 100% methanol, and 100 mM concanamycin A in 100% dimethyl
sulfoxide. The protoplasts prepared from Arabidopsis suspensioncultured cells were incubated in Murashige±Skoog's medium containing 0.4 M sucrose and 50 mM E-64d or 0.5% methanol for 1 day
under the light condition. The seedlings of the transgenic Arabidopsis were incubated in Murashige±Skoog's medium containing
each inhibitor, or the same amount of the respective solvent as a
control. Seven-day-light- plus 2-day-dark-grown seedlings were
incubated with 0.9 mM cycloheximide or 0.3% ethanol for 4 h
under the dark condition. The 7-day-light- plus 1-day-dark-grown
seedlings were incubated with 0.5 mM concanamycin A or 0.5%
DMSO for 1 day under the dark condition. The 7-day-light-grown
seedlings were incubated with 0.5 mM concanamycin A or 0.5%
DMSO for 2 days under the light condition. The 7-day-light- plus
2-day-dark-grown or the 9-day-light-grown seedlings were incubated with 50 mM E-64d or 0.5% methanol for 1 day under the light
condition.
The His-tagged GFP was inserted into pQE (Qiagen, Tokyo, Japan)
to produce pQE-GFP with which E. coli XL1-blue (Stratagene, La
Jolla, CA, USA) cells were transformed. After induction with isopropyl-b-D-thiogalactopyranoside, the cells were collected by centrifugation at 10 000 g for 5 min, gently suspended in 50 mM
phosphate buffer, pH 7.0, 300 mM NaCl and 1 mM phenylmethylsulfonyl ¯uoride, and then lysed by three bursts of sonication for
10 sec at 30-sec intervals on ice. The lysed cells were centrifuged at
10 000 g for 10 min. The supernatant was loaded on a TALON
metal af®nity resin (Clontech, Palo Alto, CA, USA) and eluted with
150 mM imidazole in the above solution. The eluted His-tagged
GFP fraction was dialyzed against 5 mM phosphate buffer, pH 7.0.
Protein concentrations in the homogenates were determined
using a protein assay kit (Bio-Rad, Hercules, CA, USA).
Protoplasts isolated from the 4-day-old Arabidopsis T87 cells
were precipitated at 420 g for 2 min, re-suspended in 2 ml of the
cold buffer solution of 50 mM HEPES-KOH, pH 8.0, and then lysed
gently by at least eight passages through a 25G needle. The lysates
were centrifuged at 10 000 g for 10 min to obtain the soluble
extracts.
The recombinant His-tagged GFP (2 mg) was incubated with the
soluble extracts (2 mg) in a reaction of 50 mM MES-KOH, pH 5.5, or
50 mM HEPES-KOH, pH 7.5, and 0.1 M dithiothreitol under the
light or dark condition at 228C. To stop the reaction, the 2 SDS
sample buffer was added and denatured at 1008C. The reactions
were subjected to an immunoblot analysis with anti-GFP antibodies.
Transformation of Arabidopsis
Protein extraction and subcellular fractionation of
Arabidopsis seedlings
Three seedlings were homogenized in 100 ml of the 2 SDS sample
buffer (100 mM Tris±HCl, pH 6.8, 4% SDS, 20% glycerol, and 5%
2-mercaptoethanol). The homogenates were heated and then
centrifuged at 15 000 g for 5 min to obtain the protein extracts
as supernatant solutions. The extracts were subjected to SDS±
PAGE followed by an immunoblot with anti-GFP antibodies.
For subcellular fractionation, 10-day-old transgenic Arabidopsis
seedlings were minced in 50 mM HEPES-KOH, pH 7.5, 0.4 M
sucrose and proteinase inhibitors (Complete, Roche, Tokyo,
Japan) on ice. The homogenate was ®ltered through cheesecloth
and centrifuged at 1000 g for 10 min at 48C to obtain the supernatant (total fraction). The supernatant was ultracentrifuged at
100 000 g for 1 h at 48C to separate into the pellet (the P100
fraction) and the supernatant (the S100 fraction). Each fraction
was subjected to an immunoblot analysis with antibodies against
each of GFP, a putative vacuolar-sorting receptor (AtELP), and
Arabidopsis aleurain homolog (AtALEU).
ß Blackwell Publishing Ltd, The Plant Journal, (2003), 35, 545±555
Spotlight irradiation
The 7-day-light- plus 2-day-dark-grown transgenic Arabidopsis
seedlings were irradiated with each of blue light, red light, and
far-red light for 1 or 4 h. Blue light (B, peak at 450 nm) was
provided by light-emitting diodes (LED-B, EYLA, Tokyo, Japan)
at 30 mmol m 2 sec 1. Red light (R, at 660 nm) was provided by red
¯uorescent tubes (FL20S/R-F, National, Osaka, Japan) at
30 mmol m 2 sec 1. Far-red light (FR, at 730 nm) was provided
by ¯uorescent tubes (FL20S FR-74, Toshiba, Tokyo, Japan) ®ltered
through plastic (Delaglass A-900, Asahi Chemical Industry, Tokyo,
Japan) at 16 mmol m 2 sec 1. These seedlings were subjected to
an immunoblot analysis with anti-GFP antibodies.
554 Kentaro Tamura et al.
Monochromatic light irradiation
The Okazaki-Large Spectrograph at the National Institute for Basic
Biology (Watanabe et al., 1982) in Okazaki, Japan, was used for
monochromatic irradiation at 20-nm interval from 400 to 700 nm to
obtain the equal-quantum action spectrum for the degradation of
the vacuolar form of GFP. The 7-day-light- plus 2-day-dark-grown
transgenic Arabidopsis seedlings were placed to irradiate at ¯uence rate of 30 mmol m 2 sec 1 for 16 h. The treated seedlings
were homogenized and were then subjected to an immunoblot
analysis with anti-GFP antibodies. The amounts of the degraded
vacuolar form of GFP were densitometrically determined. The
relative level is given as a percentage of the maximum value. Each
point is an average of three independent measurements.
Acknowledgements
We are grateful to Professor John. C. Rogers (Washington State
University) for his gift of antibodies against Arabidopsis aleurain
homolog, to Dr Yasuo Niwa of University of Shizuoka for his kind
donation of the modi®ed GFP gene with a strong ¯uorescence, to
Dr Ryo Matsushima of Kyoto University for his construct of SPNTPP-GFP, and to Ms. Eriko Yoshida for her help in crossing the
Arabidopsis det3-1 mutant and the transgenic plants expressing
SP-GFP-2SC. We also thank the Arabidopsis Biological Resource
Center for providing the seeds of Arabidopsis det3-1 mutant and
the EST clone (Accession number 153D20T7). This work was
supported by CREST of the Japan Science and Technology Corporation and Grants-in-Aid for Scienti®c Research from the Ministry of Education, Culture, Sports, Science and Technology of
Japan (nos. 12138205 and 12304049).
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