The Autophagic Degradation of Chloroplasts via
Rubisco-Containing Bodies Is Specifically Linked to
Leaf Carbon Status But Not Nitrogen Status
in Arabidopsis1[W][OA]
Masanori Izumi, Shinya Wada, Amane Makino, and Hiroyuki Ishida*
Department of Applied Plant Science, Graduate School of Agricultural Sciences, Tohoku University,
Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981–8555, Japan
Autophagy is an intracellular process facilitating the vacuolar degradation of cytoplasmic components and is important for
nutrient recycling during starvation. We previously demonstrated that chloroplasts can be partially mobilized to the vacuole
by autophagy via spherical bodies named Rubisco-containing bodies (RCBs). Although chloroplasts contain approximately
80% of total leaf nitrogen and represent a major carbon and nitrogen source for new growth, the relationship between leaf
nutrient status and RCB production remains unclear. We examined the effects of nutrient factors on the appearance of RCBs in
leaves of transgenic Arabidopsis (Arabidopsis thaliana) expressing stroma-targeted fluorescent proteins. In excised leaves, the
appearance of RCBs was suppressed by the presence of metabolic sugars, which were added externally or were produced
during photosynthesis in the light. The light-mediated suppression was relieved by the inhibition of photosynthesis. During a
diurnal cycle, RCB production was suppressed in leaves excised at the end of the day with high starch content. Starchless
mutants phosphoglucomutase and ADP-Glc pyrophosphorylase1 produced a large number of RCBs, while starch-excess mutants
starch-excess1 and maltose-excess1 produced fewer RCBs. In nitrogen-limited plants, as leaf carbohydrates were accumulated,
RCB production was suppressed. We propose that there exists a close relationship between the degradation of chloroplast
proteins via RCBs and leaf carbon but not nitrogen status in autophagy. We also found that the appearance of non-RCB-type
autophagic bodies was not suppressed in the light and somewhat responded to nitrogen in excised leaves, unlike RCBs. These
results imply that the degradation of chloroplast proteins via RCBs is specifically controlled in autophagy.
Autophagy is the major pathway by which proteins
and organelles are transported for degradation in
the vacuoles of yeast and plants or the lysosomes of
animals (for detailed mechanisms, see reviews by
Ohsumi, 2001; Levine and Klionsky, 2004; Thompson
and Vierstra, 2005; Bassham et al., 2006; Bassham,
2009). In these systems, a portion of the cytoplasm,
including entire organelles, is engulfed in a doublemembrane vesicle called an autophagosome and delivered to the vacuole/lysosome. The outer membrane
of the autophagosome then fuses with the vacuolar/
lysosomal membrane, and the inner membrane structure called the autophagic body is degraded within the
vacuole/lysosome by resident hydrolytic enzymes. A
1
This work was supported by KAKENHI (grant nos. 20200061,
20780044, and 22780054 for H.I., grant no. 21114006 for A.M., and
grant no. 21–6184 for M.I.), by Genomics for Agricultural Innovation
(grant no. GPN–0007 for A.M.), and by Research Fellowships of the
Japan Society for the Promotion of Science for Young Scientists to M.I.
* Corresponding author; e-mail [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Hiroyuki Ishida ([email protected]).
[W]
The online version of this article contains Web-only data.
[OA]
Open Access articles can be viewed online without a subscription.
www.plantphysiol.org/cgi/doi/10.1104/pp.110.158519
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recent genome-wide search confirmed that Autophagyrelated genes (ATGs) are well conserved across yeast,
plants, and animals (Meijer et al., 2007). Using ATG
knockout mutants and a monitoring system with
an autophagy marker, GFP-ATG8, numerous studies
have demonstrated the presence of the autophagy
system in plants and its importance in several biological processes (Yoshimoto et al., 2004, 2009; Liu et al.,
2005; Suzuki et al., 2005; Thompson et al., 2005; Xiong
et al., 2005, 2007; Fujiki et al., 2007; Patel and DineshKumar, 2008; Phillips et al., 2008; Hofius et al., 2009).
Plant autophagy is thought to play an important role
in nutrient recycling under starvation, similar to its
role in starvation previously noted in yeast and animals (Thompson and Vierstra, 2005; Bassham et al.,
2006). For example, the creation of autophagosomes
is induced by nitrogen, carbon, or both combined
starvation in plant heterotrophic tissues such as roots
(Yoshimoto et al., 2004; Xiong et al., 2005), hypocotyls
(Thompson et al., 2005; Phillips et al., 2008), and
suspension-cultured cells (Chen et al., 1994; Aubert
et al., 1996; Moriyasu and Ohsumi, 1996; Rose et al.,
2006). Arabidopsis (Arabidopsis thaliana) atg mutants
show accelerated leaf senescence and cannot survive
and respond to nutrient resupply after severe carbon
or nitrogen starvation (Doelling et al., 2002; Hanaoka
et al., 2002; Thompson et al., 2005; Xiong et al., 2005;
Phillips et al., 2008).
Plant PhysiologyÒ, November 2010, Vol. 154, pp. 1196–1209, www.plantphysiol.org Ó 2010 American Society of Plant Biologists
Nutrient Response of Autophagy in Leaves
The majority of plant nitrogen and other nutrients
are distributed to leaves in the vegetative growth
stage (Schulze et al., 1994; Makino et al., 1997). In C3
plants, 75% to 80% of total leaf nitrogen is distributed
to chloroplasts, primarily as photosynthetic proteins
such as Rubisco (Makino and Osmond, 1991; Makino
et al., 2003). During senescence and suboptimal environmental conditions, Rubisco and most stromal
proteins are degraded, and the released nitrogen is
remobilized to growing organs and finally stored in
seeds (Friedrich and Huffaker, 1980; Mae et al., 1983).
Chloroplast proteins can be degraded under carbonlimited conditions caused by darkness (Wittenbach,
1978), with their carbon used as a substrate for respiration. Therefore, considering the role of autophagy in
nutrient recycling, much attention should be paid to
the study of chloroplast degradation. ATG transcript
abundances are elevated during starvation-induced
leaf senescence (Yoshimoto et al., 2004; van der Graaff
et al., 2006; Chung et al., 2009). However, previous
reports have not analyzed the effects of nutrient status
on the appearance of autophagosomes or autophagic
bodies in leaves. Recently, we observed the accumulation of autophagic bodies and Rubisco-containing
bodies (RCBs), a kind of autophagic body containing
chloroplast stroma, using fluorescent markers in the
vacuole of excised leaves treated with concanamycin
A, which suppresses vacuolar lytic activity (Ishida
et al., 2008). Autophagy of chloroplast components
could be observed during senescence of individually
darkened leaves (Wada et al., 2009). However, it is not
clear how the production of autophagic bodies and
RCBs is affected by nutrient status in leaves.
In this study, we aimed to demonstrate the relationship between the nutrient status of Arabidopsis leaves
and chloroplast degradation via RCBs. We examined
the effects of nutrient conditions during leaf incubation, leaf carbohydrate contents over a diurnal cycle,
mutations affecting starch metabolism, and nitrogen
limitation on the appearance of RCBs. All analyses
showed that carbon status is a major factor controlling
the production of RCBs, while nitrogen status is less
important in the nutrient response of autophagy in
leaves. We also found that the production response of
RCBs and non-RCB-type autophagic bodies containing
cytoplasmic components other than chloroplasts to
nutrient conditions was not always the same in excised
leaves. This suggests that there is a mechanism specifically controlling RCB production in plant autophagy.
RESULTS
Responses of RCB Production to Carbon But Not
Nitrogen Supply in Excised Leaves
RCBs could be visualized in the vacuolar lumen
with laser-scanning confocal microscopy (LSCM)
when leaves of chloroplast stroma-targeted GFPexpressing Arabidopsis (named CT-GFP) were excised
and incubated in darkness in “incubation buffer,”
Plant Physiol. Vol. 154, 2010
containing concanamycin A, an inhibitor of vacuolar
H+-ATPase that suppresses vacuolar lytic activity (Fig.
1A; Ishida et al., 2008). RCBs were rarely seen when
attached leaves were excised and immediately observed during natural senescence (Ishida et al., 2008).
RCBs were also rarely observed even if excised leaves
were incubated in darkness without concanamycin
A for 20 h, suggesting that they are rapidly degraded
in the vacuolar lumen under natural cell conditions
(Ishida et al., 2008). Thus, inhibitor treatments for
suppressing vacuolar lytic activity are essential for
quantitative examination of RCB production. When
excised leaves were incubated in incubation buffer,
RCBs were detected more in mature and early senescent leaves than in young leaves, but RCBs were rarely
seen by the presence of Murashige and Skoog (MS)
medium containing Suc, even in early senescent leaves
(Ishida et al., 2008). These previous data suggested
that the production of RCBs was influenced by nutrient conditions during incubation in excised leaves.
First, we quantitatively analyzed the effects of external
nutrient supply and light conditions during incubation on the appearance of RCBs (Fig. 1E). Around 50
RCBs were detected in the field of view (188 mm 3
188 mm each) in leaves incubated in nutrient-free medium in darkness (dark MES; Fig. 1A). The addition of
MS medium containing Suc (dark MS + Suc) greatly
suppressed the appearance of RCBs. This suppression
of RCBs was not relieved by the depletion of nitrogen
(dark MS-N + Suc) but by the depletion of Suc (dark
MS-N; dark MS). Furthermore, RCB appearance was
also suppressed by the sole addition of Suc (dark +
Suc; Fig. 1B), Glc (dark + Glc), or Fru (dark + Fru).
Mannitol did not affect the appearance of RCBs
(dark + Mann); thus, it is unlikely that osmotic stress
affects RCB appearance.
The appearance of RCBs was suppressed by irradiation (light MES; Fig. 1C). The light-mediated suppression of RCB appearance was relieved by the addition of
3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an
inhibitor of photosynthetic electron transport (light +
DCMU; Fig. 1D), indicating the possibility that the
appearance of RCBs was suppressed by photosynthesis, or the products of photosynthesis, rather than by
light-mediated signals. Changes in starch and metabolic sugar contents were determined in excised leaves
incubated either in darkness or in the light (Fig. 2).
Although starch and metabolic sugars were decreased
and almost consumed after 20 h of incubation in darkness (dark MES) or in the light with DCMU (light +
DCMU), carbohydrate levels were increased during
incubation in the light (light MES). These results
suggested that RCB production was affected by photosynthetically accumulated internal carbohydrates in
the light as well as externally added sugars in darkness.
Differential RCB accumulation might be due to differences in the stability of RCBs or the maker, GFP,
in the vacuolar lumen rather than differential RCB
production between the various treatments used
here. To clarify this possibility, we used transgenic
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Izumi et al.
Figure 1. Effects of incubation conditions
on the appearance of Rubisco-containing
bodies. A to D, Visualization of RCBs by
stroma-targeted GFP in various incubation conditions. Fourth leaves of stromatargeted GFP expressing Arabidopsis at
30 d after sowing (5 d after bolting) were
excised and incubated in 10 mM MESNaOH (pH 5.5) with 1 mM concanamycin
A and 100 mM E-64d at 23°C for 20 h in
darkness (A), with 3% (w/v) Suc in darkness (B), in the light (C), or with DCMU in
the light (D). GFP appears green, and
chlorophyll fluorescence appears red. In
merged images, overlap of GFP and chlorophyll fluorescence appears yellow. In
B, C, and D, merged images are shown.
Bars = 50 mm. E, The number of RCBs
in various incubation conditions. Excised
leaves were incubated as described in A to
D in darkness (blue columns) or in the
light (yellow columns) and with various
constituents: MES, no other constituent;
MS, MS medium; MS-N, nitrogen-free MS
medium; +Suc, 3% (w/v) Suc; +Glc, 3%
(w/v) Glc; +Fru, 3% (w/v) Fru; +Mann, 3%
(w/v) mannitol; +DCMU, 10 mM DCMU.
Leaves from four independent plants were
incubated, eight quadrangular regions
(188 mm 3 188 mm each) per leaf were
monitored by LSCM, and the number of
accumulated RCBs was counted (RCB
number). Each image was considered as
an independent data point and subjected
to statistical analysis by Tukey’s test. Data
represent means 6 SE (n = 32). Columns
with the same letter were not significantly
different (P # 0.01).
Arabidopsis expressing vacuole-targeted GFP, which
is a GFP fusion of N-terminal signal peptide and
C-terminal signal propeptide of pumpkin (Cucurbita
sp.) 2S albumin (SP-GFP-2SC; Tamura et al., 2003), as
an indicator for the stability of the GFP signal in the
vacuole (Supplemental Fig. S1). When leaves of SPGFP-2SC were excised and immediately observed
with LSCM, the GFP signal was not observed in the
vacuole but in the endoplasmic reticulum and Golgi
complex, as reported previously (Supplemental Fig.
S1A; Tamura et al., 2003). It was also reported that GFP
was rapidly degraded by vacuolar proteases in the
light but that the degradation was suppressed by the
addition of concanamycin A or E-64d (Tamura et al.,
2003). When leaves of SP-GFP-2SC plants were incubated in the presence of concanamycin A and E-64d,
GFP was observed in the vacuole irrespective of the
treatment conditions used, darkness, darkness + MS
medium containing Suc, or in the light, where the
degree of RCB appearance was greatly different (Fig. 1;
Supplemental Fig. S1, B–D). This suggests that the
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treatments used did not have significant effects on
vacuolar lytic activity. If RCBs were degraded in leaves
of CT-GFP plants under conditions where RCB appearance was significantly suppressed (Fig. 1), stromatargeted GFP, which was mobilized from chloroplasts
via RCBs into the vacuole, should be distributed
uniformly as SP-GFP-2SC in the vacuolar lumen, since
GFP would not be degraded in the presence of protease inhibitors (Supplemental Fig. S1, B–D). However,
we could not observe uniformly distributed GFP in
vacuolar lumen in the leaves of CT-GFP plants under
those conditions (Fig. 1). These results suggest that the
appearance of RCBs in various incubation conditions
was mainly due to differences in RCB production.
RCB Production Is Specifically Controlled during
Autophagy of Mesophyll Cells
Previous studies have shown that autophagic bodies
accumulate under nitrogen, carbon, or nitrogen and
carbon combined starvation in heterotrophic tissues
Plant Physiol. Vol. 154, 2010
Nutrient Response of Autophagy in Leaves
production was unaffected. These data indicated that
nitrogen also affects the appearance of non-RCB-type
autophagic bodies to some extent in leaves, as reported
previously (Yoshimoto et al., 2004; Thompson et al.,
2005; Xiong et al., 2005; Phillips et al., 2008), but the
effect of nitrogen was small compared with the effect
of Suc. The appearance of non-RCB-type autophagic
bodies was not suppressed in the light (light MES),
while RCB production was consistently and markedly
suppressed. This specific suppression of RCB production related to photosynthesis suggests that RCB
production was more sensitive to increases in leaf
carbohydrate contents than autophagosome creation.
Effects of Timing of Leaf Excision in a Diurnal Cycle on
the Appearance of RCBs
Figure 2. Changes in starch, Suc, Glc, and Fru contents in incubated
leaves. Excised leaves from transgenic Arabidopsis at 30 d after sowing
were incubated at 23°C for 8 h or for 20 h in 10 mM MES-NaOH (pH
5.5) with 1 mM concanamycin A and 100 mM E-64d in darkness (black
squares), in the light (white circles), or with 10 mM DCMU in the light
(white triangles), and carbohydrate content was determined. Data
represent means 6 SE (n = 3).
(Yoshimoto et al., 2004; Thompson et al., 2005; Xiong
et al., 2005; Phillips et al., 2008). RCBs are a kind of
autophagic body that specifically contain the stromal
portion of chloroplasts. However, the pattern of RCB
accumulation was not similar to autophagic body
accumulation shown in these previous studies (Fig.
1). Therefore, we next compared RCB production with
autophagic bodies containing cytoplasmic components other than the stroma (non-RCB-type autophagic bodies) in excised leaves under various nutrient
conditions.
RCBs and entire autophagic bodies can be visualized at the same time using transgenic plants expressing stroma-targeted DsRed and the GFP-ATG8 fusion
(Fig. 3, A and B; Ishida et al., 2008). In these transgenic
plants (ecotype Wassilewskija), the number of RCBs
and non-RCB-type autophagic bodies detected in the
field of view (188 mm 3 188 mm each) was 41 6 2.7 and
20 6 1.7 (n = 64) under control conditions (dark MES;
Fig. 3C), respectively. The appearance of RCBs was
significantly suppressed by the presence of Suc or in
the light, although this was relieved by the presence of
DCMU (Fig. 3C), similar to the CT-GFP plants (ecotype
Columbia; Fig. 1). The pattern of appearance of nonRCB-type autophagic bodies was not always the same
as the pattern of RCB appearance (Fig. 3C). In darkness, the appearance of non-RCB-type autophagic
bodies was suppressed by the presence of Suc, similar
to RCBs (dark MS + Suc, dark MS-N + Suc, dark + Suc,
and light + Suc). This suggested that exogenous excess
sugars suppress entire autophagy. In Suc-free conditions, the appearance of non-RCB-type autophagic
bodies was also suppressed by the presence of MS
medium (dark MS) or nitrogen (dark + N), while RCB
Plant Physiol. Vol. 154, 2010
To examine the effects of physiological changes in
leaf carbohydrate contents on the appearance of RCBs,
we investigated RCB production during a diurnal
cycle. Fourth rosette leaves of CT-GFP plants were
excised at three time points: the end of the regular
night cycle, the end of a prolonged night, and the end
of the day, and carbohydrate contents and RCB number were determined (Fig. 4A). As expected, starch
levels were significantly higher at the end of the day
compared with the end of the night, with most of the
starch used up at the end of prolonged night (Fig. 4B).
Among soluble sugars, Suc was significantly lower at
the end of prolonged night, although no significant
changes were detected in Glc or Fru levels. Recorded
sugar levels were similar to those in previous reports
(Gibon et al., 2004; Bläsing et al., 2005; Smith and Stitt,
2007). Leaves were excised and incubated in incubation buffer for 10 h in darkness. The number of RCBs
was low in leaves excised at the end of the day, when
carbohydrate levels were highest, and high in leaves
excised at the end of prolonged night, when carbohydrate levels were at a minimum. Particularly, starch
contents seemed to be a primary factor affecting RCB
production.
Effects of Mutations Affecting Leaf Starch Content on the
Appearance of RCBs
To provide further evidence of the close relationship
between RCB production and leaf carbohydrate contents, we examined RCB production in starch content
genetic mutants, specifically starchless phosphoglucomutase (pgm-1) and ADP-Glc pyrophosphorylase1 (adg1-1)
and starch-excess starch-excess1 (sex1-1) and maltoseexcess1 (mex1-3). The mutant pgm-1 lacks chloroplast
phosphoglucomutase (Caspar et al., 1985), and adg1-1
lacks the activity of ADP-Glc pyrophosphorylase (Lin
et al., 1988). Neither mutant can accumulate starch
during the day. The mutant sex1-1 lacks glucan
water dikinase 1 and is reduced in its capacity to
degrade starch (Caspar et al., 1991; Yu et al., 2001),
while mex1-3 lacks the chloroplast maltose transporter
and contains both high levels of starch and very high
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Izumi et al.
Figure 3. Effects of incubation conditions
on the appearance of RCBs and autophagic bodies. A and B, Visualization of both
RCBs and autophagic bodies. Excised
leaves from both GFP-ATG8- and stromatargeted DsRed-expressing plants at 25 d
after sowing (5 d after bolting) were incubated at 23°C for 20 h in 10 mM MESNaOH (pH 5.5) with 1 mM concanamycin
A and 100 mM E-64d in darkness (A) or
in the light (B). GFP appears green and
DsRed appears red. In merged images,
overlap of GFP and DsRed appears yellow. Bars = 10 mm. C, The appearance of
RCBs and non-RCB-type autophagic
bodies in various incubation conditions.
Excised leaves from GFP-ATG8- and
stroma-targeted DsRed-expressing plants
were incubated as described in A and B
in darkness or in the light with various constituents: MES, no other constituent; MS,
MS medium; MS-N, nitrogen-free MS medium; +Suc, 3% (w/v) Suc; +N, nitrogen
nutrition of MS medium; +DCMU, 10 mM
DCMU. Leaves from six independent
plants were incubated, eight quadrangular
regions (188 mm 3 188 mm each) per leaf
were monitored by LSCM, and the number
of accumulated RCBs (red columns) and
non-RCB-type autophagic bodies not
containing DsRed (green columns) were
counted. Each image was considered as an
independent data point and subjected to
statistical analysis by Tukey’s test. Each
value is shown as a percentage of the level
in “dark MES.” Data represent means 6 SE
(n = 64). Values with the same letter were
not significantly different in RCBs (red
values) and non-RCB-type autophagic
bodies (green values; P # 0.05).
levels of maltose (Niittylä et al., 2004). These starchrelated mutants grow like wild-type plants in continuous light; however, their growth is progressively
impaired as the duration of the night period is increased (Caspar et al., 1985, 1991; Lin et al., 1988). We
first set up starch-related mutants expressing stromatargeted GFP and visualized the chloroplast stroma of
each mutant (Supplemental Fig. S2). In mex1-3, we
observed both normal and abnormally shaped chloroplasts, which exhibited swollen chloroplasts, as revealed by the fluorescence signals of stroma-targeted
GFP (Supplemental Fig. S2, D and E). This “swollen
chloroplast” phenotype has been noted previously in
mex1 mutant cells observed by transmission electron
microscopy (Stettler et al., 2009).
1200
In continuous-light growth conditions, both the
wild type and the mutants all took about 21 d to start
bolting. Mutants pgm-1, adg1-1, and sex1-1 were almost
the same size and had the same leaf number as the
wild type at 5 d after bolting (Fig. 5A). Only mex1-3
was smaller than the wild type; however, leaf number
was almost the same as in the wild type (Fig. 5A).
Carbohydrate contents were determined in the fourth
leaves at 5 d after bolting (Fig. 5B). As expected, starch
levels were significantly higher in starch-excess mutants than in the wild type. No starch was detected in
starchless mutants. Soluble sugar levels were similar
in wild-type and mutant plants. Leaves from each set
of plants were excised and incubated in incubation
buffer. RCBs were also observed in leaves of each of
Plant Physiol. Vol. 154, 2010
Nutrient Response of Autophagy in Leaves
Figure 4. Effects of timing of leaf excision in a diurnal cycle on the appearance of RCBs. A, Schematic representation of this
experiment. Fourth leaves of stroma-targeted GFP transgenic plants at 30 d after sowing were excised at the end of the regular
night cycle (EN) or after 4 h of prolonged night (PN) or the end of the day (ED) in a day cycle. B, The leaf carbohydrate contents
and the appearance of RCBs. The contents of starch (left) and Suc, Glc, and Fru (center) in leaves at the end of the night (gray
columns), after the prolonged night (black columns), and the end of the day (white columns) were determined. Excised leaves at
each point were incubated at 23°C for 10 h in 10 mM MES-NaOH (pH 5.5) with 1 mM concanamycin A and 100 mM E-64d in
darkness. Leaves from four independent plants were incubated, eight quadrangular regions (188 mm 3 188 mm each) per leaf
were monitored by LSCM, and the number of accumulated RCBs was counted (right). Data represent means 6 SE (n = 3–4
[carbohydrate contents] or 32 [RCB number]). Statistical analysis was performed by Tukey’s test. Columns with the same letter
were not significantly different (P # 0.05).
the starchless and starch-excess mutants, although the
number of RCBs was significantly higher in leaves of
starchless mutants and lower in starch-excess mutants
than in the wild type (Fig. 6).
Next, we analyzed RCB production throughout
the leaf life span in the wild type and starch-related
mutants under long-day growth conditions with a
14-h photoperiod, because the growth rates of each
mutant differed under long-day conditions. Here,
wild-type plants took 25 d to bolt. A further 2 to 3 d
were required in mex1-3, 3 to 4 d in sex1-1, and 5 to 6 d
in pgm1-1 and adg1-1. In wild-type and mutant plants,
leaf area of the fourth leaf rapidly increased before
bolting, and leaf expansion had almost finished as
bolting commenced (Supplemental Fig. S3). We quantified the appearance of RCBs in fourth leaves from 5 d
before bolting, during active leaf expansion, to 10 d
after bolting, the senescent stage, in each plant (Fig.
7A). In the wild type, the number of RCBs increased in
expanding, mature and early senescent leaves, as
reported previously (Ishida et al., 2008), but decreased
at the later senescent stage. In starchless mutants, the
appearance of RCBs was significantly higher than in
the wild type in expanding and mature leaves but
Plant Physiol. Vol. 154, 2010
declined during leaf senescence faster than in the wild
type. In leaves of starchless mutants, the transcription
levels of senescence-associated genes, SAG12 and
SAG13, were higher than in the wild type (Fig. 7B).
This implied rapid leaf senescence in starchless mutants, leading to a rapid decline of RCB production.
In starch-excess mutants, RCB appearance was always lower than in wild-type plants during the period examined. In senescent leaves, the appearance of
RCBs was significantly lower in starch-excess mutants
than in the wild type. The maximum number of RCBs
during the leaf life span was significantly higher in
starchless mutants and lower in starch-excess mutants
compared with the wild type (Fig. 8A). Starch could
not be detected in the leaves of starchless mutants, and
significantly higher starch contents were detected in
leaves of starch-excess mutants than in the wild type
when the number of RCBs after incubation was maximum in each plant (Fig. 8B), while soluble sugar
levels were higher in starch-related mutants than in
the wild type (Fig. 8C). The starch-mediated suppression of RCB production in starch-related mutants was
also found in the long-day growth condition used
here.
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Izumi et al.
Rubisco rapidly decreased (Fig. 9B). In spite of the
accelerated degradation of Rubisco and soluble protein, RCBs were not observed without concanamycin A
treatment in leaves of nitrogen-limited plants (data not
shown). Even if excised leaves were incubated with
incubation buffer containing concanamycin A, significantly fewer RCBs were noted in the leaves of nitrogenlimited plants compared with nitrogen-sufficient
plants from 1 d after the start of the treatment (Fig.
10A). Five days after the start of the treatment, few
RCBs could be noted in leaves of nitrogen-limited
plants even in the presence of concanamycin A (Fig.
10A). In nitrogen-limited plants, carbohydrates, starch,
Glc, and Fru were rapidly accumulated, and Suc was
also increased (Fig. 10B). When attached leaves of
nitrogen-limited plants were individually darkened to
impede the accumulation of carbohydrates, few RCBs
and faint GFP signals were observed in the vacuole,
similar to individually darkened leaves of nitrogensufficient plants (data not shown). This result supported
our conclusion that RCB production is primarily mediated by carbohydrate accumulation rather than by
nitrogen limitation in individual leaves.
DISCUSSION
Figure 5. The leaf carbohydrate contents in starch-related mutants
under continuous illumination. A, Photograph of wild-type, starchless
mutant (pgm-1, adg1-1), and starch-excess mutant (sex1-1, mex1-3)
plants expressing stroma-targeted GFP grown for 26 d after sowing (5 d
after bolting) in continuous illumination (60 mmol quanta m22 s21). B,
The leaf carbohydrate contents in the wild type and starch-related
mutants. The contents of starch (left) and Suc, Glc, and Fru (right) were
determined in leaves of stroma-targeted GFP transgenic plants of
background wild type, pgm-1, adg1-1, sex1-1, or mex1-3 at 26 d after
sowing. Data represent means 6 SE (n = 3). Statistical analysis was
performed by Tukey’s test. Columns with the same letter were not
significantly different (P # 0.05).
Recent works have demonstrated the importance of
autophagy in nutrient recycling during both nitrogen
and carbon starvation in plants (Doelling et al., 2002;
Hanaoka et al., 2002; Yoshimoto et al., 2004; Thompson
et al., 2005; Xiong et al., 2005; Phillips et al., 2008).
However, it has not been revealed how autophagy of
chloroplast proteins, which are a major source of
carbon and nitrogen for new growth, is regulated by
nutrient status. In this study, we examined the appearance of RCBs, autophagic bodies containing chloroplast proteins, under various conditions that affect
the nutrient status of leaves. Our data indicate that the
autophagy of chloroplasts via RCBs is closely and
specifically linked to leaf carbon but not to nitrogen
status in individual leaves.
Physiological Roles of RCB and Autophagy in Leaves
RCB Production Is Attenuated during
Nitrogen-Limited Senescence
Carbon or nitrogen limitation accelerates leaf senescence. We previously reported that RCBs can be observed in the vacuole without inhibitor treatment in
individually darkened leaves, where senescence was
induced by carbon limitation (Wada et al., 2009). Here,
we examined the appearance of RCBs during leaf
senescence accelerated by nitrogen limitation at the
whole plant level.
Nitrogen-limited plants were smaller than nitrogensufficient plants at 5 d after the imposition of treatments (Fig. 9A). In nitrogen-limited plant leaves, the
contents of chlorophyll, nitrogen, soluble protein, and
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The data presented here suggest one possible role of
RCBs, which is energy supply under temporal carbon
starvation. In the light, chloroplasts provide energy for
cellular processes and growth via photosynthesis. In
darkness or insufficient light conditions, which cause
energy limitation, photosynthetic carbon fixation is
reduced and the proteins may be used as an energy
source via autophagic recycling. In unicellular organisms, degradation of unnecessary cellular components is known to be an important role of autophagy.
For example, when methylotrophic yeasts such as
Pichia pastoris and Hansenula polymorpha are grown in
the presence of methanol as their sole carbon source,
they increased their peroxisome numbers, as this
organelle is essential for metabolizing methanol.
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Nutrient Response of Autophagy in Leaves
Figure 6. Effects of mutations affecting leaf starch content on the appearance of RCBs. A to E, Visualization of RCBs in leaves of
starch-related mutants. Transgenic plants expressing stroma-targeted GFP of background wild type (A), pgm-1 (B), adg1-1 (C),
sex1-1 (D), or mex1-3 (E) were incubated at 23°C for 20 h in 10 mM MES-NaOH (pH 5.5) with 1 mM concanamycin A and 100 mM
E-64d in darkness and observed with LSCM. GFP appears green and chlorophyll fluorescence appears red. Merged images are
shown. Bars = 50 mm. F, The appearance of RCBs in leaves of starch-related mutants. Leaves excised from five independent plants
of the wild type and each mutant were incubated, eight quadrangular regions (188 mm 3 188 mm each) per leaf were monitored
by LSCM, and the number of accumulated RCBs was counted. Data represent means 6 SE (n = 40). Statistical analysis was
performed by Tukey’s test. Columns with the same letter were not significantly different (P # 0.05).
When they are transferred to a Glc- or ethanol-rich
environment, the excess peroxisomes are selectively
degraded via autophagy for adaptation to the environmental change (Dunn et al., 2005; Sakai et al., 2006).
This mechanism is called pexophagy. RCBs may be a
similarly selective autophagy system for the degradation of the chloroplast component. In pexophagy
systems, whole peroxisomes are transported to the
vacuole. Chloroplasts are usually essential for carbon
assimilation in plants, while many peroxisomes are
not required for growth on most carbon sources other
than methanol in yeast. Whole chloroplast degradation would impair the resumption of photosynthesis
when light conditions improve. Partial degradation
via RCBs is possibly effective for maintaining some
basal functions of chloroplasts with a supply of
needed energy. Therefore, one possible role of RCBs
is supplying stromal proteins and chloroplast envelope as a carbon source under carbon-limited conditions without the loss of whole chloroplasts. In fact,
free amino acid levels increase during extended darkness in Arabidopsis leaves, possibly by protein degradation (Usadel et al., 2008). Several Arabidopsis atg
mutants exhibit accelerated leaf senescence and could
not survive under severe carbon limitation with exPlant Physiol. Vol. 154, 2010
tended darkness (Doelling et al., 2002; Hanaoka et al.,
2002; Thompson et al., 2005; Xiong et al., 2005; Phillips
et al., 2008). RCBs may contribute to adaptation and
survival under extended darkness by supplying respiratory carbon.
In plants, carbon starvation by darkness accelerates
leaf senescence in some cases. Weaver and Amasino
(2001) provided an experimental model of leaf senescence in Arabidopsis; senescence was induced when
individual leaves were darkened but not when whole
plants were darkened. Dark-induced senescence was
highly localized (i.e. senescence was locally accelerated in darkened but not in illuminated areas in the
same leaf). Our recent work demonstrates that chloroplast autophagy occurs in the senescence of individually darkened leaves (Wada et al., 2009). Together
with this report, our data here strongly indicate that
RCBs also have an important role in chloroplast degradation during accelerated senescence by darkness in
Arabidopsis. During senescence of individually darkened leaves, transportation of whole chloroplasts by
ATG4 gene-dependent autophagy was also observed
(Wada et al., 2009). This implied that maintenance of
basal chloroplast functions was no longer necessary in
darkened sectors, and it may be more advantageous
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Izumi et al.
The progression of leaf senescence is regulated by
several interacting environmental signals, with nutrient status being one of the most important (Lim et al.,
2003; Yoshida, 2003). Sugar is considered as an important internal signal; it has been indicated that
leaf senescence is induced not only by sugar starvation caused by darkness but also by sugar accumulation (Weaver and Amasino, 2001; Pourtau et al., 2006;
Wingler et al., 2006, 2009; van Doorn, 2008). Leaf carbon limitation can be an important factor controlling
senescence in natural conditions. For example, in a
plant canopy, upper leaves can shade older leaves,
which can be a factor accelerating loss of nitrogen and
shortening a leaf life span in shaded leaves (Oikawa
et al., 2006). Our study suggests that RCBs can function
during senescence under natural conditions depending on leaf carbon status, particularly promoted by
carbon limitation with insufficient light.
RCB Production and Carbohydrate Metabolism in
Starch-Related Mutants
Figure 7. Effects of mutations affecting leaf starch content on the
appearance of RCBs throughout leaf life span under a 14-h photoperiod. A, Changes of the appearance of RCBs in leaves of starch-related
mutants under a 14-h photoperiod. Leaves of stroma-targeted GFP
transgenic plants of background wild type (black squares), pgm-1 (black
circles), adg1-1 (white circles), sex1-1 (black triangles), or mex1-3
(white triangles) were excised 5 d before bolting and 0, 5, and 10 d after
bolting. Leaves were incubated at 23°C for 20 h in 10 mM MES-NaOH
(pH 5.5) with 1 mM concanamycin A and 100 mM E-64d in darkness.
Leaves from six independent plants were incubated, eight quadrangular
regions (188 mm 3 188 mm each) per leaf were monitored by LSCM,
and the number of accumulated RCBs was counted at each stage. Data
represent means 6 SE (n = 48). B, The transcription levels of SAG13,
SAG12, and RBCS2B as marker genes for leaf senescence. Total RNA
from third and fourth leaves of stroma-targeted GFP transgenic plants of
background wild type and each mutant during 10 d after bolting was
isolated and subjected to semiquantitative reverse transcription-PCR
using gene-specific primers. PCR products were separated by electrophoresis, stained with SYBR Green I, and quantified by a fluorescence
image analyzer. 18S ribosomal RNA was used as an internal control.
for plant growth that nutrients were remobilized to
organs under more direct illumination. In fact, in
individual leaves, dark-induced senescence was not
reversed by a return to light; conversely, whole darkened plants exhibited delayed senescence relative to
nontreated plants (Weaver and Amasino, 2001). Thus,
two roles of RCBs, supply of temporal energy source
under plant carbon limitation and effective degradation of chloroplasts under leaf carbon limitation, may
exist. RCBs that possibly contribute to the effective
degradation of chloroplast in darkened leaves cooperated with whole chloroplast degradation. Alternatively, because autophagic machinery may not have
sufficient size capacity for the transportation of normal
chloroplasts, shrinkage of chloroplast size by RCBs
may be a prerequisite for the degradation of whole
chloroplasts.
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The appearance of RCBs tended to be higher in
leaves of starchless mutants and lower in starch-excess
mutants compared with the wild type in both continuous light and day/night conditions (Figs. 5–8). These
results suggest that starch accumulation directly influences RCB production. Leaf starch content also
seemed to be a key factor of RCB production over a
diurnal cycle (Fig. 4). Accumulation of starch granules
may alter the tension of the chloroplast envelope. Two
mechanosensory proteins, MSL2 and MSL3, which
have been identified in the plastid envelope, control
plastid shape and size (Haswell and Meyerowitz,
2006). Envelope tension seems to be a signal that
regulates RCB production. However, the appearance
of RCBs was also suppressed by light during leaf
incubation in starchless mutants similar to wild-type
plants (data not shown). Additionally, in nitrogenlimited plants, the suppression of RCB appearance
could be observed from 1 d after treatment, while
starch accumulation was detected later (Fig. 10). Glc
and Fru accumulated from 1 d after the start of the
treatment (Fig. 10). Thus, we cannot attribute the
suppression of RCB production simply as results of
leaf starch accumulation. Rather, we can understand
the changes of RCB production in starch-related mutants by referring to a diurnal metabolism of leaf
carbohydrates. Carbon is assimilated during the day,
with a part of the photoassimilate retained as starch
in the chloroplast; however, at night, this starch is
degraded to support leaf respiration, allowing diurnal
maintenance of Suc levels in Arabidopsis leaves
(Gibon et al., 2004; Smith and Stitt, 2007). In starchless
mutants, soluble sugars accumulated to higher levels
than in the wild type during the day, but they rapidly
decreased and were almost completely consumed by
the middle of the night (Caspar et al., 1985; Gibon
et al., 2004). Similarly, during incubation of excised
leaves in darkness, reduced starch levels appeared to
Plant Physiol. Vol. 154, 2010
Nutrient Response of Autophagy in Leaves
Figure 8. The leaf carbohydrate contents in starch-related mutants under a 14-h photoperiod. A, The maximum number of
accumulated RCBs in incubated leaves throughout leaf life span. The maximum number of accumulated RCBs in leaves of
stroma-targeted GFP transgenic plants of background wild type, pgm-1, adg1-1, sex1-1, or mex1-3 was excerpted from Figure
7A. B and C, The leaf carbohydrate contents when the appearance of RCBs was maximum. The contents of starch (B) and Suc,
Glc, and Fru (C) were determined in fourth leaves at 4 to 6 h into the photoperiod at 5 d after bolting in wild-type and mex1-3
plants or at bolting in pgm-1, adg1-1, and sex1-1, when the appearance of RCBs after leaf incubation was maximum in Figure 7A.
Data represent means 6 SE (n = 48 [RCB number] or 4 [carbohydrate contents]). Statistical analysis was performed by Tukey’s test.
Columns with the same letter were not significantly different (P # 0.05).
allow more rapid consumption of sugars in starchless
mutants, possibly resulting in increased production of
RCBs (Figs. 5–8). During a diurnal cycle in starch- and
maltose-excess mutants, soluble sugar levels were
similar or slightly lower than wild-type levels during
the night, although the supply of soluble sugars was
partially defective by lower starch degradation capacity (Caspar et al., 1991; Trethewey and ap Rees, 1994;
Zeeman and ap Rees, 1999; Niittylä et al., 2004). During the extended night, there was a more than 30%
decrease in starch content in sex1 mutant leaves, while
most of the starch was already consumed at the start
of the extended night in the wild type (Caspar et al.,
1991). During leaf incubation in darkness, soluble
sugars may have been supplied for a longer period
by degradation of starch in starch-excess mutants than
in wild-type plants, leading to lower production of
RCBs (Figs. 5–8). We propose that available carbohydrate contents may be an important factor controlling
RCB production in leaves, although its sensing mechanisms are largely unknown. Starch contents are greatly
different from soluble sugar contents (Figs. 4, 5, and 8),
indicating that starch is the main available carbohydrate in darkness.
Recently, Stettler et al. (2009) reported that the accumulation of maltose and malto-oligosaccharides in mex1
mutants causes chloroplast dysfunction, which triggers
chloroplast degradation, possibly by autophagy. In our
study, the appearance of RCBs was low in leaves of
Plant Physiol. Vol. 154, 2010
mex1 plants (Figs. 6–8). However, we occasionally could
observe aggregated or round structures exhibiting chlorophyll fluorescence in dark-incubated leaves of mex1
plants expressing stroma-targeted GFP (data not
shown). Vacuole-located chloroplasts transported by
ATG4 gene-dependent autophagy also exhibited chlorophyll fluorescence without stroma-targeted fluorescent proteins (Wada et al., 2009). In mex1 plants,
only whole chloroplast autophagy, not RCBs, may be
induced. When naturally senescent leaves were
subjected to 20 h of darkness with concanamycin A,
autophagy of only RCBs, but not whole chloroplasts,
was observed (Fig. 1A; Ishida et al., 2008). Therefore,
the results in mex1 plants also suggest that the partial
mobilization of chloroplast components via RCBs and
autophagy of whole chloroplasts may be differentially
regulated depending upon the cellular status.
RCB Production Is Specifically Controlled in Autophagy
The effects of incubation conditions on the appearance of RCBs and non-RCB-type autophagic bodies
were not always the same (Fig. 3), indicating that there
exists a novel mechanism specifically controlling RCB
production in the progression of autophagy. In yeast
and animal cells, autophagy is induced by the starvation of either nitrogen or carbon sources (Takeshige
et al., 1992; Mizushima, 2005). Starvation-induced
autophagy has also been confirmed directly using a
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Izumi et al.
Figure 9. Nitrogen-limited senescence. A, Photographs of plants under nitrogen-sufficient condition as a control (top image) or
nitrogen-limited condition (bottom image) at 5 d after treatment. Stroma-targeted GFP-expressing plants were hydroponically
grown with nitrogen-rich solution. When plants started bolting (23 d after sowing), hydroponic solution was replaced with either
the same nitrogen-rich solution as the control or nitrogen-free solution, and plants were grown for 5 d. Bars = 10 mm. B, Changes
in the chlorophyll, nitrogen, soluble protein, and Rubisco protein contents in leaves of plants under nitrogen-sufficient (black
squares) and nitrogen-limited (white circles) conditions over the treatment period. Data represent means 6 SE (n = 3).
fluorescent marker, GFP-ATG8, in roots, hypocotyls,
and cultured plant cells (Yoshimoto et al., 2004;
Thompson et al., 2005; Xiong et al., 2005, Phillips
et al., 2008). In our study, we grew plants autotrophically and observed autophagy in leaves. Specific features of RCB induction may reflect differences in
heterotrophs and autotrophs and also in heterotrophic
and autotrophic tissues in plants. In nature, plants
have to get carbohydrates by photosynthesis and
assimilate inorganic nitrogen into organic compounds.
Chloroplasts have a central role in both photosynthesis
and nitrogen assimilation, and around 80% of total
leaf nitrogen is distributed to chloroplasts. Therefore,
although the underpinning mechanisms are still
Figure 10. Effects of nitrogen limitation on the appearance of RCBs and leaf carbohydrate contents. A, Effects of nitrogen
limitation on the appearance of RCBs. Fourth leaves of stroma-targeted GFP-expressing plants under nitrogen-sufficient (black
squares) and nitrogen-limited (white circles) conditions were excised over the treatment period and incubated at 23°C for 20 h in
10 mM MES-NaOH (pH 5.5) with 1 mM concanamycin A and 100 mM E-64d in darkness. Leaves from four independent plants
were incubated, eight quadrangular regions (188 mm 3 188 mm each) per leaf were monitored by LSCM, and the number of
accumulated RCBs was counted at each period. Data represent means 6 SE (n = 32). B, The leaf carbohydrate contents in
nitrogen-limited plants. Changes in the starch, Suc, Glc, and Fru contents were measured in fourth leaves of plants under
nitrogen-sufficient (black squares) and nitrogen-limited (white circles) conditions over the treatment period. Data represent
means 6 SE (n = 3).
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Nutrient Response of Autophagy in Leaves
unknown, it seems reasonable to consider that autophagy of chloroplast proteins must be specifically
controlled. In yeast, some selective autophagy pathways have been identified (e.g. cytoplasm-to-vacuole
targeting pathway, mitophagy, pexophagy, and also
specific ATG genes required for respective pathways
were identified; for review, see Kraft et al., 2009;
Nakatogawa et al., 2009). Our study is suggestive of
the existence of novel plant ATG genes specifically
required for the autophagic degradation of chloroplasts, and identification of such genes may be effective for elucidating the regulatory mechanism of RCB
production and specific features of plant autophagy.
MATERIALS AND METHODS
Plant Materials and Growth Conditions
Transgenic Arabidopsis (Arabidopsis thaliana) ecotype Columbia expressing
CT-GFP and ecotype Wassilewskija expressing both stroma-targeted DsRed
and the GFP-ATG8a fusion protein were described previously (Ishida et al.,
2008). Transgenic Arabidopsis ecotype Columbia expressing SP-GFP-2SC was
described previously (Tamura et al., 2003). The seeds of Arabidopsis mutant
allele pgm1-1 (Caspar et al., 1985), adg1-1 (Lin et al., 1988), sex1-1 (Caspar et al.,
1991), and the T-DNA insertion mutant mex1-3 (SAIL_574_D11) were obtained
from the Arabidopsis Biological Resource Center. mex1-3 was identified by
confirming homozygosity using PCR with gene-specific primers. Furthermore, the purported mex1-3 plants exhibited the same phenotype as the report
that first identified mex1 mutants (Niittylä et al., 2004). These mutants were
crossed to transgenic plants expressing stroma-targeted GFP. Homozygosity
of pgm-1, adg1-1, and sex1-1 was confirmed by sequencing regions including
G-to-A substitutions of PGM1 (Periappuram et al., 2000), ADG1 (Wang et al.,
1998), and SEX1 (Yu et al., 2001) in each mutant, respectively. Homozygosity
of mex1-3 was confirmed by PCR using gene-specific primers. Under long-day
conditions, plants were grown on soil (Metro-Mix 350; Sun Gro Horticulture)
on a 14-h-light/10-h-dark photoperiod with fluorescent lamps (100 mmol
quanta m22 s21) at 23°C day/18°C night temperatures. Under continuous light
conditions, plants were grown on the same soil with fluorescent lamps
(60 mmol quanta m22 s21) at 23°C. For nitrogen-limitation treatment, CT-GFP
plants were grown hydroponically on horticultural rockwools as described
previously (Wada et al., 2009). The hydroponic solution contained 3 mM KNO3,
2.5 mM potassium phosphate buffer (pH 5.5), 2.0 mM Ca(NO3)2, 2.0 mM MgSO4,
50 mM Fe-EDTA, 0.7 mM H3BO4, 170 mM MnCl2, 2.0 mM Na2MoO4, 100 mM NaCl,
5.0 mM CuSo4, 10 mM ZnSO4, and 0.1 mM CoCl2. When the plants started bolting,
the rockwools were washed with deionized water and the nutrient solution
was replaced with nitrogen-free solution. The nitrogen-free solution was
prepared by replacing KNO3 and Ca(NO3)2 with KCl and CaCl2, respectively.
replacing the KNO3 with KCl. When diurnal effects were examined, leaves
were excised at three time points representing the end of the regular night,
the end of the prolonged night, and the end of the day, and incubation time
was 10 h as described in Figure 4A. After incubation, each leaf was divided
into four parts, and two quadrangular regions (188 mm 3 188 mm each) in each
section were monitored by LSCM and images were obtained. The number
of accumulated RCBs in the images was counted. Each image was considered
an independent data point and subjected to statistical analysis.
The analysis of production of RCBs and autophagosomes in plants
expressing stroma-targeted DsRed and the GFP-ATG8 fusion was performed
in the same way, except that leaves from six independent plants at 25 d after
sowing (5 d after bolting) were used. The number of RCBs and non-RCB-type
autophagic bodies that do not exhibit DsRed was counted in each image.
Sugar Analysis
Leaves for carbohydrate quantification were rapidly frozen in liquid N2 and
then stored at 280°C until analysis. Frozen leaves were homogenized in a
liquid N2-chilled tube with a pestle. The resulting powder was immediately
extracted four times with 80% (v/v) ethanol at 80°C (Nakano et al., 1995), and
the extractant was pooled and concentrated by vacuum evaporation. The
residue was dissolved in distilled water, and the concentrations of Suc, Glc, and
Fru were determined using an F-kit for a-Glc/Suc/Fru (Roche Diagnostics).
The 80% ethanol-insoluble fraction was dried overnight at room temperature. Starch in the ethanol-insoluble fraction was extracted with 0.6 mL of
dimethyl sulfoxide and 0.15 mL of 8 M HCl at 60°C for 30 min, and the pH
was neutralized to between 4.0 and 5.0 with 0.1 mL of 2 M Na-acetate buffer
(pH 4.5) and 8 M NaOH. The solution was made up to its final volume of
1.0 mL with distilled water. The starch concentration was determined using an
F-kit for starch (Roche Diagnostics).
Quantification of Chlorophyll, Nitrogen, Soluble
Proteins, and Rubisco Protein
Frozen fourth rosette leaves were homogenized in an ice-chilled mortar
and pestle in 50 mM Na-phosphate buffer (pH 7.5) containing 2 mM iodoleacetic acid, 0.8% (v/v) 2-mercaptoethanol, and 5% (v/v) glycerol (Makino
et al., 1994). Chlorophyll was determined by the method of Arnon (1949).
Soluble protein content was measured in supernatant of part of the homogenate according to Bradford (1976) using a Bio-Rad protein assay with bovine
serum albumin as the standard. Total leaf nitrogen was determined from part
of the homogenate with Nessler’s reagent after Kjeldahl digestion. Triton
X-100 (0.1% final concentration) was added to the remaining homogenate.
After centrifugation, the supernatant was mixed with an equal volume of
SDS sample buffer containing of 200 mM Tris-HCl (pH 8.5), 2% (w/v) SDS,
0.7 M 2-mercaptoethanol, and 20% (v/v) glycerol, boiled for 3 min, and then
subjected to SDS-PAGE. The Rubisco content was determined spectrophotometrically by formamide extraction of the Coomasie Brilliant Blue R-250-stained
bands corresponding to the large and small subunits of Rubisco separated by
SDS-PAGE. Calibration curves were made with bovine serum albumin.
Image Analysis with LSCM
Semiquantitative Reverse Transcription-PCR Analysis
LSCM was performed with a Nikon C1si system equipped with a CFI Plan
Apo VC603 water-immersion objective (numerical aperture = 1.20; Nikon) as
described previously in detail (Ishida et al., 2008). The signals of GFP and
DsRed were obtained using the unmixing program of Nikon C1si software
from fluorescence spectra between 500 and 650 nm.
Total RNA was isolated from leaves using the RNeasy Plant Mini Kit
(Qiagen). Isolated RNA was treated with DNase (DNA-free; Ambion) prior to
the synthesis of first-strand cDNA with random hexamer primers (SuperScript
III first-strand synthesis system; Invitrogen). An aliquot of the synthesized
first-strand cDNA derived from 1.7 ng of total RNA was used for PCR
amplification (total volume of 6 mL) with Taq polymerase (PrimeSTAR;
TAKARA). The gene-specific primer pairs and the number of PCR cycles
were as follows: 5#-CAGCTTGCCCACCCATTGTTA-3# and 5#-GTCGTACGCACCGCTTCTTTCTTA-3#, 28 cycles for SAG13 (Barth et al., 2004);
5#-GATGAAGGCAGTGGCACACCAA-3# and 5#-TCCCACACAAACATACACAATTAAAAGC-3#, 24 cycles for SAG12 (Panchuk et al., 2005);
5#-ACCTTCTCCGCAACAAGTGG-3# and 5#-GAAGCTTGGTGGCTTGTAGG-3#, 18 cycles for RBCS2B (Acevedo-Hernández et al., 2005); and QuantumRNA 18S internal standard (Ambion), 14 cycles for 18S ribosomal RNA.
Detection of RCBs and Autophagic Bodies
Fourth rosette leaves were excised from four to six independent plants
expressing CT-GFP 4 to 6 h into the photoperiod. Incubation buffer containing
10 mM MES-NaOH (pH 5.5), 1 mM concanamycin A, and 100 mM E-64d was
infiltrated into excised leaves, which were then incubated for 20 h at 23°C in
darkness. When effects of nutrient factors during leaf incubation on RCB
production were examined, full-strength MS medium and various constituents were added to the incubation buffer, or light (50 mmol quanta m22 s21)
was irradiated. The concentration of Suc, Glc, Fru, and mannitol added was
3% (w/v), and DCMU was 10 mM. In incubation with nitrogen-free MS
medium (MS-N), MS-N medium was prepared by removal of NH4NO3 and
Plant Physiol. Vol. 154, 2010
Supplemental Data
The following materials are available in the online version of this article.
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Izumi et al.
Supplemental Figure S1. Effects of incubation conditions on the accumulation of vacuole-targeted GFP in vacuolar lumens.
Supplemental Figure S2. Visualization of stroma-targeted GFP in leaves of
starch-related mutants.
Supplemental Figure S3. Changes of leaf area in starch-related mutants
under a 14-h photoperiod.
ACKNOWLEDGMENTS
We thank Dr. Louis Irving for critical reading of the manuscript, Dr.
Maureen R. Hanson for the use of transgenic Arabidopsis expressing CTGFP and critical reading of the manuscript, Dr. Kohki Yoshimoto and Dr.
Yoshinori Ohsumi for the use of transgenic Arabidopsis expressing GFPATG8, Dr. Ikuko Hara-Nishimura for providing seeds of transgenic Arabidopsis expressing SP-GFP-2SC, and the Arabidopsis Biological Resource
Center and original donors for providing seeds of starch-related mutant
Arabidopsis.
Received April 27, 2010; accepted August 30, 2010; published August 31, 2010.
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