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LARGE-SCALE BIOLOGY ARTICLE
Global Analysis of the Role of Autophagy in Cellular
Metabolism and Energy Homeostasis in Arabidopsis
Seedlings under Carbon Starvation
Tamar Avin-Wittenberg,1 Krzysztof Bajdzienko, Gal Wittenberg, Saleh Alseekh, Takayuki Tohge, Ralph Bock,
Patrick Giavalisco, and Alisdair R. Fernie
Max-Planck-Institut für Molekulare Pflanzenphysiologie, 14476 Potsdam-Golm, Germany
Germination and early seedling establishment are developmental stages in which plants face limited nutrient supply as their
photosynthesis mechanism is not yet active. For this reason, the plant must mobilize the nutrient reserves provided by the
mother plant in order to facilitate growth. Autophagy is a catabolic process enabling the bulk degradation of cellular
constituents in the vacuole. The autophagy mechanism is conserved among eukaryotes, and homologs of many autophagyrelated (ATG) genes have been found in Arabidopsis thaliana. T-DNA insertion mutants (atg mutants) of these genes display
higher sensitivity to various stresses, particularly nutrient starvation. However, the direct impact of autophagy on cellular
metabolism has not been well studied. In this work, we used etiolated Arabidopsis seedlings as a model system for carbon
starvation. atg mutant seedlings display delayed growth in response to carbon starvation compared with wild-type seedlings.
High-throughput metabolomic, lipidomic, and proteomic analyses were performed, as well as extensive flux analyses, in
order to decipher the underlying causes of the phenotype. Significant differences between atg mutants and wild-type plants
have been demonstrated, suggesting global effects of autophagy on central metabolism during carbon starvation as well as
severe energy deprivation, resulting in a morphological phenotype.
INTRODUCTION
Macroautophagy (referred to hereafter as “autophagy”) is a conserved
eukaryotic mechanism, which is classically defined as the degradation of cytoplasmic constituents in the lytic organelle (vacuoles in
yeast and plants and lysosomes in mammals) (Reggiori and Klionsky,
2013). The general targets of autophagy vary from long-lived proteins
to protein complexes and even entire organelles (Reumann et al.,
2010). Morphologically, autophagy begins with the formation of cupshaped double membranes, which expand to form autophagosomes
engulfing malfunctioning or unneeded macromolecules and organelles and transport them for degradation inside the vacuole.
Upon arrival of the autophagosomes to the vacuoles, their outer
membranes fuse with the tonoplast, creating single-membrane
vesicles inside the vacuole, termed “autophagic bodies.” The
autophagic bodies and their contents are then degraded inside
the vacuole, providing recycled materials to build new macromolecules (Bassham, 2009). Nutrient starvation has been one of
the hallmark inducers of autophagy in yeast, mammals, and
plants (Reumann et al., 2010; Singh and Cuervo, 2011; Li and
Vierstra, 2012). The autophagy mechanism in presumed to play
a role in recycling during starvation, thus supplying the cell with
1 Address
correspondence to [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.plantcell.org) is: Tamar Avin-Wittenberg
([email protected]).
www.plantcell.org/cgi/doi/10.1105/tpc.114.134205
nutrients during the stress period (Li and Vierstra, 2012). It has
been shown to be involved in protein and lipid degradation
(Onodera and Ohsumi, 2005; Singh and Cuervo, 2011), as well as
to be associated with several metabolic disorders in animals
(Singh and Cuervo, 2011).
The genes participating in the autophagic process (termed ATG
genes) were originally discovered in yeast (Saccharomyces cerevisiae)
via the isolation of autophagy-defective mutants whose cells show
little or no accumulation of autophagic bodies during nutrient starvation (Liu and Bassham, 2012). Many ATG genes are conserved in
evolution, and homologs to the yeast genes have been found in many
organisms including mammals and plants (Avin-Wittenberg et al.,
2012; Ryter et al., 2013). Several atg mutants have been characterized
in the model plant Arabidopsis thaliana (Doelling et al., 2002; Hanaoka
et al., 2002; Yoshimoto et al., 2004; Xiong et al., 2005; Harrison-Lowe
and Olsen, 2008; Phillips et al., 2008). Among the common phenotypes of these mutants are early senescence in comparison to
wild-type plants as well as hypersensitivity to carbon and nitrogen
starvation (Bassham, 2009). Interestingly, the early senescence
phenotype of atg mutants was shown to be the consequence of
salicylic acid (SA) accumulation, apparently regulated by autophagy
(Yoshimoto et al., 2009). Autophagy in plants has also been shown
to function during abiotic stresses such as salt, drought, and
oxidative stress (Xiong et al., 2007; Liu et al., 2009) as well as in
response to pathogens (Liu and Bassham, 2012). In addition,
selective autophagic degradation of chloroplasts has been
demonstrated (Ishida et al., 2014), and evidence of peroxisome
and starch degradation by autophagy has been recently provided (Farmer et al., 2013; Kim et al., 2013; Wang et al., 2013;
The Plant Cell Preview, www.aspb.org ã 2015 American Society of Plant Biologists. All rights reserved.
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The Plant Cell
Avin-Wittenberg and Fernie, 2014). Although hypersensitivity to
nutrient starvation is a major phenotype of atg mutants, only
very few recent studies have attempted to uncover the metabolic implications underlying it (Guiboileau et al., 2012; Izumi
et al., 2013; Masclaux-Daubresse et al., 2014), and although
we are making great strides in our understanding of the metabolic impact of autophagy in plants, many questions still remain open.
Seed germination and seedling establishment are two developmental periods in which there is no carbon assimilation.
Figure 1. Carbon-Starved Etiolated atg Mutant Seedlings Display Shorter Hypocotyls in Comparison to Wild-Type Plants.
(A) atg mutant seeds and their respective wild-type controls (Col for atg5-1, atg5-3, and atg7-2; Ws for atg4a4b) were sown on 0.53 MS plates without
sucrose and grown in the dark for 7 d after imbibition. Representative pictures of the etiolated seedling are shown as well as measurement of hypocotyl
length performed using ImageJ (n = 15). Data are presented with SE; significant difference compared with the wild type (P < 0.05 in Student’s t test) is
denoted by an asterisk.
(B) atg mutant seeds and their respective wild-type controls (Col for atg5-1, atg5-3, and atg7-2; Ws for atg4a4b) were sown on 0.53 MS plates
containing 1% sucrose and grown in the dark for 7 d after imbibition. Representative pictures of the etiolated seedling are shown as well as measurement of hypocotyl length performed using ImageJ (n = 15). Data are presented with SE.
(C) Wild-type, NahG, and atg5.NahG seeds were sown on 0.53 MS plates without sucrose and grown in the dark for 7 d after imbibition. Representative
pictures of the etiolated seedling are shown as well as measurement of hypocotyl length performed using ImageJ (n = 15). Data are presented with SE;
significant difference compared with the wild type (P < 0.05 in Student’s t test) is denoted by an asterisk.
(D) atg mutant and wild-type control seeds were sown on 0.53 MS plates without sucrose, imbibed for 48 h, and transferred to continuous light
conditions. The number of germinated seedlings (defined by radical protrusion) was scored each day for 4 d, and average percent germination (n = 6) is
presented with SE. Detailed results of the assay for the depicted lines as well as additional lines are presented in Supplemental Table 1.
(E) atg mutant and wild-type control seeds were sown on 0.53 MS plates without sucrose, imbibed for 48 h, and transferred to continuous light conditions.
The number of germinated seedlings (defined by the appearance of two green cotyledons) was scored each day for 4 d, and average percent germination
(n = 6) is presented with SE. Detailed results of the assay for the depicted lines as well as additional lines are presented in Supplemental Table 1.
Autophagy in Etiolated Arabidopsis
Seeds possess storage tissues, containing reserves necessary
for the establishment of the seedling (Arc et al., 2011). Seed
germination is associated with the degradation and mobilization of these storage reserves (Fait et al., 2006). This process is
crucial in providing fuel for growth until the growing seedling
becomes photoautotrophic (Pritchard et al., 2002). Since autophagy functions as a degradation pathway facilitating nutrient mobilization, it is safe to assume it serves a function during
seedling establishment. Indeed, atg mutants have been shown
to display impaired seedling establishment (Yoshimoto et al.,
2014). However, the reasons for this phenotype, and specifically the impact on energy metabolism, have not been well
studied.
In this study, we investigated etiolated Arabidopsis seedlings
as a model for carbon starvation during seedling establishment.
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We demonstrate that under our experimental conditions, atg
mutants display delayed growth in comparison to wild-type
seedlings. Metabolic profiling revealed reduced levels of free
amino acids in the mutants, while proteomic analysis displayed
accumulation of proteins in the mutants. Metabolic flux analysis
also suggests increased respiration in atg mutants in addition to
decreased net protein biosynthesis compared with the wild
type. Examination of the lipid composition of the mutants revealed altered lipid levels, strengthening our hypothesis of
altered metabolism as well as suggesting the occurrence of
starvation stress responses in the mutants. We suggest that
only a comprehensive large-scale analysis including both steady
state metabolite levels as well as metabolic flux will enable us to
decipher such compound phenotypes as that of autophagy
deficiency.
Figure 2. atg Mutants Exhibit a Reduction in Amino Acid Levels under Carbon Starvation.
atg mutants as well as their respective wild-type and SA controls (NahG and atg5.NahG) were sown on 0.53 MS plates and grown in the dark for 6 d
after imbibition. Metabolic content was analyzed using GC-MS (n = 4 to 8). Detailed results of the assay for the depicted lines as well as additional lines
are presented in Supplemental Table 2.
(A) PCA of primary metabolite levels.
(B) Log2 values of the relative metabolic content are presented as a heat map. Significant differences compared with the wild type following Student’s
t test are denoted by one asterisk (P < 0.05) or two asterisks (P < 0.01).
(C) Relative levels of BCAA compared with the wild type. Data are presented with SE; significant differences following Student’s t test are denoted by an
asterisk (P < 0.05).
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The Plant Cell
Figure 3. Most of the Differences in Secondary Metabolism of atg Mutants Compared with the Wild Type Are SA Dependent.
atg mutants as well as their respective wild-type and SA controls (NahG and atg5.NahG) were sown on 0.53 MS plates and grown in the dark for 6 d
after imbibition. Secondary metabolite content was analyzed by LC-MS (n = 5 to 6). Detailed results of the assay for the depicted lines as well as
additional lines are presented in Supplemental Table 3.
(A) PCA of secondary metabolite levels.
(B) Log2 values of the relative metabolic content are presented as a heat map (n = 5 to 6). Significant differences compared with the wild type following
Student’s t test are denoted by one asterisk (P < 0.05) or two asterisks (P < 0.01).
RESULTS
Arabidopsis Etiolated atg Mutant Seedlings Display
a Delayed Growth Phenotype
We have chosen to use etiolated Arabidopsis seedlings as a model
system for carbon starvation since the only carbon source available
to these seedlings is that coming from the seed, rendering the
system heterotrophic. Additionally, no photosynthesis has occurred, thus eliminating the possibility of differential carbon accumulation in the atg mutants compared with the wild type. We began
our investigation by assessing the morphological phenotype of atg
mutants germinated under carbon starvation conditions in comparison to wild-type seedlings. Seed were sown on 0.53 Murashige
and Skoog (MS) plates (in the absence of a carbon source) and
grown in the dark for 7 d after imbibition. All atg mutants displayed
visibly shorter hypocotyls than their respective wild-type controls
(Columbia [Col] for atg5-1, atg5-3, and atg7-2 and Wassilewskija
[Ws] for atg4a4b). This significant difference was also evident when
hypocotyl length measurements were conducted (Figure 1A). We
were able to recover the phenotypes by supplementing the medium
with an external carbon source. The size of atg mutants grown
on 0.53 MS medium plates containing 1% sucrose was not
different from that of their respective wild-type controls (Figure
1B). In order to verify that the autophagy mechanism is indeed
functioning under our experimental conditions, we used Arabidopsis plants expressing the fusion protein GFP-ATG8f-HA
(containing ATG8f fused to green fluorescent protein and the
human influenza hemagglutinin [HA] tag) in the wild-type background to determine autophagic flux as described previously
(Sláviková et al., 2005). During autophagosome elongation, the
C terminus of ATG8 is cleaved by ATG4, and the protein is
subsequently conjugated to the lipid phosphatidylethanolamine
(PE) and inserted into the growing autophagosome membrane.
Upon fusion of the autophagosome with the vacuole, ATG8
decorating the inner membrane of the autophagosome is degraded by vacuolar proteases (Li and Vierstra, 2012). By using
a construct in which ATG8 is tagged from both termini, it is
possible to follow the rate of ATG8 cleavage by ATG4, suggesting autophagosome elongation, and the rate of ATG8
Autophagy in Etiolated Arabidopsis
degradation, suggesting autophagosome fusion with the vacuole. The ratio between these two steps is used as a measure of
autophagic flux. GFP-ATG8f-HA seeds were grown in the dark
for 6 d after imbibition on 0.53 MS plates with or without 1%
sucrose; protein extracts from the seedlings were then run on
SDS-PAGE gel, and an immunoblot assay was performed using
an antibody raised against GFP (Supplemental Figure 1). The
samples taken from seedlings grown on sucrose contained three
protein bands, corresponding to the full-length fusion protein as
well as its degradation products ATG8f-GFP and free GFP.
However, the samples taken from seedlings grown without sucrose contained only the free GFP band, suggesting increased
degradation of the GFP-ATG8f-HA protein and, thus, an increase in autophagic flux under our experimental conditions.
This observation is in agreement with a previous study showing
similar results (Sláviková et al., 2005).
It has been previously demonstrated that atg mutants accumulate SA as they age, causing an early senescence phenotype
(Yoshimoto et al., 2009). This phenotype could be recovered by
crossing atg mutant plants with plants overexpressing the
bacterial protein NahG, encoding a SA hydroxylase, thus producing stay-green atg mutants. In order to verify that the short
hypocotyl phenotype was not the result of early senescence
during seed development or of SA accumulation in the seed
tissue, we examined the hypocotyl length of 7-d-old atg5.NahG
seedlings grown on plates without sucrose. The atg5.NahG
seedlings displayed significantly shorter hypocotyls than wildtype plants, demonstrating that decreasing the SA levels in the
plants was not able to recover the morphological phenotype
(Figure 1C).
We next wished to ascertain whether the difference in size
stems from delayed germination or reduced growth following
germination. For this purpose, we compared the germination
of wild-type and atg mutants, as well as the SA control lines
(atg5.NahG seedlings; Figures 1D and 1E; Supplemental Table 1).
Two parameters were scored: radical protrusion, indicating germination; and appearance of two green cotyledons, indicative of early
seedling development (Gao et al., 2011). A delay in both parameters
was observed for the atg mutant lines in comparison to the wild type,
though eventually all lines reached the same germination percentage
(Figures 1D and 1E). This behavior was also observed for the SA
control lines (Supplemental Table 1). These results may indicate that
the difference in plants size stems, at least in part, from delayed
growth.
Differential Metabolic Response of atg Mutants under
Carbon Starvation
Autophagy has been linked to nutrient recycling under starvation
conditions (Singh and Cuervo, 2011; Guiboileau et al., 2013; Izumi
et al., 2013; Reggiori and Klionsky, 2013; Ryter et al., 2013). We
therefore set out to investigate the metabolic content of etiolated
atg mutants under carbon starvation compared with wild-type
plants. We were able to identify 30 primary metabolites using gas
chromatography-mass spectrometry (GC-MS) analysis (Supplemental
Table 2). Principal component analysis (PCA) demonstrated a good
separation between the wild-type and atg mutant lines. NahG and
atg5.NahG clustered with the wild-type line and atg mutant lines,
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respectively. A significant decrease in free amino acid levels was
observed in all atg mutants compared with the wild type (Figure 2B).
This decrease was not recovered in the SA control lines, suggesting
that the phenotype is likely to be SA independent (Figure 2B, NahG
and atg5.NahG). Of special interest were the reduced levels of lysine
and the branched-chain amino acids (BCAAs) isoleucine and valine
in atg mutants (Figure 2C). These amino acids have been shown to
function as electron donors for the tricarboxylic acid (TCA) cycle and
mitochondria electron transport chain under carbon starvation
(Araújo et al., 2010), and their decreased steady state levels in the
Figure 4. Differential Protein Accumulation in atg Mutants Compared
with the Wild Type under Carbon Starvation.
atg mutants as well as wild-type and SA controls (NahG and atg5.NahG)
were sown on 0.53 MS plates and grown in the dark for 6 d after
imbibition.
(A) Total protein amount was analyzed using BCA protein assay (n = 6).
Significant differences compared with the wild type following Student’s t
test are denoted by one asterisk (P < 0.05) or two asterisks (P < 0.01).
(B) Separation of proteins on SDS-PAGE gel. Bands displaying higher
intensity in atg mutants compared with the wild type and NahG (marked
by arrowheads) were cut from the gel for mass spectrometry identification. Mass spectrometry results are detailed in Table 1.
6 of 17
The Plant Cell
mutants likely suggest altered mitochondrial function under carbon
starvation. Additionally, reduced steady state levels of the organic
acids malate, fumarate, and dehydroascorbate were observed
(Figure 2B).
We also identified secondary metabolites using liquid
chromatography-mass spectrometry (LC-MS) analysis (Figure 3;
Supplemental Table 3). Surprisingly, while PCA demonstrated
a good separation between the wild-type and atg mutant lines,
a cross between atg5 and NahG abolished this separation (atg5.
NahG, Figure 3A). This was also evident in many of the metabolites
exhibiting a significant difference in at least one of the atg mutant
lines compared with the wild type, where the difference did not
exist in the atg5.NahG line (Figure 3B). This observation implies
a putatively considerable role for SA in determining the secondary
metabolic composition of the atg mutants. However, several secondary metabolites were, by contrast, less abundant in atg
mutants compared with the wild type in a presumably SAindependent manner (Figure 3B). However, several of these
are currently unknown with respect to their chemical identity.
Nevertheless, they may serve as interesting subjects for future research.
protein degradation. The atg5.NahG plants displayed the same
accumulation (Figure 4A); however, surprisingly, atg7-2 seedlings
did not accumulate higher protein content. This may point to
a milder phenotype and misaccumulation of fewer proteins. Indeed,
when equal amounts of protein were separated by SDS-PAGE,
several protein bands displayed stronger intensity in all atg mutants
compared with the wild type, suggesting the differential accumulation and degradation of specific proteins (Figure 4B;
Supplemental Figure 2). It is important to note that the same
protein accumulation was observed in atg5.NahG but not in the
NahG samples (Figure 4B). The bands showing a difference in
accumulation (Figure 4B, marked by arrows; Supplemental Figure
2) were excised and subjected to in-gel digestion with trypsin, and
the resulting peptides were identified using liquid chromatography
coupled to tandem mass spectrometry (Table 1). We were able to
identify an accumulation of cruciferin 3 in atg mutants compared
with the wild type. Cruciferin is the most abundant storage protein
in Arabidopsis seeds (Wan et al., 2007); thus, its accumulation in
atg mutants points to a possible impairment in protein degradation,
complying with the known function of the autophagy mechanism
(Li and Vierstra, 2012). Two other interesting proteins accumulating
in atg mutants compared with the wild type were malate synthase
(MLS) and multifunctional protein 2 (MFP2). Both proteins have
been shown to be involved in b-oxidation of fatty acids and
seedling establishment (Rylott et al., 2006; Kim et al., 2013). Additionally, MLS has been previously shown to accumulate in atg
mutant seedlings, further validating our proteomic analysis (Kim
et al., 2013). Surprisingly, we identified one protein overaccumulating in wild-type seedlings rather than atg mutants. Cytosolic triosephosphate isomerase is an enzyme involved in
glycolysis (Giegé et al., 2003), suggesting a possible upregulation of this pathway in wild-type seedlings compared with atg
mutants.
Selective Protein Accumulation Occurs in Etiolated atg
Mutant Seedlings under Carbon Starvation
Following our observation of reduction of the steady state level
of many free amino acids in atg mutants and given that proteins are
degradation targets of the autophagy machinery (Li and Vierstra,
2012), we next decided to examine the protein content of carbonstarved atg mutants compared with the wild type. We initially
measured the total protein content of carbon starved atg mutants.
Interestingly, both atg5 mutant lines presented with higher protein
content than wild-type plants, suggesting a possible impairment in
Table 1. Identification of Protein Bands from Figure 6B
Band (kD) AGI
12
20
30
50
60
80
120
Description
At1g14950 Lipid transport superfamily
protein
At3g55440 TPI; triosephosphate isomerase
At4g28520 CRU3; cruciferin 3
At3g03250 UGP1; UDP-glucose
pyrophosphorylase 1
At5g03860 MLS; malate synthase
At1g21750 PDI5
At3g06860 MFP2; multifunctional protein 2
At5g17920 ATCIMS; Cobalamin-independent
synthase family protein
At1g72150 PATELLIN1
MW
Wild Type
atg5-1
1
1
MS
2
P MS
17,881 nd
27,152 88
58,199 nd
51,706 83
63,846
55,567
78,790
84,304
nd
2
2
142 3
76 3
nd
25 1
64,007 nd
P MS
68
nd
95
2
2
atg5-3
2
P
MS
atg7-2
1
P
MS
2
P
MS
1
P
MS
2
P
MS
P
74
2 25
1 82
3 124
2 101
3 74
2
nd
63
134
nd
1 93
4 79
nd
1 51
4 129
nd
1 58
4 102
nd
1 74
5 58
nd
1 82
3 140
1
3
113 4
nd
nd
nd
446 12 445 10 456 10 450 11 430 10 418 13
176 5 151 4 342 8 292 6 384 7 399 10
209 5 250 8 66
4 46
2 106 5 144 6
57
3 173 5 101 4 130 4 67
3 71
2
nd
236
4 nd
143
5 192
7 118
4 nd
Mass spectra obtained from peptides from in-gel digestion of protein bands marked in Figure 6B were matched using Mascot and a database of
Arabidopsis proteins. Individual ions scores >22 indicate identity or extensive homology (P < 0.05). Data are presented for two biological replicates
(denoted as 1 and 2 under the line name). Band size is the size of the band corresponding to the bands marked in Figure 6B. The description is the
annotation of the matched protein. AGI is the genome accession number. MW, calculated mass of the matched protein; MS, mascot protein score; P,
number of matching peptides; nd, not detected.
Autophagy in Etiolated Arabidopsis
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Etiolated atg Mutants Display Increased Respiration in
Response to Carbon Starvation
Given that the morphological and metabolic phenotype of atg
mutants observed thus far suggests an energy deprivation phenotype, we were interested in directly checking the energy status of
the plants. As the seedlings are etiolated, photosynthesis does not
play a role in energy generation, and we therefore directly examined
the respiration parameters of carbon starved atg mutants. We
evaluated 14CO2 evolution using positionally labeled 14C-Glc in
order to assess flux through the major pathways of carbohydrate
oxidation (Nunes-Nesi et al., 2005). Wild-type and atg5-1 seedlings
were grown in the dark without sucrose for 7 d and then supplied
with [1-14C], [3:4-14C], or [6-14C]Glc (1 mM with specific activity of
1.85 MBq/mmol) for a duration of 2 h. During this time, we collected
the 14CO2 evolved at 40-min intervals. A relatively short time frame
and low glucose concentration were chosen in order not to disturb
the starvation status of the plants, thereby avoiding recording the
differences in recovery from starvation between the mutant and the
wild type. CO2 can be released from the C1 position by enzymes
that are not associated with mitochondrial respiration, unlike CO2
evolution from the C3:4 positions of Glc (Rees and Beevers, 1960).
Thus, the ratio of CO2 evolution from C1 to C3:4 positions of Glc
provides an indication of the relative rate of the TCA cycle with
respect to other processes of carbohydrate oxidation. Though the
level of C1 emission was higher than the level of C3:4 emission in
both lines (Figures 5A and 5B), the level of CO2 emission from C1
was comparable between atg5-1 and the wild type, while the level
of C3:4 emission was slightly higher in atg5-1 than in the wild type.
Thus, when considering the C1/C3:4 CO2 emission ratio, the ratio
for the wild type is higher than that of atg5-1 (1.85 and 1.66 after
120 min, respectively), suggesting a higher proportion of carbohydrate oxidation performed by the TCA cycle in atg5-1. CO2
emission from the C6 position was relatively similar between both
lines, suggesting that pentane synthesis was unaltered (Figure 5C).
Since some amino acids, such as BCAA and lysine, may be
used as substrates for respiration (Araújo et al., 2011), and since we
have observed a decrease in the steady state levels of these amino
acids in atg mutants compared with the wild type (Figure 2C), we
set out to investigate lysine metabolism in atg mutants. Wild-type
and atg5-1 seedlings were grown in the dark without sucrose for
7 d and then supplied with [U-13C]Lys for a duration of 2 h. Seedling
samples were collected every 40 min, washed, and immediately
frozen. 13C label distribution was calculated as described in
Methods in order to determine metabolic flux through several
metabolites (summarized in Table 2). Increased label distribution in
atg5-1 seedlings was demonstrated for malate, a TCA cycle intermediate, further strengthening our observation from CO2 evolution. In addition, atg5-1 showed increased label distribution in
glutamate, alanine, and aspartate compared with the wild type,
suggesting increased metabolic flux in the mutants.
Decreased Flux to Net Protein Synthesis in Carbon-Starved
atg Mutants
As the seedlings were grown under carbon starvation condition,
we hypothesized these conditions might have an effect on other
major pathways of carbon metabolism. We thus performed [U-14C]
Glc labeling of wild-type and atg5-1 seedlings. The samples were
Figure 5.
14CO
2
Evolution of Etiolated Seedlings in the Dark.
Wild-type and atg5-1 etiolated seedlings were grown in the dark for 7 d
and incubated in 10 mM MES-KOH, pH 6.5, supplemented with [1-14C]-,
[3:4-14C]-, or [6-14C]Glc ([A] to [C], respectively). The 14CO2 liberated
was captured (every 40 min) in a KOH trap, and the amount of radiolabel
released was subsequently quantified by liquid scintillation counting.
Values are presented as average 6 SE of determinations of four independent samples per line.
incubated in the presence of 1 mM [U-14C]Glc for 80 min, extracted, and fractioned into organic acids, amino acids, starch,
protein, cell wall, phosphoesters, and sucrose in order to analyze
flux alterations between wild-type and atg5-1 plants (Table 3). This
time point was chosen as an intermediate time point derived from
our CO2 evolution experiment, again to assure examination of the
response to carbon starvation rather than recovery from it. The
atg5-1 seedlings incorporated and metabolized significantly higher
amounts of label compared with wild-type seedlings, possibly
suggesting the mutants are more energy deprived, in accordance
with the observed morphological phenotype. Lower CO2 evolution
was also observed for the atg5-1 seedlings. Label redistribution in
the protein fraction was also altered in atg5-1 seedlings, with significantly lower labeling of the protein pool in atg5-1 plants. In order
to estimate absolute fluxes in atg5-1 seedlings compared with the
wild type, we calculated the specific activities of the hexose
phosphate pool and used these to calculate the fluxes to starch,
sucrose, cell wall, respiration, and protein using the assumptions documented by Geigenberger et al. (2000). Surprisingly, no
Seven-day-old etiolated seedlings grown without exogenous sucrose were incubated in the presence of 10 mM MES-KOH (pH 6.5) containing 5mM [U-13C]-lysine and collected at different time
points. 13C sum accumulation (nmol/g fresh weight) is presented as the average of four biological replicates, and variation is given as 6 SE. Significant differences following Student’s t test (P <
0.05) compared to the wild type are denoted in bold. GABA, g-aminobutyric acid.
0.03
0.10
0.56
1.41
0.07
0.99
2.29
5.35
442
8165
0.10
0.18
1.60*1025 8.36*1024
Alanine
060
060
0.05 6 0.03
060
0.22 6 0.06
0.03
Aspartate
0.71 6 0.11
0.45 6 0.09
0.50 6 0.11
0.92 6 0.10
1.86 6 1.15
2.06
GABA
0.03 6 0.01
060
0.45 6 0.09
0.37 6 0.07
0.80 6 0.18
0.27
Glutamate
1.63 6 0.12
1.15 6 0.16
2.25 6 0.14
3.93 6 0.24
6.31 6 2.91
8.45
Lysine
2.47 6 2.38
2.51 6 1.63
7115 6 932
7248 6 900
6945 6 2117
8026
0.03 6 0.03
060
060
060
0.20 6 0.19
0.31
Malate
Ornithine
9.53*1025 6 9.53*1025
060
1.26*1024 6 1.09*1024
060
4.30*1024 6 2.14*1024 2.25*1025
Wild type
Wild type
Metabolite Wild type
atg5-1
40
0
Incubation Time (min)
Table 2. Lack of Autophagy Affects Label Accumulation following
13C-Lysine
atg5-1
80
Feeding of Arabidopsis Seedlings
atg5-1
6
6
6
6
6
6
6
Wild type
120
6
6
6
6
6
6
6
0.04
0.44 6 0.11
0.15
4.41 6 0.24
0.03
1.03 6 0.19
0.34
15.50 6 1.03
2337
7748 6 2048
0.07
1.04 6 0.07
1.65*1024 4.17*1024 6 1.14*1024
The Plant Cell
atg5-1
8 of 17
difference in respiration was observed between the wild type and
atg5-1, in contrast to our observation from CO2 evolution and
[U-13C]Lys feeding experiments. However, this contradiction is
likely due to the time frame of the experiments. The major changes
in respiration were observed after 2 h in both experiments. It is
possible that after 80 min of incubation, the label was not distributed sufficiently for the difference to be evident. On the other hand,
as also seen from the label redistribution, less flux in funneled to
protein in atg5-1 mutants, suggesting lower net protein synthesis in
this line. Taken together with the higher level of total proteins observed in atg mutants (Figure 4A), we postulate that the higher level
of proteins stems from decreased degradation rather than increased synthesis.
Altered Lipid Composition of atg Mutants under
Carbon Starvation
Oil, in the form of triacylglycerol (TAG), is a major seed storage
reserve in many plant species, including Arabidopsis (Graham,
2008). Since etiolated seedlings rely on storage reserves for their
supply of energy, we set out to profile the lipid composition of
our carbon starved mutants. A total of 159 individual lipids were
identified, belonging to 12 different lipid classes. PCA revealed
a distinct separation between wild-type and atg mutant lines,
demonstrating a clear difference between the lipid compositions
of the individual lines (Figure 6A). The NahG line was also clearly
separated from the wild type; however, the lipid composition of
atg5.NahG could not be differentiated from that of atg5-3. Interestingly, not all atg mutants clustered together in the PCA, possibly pointing to a gradient in their phenotypes. In addition, an
examination of the lipid composition of Ws and atg4a4b revealed an
ecotypic difference between the wild-type lines, increasing the level
of complexity of this analysis (Supplemental Data Set 1). We first
focused on the TAG composition of the different lines, as it is the
main storage lipid in the seed. A significant accumulation of most
TAG species was observed for both atg5-1 and atg5-3 (Figure 6B).
While a mild accumulation was also observed for atg7-2, in this
instance, it was not statistically significant. During seed germination
and early seedling establishment, TAGs are hydrolyzed into fatty
acids (FAs) and glycerol. FAs are then degraded in the glyoxysome
by b-oxidation (Graham, 2008). An accumulation of FAs similar to
that of TAGs was observed for atg5-1 and atg5-3, suggesting a
possible impairment of b-oxidation. However, this accumulation
was not seen for atg7-2 (Figure 6C). Interestingly, an increase
in lysophosphatidylcholine and lysophosphatidylethanolamine
(LysoPE) was also observed for all atg mutants analyzed (Figure
6D). These lysolipids, which are editing products of the membrane
lipids PC and PE, have previously been shown to accumulate
under freezing stress in Arabidopsis (Welti et al., 2002). These
data are in keeping with the apparent general retarded lipid degradation of the mutants.
Two lipid species have been documented as being involved in
autophagosome formation. Phosphatidylinositol (PI) is phosphorylated during autophagosome vesicle nucleation by PI3-kinase and is
used as an anchor for the PI3K complex (Li and Vierstra, 2012). In
addition, PE is conjugated to ATG8 during autophagosome elongation, thus enabling its incorporation into the growing autophagosome
membrane (Liu and Bassham, 2012). We detected an increase in the
Autophagy in Etiolated Arabidopsis
9 of 17
Table 3. Lack of Autophagy Affects the Metabolism of [U-14C]Glc by Arabidopsis Seedlings
Variable
Label incorporated (Bq/gFW)
Total uptake
Total metabolized
Redistribution of radiolabel (% of total metabolized)
CO2 evolution
Organic acids
Amino acids
Sucrose
Starch
Protein
Cell wall
Fructose
Specific activity of hexose phosphates (Bq/nmol)
Metabolic flux (nmol hexose equivalents/(gFW*h)
Starch synthesis
Sucrose synthesis
Respiration
Cell wall synthesis
Protein synthesis
Wild Type
atg5-1
1286.90 6 148.38
254.23 6 58.05
1975.54 6 102.48
876.14 6 114.04
3.72
14.87
9.05
43.70
19.18
3.79
6.34
0.00
0.34
6
6
6
6
6
6
6
6
6
0.17
0.49
2.69
3.11
1.45
0.47
0.53
4.33
0.05
1225.11
2800.03
1946.11
388.15
229.22
6
6
6
6
6
218.46
525.28
274.44
46.43
28.64
2.86
14.36
12.59
42.20
13.47
1.71
4.86
7.94
0.42
6
6
6
6
6
6
6
6
6
0.06
1.04
3.06
4.27
2.29
0.16
0.37
2.00
0.06
870.70
2771.62
2017.58
311.15
112.10
6
6
6
6
6
150.49
510.00
150.19
11.30
15.62
Seven-day-old etiolated seedlings grown without exogenous sucrose were incubated in the presence of 10 mM MES-KOH (pH 6.5) containing 1 mM
[U-14C]Glc (specific activity of 1.85 MBq/mmol). Each sample was extracted with boiling ethanol, and the amount of radioactivity in each metabolic
fraction was determined as described in Methods. Values are means 6 SE (n = 3 to 4). Significant differences following Student’s t test (P < 0.05)
compared to the wild type are denoted in bold. FW, fresh weight.
PI levels of all atg mutant lines compared with the wild type, possibly
suggesting an attempt to compensate for the lack of autophagy
(Figure 7A). By contrast, the levels of large PE molecules (PE42),
containing very-long-chain fatty acids, significantly decreased in all
atg mutants examined, corresponding to the increase in LysoPE
levels (Figure 7B).
DISCUSSION
Hypersensitivity to carbon starvation is one of the most wellcharacterized phenotypes of plant autophagy mutants (Doelling
et al., 2002; Hanaoka et al., 2002; Thompson et al., 2005; Xiong
et al., 2005). We therefore selected a carbon starvation system
to study the metabolic implication of autophagy in plants. Thus far,
few studies have tackled this issue, using either nitrogen starvation
(Guiboileau et al., 2013; Masclaux-Daubresse et al., 2014) or carbon starvation in the form of darkness combined with starch accumulation defects (Izumi et al., 2013) as their experimental
systems. In this research, we chose etiolated Arabidopsis seedlings
grown on medium without supplementary sucrose as a carbon
starvation model system. The rationale behind this was that it allowed us to utilize the advantages of a “closed system” as well as
providing insights concerning the function of autophagy during
early seedling establishment, in which nutrient acquisition through
reserve mobilization plays an important role. Indeed, we were able
to see a clear growth phenotype for atg mutants compared with the
wild type, notably a phenotype that was recovered by the addition
of exogenous sucrose (Figures 1A and 1B). This phenotype is in
keeping with the previous observation that atg5 seedlings were
impaired in their ability to generate true leaves in a medium without
sucrose (Yoshimoto et al., 2014). Taken together with the results of
our germination assay (Figures 1D and 1E), we postulate that the
growth impairment phenotype of atg mutants becomes more severe as seedling growth proceeds. The contribution of nutrients
from the mother tissue has previously been shown to play an important role in seed content and also to have an impact on future
seedling establishment (Andriotis et al., 2012). Since atg mutants
additionally possess an early senescence phenotype that affects
seed set (Yoshimoto et al., 2004; Thompson et al., 2005), we used
a previously characterized stay-green atg mutant to control for
maternal tissue effects (Yoshimoto et al., 2009). The fact we could
not recover the phenotype (Figure 1C), taken alongside our documentation of increased autophagic flux under our experimental
conditions (Supplemental Figure 1), suggests the phenotype is indeed likely to stem from lack of function of the autophagy mechanism during germination and seedling establishment.
During our analysis of primary metabolites, we observed a reduction in free amino acids in atg mutants compared with wild-type
seedlings (Figure 2B). This result is in accordance with previous
results from mice and yeast (Onodera and Ohsumi, 2005; Ezaki
et al., 2011). In addition, autophagy was implicated in amino acid
release in Arabidopsis during nighttime starvation (Izumi et al.,
2013), further corroborating our observation. Surprisingly, a recent
study examining autophagy under nitrogen starvation detected, by
contrast, an amino acid accumulation in atg mutants compared
with the wild type (Masclaux-Daubresse et al., 2014). A possible
explanation for this discrepancy may stem from the age of the
studied plants. While Masclaux-Daubresse et al. (2014) analyzed
plants ranging from 30 to 75 d after sowing, we examined 6-d-old
seedlings. Not only are these two developmental systems morphologically distinct, but older plants are also autotrophic, in contrast to etiolated seedlings. Thus, the considerable differences in
10 of 17
The Plant Cell
Figure 6. Lipid Analysis Suggests Altered Lipid Degradation in atg Mutants under Carbon Starvation.
atg mutants as well as their respective wild-type and SA controls (NahG and atg5.NahG) were sown on 0.53 MS plates and grown in the dark for 6 d
after imbibition. Lipid content was analyzed by UPLC-MS (n = 4 to 6). Detailed results of the assay for the depicted lines as well as additional lines are
presented in Supplemental Data Set 1.
(A) PCA of lipid levels.
(B) Heat map of log2 values of TAG relative levels in comparison to the wild type. Significant differences following Student’s t test are denoted by one
asterisk (P < 0.05) or two asterisks (P < 0.01).
(C) Heat map of log2 values of FA relative levels in comparison to the wild type. Significant differences following Student’s t test are denoted by one
asterisk (P < 0.05) or two asterisks (P < 0.01).
(D) Heat map of log2 values of lysophosphatidylcholine (LysoPC) and LysoPE relative levels in comparison to the wild type. Significant differences
following Student’s t test are denoted by one asterisk (P < 0.05) or two asterisks (P < 0.01).
experimental systems might be responsible to the phenotypic inconsistency. The observation of context-dependent autophagy
results is not without precedence since previous studies investigating the role of autophagy in the hypersensitive response
demonstrated that shorter incubation times (Hofius et al., 2009)
yielded opposite results compared with longer incubation times (Liu
et al., 2005). Considering this previous example implies that the
opposite metabolic phenotypes observed here may additionally
arise from the longer exposure to nitrogen starvation in the previous study compared with carbon starvation in our study. It is
important to note that the time frame of the nutrient restriction
in both instances is most likely the most physiologically relevant since carbon and nitrogen starvation operate on different
timescales.
Given that the levels of free amino acids were reduced in atg
mutants, we had anticipated seeing an increase in total protein
amount in the mutants. Indeed, such an increase was observed
in atg5 mutants, but not in atg7-2 (Figure 4A). It has been
previously shown that, although all Arabidopsis atg mutants
lack autophagosomes, some display a more severe phenotype
than others for unknown reasons (Yoshimoto et al., 2009). It is
thus not unexpected to observe a milder phenotype for atg7-2
Autophagy in Etiolated Arabidopsis
Figure 7. Altered Levels of Autophagy-Related Lipids in Carbon-Starved
atg Mutants.
atg mutants as well as their respective wild-type and SA controls (NahG
and atg5.NahG) were sown on 0.53 MS plates and grown in the dark for
6 d after imbibition. Lipid content was analyzed by UPLC-MS (n = 4 to 6).
Detailed results of the assay for the depicted lines as well as additional
lines are presented in Supplemental Data Set 1.
(A) Heat map of log2 values of PI relative levels in comparison to the wild
type. Significant differences following Student’s t test are denoted by
one asterisk (P < 0.05) or two asterisks (P < 0.01).
(B) Heat map of log2 values of PE relative levels in comparison to the wild
type. Significant differences following Student’s t test are denoted by
one asterisk (P < 0.05) or two asterisks (P < 0.01).
compared with atg5 in our experimental system. This milder
atg7-2 phenotype was also observed in the lipid analysis of the
mutants (Figures 6A to 6C). On the contrary, accumulation of
selected proteins was observed in all atg mutants (Figure 4B;
Supplemental Figure 2). Proteomic analysis of the differential
bands (Table 1) revealed the accumulation of several interesting
proteins, among which was cruciferin, an Arabidopsis storage
protein (Wan et al., 2007). The higher abundance of this protein in
atg mutants, together with the increase in total protein amounts
observed in atg5 mutants, corresponds with the known role of
autophagy in protein degradation (Li and Vierstra, 2012). Also of
note is the fact that although the total protein size of cruciferin is
;60 kD, we identified the protein in the 30-kD region of the gel,
suggesting that partial degradation of the protein occurs in the
mutants. Although Arabidopsis contains storage proteins, its main
storage compound is oil, rather than proteins (Kim et al., 2013).
It would therefore be interesting in future studies to examine
11 of 17
whether seedling establishment is more severely impaired in autophagy mutants of species in which proteins are a more abundant
storage compound, such as legumes (Gallardo et al., 2008).
Since autophagy is intimately connected to nutrient recycling
and the maintenance of cellular energy status (Singh and Cuervo,
2011), we paid particular attention to the respiration of atg5-1
compared with the wild type by evaluating the TCA cycle flux as
a consequence of CO2 evolution (Figure 5). This experiment was
further prompted by the decrease in free amino acids, especially in
BCAA and lysine (Figure 2C). These amino acids were shown to
support respiration during carbon starvation both by supplying
electrons directly to the mitochondrial electron chain or by feeding
breakdown intermediates into the TCA cycle (Araújo et al., 2011).
Our results suggest that greater carbon flux is being funneled into
the TCA cycle in atg5-1 than wild-type etiolated seedlings. This is at
first glance counterintuitive given that the plants are carbon starved
and, at least to some extent, protein degradation is inhibited in the
atg mutant. Thus, to confirm that this is indeed the case, we
measured CO2 evolution following feeding starved seedlings with
positionally labeled glucose. Our conclusion is further strengthened
by our proteomic analysis and by the [U-13C]Lys feeding experiment (Tables 1 and 2, respectively). The proteomic analysis did not
detect triosephosphate isomerase, a glycolysis-related protein
(Giegé et al., 2003), in atg mutant seedlings, but was able to detect
the protein in wild-type plants, suggesting a potential shift between
glycolysis and the TCA cycle in atg mutants. Additionally, following
supply of labeled lysine, increased redistribution of label to malate
and glutamate was observed in atg5-1 compared with the wild
type. Malate is a TCA cycle intermediate (Araújo et al., 2011), and
glutamate is produced during lysine breakdown and utilization in
the electron transport chain (Araújo et al., 2010). Taken together,
these results suggest increased flux toward respiration in the mutants. Although we ascertained the concentration of fed glucose
was very low and kept the feeding time to a minimum in order to
avoid recovery from starvation, it is possible that since atg5-1 was
experiencing enhanced starvation compared with the wild type, it
was utilizing the supplied glucose faster in the TCA cycle, resulting
in higher CO2 emission. Nevertheless, the combined results indicate a differential energy homeostasis in atg5-1 compared with
the wild type and implicate the upregulation of amino acid degradation as being a likely mechanism underlying this. Supporting this
claim are the observations of greater label uptake and label metabolism in atg5-1 seedlings during the [U-14C]Glc fractionation
experiment and decreased flux to net protein synthesis in the
mutants compared with the wild type (Table 3).
Comparison of lipid contents in atg mutants and the wild type
both provides important insights and raises several interesting
questions. The main storage compounds in Arabidopsis seeds
are TAGs; these are subsequently converted to FAs, which are later
degraded by b-oxidation to supply energy for the germinating seed
and during seedling establishment (Graham, 2008). This process
takes place in the peroxisome (Hu et al., 2012); thus, it is tempting
to attribute the accumulation of TAGs and FAs in atg5-1 and atg5-3
to an impairment in b-oxidation (Figures 6B and 6C). This is further
compounded by the observation that atg5-1 mutants demonstrated impairment in seedling establishment, interpreted as a decline in peroxisomal activity (Yoshimoto et al., 2014). However, this
accumulation was not observed for the atg7-2 and atg4a4b lines
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The Plant Cell
(Figure 6C; Supplemental Data Set 1) and a pronounced difference
in TAG accumulation was observed between the wild-type samples
of the two different ecotypes (Col and Ws; Supplemental Data Set
1), suggesting peroxisomal deficiency may not be a sufficient explanation for the phenotype. Moreover, our proteomic analysis
(Table 1) revealed an accumulation of two b-oxidation enzymes in
atg mutants, MLS and MFP2 (Rylott et al., 2006; Kim et al., 2013),
rendering the assumption of impaired b-oxidation less plausible.
Further strengthening this claim is a study specifically examining
the role of autophagy in peroxisome maintenance during seedling
growth. The authors, in addition to observing MLS accumulation in
atg mutants, directly examined peroxisome function in seedlings
and did not find a difference compared with the wild type (Kim
et al., 2013). As not all atg mutants possess the exact same phenotype (Harrison-Lowe and Olsen, 2008; Yoshimoto et al., 2009),
we postulate that the lipid phenotype we present may be an ATG5specific phenotype. By contrast, increased lipid editing was a
consistent phenotype throughout the atg mutants (Figure 6D). An
increased lysolipid level has been shown to be the result of the
stress response (Welti et al., 2002; Gao et al., 2010). Taken together
with the observed energy deprivation phenotype, we can conclude
an increased stress response in the atg mutants compared with the
wild type. Another notable phenotype is the different levels of autophagy-related lipids observed in our mutants. Phosphatidylinositol 3-phosphate serves as a scaffold for the assembly of the
autophagic apparatus (Singh and Cuervo, 2011); thus, an increase
in PI levels (Figure 7A) might suggest an attempt to compensate for
the lack of autophagy. PE, on the other hand, is conjugated to
ATG8 to facilitate its incorporation into the growing autophagosome (Mizushima et al., 2011). A decrease in several PE species
(Figure 7B) may indicate the lack of ability to generate autophagosomes. These data thus suggest possible feedback mechanisms operating between the autophagy mechanism and the lipids
regulating it. Dissecting these mechanisms will be an important
area for future research into the interplay between autophagic
process and cellular metabolism.
The use of stay-green plants has proven important throughout
our analysis as a control differentiating between the direct impact of
autophagy plant metabolism and its indirect effects caused by SA
accumulation (Yoshimoto et al., 2009). This control has been previously used in the study of older plants (Guiboileau et al., 2012);
however, we suggest this might also be important when studying
seed and seedling phenotypes, as seed content and, thus, germination ability could be affected by the mother plant (Andriotis
et al., 2012). The use of SA control lines was of significant
importance when analyzing secondary metabolites in the atg mutants (Figure 3), as some of the metabolites changing in atg
mutants seem to be SA dependent. A recently published study
examining the metabolic response of Arabidopsis atg mutants
under nitrogen starvation (Masclaux-Daubresse et al., 2014) used
these stay-green lines to control for primary metabolite changes,
and the authors indeed noticed SA effects on primary metabolism.
Unfortunately, this control was not applied for secondary metabolites, which we discovered to be very important, thus necessitating a second evaluation of the secondary metabolism results.
In conclusion, energy deprivation is responsible for the delayed
growth phenotype of atg etiolated seedlings grown under carbon
starvation, highlighting the importance of autophagy in the cellular
energy economy. As mentioned above, hypersensitivity to carbon
and starvation is a hallmark of atg mutants (Bassham, 2009), and
the products of protein degradation have been shown to assist in
energy supply during carbon starvation (Araújo et al., 2010). In light
of this, and taking into account the results obtained in previous
studies (Guiboileau et al., 2013; Izumi et al., 2013; Michaeli et al.,
2014), it seems safe to assume that the autophagy mechanism
contributes to global metabolic changes not only during seedling
establishment but also during other stages of plant life. However,
when our results are compared with those of the study of Guiboileau
et al. (2013), as well as other recent works (Izumi et al., 2013;
Masclaux-Daubresse et al., 2014), it becomes evident that the role of
the autophagy mechanism alters as the plant ages and environmental
conditions change, thus rendering the underlying mechanism(s)
more difficult to elucidate. In addition, comprehensive analyses
are clearly required in order to understand the many facets of this
complex metabolic phenotype. Given the difficulties in interpreting
changes in steady state metabolite levels in such a complex system,
we demonstrate that augmenting such approaches with label redistribution studies is able to greatly enhance our understanding of
resource mobilization and energy regulation, and we thus strongly
recommend its adoption for future studies of autophagy. In applying
such an approach here, we were able to clearly demonstrate the
importance of autophagy in both nutrient mobilization and energy
homeostasis of growing seedlings. However, future studies are required to elucidate the exact role of autophagy both in other plant
tissues and other environmental circumstances.
METHODS
Plant Material and Growth Conditions
Arabidopsis thaliana ecotype Col was used in this study, apart from atg4a4b,
which is of the Ws ecotype and was therefore compared with the appropriate
wild type control. The lines used in this study are as follows: ag4a4b
(Yoshimoto et al., 2004), atg5-1 (SAIL_129B079) (Yoshimoto et al., 2009),
atg5-3 (SALK_020601) (Guiboileau et al., 2013), atg7-2 (GK-655B06)
(Hofius et al., 2009), GFP-ATG8f-HA (Honig et al., 2012), NahG overexpression (NahG) (Yoshimoto et al., 2009), and atg5.NagG (Yoshimoto
et al., 2009). Seeds were surface sterilized with bleach and sown on 0.5 MS
agar plates without additional sucrose (unless specified otherwise, in which
case 1% sucrose was added). The plates were covered with foil and
kept at 4°C for a period of 48 h and then transferred, while still covered,
to a growth chamber at 22°C. Samples were collected after 6 to 7 d.
Hypocotyl Length Measurement
Arabidopsis seedlings were grown as described above. Seven-day-old
etiolated seedlings were transferred to a plastic tray and a picture of
the seedlings was taken (15 seedlings per line). Image analysis was
performed by ImageJ (Schneider et al., 2012). The experiment was
repeated twice.
Seed Germination Assay
Arabidopsis seeds were sown as described above (six plates per line, ;100
seeds per plate). After imbibition, the foil cover was removed from the plates
and they were put in a growth chamber with continuous light and 22°C. The
number of germinated seeds was counted every day for 4 d and % germination was calculated. Germination was scored using two parameters:
radical protrusion and the appearance of two green cotyledons.
Autophagy in Etiolated Arabidopsis
Extraction, Derivatization, and Analysis of Arabidopsis Seedling
Primary Metabolites Using GC-MS
Metabolite extraction for GC-MS was performed by a method modified
from that described by Roessner-Tunali et al. (2003). Six-day-old etiolated
Arabidopsis seedlings (;50 mg) were collected and immediately frozen in
liquid nitrogen prior to storage at 280°C. The samples were then homogenized using a ball mill precooled with liquid nitrogen and extracted in
1400 mL of methanol, and 60 mL of internal standard (0.2 mg ribitol mL21
water) was subsequently added as a quantification standard. The extraction,
derivatization, standard addition, and sample injection were exactly as described previously (Lisec et al., 2006). The GC-MS system comprised a CTC
CombiPAL autosampler, an Agilent 6890N gas chromatograph, and a LECO
Pegasus III TOF-MS running in EI+ mode. Metabolites were identified in
comparison to database entries of authentic standards (Kopka et al., 2005).
Chromatograms and mass spectra were evaluated using Chroma TOF
1.0 (Leco) and TagFinder 4.0 software (Luedemann et al., 2008). Data
presentation and experimental details were provided as supplemental
data in a manner consistent with recent metabolite reporting recommendations (Araújo et al., 2011) (Supplemental Data Set 2).
Extraction, Derivatization, and Analysis of Arabidopsis Seedling
Secondary Metabolites Using LC-MS
Six-day-old etiolated Arabidopsis seedlings (;50 mg) collected and
immediately frozen in liquid nitrogen prior to storage at 280°C. Profiling of
secondary metabolite by liquid chromatography-electrospray ionization/
mass spectrometry was performed in negative and positive ion detection
mode as described (Araújo et al., 2010). All data were processed using
Xcalibur 2.1 software (Thermo Fisher Scientific). The resulting data matrix
was normalized using an internal standard (Isovitexin; CAS 29702-25-8) in
extraction buffer (5 µg/mL). Metabolites were identified and annotated
based on prior knowledge (Tohge et al., 2007; Watanabe et al., 2013) and
coelution profile of Arabidopsis leaf extracts characterized with purified
compounds from plant extracts (Nakabayashi et al., 2009). Data are reported in a manner compliant with the standards suggested by Fernie
et al. (2011) (Supplemental Data Set 2).
Protein Extraction, Quantification, and Identification by
Mass Spectrometry
Six-day-old etiolated Arabidopsis seedlings were collected and immediately frozen in liquid nitrogen prior to storage at 280°C. For immunoblotting, 20 mg of plant material was ground in liquid nitrogen and total
proteins were extracted in 100 mL protein extraction buffer (Gruis et al.,
2002). Samples were immediately incubated for 5 min at 100°C after
addition of the buffer, vortexed, and centrifuged for 10 min at 20,800g.
Supernatants were collected and 100 mL of SDS-PAGE samples buffer
was added (Laemmli, 1970). Proteins were separated on a 10% SDSPAGE and electrotransferred onto polyvinylidene difluoride membrane.
Equal loading was validated using Coomassie Brilliant Blue staining of the
membrane. For immunodetection, mouse anti-GFP antibody (Clontech;
1:20,000 dilution) was used in combination with horseradish peroxidaseconjugated rabbit anti mouse antibody (Sigma; A9044; 1:10,000 dilution).
For protein quantification and analysis, ;50 mg plant material was
ground in liquid nitrogen and suspended in protein extraction buffer
(50 mM Tris, pH 7.5, 20 mM NaCl, 10% [v/v] glycerol, 0.1% SDS, and
Complete EDTA free protease inhibitor cocktail [Roche]) and mixed well.
Samples were centrifuged for 15 min at 16,000g, 4°C, and the supernatant
was collected. Protein concentration was determined using a BCA Protein
Assay Kit (Thermo). Equal protein amounts were separated on a 10%
SDS-PAGE, followed by protein staining using a colloidal Coomassie
Brilliant Blue dye. Bands of interest were excised from the gel and
subjected to tryptic digest. The resulting peptides were resuspended in
13 of 17
5% acetonitrile and 0.1% trifluoroacetic acid, desalted using a C18 Ziptip
(Millipore), and eluted in 50% acetonitrile and 0.1% trifluoroacetic acid.
Peptides were analyzed via liquid chromatography coupled to tandem
mass spectrometry (Proxeon EASY nLC coupled to a Thermo LTQ-XL
Hybrid Ion Trap-Orbitrap mass spectrometer). For protein identification,
the Mascot search engine (Matrix Sciences) was used to match the data
generated from MS/MS spectra against the AGI proteins database (www.
arabidopsis.org). Searches were done allowing one missed cleavage,
methionine oxidation as a variable modification, peptide tolerance was set
to 10 ppm, MS/MS tolerance to 0.8 D, and peptide charge was either +2 or
+3. Standard scoring was used.
Measurement of Respiratory Parameters
Arabidopsis seeds were surface sterilized and grown in 10 mL liquid
medium in 100-mL conical flasks (20 mg seeds per nine flasks). Following
imbibition, the liquid cultures were grown in the dark at 20°C for 7 d.
Estimations of the TCA cycle flux on the basis of 14CO2 evolution were
performed following incubation of etiolated seedlings in 5 mL 10 mM
MES-KOH, pH 6.5, containing 1.85 MBq/mmol of [1-14C]-, [3:4-14C]-, or
[6-14C]Glc (Hartmann Analytic). Each flask was then sealed with Parafilm
and placed on an orbital shaker at 100 rpm. Released 14CO2 was collected
in 0.5 mL of 10% (w/v) KOH in a vial suspended in the flask (Harrison and
Kruger, 2008). Incubation was performed under green light. 14CO2 evolved
was then quantified by liquid scintillation counting. The results were interpreted following Rees and Beevers (1960).
Analysis of [U-13C]-Lys-Labeled Samples
Arabidopsis seeds were surface sterilized and grown in 10 mL liquid
medium in 100-mL conical flasks (20 mg seeds per nine flasks). Following
imbibition, the liquid cultures were grown in the dark at 20°C for 7 d.
Seedlings from four flasks were transferred under green light into one flask
containing 5 mL 10 mM MES-KOH, pH 6.5, and incubated for 1 h while
shaking. [U-13C]-Lys (Cambridge Isotope Laboratories) was added to
a final concentration of 5 mM, and samples were collected every 40 min
for 2 h. The seedlings from each flask were divided to four groups and
used as biological replicates. Each sample was placed in a sieve, washed
several times with double-distilled water, and immediately frozen in liquid
nitrogen prior to storage at 280°C. Samples were subsequently extracted
in 100% methanol, the fractional enrichment of metabolite pools was
determined exactly as described previously (Roessner-Tunali et al., 2004;
Tieman et al., 2006), and label redistribution was expressed as per
(Studart-Guimarães et al., 2007).
Incubation of Arabidopsis Seedlings with [U-14C]Glc
Arabidopsis seeds were surface sterilized and grown in 10 mL liquid
medium in 100-mL conical flasks (20 mg seeds per nine flasks). Following
imbibition, the liquid cultures were grown in the dark at 20°C for 7 d.
Etiolated seedlings were incubated under green light in 5 mL 10 mM MESKOH, pH 6.5, containing 1.85 MBq/mmol [U-14C]Glc (Hartmann Analytic)
to a final concentration of 1 mM. Samples were then incubated for 80 min,
placed in a sieve, washed several times in double-distilled water, frozen in
liquid nitrogen, and stored at 280°C until further analysis. All incubations
were performed in sealed flasks under green light and shaken at 100 rpm.
The evolved 14CO2 was collected in 0.5 mL of 10% (w/v) KOH.
Fractionation of
14C-Labeled
Tissue Extracts
Extraction and fractionation were performed as previously described
(Szecowka et al., 2012). Frozen seedling samples were extracted with
80% (v/v) ethanol at 80°C (1 mL per sample) and reextracted in two
subsequent steps with 50% (v/v) ethanol (1 mL per sample for each step),
14 of 17
The Plant Cell
and the combined supernatants were dried under an air stream at 35°C
and resuspended in 1 mL of water (Fernie et al., 2001). The soluble fraction
was subsequently separated into neutral, anionic, and basic fractions by
ion-exchange chromatography; the neutral fraction (2.5 mL) was freezedried, resuspended in 100 mL water, and further analyzed by enzymatic
digestion followed by a second ion-exchange chromatography step
(Carrari et al., 2006). To measure phosphate esters, samples (250 mL) of the
soluble fraction were incubated in 50 mL of 10 mm MES-KOH, pH 6.0, with or
without 1 unit of potato acid phosphatase (grade II; Boehringer Mannheim) for
3 h at 37°C, boiled for 2 min, and analyzed by ion-exchange chromatography
(Fernie et al., 2001) The insoluble material left after ethanol extraction was
homogenized, resuspended in 1 mL of water, and counted for starch (Fernie
et al., 2001). Fluxes were calculated as described following the assumptions
detailed by Geigenberger et al. (1997, 2000).
Extraction, Derivatization, and Analysis of Arabidopsis Seedling
Lipids Using UPLC-MS
Six-day-old etiolated Arabidopsis seedlings (;20 mg) were collected and
immediately frozen in liquid nitrogen prior to storage at 280°C. Lipid analysis
was performed as described previously (Giavalisco et al., 2011; Hummel et al.,
2011). The dried pellets were resuspended in 250 mL of a mixture of acetonitrile/isopropanol (7:3 [v/v]), thoroughly vortexed, and centrifuged. Then,
100-mL aliquots were transferred into glass vials and 2 mL was injected and
separated on an Acquity UPLC system (Waters) using a reversed-phase C8
column. The ultraperformance liquid chromatography solvents were as follows: A = water with 1% of a solution of 1 M ammonium acetate and 0.1%
acetic acid; B = 70% acetonitrile/30% isopropanol with 1% of a solution of
1 M ammonium acetate and 0.1% acetic acid. The gradient separation was
performed at a flow rate of 400 µL/min as follows: 1 min at 45% A, a 3-min
linear gradient from 45% A to 35% A, an 8-min linear gradient from 25% A to
11% A, and a 3-min linear gradient from 11% A to 1% A. After washing the
column for 3 min with 1% A, the buffer was returned to 45% A and the column
was reequilibrated for 4 min (22 min total run time). The samples were
measured in either positive or negative ion mode. The mass spectra were
acquired using an Exactive high-resolution mass spectrometer (Thermo
Fisher). The spectra were recorded using alternating full-scan and all-ion
fragmentation scan mode, covering a mass range from 100 to 1500 m/z. The
resolution was set to 10,000, with 10 scans per second, restricting the Orbitrap loading time to a maximum of 100 ms with a target value of 1 million
ions. The capillary voltage was set to 3 kV with a sheath gas flow of 60 and an
auxiliary gas flow of 35 (values are arbitrary units). The capillary temperature
was set to 150°C, and the drying gas in the heated electrospray source was
set to 350°C. The skimmer voltage was held at 25 V, and the tube lens was set
to a value of 130 V. The spectra were recorded from min 1 to 20 of the
ultraperformance liquid chromatography gradients. Obtained raw chromatograms were further processed using Excalibur software version 2.10
(Thermo Fisher). Peaks from raw chromatograms were first determined and
aligned by their parent masses, chemical noise was subtracted, and a final
alignment file of all chromatograms, which contains information about m/z
ratio, retention times, and retention time deviations for each annotated peak,
was created. Assignment of peaks, normalization, and quantification were
performed by Progenesis QI (version 1.0; Nonlinear Dynamics).
Statistical Analysis
Statistical differences between groups were analyzed by Student’s t tests
on the raw data. Results were determined to be statistically different at
a probability level of P < 0.05. Relative metabolite levels were obtained as
the ratio between the lines and the mean value of the respective wild type.
The lipid data, processed by Progenesis QI, were exported as a matrix to
Umetrics SIMCA version 13.0. The data were scaled and mean-centered
according to the vendor. PCA was performed using the Multibase Excel
add-in (Numerical Dynamics).
Accession Numbers
The Arabidopsis Genome Initiative locus numbers for the major genes
discussed in this article are as follows: ATG4a (At2g44140), ATG4b
(At3g59950), ATG5 (At5g17290), ATG7 (At5g45900), and ATG8f
(At4g16520).
Supplemental Data
Supplemental Figure 1. Autophagic Flux Is Increased in Etiolated
Arabidopsis Seedlings under Carbon Starvation.
Supplemental Figure 2. Higher Resolution Image of Protein Samples
Used for MS/MS Analysis, Detailed in Figure 4B.
Supplemental Table 1. Cumulative Percentage of Germination
(Scored by Radical Protrusion) and Seedling Establishment (Scored
by Appearance of Green Cotyledons) of atg Mutants Compared with
Wild-Type Plants.
Supplemental Table 2. Profiling of Primary Metabolites of atg Mutants
under Carbon Starvation.
Supplemental Table 3. Profiling of Secondary Metabolites of atg
Mutants under Carbon Starvation.
Supplemental Data Set 1. Lipid Profiling of atg Mutants under Carbon
Starvation.
Supplemental Data Set 2. Reporting Metabolite Data for the Compounds Presented in This Study.
ACKNOWLEDGMENTS
We thank Etienne Meyer and Toshihiro Obata for their assistance with
various aspects of data analysis during this study and Aida Maric for her
invaluable technical assistance. We also thank Gad Galili (Weizmann
Institute of Science, Rehovot, Israel), Morten Petersen (University of
Copenhagen, Copenhagen, Denmark), Celine Masclaux-Daubresse, and
Kohki Yoshimoto (Institut Jean-Pierre Bourgin, Versailles cedex, France)
for contributing the seeds used in this study. Work on autophagy in our
laboratory is supported by Minerva, Alexander von Humboldt, and EMBO
fellowships (T.A.-W.) and by the Max-Planck Society (A.R.F.).
AUTHOR CONTRIBUTIONS
T.A.-W. and A.R.F. designed the research and wrote the article. T.A.-W.
performed most of the research and data analysis. K.B., G.W., S.A., T.T.,
R.B., and P.G. performed some aspects of the research and data analysis.
Received November 12, 2014; revised December 29, 2014; accepted
January 19, 2015; published February 3, 2015.
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Global Analysis of the Role of Autophagy in Cellular Metabolism and Energy Homeostasis in
Arabidopsis Seedlings under Carbon Starvation
Tamar Avin-Wittenberg, Krzysztof Bajdzienko, Gal Wittenberg, Saleh Alseekh, Takayuki Tohge, Ralph
Bock, Patrick Giavalisco and Alisdair R. Fernie
Plant Cell; originally published online February 3, 2015;
DOI 10.1105/tpc.114.134205
This information is current as of June 17, 2017
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