LARGE-SCALE BIOLOGY ARTICLE

Global Analysis of the Role of Autophagy in CellularMetabolism and Energy Homeostasis in ArabidopsisSeedlings under Carbon StarvationOPEN

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 theirphotosynthesis mechanism is not yet active. For this reason, the plant must mobilize the nutrient reserves provided by themother plant in order to facilitate growth. Autophagy is a catabolic process enabling the bulk degradation of cellularconstituents in the vacuole. The autophagy mechanism is conserved among eukaryotes, and homologs of many autophagy-related (ATG) genes have been found in Arabidopsis thaliana. T-DNA insertion mutants (atg mutants) of these genes displayhigher sensitivity to various stresses, particularly nutrient starvation. However, the direct impact of autophagy on cellularmetabolism has not been well studied. In this work, we used etiolated Arabidopsis seedlings as a model system for carbonstarvation. 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, inorder to decipher the underlying causes of the phenotype. Significant differences between atg mutants and wild-type plantshave been demonstrated, suggesting global effects of autophagy on central metabolism during carbon starvation as well assevere energy deprivation, resulting in a morphological phenotype.

INTRODUCTION

Macroautophagy (referred to hereafter as “autophagy”) is a conservedeukaryotic mechanism, which is classically defined as the degrada-tion of cytoplasmic constituents in the lytic organelle (vacuoles inyeast and plants and lysosomes in mammals) (Reggiori and Klionsky,2013). The general targets of autophagy vary from long-lived proteinsto protein complexes and even entire organelles (Reumann et al.,2010). Morphologically, autophagy begins with the formation of cup-shaped double membranes, which expand to form autophagosomesengulfing malfunctioning or unneeded macromolecules and or-ganelles and transport them for degradation inside the vacuole.Upon arrival of the autophagosomes to the vacuoles, their outermembranes fuse with the tonoplast, creating single-membranevesicles inside the vacuole, termed “autophagic bodies.” Theautophagic bodies and their contents are then degraded insidethe vacuole, providing recycled materials to build new macro-molecules (Bassham, 2009). Nutrient starvation has been one ofthe hallmark inducers of autophagy in yeast, mammals, andplants (Reumann et al., 2010; Singh and Cuervo, 2011; Li andVierstra, 2012). The autophagy mechanism in presumed to playa role in recycling during starvation, thus supplying the cell with

nutrients during the stress period (Li and Vierstra, 2012). It hasbeen shown to be involved in protein and lipid degradation(Onodera and Ohsumi, 2005; Singh and Cuervo, 2011), as well asto 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 showlittle or no accumulation of autophagic bodies during nutrient star-vation (Liu and Bassham, 2012). Many ATG genes are conserved inevolution, and homologs to the yeast genes have been found in manyorganisms including mammals and plants (Avin-Wittenberg et al.,2012; Ryter et al., 2013). Several atgmutants have been characterizedin the model plant Arabidopsis thaliana (Doelling et al., 2002; Hanaokaet al., 2002; Yoshimoto et al., 2004; Xiong et al., 2005; Harrison-Loweand Olsen, 2008; Phillips et al., 2008). Among the common phe-notypes of these mutants are early senescence in comparison towild-type plants as well as hypersensitivity to carbon and nitrogenstarvation (Bassham, 2009). Interestingly, the early senescencephenotype of atg mutants was shown to be the consequence ofsalicylic acid (SA) accumulation, apparently regulated by autophagy(Yoshimoto et al., 2009). Autophagy in plants has also been shownto function during abiotic stresses such as salt, drought, andoxidative stress (Xiong et al., 2007; Liu et al., 2009) as well as inresponse to pathogens (Liu and Bassham, 2012). In addition,selective autophagic degradation of chloroplasts has beendemonstrated (Ishida et al., 2014), and evidence of peroxisomeand starch degradation by autophagy has been recently pro-vided (Farmer et al., 2013; Kim et al., 2013; Wang et al., 2013;

1 Address correspondence to avin@mpimp-golm.mpg.de.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Tamar Avin-Wittenberg(avin@mpimp-golm.mpg.de).OPENArticles can be viewed online without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.114.134205

The Plant Cell, Vol. 27: 306–322, February 2015, www.plantcell.org ã 2015 American Society of Plant Biologists. All rights reserved.

Avin-Wittenberg and Fernie, 2014). Although hypersensitivity tonutrient starvation is a major phenotype of atg mutants, onlyvery few recent studies have attempted to uncover the met-abolic implications underlying it (Guiboileau et al., 2012; Izumiet al., 2013; Masclaux-Daubresse et al., 2014), and although

we are making great strides in our understanding of the met-abolic impact of autophagy in plants, many questions still re-main open.Seed germination and seedling establishment are two de-

velopmental 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 withoutsucrose and grown in the dark for 7 d after imbibition. Representative pictures of the etiolated seedling are shown as well as measurement of hypocotyllength 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) isdenoted 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 platescontaining 1% sucrose and grown in the dark for 7 d after imbibition. Representative pictures of the etiolated seedling are shown as well as mea-surement of hypocotyl length performed using ImageJ (n = 15). Data are presented with SE.(C)Wild-type, NahG, and atg5.NahG seeds were sown on 0.53MS plates without sucrose and grown in the dark for 7 d after imbibition. Representativepictures 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 lightconditions. The number of germinated seedlings (defined by radical protrusion) was scored each day for 4 d, and average percent germination (n = 6) ispresented with SE. Detailed results of the assay for the depicted lines as well as additional lines are presented in Supplemental Table 1.(E) atgmutant and wild-type control seeds were sown on 0.53MS 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 307

Seeds possess storage tissues, containing reserves necessaryfor the establishment of the seedling (Arc et al., 2011). Seedgermination is associated with the degradation and mobiliza-tion of these storage reserves (Fait et al., 2006). This process iscrucial in providing fuel for growth until the growing seedlingbecomes photoautotrophic (Pritchard et al., 2002). Since au-tophagy functions as a degradation pathway facilitating nutri-ent mobilization, it is safe to assume it serves a function duringseedling establishment. Indeed, atg mutants have been shownto display impaired seedling establishment (Yoshimoto et al.,2014). However, the reasons for this phenotype, and specifi-cally the impact on energy metabolism, have not been wellstudied.

In this study, we investigated etiolated Arabidopsis seedlingsas a model for carbon starvation during seedling establishment.

We demonstrate that under our experimental conditions, atgmutants display delayed growth in comparison to wild-typeseedlings. Metabolic profiling revealed reduced levels of freeamino acids in the mutants, while proteomic analysis displayedaccumulation of proteins in the mutants. Metabolic flux analysisalso suggests increased respiration in atg mutants in addition todecreased net protein biosynthesis compared with the wildtype. Examination of the lipid composition of the mutants re-vealed altered lipid levels, strengthening our hypothesis ofaltered metabolism as well as suggesting the occurrence ofstarvation stress responses in the mutants. We suggest thatonly a comprehensive large-scale analysis including both steadystate metabolite levels as well as metabolic flux will enable us todecipher such compound phenotypes as that of autophagydeficiency.

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 dafter 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 linesare 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’st 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 anasterisk (P < 0.05).

308 The Plant Cell

RESULTS

Arabidopsis Etiolated atg Mutant Seedlings Displaya Delayed Growth Phenotype

We have chosen to use etiolated Arabidopsis seedlings as a modelsystem for carbon starvation since the only carbon source availableto these seedlings is that coming from the seed, rendering thesystem heterotrophic. Additionally, no photosynthesis has oc-curred, thus eliminating the possibility of differential carbon accu-mulation in the atgmutants compared with the wild type. We beganour investigation by assessing the morphological phenotype of atgmutants germinated under carbon starvation conditions in com-parison to wild-type seedlings. Seed were sown on 0.53Murashigeand Skoog (MS) plates (in the absence of a carbon source) andgrown in the dark for 7 d after imbibition. All atg mutants displayedvisibly 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 whenhypocotyl length measurements were conducted (Figure 1A). We

were able to recover the phenotypes by supplementing the mediumwith an external carbon source. The size of atg mutants grownon 0.53 MS medium plates containing 1% sucrose was notdifferent from that of their respective wild-type controls (Figure1B). In order to verify that the autophagy mechanism is indeedfunctioning under our experimental conditions, we used Arabi-dopsis plants expressing the fusion protein GFP-ATG8f-HA(containing ATG8f fused to green fluorescent protein and thehuman influenza hemagglutinin [HA] tag) in the wild-type back-ground to determine autophagic flux as described previously(Sláviková et al., 2005). During autophagosome elongation, theC terminus of ATG8 is cleaved by ATG4, and the protein issubsequently conjugated to the lipid phosphatidylethanolamine(PE) and inserted into the growing autophagosome membrane.Upon fusion of the autophagosome with the vacuole, ATG8decorating the inner membrane of the autophagosome is de-graded by vacuolar proteases (Li and Vierstra, 2012). By usinga construct in which ATG8 is tagged from both termini, it ispossible to follow the rate of ATG8 cleavage by ATG4, sug-gesting autophagosome elongation, and the rate of ATG8

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 dafter imbibition. Secondary metabolite content was analyzed by LC-MS (n = 5 to 6). Detailed results of the assay for the depicted lines as well asadditional 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 followingStudent’s t test are denoted by one asterisk (P < 0.05) or two asterisks (P < 0.01).

Autophagy in Etiolated Arabidopsis 309

degradation, suggesting autophagosome fusion with the vacu-ole. The ratio between these two steps is used as a measure ofautophagic flux. GFP-ATG8f-HA seeds were grown in the darkfor 6 d after imbibition on 0.53 MS plates with or without 1%sucrose; protein extracts from the seedlings were then run onSDS-PAGE gel, and an immunoblot assay was performed usingan antibody raised against GFP (Supplemental Figure 1). Thesamples taken from seedlings grown on sucrose contained threeprotein bands, corresponding to the full-length fusion protein aswell as its degradation products ATG8f-GFP and free GFP.However, the samples taken from seedlings grown without su-crose contained only the free GFP band, suggesting increaseddegradation of the GFP-ATG8f-HA protein and, thus, an in-crease in autophagic flux under our experimental conditions.This observation is in agreement with a previous study showingsimilar results (Sláviková et al., 2005).

It has been previously demonstrated that atg mutants accu-mulate SA as they age, causing an early senescence phenotype(Yoshimoto et al., 2009). This phenotype could be recovered bycrossing atg mutant plants with plants overexpressing thebacterial protein NahG, encoding a SA hydroxylase, thus pro-ducing stay-green atg mutants. In order to verify that the shorthypocotyl phenotype was not the result of early senescenceduring seed development or of SA accumulation in the seedtissue, we examined the hypocotyl length of 7-d-old atg5.NahGseedlings grown on plates without sucrose. The atg5.NahGseedlings displayed significantly shorter hypocotyls than wild-type plants, demonstrating that decreasing the SA levels in theplants was not able to recover the morphological phenotype(Figure 1C).

We next wished to ascertain whether the difference in sizestems from delayed germination or reduced growth followinggermination. For this purpose, we compared the germinationof 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 germi-nation; and appearance of two green cotyledons, indicative of earlyseedling development (Gao et al., 2011). A delay in both parameterswas observed for the atgmutant 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 SAcontrol lines (Supplemental Table 1). These results may indicate thatthe difference in plants size stems, at least in part, from delayedgrowth.

Differential Metabolic Response of atg Mutants underCarbon Starvation

Autophagy has been linked to nutrient recycling under starvationconditions (Singh and Cuervo, 2011; Guiboileau et al., 2013; Izumiet al., 2013; Reggiori and Klionsky, 2013; Ryter et al., 2013). Wetherefore set out to investigate the metabolic content of etiolatedatg mutants under carbon starvation compared with wild-typeplants. We were able to identify 30 primary metabolites using gaschromatography-mass spectrometry (GC-MS) analysis (SupplementalTable 2). Principal component analysis (PCA) demonstrated a goodseparation between the wild-type and atg mutant lines. NahG andatg5.NahG clustered with the wild-type line and atg mutant lines,

respectively. A significant decrease in free amino acid levels wasobserved in all atgmutants compared with the wild type (Figure 2B).This decrease was not recovered in the SA control lines, suggestingthat the phenotype is likely to be SA independent (Figure 2B, NahGand atg5.NahG). Of special interest were the reduced levels of lysineand the branched-chain amino acids (BCAAs) isoleucine and valinein atg mutants (Figure 2C). These amino acids have been shown tofunction as electron donors for the tricarboxylic acid (TCA) cycle andmitochondria 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 Comparedwith 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 afterimbibition.(A) Total protein amount was analyzed using BCA protein assay (n = 6).Significant differences compared with the wild type following Student’st 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 higherintensity in atg mutants compared with the wild type and NahG (markedby arrowheads) were cut from the gel for mass spectrometry identifica-tion. Mass spectrometry results are detailed in Table 1.

310 The Plant Cell

mutants likely suggest altered mitochondrial function under carbonstarvation. Additionally, reduced steady state levels of the organicacids malate, fumarate, and dehydroascorbate were observed(Figure 2B).

We also identified secondary metabolites using liquidchromatography-mass spectrometry (LC-MS) analysis (Figure 3;Supplemental Table 3). Surprisingly, while PCA demonstrateda 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 metabolitesexhibiting a significant difference in at least one of the atg mutantlines compared with the wild type, where the difference did notexist in the atg5.NahG line (Figure 3B). This observation impliesa putatively considerable role for SA in determining the secondarymetabolic composition of the atg mutants. However, several sec-ondary metabolites were, by contrast, less abundant in atgmutants compared with the wild type in a presumably SA-independent manner (Figure 3B). However, several of theseare currently unknown with respect to their chemical identity.Nevertheless, they may serve as interesting subjects for fu-ture research.

Selective Protein Accumulation Occurs in Etiolated atgMutant Seedlings under Carbon Starvation

Following our observation of reduction of the steady state levelof many free amino acids in atgmutants and given that proteins aredegradation targets of the autophagy machinery (Li and Vierstra,2012), we next decided to examine the protein content of carbon-starved atg mutants compared with the wild type. We initiallymeasured the total protein content of carbon starved atg mutants.Interestingly, both atg5 mutant lines presented with higher proteincontent than wild-type plants, suggesting a possible impairment in

protein degradation. The atg5.NahG plants displayed the sameaccumulation (Figure 4A); however, surprisingly, atg7-2 seedlingsdid not accumulate higher protein content. This may point toa 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 atgmutantscompared with the wild type, suggesting the differential accu-mulation and degradation of specific proteins (Figure 4B;Supplemental Figure 2). It is important to note that the sameprotein accumulation was observed in atg5.NahG but not in theNahG samples (Figure 4B). The bands showing a difference inaccumulation (Figure 4B, marked by arrows; Supplemental Figure2) were excised and subjected to in-gel digestion with trypsin, andthe resulting peptides were identified using liquid chromatographycoupled to tandem mass spectrometry (Table 1). We were able toidentify an accumulation of cruciferin 3 in atg mutants comparedwith the wild type. Cruciferin is the most abundant storage proteinin Arabidopsis seeds (Wan et al., 2007); thus, its accumulation inatgmutants 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 accumulatingin atg mutants compared with the wild type were malate synthase(MLS) and multifunctional protein 2 (MFP2). Both proteins havebeen shown to be involved in b-oxidation of fatty acids andseedling establishment (Rylott et al., 2006; Kim et al., 2013). Ad-ditionally, MLS has been previously shown to accumulate in atgmutant seedlings, further validating our proteomic analysis (Kimet al., 2013). Surprisingly, we identified one protein over-accumulating in wild-type seedlings rather than atg mutants. Cy-tosolic triosephosphate isomerase is an enzyme involved inglycolysis (Giegé et al., 2003), suggesting a possible upregu-lation of this pathway in wild-type seedlings compared with atgmutants.

Table 1. Identification of Protein Bands from Figure 6B

Wild Type atg5-1 atg5-3 atg7-2

1 2 1 2 1 2 1 2

Band (kD) AGI Description MW MS P MS P MS P MS P MS P MS P MS P MS P

12 At1g14950 Lipid transport superfamilyprotein

17,881 nd nd 74 2 25 1 82 3 124 2 101 3 74 2

20 At3g55440 TPI; triosephosphate isomerase 27,152 88 2 68 2 nd nd nd nd nd nd30 At4g28520 CRU3; cruciferin 3 58,199 nd nd 63 1 93 1 51 1 58 1 74 1 82 150 At3g03250 UGP1; UDP-glucose

pyrophosphorylase 151,706 83 2 95 2 134 4 79 4 129 4 102 5 58 3 140 3

60 At5g03860 MLS; malate synthase 63,846 142 3 113 4 446 12 445 10 456 10 450 11 430 10 418 13At1g21750 PDI5 55,567 76 3 nd 176 5 151 4 342 8 292 6 384 7 399 10

80 At3g06860 MFP2; multifunctional protein 2 78,790 nd nd 209 5 250 8 66 4 46 2 106 5 144 6At5g17920 ATCIMS; Cobalamin-independent

synthase family protein84,304 25 1 nd 57 3 173 5 101 4 130 4 67 3 71 2

120 At1g72150 PATELLIN1 64,007 nd 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 ofArabidopsis 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 theannotation 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 311

Etiolated atg Mutants Display Increased Respiration inResponse to Carbon Starvation

Given that the morphological and metabolic phenotype of atgmutants observed thus far suggests an energy deprivation phe-notype, we were interested in directly checking the energy status ofthe plants. As the seedlings are etiolated, photosynthesis does notplay a role in energy generation, and we therefore directly examinedthe respiration parameters of carbon starved atg mutants. Weevaluated 14CO2 evolution using positionally labeled 14C-Glc inorder to assess flux through the major pathways of carbohydrateoxidation (Nunes-Nesi et al., 2005). Wild-type and atg5-1 seedlingswere grown in the dark without sucrose for 7 d and then suppliedwith [1-14C], [3:4-14C], or [6-14C]Glc (1 mM with specific activity of1.85 MBq/mmol) for a duration of 2 h. During this time, we collectedthe 14CO2 evolved at 40-min intervals. A relatively short time frameand low glucose concentration were chosen in order not to disturbthe starvation status of the plants, thereby avoiding recording thedifferences in recovery from starvation between the mutant and thewild type. CO2 can be released from the C1 position by enzymesthat 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 Glcprovides an indication of the relative rate of the TCA cycle withrespect to other processes of carbohydrate oxidation. Though thelevel of C1 emission was higher than the level of C3:4 emission inboth lines (Figures 5A and 5B), the level of CO2 emission from C1was comparable between atg5-1 and the wild type, while the levelof 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 ratiofor the wild type is higher than that of atg5-1 (1.85 and 1.66 after120 min, respectively), suggesting a higher proportion of carbo-hydrate oxidation performed by the TCA cycle in atg5-1. CO2

emission from the C6 position was relatively similar between bothlines, suggesting that pentane synthesis was unaltered (Figure 5C).

Since some amino acids, such as BCAA and lysine, may beused as substrates for respiration (Araújo et al., 2011), andsince we have observed a decrease in the steady state levelsof these amino acids in atg mutants compared with the wildtype (Figure 2C), we set out to investigate lysine metabolism inatg mutants. Wild-type and atg5-1 seedlings were grown in thedark without sucrose for 7 d and then supplied with [U-13C]Lysfor a duration of 2 h. Seedling samples were collected every40 min, washed, and immediately frozen. 13C label distributionwas calculated as described in Methods in order to determinemetabolic flux through several metabolites (summarized in Table2). Increased label distribution in atg5-1 seedlings was demon-strated for malate, a TCA cycle intermediate, further strength-ening our observation from CO2 evolution. In addition, atg5-1showed increased label distribution in glutamate, alanine, andaspartate compared with the wild type, suggesting increasedmetabolic flux in the mutants.

Decreased Flux to Net Protein Synthesis in Carbon-Starvedatg Mutants

As the seedlings were grown under carbon starvation condition,we hypothesized these conditions might have an effect on othermajor pathways of carbon metabolism. We thus performed [U-14C]

Glc labeling of wild-type and atg5-1 seedlings. The samples wereincubated in the presence of 1 mM [U-14C]Glc for 80 min, ex-tracted, and fractioned into organic acids, amino acids, starch,protein, cell wall, phosphoesters, and sucrose in order to analyzeflux alterations between wild-type and atg5-1 plants (Table 3). Thistime point was chosen as an intermediate time point derived fromour CO2 evolution experiment, again to assure examination of theresponse to carbon starvation rather than recovery from it. Theatg5-1 seedlings incorporated and metabolized significantly higheramounts of label compared with wild-type seedlings, possiblysuggesting the mutants are more energy deprived, in accordancewith the observed morphological phenotype. Lower CO2 evolutionwas also observed for the atg5-1 seedlings. Label redistribution inthe protein fraction was also altered in atg5-1 seedlings, with sig-nificantly lower labeling of the protein pool in atg5-1 plants. In orderto estimate absolute fluxes in atg5-1 seedlings compared with thewild type, we calculated the specific activities of the hexosephosphate pool and used these to calculate the fluxes tostarch, sucrose, cell wall, respiration, and protein using the

Figure 5. 14CO2 Evolution of Etiolated Seedlings in the Dark.

Wild-type and atg5-1 etiolated seedlings were grown in the dark for 7 dand 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 liberatedwas captured (every 40 min) in a KOH trap, and the amount of radiolabelreleased was subsequently quantified by liquid scintillation counting.Values are presented as average 6 SE of determinations of four in-dependent samples per line.

312 The Plant Cell

assumptions documented by Geigenberger et al. (2000). Sur-prisingly, no difference in respiration was observed between thewild type and atg5-1, in contrast to our observation from CO2

evolution and [U-13C]Lys feeding experiments. However, thiscontradiction is likely due to the time frame of the experiments.The major changes in respiration were observed after 2 h in bothexperiments. It is possible that after 80 min of incubation, the labelwas not distributed sufficiently for the difference to be evident. Onthe other hand, as also seen from the label redistribution, less flux infunneled to protein in atg5-1 mutants, suggesting lower net proteinsynthesis in this line. Taken together with the higher level of totalproteins observed in atg mutants (Figure 4A), we postulate that thehigher level of proteins stems from decreased degradationrather than increased synthesis.

Altered Lipid Composition of atg Mutants underCarbon Starvation

Oil, in the form of triacylglycerol (TAG), is a major seed storagereserve in many plant species, including Arabidopsis (Graham,2008). Since etiolated seedlings rely on storage reserves for theirsupply of energy, we set out to profile the lipid composition ofour carbon starved mutants. A total of 159 individual lipids wereidentified, belonging to 12 different lipid classes. PCA revealeda distinct separation between wild-type and atg mutant lines,demonstrating a clear difference between the lipid compositionsof the individual lines (Figure 6A). The NahG line was also clearlyseparated from the wild type; however, the lipid composition ofatg5.NahG could not be differentiated from that of atg5-3. In-terestingly, not all atg mutants clustered together in the PCA, pos-sibly pointing to a gradient in their phenotypes. In addition, anexamination of the lipid composition of Ws and atg4a4b revealed anecotypic difference between the wild-type lines, increasing the levelof complexity of this analysis (Supplemental Data Set 1). We firstfocused on the TAG composition of the different lines, as it is themain storage lipid in the seed. A significant accumulation of mostTAG species was observed for both atg5-1 and atg5-3 (Figure 6B).While a mild accumulation was also observed for atg7-2, in thisinstance, it was not statistically significant. During seed germinationand early seedling establishment, TAGs are hydrolyzed into fattyacids (FAs) and glycerol. FAs are then degraded in the glyoxysome

by b-oxidation (Graham, 2008). An accumulation of FAs similar tothat of TAGs was observed for atg5-1 and atg5-3, suggesting apossible impairment of b-oxidation. However, this accumulationwas not seen for atg7-2 (Figure 6C). Interestingly, an increasein lysophosphatidylcholine and lysophosphatidylethanolamine(LysoPE) was also observed for all atg mutants analyzed (Figure6D). These lysolipids, which are editing products of the membranelipids PC and PE, have previously been shown to accumulateunder freezing stress in Arabidopsis (Welti et al., 2002). Thesedata are in keeping with the apparent general retarded lipid degra-dation of the mutants.Two lipid species have been documented as being involved in

autophagosome formation. Phosphatidylinositol (PI) is phosphory-lated during autophagosome vesicle nucleation by PI3-kinase and isused as an anchor for the PI3K complex (Li and Vierstra, 2012). Inaddition, PE is conjugated to ATG8 during autophagosome elonga-tion, thus enabling its incorporation into the growing autophagosomemembrane (Liu and Bassham, 2012). We detected an increase in thePI levels of all atg mutant lines compared with the wild type, possiblysuggesting 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 allatg mutants examined, corresponding to the increase in LysoPElevels (Figure 7B).

DISCUSSION

Hypersensitivity to carbon starvation is one of the most well-characterized phenotypes of plant autophagy mutants (Doellinget al., 2002; Hanaoka et al., 2002; Thompson et al., 2005; Xionget al., 2005). We therefore selected a carbon starvation systemto 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 car-bon starvation in the form of darkness combined with starch ac-cumulation defects (Izumi et al., 2013) as their experimentalsystems. In this research, we chose etiolated Arabidopsis seedlingsgrown on medium without supplementary sucrose as a carbonstarvation model system. The rationale behind this was that it al-lowed us to utilize the advantages of a “closed system” as well asproviding insights concerning the function of autophagy during

Table 2. Lack of Autophagy Affects Label Accumulation following 13C-Lysine Feeding of Arabidopsis Seedlings

Incubation Time (min)

0 40 80 120

Metabolite Wild Type atg5-1 Wild Type atg5-1 Wild Type atg5-1 Wild Type atg5-1

Alanine 0 6 0 0 6 0 0.05 6 0.03 0 6 0 0.22 6 0.06 0.03 6 0.03 0.10 6 0.04 0.44 6 0.11Aspartate 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 6 0.56 1.41 6 0.15 4.41 6 0.24GABA 0.03 6 0.01 0 6 0 0.45 6 0.09 0.37 6 0.07 0.80 6 0.18 0.27 6 0.07 0.99 6 0.03 1.03 6 0.19

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 6 2.29 5.35 6 0.34 15.50 6 1.03Lysine 2.47 6 2.38 2.51 6 1.63 7115 6 932 7248 6 900 6945 6 2117 8026 6 442 8165 6 2337 7748 6 2048

Malate 0.03 6 0.03 0 6 0 0 6 0 0 6 0 0.20 6 0.19 0.31 6 0.10 0.18 6 0.07 1.04 6 0.07Ornithine 9.53*1025 6 9.53*1025 0 6 0 1.26*1024 6 1.09*1024 0 6 0 4.30*1024 6 2.14*1024 2.25*1025 6 1.60*1025 8.36*1024 6 1.65*1024 4.17*1024 6 1.14*1024

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 biologicalreplicates, 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.

Autophagy in Etiolated Arabidopsis 313

early seedling establishment, in which nutrient acquisition throughreserve mobilization plays an important role. Indeed, we were ableto see a clear growth phenotype for atgmutants compared with thewild type, notably a phenotype that was recovered by the additionof exogenous sucrose (Figures 1A and 1B). This phenotype is inkeeping with the previous observation that atg5 seedlings wereimpaired in their ability to generate true leaves in a medium withoutsucrose (Yoshimoto et al., 2014). Taken together with the results ofour germination assay (Figures 1D and 1E), we postulate that thegrowth impairment phenotype of atg mutants becomes moresevere as seedling growth proceeds. The contribution of nu-trients from the mother tissue has previously been shown toplay an important role in seed content and also to have animpact on future seedling establishment (Andriotis et al.,2012). Since atg mutants additionally possess an early se-nescence phenotype that affects seed set (Yoshimoto et al.,2004; Thompson et al., 2005), we used a previously charac-terized stay-green atg mutant to control for maternal tissueeffects (Yoshimoto et al., 2009). The fact we could not recoverthe phenotype (Figure 1C), taken alongside our documenta-tion of increased autophagic flux under our experimentalconditions (Supplemental Figure 1), suggests the phenotypeis indeed likely to stem from lack of function of the autophagymechanism during germination and seedling establishment.

During our analysis of primary metabolites, we observed a re-duction in free amino acids in atgmutants compared with wild-typeseedlings (Figure 2B). This result is in accordance with previousresults from mice and yeast (Onodera and Ohsumi, 2005; Ezakiet al., 2011). In addition, autophagy was implicated in amino acidrelease in Arabidopsis during nighttime starvation (Izumi et al.,2013), further corroborating our observation. Surprisingly, a recent

study examining autophagy under nitrogen starvation detected, bycontrast, an amino acid accumulation in atg mutants comparedwith the wild type (Masclaux-Daubresse et al., 2014). A possibleexplanation for this discrepancy may stem from the age of thestudied plants. While Masclaux-Daubresse et al. (2014) analyzedplants ranging from 30 to 75 d after sowing, we examined 6-d-oldseedlings. Not only are these two developmental systems mor-phologically distinct, but older plants are also autotrophic, in con-trast to etiolated seedlings. Thus, the considerable differences inexperimental systems might be responsible to the phenotypic in-consistency. The observation of context-dependent autophagyresults is not without precedence since previous studies in-vestigating the role of autophagy in the hypersensitive responsedemonstrated that shorter incubation times (Hofius et al., 2009)yielded opposite results compared with longer incubation times (Liuet al., 2005). Considering this previous example implies that theopposite metabolic phenotypes observed here may additionallyarise from the longer exposure to nitrogen starvation in the pre-vious study compared with carbon starvation in our study. It isimportant to note that the time frame of the nutrient restrictionin both instances is most likely the most physiologically rele-vant since carbon and nitrogen starvation operate on differenttimescales.Given that the levels of free amino acids were reduced in atg

mutants, we had anticipated seeing an increase in total proteinamount in the mutants. Indeed, such an increase was observedin atg5 mutants, but not in atg7-2 (Figure 4A). It has beenpreviously shown that, although all Arabidopsis atg mutantslack autophagosomes, some display a more severe phenotypethan others for unknown reasons (Yoshimoto et al., 2009). It isthus not unexpected to observe a milder phenotype for atg7-2

Table 3. Lack of Autophagy Affects the Metabolism of [U-14C]Glc by Arabidopsis Seedlings

Variable Wild Type atg5-1

Label incorporated (Bq/gFW)Total uptake 1286.90 6 148.38 1975.54 6 102.48Total metabolized 254.23 6 58.05 876.14 6 114.04

Redistribution of radiolabel (% of total metabolized)CO2 evolution 3.72 6 0.17 2.86 6 0.06Organic acids 14.87 6 0.49 14.36 6 1.04Amino acids 9.05 6 2.69 12.59 6 3.06Sucrose 43.70 6 3.11 42.20 6 4.27Starch 19.18 6 1.45 13.47 6 2.29Protein 3.79 6 0.47 1.71 6 0.16Cell wall 6.34 6 0.53 4.86 6 0.37Fructose 0.00 6 4.33 7.94 6 2.00Specific activity of hexose phosphates (Bq/nmol) 0.34 6 0.05 0.42 6 0.06

Metabolic flux (nmol hexose equivalents/(gFW*h)Starch synthesis 1225.11 6 218.46 870.70 6 150.49Sucrose synthesis 2800.03 6 525.28 2771.62 6 510.00Respiration 1946.11 6 274.44 2017.58 6 150.19Cell wall synthesis 388.15 6 46.43 311.15 6 11.30Protein synthesis 229.22 6 28.64 112.10 6 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 metabolicfraction 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.

314 The Plant Cell

compared with atg5 in our experimental system. This milderatg7-2 phenotype was also observed in the lipid analysis of themutants (Figures 6A to 6C). On the contrary, accumulation ofselected proteins was observed in all atg mutants (Figure 4B;Supplemental Figure 2). Proteomic analysis of the differentialbands (Table 1) revealed the accumulation of several interestingproteins, among which was cruciferin, an Arabidopsis storageprotein (Wan et al., 2007). The higher abundance of this protein inatg mutants, together with the increase in total protein amountsobserved in atg5 mutants, corresponds with the known role ofautophagy 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 themutants. Although Arabidopsis contains storage proteins, its mainstorage compound is oil, rather than proteins (Kim et al., 2013).It would therefore be interesting in future studies to examinewhether seedling establishment is more severely impaired in au-tophagy mutants of species in which proteins are a more abundantstorage 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,

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 dafter 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 arepresented 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 oneasterisk (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 oneasterisk (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 differencesfollowing Student’s t test are denoted by one asterisk (P < 0.05) or two asterisks (P < 0.01).

Autophagy in Etiolated Arabidopsis 315

2011), we paid particular attention to the respiration of atg5-1compared with the wild type by evaluating the TCA cycle flux asa consequence of CO2 evolution (Figure 5). This experiment wasfurther prompted by the decrease in free amino acids, especially inBCAA and lysine (Figure 2C). These amino acids were shown tosupport respiration during carbon starvation both by supplyingelectrons directly to the mitochondrial electron chain or by feedingbreakdown intermediates into the TCA cycle (Araújo et al., 2011).Our results suggest that greater carbon flux is being funneled intothe TCA cycle in atg5-1 than wild-type etiolated seedlings. This is atfirst glance counterintuitive given that the plants are carbon starvedand, at least to some extent, protein degradation is inhibited in theatg mutant. Thus, to confirm that this is indeed the case, wemeasured CO2 evolution following feeding starved seedlings withpositionally labeled glucose. Our conclusion is further strengthenedby our proteomic analysis and by the [U-13C]Lys feeding experi-ment (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 detectthe protein in wild-type plants, suggesting a potential shift betweenglycolysis and the TCA cycle in atg mutants. Additionally, followingsupply of labeled lysine, increased redistribution of label to malateand glutamate was observed in atg5-1 compared with the wildtype. Malate is a TCA cycle intermediate (Araújo et al., 2011),and glutamate is produced during lysine breakdown and utili-zation in the electron transport chain (Araújo et al., 2010). Takentogether, these results suggest increased flux toward respirationin the mutants. Although we ascertained the concentration offed glucose was very low and kept the feeding time to a mini-mum in order to avoid recovery from starvation, it is possiblethat since atg5-1 was experiencing enhanced starvation com-pared with the wild type, it was utilizing the supplied glucosefaster in the TCA cycle, resulting in higher CO2 emission. Nev-ertheless, the combined results indicate a differential energyhomeostasis in atg5-1 compared with the wild type and impli-cate the upregulation of amino acid degradation as being a likelymechanism underlying this. Supporting this claim are the ob-servations of greater label uptake and label metabolism in atg5-1 seedlings during the [U-14C]Glc fractionation experiment anddecreased flux to net protein synthesis in the mutants comparedwith the wild type (Table 3).Comparison of lipid contents in atg mutants and the wild type

both provides important insights and raises several interestingquestions. The main storage compounds in Arabidopsis seedsare TAGs; these are subsequently converted to FAs, which are laterdegraded by b-oxidation to supply energy for the germinating seedand during seedling establishment (Graham, 2008). This processtakes place in the peroxisome (Hu et al., 2012); thus, it is temptingto attribute the accumulation of TAGs and FAs in atg5-1 and atg5-3to an impairment in b-oxidation (Figures 6B and 6C). This is furthercompounded by the observation that atg5-1 mutants demon-strated impairment in seedling establishment, interpreted as a de-cline in peroxisomal activity (Yoshimoto et al., 2014). However, thisaccumulation was not observed for the atg7-2 and atg4a4b lines(Figure 6C; Supplemental Data Set 1) and a pronounced differencein TAG accumulation was observed between the wild-type samplesof the two different ecotypes (Col and Ws; Supplemental Data Set1), suggesting peroxisomal deficiency may not be a sufficient ex-planation for the phenotype. Moreover, our proteomic analysis(Table 1) revealed an accumulation of two b-oxidation enzymes inatg 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 examiningthe role of autophagy in peroxisome maintenance during seedlinggrowth. The authors, in addition to observing MLS accumulation inatg mutants, directly examined peroxisome function in seedlingsand did not find a difference compared with the wild type (Kimet al., 2013). As not all atg mutants possess the exact same phe-notype (Harrison-Lowe and Olsen, 2008; Yoshimoto et al., 2009),we postulate that the lipid phenotype we present may be an ATG5-specific phenotype. By contrast, increased lipid editing was aconsistent phenotype throughout the atg mutants (Figure 6D). Anincreased lysolipid level has been shown to be the result ofthe stress response (Welti et al., 2002; Gao et al., 2010). Takentogether with the observed energy deprivation phenotype, we

Figure 7. Altered Levels of Autophagy-Related Lipids in Carbon-Starvedatg Mutants.

atg mutants as well as their respective wild-type and SA controls (NahGand atg5.NahG) were sown on 0.53 MS plates and grown in the dark for6 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 additionallines are presented in Supplemental Data Set 1.(A) Heat map of log2 values of PI relative levels in comparison to the wildtype. Significant differences following Student’s t test are denoted byone asterisk (P < 0.05) or two asterisks (P < 0.01).(B) Heat map of log2 values of PE relative levels in comparison to the wildtype. Significant differences following Student’s t test are denoted byone asterisk (P < 0.05) or two asterisks (P < 0.01).

316 The Plant Cell

can conclude an increased stress response in the atg mutantscompared with the wild type. Another notable phenotype isthe different levels of autophagy-related lipids observed inour mutants. Phosphatidylinositol 3-phosphate serves as ascaffold for the assembly of the autophagic apparatus (Singhand Cuervo, 2011); thus, an increase in PI levels (Figure 7A)might suggest an attempt to compensate for the lack ofautophagy. PE, on the other hand, is conjugated to ATG8 tofacilitate 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 au-tophagosomes. These data thus suggest possible feedbackmechanisms operating between the autophagy mechanismand the lipids regulating it. Dissecting these mechanisms willbe an important area for future research into the interplay be-tween autophagic process and cellular metabolism.

The use of stay-green plants has proven important throughoutour analysis as a control differentiating between the direct impact ofautophagy plant metabolism and its indirect effects caused by SAaccumulation (Yoshimoto et al., 2009). This control has been pre-viously used in the study of older plants (Guiboileau et al., 2012);however, we suggest this might also be important when studyingseed and seedling phenotypes, as seed content and, thus, ger-mination ability could be affected by the mother plant (Andriotiset al., 2012). The use of SA control lines was of significantimportance when analyzing secondary metabolites in the atg mu-tants (Figure 3), as some of the metabolites changing in atgmutants seem to be SA dependent. A recently published studyexamining the metabolic response of Arabidopsis atg mutantsunder nitrogen starvation (Masclaux-Daubresse et al., 2014) usedthese 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 metab-olites, which we discovered to be very important, thus necessi-tating a second evaluation of the secondary metabolism results.

In conclusion, energy deprivation is responsible for the delayedgrowth phenotype of atg etiolated seedlings grown under carbonstarvation, highlighting the importance of autophagy in the cellularenergy economy. As mentioned above, hypersensitivity to carbonand starvation is a hallmark of atg mutants (Bassham, 2009), andthe products of protein degradation have been shown to assist inenergy supply during carbon starvation (Araújo et al., 2010). In lightof this, and taking into account the results obtained in previousstudies (Guiboileau et al., 2013; Izumi et al., 2013; Michaeli et al.,2014), it seems safe to assume that the autophagy mechanismcontributes to global metabolic changes not only during seedlingestablishment but also during other stages of plant life. However,when our results are compared with those of the study of Guiboileauet al. (2013), as well as other recent works (Izumi et al., 2013;Masclaux-Daubresse et al., 2014), it becomes evident that therole of the autophagy mechanism alters as the plant ages andenvironmental conditions change, thus rendering the underlyingmechanism(s) more difficult to elucidate. In addition, compre-hensive analyses are clearly required in order to understand themany facets of this complex metabolic phenotype. Given thedifficulties in interpreting changes in steady state metabolitelevels in such a complex system, we demonstrate that aug-menting such approaches with label redistribution studies is

able to greatly enhance our understanding of resource mobi-lization and energy regulation, and we thus strongly recom-mend its adoption for future studies of autophagy. In applyingsuch an approach here, we were able to clearly demonstratethe importance of autophagy in both nutrient mobilization and en-ergy homeostasis of growing seedlings. However, future studiesare required to elucidate the exact role of autophagy both in otherplant 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 theWs ecotype and was therefore compared with the appropriatewild 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 over-expression (NahG) (Yoshimoto et al., 2009), and atg5.NagG (Yoshimotoet al., 2009). Seeds were surface sterilized with bleach and sown on 0.5MS agar plates without additional sucrose (unless specified otherwise,in which case 1% sucrose was added). The plates were covered withfoil and kept at 4°C for a period of 48 h and then transferred, while stillcovered, to a growth chamber at 22°C. Samples were collected after6 to 7 d.

Hypocotyl Length Measurement

Arabidopsis seedlings were grown as described above. Seven-day-oldetiolated seedlings were transferred to a plastic tray and a picture ofthe seedlings was taken (15 seedlings per line). Image analysis wasperformed by ImageJ (Schneider et al., 2012). The experiment wasrepeated 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 fromthe plates and they were put in a growth chamber with continuous lightand 22°C. The number of germinated seeds was counted every day for4 d and % germination was calculated. Germination was scored usingtwo parameters: radical protrusion and the appearance of two greencotyledons.

Extraction, Derivatization, and Analysis of Arabidopsis SeedlingPrimary Metabolites Using GC-MS

Metabolite extraction for GC-MS was performed by a method modifiedfrom that described by Roessner-Tunali et al. (2003). Six-day-old etiolatedArabidopsis seedlings (;50 mg) were collected and immediately frozen inliquid nitrogen prior to storage at 280°C. The samples were then ho-mogenized using a ball mill precooled with liquid nitrogen and extracted in1400 mL of methanol, and 60 mL of internal standard (0.2 mg ribitol mL21

water) was subsequently added as a quantification standard. The extrac-tion, derivatization, standard addition, and sample injection were exactly asdescribed previously (Lisec et al., 2006). The GC-MS system compriseda CTC CombiPAL autosampler, an Agilent 6890N gas chromatograph,and a LECO Pegasus III TOF-MS running in EI+ mode. Metaboliteswere identified in comparison to database entries of authentic standards(Kopka et al., 2005). Chromatograms and mass spectra were evaluatedusing Chroma TOF 1.0 (Leco) and TagFinder 4.0 software (Luedemann

Autophagy in Etiolated Arabidopsis 317

et al., 2008). Data presentation and experimental details were providedas supplemental data in a manner consistent with recent metabolitereporting recommendations (Araújo et al., 2011) (Supplemental Data Set 2).

Extraction, Derivatization, and Analysis of Arabidopsis SeedlingSecondary Metabolites Using LC-MS

Six-day-old etiolated Arabidopsis seedlings (;50 mg) collected andimmediately frozen in liquid nitrogen prior to storage at280°C. Profiling ofsecondary metabolite by liquid chromatography-electrospray ionization/mass spectrometry was performed in negative and positive ion detectionmode as described (Araújo et al., 2010). All data were processed usingXcalibur 2.1 software (Thermo Fisher Scientific). The resulting datamatrix was normalized using an internal standard (Isovitexin; CAS29702-25-8) in extraction buffer (5 mg/mL). Metabolites were identifiedand annotated based on prior knowledge (Tohge et al., 2007; Watanabeet al., 2013) and coelution profile of Arabidopsis leaf extracts characterizedwith purified compounds from plant extracts (Nakabayashi et al., 2009).Data are reported in a manner compliant with the standards suggested byFernie et al. (2011) (Supplemental Data Set 2).

Protein Extraction, Quantification, and Identification byMass Spectrometry

Six-day-old etiolated Arabidopsis seedlings were collected and imme-diately frozen in liquid nitrogen prior to storage at 280°C. For immuno-blotting, 20 mg of plant material was ground in liquid nitrogen and totalproteins were extracted in 100 mL protein extraction buffer (Gruis et al.,2002). Samples were immediately incubated for 5 min at 100°C afteraddition of the buffer, vortexed, and centrifuged for 10 min at 20,800g.Supernatants were collected and 100 mL of SDS-PAGE samples bufferwas added (Laemmli, 1970). Proteins were separated on a 10% SDS-PAGE and electrotransferred onto polyvinylidene difluoride membrane.Equal loading was validated using Coomassie Brilliant Blue stainingof the membrane. For immunodetection, mouse anti-GFP antibody(Clontech; 1:20,000 dilution) was used in combination with horse-radish peroxidase-conjugated rabbit anti mouse antibody (Sigma;A9044; 1:10,000 dilution).

For protein quantification and analysis, ;50 mg plant material wasground 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, andComplete EDTA free protease inhibitor cocktail [Roche]) and mixed well.Samples were centrifuged for 15min at 16,000g, 4°C, and the supernatantwas collected. Protein concentration was determined using a BCAProteinAssay Kit (Thermo). Equal protein amounts were separated on a 10%SDS-PAGE, followed by protein staining using a colloidal CoomassieBrilliant Blue dye. Bands of interest were excised from the gel andsubjected to tryptic digest. The resulting peptides were resuspended in5% 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 tandemmass spectrometry (Proxeon EASY nLC coupled to a Thermo LTQ-XLHybrid Ion Trap-Orbitrap mass spectrometer). For protein identification,the Mascot search engine (Matrix Sciences) was used to match the datagenerated 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 setto 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 liquidmedium 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 wereperformed following incubation of etiolated seedlings in 5 mL 10 mMMES-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 Parafilmand placed on an orbital shaker at 100 rpm. Released 14CO2 was collectedin 0.5 mL of 10% (w/v) KOH in a vial suspended in the flask (Harrison andKruger, 2008). Incubation was performed under green light. 14CO2 evolvedwas then quantified by liquid scintillation counting. The results were in-terpreted following Rees and Beevers (1960).

Analysis of [U-13C]-Lys-Labeled Samples

Arabidopsis seeds were surface sterilized and grown in 10 mL liquidmedium in 100-mL conical flasks (20 mg seeds per nine flasks). Followingimbibition, 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 flaskcontaining 5 mL 10 mM MES-KOH, pH 6.5, and incubated for 1 h whileshaking. [U-13C]-Lys (Cambridge Isotope Laboratories) was added toa final concentration of 5 mM, and samples were collected every 40 minfor 2 h. The seedlings from each flask were divided to four groups andused as biological replicates. Each sample was placed in a sieve, washedseveral times with double-distilled water, and immediately frozen in liquidnitrogen prior to storage at280°C. Samples were subsequently extractedin 100% methanol, the fractional enrichment of metabolite pools wasdetermined 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 liquidmedium in 100-mL conical flasks (20 mg seeds per nine flasks). Fol-lowing imbibition, the liquid cultures were grown in the dark at 20°C for7 d. Etiolated seedlings were incubated under green light in 5 mL 10 mMMES-KOH, pH 6.5, containing 1.85 MBq/mmol [U-14C]Glc (HartmannAnalytic) to a final concentration of 1 mM. Samples were then incubatedfor 80 min, placed in a sieve, washed several times in double-distilledwater, frozen in liquid nitrogen, and stored at 280°C until furtheranalysis. All incubations were performed in sealed flasks under greenlight and shaken at 100 rpm. The evolved 14CO2 was collected in 0.5 mLof 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 with80% (v/v) ethanol at 80°C (1 mL per sample) and reextracted in twosubsequent steps with 50% (v/v) ethanol (1 mL per sample for each step),and the combined supernatants were dried under an air stream at 35°Cand resuspended in 1 mL of water (Fernie et al., 2001). The solublefraction was subsequently separated into neutral, anionic, and basicfractions by ion-exchange chromatography; the neutral fraction (2.5mL) was freeze-dried, resuspended in 100 mL water, and further an-alyzed by enzymatic digestion followed by a second ion-exchangechromatography step (Carrari et al., 2006). To measure phosphate es-ters, samples (250 mL) of the soluble fraction were incubated in 50 mL of10 mm MES-KOH, pH 6.0, with or without 1 unit of potato acid phos-phatase (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 detailedby Geigenberger et al. (1997, 2000).

318 The Plant Cell

Extraction, Derivatization, and Analysis of Arabidopsis SeedlingLipids Using UPLC-MS

Six-day-old etiolated Arabidopsis seedlings (;20 mg) were collected andimmediately frozen in liquid nitrogen prior to storage at280°C. Lipid analysiswas performed as described previously (Giavalisco et al., 2011; Hummel et al.,2011). The dried pellets were resuspended in 250 mL of a mixture of ace-tonitrile/isopropanol (7:3 [v/v]), thoroughly vortexed, and centrifuged. Then,100-mL aliquots were transferred into glass vials and 2 mL was injected andseparated on an Acquity UPLC system (Waters) using a reversed-phase C8column. The ultraperformance liquid chromatography solvents were as fol-lows: 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 of1 M ammonium acetate and 0.1% acetic acid. The gradient separation wasperformed at a flow rate of 400 mL/min as follows: 1 min at 45% A, a 3-minlinear gradient from 45%A to 35% A, an 8-min linear gradient from 25%A to11% A, and a 3-min linear gradient from 11% A to 1% A. After washing thecolumn for 3minwith 1%A, the buffer was returned to 45%Aand the columnwas reequilibrated for 4 min (22 min total run time). The samples weremeasured in either positive or negative ion mode. The mass spectra wereacquired using an Exactive high-resolution mass spectrometer (ThermoFisher). The spectra were recorded using alternating full-scan and all-ion fragmentation scan mode, covering a mass range from 100 to1500 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 witha target value of 1 million ions. The capillary voltage was set to 3 kV witha sheath gas flow of 60 and an auxiliary gas flow of 35 (values arearbitrary units). The capillary temperature was set to 150°C, and thedrying gas in the heated electrospray source was set to 350°C. Theskimmer voltage was held at 25 V, and the tube lens was set to a value of130 V. The spectra were recorded from min 1 to 20 of the ultra-performance liquid chromatography gradients. Obtained raw chroma-tograms were further processed using Excalibur software version 2.10(Thermo Fisher). Peaks from raw chromatograms were first determinedand aligned by their parent masses, chemical noise was subtracted, anda final alignment file of all chromatograms, which contains informationabout m/z ratio, retention times, and retention time deviations for eachannotated peak, was created. Assignment of peaks, normalization, andquantification were performed by Progenesis QI (version 1.0; NonlinearDynamics).

Statistical Analysis

Statistical differences between groups were analyzed by Student’s t testson the raw data. Results were determined to be statistically different ata probability level of P < 0.05. Relative metabolite levels were obtained asthe 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 toUmetrics SIMCA version 13.0. The data were scaled and mean-centeredaccording to the vendor. PCA was performed using the Multibase Exceladd-in (Numerical Dynamics).

Accession Numbers

The Arabidopsis Genome Initiative locus numbers for the major genesdiscussed 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 EtiolatedArabidopsis Seedlings under Carbon Starvation.

Supplemental Figure 2. Higher Resolution Image of Protein SamplesUsed for MS/MS Analysis, Detailed in Figure 4B.

Supplemental Table 1. Cumulative Percentage of Germination(Scored by Radical Protrusion) and Seedling Establishment (Scored byAppearance of Green Cotyledons) of atg Mutants Compared withWild-Type Plants.

Supplemental Table 2. Profiling of Primary Metabolites of atgMutantsunder Carbon Starvation.

Supplemental Table 3. Profiling of Secondary Metabolites of atgMutants under Carbon Starvation.

Supplemental Data Set 1. Lipid Profiling of atgMutants under CarbonStarvation.

Supplemental Data Set 2. Reporting Metabolite Data for the Com-pounds Presented in This Study.

ACKNOWLEDGMENTS

We thank Etienne Meyer and Toshihiro Obata for their assistance withvarious aspects of data analysis during this study and Aida Maric for herinvaluable technical assistance. We also thank Gad Galili (WeizmannInstitute of Science, Rehovot, Israel), Morten Petersen (University ofCopenhagen, Copenhagen, Denmark), Celine Masclaux-Daubresse, andKohki Yoshimoto (Institut Jean-Pierre Bourgin, Versailles cedex, France)for contributing the seeds used in this study. Work on autophagy in ourlaboratory is supported by Minerva, Alexander von Humboldt, and EMBOfellowships (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; acceptedJanuary 19, 2015; published February 3, 2015.

REFERENCES

Andriotis, V.M., Pike, M.J., Schwarz, S.L., Rawsthorne, S., Wang, T.L.,and Smith, A.M. (2012). Altered starch turnover in the maternal planthas major effects on Arabidopsis fruit growth and seed composition.Plant Physiol. 160: 1175–1186.

Araújo, W.L., Ishizaki, K., Nunes-Nesi, A., Larson, T.R., Tohge, T.,Krahnert, I., Witt, S., Obata, T., Schauer, N., Graham, I.A., Leaver, C.J.,and Fernie, A.R. (2010). Identification of the 2-hydroxyglutarateand isovaleryl-CoA dehydrogenases as alternative electron donorslinking lysine catabolism to the electron transport chain of Arabidopsismitochondria. Plant Cell 22: 1549–1563.

Araújo, W.L., Tohge, T., Ishizaki, K., Leaver, C.J., and Fernie, A.R.(2011). Protein degradation - an alternative respiratory substrate forstressed plants. Trends Plant Sci. 16: 489–498.

Arc, E., Galland, M., Cueff, G., Godin, B., Lounifi, I., Job, D., andRajjou, L. (2011). Reboot the system thanks to protein post-translational modifications and proteome diversity: How quiescentseeds restart their metabolism to prepare seedling establishment.Proteomics 11: 1606–1618.

Avin-Wittenberg, T., and Fernie, A.R. (2014). At long last: evidencefor pexophagy in plants. Mol. Plant 7: 1257–1260.

Avin-Wittenberg, T., Honig, A., and Galili, G. (2012). Variations ona theme: plant autophagy in comparison to yeast and mammals.Protoplasma 249: 285–299.

Autophagy in Etiolated Arabidopsis 319

Bassham, D.C. (2009). Function and regulation of macroautophagy inplants. Biochim. Biophys. Acta 1793: 1397–1403.

Carrari, F., Baxter, C., Usadel, B., Urbanczyk-Wochniak, E., Zanor,M.-I., Nunes-Nesi, A., Nikiforova, V., Centero, D., Ratzka, A.,Pauly, M., Sweetlove, L.J., and Fernie, A.R. (2006). Integratedanalysis of metabolite and transcript levels reveals the metabolicshifts that underlie tomato fruit development and highlight regula-tory aspects of metabolic network behavior. Plant Physiol. 142:1380–1396.

Doelling, J.H., Walker, J.M., Friedman, E.M., Thompson, A.R., andVierstra, R.D. (2002). The APG8/12-activating enzyme APG7 isrequired for proper nutrient recycling and senescence in Arabi-dopsis thaliana. J. Biol. Chem. 277: 33105–33114.

Ezaki, J., et al. (2011). Liver autophagy contributes to the maintenance ofblood glucose and amino acid levels. Autophagy 7: 727–736.

Fait, A., Angelovici, R., Less, H., Ohad, I., Urbanczyk-Wochniak, E.,Fernie, A.R., and Galili, G. (2006). Arabidopsis seed developmentand germination is associated with temporally distinct metabolicswitches. Plant Physiol. 142: 839–854.

Farmer, L.M., Rinaldi, M.A., Young, P.G., Danan, C.H., Burkhart,S.E., and Bartel, B. (2013). Disrupting autophagy restores peroxi-some function to an Arabidopsis lon2 mutant and reveals a role forthe LON2 protease in peroxisomal matrix protein degradation. PlantCell 25: 4085–4100.

Fernie, A.R., Aharoni, A., Willmitzer, L., Stitt, M., Tohge, T., Kopka,J., Carroll, A.J., Saito, K., Fraser, P.D., and DeLuca, V. (2011).Recommendations for reporting metabolite data. Plant Cell 23:2477–2482.

Fernie, A.R., Roscher, A., Ratcliffe, R.G., and Kruger, N.J. (2001).Fructose 2,6-bisphosphate activates pyrophosphate: fructose-6-phosphate 1-phosphotransferase and increases triose phosphateto hexose phosphate cycling in heterotrophic cells. Planta 212:250–263.

Gallardo, K., Thompson, R., and Burstin, J. (2008). Reserve accu-mulation in legume seeds. C. R. Biol. 331: 755–762.

Gao, G., Zhang, S., Wang, C., Yang, X., Wang, Y., Su, X., Du, J.,and Yang, C. (2011). Arabidopsis CPR5 independently regulatesseed germination and postgermination arrest of developmentthrough LOX pathway and ABA signaling. PLoS ONE 6: e19406.

Gao, W., Li, H.Y., Xiao, S., and Chye, M.L. (2010). Acyl-CoA-bindingprotein 2 binds lysophospholipase 2 and lysoPC to promote toler-ance to cadmium-induced oxidative stress in transgenic Arabi-dopsis. Plant J. 62: 989–1003.

Geigenberger, P., Fernie, A.R., Gibon, Y., Christ, M., and Stitt, M.(2000). Metabolic activity decreases as an adaptive response tolow internal oxygen in growing potato tubers. Biol. Chem. 381:723–740.

Geigenberger, P., Reimholz, R., Geiger, M., Merlo, L., Canale, V.,and Stitt, M. (1997). Regulation of sucrose and starch metabolismin potato tubers in response to short-term water deficit. Planta 201:502–518.

Giavalisco, P., Li, Y., Matthes, A., Eckhardt, A., Hubberten, H.M.,Hesse, H., Segu, S., Hummel, J., Kohl, K., and Willmitzer, L.(2011). Elemental formula annotation of polar and lipophilic me-tabolites using (13) C, (15) N and (34) S isotope labelling, in com-bination with high-resolution mass spectrometry. Plant J. 68:364–376.

Giegé, P., Heazlewood, J.L., Roessner-Tunali, U., Millar, A.H.,Fernie, A.R., Leaver, C.J., and Sweetlove, L.J. (2003). Enzymes ofglycolysis are functionally associated with the mitochondrion inArabidopsis cells. Plant Cell 15: 2140–2151.

Graham, I.A. (2008). Seed storage oil mobilization. Annu. Rev. PlantBiol. 59: 115–142.

Gruis, D.F., Selinger, D.A., Curran, J.M., and Jung, R. (2002).Redundant proteolytic mechanisms process seed storage pro-teins in the absence of seed-type members of the vacuolar pro-cessing enzyme family of cysteine proteases. Plant Cell 14:2863–2882.

Guiboileau, A., Avila-Ospina, L., Yoshimoto, K., Soulay, F.,Azzopardi, M., Marmagne, A., Lothier, J., and Masclaux-Daubresse, C. (2013). Physiological and metabolic consequencesof autophagy deficiency for the management of nitrogen and proteinresources in Arabidopsis leaves depending on nitrate availability.New Phytol. 199: 683–694.

Guiboileau, A., Yoshimoto, K., Soulay, F., Bataillé, M.P., Avice,J.C., and Masclaux-Daubresse, C. (2012). Autophagy machinerycontrols nitrogen remobilization at the whole-plant level under bothlimiting and ample nitrate conditions in Arabidopsis. New Phytol.194: 732–740.

Hanaoka, H., Noda, T., Shirano, Y., Kato, T., Hayashi, H., Shibata, D.,Tabata, S., and Ohsumi, Y. (2002). Leaf senescence and starvation-induced chlorosis are accelerated by the disruption of an Arabidopsisautophagy gene. Plant Physiol. 129: 1181–1193.

Harrison, P.W., and Kruger, N.J. (2008). Validation of the designof feeding experiments involving [(14)C]substrates used to mo-nitor metabolic flux in higher plants. Phytochemistry 69: 2920–2927.

Harrison-Lowe, N.J., and Olsen, L.J. (2008). Autophagy protein 6(ATG6) is required for pollen germination in Arabidopsis thaliana.Autophagy 4: 339–348.

Hofius, D., Schultz-Larsen, T., Joensen, J., Tsitsigiannis, D.I.,Petersen, N.H., Mattsson, O., Jørgensen, L.B., Jones, J.D.,Mundy, J., and Petersen, M. (2009). Autophagic componentscontribute to hypersensitive cell death in Arabidopsis. Cell 137:773–783.

Honig, A., Avin-Wittenberg, T., Ufaz, S., and Galili, G. (2012). A newtype of compartment, defined by plant-specific Atg8-interactingproteins, is induced upon exposure of Arabidopsis plants to carbonstarvation. Plant Cell 24: 288–303.

Hu, J., Baker, A., Bartel, B., Linka, N., Mullen, R.T., Reumann, S.,and Zolman, B.K. (2012). Plant peroxisomes: biogenesis andfunction. Plant Cell 24: 2279–2303.

Hummel, J., Segu, S., Li, Y., Irgang, S., Jueppner, J., andGiavalisco, P. (2011). Ultra performance liquid chromatographyand high resolution mass spectrometry for the analysis of plantlipids. Front. Plant Sci. 2: 54.

Ishida, H., Izumi, M., Wada, S., and Makino, A. (2014). Roles ofautophagy in chloroplast recycling. Biochim. Biophys. Acta 1837:512–521.

Izumi, M., Hidema, J., Makino, A., and Ishida, H. (2013). Autophagycontributes to nighttime energy availability for growth in Arabi-dopsis. Plant Physiol. 161: 1682–1693.

Kim, J., Lee, H., Lee, H.N., Kim, S.H., Shin, K.D., and Chung, T.(2013). Autophagy-related proteins are required for degradation ofperoxisomes in Arabidopsis hypocotyls during seedling growth.Plant Cell 25: 4956–4966.

Kopka, J., et al. (2005). GMD@CSB.DB: the Golm Metabolome Database.Bioinformatics 21: 1635–1638.

Laemmli, U.K. (1970). Cleavage of structural proteins during the as-sembly of the head of bacteriophage T4. Nature 227: 680–685.

Li, F., and Vierstra, R.D. (2012). Autophagy: a multifaceted in-tracellular system for bulk and selective recycling. Trends Plant Sci.17: 526–537.

Lisec, J., Schauer, N., Kopka, J., Willmitzer, L., and Fernie, A.R.(2006). Gas chromatography mass spectrometry-based metaboliteprofiling in plants. Nat. Protoc. 1: 387–396.

320 The Plant Cell

Liu, Y., and Bassham, D.C. (2012). Autophagy: pathways for self-eating in plant cells. Annu. Rev. Plant Biol. 63: 215–237.

Liu, Y., Xiong, Y., and Bassham, D.C. (2009). Autophagy is requiredfor tolerance of drought and salt stress in plants. Autophagy 5:954–963.

Liu, Y., Schiff, M., Czymmek, K., Tallóczy, Z., Levine, B., andDinesh-Kumar, S.P. (2005). Autophagy regulates programmedcell death during the plant innate immune response. Cell 121:567–577.

Luedemann, A., Strassburg, K., Erban, A., and Kopka, J. (2008).TagFinder for the quantitative analysis of gas chromatography—mass spectrometry (GC-MS)-based metabolite profiling experi-ments. Bioinformatics 24: 732–737.

Masclaux-Daubresse, C., Clément, G., Anne, P., Routaboul, J.M.,Guiboileau, A., Soulay, F., Shirasu, K., and Yoshimoto, K. (2014).Stitching together the multiple dimensions of autophagy usingmetabolomics and transcriptomics reveals impacts on metabolism,development, and plant responses to the environment in Arabi-dopsis. Plant Cell 26: 1857–1877.

Michaeli, S., Avin-Wittenberg, T., and Galili, G. (2014). Involvementof autophagy in the direct ER to vacuole protein trafficking route inplants. Front. Plant Sci. 5: 134.

Mizushima, N., Yoshimori, T., and Ohsumi, Y. (2011). The role of Atgproteins in autophagosome formation. Annu. Rev. Cell Dev. Biol. 27:107–132.

Nakabayashi, R., Kusano, M., Kobayashi, M., Tohge, T., Yonekura-Sakakibara, K., Kogure, N., Yamazaki, M., Kitajima, M., Saito,K., and Takayama, H. (2009). Metabolomics-oriented isolation andstructure elucidation of 37 compounds including two anthocyaninsfrom Arabidopsis thaliana. Phytochemistry 70: 1017–1029.

Nunes-Nesi, A., Carrari, F., Lytovchenko, A., Smith, A.M., Loureiro,M.E., Ratcliffe, R.G., Sweetlove, L.J., and Fernie, A.R. (2005).Enhanced photosynthetic performance and growth as a conse-quence of decreasing mitochondrial malate dehydrogenase activityin transgenic tomato plants. Plant Physiol. 137: 611–622.

Onodera, J., and Ohsumi, Y. (2005). Autophagy is required formaintenance of amino acid levels and protein synthesis under ni-trogen starvation. J. Biol. Chem. 280: 31582–31586.

Phillips, A.R., Suttangkakul, A., and Vierstra, R.D. (2008). TheATG12-conjugating enzyme ATG10 Is essential for autophagicvesicle formation in Arabidopsis thaliana. Genetics 178: 1339–1353.

Pritchard, S.L., Charlton, W.L., Baker, A., and Graham, I.A. (2002).Germination and storage reserve mobilization are regulated in-dependently in Arabidopsis. Plant J. 31: 639–647.

Rees, T.A., and Beevers, H. (1960). Pathways of glucose dissimila-tion in carrot slices. Plant Physiol. 35: 830–838.

Reggiori, F., and Klionsky, D.J. (2013). Autophagic processes in yeast:mechanism, machinery and regulation. Genetics 194: 341–361.

Reumann, S., Voitsekhovskaja, O., and Lillo, C. (2010). From signaltransduction to autophagy of plant cell organelles: lessons fromyeast and mammals and plant-specific features. Protoplasma 247:233–256.

Roessner-Tunali, U., Liu, J., Leisse, A., Balbo, I., Perez-Melis, A.,Willmitzer, L., and Fernie, A.R. (2004). Kinetics of labelling of or-ganic and amino acids in potato tubers by gas chromatography-mass spectrometry following incubation in (13)C labelled isotopes.Plant J. 39: 668–679.

Roessner-Tunali, U., Hegemann, B., Lytovchenko, A., Carrari, F.,Bruedigam, C., Granot, D., and Fernie, A.R. (2003). Metabolicprofiling of transgenic tomato plants overexpressing hexokinasereveals that the influence of hexose phosphorylation diminishesduring fruit development. Plant Physiol. 133: 84–99.

Rylott, E.L., Eastmond, P.J., Gilday, A.D., Slocombe, S.P., Larson,T.R., Baker, A., and Graham, I.A. (2006). The Arabidopsis thalianamultifunctional protein gene (MFP2) of peroxisomal beta-oxidationis essential for seedling establishment. The Plant journal: for celland molecular biology 45, 930–941.

Ryter, S.W., Cloonan, S.M., and Choi, A.M. (2013). Autophagy:a critical regulator of cellular metabolism and homeostasis. Mol.Cells 36: 7–16.

Schneider, C.A., Rasband, W.S., and Eliceiri, K.W. (2012). NIHImage to ImageJ: 25 years of image analysis. Nat. Methods 9:671–675.

Singh, R., and Cuervo, A.M. (2011). Autophagy in the cellular ener-getic balance. Cell Metab. 13: 495–504.

Sláviková, S., Shy, G., Yao, Y., Glozman, R., Levanony, H.,Pietrokovski, S., Elazar, Z., and Galili, G. (2005). The autophagy-associated Atg8 gene family operates both under favourable growthconditions and under starvation stresses in Arabidopsis plants.J. Exp. Bot. 56: 2839–2849.

Studart-Guimarães, C., Fait, A., Nunes-Nesi, A., Carrari, F.,Usadel, B., and Fernie, A.R. (2007). Reduced expression of succinyl-coenzyme A ligase can be compensated for by up-regulation of thegamma-aminobutyrate shunt in illuminated tomato leaves. Plant Physiol.145: 626–639.

Szecowka, M., Osorio, S., Obata, T., Araújo, W.L., Rohrmann, J.,Nunes-Nesi, A., and Fernie, A.R. (2012). Decreasing the mito-chondrial synthesis of malate in potato tubers does not affectplastidial starch synthesis, suggesting that the physiological regu-lation of ADPglucose pyrophosphorylase is context dependent.Plant Physiol. 160: 2227–2238.

Thompson, A.R., Doelling, J.H., Suttangkakul, A., and Vierstra,R.D. (2005). Autophagic nutrient recycling in Arabidopsis directedby the ATG8 and ATG12 conjugation pathways. Plant Physiol. 138:2097–2110.

Tieman, D., Taylor, M., Schauer, N., Fernie, A.R., Hanson, A.D., andKlee, H.J. (2006). Tomato aromatic amino acid decarboxylasesparticipate in synthesis of the flavor volatiles 2-phenylethanol and2-phenylacetaldehyde. Proc. Natl. Acad. Sci. USA 103: 8287–8292.

Tohge, T., Yonekura-Sakakibara, K., Niida, R., Watanabe-Takahashi, A., and Saito, K. (2007). Phytochemical genomicsin Arabidopsis thaliana: A case study for functional identifica-tion of flavonoid biosynthesis genes. Pure Appl. Chem. 79:811–823.

Wan, L., Ross, A.R., Yang, J., Hegedus, D.D., and Kermode, A.R.(2007). Phosphorylation of the 12 S globulin cruciferin in wild-typeand abi1-1 mutant Arabidopsis thaliana (thale cress) seeds. Bio-chem. J. 404: 247–256.

Wang, Y., Yu, B., Zhao, J., Guo, J., Li, Y., Han, S., Huang, L., Du, Y.,Hong, Y., Tang, D., and Liu, Y. (2013). Autophagy contributes toleaf starch degradation. Plant Cell 25: 1383–1399.

Watanabe, M., Balazadeh, S., Tohge, T., Erban, A., Giavalisco, P.,Kopka, J., Mueller-Roeber, B., Fernie, A.R., and Hoefgen, R.(2013). Comprehensive dissection of spatiotemporal metabolicshifts in primary, secondary, and lipid metabolism during de-velopmental senescence in Arabidopsis. Plant Physiol. 162: 1290–1310.

Welti, R., Li, W., Li, M., Sang, Y., Biesiada, H., Zhou, H.E.,Rajashekar, C.B., Williams, T.D., and Wang, X. (2002). Profilingmembrane lipids in plant stress responses. Role of phospholipase Dalpha in freezing-induced lipid changes in Arabidopsis. J. Biol.Chem. 277: 31994–32002.

Xiong, Y., Contento, A.L., and Bassham, D.C. (2005). AtATG18a isrequired for the formation of autophagosomes during nutrient

Autophagy in Etiolated Arabidopsis 321

stress and senescence in Arabidopsis thaliana. Plant J. 42:535–546.

Xiong, Y., Contento, A.L., Nguyen, P.Q., and Bassham, D.C. (2007).Degradation of oxidized proteins by autophagy during oxidativestress in Arabidopsis. Plant Physiol. 143: 291–299.

Yoshimoto, K., Hanaoka, H., Sato, S., Kato, T., Tabata, S., Noda,T., and Ohsumi, Y. (2004). Processing of ATG8s, ubiquitin-likeproteins, and their deconjugation by ATG4s are essential for plantautophagy. Plant Cell 16: 2967–2983.

Yoshimoto, K., Jikumaru, Y., Kamiya, Y., Kusano, M., Consonni,C., Panstruga, R., Ohsumi, Y., and Shirasu, K. (2009). Autophagynegatively regulates cell death by controlling NPR1-dependentsalicylic acid signaling during senescence and the innate immuneresponse in Arabidopsis. Plant Cell 21: 2914–2927.

Yoshimoto, K., Shibata, M., Kondo, M., Oikawa, K., Sato, M.,Toyooka, K., Shirasu, K., Nishimura, M., and Ohsumi, Y. (2014).Organ-specific quality control of plant peroxisomes is mediated byautophagy. J. Cell Sci. 127: 1161–1168.

322 The Plant Cell

DOI 10.1105/tpc.114.134205; originally published online February 3, 2015; 2015;27;306-322Plant Cell

Bock, Patrick Giavalisco and Alisdair R. FernieTamar Avin-Wittenberg, Krzysztof Bajdzienko, Gal Wittenberg, Saleh Alseekh, Takayuki Tohge, Ralph

Arabidopsis Seedlings under Carbon StarvationGlobal Analysis of the Role of Autophagy in Cellular Metabolism and Energy Homeostasis in

 This information is current as of January 25, 2021

 

Supplemental Data /content/suppl/2015/01/20/tpc.114.134205.DC1.html

References /content/27/2/306.full.html#ref-list-1

This article cites 75 articles, 33 of which can be accessed free at:

Permissions https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X

eTOCs http://www.plantcell.org/cgi/alerts/ctmain

Sign up for eTOCs at:

CiteTrack Alerts http://www.plantcell.org/cgi/alerts/ctmain

Sign up for CiteTrack Alerts at:

Subscription Information http://www.aspb.org/publications/subscriptions.cfm

is available at:Plant Physiology and The Plant CellSubscription Information for

ADVANCING THE SCIENCE OF PLANT BIOLOGY © American Society of Plant Biologists