autophagy deficiency compromises alternative pathways of ... · autophagy deﬁciency compromises...

Autophagy Deficiency Compromises AlternativePathways of Respiration following Energy Deprivation inArabidopsis thaliana1

Jessica A. S. Barros ,a João Henrique F. Cavalcanti ,a David B. Medeiros,a Adriano Nunes-Nesi,a

Tamar Avin-Wittenberg,b,c Alisdair R. Fernie,b and Wagner L. Araújoa,2

aMax Planck Partner Group at the Departamento de Biologia Vegetal, Universidade Federal de Viçosa,36570-900 Viçosa, Minas Gerais, BrazilbMax Planck Institute of Molecular Plant Physiology, D-14476 Potsdam-Golm, GermanycDepartment of Plant and Environmental Sciences, Alexander Silberman Institute of Life Sciences, HebrewUniversity of Jerusalem, Givat Ram, Jerusalem 9190401, Israel

ORCID IDs: 0000-0002-1136-6564 (J.H.F.C.); 0000-0001-9086-730X (D.B.M.); 0000-0002-4796-2616 (W.L.A.).

Under heterotrophic conditions, carbohydrate oxidation inside the mitochondrion is the primary energy source forcellular metabolism. However, during energy-limited conditions, alternative substrates are required to supportrespiration. Amino acid oxidation in plant cells plays a key role in this by generating electrons that can be transferredto the mitochondrial electron transport chain via the electron transfer flavoprotein/ubiquinone oxidoreductase system.Autophagy, a catabolic mechanism for macromolecule and protein recycling, allows the maintenance of amino acid poolsand nutrient remobilization. Although the association between autophagy and alternative respiratory substrates has beensuggested, the extent to which autophagy and primary metabolism interact to support plant respiration remains unclear. Toinvestigate the metabolic importance of autophagy during development and under extended darkness, Arabidopsis(Arabidopsis thaliana) mutants with disruption of autophagy (atg mutants) were used. Under normal growth conditions, atgmutants showed lower growth and seed production with no impact on photosynthesis. Following extended darkness, atgmutants were characterized by signatures of early senescence, including decreased chlorophyll content and maximumphotochemical efficiency of photosystem II coupled with increases in dark respiration. Transcript levels of genes involvedin alternative pathways of respiration and amino acid catabolism were up-regulated in atg mutants. The metabolite profiles ofdark-treated leaves revealed an extensive metabolic reprogramming in which increases in amino acid levels were partiallycompromised in atg mutants. Although an enhanced respiration in atg mutants was observed during extended darkness,autophagy deficiency compromises protein degradation and the generation of amino acids used as alternative substrates tothe respiration.

Energy availability is a key factor influencing bothplant growth and development. Accordingly, plantsobtain their energy both by harvesting light energyduring photosynthesis and by oxidation of organicsubstrates via mitochondrial respiration. The latterprocess is mostly dependent on carbohydrates throughthe oxidation of organic acids by the tricarboxylic acidcycle (Plaxton and Podesta, 2006; Sweetlove et al., 2010;Araújo et al., 2011). In situations where carbohydratesbecome limiting, including environmental stress condi-tions and natural plant senescence, plant cells are forcedto use alternative substrates, such as lipids and aminoacids, to provide energy and maintain active mitochon-drial metabolism (Buchanan-Wollaston et al., 2005;Araújo et al., 2011; Kirma et al., 2012).

Compelling evidence has demonstrated that aminoacids produced from protein degradation can be an im-portant source of alternative substrates for plant respira-tion, supporting ATP synthesis through a route that differsfrom the classical respiratory pathway (Araújo et al., 2010;Engqvist et al., 2011; Peng et al., 2015). The electron transferflavoprotein (ETF)/ETF:ubiquinone oxidoreductase (ETF/

ETFQO) system has been characterized substantially inmammals for the catabolism of fatty acids, amino acids,and choline (Watmough and Frerman, 2010). Although theentire pathway for the degradation of branched-chainamino acids (BCAAs) has been found in plant mitochon-dria (Salvato et al., 2014), only two dehydrogenases havebeen identified to date in plants as able to donate electronsto the ETF/ETFQO complex. These two enzymes are (1)isovaleryl-CoAdehydrogenase (IVDH),which is involvedin the degradation of BCAAs, and (2) 2-hydroxyglutaratedehydrogenase (D2HGDH), which uses Lys as an alter-native substrate (Engqvist et al., 2009, 2011; Araújo et al.,2010). These alternative pathways can provide electronsfrom amino acid oxidation directly to the mitochondrialelectron transport chain via the ETF complex as well as bythe direct feeding of catabolic products into the tricar-boxylic acid cycle (Ishizaki et al., 2005, 2006; Araújo et al.,2010, 2011; Kirma et al., 2012). Notably, not only the de-hydrogenases related to the ETF/ETFQO complex butalso enzymes associated with amino acid catabolism ingeneral (Hildebrandt et al., 2015), including the oxi-dation of sulfur-containing amino acids such as Cys

62 Plant Physiology�, September 2017, Vol. 175, pp. 62–76, www.plantphysiol.org � 2017 American Society of Plant Biologists. All Rights Reserved. www.plantphysiol.orgon June 27, 2020 - Published by Downloaded from

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http://orcid.org/0000-0002-1136-6564

http://orcid.org/0000-0002-1136-6564

http://orcid.org/0000-0002-1136-6564

http://orcid.org/0000-0002-1136-6564

http://orcid.org/0000-0002-1136-6564

http://orcid.org/0000-0001-9086-730X

http://orcid.org/0000-0001-9086-730X

http://orcid.org/0000-0001-9086-730X

http://orcid.org/0000-0001-9086-730X

http://orcid.org/0000-0001-9086-730X

http://orcid.org/0000-0002-4796-2616

http://orcid.org/0000-0002-4796-2616

http://orcid.org/0000-0002-4796-2616

http://orcid.org/0000-0002-4796-2616

http://orcid.org/0000-0002-4796-2616

http://orcid.org/0000-0002-1136-6564

http://orcid.org/0000-0001-9086-730X

http://orcid.org/0000-0002-4796-2616

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and Met by the Ethylmalonic Encephalopathy Pro-tein1 (Krüßel et al., 2014), have been associated withmitochondrial metabolism.The physiological role of the ETF/ETFQO system

during plant stress responses has been demonstratedunequivocally (for review, see Araújo et al., 2011). Addi-tionally, several studies have demonstrated the inductionof transcripts of these proteins during dark-induced se-nescence (Buchanan-Wollaston et al., 2005), oxidativestress (Lehmann et al., 2009), and under conditions inwhich free amino acids are plentiful (Weigelt et al., 2008).Furthermore, the function of this alternative pathway,and by corollary of BCAA catabolism in stress tolerancemechanisms including drought and carbon starvation,have been demonstrated (Ishizaki et al., 2005, 2006;Araújo et al., 2010; Engqvist et al., 2011; Peng et al.,2015; Pires et al., 2016). Although these studies haveclearly enhanced our understanding concerning theuse of amino acids as respiratory substrates, the func-tional linkage between protein degradation, amino acidturnover, and the alternative pathways of respirationremains to be fully elucidated.Macroautophagy (referred to hereafter as autophagy)

is a highly conserved and regulated catabolic processinvolved in the degradation of cytoplasmic constituentsincluding soluble proteins, protein aggregates, or evenentire organelles, allowing the recycling of cell com-ponents into primary molecules (Li and Vierstra, 2012;Liu and Bassham, 2012; Zientara-Rytter and Sirko,2016). Briefly, during this process, cell components aresequestered by the autophagosomes and deliveredinto the vacuoles, where this material is then degradedand macromolecules are released back into the cytosolfor reuse (Feng et al., 2014). The knowledge of autophagicmechanisms has been expanded primarily through ge-netic analyses in Saccharomyces cerevisiae, leading to the

identification of autophagy-related (ATG) genes that en-code the central autophagymachinery. Homologs of ATGgenes also have been identified in plants, and their func-tions are similar to those in yeast cells (Liu and Bassham,2012; Lv et al., 2014; Michaeli et al., 2016). The physio-logical importance of autophagy has been demonstratedextensively through the characterization of several Ara-bidopsis (Arabidopsis thaliana) loss-of-functionmutants (atgmutants). Early senescence and hypersensitivity to carbonstarvation are commonly observed phenotypes in atgmutants, providing evidence of a significant role of theautophagic process in nutrient recycling, particularly un-der stress conditions (Doelling et al., 2002; Bassham, 2009;Wada et al., 2009; Li and Vierstra, 2012; Liu and Bassham,2012; Yoshimoto et al., 2014). Notably, the involvement ofautophagy in providing respiratory substrates followingstress conditionswas demonstrated only recently. Growthimpairments under both long- and short-day conditionshave been observed in the Arabidopsis starchless atgdouble mutant, which was associated with reduced sol-uble sugar availability during the night (Izumi et al., 2013).Since amino acids can be used as alternative substrates forenergy supply following carbon starvation (Araújo et al.,2010), autophagy seems to have a potential role in ener-getic maintenance under this condition. Decreased levelsof free amino acids also have been reported in etiolatedArabidopsis seedlings following carbon starvation (Avin-Wittenberg et al., 2015). The reduced levels of BCAA andLys coupled with the increased redistribution of labeledLys to malate indicate a potential role for amino acids insupporting the respiratory flux observed in atg mutants(Avin-Wittenberg et al., 2015).

Although the importance of autophagy undernutrient-starved and other stressful conditions has beendemonstrated, the exact linkage between autophagy andalternative pathways of respiration remains unclear.Here, we investigated how primary metabolism andphysiological aspects are impaired in three indepen-dent atg T-DNA insertion mutant lines during dark-induced senescence. We used mutants for ATG5 andATG7 genes that have a full inhibition of autophagy(Thompson et al., 2005; Phillips et al., 2008; Shin et al.,2014) and the atg9-1 mutant that displayed a milderreduction of the autophagic process (Shin et al., 2014;Zhuang et al., 2017). Our results demonstrate an early-senescence phenotype coupled with an accumulationof organic acids following extended darkness in all atgmutants. Reduced levels of several amino acids in theatg mutants were associated with an induction at thetranscriptional level of enzymes of the ETF/ETFQOpathway following extended darkness. Collectively,the data obtained indicate that metabolite recyclingduring autophagy and alternative pathways of respi-ration are both required to provide correct respiratoryfunction under conditions of carbon starvation. Theresults are discussed in the context of the importanceof the autophagic process and the current models ofreserve mobilization and alternative pathways of res-piration during extended dark-induced senescence inleaves.

1 This work was supported by funding from the Max Planck So-ciety, the National Council for Scientific and Technological Develop-ment (CNPq-Brazil grant no. 402511/2016-6), and the Foundation forResearch Assistance of the Minas Gerais State (FAPEMIG-Brazilgrant no. APQ-01078-15) to W.L.A. Scholarships granted by theAgency for the Support and Evaluation of Graduate Education(CAPES-Brazil) to J.A.S.B., FAPEMIG to J.H.F.C (grant no BDP-00018-16) and D.B.M., and research fellowships granted by CNPqto A.N.-N. and W.L.A. are gratefully acknowledged. The work per-formed by T.A.-W. was supported by Minerva, Alexander vonHumboldt, and EMBO postdoctoral fellowships.

2 Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Wagner L. Araújo ([email protected]).

J.A.S.B., J.H.F.C., T.A.-W., and W.L.A. designed the research; J.A.S.B.performed most of the research; J.H.F.C. and W.L.A. supervised theproject; D.B.M. and A.N.-N contributed new reagents/analytic tools;A.N.-N. and A.R.F. analyzed the data, discussed the results, andcomplemented the writing; J.A.S.B., J.H.F.C., T.A.-W., A.R.F., andW.L.A. analyzed the data and wrote the article with contributionsof all the authors.

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Autophagy and Alternative Respiratory Pathways

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RESULTS

Characterization of T-DNA Insertional Mutants ofATG Genes

To examine the involvement of autophagy in meta-bolic responses during plant development and carbonlimitation, we analyzed three previously describedloss-of-function mutants of the autophagy pathway,namely (1) ATG5 (atg5-1; Thompson et al., 2005), (2)ATG7 (atg-7-2; Hofius et al., 2009), and (3) ATG9 (atg-9-1;Hanaoka et al., 2002). To this end, the homozygosity ofeach mutant line was confirmed using primer pairsdesigned to span the T-DNA insertion sites of each locus.The Arabidopsis glyceraldehyde 3-phosphate dehy-drogenase (GAPDH) gene was used as a control todemonstrate the integrity and quantity of the RNApreparation (Supplemental Fig. S1D). ATG5, ATG7,andATG9mRNAswere detected in the wild-type control(Columbia-0) using the primer sets L1/R1, L2/R2, andL3/R3, respectively (Supplemental Fig. S1, A–C). How-ever, no amplification products for the gene correspond-ing to the mutation were observed in the atg mutants(Supplemental Fig. S1D), confirming that transcriptsspanning the T-DNA insertion site are absent in thesemutant lines.

Importance of Autophagy for Both Growth and Seed Yield

In order to obtain further insight into the consequencesof the lack of ATG genes under optimal short-day con-ditions, mutant plants were grown side by side with theirrespective wild-type controls in order to evaluate theirmorphological and physiological characteristics. Mor-phological analysis revealed a reduction of rosette areaand a decrease in both fresh and dry weight matter butonly in atg7-2 mutants (Table I). The specific leaf area,however, was invariant between wild-type plants and allatg mutant lines (Table I).

Given that the disruption of autophagy seems to resultin minor growth impairments, we next evaluated a rangeof physiological parameters in order to assess whetherchanges in growthmay be associatedwith an alteration inthose parameters. In 4-week-old plants, no differenceswere observed in net assimilation rate (SupplementalFig. S2A), stomatal conductance (Supplemental Fig.S2B), or internal CO2 concentration (Supplemental Fig.S2C). Thus, although autophagy seems to be a limiting

factor for normal growth in Arabidopsis plants, this isunlikely to be related to the photosynthetic efficiencyof the lines.

We next investigated the impact of the autophagicprocess during the reproductive stage. Although a re-duction in the number of seeds per siliquewas observed(Fig. 1A), nomajor changes in 1,000 seeds weight (Fig. 1B)or in silique length (Fig. 1C) were found in atg mutants.The total number of siliques per plant was reduced in atgmutants (Fig. 1E)with no changes in branch numbers (Fig.1F), leading to a reduction in seed yield per plant in atgmutants (Fig. 1D), in agreement with a previous obser-vation of lower total seed weight in Arabidopsis plantslacking core components of the ATG system (Guiboileauet al., 2012).

atg Mutants Are More Susceptible to Energy Deprivation

Following the confirmation of the molecular identityof the T-DNA insertionalmutants and given that previousstudies have implied the function of autophagy understress conditions (Izumi et al., 2013;Avin-Wittenberg et al.,2015), we next transferred 4-week-old mutant plants,alongside wild-type plants, to carbon starvation inducedby extended dark conditions. Under these conditions, arange of phenotypes became apparent (Fig. 2). The atg5-1and atg7-2 mutants started to wilt and show signs of se-nescence after only 6 d of continuous darkness, and bothmutants were apparently dead after 12 d of continuousdarkness (Supplemental Fig. S3). It should be pointed outthat wild-type plants were still alive and exhibited onlylimited signs of senescence and no visible abnormalitiesafter 12 d of continuous darkness. The atg9-1mutant alsoshowed signs of senescence after 9 d of continuous dark-ness, but with a less severe phenotype compared withatg5-1 and atg7-2 mutants, thus showing an intermediatesenescence phenotype between wild-type plants and theother two atg mutant lines (Fig. 2A).

In order to further investigate the accelerated senes-cence symptoms, two parameters related to chloroplastfunction, chlorophyll content and maximum photochem-ical efficiency of PSII (maximum variable fluorescence/maximum yield of fluorescence [Fv/Fm]), were analyzed(Fig. 2, B and C). During the extended dark treatment,chlorophyll content declined more rapidly in the mutantsthan in the wild type (Fig. 2B). Accordingly, these resultswere associated with a more rapid decline in Fv/Fm in

Table I. Growth parameters of atg mutants (4-week-old plants)

Values presented are means 6 SE of at least six independent biological replicates per genotype. Values in boldface were determined by Student’st test to be significantly different (P , 0.05) from the wild type.

ParameterGenotype

Wild Type atg5-1 atg7-2 atg9-1

Fresh weight (mg) 88.4 6 9.10 77.8 6 3.94 62.3 6 4.33 91.7 6 10.91Dry weight (mg) 9.9 6 1.19 7.6 6 0.44 5.9 6 0.30 8.62 6 0.92Rosette area (cm2) 30.0 6 2.15 29.0 6 1.28 24.6 6 0.30 30.5 6 1.88Specific leaf area (cm2 g21) 426 6 15.4 460 6 19.9 482 6 20.8 449 6 34.7

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atg5-1 and atg7-2 mutants after 6 d of darkness (Fig. 2C).By contrast, Fv/Fm values in wild-type and atg9-1 linesremained similar throughout the entire time period of theexperiment (Fig. 2C). Thus, these parameters are in goodagreement with an early-senescence phenotype observedin both atg5-1 and atg7-2mutants in comparison with thewild type and atg9-1.

The Respiratory Response of atg Mutants UnderDark-Induced Starvation

Given the severe phenotype of atg mutants undercarbon starvation and that alternative pathways ofrespiration are important for plant survival duringdarkness (Ishizaki et al., 2005, 2006; Araújo et al., 2010),we next investigated the connection between autoph-agy and plant respiration. To this end, we evaluated theCO2 gas-exchange rate, since leaf respiration representsa major source of CO2 release in plants (Atkin et al.,2000). Dark respiration rates measured immediately atthe start of the dark treatment (day 0) were virtuallyinvariant between the genotypes, indicating that au-tophagy deficiency has a minor impact on plant respi-ration under nonstressed conditions prior to bolting(Fig. 3). Interestingly, dark respiration rates were re-duced after 3 d under darkness in all genotypes, andsuch low levels of dark respiration were maintained inwild-type plants during extended darkness. Following

darkness, the atgmutants also presented a reduction ofdark respiration, but this reduction seemed to be lowerthan the one observed in wild-type plants. In addition,we observed that, after 6 d of treatment, dark respira-tion had increased again in atg5-1 and atg7-2 mutants,reaching higher levels after 9 d under darkness. It wasalso observed that dark respiration rates remainedsimilar to those of the wild type in the atg9-1 mutants,with a significant increase starting only from 9 d afterdarkness (Fig. 3).

Deficiency of Autophagy Leads to a Differential MetabolicResponse following Carbon Starvation

To elucidate the connection between autophagy andnutrient recycling following carbon starvation, we fur-ther conducted a detailed metabolic analysis in leavesduring the extended dark treatment. It is important tomention that all genotypes used in this study showedsimilar levels of total soluble proteins, total amino acids,organic acids, soluble sugars, and starch in samples har-vested immediately prior to the start of the dark treatment(day 0; Fig. 4; Supplemental Fig. S4).

As might be expected, starch levels declined rapidlyfrom 3 d of dark treatment onward (Fig. 4A). Interest-ingly, after 9 d of darkness, higher starch content wasobserved in atg5-1 and atg7-2 mutants coupled withlower levels of Suc and Glc in atg mutants compared

Figure 1. Seed and silique phenotypesobserved in Arabidopsis atg mutants. A,Number of seeds per silique. B, Seedweight. C, Silique length. D, Seed yield. E,Total siliques per plant. F, Branch number.Seed weight was obtained by measuring500 seeds (n = 10). Silique length (C) wasdetermined in images taken with a digitalcamera (Canon Powershot A650 IS) at-tached to a stereomicroscope (Zeiss Stemi2000-C). The measurements were per-formed on the images using ImageJ soft-ware. Values presented are means6 SE ofat least 10 biological replicates per geno-type. Asterisks designate values that weredetermined by Student’s t test to be sig-nificantly different (P , 0.05) from thewild type (WT).






with wild-type plants (Supplemental Fig. S4). Whilethese changes are striking, it was suggested previouslythat autophagy contributes to leaf starch degradation(Wang et al., 2013).

Given that proteins are degradation targets of theautophagy machinery (Li and Vierstra, 2012), we nextdecided to examine the protein content during the ex-tended dark treatment. Thus, while total protein con-tent decreased during dark treatment in all genotypes(Fig. 4B), the levels were reduced to a lesser extent in themutants, especially in atg5-1 and atg9-1 lines, in com-parison with wild-type plants. Increases in the levels oftotal amino acids were observed throughout the darktreatment, most likely as a result of enhanced proteindegradation prompted by the carbon starvation con-ditions (Fig. 4C). Accordingly, after 9 d of darkness, theamino acid content was significantly higher in atg5-1and atg7-2 mutants, while no change was observed inthe atg9-1 mutant.

In order to obtain a more detailed characterization ofchanges in the metabolism of atg mutants, we next de-cided to extend this study to encompass the majorpathways of metabolism bymetabolite profiling, whichwas able to provide information on 30 primary me-tabolites across the experiment. We observed consid-erable changes in the levels of a wide range of organicacids, amino acids, and sugars in atg mutants in re-sponse to dark treatment (Figs. 5 and 6). The tricar-boxylic acid cycle intermediates citrate, fumarate,malate, and pyruvate were increased in atg mutants

at the end of the dark treatment, mainly in atg5-1 andatg7-2 genotypes (Fig. 5). By contrast, in wild-typeplants, the levels of these organic acids were constant

Figure 3. Dark respiration during extended dark treatment. The CO2

efflux rates of 4-week-old Arabidopsis plants immediately after light wasturned off (0 d) and during further treatment for 9 d in darkness wereanalyzed. Gas-exchange measurements were performed with anopen-flow infrared gas-exchange analyzer system with a portablephotosynthesis system to fit a whole-plant cuvette. Values presentedare means 6 SE of seven biological replicates per genotype. Asterisksdesignate values that were determined by Student’s t test to be sig-nificantly different (P , 0.05) from the wild type (WT).

Figure 2. Phenotypes of Arabidopsis atgmutants under extended dark treatment.A, Images of 4-week-old, short-day-grownArabidopsis plants immediately light wasturned off (0 d) and after further treatmentfor 9 d in darkness conditions. B and C,Chlorophyll (Chl) content (B) and Fv/Fm (C)of leaves of 4-week-old, short-day-grownArabidopsis plants after further treatmentfor 0, 3, 6, and 9 d in extended darkness.Values are means6 SE of five independentsamplings. Asterisks indicate values thatwere determined by Student’s t test to besignificantly different (P , 0.05) from thewild type (WT) at each time point ana-lyzed. FW, Fresh weight.


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or tended to be reduced, indicating an altered opera-tion of the tricarboxylic acid cycle in the atg mutantsduring darkness. Although not different from wild-type plants, the levels of oxaloacetate clearly increasedwhereas the levels of dehydroascorbate were reduceddramatically at the end of the dark treatment, decliningto as low as 35% of the levels measured at the start oftreatment. In agreement with our spectrometric assays(Supplemental Fig. S4), reduced levels of sugars wereobserved in all genotypes starting after 3 d of darkness. Itis important to mention that minor differences, includ-ing significantly reduced levels of Suc, Glc, and Fru inatg mutants, were observed after 3 d of darkness.

Additionally, increased trehalose levels starting from 6 dof darkness were observed in all mutant lines coupledwith the absence of changes in maltose levels (Fig. 5).

Analyses of the levels of individual amino acidsrevealed that Asn, Ile, Leu, Lys, Met, Phe, Trp, Tyr, andVal increased significantly in all genotypes following darktreatment, while the levels of Ala were reduced (Fig. 6).Intriguingly, some metabolites showed distinct directionof changes with respect to the wild type, as in the case ofb-Ala, Orn, and Ser, which only increased in the wild typeand atg9-1, whereas Asp, His, and Gly increased more inatg5-1 and atg7-2mutants (Fig. 6). Although the Gln levelswere virtually constant in wild-type plants following darktreatment, reductions in the levels of this amino acid wereobserved for both atg5-1 and atg7-2 mutant lines. Interest-ingly, increases in BCAAs (Leu, Ile, and Val), Lys, and Tyrobserved in wild-type plants following dark treatmentwere less pronounced in the atgmutants.

Carbon Starvation Leads to the Induction of AlternativePathways in atg Mutants

In order to investigate whether autophagic impair-ment coupled with amino acid degradation has an in-fluence on alternative respiratory pathways, expressionanalysis of genes related to the ETF/ETFQO systemwas performed by quantitative reverse transcription(RT)-PCR (Fig. 7). During carbon starvation, the im-portance of BCAAs, aromatic amino acids, and Lys asalternative respiratory substrates was demonstratedvia the characterization of loss-of-function mutantsfor IVDH, D2HGDH, ETF, and ETFQO (Ishizaki et al.,2005, 2006; Araújo et al., 2010). Here, we demonstratedthat the transcript levels of IVDH, ETFQO, ETFb, andD2HGDH were generally induced in atg5-1 and atg7-2in comparison with the levels observed in wild-typeplants, while a mild induction was observed in atg9-1mutants when compared with the other atg mutantsunder extended dark treatment (Fig. 7, A–D). Morespecifically, ETFb was only up-regulated in atg5-1 andatg7-2 plants after 6 d of darkness, with no changesobserved in either wild-type or atg9-1mutant plants (Fig.7A). In addition, ETFQO was up-regulated followingdark treatment in all genotypes, but with higher expres-sion levels in atg mutants after 6 d of darkness (Fig. 7B).Also, there was an early induction of IVDH transcripts inboth the wild type and atg mutants after 3 d of darkness(Fig. 7C). Such strong induction of IVDH, reachingincrements higher than 20-fold after 3 d of dark tran-sition, reinforces claims of its pivotal role in amino aciddegradation (Araújo et al., 2010; Peng et al., 2015). Giventhat Lys catabolism can occur by either D2HGDH or Lys-ketoglutarate reductase/saccharopine dehydrogenase(LKR/SDH; Engqvist et al., 2009, 2011; Galili, 2011; Kirmaet al., 2012), we next decided to investigate the changes inthe expression of these genes. The expression ofD2HGDHwas only up-regulated in atg5-1 and atg7-2 plants after 6 dof darkness,with no changes in the other genotypes studiedhere (Fig. 7D). Interestingly, the expression of LKR/SDHwas induced strongly in all genotypes following dark

Figure 4. Metabolite levels in Arabidopsis atgmutants. Starch (A), protein(B), and amino acids (C)weremeasured usingwhole rosettes of 4-week-oldshort-day-grown Arabidopsis plants after further treatment for 0, 3, 6, and9d in extendeddarkness. Values presented aremeans6 SE of fivebiologicalreplicates per genotype. Asterisks designate values thatwere determined byStudent’s t test to be significantly different (P , 0.05) from the wild type(WT) at each time point analyzed. FW, Fresh weight.






treatment, with higher induction being observed in atg5-1 and atg7-2 following 6 and 9 d of darkness (Fig. 7E).Our data showed a higher induction of LKR/SDH (about200-fold) than of D2HGDH (10-fold; Fig. 7, D and E).

Impairment of Autophagy Induces Senescence andChloroplast Degradation Events

Given that several senescence parameters were in-duced in response to darkness, we next investigated theexpression of the commonly known senescence-associatedgenes SAG12 and SAG13 during dark-induced senescence.Interestingly, although no changes in the transcript levels

of SAG12 or SAG13 were observed in either wild-type oratg9-1 mutant plants following darkness, it was observedthat, in atg5-1 and atg7-2 mutants, the transcript levels ofSAG12were highly induced at all time points coupledwithan up-regulation of SAG13 after 6 d of darkness (Fig. 7, FandG). Taken togetherwith chlorophyll content andFv/Fmvalues, these results are in good agreement with an early-senescence phenotype observed in those genotypes.

The degradation of chloroplasts is a hallmark of bothnatural and stress-induced plant senescence (Ishida et al.,2014), and autophagy is an established cellular pathwayinvolved in targeting chloroplast proteins for degradation(Ishida et al., 2008, 2014; Michaeli et al., 2014; Izumi et al.,

Figure 5. Relative levels of sugars and organic acids in Arabidopsis atgmutants during extended dark conditions as measured bygas chromatography-mass spectrometry (GC-MS). The y axis values represent the metabolite level relative to the wild type (WT).Data were normalized relative to the mean content calculated for the 0-d dark-treated leaves of the WT (in case no response wasdetected at 0 d, normalizationwas performed against 3-d dark-treated leaves of the wild type). Values presented aremeans6 SE offive biological replicates per genotype. Asterisks designate values that were determined by Student’s t test to be significantlydifferent (P , 0.05) from the WT at each time point analyzed.


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Figure 6. Relative levels of amino acids in Arabidopsis atgmutants during extended dark conditions as measured by GC-MS.The y axis values represent the metabolite level relative to the wild type (WT). Data were normalized to the mean responsecalculated for the 0-d dark-treated leaves of the WT (in case no response was detected at 0 d, normalization was performedagainst 3-d dark-treated leaves of the wild type). Values presented are means 6 SE of five biological replicates per genotype.Asterisks designate values that were determined by Student’s t test to be significantly different (P, 0.05) from theWTat eachtime point analyzed.

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2017). Recently, an autophagy-independent pathway forchloroplast degradation, the chloroplast vesiculation (CV)pathway, which is associated with thylakoid and stromaprotein degradation, was demonstrated unequivocally(Wang and Blumwald, 2014). Thus, we next investigatedthe expression of the CV gene during our experimentalconditions. Interestingly, a higher induction of CV geneexpression was observed in atg5-1 and atg7-2 mutantsunder dark-induced senescence (110- and 60-fold at 9 d ofdarkness, respectively), while the transcript levels wereessentially invariant inwild-type plants and showedonly aminor induction in atg9-1 mutants (Fig. 7H).

DISCUSSION

During the last decade, a growing body of evidence hasemerged showing the function of autophagy in nutrientrecycling under energy-limited conditions (Thompson

et al., 2005; Phillips et al., 2008; Chung et al., 2010; Izumiet al., 2010). Although the connection between autophagy,protein degradation, and amino acid availability duringenergetic limitation has been demonstrated (Izumi et al.,2013; Avin-Wittenberg et al., 2015), our current under-standing of the metabolic process associated with energysupply following carbon starvation remains fragmentary.Here, we provide further evidence of the importance ofautophagy in governing an exquisite metabolic reprog-ramming during both dark-induced carbon starvation anddevelopmental transitions in the plant life cycle.

The Significance of Autophagy during Plant Development

By using well-characterized autophagy-deficient mu-tants, we provide further evidence that this process im-pacts both vegetative and reproductive development(Fig. 1; Table I). A reduction of growth was observed forthe atg7-2 mutant (Table I). Growth inhibition also has

Figure 7. Changes in transcript levelsin 4-week-old, short-day-grownArabidopsis plants after further treat-ment for 0, 3, 6, and 9 d in extendeddarkness. Transcript abundance isshown for genes associated with thealternative pathways of respiration,including ETFb (A), ETFQO (B),IVDH (C), D2HGDH (D), LKR/SDH(E), SAG12 (F), SAG13 (G), and CV(H). The y axis values represent themetabolite level relative to the wildtype (WT). Data were normalizedwith respect to the mean responsecalculated for the 0-d dark-treatedleaves of the wild type. Values pre-sented are means 6 SE of three in-dependent biological replicates.Asterisks indicate values that weredetermined by Student’s t test to besignificantly different (P , 0.05)from the wild type at each timepoint analyzed.


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been observed in atg mutants grown under both short-day conditions and mineral-rich medium without Suc,providing a mechanism where the autophagic processparticipates in nighttime energy availability and sustainsgrowth (Izumi et al., 2013). Furthermore, it was observedthat the lack of an autophagic process culminates in anegative impact on seed production (Fig. 1D). Lowerseed yield already has been demonstrated in atgmutants(Avila-Ospina et al., 2014), and impairments of nutrientremobilization also have been associatedwith the lack orreduction of autophagy (Avila-Ospina et al., 2014; Li et al.,2015). Therefore, it is highly tempting to suggest that theimpaired reproductive growth phenotype of atg mutantscan be at least partly related to the impairment of proteindegradation and remobilization processes during seedformation (Guiboileau et al., 2012; Li et al., 2015). Thatsaid, the exact mechanistic relationship between energeticmetabolism, seed production, and autophagy itself will beexamined in detail in future studies.

Autophagy Plays a Pivotal Role in Plant Survival andRespiration during Extended Darkness

In addition to the function of autophagy during devel-opmental processes, autophagy seems to be strictly nec-essary for plant cell survival following carbon starvation.The first evidence was the early onset of dark-inducedsenescence observed in atg mutants accompanied by theloss of chlorophyll and photosynthetic competence(Fig. 2). In agreement with the observed phenotype,senescence-associated genes such as SAG12 and SAG13were up-regulated mainly in atg5-1 and atg7-2 mutantsduring dark treatment (Fig. 7, F and G). Since a largenumber of reporters showed that SAG12 is usuallyup-regulated in natural senescence but not in dark-induced senescence (Noh and Amasino, 1999; Weaverand Amasino, 2001; Grbi�c, 2003), it is reasonable to as-sume that the mildly induced senescence observed inwild-type plants was not sufficient to induce the tran-scription of SAG12 and SAG13. However, transcriptinduction of SAG12 and SAG13 in atg5-1 and atg7-2mutants can be at least partially associated with changesin the salicylic acid signaling and accumulation usuallyobserved in atg mutants (Morris et al., 2000; Yoshimotoet al., 2009; Zhao et al., 2016).To decipher the autophagy function in the mainte-

nance of cellular energy status, we paid particular at-tention to the respiration of atgmutants. The respiratoryrates were reduced in all genotypes as a consequence ofcarbon depletion with extended treatment (Fig. 3), and itwas kept low in wild-type plants during darkness. Inagreement, a lower respiratory rate was observed pre-viously in dark-treated Arabidopsis plants (Keech et al.,2007) as well as in cell culture under Suc starvation(Contento et al., 2004). The atg mutants also showed re-duction of total dark respiration after 3 d in darkness;however, increaseswere observed during extended darktreatment (after 6 and 9 d) in atg 5-1 and atg7-2 and rel-atively later and to a lesser extent in atg9-1 plants (Fig. 3).

It seems reasonable to assume that wild-type plants arewaiting in a so-called standby mode for better subopti-mal environmental conditions (Keech et al., 2007). Insharp contrast, atg mutants are not able to fine-tune thisbasal metabolism.

Interestingly, we also observed higher levels of tri-carboxylic acid cycle intermediates from 6 d of darknessin atg 5-1, atg7-2, and, to a lesser extent, atg9-1 plants(Fig. 5). When considered together with higher CO2rates, these results suggest a misregulation of respira-tory metabolism characterized by a higher flux throughthe tricarboxylic acid cycle as a consequence of the res-piratory activity in atg mutants. This conclusion is con-sistent with previous observations of the flow of labeledcarbon showing that (1) a higher proportion of carbo-hydrate oxidation occurs via the tricarboxylic acid cycleand (2) increased label distribution in malate pools from[13C]Lys occurs in atg5-1 etiolated seedlings, indicating apossible higher flux for respiration under carbon deple-tion (Avin-Wittenberg et al., 2015).

Autophagy Deficiency Compromises Amino Acid Releaseand Impacts Alternative Pathways of Respiration

The levels of the majority of the amino acids gener-ally increased within the first 3 d of darkness, albeit to alesser extent in atg mutants (Fig. 6). BCAAs, aromaticamino acids, and Lys have been characterized previ-ously as alternative substrates able to sustain respirationduring carbon starvation (Ishizaki et al., 2005, 2006;Araújo et al., 2010). Our findings demonstrate that, fol-lowing dark treatment, increases in the amounts of theseamino acids were partially compromised in atgmutantsin comparisonwithwild-type plants (Fig. 6). It should bementioned that a minor impact on the levels of BCAAsand Lys (Fig. 6) as well as amilder sensitivity phenotypeunder darkness were observed in atg9-1 in relation toatg5-1 and atg7-2 mutants (Fig. 2). Accordingly, bothATG5 and ATG7 participate in autophagosome forma-tion by ubiquitin-like conjugating systems, and as such,correspondentArabidopsismutants fail to formautophagicstructures (Li andVierstra, 2012; Liu andBassham, 2012; Lvet al., 2014). On the other hand, althoughATG9 operates inlipid delivery for phagophore growth, it seems not to bestrictly necessary for the whole autophagic flux (Shin et al.,2014; Zhuang et al., 2017). Thus, autophagic activity is notfully blocked in the atg9 mutants, which can explain therelatively minor phenotype observed in this mutant line.Nonetheless, our data indicate that functional autophagy isprobably required to allow the precise provision of ener-getic substrates, since the same pattern of reduced levels ofmost amino acids also was observed in other reports thatalso used atg mutants following carbon starvation (Izumiet al., 2013; Avin-Wittenberg et al., 2015).

The use of amino acids under energy-limited condi-tions is seemingly related to the operation of alternativepathways of respiration. In this scenario, we observed asignificant transcript induction of genes involved withalternative respiration (ETFb, ETFQO, IVDH, and





D2HGDH) and Lys catabolism (LKR/SDH) mainly inatg5-1 and atg7-2 mutants following extended darkness(Fig. 7, A–E). The induction of amino acid degradationand alternative pathways of respiration has been ob-served during prolonged darkness and developmentalleaf senescence (Ishizaki et al., 2005, 2006; Peng et al.,2015; Chrobok et al., 2016). The transcriptional regula-tion seems to be dependent on the energy status of plantsandmight contribute to the reduced amino acid levels ofatg mutants. Thus, when there is autophagy deficiency,the generation of amino acids following protein degra-dation is compromised but the alternative pathwaysof respiration are induced, leading to an even higherreduction in the levels of amino acids that is likelyassociated with a complex regulation of respiratorymetabolism in plants.

Although the pathways of essential amino acids andtheir interactions with the regulatory networks in plantshave long been established, certain gaps remain unfilled,such as how amino acid metabolism is associated withthe autophagic process.We cannot formally exclude thatthe decreased levels in amino acids (Fig. 6) can be, at leastpartially, related to their increased utilization as carbonskeletons, allowing the synthesis of specific compoundsrequired under carbon limitation in atg mutants. In fact,amino acids have a plethora of functions in addition to

their role as a catabolic substrate. The dynamic of aminoacid levels depends on both catabolic and biosyntheticreactions, and as such, several amino acids representprecursors of nucleotides, phytohormones, or secondarymetabolites (for review, see Hildebrandt et al., 2015).Therefore, the accurate recycling of amino acids, lipids,carbohydrates, or micronutrients and macronutrientsavailable in the plant cell becomes a critical factor thatensures plant survival and growth. This is particularlytrue given that, in the functional deficiency of autoph-agy, plants are still likely able to have a complete andnormal development (Figs. 1 and 2A).

The Chloroplast Degradation Pathway Is Induced duringAutophagy Deficiency

The differential respiratory response observed betweenwild-type and atg plants is seemingly able to explain par-tially the plant survival following carbon starvation con-ditions. Generally speaking, low cellularmetabolic activityprovides longer maintenance of cell viability duringextended dark conditions. This response may be re-lated directly to the inhibition of energy consumptionthat allows preservation of the photosynthetic ma-chinery (Keech et al., 2007). While wild-type plantsstarted to show a few signs of senescence from 12 d of

Figure 8. Schematic model showing the catabolic process involved in macromolecule degradation leading to electron donationto the ETF-ETFQO pathway during dark-induced senescence in wild-type plants (A) and atg mutants (B). Carbon starvationconditions promoted by extended darkness are associated with macromolecule degradation via several catabolic pathways (e.g.autophagy), releasing amino acids to be oxidized. The electrons generated are transferred, via the ETF/ETFQO system, to therespiratory chain through the ubiquinol pool, promoting plant survival. In atg mutants, there is a compromised amino acid (aa)supply, particularly BCAAs and Lys, recognized previously to be able to feed electrons to the ETF/ETFQO system. Simultaneously,there is a higher induction of genes associated with the ETF/ETFQO pathways and autophagy independent of chloroplast deg-radation, CV, which leads to a hypersensitivity response to energetic limitations in atg mutants.


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darkness onward (Supplemental Fig. S3), atg mutantspresented a markedly early-senescence phenotype,with faster loss of photochemical efficiency (Fig. 2) andslower reduction in protein levels (Fig. 4). Whereas allour data point to a dominant role of autophagy in re-ducing protein levels during carbon deprivation, wecannot yet rule out a simultaneous induction of othercatabolic pathways. In this scenario, we observed anup-regulation of the CV gene only in atg mutants follow-ing carbon starvation (Fig. 7H). The role of autophagy inchloroplast degradation is well known (Ishida et al., 2008;Liu and Bassham, 2012; Xie et al., 2015; Izumi et al., 2017).However, recently, an autophagy-independent process ofchloroplast degradation associated with the CV pathwaywas reported (Wang and Blumwald, 2014). Our findingssuggest that CV is highly induced in the absence of au-tophagy, contributing to the early-senescence phenotypeobserved in atg mutants.Although our results provide circumstantial evidence,

we are not able to ascertain whether autophagy is linkeddirectly with the provision of amino acids or the extent towhich the absence of autophagy is able to induce othercatabolic pathways. It seems tempting to suggest that theincreased stress response observed in atgmutants is likelydue to multiple feedback mechanisms able not only toregulate autophagy but also to impact amino acid andprotein catabolism as well as plant respiration undercarbon limitation (Fig. 8). The molecular mechanismsinvolved in the regulation of amino acid catabolism arelargely unknown (Hildebrandt et al., 2015; Galili et al.,2016), and as such, the exact mechanism connecting au-tophagy and dark respiration in plants seems to be un-clear from this study. It will be interesting to determinethis linkage in a manner that does not affect any otherconnected pathway, potentially by the use of conditionalinhibition (inducible artificial microRNA lines) of theautophagic pathway.

CONCLUSION

Wehave presented compelling evidence that autophagyhas an important role during various stages of Arabidopsisdevelopment or during carbon deprivation condi-tions. Although the impairment of growth observed isrelated to neither changes in photosynthesis nor darkrespiration under optimal conditions, it is noteworthythat the reduction in seed yield in atgmutants stronglyreinforces the significant contribution of autophagy tometabolic processes affecting plant developmental fitness.Furthermore, during conditions of prolonged darkness,the impairment of protein degradation and thereby aminoacid remobilization experienced by atg mutants, coupledwith an increased use of amino acids as alternativesubstrates to mitochondrial respiration, culminate in ahypersensitive phenotype. Thus, despite the higherup-regulation of genes related to alternative pathwaysof respiration, the supply of amino acids seems to beinsufficient tomatch the enhanced respiration rates of atgmutants (Fig. 8). Collectively, this energetic depletion

may favor an induction of other catabolic pathways,including the degradation of chloroplastic proteins viaautophagy-independent routes such as the CV. Takentogether, the results presented here highlight the com-plexity and specificity of plant metabolism in responseto carbon limitation and suggest that myriad interplaysare involved between autophagy and plant respiration.Dissecting the intertwinedmechanisms involved in theircoregulation will be required to fully understand theimplications of autophagy for the regulation of plantmetabolism.

MATERIALS AND METHODS

Plant Material and Dark Treatment

All Arabidopsis (Arabidopsis thaliana) plants used in this study were of theColumbia-0 ecotype. The T-DNAmutant lines atg9-1 (Hanaoka et al., 2002), atg5-1 (SAIL_129B079; Yoshimoto et al., 2009), and atg7-2 (GK-655B06; Hofius et al.,2009) were used in this study. Seeds were surface sterilized and imbibed for 4 d at4°C in the dark on 0.7% (w/v) agar plates containing one-half-strengthMurashigeand Skoog medium (Sigma-Aldrich; pH 5.7). Seeds were subsequently germi-nated and grown at 22°C under short-day conditions (8 h of light/16 h of dark),60% relative humidity,with 150mmol photonsm22 s21. For dark treatments, 10- to14-d-old seedlings were transferred to soil and then grown at 22°C under short-day conditions (8 h of light/16 h of dark). Four-week-old plants were transferredto darkness in the same growth cabinet. Whole rosettes of two different plants ofsix independent samples bygenotypewere harvested at intervals of 0, 3, 6, and 9 dafter the transition todarkness and immediately frozen in liquidnitrogen and thenstored at ‒80°C until further analysis.

T-DNA Insertion Mutants and Genotype Characterization

Homozygous mutant lines were confirmed by PCR using ATG7-specificprimers (forward, 59-GACTGTACCTAACTCAGTGGGATG-39, and reverse, 59-GCTCCTGCAATAGGAGCTAGAC-39) in combinationwith theT-DNAleft borderprimer (GABI-08474, forward, 59-ATAATAACGCTGCGGACATCTACATTTT-39)for the atg7-2 mutant; ATG5-specific primers (forward, 59-TTAGCACCAAGAA-TAGGATATTTGC-39, and reverse, 59-TGCAATTTCCATTGATGATATATTG-39)in combination with the T-DNA left border primer (LB1, forward, 59-GCCTTTTCAGAAATGGATAAATAGCCTTGCTTCC-39) for the atg5-1mutant;and ATG9- specific primers (forward, 59-CTAAGAGATGGCGTGGAAAGG-39,and reverse, 59-CTTGAGGTTTGAGGCATTTCA-39) with the T-DNA left borderprimer (LB1, 59-GCCTTTTCAGAAATGGATAAATAGCCTTGCTTC-39) forthe atg9-1 mutant. Glyceraldehyde-3-phosphate dehydrogenase (forward, 59-TGGTTGATCTCGTTGTGCAGGTCTC-39, and reverse, 59-GTCAGCCAAGT-CAACAACTCTCTG-39) was used for the normalization of gene transcript levels.

Evaluation of Biometric Parameters of Seeds

To phenotype reproductive tissues, seeds were submitted to the proceduredescribedabove, and the seedlingswere transferred to commercial substrate andkept in a growth chamber at 22°C6 2°C, 60% relative humidity, and irradianceof 150mmol photonsm22 s21 with a photoperiod of 12 h of light and 12 h of darkfor seed production. Siliques were harvested and cleared with 0.2 M NaOH and1% SDS solution to remove pigments. For length determination, images of atleast 50 Arabidopsis siliques were taken with a digital camera (Canon Power-shot A650 IS) attached to a stereomicroscope (Zeiss Stemi 2000-C). The mea-surement of silique length was performed on the images using ImageJ software.Seed weight was determined by weighing aliquots of a known number of seeds(500 seeds per aliquot). Total seed yield was determined by weighing seedscollected from at least 10 individual plants.

Biochemical Assays

Leaf sampleswereharvestedat the timepoints indicated, immediately frozenin liquid nitrogen, and stored at 280°C until further analysis. Extraction wasperformed by rapid grinding of tissue in liquid nitrogen and immediate






addition of ethanol as described by Gibon et al. (2004). Photosynthetic pigmentswere determined exactly as described by Porra et al. (1989). The levels of starch,Suc, Glc, and Fruwere determined exactly as described previously (Fernie et al.,2001). Malate and fumarate contents were determined as described before(Nunes-Nesi et al., 2007). Protein and total amino acid levels were determinedas described previously (Cross et al., 2006).

Measurements of Photosynthetic Parameters

Gas-exchange measurements were performed with an open-flow infraredgas-exchange analyzer system (Li-Cor 6400XT) with a portable photosynthesissystem. Light was supplied from a series of light-emitting diodes located abovethe cuvette, providing an irradiance of 150 mmol photons m22 s21, exactly as inour growth conditions. The reference CO2 concentration was set at 400 mmolCO2 mol21 air. All measurements were performed at 25°C, and vapor pressuredeficit was maintained at 26 0.2 kPa, while the amount of blue light was set to10% of photon flux density to optimize stomatal aperture. The determination ofthe photosynthetic parameters was performed in 4-week-old plants. Fv/Fm,which corresponds to the potential quantum yield of the photochemical reac-tions of PSII and represents a measure of photochemical efficiency, was mea-sured as described previously (Oh et al., 1996) during the dark treatment. Darkrespiration was measured using the same gas exchange described above in fullrosettes of plants maintained in darkness.

Metabolite Profiling

Metabolite profiling was performed using approximately 50 mg of wholerosette material. The extraction, derivatization, standard addition, andsample injection were performed based on the GC-MS-based metaboliteprofiling protocol of Lisec et al. (2006). Peak detection, retention timealignment, and library matching were performed using the Target Search Rpackage (Cuadros-Inostroza et al., 2009). Metabolites were identified incomparison with database entries of authentic standards (Kopka et al., 2005;Schauer et al., 2005). Identification and annotation of detected peaks followedthe recommendations for reporting metabolite data described by Fernie et al.(2011). The full data set from the metabolite profiling study is additionallyavailable as Supplemental Table S1.

Expression Analysis by qRT-PCR

Total RNA was isolated using TRIzol reagent (Ambion, Life Technology)according to themanufacturer’s recommendations. Total RNAwas treatedwithDNase I (RQ1 RNase free DNase I; Promega). The integrity of the RNA waschecked on 1% (w/v) agarose gels, and the concentration wasmeasured using aNanodrop spectrophotometer. Finally, 2 mg of total RNA were reverse tran-scribed with SuperScript II RNase H2 reverse transcriptase (Invitrogen) andoligo(dT) primer according to the manufacturer’s recommendations. Real-timePCR was performed on a 96-well microtiter plate with an ABI PRISM 7900 HTsequence detection system (Applied Biosystems Applera) using Power SYBRGreen PCRMasterMix according to Piques et al. (2009). The primers used herewere designed using the open-source program QuantPrime-qPCR primerdesign tool (Arvidsson et al., 2008) and are described in Supplemental TableS2. The transcript abundance was calculated by the standard curves of eachselected gene and normalized using the constitutively expressed gene ACTIN(AT2G37620). Data analyses were performed as described by Caldana et al.(2007). Three biological replicates were processed for each experimentalcondition.

Statistical Analyses

The experiments were conducted in a completely randomized design withthree to seven replicates of eachgenotype.Datawere statisticallyexaminedusingANOVA and tested for significant (P , 0.05) differences using Student’s t test.All statistical analyses were performed using the algorithm embedded intoMicrosoft Excel.

Accession Numbers

The Arabidopsis Genome Initiative locus numbers for the major genes dis-cussed in this article are as follows: ATG5 (At5g17290), ATG7 (At5g45900), andATG9 (At2g31260).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Schematic representation of the sites of T-DNAinsertion in atg mutants.

Supplemental Figure S2. Gas-exchange parameters are not affected in atgmutants.

Supplemental Figure S3. Phenotypes of Arabidopsis mutants under ex-tended dark treatment.

Supplemental Figure S4. Metabolite levels in atg Arabidopsis mutants.

Supplemental Table S1. Relative metabolite contents of leaves of Arabi-dopsis knockout mutants atg5-1, atg7-2, and atg9-1 and wild-type plantsafter further treatment for 0, 3, 6, and 9 d in darkness.

Supplemental Table S2. Primers utilized for quantitative RT-PCR.

ACKNOWLEDGMENTS

Discussions with DimasM. Ribeiro andAgustin Zsögön (Universidade Fed-eral de Viçosa) were highly valuable in the development of this work. We alsothank Gad Galili (Weizmann Institute of Science) for contributing the seedsused in this study.

Received February 13, 2017; accepted July 11, 2017; published July 14, 2017.

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