Metabolite Regulatory Interactions Control Plant RespiratoryMetabolism via Target of Rapamycin (TOR)Kinase Activation[OPEN]

Brendan M. O’Leary, Glenda Guek Khim Oh, Chun Pong Lee, and A. Harvey Millar1

Australian Research Council Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, University of WesternAustralia, Perth, Western Australia, Australia 6009

ORCID IDs: 0000-0002-8770-155X (B.M.O.); 0000-0001-6102-061 (G.G.K.O.); 0000-0002-8760-5779 (C.P.L.); 0000-0001-9679-1473(A.H.M.).

Respiration rate measurements provide an important readout of energy expenditure and mitochondrial activity in plant cellsduring the night. As plants inhabit a changing environment, regulatory mechanisms must ensure that respiratory metabolismrapidly and effectively adjusts to the metabolic and environmental conditions of the cell. Using a high-throughput approach,we have directly identified specific metabolites that exert transcriptional, translational, and posttranslational control over thenighttime O2 consumption rate (RN) in mature leaves of Arabidopsis (Arabidopsis thaliana). Multi-hour RN measurementsfollowing leaf disc exposure to a wide array of primary carbon metabolites (carbohydrates, amino acids, and organic acids)identified phosphoenolpyruvate (PEP), Pro, and Ala as the most potent stimulators of plant leaf RN. Using metabolitecombinations, we discovered metabolite-metabolite regulatory interactions controlling RN. Many amino acids, as well as Glcanalogs, were found to potently inhibit the RN stimulation by Pro and Ala but not PEP. The inhibitory effects of amino acids onPro- and Ala-stimulated RN were mitigated by inhibition of the Target of Rapamycin (TOR) kinase signaling pathway.Supporting the involvement of TOR, these inhibitory amino acids were also shown to be activators of TOR kinase. This workprovides direct evidence that the TOR signaling pathway in plants responds to amino acid levels by eliciting regulatory effectson respiratory energy metabolism at night, uniting a hallmark mechanism of TOR regulation across eukaryotes.

INTRODUCTION

Plant respiratory metabolism is a robust and flexible network ofreactions that is capable of oxidizing many different carbonsubstrates to produce not only ATP and reducing power but alsocarbon skeletons to support biosynthetic processes such asprotein synthesis (Plaxton and Podestá, 2006; O’Leary andPlaxton, 2016). Although the main pathways of carbohydraterespiration include glycolysis, the oxidative pentose phosphatepathway, the tricarboxylic acid cycle (TCA cycle), and the mito-chondrial electron transport chain (mETC), many other pathwayscatabolizing amino acids, fatty acids, and other compounds arecapable of feeding carbon into the respiratory system (Millar et al.,2011;RasmussonandMøller, 2011). Theactivity of the respiratorypathways underpins a plant cell’s energy use and biosyntheticperformance under various heterotrophic conditions. Respirationalso functions to maximize the rate of photosynthesis in leaf cellsduring the day (Tcherkez et al., 2017; O’Leary et al., 2019).Therefore, understanding how respiration is controlled and reg-ulated under differentmetabolic conditions is ofmajor importancefor understanding the evolution of carbon and energy use strat-egies of plants.

Decades of research into the various pathways of plant respi-ration have revealed a decidedly complex, multi-level system ofregulatory mechanisms. These mechanisms can generally becategorized as posttranslational, thus affecting the activity ofexisting enzymes, or as changes in gene expression, which alterthe amount and type of enzymes present (Plaxton and Podestá,2006;O’LearyandPlaxton,2014,2017).Overshortperiodsof time(minutes), metabolism is mostly regulated by posttranslationalmechanisms, including substrate levels, feedback inhibition, al-losteric effector levels, and protein modifications, which all in-fluence the activity of existing enzymes. Over longer periods oftime (hours to days), perceived changes in cellular and environ-mental conditions, including diurnal cycles, lead to changes ingene expression and protein levels via signal transductionpathways (Giraud et al., 2010; Lee et al., 2010).Metabolite concentrations are a crucial aspect of the post-

translational control of respiration in all organisms by acting assubstrates, allosteric effectors, and feedback inhibitors of variousmetabolic enzymes (Plaxton and Podestá, 2006). Likewise, manymetabolites induce signal transduction cascades that link nutri-tional or environmental status to changes in enzyme expressionlevels andposttranslationalmodifications (WangandLei, 2018). Inplants, the best understood examples of nutrient signaling me-tabolites are carbohydrates. For example, in Arabidopsis (Arabi-dopsis thaliana), Glc levels are partly signaled via hexokinase andalso activate the Target of Rapamycin (TOR) pathway (Xiong et al.,2013),whileSuc levels aresignaledpartly via theSuc-trehalose-6-phosphate nexus (Figueroa et al., 2016; Figueroa andLunn, 2016).Both of these metabolites convey crucial information about

1 Address correspondence to harvey.millar@uwa.edu.au.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: A. Harvey Millar (harvey.millar@uwa.edu.au).[OPEN]Articles can be viewed without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.19.00157

The Plant Cell, Vol. 32: 666–682, March 2020, www.plantcell.org ã 2020 ASPB.

a plant’s carbon status that affects downstream energy utilizationand respiration. Carbohydrates are the predominant respiratorysubstrates in plants, and cellular sugar levels correlate with res-piration rates (Azcón-Bieto et al., 1983; Noguchi, 2005; O’Learyet al., 2019).

Recently, the level of specific amino acids and organic acidshave also been shown to correlate with leaf respiration rates inArabidopsis (O’Leary et al., 2017). However, very little is knownabout nutritional signals emanating from amino acids or organicacids in plants (Hannah et al., 2010). A reliance on amino acids orfatty acids as respiratory substrates in plants is generally thoughtto be limited to situations of carbohydrate deprivation such asextended darkness and seed germination (Graham, 2008; Kunzet al., 2009; Araújo et al., 2011; Hildebrandt et al., 2015). However,in animals and yeast, amino acids are key regulators of the TORkinase signaling pathway, which is a central hub integrating in-formation about nutritional and environmental status (Dobrenelet al., 2016; Wolfson and Sabatini, 2017). In plants, this potentialaspect of nutrient signaling has not been demonstrated.

The flux of electrons through themETC is amenable to dynamicflux measurements because it terminates in the consumption ofO2, a process that ismeasurable in real timeby several techniques(Scafaro et al., 2017). So far, measurements of respiration ratedynamics in response to metabolite level changes have mostlybeen performed in isolated mitochondria that lack both de novogeneexpressionandcytosolic/plastid enzymes, andare thereforecapable of revealing only part of the metabolic control of respi-ration that occurs in plant cells. Few studies have observed dy-namic respiratory responses to metabolites within intact planttissues, and these have all focused on the influence of carbo-hydrates (Brouquisse et al., 1991; Noguchi, 2005). Here, we

explored the regulatory effects of a broad range of metabolites onleaf nighttime O2 consumption rate (RN) using multi-hour mea-surements ofO2 consumption. AlthoughSuc,Glc, Fru, andmaltosecould stimulate RN, it was phosphoenolpyruvate (PEP), Pro, andAla that displayed the strongest time-dependent stimulatory ef-fects among the metabolites tested. Metabolite-metabolite reg-ulatory interactions were also discovered in which the stimulationof RN by Ala and Pro is influenced by the levels of other metab-olites. The patterns of metabolite-metabolite regulatory inter-actions were reminiscent of metabolic control by the TOR kinasepathway in animal and yeast cells. To validate this connection, weshowed that amino acid interactions with Pro and Alametabolismwere sensitive to three independent typesof TORkinase inhibitorsand that amino acid treatments activate TOR kinase in matureleaves to a similar extent to Suc. Our results indicate that the TORkinase pathway is involved in mediating amino acid-derivedmetabolic regulatory signals in mature plant leaves that in-fluence respiratory activity and thus plant metabolic rate.

RESULTS

The Stimulation of RN by Metabolites Is Time Dependent

As a tightly regulated metabolic network, respiration rateresponds to various changes in the metabolic conditions ofthe cell. We therefore established a nighttime O2 consumptionmeasurement to observe tissue level changes in RN over multiplehours following metabolic perturbations induced by floating leafdiscs on buffered solutions. Leaf discs floated on the buffersolution alone without further chemical additions (control

TOR Control of Plant Respiratory Metabolism 667

treatments) produced a respiratory trace where RN decreasedduring the first several hours before gradually stabilizing towardthe end of a 14-h measurement (Figure 1).

To uncover the regulatory properties of the respiratory network,we performed a high-throughput screen of themulti-hour effect ofexogenous metabolites on leaf disc RN. Many metabolically rel-evant carbohydrates and organic acids were tested, includingselected TCA cycle intermediates. Citrate and isocitrate wereexcluded, as they have been shown to strongly chelate metalcations and thuscandisrupt normal cellular behavior (Ränbyet al.,1999; Sul et al., 2016). Most proteinogenic amino acids andg-aminobutyric acid (GABA) were also tested. Trp and Tyr wereexcluded because they lack sufficient solubility, and Cys wasexcluded because it undergoes spontaneous oxidation, dis-rupting the assay. While most exogenous metabolite treatmentsdid not have an appreciable effect on RN, certain metabolitesdisplayed strong time-dependent stimulations of RN (Figure 1). In

particular, PEP, Ala, and Pro had large stimulatory effects on RN

that began after;2 to 4 h of incubation and led to an approximatedoubling of RN compared with control after 14 h, which corre-sponds to an;50% increase in RN compared with 0 h. Suc, Glc,maltose, Ser, Thr, and Gly showed a less pronounced but stillsubstantial stimulation of RN. By contrast, pyruvate, which isa direct product of both Ala and PEP catabolism within the re-spiratory pathways, had only aminor effect on RN. In addition, thenitrogensourcesNH4

1andNO32at10mMalsohadnoeffectonRN

over the time course. In combination, these results demonstrateclear and unexpected differences in the effects of exogenousmetabolites on RN.Metabolism likely adjusts diurnally in response to changing

output demands and levels of substrates, including carbonreserves (e.g., starch), which may cause some external metab-olites to bemore or less stimulatory at certain times. To assess theeffect of time of night on the stimulation of RN by various

Figure 1. Time-Dependent Stimulation of RN by Exogenous Metabolites.

Single leaf discs were floated on respiration buffer in tubes, and the RN was captured as a moving average over the time course. Graphs show the effect of10 mM metabolite additions to the buffer medium. The control treatment is leaf RN in the absence of exogenous metabolites. In each graph, the linesrepresentmeanRNafter additionof themetabolite indicated, and theblueareashows the95%confidence intervalsofuntreatedcontrols.RN ratesareshownas fold change relative to untreated controls. All traces represent the average of at least six different leaf disc measurements.

668 The Plant Cell

metabolites, assays of RN in the presence of Suc, Glc, PEP, py-ruvate, Ala, Pro, Ser, and Gly were commenced at 5 and 10 h intothe usual 16-h dark period. RNmeasurementswere then recordedfor a further 14 h in each case. The metabolite stimulations of RN

were observed to be largely independent of time of night, whichindicates that depleted starch reserves are not the major de-terminant of the extent of stimulation by exogenousmetabolites inthis system (Supplemental Figure 1).

Accumulation Patterns of Metabolites in Leaf Discsfollowing Exogenous Exposure

It was not knownwhethermetaboliteswould be appreciably takenup into leaf tissue from external solutions. Therefore, we assayedthe accumulation of metabolites in leaf discs following 4-h in-cubations on metabolite solutions (Table 1). All amino acids andglycolytic intermediates accumulated substantially except forAspand Glu. TCA cycle intermediates displayed modest or no ac-cumulation. Suc, Glc, and Fru did not display significant accu-mulation at 4 h, but when the incubation was extended to 8 h,significant accumulation of these metabolites was observed(Supplemental Table 1). The delayed accumulation of thesesugarsmay in part reflect their metabolism by the leaf discs. In thecase of compounds that do not appreciably accumulate and donot stimulate RN (e.g., malate, fumarate, Asp, and Glu), it remainsunclear whether they were not appreciably taken up by leaf tissueor they were taken up but changes in total concentration were notobserved due to metabolism.

Stimulation of Respiration by Pro and Ala Is Disrupted by thePresence of Other Metabolites

To observe whether interactions between metabolites would af-fect RN, we focused on whether the large RN stimulations causedby PEP, Pro, andAlawould be influencedby simultaneousprovisionof any additional metabolite (referred to as the co-metabolite). Re-spiratory substrates including carbohydrates, glycolytic inter-mediates, TCA cycle dicarboxylic acids, and amino acids wereapplied exogenously at 10 mM alone and in combination withPEP, Pro, and Ala, and leaf disc RN was measured over time. Bycomparing the relative RN at 14 h for these incubations, it wasobserved that many amino acids (Figure 2) as well as malate(Figures 3A to 3C) had the effect of blocking Pro and Ala stimu-lation of RN. By contrast, only the addition of Lys significantlydiminished PEP-stimulated RN. Carbohydrate substrates andglycolytic intermediates did not affect Pro-, Ala-, or PEP-stimulated RN, with the exception that pyruvate modestly in-creased the stimulatory effect of PEP on RN (Figures 3D to 3F;Supplemental Figure 2).

Three Glc analogs, glucosamine, 2-deoxyglucose, and man-nose, which are inhibitors of hexokinase and glucose-6-phos-phate dehydrogenase of the oxidative pentose phosphatepathway, were also tested. These three co-metabolites, which arethemselvespoor respiratory substrates (Pegoetal., 1999), had theeffect of inhibiting RN and strongly inhibiting Ala and Pro stimu-lation of RN, but they were less effective at inhibiting PEP-stimulated RN (Figures 3D to 3F).

PEP, Pro, and Ala Accumulation in Leaf Tissue AccompaniesRespiratory Stimulation

Themechanism of RN stimulation by Pro and Ala and suppressionof stimulation by certain external metabolites could involvetranscriptional, translational, or posttranslational factors. Post-translational, time-dependent stimulation of RN by Pro and Alacould be due to the gradual accumulation of these metabolites

Table 1. Accumulation of Exogenous Metabolites in Leaf Discs

CompoundMetabolite Level Ratio(Exposed/Nonexposed 6 SE)

Amino acidsGly 5.2 6 1.1a

Ala 5.6 6 0.2a

Val 50.4 6 5.1a

Leu 105.6 6 5.4a

Ile 98.9 6 9.1a

Met 47.8 6 4.3a

Phe 39.9 6 3.9a

Pro 39.9 6 4.2a

Ser 15.0 6 1.6a

Thr 13.2 6 1.0a

Asn 6.0 6 2.0a

Gln 8.8 6 0.9a

Glu 1.4 6 0.3a

Asp 1.2 6 0.1a

Lys 34.8 6 4.3a

Arg 8.2 6 2.5a

GABA 8.3 6 0.8a

CarbohydratesSuc 1.5 6 0.6a

Glc 1.2 6 0.5a

Fru 1.5 6 0.2a

Mannitol 101.3 6 10.3a

Maltose 5.0 6 1.0a

TCA cycle organic acidsCitrate 1.1 6 0.1a

Aconitate 1.0 6 0.1a

Isocitrate 1.4 6 0.2a

a-Ketoglutarate 2.0 6 0.3a

Succinate 1.4 6 0.1a

Fumarate 1.0 6 0.04a

Malate 1.0 6 0.01a

Glycolytic metabolitesGluconate 26.0 6 3.2a

Glycerol 13.3 6 0.8a

Glucose-6-phosphate 2.7 6 0.2b

Fructose-6-phosphate 5.3 6 0.8b

3-Phosphoglycerate 2.0 6 0.1a

PEP 8.0 6 2.1c

Pyruvate 3.1 6 0.5c

Leaf disc samples were incubated for 4 h in the presence of the indicatedexogenous metabolite, and the fold increase of that metabolite versuscontrol incubations was determined. Significant increases are indicatedin boldface (t test, P < 0.05).aDetermined by gas chromatography-mass spectrometry.bDetermined by liquid chromatography-quadrupole/time-of-flight-massspectrometry.

cDetermined by enzymatic analysis.

TOR Control of Plant Respiratory Metabolism 669

within the leaf cells and their use as substrates to increase met-abolic fluxes linked to RN. Titrations of exogenous Pro revealedthat higher external Pro concentrations caused greater RN stim-ulation, consistent with a substrate-driven RN stimulation (Fig-ure 4A). By contrast, titrations of exogenous Ala showeda respiratory stimulation peaking at ;5 to 10 mM and sub-sequentlydecreasingathigher concentrations (Figure4A).Assaysof metabolite accumulation in leaf discs revealed that Pro and Alalevels increased markedly during the time course, although tovaryingabsoluteamounts (Figure4B). Ineachcase, the increase inPro or Ala levels preceded the increase in tissue RN by severalhours, suggesting thatmetabolite level changes are related to butnot solely responsible for stimulation of RN.

Wenextassessedwhether theco-metabolites that inhibitedProand Ala stimulation of RN did so by blocking Pro/Ala uptake intoleaf tissue. We assayed the effect of Suc and five inhibitory co-metabolites on the uptake of Ala and Pro (Figure 4C). After 4 h of

incubation, the presence of Ile and Met partly reduced the ac-cumulationofProby31and48%, respectively, comparedwith thesamples incubated in Pro alone. Similarly, Ala accumulation waspartly reduced by Met (37% reduction). The other metabolitestested (Suc, glucosamine, Asp, and malate) had insignificant orminor (<20%) effects on Pro and Ala accumulation. Given thetitratable influence of Pro andAla onRN, thesemodest differencesin uptake appear insufficient to account for the strong inhibitoryeffect of these co-metabolites on Pro- and Ala-stimulated RN.

Ala and Pro Stimulation of Respiration Occurs via Changesin Gene Expression

We next evaluated whether differences in gene expression couldbe involved in the observed patterns of RN regulation. The ap-plication of cycloheximide, which inhibits cytosolic ribosomal

Figure 2. The Effect of Exogenous Amino Acids on Pro-, Ala-, and PEP-Stimulated RN.

(A) to (C)Amino acidswere addedby themselves or in combinationwith Pro (A), Ala (B), or PEP (C) to the respiration buffer followedbymeasurement of leafdisc RN. The values represent averaged RN at 14 h of incubation expressed relative to two control treatments, with no metabolite addition set at 0%stimulation and RN stimulation caused by Ala, PEP, and Pro alone set at 100% stimulation, respectively. Asterisks indicate significant differences betweenthe metabolite combination treatments versus the corresponding Ala-, PEP-, or Pro-only control treatment (ANOVA, P < 0.05; n 5 6). Among thosetreatments found to be significantly different, a second statistical test was conducted (indicated by n.s.) identifying those treatments where the addition ofAla, PEP, or Pro did not significantly stimulate respiration in comparison with the co-metabolite on its own (paired one-tailed t test, P < 0.05).

670 The Plant Cell

translation, completely blocked the stimulatory effects of Pro andAla on RN but did not affect Pro or Ala uptake into leaf discs(Supplemental Figure 3). The effect of cycloheximide indicatesthat changes in gene expression are necessary for the dynamicincreases in RN caused by Pro and Ala. As observed previously,this 2.5 mMcycloheximide treatment, by itself, has no appreciableeffect on RN (O’Leary et al., 2017).

To determine if the effect of Pro on RN was the result of prolinedehydrogenase (PDH) activity, which oxidizes Pro in the mito-chondrial matrix and feeds electrons into the mETC (Cabassa-Hourton et al., 2016), we assessed whether the competitive PDHinhibitor L-thiazolidine-4-carboxylic acid (T4C) would reduce thestimulation of RN by Pro. At 4 mM, T4C reduced the stimulatory

effect of Pro by 55% at 14 h (Figure 5A). This is consistent withprevious reports that T4C is capable of reducing Pro-dependentO2 uptake by 67% in isolated barley (Hordeum vulgare) mito-chondria (Elthon and Stewart, 1984). We next observed thattranscripts for both PDH isoforms in Arabidopsis, PDH1 andPDH2, increasedduring leaf disc incubation inPro (Figures5Band5C). PDH1 is expressed at a much higher level than PDH2 and isthought to represent the bulk of PDH activity (Funck et al., 2010;Cabassa-Hourton et al., 2016). Using confirmed T-DNA insertionlines (Funck et al., 2010), we assessed whether disruption of PDHgene expression would inhibit the ability of Pro to stimulate RN.Both pdh1-1 and pdh1-2 displayed modest reductions in RN

stimulation by Pro (Figure 5D; Supplemental Figure 4), while pdh2-1

Figure 3. The Effect of Exogenous TCA Cycle Intermediates and Carbohydrates on Pro-, Ala-, and PEP-Stimulated RN.

(A) to (C) Experiments were performed with TCA cycle intermediates as co-metabolites.(D) to (F) Experiments were performed with carbohydrates and related compounds as co-metabolites.See Figure 2 for details. 2-DG, 2-deoxyglucose; Glc-N, glucosamine; a-KG, a-ketoglutarate; OAA, oxaloacetate.

TOR Control of Plant Respiratory Metabolism 671

was stimulated by Pro similarly to the wild type (Figure 5E). Bycontrast, pdh1-1 pdh2-1 displayed a clear reduction of RN in thepresence of Pro (Figure 5F). pdh1-1 pdh2-1 leaf discs displayed analmost complete loss of PDH1 transcripts, and PDH2 transcriptswere undetectable (Supplemental Figure 4B). Furthermore, pdh1-1pdh2-1wasnolongersensitivetoRNinhibitionbyT4Cinthepresenceof Pro (Supplemental Figure 4). Together, these results indicate thatexternal Pro accumulates in leaf disc tissue and induces the ex-pression of PDH isoforms that in turn increase the mitochondrialcatabolism of Pro, thus increasing RN.

The precise mechanism or metabolic route by which Ala mightstimulate RNwas somewhat unclear, given that pyruvate does notalso stimulate RN. Therefore, we decided to establish whethermetabolism of Ala to acetyl-CoA via an alanine aminotransferase(AlaAT) and the pyruvate dehydrogenase complex (PDC) wasnecessary for Ala’s stimulatory effect on RN. The AlaAT inhibitorcycloserine inhibited the Ala stimulation of RN by ;65%(Figure 6A). During incubation in Ala, the total AlaAT activity of theleaf discs increased with time (Figure 6B). The transcript abun-dance of neither Arabidopsis AlaAT isoform, ALAAT1 or ALAAT2,

Figure 4. The Influence of Pro and Ala Concentrations on RN.

(A)Different external concentrations of Pro (left) and Ala (right) were applied exogenously, and leaf discRNwasmeasured over 14 h. The average relative RN

comparedwith control treatments is shown (n56). Thepanelsbelowshow the relative respiration ratesat 14h.Datapoints are shown; error bars indicate SE.(B)The amount of Pro (left) andAla (right) in leaf discs during incubation in the presence or absence of 10mMPro or Ala, respectively. Data points are shown(n5 4). Lines representmean values. Error bars indicate SE. Asterisks indicate significant differences between control and Pro or Ala treatments at that time(ANOVA, P < 0.05).(C)Theeffectof exogenousco-metaboliteson theuptakeofProandAla fromthemedia into leafdiscs.Leafdiscswerefloated in respirationbuffer containingmetabolites for4h, then theamountofPro (left) orAla (right) per the leafdiscswasquantified.Datapointsareshown (n$4).Solidbars representmeanvalues;error bars indicate SE. Asterisks indicate significant differences compared with Ala or Pro alone (ANOVA, P < 0.05).

672 The Plant Cell

correlated with the time-dependent stimulation of RN by Ala(Figure 6C). However, the characterized alaat1-1 T-DNA insertionline (Miyashita et al., 2007) displayed greatly reduced Ala stimu-lation of RN (Figure 6D) and also lowered total extractable AlaATactivity (Supplemental Figure 5). Furthermore, mab1, which isdeficient in the PDC E1b subunit and displays greatly reducedmitochondrial PDCactivity (Ohbayashi et al., 2019), alsodisplayeda lack of RN stimulation by Ala (Figure 6D). These results indicatethat Ala stimulation of RN involves the conversion of Ala to acetyl-CoA in the mitochondria.

PDH Expression Is Repressed by Metabolites That AlsoSuppress Pro-Stimulated RN

Todeterminewhether the regulatoryeffectsofco-metabolites thatreduceProandAla stimulationofRNcouldbemediatedat the levelof gene expression, we evaluated whether select repressive co-metabolites (namely glucosamine, malate, Asp, Ile, and Met)

inhibited the induction of PDH or AlaAT isoforms when appliedtogether with Pro or Ala, respectively. External Suc treatment waspreviously shown to inhibit PDH expression, as described byHanson et al. (2008) and Funck et al. (2010) andwas therefore alsoinvestigated. In the case of Pro incubations, glucosamine com-pletely blocked PDH1 transcript accumulation, while Suc causeda partial decrease in PDH1 accumulation relative to controltreatments (Figure 5G). The pattern of PDH2 transcript accumu-lation was different, with the Suc, malate, Asp, Met, and Ile sig-nificantly inhibiting PDH2 accumulation in the presence of Pro(Figure 5H). In the case of Ala incubations, the presence of thesame co-metabolites had no significant effect on the transcriptabundances of ALAAT1 or ALAAT2 (Supplemental Figure 5).

Metabolite-RN Interactions Are Sensitive to TOR Inhibitors

A clear pattern within the co-metabolite interactions was thatmany amino acids caused a similar repressive effect on RN

Figure 5. Pro Stimulation of RN Is Linked to Increased PDH Expression and Activity.

(A) Averaged traces of fold stimulation of leaf disc RN by Pro in the presence and absence of the PDH inhibitor T4C (n 5 6).(B) and (C)Transcript levels ofPDH1 (B) andPDH2 (C) upon exposure of leaf discs to external Pro control treatment. Replicate data points are shown. Linesrepresent mean values; error bars indicate SE (n 5 3).(D) to (F) Averaged traces of RN of wild-type and PDH knockdown lines in the presence and absence of external Pro (n 5 8).(G) and (H) The effect of external metabolite combinations on the expression ofPDH genes. Transcript analysis forPDH1 (G) andPDH2 (H)was performedfollowing 10-h incubations with or without Pro and additional co-metabolites. Bars represent average values relative to control treatments. Replicate datapoints are shown. Dashed lines indicate separate analyses. Asterisks indicate significant differences in transcript abundance compared with the cor-responding treatment with Pro alone (ANOVA, P < 0.05; n 5 4).

TOR Control of Plant Respiratory Metabolism 673

induction by both Pro and Ala. Relatively little information isavailable about the sensing of amino acids in plants (Gent andForde, 2017;Dinkeloo et al., 2018), but in fungi andanimals, aminoacids are strong inducers of the TOR pathway, which is a masterregulator of nutrient sensing and energymetabolism (Jewell et al.,2013). We therefore hypothesized that the TOR pathway in plantswas responsible for inhibiting the respiration of amino acids in thepresence of elevated amino acid levels. If this were true, theninhibiting the TOR signaling pathway at the time of metabolicinteractionwould block the ability of amino acid co-metabolites tosuppress Ala- and Pro-stimulated RN. To test this hypothesis, we

first examined theeffect of theTOR inhibitor AZD8055 (AZD) onRN

in thepresenceofmetabolites (Figures 7A to7C). Interestingly, theeffect of 2 mM AZD on RN was dependent on which metaboliteswere present in the incubation mixture. AZD caused strong RN

stimulation in the presence of Asp and malate and weaker RN

stimulationon itsownor in thepresenceofMet.Next,weobservedthat thepresenceofAZDpartly restored theabilityofProandAla tostimulate RN while in the presence of Ile and Met. Two otherchemical inhibitors of TOR that have been demonstrated to beeffective in plants are TORIN2 and WYE-125132 (WYE-125;Montané and Menand, 2013, 2019). Both of these inhibitors

Figure 6. Ala Stimulation of RN Is Linked to AlaAT and Pyruvate Dehydrogenase Activity.

(A) Average traces of fold stimulation of leaf disc RN by Ala in the presence and absence of 50 mM of the AlaAT inhibitor cycloserine (n 5 6).(B)Total AlaAT extractable activity relative to 0-h treatment. Asterisks indicate significant differencescomparedwith 0 h (ANOVA,P<0.05;n53). Error barsindicate SE.(C) Transcript levels ofALAAT1 (left) andALAAT2 (right) upon exposure of leaf discs to external Ala. Replicate data points are shown; line representsmeanvalues (n 5 3).(D) Representative traces of average RN of the wild type alongside alaat1-1 (left) or mab1 (right) in the presence and absence of external Ala (n 5 8).

674 The Plant Cell

behaved similarly to AZD in partly restoring the ability of Pro tostimulateRN in thepresence of Ile (Figure 7D).While TOR is namedfor its sensitivity to rapamycin in animals, this inhibitor often haslittle effect on TOR in plants (Dobrenel et al., 2016), except in theisolated case of a BP12 expression line that reportedly showedincreased sensitivity of plant TOR to rapamycin (Ren et al., 2012).In our hands, rapamycin displayed no effect on respiration ormetabolite interactions with RN in leaf discs from either wild-typeplants or a BP12 expression line (Supplemental Figure 6).

Todeterminewhether the regulatoryeffectsof Ilewereunique toPro and Ala respiration or a general property of amino acid ca-tabolism, we assessed whether Thr-stimulated RN (the next

strongest aminoacidstimulation followingAlaandPro)wouldalsobe affected by Ile and AZD. Thr-stimulated RNwas inhibited by Ile,and this repression was mitigated by AZD (Figure 7E); Thrtherefore behaves similar to Ala and Pro even though all threeamino acids are catabolized by distinct pathways.These results suggested that certain elevated amino acid levels

signal to restrict aspects of respiratory catabolism via the TORpathway. We therefore used an established immunoblot assay ofthe phosphorylation of the downstreamTOR target S6K in anS6Koverexpression line to ascertain whether amino acids could ac-tivate TOR. Arabidopsis S6K is subject to multiple phosphoryla-tion events, with T449 being phosphorylated by TOR (Xiong and

Figure 7. TOR Regulates the Amino Acid Interactions Controlling RN in Leaf Discs.

(A) to (C)AverageRN ratesweredeterminedafter14hof incubationwithvariousmetabolite treatments in thepresenceandabsenceof2mMAZD.Treatmentswere performed without metabolite combinations (A), as metabolite combinations with Ala (B), and as metabolite combinations with Pro (C). Error barsindicate SE. Asterisks indicate significant effects of AZDonRN rate for thatmetabolite treatment (t test, P < 0.05; n> 14). Double daggers indicate significantstimulation by Ala or Pro compared with the same treatment without Ala or Pro (t test, P < 0.05; n > 14). Glc-N, glucosamine.(D)Comparisonof theeffectsof2mMAZD,TORIN2,orWYE-125onRNstimulationbyProandAla.AverageRN ratesweredeterminedafter12hof incubation.Error bars indicate SE. Asterisks indicate significant effects of AZD, TORIN2, orWYE-125 compared with no inhibitor for that metabolite treatment (ANOVA,P < 0.05; n > 23).(E)AverageRN ratesweredeterminedafter 14hof incubation in thepresence andabsenceof combinationsof Thr, Leu, and2mMAZD.Replicate datapointsare shown. Error bars indicate SE. Asterisks represent significant differences among corresponding treatmentswith andwithout AZD (t test, P < 0.05; n5 7).(F) Leaf discs from Arabidopsis expressing exogenous HA-tagged S6K were incubated in the corresponding treatments for 4 h prior to SDS-PAGEimmunoblotting with anti-HA tag or anti-S6K-phospho-T449 antibodies. Wild-type control treatment leaf discs are included as a control. The top bandcorresponds to a phosphorylated version of S6K.

TOR Control of Plant Respiratory Metabolism 675

Sheen, 2012). Upon phosphorylation, S6K migrates slower onSDS-PAGE, leading to a phosphorylation-dependent bandingpattern (VanLeeneetal., 2019).Using thisassay,SucandGlchavepreviously been identifiedasmetabolic stimulators of TORactivityin Arabidopsis seedlings (Xiong et al., 2013; Van Leene et al.,2019). Leaf disc incubation in Ile, Gln, or Suc led to a similar in-crease in the T449-phosphorylated S6K compared with thecontrol, indicating that they are each capable of activating TOR inmature leaves (Figure 7F). The inclusion of AZD completely in-hibited S6K phosphorylation.

TOR signaling alters the abundance of transcripts for manymetabolic genes. We therefore tested whether AZD was influ-encing the expression of PDH or AlaAT genes in our system.ALAAT1andALAAT2 transcriptswereonlymodestly increasedbyAZD treatment (Supplemental Figure 5C), again indicating thatALAAT transcript induction is not a major site of control of Alacatabolism. By contrast, AZD treatment strongly increased PDH1and PDH2 expression in the presence or absence of Pro(Figure 8A). Furthermore, during leaf disc incubationswithPro andinteracting co-metabolites, AZD was shown to mitigate the in-hibitionofPDH1andPDH2expressioncausedbymost interactingco-metabolites tested except glucosamine (Figure 8B).

Tohelp further clarify themechanismofPro catabolic regulationby TOR, we tested whether TOR inhibition by AZD affected Pro

uptake into leaf disc tissue. After a 5-h incubation, AZD treatmentdid not affect Pro uptake into leaf discs (Figure 8C). TOR inhibitionalso activates autophagy. To assess whether autophagy wasrequired for Pro- or Ala-dependent stimulation of RN, we treatedleaf discswith the autophagy inhibitorwortmannin in thepresenceand absence of Pro or Ala. Wortmannin did not block the stim-ulation of RN by Pro or Ala (Figure 8D). Therefore, the interaction ofTOR signaling with Pro-induced respiration does not appear torequire changes in Pro uptake or autophagy.

DISCUSSION

The results from this study demonstrate that exogenously sup-plied metabolites, on their own and in combinations, can greatlyinfluence leaf respiratory rates when observations are continuedover hours. The time dependencies of the respiratory responsesare likely to be a consequence of both metabolite uptake and denovo gene expression, which both exert change on the systemover thedurationof the14-hmeasurement. Thismeans that theRN

dynamics observed here are due to both the transcriptional/translational and posttranslational effects of metabolite levels onrespiratory enzyme activities. Because the transcriptional/trans-lational and posttranslational machinery of the whole cell re-mained intact, the information gained in these RN experiments is

Figure 8. TOR Inhibition Regulates PDH Gene Expression.

(A) The effect of 2 mMAZD on leaf disc PDH1 (left) and PDH2 (right) transcript levels determined after 9 h of incubation in the presence and absence of Pro.Bars represent average transcript levels relative to control incubations with no additions. Replicate data points are shown. Asterisks represent significantdifferences between treatments with and without AZD (ANOVA, P < 0.05; n 5 4).(B) The effect of 2 mM AZD on leaf disc PDH1 (left) and PDH2 (right) transcript levels was determined after 9 h of incubation in the presence of Pro incombination with select co-metabolites. Bars represent average transcript levels relative to control incubations with Pro only. Replicate data points areshown. Asterisks represent significant differences with respect to the corresponding control treatment with or without AZD (two-way ANOVA, P < 0.05;n 5 3).(C) The effect of AZDon the uptake of Pro from themedia into leaf discs following a 4-h incubation. Data points are shown (n5 5); solid bars representmeanvalues. The effect of AZD was not significant (t test, P > 0.05).(D) The effect of wortmannin on Ala- and Pro-stimulated RN. Averaged traces of fold stimulation of leaf disc RN by Ala (left) or Pro (right) are shown in thepresenceandabsenceof theautophagy inhibitorwortmannin (n57). The right panel shows the relative respiration ratesat 14h.Datapointsare shown; errorbars indicate SE. Significant effects of wortmannin are indicated with the asterisk (t test, P < 0.05; n 5 7).

676 The Plant Cell

distinct from and complementary to information about the met-abolic regulation of respiration derived from studying isolatedmitochondria. The combination of high-throughput and long-duration RN assays upon intact cells (tissue) succeeded in un-covering novel aspects of respiratory regulation, including theinvolvement of the TOR pathway, which we discuss below withregard to its importance for control of plant metabolism.

The O2 consumption rates that we measure in this study, anddefine as RN, indicate that the mETC fluxes are varying in re-sponse to changing metabolite concentrations. However, re-spiratory activity can also be measured as CO2 production,which provides different and complementary information aboutrespiratory fluxes. The ratio of respiratory CO2 production to O2

consumption, known as the respiratory quotient (RQ), variesdepending on the oxidation status of the substrate being me-tabolized. Therefore, the relative magnitude of respiratorystimulation by various metabolites would likely be different ifconsidered in terms of CO2 release versus O2 consumption. Forexample, carbohydrates, with an RQ of 1, would release 25%moreCO2per unit ofO2 consumeduponcompleteoxidation thanthe average amino acid, with an RQ of 0.8.

Why Some Respiratory Substrates Stimulate RN and OthersDo Not

Certain oxidizable metabolites can clearly have a dispropor-tionate effect on Arabidopsis leaf RN that only becomes ob-servable after several hours of exposure. The immediatequestion that arises from the screen of central carbon metab-olites (Figure 1) is, why are some of these respiratory substrateseffective stimulators of total RN whereas others are not? Theexistenceandorganizationof the various respiratorypathways isclearly not a sufficient explanation for the varied effects ofmetabolites on RN.

Mitochondria are highly involved in the metabolism of aminoacids inplants bothduringnormal processes likephotorespiration(Douce et al., 2001) and more broadly under stress conditions(Araújo et al., 2011; Hildebrandt et al., 2015). Gly is the pre-dominant amino acid substrate supplied to leaf mitochondriabecause of photorespiration in the light. The induced use of otheramino acids as respiratory substrates under carbon starvation orsenescence, in particular Ile, Leu, Val, and Lys, is well studied andhasbeen linked toaSnRK1-bZIPsignal transductionpathwayandthe upregulation of the ETF/ETFQO complex (Araújo et al., 2010;Cavalcanti et al., 2017; Pedrotti et al., 2018). By contrast, a con-nection that distinguishes Ala and Pro from most other aminoacids is that theyarebothwell known toaccumulate in response tocertain stresses; furthermore, this accumulation occurs at leastpartly in the cytosol in the case of Pro (Ketchum et al., 1991;Gagneul et al., 2007). Pro accumulates following salt and osmoticstress and may play several protective roles (Verbruggen andHermans, 2008). Ala accumulates under hypoxic stress, includingflooding conditions, allowing continued ATP generation by gly-colysis by acting as an alternate sink for pyruvate, whosedownstream oxidation is inhibited because of a lack of O2

(Miyashita et al., 2007;Rochaet al., 2010;Antónioet al., 2016). Thereason Ala and Pro in particular stimulate RN strongly (Figure 1)may be because plants have evolved metabolic and gene

regulatory responses that act to rapidly metabolize high levels ofAla and Pro when the relevant cellular conditions that cause theiraccumulation are not present (Miyashita et al., 2007; Szabadosand Savouré, 2010). By contrast, plants likely have not evolvedequally effective and coordinated regulatory responses to alle-viate excess amounts of other amino acids, which do not typicallyaccumulate to high levels in the environment.Diurnal variations in leafProcontent suggest thatdegradationof

Pro naturally occurs during the night (Hayashi et al., 2000). Fur-thermore, external Pro is known to stimulate the expression ofPDH isoforms under nonstress conditions (Verbruggen andHermans, 2008; Funck et al., 2010). Pro also induces the upre-gulation of the expression of D1-pyrroline-5-carboxylate de-hydrogenase,which togetherwithPDHconstitutes thepathwayofPro oxidation to Glu (Deuschle et al., 2004). This mitochondrion-localized pathway generates reducing power at both enzymaticsteps that can then enter the mETC, fueling O2 consumption.Respiration measurements on mitochondria isolated from 15-d-old seedlings indicated that only PDH1 can mediate measurablePro-dependent O2 consumption (Cabassa-Hourton et al., 2016).Our results indicate that in mature leaves both PDH isoforms cancontribute to Pro-dependent RN, consistent with their shared rolein regulating Pro accumulation in senescing leaves (Launay et al.,2019). For Ala, the cessation of hypoxia stimulates Ala catabolismvia a pathway that involves the mitochondria-localized AlaAT1(Figure 6D; Miyashita et al., 2007). The fate of pyruvate formed inthis reaction has not been experimentally characterized, althoughour data suggest that it is largely oxidized via the mitochondrialPDC (Figure 6D) and presumably further oxidized by the TCAcycle.Other metabolites that stimulated RN, such as PEP, 3-

phosphoglycerate, Suc, Glc, and Fru (Figure 1), would not stim-ulate R in isolated mitochondria because they lack carbohydrateor glycolytic metabolic enzymes. These results highlight how thelarger multi-compartmented cellular metabolic network contrib-utes to the control of RN outside the realm of mitochondrion-localizedmetabolism. The long-duration assaysof respiration ratedynamics outlined in this study offer the potential to continue touncover mechanisms controlling mitochondrial and cellular me-tabolism and to identify metabolic phenotypes that clarify genefunction.

The Regulatory Pathway Involved in Respiratory CataboliteRepression in Plants

Novel regulatory interactions among metabolites were identifiedwherein the presence of a co-metabolite in the external mediuminhibited RN simulation by another metabolite. Several aminoacids, malate and theGlc analogs glucosamine, 2-deoxyglucose,andmannose, were capable of this effect. A clear patternwas thatthese co-metabolites each had similar effects on Pro- and Ala-stimulated RN but were less effective or ineffective at reducingPEP-stimulatedRN (Figures 2 and3). In the caseof Pro, incubationwith inhibitory co-metabolites was accompanied by reducedexpression of PDH transcripts, clearly implicating manipulationsof gene expression as a likely mechanism of action (Figure 5).Posttranslational effects of co-metabolites upon the specificaspects of respiratory metabolism that concern Ala and Pro but

TOR Control of Plant Respiratory Metabolism 677

not PEP metabolism cannot be ruled out; however, there is noindication of such a mechanism in the literature. Therefore, ourevidence led us to further investigate a transcriptional/trans-lational mechanism of action.

Although the regulation of amino acid metabolism is complex(Pratelli and Pilot, 2014), it seemed implausible that the differentinhibitory co-metabolites could each be an allosteric inhibitor orotherwise affect the transcriptional, translational, or post-translational control of Rn by independent mechanisms. There-fore, we hypothesized that the effects of these metabolitesconverge on a signaling pathway that elicits multiple changes inenzyme activities sufficient to inhibit the respiration of both Proand Ala by mitochondria. A pertinent example of a metabolicsignaling pathway that receives nutritional inputs from multiplerespiratory substrates is theTORkinasepathway. TheTORkinasepathway is a master regulator of eukaryotic carbon metabolism,where its activation generally serves to promote anabolic me-tabolismand inhibit catabolicmetabolism (Dobrenel et al., 2016). Itwas plausible that the TOR kinase pathway would be involved inthe metabolite regulation observed in this study because aminoacids are one of the primary signals activating the TORpathway inother eukaryotes (Jewell et al., 2013). In animals and yeast, thepresence of amino acids promotes TOR activity, which activatesprotein synthesis and growth, while the absence of amino acidsinhibits TORactivity, which activates catabolic processes such asautophagy (González and Hall, 2017). Consistently, we observedhere that certain plant respiratory catabolic processes weresuppressed by elevated amino acid levels. TOR activation inplants has so far only been linked to the presence of Glc and Suc,and the involvement of amino acids in plant TOR pathway reg-ulation has not been reported (Xiong et al., 2013; Van Leene et al.,2019). Interestingly, plants lack all the components of amino acidsensing upstream of TOR that are described in animals and fungi(Wolfson and Sabatini, 2017). Hence TOR-dependent amino acidsensing in plants is likely to involve new or divergent componentsthat await identification.

Despite these deficits, we have provided clear evidence thatamino acid levels do influencemetabolic activities in plants via theTOR kinase signaling pathway. The inhibitory effects of aminoacids on both Pro- and Ala-stimulated RN are largely removedwhen TOR inhibitors are present (Figure 7). Furthermore, aminoacids were shown to be activators of TOR kinase activity(Figure 7F). TOR inhibition counteracted the effect of several in-hibitory co-metabolites on PDH transcript levels (Figure 8B).Therefore, in the case of Pro, these results provide a mechanisticbasis for the regulatory interaction between certain amino acidsand Pro-stimulated RN (Figure 9A). Clearly, other gene targetsbesides PDH isoforms are involved to account for the observedcontrol ofotheraminoacid respiration (e.g., AlaandThr) byTOR. Inthis regard, the general upregulation of amino acid catabolicgenes, including PDH1, by inactivation of TOR has been re-peatedly observed in previous plant transcriptome analyses (Renet al., 2012; Xiong et al., 2013; De Vleesschauwer et al., 2018). Inaddition, disruption of TOR signaling in plants leads to the ac-cumulation of high levels of amino acids and TCA cycle organicacids (Moreau et al., 2012; Ren et al., 2012; Caldana et al., 2013),potentially due to activation of amino acid synthesis (Mubeenet al., 2018). Our findings provide evidence that TOR activity in

plants is influenced by amino acid levels leading to downstreameffects on respiratory substrate use, including that of amino acids.A model of this general mechanism of action is depicted inFigure 9B.

Figure 9. Hypothetical Model of TORKinase Function in the Regulation ofPro Metabolism and Amino Acid Respiration in General.

(A) Regulation of Pro metabolism.(B) Amino acid respiration in general.Amino acids activate the plant TOR pathway by yet-undiscoveredmechanisms. Activation of TOR inhibits amino acid catabolism and res-piration by targeting the expression of amino acid catabolic genes, con-sistent with previous transcriptomic results. At the same time, TORactivationpromotesanabolicpathways, partly viaS6Kphosphorylation, asametabolic destination for amino acids. In this way, the presence of aminoacids contributes to an overall signal of nutrient abundance in matureleaves.

678 The Plant Cell

Differences in TOR Regulation between Eukaryotes HelpExplain the Functional Behavior of Plant Respiration

The implication of the TOR pathway in regulating respiratoryactivity in plants unites a function of the TOR pathway acrossa wider set of eukaryotes. Yet, differences between TOR regu-latory properties also indicate additional ways in which the plantrespiratory regulatory network has adapted to unique metabolicdemands.Asshown inFigure7, inhibitionofTORincreasesRNratein leaves. In yeast, TOR inhibition also increases respiratory O2

consumption, although only during growth on certain substrates(i.e., Glc but not glycerol; Bonawitz et al., 2007). From ametabolicstandpoint, the inhibition of the yeast TOR pathway promotesa shift from fermentation to the more energetically efficient res-piration of carbon substrates alongwith the usageof nonpreferredN sources, including Pro (Hardwick et al., 1999). By contrast, inanimal cells, inhibition of TOR decreases respiratory O2 con-sumption and increases aerobic glycolysis, shifting Glc metab-olism away from mitochondrial respiration (Schieke et al., 2006;Cunningham et al., 2007; Ramanathan and Schreiber, 2009). In-hibition of TOR also leads to opposite responses in plant andanimal cells with regard to TCA cycle organic acid levels; levelsincrease and decrease in plant and animal cells, respectively, inline with changes in respiration rate (Ramanathan and Schreiber,2009; Caldana et al., 2013).

Mechanistically and genetically, much less is known about thefunctioning of the TOR pathway in plants compared with ourdetailedunderstanding inanimalsand fungi (Dobrenel etal., 2016).This has largely been due to experimental constraints in plantsystems, including the embryo lethality of TOR mutations, thevariable efficiency of rapamycin as a plant TOR inhibitor, and therelativescarcityofassays forTORactivity (XiongandSheen,2012;Dobrenel et al., 2016). The development of additional TOR in-hibitors has helped in this regard. AZD has been demonstrated tospecifically inhibit TOR compared with the most closely relatedphosphatidylinositol 3-kinases (Chrestaetal., 2010).Furthermore,theeffectofAZDwasshowntobe related toTORexpression levelsin Arabidopsis (Montané andMenand, 2013). Therefore, the long-term respiration measurements presented here, in combinationwith specific TOR inhibitors, represent an additional means bywhich to probe TOR function in plants, in particular the responsesto various metabolic conditions where TORmay have specializedfunctions in plants compared with animals or yeast. A key area offuture research is whether the regulatory properties of RN asdescribed here for leaves, suchasRN stimulation byPEP,Pro, andAla, also occur in other plant tissues. It has already been observedthat the regulatory properties of the plant TOR pathway are likelydifferent between tissues and developmental stages (Xiong andSheen, 2014).

METHODS

Plant Material and Growth Conditions

Arabidopsis (Arabidopsis thaliana) accession Col-0 (N6000) was used asthe wild type. The following mutant lines were used: alaat1-1 (At1g17290;Miyashita et al., 2007); pdh1-2, pdh2-1, and pdh1-1 (At3g30775 andAt5g38710; Funck et al., 2010);mab1 (At5g50850; Ohbayashi et al., 2019);and35S-S6K1-HA (At3g08730;VanLeeneet al., 2019). Thepdh1-1mutant

was backcrossed three times and the pdh2-1mutant five times to Col-0 toobtain near isogenic lines before pdh1-1 pdh2-1 double mutants wereselected in the F2 population of a cross between the two mutant lines.

Seedswere sown into a 3:1:1mix of potting soil, perlite, and vermiculite,supplemented with slow-release fertilizer, and covered with a transparentplastic cover until established. Plants were grown in a controlled-environment growth chamber maintaining a short-day photoperiod of8hof lightand16ofhdark (11:00PM to7:00 AM light)with tubularfluorescentlighting with a photon flux of 120 mmolm22 s21, a relative humidity of 75%,and a day/night temperature cycle of 22°C to 17°C. Soil was kept wellmoistened with regular watering.

Respiration Measurements

RespirationmeasurementswereperformedonaQ2oxygen sensor (Astec-Global) in sealed 850-mL capacity tubes at 21°C. Leaf discs (7 mm di-ameter) harvested from the mature leaves of 7- to 9-week-old plants at 4 hinto thenight period (11:00 AM)werefloatedadaxial sideupon topof600mLof respiration buffer (50 mM HEPES, 10 mM MES, pH 6.6, and 200 mMCaCl2) with or without additional metabolites or chemicals. All metaboliteconcentrations were 10 mM unless otherwise indicated. A minimum of sixleaf discs from different plants were assayed for each treatment. O2

concentration measurements were made at the shortest interval permis-sible by the Q2 sensor, which depended on the number of samples beingmeasured, up to a maximum interval of 5 min. To generate RN traces,amoving slopeofO2 consumptionwas calculatedusing a2-hwindow. TheO2 partial pressure was estimated to be 20.95% of an atmosphericpressure of 101 kPa, and the ideal gas law was used to calculate molar O2

consumption rates within the tubes (Scafaro et al. 2017).

Transcript Analysis

Leaf discswere subjected to the samemetabolite-exposure treatments aswith the respiration assays. At the specified time, the leaf discs were re-moved from the surface of the media and snap-frozen in liquid nitrogen.Two leaf discs were combined for each sample and powdered in a beadmill. RNA isolation, cDNA generation, and qPCR were performed as de-scribedbyVanAkenet al. (2016). Amplification curveswere analyzed usingLineRegPCR (Ruijter et al., 2009). Average N0 values of two technicalreplicates were calculated for each biological replicate sample. RelativemRNA expression levels in each biological replicate sample were thendetermined by normalizing the N0 of each replicate sample separatelyagainst each of the two reference genes (AtACT2 and AtUCP5) andcombining the two normalized values by using the geometric mean. Pri-mers for RT-qPCR are shown in Supplemental Table 2.

Metabolite Analyses

Samples of two leaf discs were rinsed briefly in distilled water, patted dryon a paper towel, then snap-frozen in liquid N2 and powdered usinga bead mill. For Ala and PEP measurements, metabolites were extractedin 250 mL of 3.5% (v/v) perchloric acid containing 1.8% (w/v) poly-vinylpolypyrrolidone and 1.6% (w/v) powdered activated charcoal. Sam-ples were centrifuged, and 200 mL of supernatant was neutralized with45 mL of 2 M KOH, followed by centrifugation. Ala was measured using anAlaATand lactatedehydrogenase (LDH) coupledassay.A total of 200mLofsamplewasmixedwith 50mLof assaymix (300mMHEPES, pH7.2, 25mMa-ketoglutarate, 400 mM NADH, and 0.3% [w/v] BSA [added to cause theformationofauniformmeniscus]) ona96-well plate.Themeasurementwasstarted by adding 0.55 units of LDH and 0.25 units of AlaAT, and the rate ofNADHoxidationwasdeterminedand interpolatedagainst astandardcurveofAlaconcentrations.PEPwasmeasuredusingapyruvatekinaseandLDHcoupled assay. A total of 200 mL of sample was mixed with 50 mL of PEP

TOR Control of Plant Respiratory Metabolism 679

assaymix (300mMHEPES,pH7.2,5mMNa3PO4,5mMADP,5mMMgCl2,400 mM NADH, and 0.3% [w/v] BSA) and 0.5 units of LDH. The mea-surement was started by adding 1.2 units of pyruvate kinase, and theamount of PEP was deduced from the amount of NADH oxidized to NAD1

(calculated with a calibrated molar extinction coefficient of 0.054 absor-bance units per mmol of NADH) assuming a 1:1 PEP-to-NAD1 stoichi-ometry. For Pro measurements, the samples were extracted twice in100 mL of 80% ethanol for 20 min at 95°C. Pro measurements wereperformed using a modified version of the assay of Carillo et al. (2008).Sampleswerespundown,and50mLofextractwasadded to50mLofwaterand 200 mL of assay mix (1% [w/v] ninhydrin, 60% [v/v] acetic acid, and20% [v/v] ethanol). The samples were incubated at 95°C for 20 min andcooled on ice, followed by measurement of absorbance at 520 nm. Proconcentrations were determined by interpolation from a standard curve.

Gas chromatography-mass spectrometry metabolite analysis wasperformedasdescribedpreviously (O’Leary et al., 2017). Analyses of sugarphosphates were performed using an Agilent 1100 HPLC system coupledto an Agilent 6510 Quadrupole/Time-of-Flight mass spectrometerequipped with an electrospray ion source. Data acquisition and liquidchromatography-mass spectrometry control were performed using theAgilent MassHunter Data Acquisition software (version B.02.00). Sepa-ration of metabolites was performed using a Luna C18 column (Phe-nomenex; 1503 2mm, 3 mmparticle size). The mobile phase consisted of97:3 (v/v) water:methanol with 10mM tributylamine and 15mMacetic acid(solvent A) and 100%methanol (solvent B). The gradient programwas 0%B,0min; 1%B,5min; 5%B,15min; 10%B,22min; 15%B,24min; 27%B,35 min; 60%B, 40 min; 95%B, 47 min; 95%B, 50min; 0%B, 52 min; and0%B, 68min. The flow ratewas 0.2mL/min,with column temperature keptat 35°C, autosampler was cooled to 10°C, and injection volumewas 30 mL.TheQuadrupole/Time-of-Flight was operated inmass spectrometrymodewith negative ion polarity using the following operation settings: capillaryvoltage, 4000 V; drying N2 gas and temperature, 10 L/min and 250°C,respectively; nebulizer, 30 p.s.i. Fragmentor, skimmer, and octopole radiofrequency (Oct1 RF Vpp) voltages were set to 110, 65, and 750 V, re-spectively. The scan rangewas 70 to 1200m/z, and spectrawere collectedat 4.4 spectra/s, which corresponded to 2148 transients per spectrum. Allmass spectrometry scan data were analyzed using MassHunter Quanti-tative Analysis Software (version B.07.01, Build 7.1.524.0).

Enzyme Activity Assays

Samples of two leaf discs were snap-frozen in liquid N2 and powderedusing a bead mill. Protein was then extracted in 500 mL of buffer solution(100 mM HEPES, pH 7.0, 1 mM DTT, 0.1% [v/v] Triton X-100, 2% [w/v]polyvinylpolypyrrolidone, and 1mMEDTA), and samples were centrifugedfor 5 min at 4°C and 20,000g. AlaAT activity was subsequently assayedenzymatically by adding 20 mL of supernatant to 230 mL of 150 mMNADH,10 mM Ala, 5 mM a-ketoglutarate, 0.4 mMMgCl2, 50 mMHEPES, pH 7.0,and 1 unit of LDH and monitoring NADH oxidation over time photomet-rically on a 96-well plate.

Immunoblotting

Samples of two leaf discs from S6K-HA plants were extracted in 50 mL of100 mMHEPES, pH 7.5, 25 mM glycerol-2-phosphate, 10 mMNaF, 0.1%(v/v) Triton X-100, 2 mM PMSF, and cOmplete protease inhibitor cocktail(Roche) according to the manufacturer’s instructions. Samples were di-luted and boiled in SDS sample buffer, run on an Any kD precast gel (Bio-Rad) for60minat150V, transferredontoaPVDFmembrane,blocked in2%skim milk powder, and probed overnight with anti-HA antibody (Pro-teintech, catalog No. 66006-2-lg) at 1:20,000 dilution or anti-S6K-phos-phoT449 antibody (Abcam, catalog No. 207399, lot No. GR243231-22) at1:1000. Following secondary antibody incubation, the blots were exposed

using Clarity Western ECL Substrate (Bio-Rad) with an Amersham 680Imager CCD camera (GE).

Statistical Analysis

All statistical analyses were performed with Sigma Plot v. 13. Statisticaltests and replicate number are as indicated in figure legends. Biologicalreplicates indicate samples thatwere collected fromdifferent plants grownat the same time. Posthoc testing following ANOVA was performed usingthe Holm-Sidak method. All experimental results were repeated at leastonce with separate batches of plants.

Accession Numbers

Sequence data from this article can be found in the Arabidopsis GenomeInitiative or GenBank/EMBL databases under the following accessionnumbers: AtALAAT1 (At1g17290), AtALAAT2 (At1g72330), AtPDH1(At3g30775), AtPDH2 (At5g38710), mab1 (At5g50850), and AtS6K1(At3g08730).

Supplemental Data

Supplemental Figure 1. The time dependent metabolite stimulation ofRN is independent of time of night.

Supplemental Figure 2. The effect of exogenous glycolytic inter-mediates on Pro-, Ala- and PEP-stimulated RN.

Supplemental Figure 3. Cycloheximide completely blocks RN stimu-lation by external Ala and Pro.

Supplemental Figure 4. Proline stimulation of RN in PDHdeficient lines

Supplemental Figure 5. The effect of external metabolites and AZDon the expression of AlaAT.

Supplemental Figure 6. The effect of rapamycin on RN and repressionof Pro-stimulated RN by Ile.

Supplemental Table 1. Accumulation of exogenous carbohydrates inleaf discs upon 8 h exposure.

Supplemental Table 2. Primers used

ACKNOWLEDGMENTS

We thank Dietmar Funck for the kind gift of the pdh knockout lines, foradvice, and for a critical evaluation of the article. We thank Jelle Van Leeneand Geert De Jaeger for kindly providing the S6K overexpression line. Wethank Santiago Signorelli for a critical reading of the article. This work wassupported by the Australian Research Council (ARC grants CE140100008andDP180104136 toB.M.O.,G.G.K.O.,C.P.L., andA.H.M.) andbyanARCDiscovery Early Career Research Award Fellowship (DE150100130to B.M.O.).

AUTHOR CONTRIBUTIONS

B.M.O. and A.H.M. designed the research; B.M.O., G.G.K.O., and C.P.L.performed the research; B.M.O. analyzed the data; B.M.O. and A.H.M.wrote the article.

Received August 23, 2019; revised November 18, 2019; accepted De-cember 23, 2019; published December 30, 2019.

680 The Plant Cell

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682 The Plant Cell

DOI 10.1105/tpc.19.00157; originally published online December 30, 2019; 2020;32;666-682Plant Cell

Brendan M. O'Leary, Glenda Guek Khim Oh, Chun Pong Lee and A. Harvey MillarRapamycin (TOR) Kinase Activation

Metabolite Regulatory Interactions Control Plant Respiratory Metabolism via Target of

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