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. 2019 Dec 30;32(3):666–682. doi: 10.1105/tpc.19.00157

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

Brendan M O’Leary 1, Glenda Guek Khim Oh 1, Chun Pong Lee 1, A Harvey Millar 1,1
PMCID: PMC7054028  PMID: 31888967

The TOR signaling pathway in plants responds to amino acid levels by eliciting regulatory effects on respiratory energy metabolism at night, uniting a hallmark mechanism of TOR regulation across eukaryotes.

Abstract

Respiration rate measurements provide an important readout of energy expenditure and mitochondrial activity in plant cells during the night. As plants inhabit a changing environment, regulatory mechanisms must ensure that respiratory metabolism rapidly 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 the nighttime O2 consumption rate (RN) in mature leaves of Arabidopsis (Arabidopsis thaliana). Multi-hour RN measurements following 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 metabolite combinations, we discovered metabolite-metabolite regulatory interactions controlling RN. Many amino acids, as well as Glc analogs, were found to potently inhibit the RN stimulation by Pro and Ala but not PEP. The inhibitory effects of amino acids on Pro- 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 work provides direct evidence that the TOR signaling pathway in plants responds to amino acid levels by eliciting regulatory effects on respiratory energy metabolism at night, uniting a hallmark mechanism of TOR regulation across eukaryotes.

INTRODUCTION

Plant respiratory metabolism is a robust and flexible network of reactions that is capable of oxidizing many different carbon substrates to produce not only ATP and reducing power but also carbon skeletons to support biosynthetic processes such as protein synthesis (Plaxton and Podestá, 2006; O’Leary and Plaxton, 2016). Although the main pathways of carbohydrate respiration include glycolysis, the oxidative pentose phosphate pathway, the tricarboxylic acid cycle (TCA cycle), and the mitochondrial electron transport chain (mETC), many other pathways catabolizing amino acids, fatty acids, and other compounds are capable of feeding carbon into the respiratory system (Millar et al., 2011; Rasmusson and Møller, 2011). The activity of the respiratory pathways underpins a plant cell’s energy use and biosynthetic performance under various heterotrophic conditions. Respiration also functions to maximize the rate of photosynthesis in leaf cells during the day (Tcherkez et al., 2017; O’Leary et al., 2019). Therefore, understanding how respiration is controlled and regulated under different metabolic conditions is of major importance for understanding the evolution of carbon and energy use strategies of plants.

Decades of research into the various pathways of plant respiration have revealed a decidedly complex, multi-level system of regulatory mechanisms. These mechanisms can generally be categorized as posttranslational, thus affecting the activity of existing enzymes, or as changes in gene expression, which alter the amount and type of enzymes present (Plaxton and Podestá, 2006; O’Leary and Plaxton, 2014, 2017). Over short periods of time (minutes), metabolism is mostly regulated by posttranslational mechanisms, including substrate levels, feedback inhibition, allosteric effector levels, and protein modifications, which all influence the activity of existing enzymes. Over longer periods of time (hours to days), perceived changes in cellular and environmental conditions, including diurnal cycles, lead to changes in gene expression and protein levels via signal transduction pathways (Giraud et al., 2010; Lee et al., 2010).

Metabolite concentrations are a crucial aspect of the posttranslational control of respiration in all organisms by acting as substrates, allosteric effectors, and feedback inhibitors of various metabolic enzymes (Plaxton and Podestá, 2006). Likewise, many metabolites induce signal transduction cascades that link nutritional or environmental status to changes in enzyme expression levels and posttranslational modifications (Wang and Lei, 2018). In plants, the best understood examples of nutrient signaling metabolites are carbohydrates. For example, in Arabidopsis (Arabidopsis thaliana), Glc levels are partly signaled via hexokinase and also activate the Target of Rapamycin (TOR) pathway (Xiong et al., 2013), while Suc levels are signaled partly via the Suc-trehalose-6-phosphate nexus (Figueroa et al., 2016; Figueroa and Lunn, 2016). Both of these metabolites convey crucial information about a plant’s carbon status that affects downstream energy utilization and respiration. Carbohydrates are the predominant respiratory substrates in plants, and cellular sugar levels correlate with respiration rates (Azcón-Bieto et al., 1983; Noguchi, 2005; O’Leary et al., 2019).

graphic file with name TPC_201900157R1_fx1.jpg

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Recently, the level of specific amino acids and organic acids have also been shown to correlate with leaf respiration rates in Arabidopsis (O’Leary et al., 2017). However, very little is known about nutritional signals emanating from amino acids or organic acids in plants (Hannah et al., 2010). A reliance on amino acids or fatty acids as respiratory substrates in plants is generally thought to be limited to situations of carbohydrate deprivation such as extended darkness and seed germination (Graham, 2008; Kunz et al., 2009; Araújo et al., 2011; Hildebrandt et al., 2015). However, in animals and yeast, amino acids are key regulators of the TOR kinase signaling pathway, which is a central hub integrating information about nutritional and environmental status (Dobrenel et al., 2016; Wolfson and Sabatini, 2017). In plants, this potential aspect of nutrient signaling has not been demonstrated.

The flux of electrons through the mETC is amenable to dynamic flux measurements because it terminates in the consumption of O2, a process that is measurable in real time by several techniques (Scafaro et al., 2017). So far, measurements of respiration rate dynamics in response to metabolite level changes have mostly been performed in isolated mitochondria that lack both de novo gene expression and cytosolic/plastid enzymes, and are therefore capable of revealing only part of the metabolic control of respiration that occurs in plant cells. Few studies have observed dynamic respiratory responses to metabolites within intact plant tissues, and these have all focused on the influence of carbohydrates (Brouquisse et al., 1991; Noguchi, 2005). Here, we explored the regulatory effects of a broad range of metabolites on leaf nighttime O2 consumption rate (RN) using multi-hour measurements of O2 consumption. Although Suc, Glc, Fru, and maltose could stimulate RN, it was phosphoenolpyruvate (PEP), Pro, and Ala that displayed the strongest time-dependent stimulatory effects among the metabolites tested. Metabolite-metabolite regulatory interactions were also discovered in which the stimulation of RN by Ala and Pro is influenced by the levels of other metabolites. The patterns of metabolite-metabolite regulatory interactions were reminiscent of metabolic control by the TOR kinase pathway in animal and yeast cells. To validate this connection, we showed that amino acid interactions with Pro and Ala metabolism were sensitive to three independent types of TOR kinase inhibitors and that amino acid treatments activate TOR kinase in mature leaves to a similar extent to Suc. Our results indicate that the TOR kinase pathway is involved in mediating amino acid-derived metabolic regulatory signals in mature plant leaves that influence respiratory activity and thus plant metabolic rate.

RESULTS

The Stimulation of RN by Metabolites Is Time Dependent

As a tightly regulated metabolic network, respiration rate responds to various changes in the metabolic conditions of the cell. We therefore established a nighttime O2 consumption measurement to observe tissue level changes in RN over multiple hours following metabolic perturbations induced by floating leaf discs on buffered solutions. Leaf discs floated on the buffer solution alone without further chemical additions (control treatments) produced a respiratory trace where RN decreased during the first several hours before gradually stabilizing toward the end of a 14-h measurement (Figure 1).

Figure 1.

Figure 1.

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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 of 10 mM metabolite additions to the buffer medium. The control treatment is leaf RN in the absence of exogenous metabolites. In each graph, the lines represent mean RN after addition of the metabolite indicated, and the blue area shows the 95% confidence intervals of untreated controls. RN rates are shown as fold change relative to untreated controls. All traces represent the average of at least six different leaf disc measurements.

To uncover the regulatory properties of the respiratory network, we performed a high-throughput screen of the multi-hour effect of exogenous metabolites on leaf disc RN. Many metabolically relevant carbohydrates and organic acids were tested, including selected TCA cycle intermediates. Citrate and isocitrate were excluded, as they have been shown to strongly chelate metal cations and thus can disrupt normal cellular behavior (Ränby et al., 1999; Sul et al., 2016). Most proteinogenic amino acids and γ-aminobutyric acid (GABA) were also tested. Trp and Tyr were excluded because they lack sufficient solubility, and Cys was excluded because it undergoes spontaneous oxidation, disrupting the assay. While most exogenous metabolite treatments did not have an appreciable effect on RN, certain metabolites displayed 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 approximate doubling of RN compared with control after 14 h, which corresponds to an ∼50% increase in RN compared with 0 h. Suc, Glc, maltose, Ser, Thr, and Gly showed a less pronounced but still substantial stimulation of RN. By contrast, pyruvate, which is a direct product of both Ala and PEP catabolism within the respiratory pathways, had only a minor effect on RN. In addition, the nitrogen sources NH4+ and NO3− at 10 mM also had no effect on RN over the time course. In combination, these results demonstrate clear and unexpected differences in the effects of exogenous metabolites on RN.

Metabolism likely adjusts diurnally in response to changing output demands and levels of substrates, including carbon reserves (e.g., starch), which may cause some external metabolites to be more or less stimulatory at certain times. To assess the effect of time of night on the stimulation of RN by various metabolites, assays of RN in the presence of Suc, Glc, PEP, pyruvate, Ala, Pro, Ser, and Gly were commenced at 5 and 10 h into the usual 16-h dark period. RN measurements were then recorded for a further 14 h in each case. The metabolite stimulations of RN were observed to be largely independent of time of night, which indicates that depleted starch reserves are not the major determinant of the extent of stimulation by exogenous metabolites in this system (Supplemental Figure 1).

Accumulation Patterns of Metabolites in Leaf Discs following Exogenous Exposure

It was not known whether metabolites would be appreciably taken up into leaf tissue from external solutions. Therefore, we assayed the accumulation of metabolites in leaf discs following 4-h incubations on metabolite solutions (Table 1). All amino acids and glycolytic intermediates accumulated substantially except for Asp and Glu. TCA cycle intermediates displayed modest or no accumulation. Suc, Glc, and Fru did not display significant accumulation 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 these sugars may in part reflect their metabolism by the leaf discs. In the case of compounds that do not appreciably accumulate and do not stimulate RN (e.g., malate, fumarate, Asp, and Glu), it remains unclear whether they were not appreciably taken up by leaf tissue or they were taken up but changes in total concentration were not observed due to metabolism.

Table 1. Accumulation of Exogenous Metabolites in Leaf Discs.

Compound Metabolite Level Ratio (Exposed/Nonexposed ± se)
Amino acids
 Gly 5.2 ± 1.1a
 Ala 5.6 ± 0.2a
 Val 50.4 ± 5.1a
 Leu 105.6 ± 5.4a
 Ile 98.9 ± 9.1a
 Met 47.8 ± 4.3a
 Phe 39.9 ± 3.9a
 Pro 39.9 ± 4.2a
 Ser 15.0 ± 1.6a
 Thr 13.2 ± 1.0a
 Asn 6.0 ± 2.0a
 Gln 8.8 ± 0.9a
 Glu 1.4 ± 0.3a
 Asp 1.2 ± 0.1a
 Lys 34.8 ± 4.3a
 Arg 8.2 ± 2.5a
 GABA 8.3 ± 0.8a
Carbohydrates
 Suc 1.5 ± 0.6a
 Glc 1.2 ± 0.5a
Fru 1.5 ± 0.2a
 Mannitol 101.3 ± 10.3a
 Maltose 5.0 ± 1.0a
TCA cycle organic acids
 Citrate 1.1 ± 0.1a
 Aconitate 1.0 ± 0.1a
 Isocitrate 1.4 ± 0.2a
 α-Ketoglutarate 2.0 ± 0.3a
 Succinate 1.4 ± 0.1a
 Fumarate 1.0 ± 0.04a
 Malate 1.0 ± 0.01a
Glycolytic metabolites
 Gluconate 26.0 ± 3.2a
 Glycerol 13.3 ± 0.8a
 Glucose-6-phosphate 2.7 ± 0.2b
 Fructose-6-phosphate 5.3 ± 0.8b
 3-Phosphoglycerate 2.0 ± 0.1a
 PEP 8.0 ± 2.1c
 Pyruvate 3.1 ± 0.5c
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Leaf disc samples were incubated for 4 h in the presence of the indicated exogenous metabolite, and the fold increase of that metabolite versus control incubations was determined. Significant increases are indicated in boldface (t test, P < 0.05).

a

Determined by gas chromatography-mass spectrometry.

b

Determined by liquid chromatography-quadrupole/time-of-flight-mass spectrometry.

c

Determined by enzymatic analysis.

Stimulation of Respiration by Pro and Ala Is Disrupted by the Presence of Other Metabolites

To observe whether interactions between metabolites would affect RN, we focused on whether the large RN stimulations caused by PEP, Pro, and Ala would be influenced by simultaneous provision of any additional metabolite (referred to as the co-metabolite). Respiratory substrates including carbohydrates, glycolytic intermediates, TCA cycle dicarboxylic acids, and amino acids were applied exogenously at 10 mM alone and in combination with PEP, Pro, and Ala, and leaf disc RN was measured over time. By comparing the relative RN at 14 h for these incubations, it was observed that many amino acids (Figure 2) as well as malate (Figures 3A to 3C) had the effect of blocking Pro and Ala stimulation of RN. By contrast, only the addition of Lys significantly diminished PEP-stimulated RN. Carbohydrate substrates and glycolytic intermediates did not affect Pro-, Ala-, or PEP-stimulated RN, with the exception that pyruvate modestly increased the stimulatory effect of PEP on RN (Figures 3D to 3F; Supplemental Figure 2).

Figure 2.

Figure 2.

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

(A) to (C) Amino acids were added by themselves or in combination with Pro (A), Ala (B), or PEP (C) to the respiration buffer followed by measurement of leaf disc 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 between the metabolite combination treatments versus the corresponding Ala-, PEP-, or Pro-only control treatment (ANOVA, P < 0.05; n = 6). Among those treatments found to be significantly different, a second statistical test was conducted (indicated by n.s.) identifying those treatments where the addition of Ala, 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).

Figure 3.

Figure 3.

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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; α-KG, α-ketoglutarate; OAA, oxaloacetate.

Three Glc analogs, glucosamine, 2-deoxyglucose, and mannose, which are inhibitors of hexokinase and glucose-6-phosphate dehydrogenase of the oxidative pentose phosphate pathway, were also tested. These three co-metabolites, which are themselves poor respiratory substrates (Pego et al., 1999), had the effect of inhibiting RN and strongly inhibiting Ala and Pro stimulation of RN, but they were less effective at inhibiting PEP-stimulated RN (Figures 3D to 3F).

PEP, Pro, and Ala Accumulation in Leaf Tissue Accompanies Respiratory Stimulation

The mechanism of RN stimulation by Pro and Ala and suppression of stimulation by certain external metabolites could involve transcriptional, translational, or posttranslational factors. Posttranslational, time-dependent stimulation of RN by Pro and Ala could be due to the gradual accumulation of these metabolites within the leaf cells and their use as substrates to increase metabolic fluxes linked to RN. Titrations of exogenous Pro revealed that higher external Pro concentrations caused greater RN stimulation, consistent with a substrate-driven RN stimulation (Figure 4A). By contrast, titrations of exogenous Ala showed a respiratory stimulation peaking at ∼5 to 10 mM and subsequently decreasing at higher concentrations (Figure 4A). Assays of metabolite accumulation in leaf discs revealed that Pro and Ala levels increased markedly during the time course, although to varying absolute amounts (Figure 4B). In each case, the increase in Pro or Ala levels preceded the increase in tissue RN by several hours, suggesting that metabolite level changes are related to but not solely responsible for stimulation of RN.

Figure 4.

Figure 4.

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The Influence of Pro and Ala Concentrations on RN.

(A) Different external concentrations of Pro (left) and Ala (right) were applied exogenously, and leaf disc RN was measured over 14 h. The average relative RN compared with control treatments is shown (n = 6). The panels below show the relative respiration rates at 14 h. Data points are shown; error bars indicate se.

(B) The amount of Pro (left) and Ala (right) in leaf discs during incubation in the presence or absence of 10 mM Pro or Ala, respectively. Data points are shown (n = 4). Lines represent mean values. Error bars indicate se. Asterisks indicate significant differences between control and Pro or Ala treatments at that time (ANOVA, P < 0.05).

(C) The effect of exogenous co-metabolites on the uptake of Pro and Ala from the media into leaf discs. Leaf discs were floated in respiration buffer containing metabolites for 4 h, then the amount of Pro (left) or Ala (right) per the leaf discs was quantified. Data points are shown (n ≥ 4). Solid bars represent mean values; error bars indicate se. Asterisks indicate significant differences compared with Ala or Pro alone (ANOVA, P < 0.05).

We next assessed whether the co-metabolites that inhibited Pro and Ala stimulation of RN did so by blocking Pro/Ala uptake into leaf 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 accumulation of Pro by 31 and 48%, respectively, compared with the samples incubated in Pro alone. Similarly, Ala accumulation was partly reduced by Met (37% reduction). The other metabolites tested (Suc, glucosamine, Asp, and malate) had insignificant or minor (<20%) effects on Pro and Ala accumulation. Given the titratable influence of Pro and Ala on RN, these modest differences in uptake appear insufficient to account for the strong inhibitory effect of these co-metabolites on Pro- and Ala-stimulated RN.

Ala and Pro Stimulation of Respiration Occurs via Changes in Gene Expression

We next evaluated whether differences in gene expression could be involved in the observed patterns of RN regulation. The application of cycloheximide, which inhibits cytosolic ribosomal translation, completely blocked the stimulatory effects of Pro and Ala on RN but did not affect Pro or Ala uptake into leaf discs (Supplemental Figure 3). The effect of cycloheximide indicates that changes in gene expression are necessary for the dynamic increases in RN caused by Pro and Ala. As observed previously, this 2.5 µM cycloheximide treatment, by itself, has no appreciable effect on RN (O’Leary et al., 2017).

To determine if the effect of Pro on RN was the result of proline dehydrogenase (PDH) activity, which oxidizes Pro in the mitochondrial matrix and feeds electrons into the mETC (Cabassa-Hourton et al., 2016), we assessed whether the competitive PDH inhibitor l-thiazolidine-4-carboxylic acid (T4C) would reduce the stimulation of RN by Pro. At 4 mM, T4C reduced the stimulatory effect of Pro by 55% at 14 h (Figure 5A). This is consistent with previous reports that T4C is capable of reducing Pro-dependent O2 uptake by 67% in isolated barley (Hordeum vulgare) mitochondria (Elthon and Stewart, 1984). We next observed that transcripts for both PDH isoforms in Arabidopsis, PDH1 and PDH2, increased during leaf disc incubation in Pro (Figures 5B and 5C). PDH1 is expressed at a much higher level than PDH2 and is thought to represent the bulk of PDH activity (Funck et al., 2010; Cabassa-Hourton et al., 2016). Using confirmed T-DNA insertion lines (Funck et al., 2010), we assessed whether disruption of PDH gene 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 was stimulated by Pro similarly to the wild type (Figure 5E). By contrast, pdh1-1 pdh2-1 displayed a clear reduction of RN in the presence of Pro (Figure 5F). pdh1-1 pdh2-1 leaf discs displayed an almost complete loss of PDH1 transcripts, and PDH2 transcripts were undetectable (Supplemental Figure 4B). Furthermore, pdh1-1 pdh2-1 was no longer sensitive to RN inhibition by T4C in the presence of Pro (Supplemental Figure 4). Together, these results indicate that external Pro accumulates in leaf disc tissue and induces the expression of PDH isoforms that in turn increase the mitochondrial catabolism of Pro, thus increasing RN.

Figure 5.

Figure 5.

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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 = 6).

(B) and (C) Transcript levels of PDH1 (B) and PDH2 (C) upon exposure of leaf discs to external Pro control treatment. Replicate data points are shown. Lines represent mean values; error bars indicate se (n = 3).

(D) to (F) Averaged traces of RN of wild-type and PDH knockdown lines in the presence and absence of external Pro (n = 8).

(G) and (H) The effect of external metabolite combinations on the expression of PDH genes. Transcript analysis for PDH1 (G) and PDH2 (H) was performed following 10-h incubations with or without Pro and additional co-metabolites. Bars represent average values relative to control treatments. Replicate data points are shown. Dashed lines indicate separate analyses. Asterisks indicate significant differences in transcript abundance compared with the corresponding treatment with Pro alone (ANOVA, P < 0.05; n = 4).

The precise mechanism or metabolic route by which Ala might stimulate RN was somewhat unclear, given that pyruvate does not also stimulate RN. Therefore, we decided to establish whether metabolism of Ala to acetyl-CoA via an alanine aminotransferase (AlaAT) and the pyruvate dehydrogenase complex (PDC) was necessary for Ala’s stimulatory effect on RN. The AlaAT inhibitor cycloserine inhibited the Ala stimulation of RN by ∼65% (Figure 6A). During incubation in Ala, the total AlaAT activity of the leaf discs increased with time (Figure 6B). The transcript abundance of neither Arabidopsis AlaAT isoform, ALAAT1 or ALAAT2, correlated with the time-dependent stimulation of RN by Ala (Figure 6C). However, the characterized alaat1-1 T-DNA insertion line (Miyashita et al., 2007) displayed greatly reduced Ala stimulation of RN (Figure 6D) and also lowered total extractable AlaAT activity (Supplemental Figure 5). Furthermore, mab1, which is deficient in the PDC E1β subunit and displays greatly reduced mitochondrial PDC activity (Ohbayashi et al., 2019), also displayed a lack of RN stimulation by Ala (Figure 6D). These results indicate that Ala stimulation of RN involves the conversion of Ala to acetyl-CoA in the mitochondria.

Figure 6.

Figure 6.

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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 µM of the AlaAT inhibitor cycloserine (n = 6).

(B) Total AlaAT extractable activity relative to 0-h treatment. Asterisks indicate significant differences compared with 0 h (ANOVA, P < 0.05; n = 3). Error bars indicate se.

(C) Transcript levels of ALAAT1 (left) and ALAAT2 (right) upon exposure of leaf discs to external Ala. Replicate data points are shown; line represents mean values (n = 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 = 8).

PDH Expression Is Repressed by Metabolites That Also Suppress Pro-Stimulated RN

To determine whether the regulatory effects of co-metabolites that reduce Pro and Ala stimulation of RN could be mediated at the level of 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 applied together with Pro or Ala, respectively. External Suc treatment was previously shown to inhibit PDH expression, as described by Hanson et al. (2008) and Funck et al. (2010) and was therefore also investigated. In the case of Pro incubations, glucosamine completely blocked PDH1 transcript accumulation, while Suc caused a partial decrease in PDH1 accumulation relative to control treatments (Figure 5G). The pattern of PDH2 transcript accumulation was different, with the Suc, malate, Asp, Met, and Ile significantly inhibiting PDH2 accumulation in the presence of Pro (Figure 5H). In the case of Ala incubations, the presence of the same co-metabolites had no significant effect on the transcript abundances of ALAAT1 or ALAAT2 (Supplemental Figure 5).

Metabolite-RN Interactions Are Sensitive to TOR Inhibitors

A clear pattern within the co-metabolite interactions was that many amino acids caused a similar repressive effect on RN induction by both Pro and Ala. Relatively little information is available about the sensing of amino acids in plants (Gent and Forde, 2017; Dinkeloo et al., 2018), but in fungi and animals, amino acids are strong inducers of the TOR pathway, which is a master regulator of nutrient sensing and energy metabolism (Jewell et al., 2013). We therefore hypothesized that the TOR pathway in plants was responsible for inhibiting the respiration of amino acids in the presence of elevated amino acid levels. If this were true, then inhibiting the TOR signaling pathway at the time of metabolic interaction would block the ability of amino acid co-metabolites to suppress Ala- and Pro-stimulated RN. To test this hypothesis, we first examined the effect of the TOR inhibitor AZD8055 (AZD) on RN in the presence of metabolites (Figures 7A to 7C). Interestingly, the effect of 2 µM AZD on RN was dependent on which metabolites were present in the incubation mixture. AZD caused strong RN stimulation in the presence of Asp and malate and weaker RN stimulation on its own or in the presence of Met. Next, we observed that the presence of AZD partly restored the ability of Pro and Ala to stimulate RN while in the presence of Ile and Met. Two other chemical inhibitors of TOR that have been demonstrated to be effective in plants are TORIN2 and WYE-125132 (WYE-125; Montané and Menand, 2013, 2019). Both of these inhibitors behaved similarly to AZD in partly restoring the ability of Pro to stimulate RN in the presence of Ile (Figure 7D). While TOR is named for its sensitivity to rapamycin in animals, this inhibitor often has little effect on TOR in plants (Dobrenel et al., 2016), except in the isolated case of a BP12 expression line that reportedly showed increased sensitivity of plant TOR to rapamycin (Ren et al., 2012). In our hands, rapamycin displayed no effect on respiration or metabolite interactions with RN in leaf discs from either wild-type plants or a BP12 expression line (Supplemental Figure 6).

Figure 7.

Figure 7.

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

(A) to (C) Average RN rates were determined after 14 h of incubation with various metabolite treatments in the presence and absence of 2 µM AZD. Treatments were performed without metabolite combinations (A), as metabolite combinations with Ala (B), and as metabolite combinations with Pro (C). Error bars indicate se. Asterisks indicate significant effects of AZD on RN rate for that metabolite treatment (t test, P < 0.05; n > 14). Double daggers indicate significant stimulation by Ala or Pro compared with the same treatment without Ala or Pro (t test, P < 0.05; n > 14). Glc-N, glucosamine.

(D) Comparison of the effects of 2 µM AZD, TORIN2, or WYE-125 on RN stimulation by Pro and Ala. Average RN rates were determined after 12 h of incubation. Error bars indicate se. Asterisks indicate significant effects of AZD, TORIN2, or WYE-125 compared with no inhibitor for that metabolite treatment (ANOVA, P < 0.05; n > 23).

(E) Average RN rates were determined after 14 h of incubation in the presence and absence of combinations of Thr, Leu, and 2 µM AZD. Replicate data points are shown. Error bars indicate se. Asterisks represent significant differences among corresponding treatments with and without AZD (t test, P < 0.05; n = 7).

(F) Leaf discs from Arabidopsis expressing exogenous HA-tagged S6K were incubated in the corresponding treatments for 4 h prior to SDS-PAGE immunoblotting with anti-HA tag or anti-S6K-phospho-T449 antibodies. Wild-type control treatment leaf discs are included as a control. The top band corresponds to a phosphorylated version of S6K.

To determine whether the regulatory effects of Ile were unique to Pro and Ala respiration or a general property of amino acid catabolism, we assessed whether Thr-stimulated RN (the next strongest amino acid stimulation following Ala and Pro) would also be affected by Ile and AZD. Thr-stimulated RN was inhibited by Ile, and this repression was mitigated by AZD (Figure 7E); Thr therefore behaves similar to Ala and Pro even though all three amino acids are catabolized by distinct pathways.

These results suggested that certain elevated amino acid levels signal to restrict aspects of respiratory catabolism via the TOR pathway. We therefore used an established immunoblot assay of the phosphorylation of the downstream TOR target S6K in an S6K overexpression line to ascertain whether amino acids could activate TOR. Arabidopsis S6K is subject to multiple phosphorylation events, with T449 being phosphorylated by TOR (Xiong and Sheen, 2012). Upon phosphorylation, S6K migrates slower on SDS-PAGE, leading to a phosphorylation-dependent banding pattern (Van Leene et al., 2019). Using this assay, Suc and Glc have previously been identified as metabolic stimulators of TOR activity in Arabidopsis seedlings (Xiong et al., 2013; Van Leene et al., 2019). Leaf disc incubation in Ile, Gln, or Suc led to a similar increase in the T449-phosphorylated S6K compared with the control, indicating that they are each capable of activating TOR in mature leaves (Figure 7F). The inclusion of AZD completely inhibited S6K phosphorylation.

TOR signaling alters the abundance of transcripts for many metabolic genes. We therefore tested whether AZD was influencing the expression of PDH or AlaAT genes in our system. ALAAT1 and ALAAT2 transcripts were only modestly increased by AZD treatment (Supplemental Figure 5C), again indicating that ALAAT transcript induction is not a major site of control of Ala catabolism. By contrast, AZD treatment strongly increased PDH1 and PDH2 expression in the presence or absence of Pro (Figure 8A). Furthermore, during leaf disc incubations with Pro and interacting co-metabolites, AZD was shown to mitigate the inhibition of PDH1 and PDH2 expression caused by most interacting co-metabolites tested except glucosamine (Figure 8B).

Figure 8.

Figure 8.

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TOR Inhibition Regulates PDH Gene Expression.

(A) The effect of 2 µM AZD 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 significant differences between treatments with and without AZD (ANOVA, P < 0.05; n = 4).

(B) The effect of 2 µM AZD on leaf disc PDH1 (left) and PDH2 (right) transcript levels was determined after 9 h of incubation in the presence of Pro in combination with select co-metabolites. Bars represent average transcript levels relative to control incubations with Pro only. Replicate data points are shown. Asterisks represent significant differences with respect to the corresponding control treatment with or without AZD (two-way ANOVA, P < 0.05; n = 3).

(C) The effect of AZD on the uptake of Pro from the media into leaf discs following a 4-h incubation. Data points are shown (n = 5); solid bars represent mean values. 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 the presence and absence of the autophagy inhibitor wortmannin (n = 7). The right panel shows the relative respiration rates at 14 h. Data points are shown; error bars indicate se. Significant effects of wortmannin are indicated with the asterisk (t test, P < 0.05; n = 7).

To help further clarify the mechanism of Pro catabolic regulation by TOR, we tested whether TOR inhibition by AZD affected Pro uptake into leaf disc tissue. After a 5-h incubation, AZD treatment did not affect Pro uptake into leaf discs (Figure 8C). TOR inhibition also activates autophagy. To assess whether autophagy was required for Pro- or Ala-dependent stimulation of RN, we treated leaf discs with the autophagy inhibitor wortmannin in the presence and absence of Pro or Ala. Wortmannin did not block the stimulation of RN by Pro or Ala (Figure 8D). Therefore, the interaction of TOR signaling with Pro-induced respiration does not appear to require changes in Pro uptake or autophagy.

DISCUSSION

The results from this study demonstrate that exogenously supplied metabolites, on their own and in combinations, can greatly influence leaf respiratory rates when observations are continued over hours. The time dependencies of the respiratory responses are likely to be a consequence of both metabolite uptake and de novo gene expression, which both exert change on the system over the duration of the 14-h measurement. This means that the RN dynamics observed here are due to both the transcriptional/translational and posttranslational effects of metabolite levels on respiratory enzyme activities. Because the transcriptional/translational and posttranslational machinery of the whole cell remained intact, the information gained in these RN experiments is distinct from and complementary to information about the metabolic regulation of respiration derived from studying isolated mitochondria. The combination of high-throughput and long-duration RN assays upon intact cells (tissue) succeeded in uncovering novel aspects of respiratory regulation, including the involvement of the TOR pathway, which we discuss below with regard to its importance for control of plant metabolism.

The O2 consumption rates that we measure in this study, and define as RN, indicate that the mETC fluxes are varying in response to changing metabolite concentrations. However, respiratory activity can also be measured as CO2 production, which provides different and complementary information about respiratory fluxes. The ratio of respiratory CO2 production to O2 consumption, known as the respiratory quotient (RQ), varies depending on the oxidation status of the substrate being metabolized. Therefore, the relative magnitude of respiratory stimulation by various metabolites would likely be different if considered in terms of CO2 release versus O2 consumption. For example, carbohydrates, with an RQ of 1, would release 25% more CO2 per unit of O2 consumed upon complete oxidation than the average amino acid, with an RQ of 0.8.

Why Some Respiratory Substrates Stimulate RN and Others Do Not

Certain oxidizable metabolites can clearly have a disproportionate effect on Arabidopsis leaf RN that only becomes observable after several hours of exposure. The immediate question that arises from the screen of central carbon metabolites (Figure 1) is, why are some of these respiratory substrates effective stimulators of total RN whereas others are not? The existence and organization of the various respiratory pathways is clearly not a sufficient explanation for the varied effects of metabolites on RN.

Mitochondria are highly involved in the metabolism of amino acids in plants both during normal processes like photorespiration (Douce et al., 2001) and more broadly under stress conditions (Araújo et al., 2011; Hildebrandt et al., 2015). Gly is the predominant amino acid substrate supplied to leaf mitochondria because of photorespiration in the light. The induced use of other amino acids as respiratory substrates under carbon starvation or senescence, in particular Ile, Leu, Val, and Lys, is well studied and has been linked to a SnRK1-bZIP signal transduction pathway and the upregulation of the ETF/ETFQO complex (Araújo et al., 2010; Cavalcanti et al., 2017; Pedrotti et al., 2018). By contrast, a connection that distinguishes Ala and Pro from most other amino acids is that they are both well known to accumulate in response to certain stresses; furthermore, this accumulation occurs at least partly in the cytosol in the case of Pro (Ketchum et al., 1991; Gagneul et al., 2007). Pro accumulates following salt and osmotic stress and may play several protective roles (Verbruggen and Hermans, 2008). Ala accumulates under hypoxic stress, including flooding conditions, allowing continued ATP generation by glycolysis by acting as an alternate sink for pyruvate, whose downstream oxidation is inhibited because of a lack of O2 (Miyashita et al., 2007; Rocha et al., 2010; António et al., 2016). The reason 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 of Ala and Pro when the relevant cellular conditions that cause their accumulation are not present (Miyashita et al., 2007; Szabados and Savouré, 2010). By contrast, plants likely have not evolved equally effective and coordinated regulatory responses to alleviate excess amounts of other amino acids, which do not typically accumulate to high levels in the environment.

Diurnal variations in leaf Pro content suggest that degradation of Pro naturally occurs during the night (Hayashi et al., 2000). Furthermore, external Pro is known to stimulate the expression of PDH isoforms under nonstress conditions (Verbruggen and Hermans, 2008; Funck et al., 2010). Pro also induces the upregulation of the expression of Δ1-pyrroline-5-carboxylate dehydrogenase, which together with PDH constitutes the pathway of Pro oxidation to Glu (Deuschle et al., 2004). This mitochondrion-localized pathway generates reducing power at both enzymatic steps 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 measurable Pro-dependent O2 consumption (Cabassa-Hourton et al., 2016). Our results indicate that in mature leaves both PDH isoforms can contribute to Pro-dependent RN, consistent with their shared role in regulating Pro accumulation in senescing leaves (Launay et al., 2019). For Ala, the cessation of hypoxia stimulates Ala catabolism via a pathway that involves the mitochondria-localized AlaAT1 (Figure 6D; Miyashita et al., 2007). The fate of pyruvate formed in this reaction has not been experimentally characterized, although our data suggest that it is largely oxidized via the mitochondrial PDC (Figure 6D) and presumably further oxidized by the TCA cycle.

Other metabolites that stimulated RN, such as PEP, 3-phosphoglycerate, Suc, Glc, and Fru (Figure 1), would not stimulate R in isolated mitochondria because they lack carbohydrate or glycolytic metabolic enzymes. These results highlight how the larger multi-compartmented cellular metabolic network contributes to the control of RN outside the realm of mitochondrion-localized metabolism. The long-duration assays of respiration rate dynamics outlined in this study offer the potential to continue to uncover mechanisms controlling mitochondrial and cellular metabolism and to identify metabolic phenotypes that clarify gene function.

The Regulatory Pathway Involved in Respiratory Catabolite Repression in Plants

Novel regulatory interactions among metabolites were identified wherein the presence of a co-metabolite in the external medium inhibited RN simulation by another metabolite. Several amino acids, malate and the Glc analogs glucosamine, 2-deoxyglucose, and mannose, were capable of this effect. A clear pattern was that these co-metabolites each had similar effects on Pro- and Ala-stimulated RN but were less effective or ineffective at reducing PEP-stimulated RN (Figures 2 and 3). In the case of Pro, incubation with inhibitory co-metabolites was accompanied by reduced expression of PDH transcripts, clearly implicating manipulations of gene expression as a likely mechanism of action (Figure 5). Posttranslational effects of co-metabolites upon the specific aspects of respiratory metabolism that concern Ala and Pro but not PEP metabolism cannot be ruled out; however, there is no indication of such a mechanism in the literature. Therefore, our evidence led us to further investigate a transcriptional/translational mechanism of action.

Although the regulation of amino acid metabolism is complex (Pratelli and Pilot, 2014), it seemed implausible that the different inhibitory co-metabolites could each be an allosteric inhibitor or otherwise affect the transcriptional, translational, or posttranslational control of Rn by independent mechanisms. Therefore, we hypothesized that the effects of these metabolites converge on a signaling pathway that elicits multiple changes in enzyme activities sufficient to inhibit the respiration of both Pro and Ala by mitochondria. A pertinent example of a metabolic signaling pathway that receives nutritional inputs from multiple respiratory substrates is the TOR kinase pathway. The TOR kinase pathway is a master regulator of eukaryotic carbon metabolism, where its activation generally serves to promote anabolic metabolism and inhibit catabolic metabolism (Dobrenel et al., 2016). It was plausible that the TOR kinase pathway would be involved in the metabolite regulation observed in this study because amino acids are one of the primary signals activating the TOR pathway in other eukaryotes (Jewell et al., 2013). In animals and yeast, the presence of amino acids promotes TOR activity, which activates protein synthesis and growth, while the absence of amino acids inhibits TOR activity, which activates catabolic processes such as autophagy (González and Hall, 2017). Consistently, we observed here that certain plant respiratory catabolic processes were suppressed by elevated amino acid levels. TOR activation in plants has so far only been linked to the presence of Glc and Suc, and the involvement of amino acids in plant TOR pathway regulation has not been reported (Xiong et al., 2013; Van Leene et al., 2019). Interestingly, plants lack all the components of amino acid sensing upstream of TOR that are described in animals and fungi (Wolfson and Sabatini, 2017). Hence TOR-dependent amino acid sensing in plants is likely to involve new or divergent components that await identification.

Despite these deficits, we have provided clear evidence that amino acid levels do influence metabolic activities in plants via the TOR kinase signaling pathway. The inhibitory effects of amino acids on both Pro- and Ala-stimulated RN are largely removed when TOR inhibitors are present (Figure 7). Furthermore, amino acids were shown to be activators of TOR kinase activity (Figure 7F). TOR inhibition counteracted the effect of several inhibitory co-metabolites on PDH transcript levels (Figure 8B). Therefore, in the case of Pro, these results provide a mechanistic basis for the regulatory interaction between certain amino acids and Pro-stimulated RN (Figure 9A). Clearly, other gene targets besides PDH isoforms are involved to account for the observed control of other amino acid respiration (e.g., Ala and Thr) by TOR. In this regard, the general upregulation of amino acid catabolic genes, including PDH1, by inactivation of TOR has been repeatedly observed in previous plant transcriptome analyses (Ren et al., 2012; Xiong et al., 2013; De Vleesschauwer et al., 2018). In addition, disruption of TOR signaling in plants leads to the accumulation of high levels of amino acids and TCA cycle organic acids (Moreau et al., 2012; Ren et al., 2012; Caldana et al., 2013), potentially due to activation of amino acid synthesis (Mubeen et al., 2018). Our findings provide evidence that TOR activity in plants is influenced by amino acid levels leading to downstream effects on respiratory substrate use, including that of amino acids. A model of this general mechanism of action is depicted in Figure 9B.

Figure 9.

Figure 9.

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Hypothetical Model of TOR Kinase Function in the Regulation of Pro 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-undiscovered mechanisms. Activation of TOR inhibits amino acid catabolism and respiration by targeting the expression of amino acid catabolic genes, consistent with previous transcriptomic results. At the same time, TOR activation promotes anabolic pathways, partly via S6K phosphorylation, as a metabolic destination for amino acids. In this way, the presence of amino acids contributes to an overall signal of nutrient abundance in mature leaves.

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

The implication of the TOR pathway in regulating respiratory activity in plants unites a function of the TOR pathway across a wider set of eukaryotes. Yet, differences between TOR regulatory properties also indicate additional ways in which the plant respiratory regulatory network has adapted to unique metabolic demands. As shown in Figure 7, inhibition of TOR increases RN rate in 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 a metabolic standpoint, the inhibition of the yeast TOR pathway promotes a shift from fermentation to the more energetically efficient respiration of carbon substrates along with the usage of nonpreferred N sources, including Pro (Hardwick et al., 1999). By contrast, in animal cells, inhibition of TOR decreases respiratory O2 consumption and increases aerobic glycolysis, shifting Glc metabolism away from mitochondrial respiration (Schieke et al., 2006; Cunningham et al., 2007; Ramanathan and Schreiber, 2009). Inhibition of TOR also leads to opposite responses in plant and animal cells with regard to TCA cycle organic acid levels; levels increase and decrease in plant and animal cells, respectively, in line with changes in respiration rate (Ramanathan and Schreiber, 2009; Caldana et al., 2013).

Mechanistically and genetically, much less is known about the functioning of the TOR pathway in plants compared with our detailed understanding in animals and fungi (Dobrenel et al., 2016). This has largely been due to experimental constraints in plant systems, including the embryo lethality of TOR mutations, the variable efficiency of rapamycin as a plant TOR inhibitor, and the relative scarcity of assays for TOR activity (Xiong and Sheen, 2012; Dobrenel et al., 2016). The development of additional TOR inhibitors has helped in this regard. AZD has been demonstrated to specifically inhibit TOR compared with the most closely related phosphatidylinositol 3-kinases (Chresta et al., 2010). Furthermore, the effect of AZD was shown to be related to TOR expression levels in Arabidopsis (Montané and Menand, 2013). Therefore, the long-term respiration measurements presented here, in combination with specific TOR inhibitors, represent an additional means by which to probe TOR function in plants, in particular the responses to various metabolic conditions where TOR may have specialized functions in plants compared with animals or yeast. A key area of future research is whether the regulatory properties of RN as described here for leaves, such as RN stimulation by PEP, Pro, and Ala, also occur in other plant tissues. It has already been observed that the regulatory properties of the plant TOR pathway are likely different between tissues and developmental stages (Xiong and Sheen, 2014).

METHODS

Plant Material and Growth Conditions

Arabidopsis (Arabidopsis thaliana) accession Col-0 (N6000) was used as the wild type. The following mutant lines were used: alaat1-1 (At1g17290; Miyashita et al., 2007); pdh1-2, pdh2-1, and pdh1-1 (At3g30775 and At5g38710; Funck et al., 2010); mab1 (At5g50850; Ohbayashi et al., 2019); and 35S-S6K1-HA (At3g08730; Van Leene et al., 2019). The pdh1-1 mutant was backcrossed three times and the pdh2-1 mutant five times to Col-0 to obtain near isogenic lines before pdh1-1 pdh2-1 double mutants were selected in the F2 population of a cross between the two mutant lines.

Seeds were sown into a 3:1:1 mix of potting soil, perlite, and vermiculite, supplemented with slow-release fertilizer, and covered with a transparent plastic cover until established. Plants were grown in a controlled-environment growth chamber maintaining a short-day photoperiod of 8 h of light and 16 of h dark (11:00 pm to 7:00 am light) with tubular fluorescent lighting with a photon flux of 120 µmol m−2 s−1, a relative humidity of 75%, and a day/night temperature cycle of 22°C to 17°C. Soil was kept well moistened with regular watering.

Respiration Measurements

Respiration measurements were performed on a Q2 oxygen sensor (Astec-Global) in sealed 850-μL capacity tubes at 21°C. Leaf discs (7 mm diameter) harvested from the mature leaves of 7- to 9-week-old plants at 4 h into the night period (11:00 am) were floated adaxial side up on top of 600 μL of respiration buffer (50 mM HEPES, 10 mM MES, pH 6.6, and 200 µM CaCl2) with or without additional metabolites or chemicals. All metabolite concentrations were 10 mM unless otherwise indicated. A minimum of six leaf discs from different plants were assayed for each treatment. O2 concentration measurements were made at the shortest interval permissible by the Q2 sensor, which depended on the number of samples being measured, up to a maximum interval of 5 min. To generate RN traces, a moving slope of O2 consumption was calculated using a 2-h window. The O2 partial pressure was estimated to be 20.95% of an atmospheric pressure 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 discs were subjected to the same metabolite-exposure treatments as with the respiration assays. At the specified time, the leaf discs were removed from the surface of the media and snap-frozen in liquid nitrogen. Two leaf discs were combined for each sample and powdered in a bead mill. RNA isolation, cDNA generation, and qPCR were performed as described by Van Aken et al. (2016). Amplification curves were analyzed using LineRegPCR (Ruijter et al., 2009). Average N0 values of two technical replicates were calculated for each biological replicate sample. Relative mRNA expression levels in each biological replicate sample were then determined by normalizing the N0 of each replicate sample separately against each of the two reference genes (AtACT2 and AtUCP5) and combining the two normalized values by using the geometric mean. Primers for RT‐qPCR are shown in Supplemental Table 2.

Metabolite Analyses

Samples of two leaf discs were rinsed briefly in distilled water, patted dry on a paper towel, then snap-frozen in liquid N2 and powdered using a bead mill. For Ala and PEP measurements, metabolites were extracted in 250 μL of 3.5% (v/v) perchloric acid containing 1.8% (w/v) polyvinylpolypyrrolidone and 1.6% (w/v) powdered activated charcoal. Samples were centrifuged, and 200 μL of supernatant was neutralized with 45 μL of 2 M KOH, followed by centrifugation. Ala was measured using an AlaAT and lactate dehydrogenase (LDH) coupled assay. A total of 200 μL of sample was mixed with 50 μL of assay mix (300 mM HEPES, pH 7.2, 25 mM α-ketoglutarate, 400 µM NADH, and 0.3% [w/v] BSA [added to cause the formation of a uniform meniscus]) on a 96-well plate. The measurement was started by adding 0.55 units of LDH and 0.25 units of AlaAT, and the rate of NADH oxidation was determined and interpolated against a standard curve of Ala concentrations. PEP was measured using a pyruvate kinase and LDH coupled assay. A total of 200 μL of sample was mixed with 50 μL of PEP assay mix (300 mM HEPES, pH 7.2, 5 mM Na3PO4, 5 mM ADP, 5 mM MgCl2, 400 µM NADH, and 0.3% [w/v] BSA) and 0.5 units of LDH. The measurement was started by adding 1.2 units of pyruvate kinase, and the amount of PEP was deduced from the amount of NADH oxidized to NAD+ (calculated with a calibrated molar extinction coefficient of 0.054 absorbance units per µmol of NADH) assuming a 1:1 PEP-to-NAD+ stoichiometry. For Pro measurements, the samples were extracted twice in 100 μL of 80% ethanol for 20 min at 95°C. Pro measurements were performed using a modified version of the assay of Carillo et al. (2008). Samples were spun down, and 50 μL of extract was added to 50 μL of water and 200 μL of assay mix (1% [w/v] ninhydrin, 60% [v/v] acetic acid, and 20% [v/v] ethanol). The samples were incubated at 95°C for 20 min and cooled on ice, followed by measurement of absorbance at 520 nm. Pro concentrations were determined by interpolation from a standard curve.

Gas chromatography-mass spectrometry metabolite analysis was performed as described previously (O’Leary et al., 2017). Analyses of sugar phosphates were performed using an Agilent 1100 HPLC system coupled to an Agilent 6510 Quadrupole/Time-of-Flight mass spectrometer equipped with an electrospray ion source. Data acquisition and liquid chromatography-mass spectrometry control were performed using the Agilent MassHunter Data Acquisition software (version B.02.00). Separation of metabolites was performed using a Luna C18 column (Phenomenex; 150 × 2 mm, 3 µm particle size). The mobile phase consisted of 97:3 (v/v) water:methanol with 10 mM tributylamine and 15 mM acetic acid (solvent A) and 100% methanol (solvent B). The gradient program was 0% B, 0 min; 1% B, 5 min; 5% B, 15 min; 10% B, 22 min; 15% B, 24 min; 27% B, 35 min; 60% B, 40 min; 95% B, 47 min; 95% B, 50 min; 0% B, 52 min; and 0% B, 68 min. The flow rate was 0.2 mL/min, with column temperature kept at 35°C, autosampler was cooled to 10°C, and injection volume was 30 µL. The Quadrupole/Time-of-Flight was operated in mass spectrometry mode with negative ion polarity using the following operation settings: capillary voltage, 4000 V; drying N2 gas and temperature, 10 L/min and 250°C, respectively; nebulizer, 30 p.s.i. Fragmentor, skimmer, and octopole radio frequency (Oct1 RF Vpp) voltages were set to 110, 65, and 750 V, respectively. The scan range was 70 to 1200 m/z, and spectra were collected at 4.4 spectra/s, which corresponded to 2148 transients per spectrum. All mass spectrometry scan data were analyzed using MassHunter Quantitative 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 powdered using a bead mill. Protein was then extracted in 500 μL of buffer solution (100 mM HEPES, pH 7.0, 1 mM DTT, 0.1% [v/v] Triton X-100, 2% [w/v] polyvinylpolypyrrolidone, and 1 mM EDTA), and samples were centrifuged for 5 min at 4°C and 20,000g. AlaAT activity was subsequently assayed enzymatically by adding 20 μL of supernatant to 230 μL of 150 µM NADH, 10 mM Ala, 5 mM α-ketoglutarate, 0.4 mM MgCl2, 50 mM HEPES, pH 7.0, and 1 unit of LDH and monitoring NADH oxidation over time photometrically on a 96-well plate.

Immunoblotting

Samples of two leaf discs from S6K-HA plants were extracted in 50 μL of 100 mM HEPES, pH 7.5, 25 mM glycerol-2-phosphate, 10 mM NaF, 0.1% (v/v) Triton X-100, 2 mM PMSF, and cOmplete protease inhibitor cocktail (Roche) according to the manufacturer’s instructions. Samples were diluted and boiled in SDS sample buffer, run on an Any kD precast gel (Bio-Rad) for 60 min at 150 V, transferred onto a PVDF membrane, blocked in 2% skim milk powder, and probed overnight with anti-HA antibody (Proteintech, catalog No. 66006-2-lg) at 1:20,000 dilution or anti-S6K-phosphoT449 antibody (Abcam, catalog No. 207399, lot No. GR243231-22) at 1:1000. Following secondary antibody incubation, the blots were exposed using Clarity Western ECL Substrate (Bio-Rad) with an Amersham 680 Imager CCD camera (GE).

Statistical Analysis

All statistical analyses were performed with Sigma Plot v. 13. Statistical tests and replicate number are as indicated in figure legends. Biological replicates indicate samples that were collected from different plants grown at the same time. Posthoc testing following ANOVA was performed using the Holm-Sidak method. All experimental results were repeated at least once with separate batches of plants.

Accession Numbers

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

Supplemental Data

DIVE Curated Terms

The following phenotypic, genotypic, and functional terms are of significance to the work described in this paper:

Acknowledgments

We thank Dietmar Funck for the kind gift of the pdh knockout lines, for advice, and for a critical evaluation of the article. We thank Jelle Van Leene and Geert De Jaeger for kindly providing the S6K overexpression line. We thank Santiago Signorelli for a critical reading of the article. This work was supported by the Australian Research Council (ARC grants CE140100008 and DP180104136 to B.M.O., G.G.K.O., C.P.L., and A.H.M.) and by an ARC Discovery Early Career Research Award Fellowship (DE150100130 to 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.

Footnotes

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