Target of rapamycin (TOR) is a conserved eukaryotic serine/threonine protein kinase that regulates metabolism by promoting anabolic processes when nutrients are available (Liu and Sabatini, 2020). In the most well-studied pathway, mammalian TOR is stimulated by amino acids to promote translation, acting as a rheostat to couple free amino acid availability with rates of amino acid incorporation into proteins (Valvezan and Manning, 2019). TOR is under intense biomedical investigation because dysregulation of the TOR network causes or contributes to a wide range of human diseases, prominently including cancer (Saxton and Sabatini, 2017). Therefore, many details of the TOR signaling pathway have been elucidated in mammals and yeast; less is known, however, about the TOR network in other eukaryotic lineages. In the plant model for genetics and molecular biology, Arabidopsis thaliana, TOR is essential for the earliest stages of embryogenesis (Menand et al., 2002), and inhibiting TOR strongly represses growth and development (Deprost et al., 2007; Xiong and Sheen, 2012). Plant TOR activity is controlled by a number of upstream signals, such as glucose (Xiong et al., 2013), light (Chen et al., 2018a; Li et al., 2017b), nucleotides (Busche et al., 2020), and phytohormones including auxin (Li et al., 2017b; Schepetilnikov et al., 2017), brassinosteroids (Zhang et al., 2016), and abscisic acid (Wang et al., 2018). When plant TOR is active, it promotes the transcription of genes involved in cell-cycle progression, ribosome biogenesis, and various other metabolic processes, depending on developmental context (Xiong et al., 2013). As in other eukaryotes, plant TOR associates with at least two additional proteins, RAPTOR (Regulatory-associated protein of TOR) and LST8 (Lethal with SEC13 8), to form an active complex called TORC1 (TOR COMPLEX 1) (Deprost et al., 2005; Mahfouz et al., 2006; Moreau et al., 2012); it is not known whether TOR acts in any RAPTOR- or LST8-independent complexes in plants (Van Leene et al., 2019). Very little is understood about the signal transduction networks downstream from TOR in plants; elucidating these signaling pathways is a major goal to understand how TOR signaling evolved in eukaryotes and how TOR signaling networks could be manipulated to promote agricultural yields while reducing reliance on expensive and environmentally-harmful fertilizer inputs (Busche et al., 2020).

Rapamycin, an inhibitor of TORC1 first isolated from Streptomyces hygroscopicus cultures (Vézina et al., 1975), represses growth largely by inhibiting mRNA translation (Thomas and Hall, 1997), which has led to extensive studies of the mechanisms underlying the regulation of mRNA translation by TOR. As a simplified model, in mammals, TOR specifically promotes the translation of mRNAs that encode ribosomal proteins (RPs) and a handful of other components of the translation apparatus; in turn, these additional ribosomes increase global rates of mRNA translation. Vertebrate RP mRNAs have evolved a regulatory motif, called a 5′ terminal oligopyrimidine tract (5′TOP), which begins with a cytosine at the 5′ cap and is followed by several uracils and/or cytosines, but no (or very few) adenines or guanines (Meyuhas et al., 1996). Approximately 100 mammalian transcripts are classically considered 5′TOP mRNAs, including all ~80 cytosolic RP mRNAs (Philippe et al., 2020). The 5′TOP motif is crucial for the TOR-mediated regulation of RP mRNA translation initiation in vertebrates: when TOR is inactive, 5′TOP motifs are sufficient to strongly repress translation initiation (Meyuhas and Kahan, 2015). Multiple TOR substrates have been proposed to mediate TOR-5′TOP mRNA translation regulation (Berman et al., 2020; Meyuhas and Kahan, 2015; Philippe et al., 2020; Thoreen et al., 2012), including eS6 kinases (S6Ks), eIF4E-binding proteins (4E-BPs), eIF4G initiation factors, and La-related protein 1 (LARP1), among others. The precise mechanisms remain contested, however, and only S6K, eIF4G, and LARP1 are conserved across eukaryotes.

LARP1’s role in translation was first discovered in a screen for proteins that differentially associate with the 5′ cap in response to TOR activity (Tcherkezian et al., 2014). Subsequent mechanistic studies have alternatively proposed that LARP1 promotes translation of 5′TOP mRNAs (Tcherkezian et al., 2014), represses translation of 5′TOP mRNAs (Fonseca et al., 2015; Philippe et al., 2020; Philippe et al., 2018), has dual and opposing roles in controlling translation of 5′TOP mRNAs depending on its phosphorylation status (Hong et al., 2017), or has no role in regulating translation, but instead stabilizes 5′TOP mRNAs (Gentilella et al., 2017). At a molecular level, LARP1 can bind directly to the 7-methylguanosine (m⁷G) and 5′TOP (Cassidy et al., 2019; Lahr et al., 2017), but may also associate with pyrimidine-enriched sequences elsewhere in a transcript (Hong et al., 2017), and is often found in complex with the polyA tail via protein-protein interactions with polyA binding proteins (PABPs) and/or direct interaction with the polyA RNA (Al-Ashtal et al., 2019; Aoki et al., 2013). These apparently conflicting models (Berman et al., 2020) may reflect differences in the physiological status or genetics of the cell types used, multiple protein- and RNA-binding sites on the LARP1 protein with distinct affinities, or confounding effects of different techniques to identify LARP1 binding partners with distinct inherent biases. LARP1 is under increasing scrutiny because of its clinical significance: in addition to contributions to the progression of some cancers (Mura et al., 2015) and a possible link to Zika virus pathogenesis (Scaturro et al., 2018), LARP1 was recently found to physically associate with the RNA-binding nucleocapsid of the recently-emerged zoonotic coronavirus SARS-CoV-2 (Gordon et al., 2020). Therefore, in light of evidence that inhibiting TOR limits replication of a closely related coronavirus (MERS-CoV) (Kindrachuk et al., 2015) and the availability of FDA-approved TOR inhibitors (especially rapamycin and related, rapamycin-like compounds) and several additional TOR inhibitors in clinical trials, ablating TOR-LARP1 signaling has been proposed as a possible pharmacological target to treat severe coronavirus infections (Gordon et al., 2020; Zhou et al., 2020).

Most strikingly, although LARP1 is deeply conserved in eukaryotes, the ubiquitous 5′TOP motif in the 5′ leader of cytosolic RP mRNAs only recently evolved in animals, suggesting that LARP1 did not co-evolve with its proposed current primary function in humans, the direct regulation of cytosolic RP mRNA translation. In plants, LARP1 has been reported to associate with the cytosolic exoribonuclease, XRN4, and recruit XRN4 to specific transcripts during heat shock to promote their degradation (Merret et al., 2013), but it is not known whether LARP1 regulates mRNA stability and/or translation under standard physiological conditions. Pioneering efforts to identify the mechanisms regulating translation of 5′TOP mRNAs, however, did show that wheat germ extracts contain a repressor that specifically limits translation of 5′TOP mRNAs in cell-free translation assays (Biberman and Meyuhas, 1999; Shama and Meyuhas, 1996), suggesting that plants also discriminately regulate translation of 5′TOP mRNAs. Here, we show that the TOR-LARP1-5′TOP signaling axis regulates translation in Arabidopsis, impacting expression of a set of deeply conserved 5′TOP genes, including translation elongation factors, polyA-binding proteins, karyopherins/importins, and the translationally-controlled tumor protein. Moreover, we identify new, plant-specific genes regulated by TOR-LARP1-5′TOP signaling, including several genes involved in auxin signaling, developmental patterning, and chromatin modifications. Significantly, many of the 5′TOP mRNAs do encode proteins that contribute to ribosome biogenesis, although only a handful of cytosolic RP mRNAs themselves have 5′TOP motifs. We propose that TOR-LARP1-5′TOP mRNA signaling arose early during eukaryotic evolution to coordinate translation and cell division with cellular metabolic status and nutrient availability, and we argue that TOR-LARP1-5′TOP signaling has since evolved new, plant-specific targets that regulate plant physiology, growth, and development.

To identify novel downstream components of the plant TOR signaling network, we took orthogonal global approaches to quantify how deactivating TOR impacts the plant transcriptome (RNA-Seq), translatome (Ribo-Seq), proteome, and phosphoproteome (Figure 1A). Seedlings were grown to quiescence in half-strength MS media for 3 days under photosynthesis-limiting conditions (Xiong et al., 2013) with a 12 hr light/12 hr dark diurnal cycle. Media were then replaced with half-strength MS media plus 15 mM glucose to activate TOR. 24 hr later, media were replaced again with half-strength MS media plus 15 mM glucose (to maintain TOR activity) or half-strength MS media plus 15 mM glucose and 5.0 μM Torin2 (to attenuate TOR activity) (Li et al., 2017b; Montané and Menand, 2013). Seedlings were collected 2 hr after these treatments. At least 600 seedlings were pooled for each treatment and considered one sample, and the entire experiment was replicated three times. After collection, portions of each sample were divided for different analyses. Total RNA was extracted from one set of samples in polysome buffer with cycloheximide; an aliquot of this RNA was used to build RNA-Seq libraries after protease treatment and depletion of rRNA, and the rest of the RNA was used for ribosome footprint profiling (Hsu et al., 2016; Ingolia et al., 2009). Total protein was extracted from a parallel set of samples and digested with trypsin; an aliquot of this digested protein was TMT-labeled and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the rest was enriched for phosphopeptides and then TMT-labeled and analyzed by LC/MS-MS. In summary, we quantified transcripts from 19,087 genes, ribosome footprints from 14,010 genes, peptides from 2814 proteins, and phosphopeptides from 657 phosphoproteins (Supplementary file 1).

Figure 1

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For our assays, we attenuated TOR activity using Torin2, a potent ATP-competitive TOR inhibitor that is effective at reducing TOR activity in Arabidopsis thaliana (Li et al., 2017b; Montané and Menand, 2013). TOR is a member of the atypical phosphatidylinositol-3-kinase (PI3K)-like protein kinases (PIKKs) that evolved from lipid kinases (PI3Ks) and were present in the last eukaryotic common ancestor (Brunkard, 2020; Keith and Schreiber, 1995). PIKKs are a small family of only five kinases involved in metabolic regulation (TOR), nonsense-mediated decay (SMG1), and the DNA damage response (ATM, ATR, and DNA-PKcs). Although all of these were present in the last eukaryotic common ancestor, they are not conserved in all extant eukaryotic lineages; the A. thaliana genome, for example, only encodes TOR, ATM, and ATR. ATP-competitive TOR inhibitors, which have been developed for pharmacological treatment of TOR-associated human diseases, show a range of selectivity for TOR, with some exhibiting low selectivity (e.g. PP242) (Liu et al., 2012) or extremely high selectivity (e.g. AZD8055, which is ~1,000-fold selective for TOR) (Chresta et al., 2010). In between these extremes, Torin2 is at least 100-fold more selective for TOR than other PIKKs in in vitro assays (Liu et al., 2013). In cell types that have strongly induced the DNA damage response, however, such as some breast cancers, Torin2 can have synergistic cellular effects by inhibiting both TOR and another PIKK (Chopra et al., 2020). For example, a recent report argues that Torin2 causes cytotoxicity in some triple-negative breast cancer cell lines with highly elevated ATR activity by inhibiting TOR and attenuating ATR (Chopra et al., 2020). AZD8055 can also cause cytotoxicity, but only at higher concentrations (Chopra et al., 2020). Since ATR and ATM are not induced under our growing conditions, we expect that Torin2 specifically inhibits TOR in our assays. We also chose a near-minimal concentration of Torin2 to attenuate, rather than completely abolish, TOR activity, which also reduces the likelihood of non-specific activity. Nonetheless, it is possible that Torin2 could have minor off-target effects, which should be considered throughout our analyses below.

The seedling TOR-regulated proteome and phosphoproteome

We conducted quantitative global proteomics to define changes in protein abundance 2 hr after inactivating TOR with Torin2. Of 2814 proteins quantified in our analysis, 331 showed significant differences in abundance between Torin2- and mock-treated seedlings (Supplementary file 1). We observed only small differences in protein abundance between treatments (~1.2-fold changes, on average, for the significantly impact proteins), but this is likely because protein half-lives (~hours to days) are typically an order of magnitude longer than mRNA half-lives (~minutes to hours) in eukaryotes (Li et al., 2017a; Toyama and Hetzer, 2013). Strikingly, we observed a statistically- significant decrease in the abundance of 20 of the 116 RPs detected in the seedling proteome (down 1.2-fold on average) (Figure 1E, Supplementary file 4). The only other biological process detected as significantly impacted in the Torin2-treated quantitative proteome was an induction of various solute transporters.

The Torin2-sensitive phosphoproteome revealed 85 phosphoproteins that accumulated to significantly different levels in Torin2-treated seedlings compared to mock-treated controls (Figure 2A, Supplementary file 4). About half of these were abundant enough that they were also quantified in the global proteome (44 proteins), of which eight showed significant differences in total protein levels after Torin2 treatment (Supplementary file 1). mRNAs encoding all of these significantly-impacted phosphoproteins were detected by RNA-Seq (Supplementary file 1), except for DF1, a seed-specific transcription factor that we speculate may persist as a stable protein for some time after germination, but is no longer transcriptionally expressed. Levels of 25 of these mRNAs showed significant differences after Torin2 treatment (Supplementary file 1). Although the transcripts and/or total protein levels of several of the phosphoproteins changed, these differences could not readily explain the magnitude of difference in protein phosphorylation we detected. Moreover, phosphorylation often impacts protein stability, and this global experiment cannot distinguish whether changes in protein level are caused by differential phosphorylation or if changes in phosphoprotein levels reflect unrelated changes in protein stability. Thus, while we include these parallel data, we proceeded to analyze all 85 putative TOR-sensitive phosphoproteins identified in these experiments as presumptive substrates or downstream targets of TOR.

Figure 2

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We first focused on proteins whose phosphorylation decreases upon Torin2 treatment, since these could be direct substrates of TOR (Figure 2). The strongest effect was on eS6b, a canonical phosphorylation target of the TOR-S6K signaling axis; phosphorylated eS6b was readily detected in mock-treated controls but was undetectable in Torin2-treated samples (Figure 2C). Ser240, which is near the C-terminus of eS6b, became dephosphorylated upon Torin2 treatment, which is in agreement with previous reports that eS6b-Ser240 phosphorylation is dependent on TOR-S6K activity (Figure 2B). This clear result served as robust internal validation for our Torin2-treated phosphoproteome.

We next compared the Torin2-sensitive seedling phosphoproteome to the TOR-regulated cell suspension culture phosphoproteome (Van Leene et al., 2019). Eight targets overlapped between our datasets: in both experiments, inhibiting TOR decreased phosphorylation of eS6b, LARP1, eukaryotic initiation factor eIF4B1, the plant-specific CONSERVED BINDING OF EIF4E one protein (CBE1), and a plant ubiquitin regulatory X domain-containing protein (PUX5), whereas inhibiting TOR increased phosphorylation of eukaryotic initiation factor eIF4G, the universal stress protein PHOS32, and a plant-specific DUF1421 protein of unknown function (At4g28300). Of the remaining 77 phosphoproteins that we found were sensitive to Torin2, 58 were detected in the cell suspension culture phosphoproteomes, and in many cases their phosphorylation was similarly impacted by TOR inhibition, but not at statistically-significant levels in those experiments (Van Leene et al., 2019). These corroborating results further confirmed that our experimental approach identified bona fide phosphorylation targets of the TOR signaling network. To confirm that Torin2 did not inhibit ATM or ATR activity under our experimental conditions, we compared the Torin2-sensitive seedling phosphoproteome to the Arabidopsis ATM and ATR-dependent phosphoproteome, a set of 108 phosphoproteins that were differentially phosphorylated in atm;atr double mutants compared to wild-type in response to irradiation, that is ATM/ATR-activating conditions (Roitinger et al., 2015). Only 9 of those phosphoproteins were detected with the same phosphosites in any of our phosphoproteome experiments, and none of these phosphosites were significantly inhibited by Torin2. These results support our hypothesis that Torin2 selectively acts only to inhibit TOR in Arabidopsis seedlings grown under our physiological conditions.

The Torin2-sensitive seedling phosphoproteome is strongly enriched for proteins involved in ribosome biogenesis, including RP subunits (PANTHER GO cellular component cytosolic RPs were 8.6-fold overrepresented, p<0.01, Fisher’s exact test with Bonferroni correction) (Figure 2F). Enrichment analysis was performed with PANTHER gene ontologies, as described in detail in the methods section. In addition to eS6b, phosphorylation of the large ribosomal subunit uL18a decreased in Torin2-treated seedlings, as did phosphorylation of the acidic stalk proteins uL10b, P2b, and P3a, and the uL10/acidic stalk-like RPD1/Mrt4 protein involved in nuclear ribosome assembly. Oppositely, uL30a phosphorylation somewhat increased, despite a significant decrease in uL30a transcripts. uL18a is a component of the 5S ribonucleoprotein particle, along with uL5 and the 5S rRNA, that is assembled as a subcomplex prior to incorporation into the large subunit. Recessive alleles of uL18a, including piggyback 3 (Pinon et al., 2008) and oligocellula 5 (Yao et al., 2008), impact Arabidopsis leaf development (Fujikura et al., 2009) and enhance phenotypes of asymmetric leaves 1 (Pinon et al., 2008; Yao et al., 2008) and angustifolia three mutants that drastically disrupt leaf patterning. The differentially phosphorylated residue in uL18a, Ser131, is not conserved in humans but is deeply conserved in the plant lineage. Beyond RPs per se, several proteins involved in ribosome biogenesis, such as the nucleolar proteins GAR2-Like and nucleolin, were significantly less phosphorylated in Torin2-treated seedlings (Supplementary file 1).

Multiple RNA-binding proteins are differentially phosphorylated in the Torin2-sensitive seedling phosphoproteome (PANTHER GO molecular function RNA-binding proteins were 5.3-fold overrepresented, p<10⁻⁵, Fisher’s exact test with Bonferroni correction) (Figure 2F). Several of these have been assigned functions in mRNA splicing or maturation (PANTHER GO-Slim biological process RNA splicing were 9.5-fold overrepresented, p<0.05, Fisher’s exact test with Bonferroni correction), including RNA-binding protein 25 (RBM25), serrate (SE), arginine/serine-rich splicing factor 40 and 41 (RS40 and RS41), binding to TOMV RNA 1L (BTR1L, orthologous to human NOVA), defectively organised tributaries 2 (DOT2, orthologous to human SART-1), and FIP1, which are all significantly dephosphorylated upon TOR inactivation by Torin2, and splicing factor SC35 (SC35), which is slightly more phosphorylated in Torin2-treated seedlings. Proteins that are known or predicted to regulate translation initiation are also enriched in the Torin2-sensitive seedling phosphoproteome, including eIF5B1, eIF4B1, CBE1, and LARP1a, which are dephosphorylated in Torin2-treated seedlings, and eIF4G, which is hyperphosphorylated in Torin2-treated seedlings (Figure 2A and E). These results suggest that TOR plays a major role in the regulation of transcript processing and translation, and that TOR influences mRNA expression through multiple, distinct signaling axes.

Beyond these general trends, we noticed a Torin2-sensitive change in the phosphorylation status of two critical proteins involved in signal transduction: RAPTOR1 and TOPLESS (Figure 2). RAPTOR1 is an essential protein that complexes with TOR and LST8 to form TORC1. In Arabidopsis and humans, RAPTOR is post-translationally modified to modulate TORC1 activity in response to various cues (Carrière et al., 2008; Dunlop et al., 2011; Foster et al., 2010; Gwinn et al., 2008; Wang et al., 2009; Wang et al., 2018). We found that phosphorylation of RAPTOR1 Thr912 and Ser916, residues between the conserved RAPTOR HEAT repeats and WD40 repeats, significantly decreased in response to Torin2 treatment. These residues are conserved in most land plant RAPTOR sequences, including Physcomitrella patens and Marchantia polymorpha RAPTOR orthologues, but are not found in other eukaryotic lineages (Figure 2D). Torin2-sensitive phosphorylation of RAPTOR1 residues suggests that TORC1 could regulate its own activity, although further studies will be needed to determine whether RAPTOR1 is a direct substrate of TORC1 and how phosphorylation of these residues affects TORC1 activity.

TOPLESS is a transcriptional regulator that mediates multiple phytohormone pathways (Long et al., 2002; Oh et al., 2014; Pauwels et al., 2010; Szemenyei et al., 2008); several studies have proposed that TOR signaling participates in phytohormone signaling networks (Li et al., 2017b; Schepetilnikov et al., 2017; Wang et al., 2018; Zhang et al., 2016), although most have focused on whether TOR activity is sensitive to phytohormones as upstream cues, rather than whether TOR modulates downstream phytohormone responses. We found that TOPLESS Thr286 and Ser214 phosphorylation decreased after Torin2 treatment in wild-type seedlings (Figure 2B and C). TOPLESS is a member of the Groucho/Tup1 family of co-repressors (Cavallo et al., 1998; Keleher et al., 1992), which are recruited to promoters by diverse transcription factors where they repress transcription. TOPLESS is most famous in plants for its interactions with phytohormone-regulated transcription factors, including direct interaction with auxin-responsive Aux/IAA-ARF complexes (Szemenyei et al., 2008), jasmonate-responsive NINJA-JAZ complexes (Pauwels et al., 2010), and brassinosteroid-responsive BZR1 complexes (Oh et al., 2014). TOPLESS-Ser286 is deeply conserved in land plants (Figure 2D), and TOPLESS-Ser214 is broadly conserved in angiosperms, including Amborella trichocarpa, monocots, and dicots, but is not found in non-flowering plants (Figure 2D). Future studies of the functional impact of these phosphorylation events could illuminate a role for TOR-TOPLESS regulation of the complex crosstalk among metabolic, developmental, and stress-response signaling networks.

The seedling Torin2-sensitive phosphoproteome also revealed that the phosphorylation of two actin-binding VILLIN proteins, VILLIN 2 (VLN2) and VLN3, is regulated by TOR (Figure 2). In mammals and yeast, the RAPTOR-independent TORC2 complex regulates actin filamentation through multiple pathways (Jacinto et al., 2004; Loewith et al., 2002; Rispal et al., 2015; Sarbassov and Kim, 2004; Schmidt et al., 1996). Recently, a forward genetic screen in Arabidopsis found that a recessive allele of isopropylmalate synthase 1 (IPMS1) promotes actin filamentation in a TOR-dependent pathway (Cao et al., 2019). IPMS1 is the first committed step of leucine biosynthesis; accordingly, ipms1 mutants exhibit broad defects in free amino acid accumulation (Cao et al., 2019). While the exact cause remains undefined, the disruption of amino acid metabolism in ipms1 mutants apparently increases cellular TOR activity (Cao et al., 2019; Schaufelberger et al., 2019). Reducing TOR activity by treating ipms1 mutants with ATP-competitive TOR inhibitors rescues the ipms1 actin organization phenotype, which suggests that TOR regulates actin filamentation in plants. We found that Torin2 decreases phosphorylation of VLN2 and VLN3, closely related VILLINs, at the same sites near the C-terminus in both proteins. VLN2 and VLN3 are required for normal plant development and actin bundling in Arabidopsis (Bao et al., 2012; Qu et al., 2013; van der Honing et al., 2012; Wu et al., 2015), but their post-translational regulation remains underexplored in plants. Functional studies of TOR-promoted VLN2/VLN3 phosphorylation could reveal new links between TOR and the cytoskeleton in plants.

LARP1 promotes growth, chloroplast biogenesis, and TOR signaling in plants

We chose to focus further studies on LARP1 for several reasons. LARP1 is deeply conserved in eukaryotes and is a consistent target of TOR in mammalian and plant phosphoproteomic screens, but the functional significance of TOR-LARP1 signaling remains unresolved (Berman et al., 2020), even in biomedical model systems. We began by investigating the role of LARP1 in normal plant development and physiology. In some eukaryotes, such as Drosophila melanogaster, LARP1 is essential for early stages of development (Burrows et al., 2010). In contrast, LARP1 is not essential in C. elegans, although larp1 mutants exhibit delayed growth and very low-frequency arrest of embryogenesis (Nykamp et al., 2008). LARP1 contributes to heat stress recovery in Arabidopsis (Merret et al., 2013), but its role in development under standard growing conditions has not been thoroughly characterized. To address this, we grew larp1 and wild-type plants for 15 days on half-strength MS media plus 0.8% agar, and then measured root growth (Figure 3A). We observed 36% shorter roots in larp1 than in wild-type plants, along with a reduction in the number of secondary roots (although this is possibly a consequence of the defect in primary root growth) (Figure 3B). The first true leaves of larp1 plants were also significantly smaller than in wild-type plants grown under these conditions (Figure 3C). We next assayed shoot growth in soil-grown plants four weeks post-germination. We did not observe any obvious defects in leaf morphology (Figure 3D), but again, the most recently-emerged leaf (L12) was significantly smaller in larp1 plants than in wild-type (Figure 3E). Given the growth defect in larp1 and that LARP1 is proposed to regulate translation in human cells, we next tested whether free amino acid levels are different between larp1 and wild-type shoots, which could indicate a global defect in protein biosynthesis (Figure 3F). We found virtually no differences between larp1 and wild-type plants, however, except that alanine accumulated to 38% higher levels than in larp1 shoots (Figure 3F).

Figure 3

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We noticed that larp1 mutants appear relatively chlorotic as seedlings under the conditions described above for global profiling experiments, so we next measured chlorophyll accumulation in larp1 and wild-type seedlings after supplying them with glucose or glucose and Torin2 to attenuate TOR activity (Figure 3G). Total chlorophyll levels were significantly lower in larp1 mutants than in wild-type, whether or not the seedlings were treated with Torin2 (Figure 3G). In wild-type seedlings, treatment with Torin2 for 2 hr caused a slight but significant decrease in chlorophyll levels (Figure 3G). There was no significant difference in chlorophyll levels between treatments in larp1 seedlings, however (Figure 3G).

To determine how LARP1 promotes growth and test whether LARP1 genetically interacts with the glucose-TOR signaling pathway, we conducted all of the global profiling experiments described above (Figure 1A) using larp1 mutants and otherwise identical treatments. RNA-Seq revealed that several processes are disrupted in larp1 mutants compared to wild-type in glucose-supplied seedlings (Figure 4A). Most prominently, in larp1, we observed significant repression of genes involved in photosynthesis, especially genes encoding components of the photosynthetic electron transport chain (pETC) complexes (Figure 4A, Supplementary file 5). Repression of photosynthesis-associated nuclear gene expression is consistent with our observation that chlorophyll levels were significantly lower in these larp1 seedlings than in wild-type (Figure 3G). Global proteomic analysis confirmed that photosynthesis-associated proteins are less abundant in larp1 mutants (Figure 4B, Supplementary file 6). In addition to photosynthesis-associated transcripts, E3 ubiquitin ligase mRNAs, biotic stress-response transcripts, and cell wall glycoprotein mRNAs (including those that encode arabinogalactan proteins) were also repressed in larp1 (Figure 4A). Several biological processes are transcriptionally induced in larp1 compared to wild-type, including genes involved in glucosinolate biosynthesis, HSP70/HSP90 chaperones, microtubule cytoskeleton, lipid degradation, and lipid body-associated genes (oleosins and caleosins), flavonoid biosynthesis, and cell-cycle genes (especially those involved in cell division) (Figure 4A). In response to Torin2, larp1 mutants showed largely similar responses as wild-type seedlings, including extensive repression of cytosolic ribosome biogenesis, nucleocytoplasmic trafficking, cell wall biosynthesis, and cell-cycle progression, as well as broad induction of genes involved in protein catabolism (including autophagy and E3 ubiquitin ligases), biotic stress-response genes, protein chaperoning, and receptor-like kinases (Figure 4D, Supplementary file 8).

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Even in glucose-supplied seedlings, larp1 mutants showed several differences in TE compared to wild-type (Figure 4C, Supplementary file 7), demonstrating that LARP1 contributes to plant physiology even when TOR is active. We observed at least a two-fold change in TE for 835 transcripts in larp1 compared to wild-type when seedlings were supplied glucose (Figure 4C). The TE of several categories of mRNAs was relatively repressed in larp1 mutants, including mRNAs that encode cytoskeletal kinesins, proteins involved in chromatin modifications and organization, and proteins involved in translation (Figure 4C). Inhibiting TOR with Torin2 impacted the TE of 239 transcripts in the larp1 background compared to glucose-supplied larp1 seedlings (Figure 4E, Supplementary file 9). As in wild-type, MapMan analysis showed that translation of cytosolic RP mRNAs was broadly repressed by Torin2 in larp1. Overall, these results demonstrate that LARP1 is not required for all effects of Torin2 on mRNA translation in plants, suggesting that other proteins may exert translational control in response to TOR deactivation.

Knocking down LARP1 expression can reduce TOR activity in some human cell lines (Mura et al., 2015), which led us to investigate whether TOR activity is altered in Arabidopsis larp1 mutants by comparing the phosphoproteomes of larp1 and wild-type plants. Under mock conditions, 96 phosphoproteins accumulated to significantly different levels in larp1 than wild-type seedlings. 36 of these overlapped with the Torin2-sensitive phosphoproteome, more than twice the number expected (p<10⁻¹¹; at most 17 overlapping phosphoproteins would be expected, p>0.05). Remarkably, all but one of these phosphoproteins was affected in the same pattern in mock-treated larp1 compared to mock-treated wild-type as in the Torin2-treated wild-type compared to mock-treated wild-type (Figure 4F, Supplementary file 6). To validate these results, we assayed phosphorylation of the well-established TOR substrate, S6K-T449, in wild-type and larp1 plants using phosphospecific polyclonal antibodies against S6K-pT449 and monoclonal antibodies against total S6K (Figure 4G). As predicted by the global phosphoproteomic results, S6K-pT449/S6K ratios are noticeably lower in larp1 (Figure 4G). Thus, LARP1 is required to maintain high levels of TOR activity in actively growing Arabidopsis seedlings, which could contribute to the growth defects we observed.

TOPscore analysis reveals conserved TOR-LARP1-5′TOP signaling axis

In mammals, TOR specifically controls the translation of a canonical set of ~100 mRNAs via the 5′TOP motif, which is present in all cytosolic RP mRNAs and a few other transcripts involved in translation initiation and elongation (Berman et al., 2020; Jefferies et al., 1994). In plants, however, it has been reported that RP mRNAs do not have 5′TOP or pyrimidine-enriched motifs, although, to our knowledge, there has been no comprehensive effort to define plant 5′TOP mRNAs. We sought to annotate Arabidopsis transcripts to identify likely 5′TOP mRNAs using a modified version of the recently-described TOPscore approach (Philippe et al., 2020; Figure 5A). TOPscores are calculated using quantitative transcription start site-sequencing (TSS-Seq), which provides single-nucleotide resolution of 5′ mRNA ends genome-wide (Figure 5A). Every TSS-Seq read in the 5′ leader of an mRNA is scored by whether it is part of an oligopyrimidine tract: all 5′ ends starting with purines are scored as 0, and 5′ ends starting with a pyrimidine are scored as one plus the distance to the next purine in the 5′ leader (thus, for example, a TSS-Seq read that starts with a pyrimidine followed by four consecutive pyrimidines and then a purine is scored as 5). The sum of these scored reads is then divided by the total number of TSS-Seq reads that mapped to the 5′ leader.

Figure 5

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Figure 5—source data 1

TOR-LARP1-5′TOP signaling in Arabidopsis seedlings regulates translation of mRNAs that encode deeply conserved eukaryotic proteins, plant lineage-specific proteins, and diverse proteins involved in ribosome biogenesis.

(A) Several mRNAs are translationally regulated by TOR-LARP1-5′TOP signaling in both humans (Philippe et al., 2020) and Arabidopsis. (B) Some 5′TOP mRNAs in Arabidopsis encode proteins that evolved in the plant lineage, including genes involved in chloroplast physiology and developmental patterning. (C) Although the translation of only a few RP transcripts is directly controlled by TOR-LARP1-5′TOP signaling in Arabidopsis seedlings, multiple other steps of ribosome biogenesis are subject to TOR-LARP1-5′TOP regulation.

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We took advantage of published paired-end analysis of TSS (PEAT) TSS-Seq data (Morton et al., 2014) to quantify TOPscores in Arabidopsis (Figure 5). Using stringent parameters to ensure high-quality results, we calculated TOPscores for transcripts encoded by 9143 genes (Supplementary file 1). The median TOPscore in this dataset is 1.3, with 90% of TOPscores between 0.38 and 4.3 (Figure 5E). We next used MapMan to conduct Wilcoxon rank-sum analyses to determine if gene TOPscores broadly correlate with any biological functions (Figure 5E). We found that genes involved in RNA biosynthesis, especially transcription factors in the Zn finger C2C2 superfamily, have higher TOPscores. Genes encoding proteins involved in nucleocytoplasmic transport, including importin/karyopherins, nucleoporins, and Ran GTPases, also have significantly higher TOPscores. Genes involved in cell wall remodeling and biochemistry, however, have significantly lower TOPscores. Glycosyltransferase genes, including UDP-glucosyl transferase (UGT) and xyloglucan endotransglucosylase/transferase (XTH) genes, have remarkably low TOPscores, as do genes involved in lignin biosynthesis (Figure 5E).

Next, we tested whether TOPscores correlate with changes in TE in response to TOR inhibition. The median TOPscore for all genes expressed across Ribo-Seq experiments (with RPKM ≥10) is 1.4 (Figure 5F), whereas the median TOPscore of genes that are specifically translationally repressed upon Torin2 treatment (at least two-fold decrease in relative translational efficiency) is 2.0 (Figure 5F), significantly higher (Mann–Whitney U test, p<0.01). This result demonstrates that inhibiting TOR represses translation of mRNAs with high TOPscores. Strikingly, this effect is dependent on LARP1: in larp1 mutants, the median TOPscore of genes that are specifically translationally repressed upon Torin2 treatment is only 1.0 (Figure 5F), significantly lower than the median TOPscore for all genes (Mann-Whitney U test, p<0.01). Thus, in aggregate, LARP1 is required to repress 5′TOP mRNA translation in response to TOR inhibition in plants. We therefore propose that the TOR-LARP1-5′TOP signaling axis is conserved between plants and mammals.

Sequencing data are available at NCBI SRA, project PRJNA639161. Proteome data are available via PRIDE and ProteomeXchange, doi:https://doi.org/10.6019/PXD019942.

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Author details

1. M Regina Scarpin

   1. Department of Plant and Microbial Biology, University of California at Berkeley, Berkeley, United States
   2. Plant Gene Expression Center, U.S. Department of Agriculture Agricultural Research Service, Albany, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing

   Competing interests

   No competing interests declared

2. Samuel Leiboff

   1. Department of Plant and Microbial Biology, University of California at Berkeley, Berkeley, United States
   2. Plant Gene Expression Center, U.S. Department of Agriculture Agricultural Research Service, Albany, United States
   3. Department of Botany and Plant Pathology, Oregon State University, Corvallis, United States

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Writing - original draft

   Competing interests

   No competing interests declared

3. Jacob O Brunkard

   1. Department of Plant and Microbial Biology, University of California at Berkeley, Berkeley, United States
   2. Plant Gene Expression Center, U.S. Department of Agriculture Agricultural Research Service, Albany, United States
   3. Laboratory of Genetics, University of Wisconsin—Madison, Madison, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   brunkard@berkeley.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6407-9393

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