Update on Target of Rapamycin Signaling in Plant Stress Responses

Target of Rapamycin Signaling in PlantStress Responses1[OPEN]

Liwen Fu,a Pengcheng Wang,b,2 and Yan Xionga,2,3

aBasic Forestry and Proteomics Research Centre, Haixia Institute of Science and Technology, Fujian Agricultureand Forestry University, Fujian Province 350002, People’s Republic of ChinabShanghai Centre for Plant Stress Biology, Chinese Academy of Sciences Centre for Excellence in MolecularPlant Sciences, Chinese Academy of Sciences, Shanghai 201602, People’s Republic of China

ORCID IDs: 0000-0002-7941-0728 (L.F.); 0000-0001-6043-4132 (P.W.); 0000-0003-0676-8267 (Y.X.).

Target of Rapamycin (TOR) is an atypical Ser/Thr protein kinase that is evolutionally conserved among yeasts, plants, andmammals.In plants, TOR signaling functions as a central hub to integrate different kinds of nutrient, energy, hormone, and environmentalsignals. TOR thereby orchestrates every stage of plant life, from embryogenesis, meristem activation, root, and leaf growth toflowering, senescence, and life span determination. Besides its essential role in the control of plant growth and development, recentresearch has also shed light on its multifaceted roles in plant environmental stress responses. Here, we review recent findings on theinvolvement of TOR signaling in plant adaptation to nutrient deficiency and various abiotic stresses. We also discuss the mechanismsunderlying how plants cope with such unfavorable conditions via TOR–abscisic acid crosstalk and TOR-mediated autophagy, both ofwhich play crucial roles in plant stress responses. Until now, little was known about the upstream regulators and downstreameffectors of TOR in plant stress responses. We propose that the Snf1-related protein kinase–TOR axis plays a role in sensing variousstress signals, and predict the key downstream effectors based on recent high-throughput proteomic analyses.

Plants are challenged throughout their life cycles byvarious types of environmental stresses, such as nutri-ent deficiencies, extreme temperatures, drought, andhigh salinity. To deal with such unfavorable growthconditions, plants have evolved elaborate and efficientstress perception and signal transduction systems.Furthermore, plant stress responses are always ac-companied by extensive transcriptional, translational,and metabolic changes to redirect energy and nutrientresources for stress adaptation. Increasing evidence hasrevealed an essential role of target of rapamycin (TOR),a master regulator of energy maintenance and meta-bolic homeostasis in all eukaryotic organisms, in plantstress responses and stress adaptation.

TOR was first identified in budding yeast throughgenetic mutant screens for resistance to rapamycin,

a chemical molecule produced by the bacteriumStreptomyces hygroscopicus (Heitman et al., 1991).Subsequent studies identified TOR genes in almost all

1This work was supported by the Recruitment Program of GlobalExperts, People’s Republic of China, the National Natural ScienceFoundation of China (grant no. 31870269 to Y.X. and grant no.31771358 to P.W.), Strategic Priority Research Program of theChinese Academy of Sciences (grant no. XDB27040106 to P.W.),and the Basic Forestry and Proteomics Research Centre, Haixia Insti-tute of Science and Technology, Fujian Agriculture and ForestryUniversity.

2Senior authors3Author for contact: yanxiong@fafu.edu.cn.L.F. analyzed the phosphoproteomic data; L.F., P.W., and Y.X.

wrote the article.[OPEN]Articles can be viewed without a subscription.www.plantphysiol.org/cgi/doi/10.1104/pp.19.01214

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eukaryotes, including animals and plants (Kunzet al., 1993; Menand et al., 2002; Sabatini et al.,1994). TOR is an atypical Ser/Thr protein kinase, re-sembling phosphatidylinositol lipid kinases, that isboth structurally and functionally conserved amongall eukaryotes (Xiong and Sheen, 2014). TOR exerts itsfunction in complex forms. In mammals and yeasts,TOR forms at least two structurally and functionallydistinct protein complexes (TORCs) with both shared(LST8) and distinct (Raptor in TORC1; Rictorand mSIN1 in TORC2) TOR-interacting partners. Inplants, the precise compositions of the TOR kinasecomplexes have not been characterized. TOR, Raptor,and LST8 (but neither Rictor nor mSIN1) gene ortho-logs could be identified in all available plant ge-nomes, indicating that only classical TORC1 exists inplants. One copy of TOR, two copies of Raptor (Rap-torA and RaptorB), and two copies of LST8 (LST8-1 and LST8-2) exist in the Arabidopsis (Arabidopsisthaliana) genome, although LST8-2 might be a pseu-dogene due to its undetectable transcript level(Anderson et al., 2005; Deprost et al., 2005; Moreauet al., 2012). Gain-of-function and loss-of-functionanalyses have revealed that Arabidopsis TOR, Rap-tor, and LST8 are all essential for regulating multipleaspects of plant growth, development, and stressadaptation (Anderson et al., 2005; Deprost et al., 2005;Ren et al., 2011; Moreau et al., 2012; Xiong et al.,2013). It is worth noting that, based on these func-tional analyses, TOR appears to regulate a muchbroader spectrum of biological functions than Raptoror LST8. For example, the null tor mutant is embryo-lethal, while the raptora/b double mutant exhibitsnormal embryonic development but is arrested dur-ing seedling development, and the lst8-1mutant onlyexhibits modest dwarf growth and early senescencephenotypes. Interestingly, although most eukaryoteshave only one copy of the TOR gene, two TOR geneshave been identified in three polyploids (Glycine max,Populus trichocarpa, and Brassica rapa) and four TORgenes have been identified in allotetraploid cotton(Gossypium hirsutum; Song et al., 2019). Sessile plantsmight possess unique TOR complexes with plant-specific components that serve as a functional equiv-alent of TORC2 or may even have plant-specializedfunctions for adaptation to constant environmentalchallenges.

In plants, TOR functions as a central hub that in-tegrates signals, including nutrient, hormone, light,energy, and other environmental cues to orchestrategrowth and development. TOR modulates a myriadof cellular activities, including cell division, cell ex-pansion, transcription, mRNA translation, ribosomebiogenesis, metabolism, nutrient assimilation andtransport, and signaling via multiple partners andeffectors in complex signaling networks, which havebeen extensively discussed in several excellent re-cent reviews (Shi et al., 2018; Jamsheer K et al.,2019; Ryabova et al., 2019; Wu et al., 2019). Besidesits essential role in the control of plant growth and

development, recent research also suggests an indis-pensable role for TOR in plant environmental stressresponses. Plants with TOR dysfunctions behave as ifthey are stressed, even in the absence of a stressor.Transcriptome and metabolomics analyses in lst8-1and the conditionally inducible tor-es, amiR-tor mu-tant revealed a broad regulation of plant stress- andautophagy-related genes, and diverse plant meta-bolic pathways modulating myo-inositol, raffinose,and galactinol, which usually accumulate understress conditions such as high light, nutrient starva-tion, cold, drought, and high salt (Moreau et al., 2012;Caldana et al., 2013; Xiong et al., 2013). Down-regulated TOR signaling by chemical inhibitor AZD-8055 also activates genes involved in stress hormone(e.g. ethylene, jasmonic acid, and abscisic acid [ABA])signaling pathways (Dong et al., 2015). Intriguingly,modulating TOR expression can cause either stress-sensitive or stress-tolerant phenotypes depending onthe type of stress encountered, further supporting themultifaceted roles of TOR in plant responses to abioticstress (Deprost et al., 2007; Bakshi et al., 2017; Wanget al., 2017; Dong et al., 2019). Here, we focus on re-cent advances that enable a more thorough under-standing of TOR’s many functions in plant responsesto different nutrient deficiencies and various abioticstresses, and discuss potential upstream regulatorsand downstream effectors of TOR.

TOR SIGNALING IN NUTRIENT SENSINGAND DEFICIENCY

Plants obtain different kinds of nutrients from above-ground photosynthesis and below-ground soil nutrientassimilation. The ability to sense, assimilate, transport,and utilize various nutrients between sink and sourceorgans is vital for plant survival and growth. TOR is acore component in plant nutrient sensing and com-munication networks.

In plants, Glc derived from photosynthesis in leafsources provides carbon-based energy and buildingblocks (Sheen, 2014; Li and Sheen, 2016). Depletionof Glc completely blocks the kinase activity of TOR,and increases the expression of sets of autophagy-and protein degradation-related genes, indicating thatrecycling processes are activated to overcome thenutrient-deficient conditions (Xiong and Sheen, 2012;Xiong et al., 2013). Glc can quickly reactivate TOR ac-tivity via the glycolysis–mitochondria–electron trans-port chain energy relay, as chemical inhibitors targetingthe first step of glycolysis and different steps of theelectron transport chain completely prevent TOR ac-tivation by Glc. Thus, sugar-mediated TOR can sensethe cellular metabolic and bioenergetic status to ma-nipulate energy signaling in plants. Glc-activated TORthen phosphorylates and activates transcription factorE2Fa/E2Fb to promote root growth and true leaf for-mation by enhancing cell division activity in the rootmeristem and shoot apex, respectively (Xiong and

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Sheen, 2012; Xiong et al., 2013; Li et al., 2017). Inter-estingly, in the shoot apex, Glc alone is not enough toactivate cell proliferation; the Rho-like small GTPaseROP2 was shown to bind to and activate TOR in asynergistic action along with Glc and auxin signaling(Xiong and Sheen, 2012; Xiong et al., 2013; Li et al.,2017). TOR also mediates crosstalk between sugarsignaling and brassinosteroid signaling. Glc-activatedTOR can inhibit autophagy to stabilize BZR1, which isa positive regulator in brassinosteroid signaling, topromote cell growth in hypocotyls (Zhang et al., 2016).Sulfur is another important nutrient for plants. Sulfur

assimilation begins with SO422 that is absorbed by sul-

fate transporters in the roots and transformed intoadenosine 59-phosphosulfate (APS), SO3

22, and S22,which are catalyzed by ATP sulfurylase, APS reductase(APR), and sulfite reductase (SIR), respectively (Jobeet al., 2019). S22 then reacts with O-acetyl-Ser to pro-duce Cys, which serves as the donor for either proteinsynthesis or sulfur-containing compounds includingglutathione (GSH) and various glucosinolates. Re-cently, the relationship between TOR and sulfur sig-naling has become evident. In the sir1-1 mutant, whichcould not produce S22, TOR activity is abolished, andGlc content is significantly lower than that in wild-typeArabidopsis (Dong et al., 2017). Interestingly, exoge-nous supply of Glc or grafting the wild-type shoot ontothe sir1-1 root rescues TOR activity, cell division in rootapical meristem, and the growth arrest phenotype inthe sir1-1 mutant (Dong et al., 2017), suggesting thatsulfur availability does not affect TOR signaling inde-pendently, but acts through Glc energy signaling.Moreover, reducing GSH synthesis by inhibiting Glu-Cys ligase activity partially restores the dwarf pheno-type and increases TOR activity in the sir1-1mutant,suggesting that reallocation of sulfur flux from GSHbiosynthesis to protein translation can promote plantgrowth via the regulation of TOR (Speiser et al.,2018). In addition, Malinovsky et al. (2017) reportedthat a distinct plant defense-related glucosinolate,3-hydroxypropylglucosinolate, can function like aTOR inhibitor to block Glc-TOR–promoted rootmeristem activation and root elongation. Thus, thedirection of sulfur flux and its derived metabolitesappear to serve key roles in balancing plant growthand stress responses via TOR regulation in responseto environmental cues.Organic nitrogen-containingmolecules (amino acids)

are key upstream signals for mammalian TOR activa-tion. A very recent study showed that the accumulationof branched-chain amino acids could also upregulateTOR activity in Arabidopsis, causing reorganization ofthe actin cytoskeleton and actin-associated endo-membranes (Cao et al., 2019). Although amino acidsensors for Leu, Arg, and Gln have been discovered inmammalian systems in the past decades (Saxton andSabatini, 2017), no orthologs have been identified inplant genomes. Plants obtain organic nitrogen throughnitrogen assimilation. Plants take in nitrate/ammoniumfrom the soil and convert these compounds to Gln, and

then into other amino acids via the Gln synthetase/Gln-2-oxoglutarate aminotransferase cycle (Krapp,2015). It has been reported that Arabidopsis seed-lings overexpressing TOR are hypersensitive to highnitrate inhibition of root growth (Deprost et al., 2007).Recent studies showed that TOR is inhibited innitrogen-deprived seedlings, and that resupply of eithernitrate, ammonium, or amino acids quickly reactivatesTOR (Liu et al., 2018). However, nitrogen starvation isoften associatedwith higher level of sugars. It remains tobe examined whether inhibition of TOR by nitrogenstarvation, like sulfur deprivation, is related tometabolicand energy generation processes, or if plants haveevolved unique nitrogen-sensing systems for TORactivation.A direct link between other essential inorganic

nutrients and TOR is also being established. Cousoet al. (2020) reported that in Chlamydomonas rein-hardtii, phosphorus deprivation negatively affectedLST8 protein stability, resulting in a downregulationof TORC1 activity. Interestingly, in addition to thedirect influence of carbon, nitrogen, sulfur, and phos-phorus availability on TORkinase activity, genome-widetranscriptional profiling has revealed that Glc-TOR sig-naling activates transcription of genes involved in sulfurassimilation and transport includingAPS1, APS3, APK1,APK2, APR1, APR2, APR3, SIR, SULTR1.2, SULTR2.2,SULTR3.5, and SULTR4.2, as well as genes involved innitrogen assimilation and transport, including NIA1,NIA2, NIR1, NRT1.1, NRT1.2, NRT1.5, NRT2.2, andNRT 3.1 (Xiong et al., 2013). Therefore, there is a re-ciprocal positive feedback regulation loop among Glc,sulfur, and nitrogen signaling, and TOR may functionas a central hub that orchestrates nutrient acquisition,shuttling, and communication between above-groundand below-ground tissues (Fig. 1).

TOR SIGNALING IN ABIOTIC STRESSES

Advancing research has shown that TOR playsmultifaceted roles in the plant response to various kindsof abiotic stress, andmay function as either a positive ora negative regulator depending on the type and dura-tion of stress encountered.Temperature is a major factor in plant metabolism

and growth. Wang et al. (2017) showed that Arabi-dopsis TOR activity is quickly abolished by cold treat-ment at time points as early as 10min, but recovers after2 h of treatment. Furthermore, cold treatment com-promises enhanced anthocyanin accumulation in theinducible tor-es mutant under normal temperature, in-dicating that TOR is likely to be a negative regulator incold acclimation. Because inhibition of translation isessential for cold tolerance, inactive TOR might de-crease translation in plants to prepare them for unfa-vorable cold conditions (Wang et al., 2017). However,another independent study suggested that TOR seemsto positively regulate the plant cold response (Donget al., 2019). Depletion of AtTHADA (which codes for

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AtTHADA, the plant protein ortholog of the cold re-sponse regulator HsTHADA in humans) lowers energystatus, decreases TOR activity, and causes growth ar-rest in Arabidopsis (Dong et al., 2019). Meanwhile, theAtthada mutant and TOR-RNAi (35-7) lines are hyper-sensitive to cold conditions (Dong et al., 2019). Thedifferences between these studies might be caused bydifferent silencing efficiencies in different TOR-RNAilines or by different growth conditions, further indi-cating the complexity and dynamic nature of the TOR-regulated cold response.

In addition to cold stress, TOR is involved in hightemperature tolerance. Exogenous application of Glc,overexpression of TOR, and overexpression of E2Fa allresult in higher heat shock gene expression and seed-ling survival rates after recovery from heat stresstreatment. Downregulation of TOR, downregulation ofE2Fa, and treatment with the TOR inhibitor AZD-8055or Torin1 lead to decreased seedling survival (Sharmaet al., 2019). HIKESHI-LIKE PROTEIN1 (AtHLP1) is anortholog of HsHikeshi, which imports HSP70 into thenucleus to promote thermo-tolerance in humans (Koseet al., 2012; Koizumi et al., 2014; Sharma et al., 2019).Glc-TOR-activated E2Fa directly binds to the promoterof AtHLP1 to activate AtHLP1. AtHLP1 binds directlyto the promoters of many heat shock genes, which inturn leads to histone acetylation and H3K4me3 accu-mulation to activate and maintain thermo-memory,eventually enhancing thermo-tolerance (Sharma et al.,2019). Interestingly, proHLP1::GUS exhibits strong GUSinduction in the proliferation zone of the shoot apexafter 24 h of heat stress recovery in the presence of Glc.These results suggest that cell proliferation in the shootapex must be coordinated with internal and externalcues to maintain growth and survival, and is mediatedby Glc-TOR energy signaling.

TOR positively regulates the plant response todrought and osmotic stresses. In Arabidopsis, TORoverexpression lines have a longer primary root thancontrol lines exposed to a high concentration of potas-sium chloride (Deprost et al., 2007). Ectopic expressionof Arabidopsis TOR gene in rice (Oryza indica) enhanceswater-use efficiency, growth, and yield under water-limiting conditions (Bakshi et al., 2017). These trans-genic rice lines also show seed germination insensitivityto ABA treatment (Bakshi et al., 2017, 2019). These ob-servations suggest that constitutive TOR expressionmight alleviate the effect of drought or osmotic stress onplant growth.

In contrast, TOR negatively regulates the plant re-sponse to oxidative stress and DNA/RNA damage.Maf1 is a conserved repressor of RNA polymerase III,which is responsible for synthesizing small RNAs, 5Sribosomal RNA, and tRNAs. Maf1’s activity is medi-ated by phosphorylation/dephosphorylation, and de-phosphorylation of Maf1 promotes its repressoractivity. Both oxidative stress or DNA/RNA damageand TOR silencing stimulate Maf1 dephosphorylation(Ahn et al., 2019). It is very likely that these stressesinhibit TOR activity to enhance the dephosphorylation

of Maf1 and activate its repressor function. In this way,plants may slow down protein synthesis and cellgrowth or division to overcome these environmentalstresses.

CROSSTALK BETWEEN TOR SIGNALING ANDABA SIGNALING

The phytohormone ABA plays a key role in inte-grating a wide range of stress signals and controllingdownstream stress responses. Upon stress, ABA accu-mulates rapidly and binds to its intracellular PYR/PYL/RCAR receptors. The ABA-receptor complexbinds to and inhibits the clade A PP2C protein phos-phatases. PP2C inhibition releases the activity of Snf1-related protein kinase 2s (SnRK2s), which phosphorylatedownstream targets to mediate protective responsessuch as stomatal closure and the expression of ABA-responsive genes (Chen et al., 2020).

TOR signaling has been found to regulate ABA bio-synthesis and distribution. ABA content is decreased inraptorb seedlings, lst8-1 seedlings, and seedlings treatedwith the TOR inhibitor AZD-8055 (Kravchenko et al.,2015). Some genes that encode critical enzymes in ABAbiosynthesis, such as NECD3 and AOO3, show de-creased expression in the raptorb mutant (Kravchenkoet al., 2015). However, the ABA content of raptorb

Figure 1. TOR signaling networks mediate nutrient interorgan dia-logues to drive plant growth. Plant obtain carbon, nitrogen, sulfur,phosphate, and other micronutrients from above-ground photosynthe-sis and below-ground soil nutrient assimilation. There is a reciprocalpositive feedback regulation loop among Glc, sulfur, and nitrogen sig-naling, and TOR functions as a central hub that orchestrates nutrientacquisition, shuttling, and communication between interorgan coor-dination. ETC, electron transport chain; NIA, nitrate reductase; NIR,nitrite reductase; NRT, nitrate transporter; SULTR, sulfate transporter;SWEET, Suc transporter; TPS, trehalose-6-phosphate synthase.

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mutant seeds is elevated (Salem et al., 2017), suggestingthat TOR may also be involved in the distributionof ABA.TOR signaling and ABA signaling converge on two

Protein Phosphatase 2A (PP2A)-associated proteins,TAP46 and TIP41 (Ahn et al., 2011; Hu et al., 2014;Punzo et al., 2018a, 2018b). TAP46 is directly phos-phorylated by TOR kinase, and functions as a positiveeffector in TOR signaling (Ahn et al., 2011). Meanwhile,TAP46 negatively regulates the phosphatase activityof PP2A, prevents it from dephosphorylating ABI5(thereby stabilizes ABI5), and finally enhances ABAsensitivity in plants (Hu et al., 2014). TIP41 interactswith the catalytic subunit of PP2A and negativelyregulates ABA sensitivity (Punzo et al., 2018a, 2018b).TIP41 is also involved in TOR signaling. The tip41mutants display growth retardation, similar to thephenotype caused by TOR silencing, and are hyper-sensitive to the TOR inhibitor AZD-8055 (Punzo et al.,2018a, 2018b). Recent large-scale genetic screens forinsensitivity to the TOR inhibitor AZD-8055 identifiedtwo important mediators of ABA signaling, YAK1 andABI4, as the key downstream regulators of TOR sig-naling to control root growth, meristem activation,and seed germination (Li et al., 2015; Kim et al., 2016;Barrada et al., 2019).Upon sensing environmental stresses, plants usually

transiently sacrifice growth and activate protectivestress responses. Recently, a reciprocal negative cross-talk between TOR and ABA signaling has been shownto regulate such a trade-off between plant growth andstress adaptation (Fig. 2; Wang et al., 2018). In un-stressed Arabidopsis, TOR phosphorylates ABA re-ceptors at a highly conserved Ser, corresponding toSer-119 in PYL1, to compromise ABA signaling by abol-ishing PYL binding activity to ABA, thereby inhibitingPP2C phosphatase. Expression of phosphor-mimickingPYL1S119D in multiple ABA receptor mutants does notcomplement theABA-insensitivephenotype (Wang et al.,2018). The raptorb and lst8-1 mutants display hypersen-sitivity to exogenous ABA application (Salem et al., 2017;Wang et al., 2018). On the other hand, ABA also antag-onizes TOR signaling. ABA-activated SnRK2s directlyinteract and phosphorylate RaptorB. This phospho-rylation triggers the disassociation of RaptorB fromthe TOR complex, and thereby inhibits TOR’s kinaseactivity (Wang et al., 2018). Therefore, under nutrient-rich conditions, active TOR inhibits ABA signalingto direct resources to growth, whereas under stressconditions, ABA signaling is activated, and ABA-activated SnRK2 inhibits TOR activity to sacrificegrowth for survival during stress. Importantly, the Serresidue corresponding to Ser-119 in PYL1 is highlyconserved across all 121 PYLs from 12 different plantspecies, suggesting that this phosphor-regulatoryfeedback loop is a conserved mechanism that landplants utilize to optimize the balance of growth andstress responses. Strikingly, several PYLs (PYL5 toPYL12) in Arabidopsis can bind to and inhibit PP2Cseven in the absence of ABA (Hao et al., 2011; Fujii and

Zhu, 2012), while the phosphor-mimicking mutation ofthe TOR phosphorylation site within PYL10 abolishesthis ABA-independent interaction with PP2Cs (Wanget al., 2018). Therefore, TOR might also inhibit the ac-tivation of the ABA-independent PYLs under nonstressconditions to promote growth and development.

TOR NEGATIVELY REGULATES AUTOPHAGY

Autophagy is a process in which harmful or un-wanted cellular components are delivered into lyticvacuoles to be recycled (Zhuang et al., 2018; Signorelliet al., 2019). Autophagy promotes plant resistance tonutrient deficiency, salt stress, drought stress, oxidativestress, and endoplasmic reticulum stress (Pu et al.,2017a). TOR is one of the key negative regulators ofautophagy. Downregulation of TOR expression orkinase activity leads to constitutive activation of au-tophagy (Liu and Bassham, 2010). However, TORantagonizes some, but not all, of the abiotic stress-triggered autophagy process (Liu and Bassham,2010; Pu et al., 2017a, 2017b; Soto-Burgos and Bas-sham, 2017). Nutrient deficiency, salt stress, anddrought stress all induce autophagy through TORkinase, as overexpression of TOR under these con-dition significantly reduces the autophagy caused bythese stresses (Pu et al., 2017a, 2017b; Soto-Burgosand Bassham, 2017). However, oxidative stress and

Figure 2. A Tai-Chi model of the phospho-reciprocal regulation ofthe TOR kinase and ABA signaling to balance plant growth and stressresponse. Under growth-promoting conditions, active TOR phosphor-ylates ABA receptors PYR/PYLs to inhibit ABA signaling, and directsresources toward growth; under stress conditions, ABA-activatedSnRK2s phosphorylate Raptor to decrease TOR activity, and sacrificegrowth for survival during stress.

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Table 1. TOR-regulated stress-related proteins

Protein AGI No.a Related Plant Stress Responses Methods

VIP1 At1g43700 Wound, cold, heavy metal, salt, osmotic, oxidative, and mechanical stress; sulfur deficiency(Pitzsche et al., 2009; Wu et al., 2010; Tsugama et al., 2012, 2018)

Phosphoproteomics

OZF1 At2g19810 Sugar and nitrogen deficiency; oxidative, drought, salt, and osmotic stress (Contento et al.,2004; Peng et al., 2007; Huang et al., 2011; Lee et al., 2012; Ding et al., 2013)

Phosphoproteomics

ATG1c At2g37840 Autophagy-related stress (Qi et al., 2017) PhosphoproteomicsBAM1 At3g23920 Drought, osmotic, salt, and heat stress (Simpson et al., 2003; Monroe et al., 2014; Prasch

et al., 2015; Liu et al., 2019b)Phosphoproteomics

At3g26730 At3g26730 ABA-related stress (Bang et al., 2008) PhosphoproteomicsATG13 At3g49590 Autophagy-related stress (Son et al., 2018) PhosphoproteomicsATG1b At3g53930 Autophagy-related stress (Qi et al., 2017) PhosphoproteomicsGCN5 At3g54610 Cold and salt stress (Pavangadkar et al., 2010; Zheng et al., 2019) PhosphoproteomicsEIF4G At3g60240 Heat stress (Wu et al., 2013) PhosphoproteomicsATG1a At3g61960 Autophagy-related stress (Qi et al., 2017) PhosphoproteomicsATHD1 At4g38130 Salt, drought, and heat stress (Ueda et al., 2018) PhosphoproteomicsLARP1a At5g21160 Heat stress (Merret et al., 2013) PhosphoproteomicsSGS3 At5g23570 Heat stress (Liu et al., 2019a) PhosphoproteomicsYAK1 At5g35980 ABA-related and drought stress (Kim et al., 2016) PhosphoproteomicsPLDRP1 At5g39570 Drought and salt stress (Ufer et al., 2017) PhosphoproteomicsPAH2 At5g42870 Phosphorus depletion (Nakamura et al., 2009) PhosphoproteomicsAKS2 At1g05805 ABA-related stress (Takahashi et al., 2013) InteractomePFD4 At1g08780 ABA-related and cold stress (Kurup et al., 2000; Perea-Resa et al., 2017) InteractomeKINbg At1g09020 Sugar deficiency (Emanuelle et al., 2015) InteractomeFHY2 At1g09570 UV and cold stress (Rusaczonek et al., 2015) InteractomeHOP1 At1g12270 Heat stress (Fernandez-Bautista et al., 2018) InteractomeHSP70B At1g16030 Heat stress (Sung et al., 2001) InteractomeCAT1 At1g20630 Drought stress (Hsieh et al., 2002; Xing et al., 2008) InteractomeCPK11 At1g35670 ABA-related stress (Zhu et al., 2007) InteractomeTUA2 At1g50010 Wounding, osmotic, and cold stress (Testerink et al., 2004) InteractomeFYPP1 At1g50370 ABA-related stress (Dai et al., 2013) InteractomeMKK4 At1g51660 Wounding and osmotic stress (Li et al., 2018) InteractomeCPN60B At1g55490 Cold stress (Goulas et al., 2006) InteractomePP2A-1 At1g59830 ABA-related stress (Punzo et al., 2018b) InteractomeHOP2 At1g62740 Heat stress (Fernandez-Bautista et al., 2018) InteractomePP2A At1g69960 ABA-related and salt stress (Hu et al., 2017) InteractomePP5 At2g42810 Heat stress (Park et al., 2011) InteractomeKIN10 At3g01090 Autophagy-related and ABA-related stress; low-energy, carbon, and phosphorus deficiency

(Hamasaki et al., 2019)Interactome

S6K1 At3g08730 Cold, salt, and osmotic stress (Mahfouz et al., 2006) InteractomeHSP70 At3g09440 Cold and heat stress (Sharma et al., 2007) Interactome2CPA At3g11630 Cold and oxidative stress (Goulas et al., 2006; Pulido et al., 2010; Juszczak et al., 2016) InteractomeHSC70-4 At3g12580 Heat, salt, osmotic, and oxidative stress (Montero-Barrientos et al., 2010) InteractomeKIN11 At3g29160 Sugar deficiency (Baena-Gonzalez et al., 2007; Sheen, 2014) InteractomeATJ3 At3g44110 Salt and osmotic stress (Salas-Munoz et al., 2016) InteractomeATG13 At3g49590 Autophagy-related stress (Son et al., 2018) InteractomeATG1b At3g53930 Autophagy-related stress (Qi et al., 2017) InteractomeFER3 At3g56090 Oxidative stress (Ravet et al., 2009) InteractomeMPK4 At4g01370 Salt and heat stress (Andrasi et al., 2019) InteractomeGRXS17 At4g04950 Cold, heat, and drought stress (Wu et al., 2017) InteractomeTUA6 At4g14960 Salt stress (Dinneny et al., 2008) InteractomeATPDX1 At5g01410 Chilling, drought, salt, osmotic, and ozone stress (Denslow et al., 2007) InteractomeHSP70-1 At5g02500 Cold, heat, salt, osmotic, and heavy metal stress (Lee and Schoffl, 1996; Zhang et al., 2003;

Leng et al., 2017)Interactome

UBP12 At5g06600 UV stress (Al Khateeb et al., 2019) InteractomeTSN1 At5g07350 Heat and salt stress (Gutierrez-Beltran et al., 2015) InteractomeGDH2 At5g07440 Salt stress (Jiang et al., 2007) InteractomeASN3 At5g10240 Nitrogen deficiency (Bi et al., 2007) InteractomeGDH1 At5g18170 Low oxygen stress (Sarry et al., 2006) InteractomeATJ2 At5g22060 Heat and cold stress (Li et al., 2005) InteractomePFD5 At5g23290 Salt stress (Rodrıguez-Milla and Salinas, 2009) InteractomeYAK1 At5g35980 ABA-related and drought stress (Kim et al., 2016) InteractomePFD3 At5g49510 Salt stress (Rodrıguez-Milla and Salinas, 2009) Interactome

(Table continues on following page.)

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ER stress trigger autophagy in a TOR-independentmanner (Pu et al., 2017a, 2017b; Soto-Burgos andBassham, 2017).The autophagy-related1 (ATG1)/ATG13/ATG17

kinase complex plays an essential role in the onsetof autophagy, and is the direct TORC1 substrate inmammals and yeast. In Arabidopsis, there are threeATG1 and two ATG13 homologs; their roles in theregulation of autophagy in response to nutrient star-vation have been uncovered (Suttangkakul et al., 2011).Son et al. (2018) found that ATG13 contains a motif thatcould be phosphorylated by TOR kinase, and that de-letion of this TOR-recognized motif in ATG13 enhancesautophagy in Arabidopsis protoplasts. Recent high-throughput phosphoproteomics analysis using Arabi-dopsis suspension cell culture also revealed that ATG1sand ATG13s are direct TOR substrates. These studiesreinforce that TOR-regulated phosphorylation of theATG1/ATG13/ATG17 complex is essential for inhib-iting autophagy in plants.The upstream signals of TOR signaling also regulate

autophagy. As a growth hormone, auxin stimulatesTOR activity through a physical interaction betweenTOR and auxin-activated ROP2 to promote the activa-tion of shoot apex cell proliferation (Schepetilnikovet al., 2013; Li et al., 2017). Interestingly, auxin alsoacts upstream of TOR in the regulation of autophagy.As mentioned above, nutrient deficiency, salt stress,and drought stress induce autophagy via TOR signal-ing, but the addition of auxin prevents the autophagyphenomenon induced by these stress conditions(Pu et al., 2017a). Meanwhile, auxin has no effect onoxidative or ER stress-induced autophagy, indicatingthat auxin specifically affects TOR-dependent autoph-agy (Pu et al., 2017a).

UPSTREAM REGULATORS OF PLANTTOR-STRESS SIGNALING

In contrast with the significant progress made indiscovering the various molecular functions of TORsignaling in plant stress responses, the upstream regu-lators of TOR remain poorly understood. Plants possessa family of unique Rho-like small GTPases with 11members that function as central hubs in signalingnetworks (Nagawa et al., 2010). As mentioned above,ROP2/3/6 has been shown to bind to and activate TORstimulated by auxin signaling (Schepetilnikov et al.,2013; Li et al., 2017). Whether other ROPs are in-volved in stress sensing and regulation in TOR signal-ing remains a worthwhile question to be studied.

SnRKs are a group of kinases that play vital roles in awide range of plant stress responses. Plants containthree SnRK families: SnRK1s, SnRK2s, and SnRK3s(Halford and Hey, 2009). Increasing evidence suggeststhat part of the SnRK-regulated stress response is ach-ieved by the SnRKs-TOR module.SnRK1 complex functions as a conserved energy

sensor, which is activated under low energy conditionsand is repressed under energy-rich conditions. In yeastand animal cells, nutrient starvations stimulate SNF1/AMPK,which repress TOR activity by phosphorylatingRaptor proteins to suppresses cell growth and biosyn-thetic processes (Gwinn et al., 2008). In Arabidopsis,KIN10/11 protein kinases provide catalytic activities inthe SnRK1 complex, and act antagonistically to TOR inthe regulation of convergent primary sugar-responsivegenes (Baena-González et al., 2007; Xiong et al., 2013; Liand Sheen, 2016), indicating that KIN10/11 functionsupstream of TOR to regulate energy starvation pro-cesses. Furthermore, it was reported that KIN10 inter-acts with and phosphorylates Raptor in the TORcomplex, providing a biochemical basis for the SnRK1-TOR regulation module (Nukarinen et al., 2016). No-tably, KIN10 also functions upstream of TOR to activateautophagy (Pu et al., 2017b; Soto-Burgos and Bassham,2017).The SnRK2s are a group of plant-specific Ser/Thr

kinases with 10 members (Kulik et al., 2011). SnRK2.2,SnRK2.3, and SnRK2.6 are key regulators in ABA sig-naling, where all 10 members are essential for osmoticstress responses (Zhu, 2016). As discussed above, ABA-dependent SnRK2.6 phosphorylates RaptorB and dis-sociates it from the TOR complex (Fig. 2). In this way,SnRK2s shut down TOR-promoted growth and en-hance stress adaptation responses (Wang et al., 2018).Osmotic stresses also repress TOR activity (Wanget al., 2018), and PYR1/PYLs/RCARs could interactwith SnRK2s to inhibit activation of SnRK2s uponosmotic stress condition (Zhao et al., 2018). WhetherTOR phosphorylation of PYLs regulates osmoticstress-induced SnRK2 activation or vice versa is notknown yet.SnRK3 is also known as Calcineurin B-like protein-

interacting protein kinase (CIPK; Manik et al., 2015).Arabidopsis has 26 CIPKs in total (Kolukisaoglu et al.,2004). The majority of stresses trigger rapid, transientCa21 signatures; and consequently, as a Ca21 sensor,the Calcineurin B-like protein-CIPK module partici-pates broadly in various kinds of stress responses, es-pecially in ion homeostasis (Liu et al., 2000; Zhu, 2016;Sardar et al., 2017). Interestingly, the expression ofSnRK3.24 (CIPK5) is downregulated after long-term

Table 1. (Continued from previous page.)

Protein AGI No.a Related Plant Stress Responses Methods

TAP46_2A At5g53000 Cold stress (Harris et al., 1999) InteractomeATG101 At5g66930 Autophagy-related stress (Li et al., 2014) Interactome

aAGI, Arabidopsis Gene-Initiative Identifier

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TOR inhibition (Dong et al., 2015), and the cipk5 mu-tant exhibits decreased TOR activity (Meteignier et al.,2017), suggesting that SnRK3s might, like KIN10and SnRK2s, phosphorylate Raptor to regulate TORactivity and signaling.

DOWNSTREAM EFFECTORS OF PLANTTOR-STRESS SIGNALING

TOR is a core merging point in the plant stress sig-naling network. However, until now, only a very lim-ited number of TOR substrates or TOR-regulatedproteins have been identified. Very recently, van Leeneet al. (2019) performed quantitative phosphoproteo-mics and interactome analysis using Arabidopsis cellcultures with or without AZD8055 treatment. A total of83 TOR-regulated phosphoproteins and 215 proteinsinteracting with the TOR complex (TOR, LST8-1, Rap-torA, or RaptorB) were identified (van Leene et al.,2019). Some of these proteins may be direct TOR sub-strates.We performed a literature search to examine thebiological functions of these proteins, and found that19% of TOR-regulated phosphoproteins and 20% ofTOR complex interacting proteins participate in variousstress responses (Table 1). These TOR signaling-relatedtargets include VirE2-Interacting Protein1 involved inosmotic and sulfate deprivation response, GeneralControl Nonderepressible5 affecting histone acetyla-tion under cold and salt stress, ATG1/13 for autophagy

induction, and La-related protein1 involved in the heatstress-triggered mRNA degradation process (Pitzschkeet al., 2009; Merret et al., 2013; Qi et al., 2017; Son et al.,2018; Zheng et al., 2019). These putative TOR sub-strates provide valuable directions for future studiesof TOR-regulated stress responses.

CONCLUSION

During the last decade, our knowledge of plant TORsignaling has increased significantly. It is now clear thatTOR acts as a master regulator to sense and transducenutrient, energetic, hormonal, metabolic, and environ-mental stress inputs into physiological, molecular, anddevelopmental responses for growth and stress adap-tation. Despite the great wealth of information that hasbecome available, several questions still remain to beanswered, and many others are emerging (see Out-standingQuestions). In addition to its well-known rolesin regulation of protein translation, it will be fruitful todissect how TOR signaling represses a vast spectrum ofprimary target gene pathways in stress and immuneresponses. As a protein kinase, the phosphorylation ofThr-449 in the TOR-substrate protein ribosomal S6kinase1 is used as a conserved indicator of endoge-nous TOR activity. Developing tissue-specific andfluorescence-visualized TOR kinase activity markerswill help to quantitatively measure TOR activity andspecific signaling output in different organs, e.g. sinkand source tissues, thereby facilitating a more accurateinterpretation of the different or even opposite phe-notypes when TOR signaling is perturbed under var-ious environmental conditions.Received October 7, 2019; accepted December 21, 2019; published January 16,2020.

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TOR Signaling In Response to Plant Stress

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