Live long and praise TOR: a role for TOR signaling in every stage of plant life

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Live long and praise TOR: a role for TOR signaling in every stage of

plant life

Quilichini, Teagen D.; Gao, Peng; Pandey, Prashant K.; Xiang, Daoquan;

Ren, Maozhi; Datla, Raju

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Live long and praise TOR: A role for TOR signaling in every stage of plant life

Teagen D. Quilichini1, Peng Gao1, Prashant K. Pandey1, Daoquan Xiang1, Maozhi Ren2, Raju Datla1

1

National Research Council of Canada, Saskatoon, SK S7N 0W9, Canada

2

School of Life Sciences, Chongqing University, Chongqing 401331, China Corresponding author: Raju Datla

Phone: (306) 975-5267 Fax: (306) 975-4839

Email: raju.datla@nrc-cnrc.gc.ca

Abstract:

From scientific advances in medical research to the plethora of anti-aging products available, our obsession with slowing the aging process and increasing lifespan is indisputable. A large research effort has been levied towards this perpetual search for the fountain of youth, yet the molecular mechanisms go er i g a orga is s lifespa a d the auses of agi g are only beginning to emerge in animals and remain largely unanswered in plants. One central pathway in eukaryotes controlling cell growth, development and metabolism, the target of rapamycin (TOR), plays an evolutionarily conserved role in aging and the determination of lifespan. The modulation of TOR pathway components in a wide range of species, including the model plant Arabidopsis thaliana have effects on lifespan. However, the mechanisms enabling some of the longest living species to endure, including trees that can live for millennia, have not been defined. Here, we introduce key TOR research from plant systems and discuss its implications in the plant life cycle and the broader field of lifespan research. TOR pathway functions in plant life cycle progression and lifespan determination are discussed, noting key differences from yeast and animal systems and the i porta e of o i s resear h for the continued progression of TOR signaling research.

Keywords: Arabidopsis; development; kinase; lifespan; meristems; signaling; target of rapamycin (TOR)

Downloaded from ht tps: //academic. oup. com/ jxb/ advance-art icle-abst ract /doi/ 10. 1093/ jxb/ erz125/ 5418542 by Nat ional Research Council Canada user on 09 A pril 2019

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Lifespan in the plant kingdom

The duratio of a li i g orga is s e iste e, from a viable state to death, defines its lifespan. Plants exhibit tremendous lifespan variation, from several weeks or months, as in the model species Arabidopsis thaliana (Arabidopsis), to some of the longest-lived organisms known, such as the coast redwood (Sequoia sempervirens; Figure 1A) that can live 2,000 years, the giant sequoias (Sequoiadendron giganteum) that can live over 3,200 years and the bristlecone pine (Pinus longaeva) believed to live up to 4,600 years (Thomas, 2012; Russell et al., 2014). Variation in plant lifespan is influenced in part by the number of reproductive cycles accomplished before death. Monocarpic species flower and set seed once in their life cycle, while polycarpic species reproduce several times (Amasino, 2009). Plant lifespan is also influenced by the life strategy employed and the regulation of meristems, especially in the context of determinate and/or indeterminate growth. For example, in annual plants that live up to one year, the transition from vegetative to reproductive growth is associated with determinate growth accompanied by senescence and death. In contrast, the majority of perennial species, capable of living for more than two years, resume vegetative growth after flowering by maintaining vegetative meristems or by reverting reproductive meristems to a vegetative state (Tooke et al., 2005; Wang et al., 2009). The diversity in plant longevity is impressive, but our understanding of the mechanisms controlling the growth and lifespan of plants is in its early stages.

The underpinning mechanisms governing lifespan and its extension is an active field of research in which major advancements have been made, particularly in animal model systems. Lifespan research in humans and many animals often focuses on life after growth and reproduction, and on the process of aging, in which cellular damage accumulates over time (Cornu et al., 2013). In contrast, several species in the plant kingdom are capable of indeterminate growth through perpetual maintenance of meristems and their stem cells, enabling a plethora of life strategies, multiple phase changes, and modular growth habit and thus conferring extreme lifespan diversity. Plant aging is further differentiated from animal systems by the process of senescence, an ordered deterioration process that enables nutrient remobilization in the final stages of organ or whole plant development (Khan et al., 2014). The unique capabilities of plants and astounding lifespan achievements suggest that the mechanisms governing plant growth and development may also have distinct regulatory controls and effects on lifespan determination. Expanding critical knowledge of the regulatory mechanisms controlling plant lifespan offers applied potential for the manipulation of agricultural crops.

TOR: a key regulator in eukaryote life

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Organisms must adapt their growth and development based on the state of their surroundings and ever-changing nutrient availability. From unicellular forms to complex multicellular organisms, the evolutionarily conserved target of rapamycin (TOR) appears common to all eukaryotes (Fingar and Blenis, 2004; Kapahi et al., 2010). As a member of the phosphatidylinositol 3-kinase related protein kinase superfamily, TOR is a serine-threonine protein kinase that senses and integrates cellular status information from numerous stimuli, including hormone signals, nutrient and energy availability, and stress information. In response to status inputs, TOR mediates changes in protein phosphorylation that affect important cellular and metabolic processes including nutrient sensing, ribosome biogenesis, polysome integrity, protein synthesis, metabolism, development and autophagy (Proud, 2004; Tavernarakis, 2008; Laplante and Sabatini, 2009; Shi et al., 2018; Schepetilnikov and Ryabova, 2018). Through diverse regulatory controls, TOR signaling promotes anabolic processes including protein, membrane lipid and nucleotide biosynthesis under nutrient rich conditions, or triggers catabolic processes such as autophagy when faced with stress or starvation.

In addition to the fundamental role TOR plays in linking nutrient status to cell growth and metabolic homeostasis, an emerging body of literature in diverse species, including yeast, invertebrates, mammals, and plants has uncovered a role for the TOR pathway in lifespan determination and aging (Kapahi et al., 2010; Ren et al., 2012; Kennedy and Lamming, 2016; Weichhart, 2018). To explore TOR functions in the distinct autotrophic plant kingdom, this review considers TO‘ s regulation of growth and development in the life cycle and emerging data on the lifespan of plants. Focus is placed on the mechanisms governing lifespan in the monocarpic annual herb Arabidopsis, as the wealth of research on this topic has been performed with this model plant species.

The TOR pathway from a plants perspective

As sessile, photoautotrophic organisms, the needs of plants in sensing and responding to environmental changes differ from heterotrophic eukaryotic lineages. The importance of TOR in plants has been demonstrated by early embryo lethality in T-DNA insertion mutations of Arabidopsis TOR (Menand et al., 2002; Ren et al., 2011). Some of the key TOR pathway components, including Raptor, LST8, S6K, TAP46, E2Fa and RPS6 confer important functions in these photoautotrophic organisms and differ considerably in several aspects from their heterotrophic counterparts.

Most eukaryotes contain a single TOR ortholog, such as the well-studied mechanistic TOR (mTOR) in mammals (Helliwell et al., 1994; Laplante and Sabatini, 2012; Saldivia et al., 2013; Shimobayashi and Hall, 2014; Eltschinger and Loewith, 2016). In animals, TOR interacts with two key proteins, regulatory-associated protein of mTOR (RAPTOR) and rapamycin-insensitive companion of mTOR (RICTOR), defining distinct complexes with unique regulatory functions, TOR Complex 1 (TORC1) and TOR Complex 2 (TORC2), respectively (Wullschleger et al., 2006). A plant TORC1 equivalent complex, composed of the TOR kinase, RAPTOR and Lethal with Sec Thirteen 8 (LST8), has been identified, but it is not clear whether a second TOR complex or RICTOR ortholog exists in plants (Dobrenel et al., 2016). Two isoforms

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of both RAPTOR and LST8 have been identified in plants, but whether these proteins are functionally redundant and/or assemble into distinct complexes with TOR is not known (Deprost et al., 2007; Moreau et al., 2012). From the N-terminus to the C-terminus, the large TOR kinase protein consists of several HEAT (Huntington, Elongation Factor 3 regulatory subunit A of PP2A TOR1) repeats, a FAT (FRAP-ATM-TTRAP) domain that interacts with RAPTOR, and a kinase domain containing an FRB (fkbp-rapamycin-binding) domain and FAT carboxy-terminal (FATC) domain (Yang et al., 2013). TOR protein sequence shows high conservation across eukaryotes, particularly in the kinase domain (Menand et al., 2002). TOR domain studies in Arabidopsis have revealed some functional differences from other eukaryotes, including a non-essential role for the FATC domain in embryo development, in contrast to its essential role in yeast and mammals (Ren et al., 2011). The properties of the TOR kinase protein and its complex formations in plants likely support its specialized and diverse roles in plant life.

The network of upstream regulators and downstream effectors associated with TOR signaling is enormous, complex and exceeds the scope of this review. However, some of the known differences in the plant TOR signaling network are highlighted herein. As discussed in the vegetative growth phase section, a primary function of TOR is in the regulation of ribosome biogenesis and protein synthesis. Two major downstream effectors of TOR, ribosomal protein S6 kinase (S6K) and eukaryotic translation initiation factor4E (eIF4E)-binding protein1 (4E-BP1), mediate ribosome biogenesis and protein synthesis in heterotrophic eukaryotes, although S6K has unique roles in plants, and until recently 4E-BP1 appeared to lack orthologs in plants (Schepetilnikov and Ryabova, 2018). The identification and characterization of Conserved Binding of eIF4E 1 (CBE1) in Arabidopsis, which has eIF4E binding activity and forms cap-binding complexes, suggests that plant translation initiation may be regulated by 4E-BP-like protein(s) in a similar manner to heterotrophic species (Patrick et al., 2018). In Arabidopsis, one of the six E2F transcription factors, E2Fa, has been identified as a direct TOR phosphorylation target in the presence of glucose (Xiong et al., 2013; Li et al., 2017). This unique TOR response to glucose enables transcriptional control of cell cycle genes and represents the first such process identified in animals or plants (Xiong et al., 2013; Xiong and Sheen, 2013).

Considering the immobile and autotrophic life strategy of plants, it is likely that unique upstream inputs to TOR have evolved in plants to ensure that environmental conditions are perceived and integrated into biological responses. The upstream regulators of plant TOR have not been characterized as extensively as the downstream effectors (Dobrenel et al., 2016). Plants appear to lack orthologs for several of the upstream regulators of TOR known in other eukaryotes, including the tuberous sclerosis complex (TSC1 and TSC2), Akt/protein kinase B, RHEB and RAG small GTPases (Tee et al., 2002; Sarbassov et al., 2005; Xiong and Sheen, 2014; Dobrenel et al., 2016). However, the plant-specific Rho-related GTPase protein 2 (ROP2) functions as an upstream regulator of TOR in response to light and auxin signals (Li et al., 2017; Schepetilnikov et al., 2017). Due to their unique and diverse functions in plant growth and development, phytohormones are likely to be a major component of plant TOR regulation, although little is known about the underpinning signaling mechanisms (Dobrenel et al., 2016). Several studies have linked the TOR pathway with phytohormone signaling, including abscisic acid, brassinosteroids, cytokinin, jasmonic acid, and salicylic acid (as recently summarized by Schepetilnikov and Ryabova, 2018). Mediated by the physical interaction of ROP2 GTPase with TOR, auxin acts as an upstream effector of TOR, driving polysome loading and the translation of selected mRNAs, and represents one of the best characterized upstream regulatory pathways of plant TOR to date (Schepetilnikov et al., 2017; Schepetilnikov and Ryabova, 2018). Additionally, auxin-mediated TOR

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activation has been linked to the inhibition of autophagy and meristem activation (Li et al., 2017; Pu et al., 2017). Discoveries related to phytohormone-induced TOR regulation are expected to be forefront in future research efforts.

The discovery of TOR was made possible by the sensitivity of yeast and mammals to rapamycin, a potent immunosuppressive and anti-proliferative metabolite produced by Streptomyces hygroscopicus (Sehgal et al., 1975; Segall et al., 1986; Heitman et al., 1991; Morris 1992; Chantranupong et al., 2015). Rapamycin-mediated inhibition operates through the FRB domain of TOR, where the FK506 binding protein 12 (FKBP12) binds in the presence of rapamycin and specifically inactivates TOR (Heitman et al., 1991; Stan et al., 1994; Zheng et al., 1995). Rapamycin insensitivity in flowering species has been widely reported. The lack of or weak interaction between the Arabidopsis TOR FRB and the FKBP12-rapamycin complex (Menand et al., 2002; Mahfouz et al., 2006; Ren et al., 2012) further supports this insensitivity in plants, however in vivo protein interaction assays found rapamycin could induce FKBP12 interactions ith Ara idopsis TO‘ s F‘B do ai (Xiong and Sheen, 2012). Altogether, ineffective rapamycin treatment and early embryo lethality in tor mutant lines have slowed progress in the identification and characterization of upstream interactors and downstream targets of TOR in plants especially in post-embryonic phases of development.

Despite unique challenges faced in plant research, the appli atio of o i approaches has facilitated TOR signaling studies. Chemical and ATP-competitive TOR inhibitors, such as AZD-8055, KU63794, PP242, Torin1 and 2, and WYE 354 and 132, offer an effective alternative to rapamycin for TOR functional studies in plants (Schepetilnikov et al., 2013; Montané and Menand, 2013; Li et al., 2017). Using RNA interference to repress TOR function in plants, particularly under inducible control, has proven effective for circumventing the embryo lethality of complete TOR gene knockouts (Deprost et al., 2007; Caldana et al., 2013; Xiong et al., 2013). Using yeast or mammalian FKBP12 overexpression in Arabidopsis conditionally induces rapamycin sensitivity and offers an additional approach to study TOR functions in plants (Sormani et al., 2007; Leiber et al., 2010; Ren et al., 2011; Xiong and Sheen, 2012; Ren et al., 2012). This approach has uncovered a role for TOR in plant lifespan determination (Figure 1B). Specifically, the overexpression of a single copy of yeast FKBP12 (BP12-2) in Arabidopsis yielded an average lifespan extension of over two weeks, equivalent to over 25% longer life, when grown on rapamycin-enriched media (Ren et al., 2012). In this transgenic line, inducing rapamycin sensitivity extended longevity, with a reduced rate of growth and delayed developmental transitions such as emergence and time to flowering (Ren et al., 2012; Figure 1B). Conversely, the overexpression of plant TOR (P35S:TOR) accelerated the progression of Arabidopsis development, causing early flowering, senescence and overall shortened lifespan (Ren et al., 2011; 2012; Figure 1B). Using an ethanol inducible system, significant reductions in TOR expression have also been achieved at a specific stage of Arabidopsis development (Deprost et al., 2007). In this study, inducing reduction of TOR mRNA to less than 20% of its normal levels at the bolting stage resulted in disturbed leaf growth and nutrient recycling, apparently inducing early senescence (Deprost et al., 2007). These findings emphasize the fundamental role played by TOR in regulating growth and metabolic homeostasis, as significant reductions in TOR expression produced severe consequences. Altogether, these observations revealed that modulating TOR expression and/or activity could lead to extended lifespan in Arabidopsis and fine-tuning the level of TOR expression may be required to balance essential roles in plant growth and development with lifespan extension.

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A role for TOR signaling in every stage of plant life

After successful fertilization, an annual pla t s life cycle begins with embryogenesis, seed germination and seedling establishment, and proceeds through vegetative growth, the reproductive phase, seed production, the cessation of growth, senescence and death (as modeled in Figure 2). The broad scope of TOR functions in plant development and during phase changes has revealed roles for this master regulator in lifespan determination. The complex network of signals and effectors of TOR makes this pathway central and critical in the coordination of plant growth and development from the beginning to the end of the life cycle. We egi our dis ussio of TO‘ s role i the pla t life le ith embryogenesis.

1. Embryogenesis

The essential role of TOR in plants became evident as mutations in TOR produced early embryo lethality (Menand et al., 2002; Ren et al., 2011). In two Arabidopsis T-DNA insertion lines upstream of the FRB domain, tor homozygous mutants presented as aborted seeds with arrest occurring in the embryo and endosperm (Menand et al., 2002). Intriguingly, developmental arrest was reported to occur earlier in endosperm development, prior to its transition from syncytial growth to cellularization; while embryos aborted by the dermatogen stage of development (Menand et al., 2002). These data suggest that TOR plays a critical role in embryo and endosperm development.

To further characterize the critical role of TOR in embryogenesis, a T-DNA insertion and complementation approach was used to define key domains of TOR, including the HEAT, FAT and kinase domains, and distinguish the roles of these domains in plant growth and development (Ren et al., 2011). A critical function for all of the domains tested in embryogenesis was identified, with the exception of FATC. The stage reached by aborting embryos varied, with arrest in the 16-32 cell stage occurring in severe alleles, providing evidence that up to five cell division cycles can occur without expression of functional TOR after fertilization. The production of tor null mutant embryos and apparent lack of segregation distortion suggest that post-meiotic events in gametes progress normally in the absence of expression of a functional copy of TOR, raising the possibility for compensation of its functionality derived through the heterozygous gamete mother cell. Further studies are required to clarify mechanistically TOR functions in gametogenesis and fertilization. TOR mutant embryos also exhibited severe reductions in rRNA, suggesting that cellular growth and development beyond the 32-cell stage requires a sufficient supply of ribosomes to support protein synthesis and other TOR mediated essential functions. Moreover, the critical role of the kinase domain in embryogenesis was established by the

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embryo arrest phenotype in TOR mutant lines carrying a defective kinase domain, and by genetic complementation tests, where the kinase domain rescued early embryo lethality. Reintroduction of the kinase domain enabled embryo development to proceed to the late heart stage, but it was not sufficient for complete rescue of embryo lethality in the most severe mutant lines tested. Full restoration of embryogenesis required genetic complementation with full length TOR (Ren et al., 2011). An interesting exception to kinase domain complementation efficacy was observed in a TOR kinase domain mutant (tor-10), in which a less severe mutant phenotype suggested a partial TOR protein product was present. In tor-10, full complementation with the kinase domain transgene was achieved. Together with TOR domain interaction assay data, these results suggest partial TOR peptides may assemble and restore native functions and emphasizes the essential role of the kinase domain in TOR functionality (Ren et al., 2011).

2. Transitioning from a heterotrophic embryo to a photoautotrophic seedling

For many plant species, the transition from an embryonic mature seed to a seedling after its

germination marks a dramatic shift in the source of plant sustenance, from maternal seed

reserves and heterotrophic growth to photosynthesis-sourced sugars and photoautotrophic

growth. Studies on the transition from embryonic to post-embryonic plant growth and the

mechanisms that govern meristem activation have uncovered several pivotal roles for TOR

(Xiong et al., 2013; Pfeiffer et al., 2016; Xiong et al., 2017). Stimulated by glucose, seedling

photosynthesis was shown to control TOR signaling, promoting transcriptional activation of

S-phase cell cycle genes and meristem activation in the root through direct phosphorylation of the

E2Fa transcription factor (Xiong et al., 2013). This study provides insight into

TOR’s ability to

affect global changes through transcriptional control, rather than translational control alone.

After seed germination, all plant organs continue to develop from the activities of meristems. In

the shoot apical meristem, sugar and light signals are integrated by TOR to activate stem cells,

with TOR mediating the light-dependent activation of the WUSCHEL transcription factor

(Pfeiffer et al., 2016). In dark growth conditions, exposure of the shoot apical meristem to

sucrose promoted cell proliferation in a TOR pathway-dependent manner (Mohammed et al.,

2018). These discoveries identify TOR as a central regulator of post-embryonic seedling

establishment, linking light and photosynthesis-derived nutrient signals to stem cell activation

and growth. For more information on the roles of glucose and light on TOR signaling and

meristem regulation, readers are directed to Xiong et al., 2013; Xiong and Sheen, 2013; Pfeiffer

et al., 2017; Li et al., 2017; and Shi et al., 2018.

A recent study examined the role of Arabidopsis TOR in the transition from heterotrophic to photoautotrophic growth by testing the effect of TOR repression on seedlings during the transition from dark to light (Zhang et al., 2018). Upon illumination, etiolated TOR repressed seedlings (through RAPTOR1B mutations or TOR kinase active site chemical inhibition) had increased rates of greening and alterations in the GA-DELLA signaling pathway including down-regulation of all gibberellin receptors. This study implicates the TOR complex in chlorophyll biosynthesis, photomorphogenesis and gibberellin-DELLA pathway regulation (Zhang et al., 2018), in contrast to previous reports where TOR functions as

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a positive regulator of chlorophyll biosynthesis and inhibition of RAPTOR1B is associated with germination delay (Li et al., 2015; Sun et al., 2016; Salem et al., 2017). Further, altered carbon partitioning and accumulation of primary and secondary metabolites have been associated with delays in developmental stage transitions (Salem et al., 2018). It is therefore important to consider the developmental context in which genes are studied. Using active-site TOR inhibitors alone and in combination with induced rapamycin-sensitive Arabidopsis (due to the overexpression of a single copy of the FKBP12 gene from yeast), Xiong et al., 2017 identified S6K2 as a direct downstream target of TOR. In addition, a novel phosphorylation target of TOR-S6K2, the brassinosteroid insensitive 2 (BIN2) was characterized, uncovering a role for TOR-S6K2-BIN2 signaling in heterotrophic to photoautotrophic transitioning (Xiong et al., 2017). Altogether, these findings showed TOR signaling plays a central role in the transition of mature quiescent seeds into photosynthetically proficient seedlings.

In addition to enabling a seedlings transition to photoautotrophic growth, transcriptomic studies in Arabidopsis and potato have suggested that TOR plays a crucial role in photosynthesis and phytohormone signaling (Dong et al., 2015; Deng et al., 2017). Under conditions of TOR inhibition in rice, the formation of chloroplast thylakoid grana was perturbed, suggesting reduced photosynthetic capacity (Sun et al., 2016). Arabidopsis plants with yeast FKBP12 (BP12-2), rapamycin treatment alone or with active site TOR inhibitors exhibit decreased leaf chloroplast abundance and size, amyloplast formation and the repressed expression of several genes with roles in photosynthesis (Xiong et al., 2017). Further, in chlorotic Arabidopsis TOR inactivated plants, a reduction in transcripts encoding plastid-localized ribosomal proteins was identified (Dobrenel et al., 2016b). These findings support a role for TOR in controlling critical plant life sustaining processes including chloroplast formation and photosynthesis.

3. The vegetative growth phase

The growth and differentiation of cells is a complex process requiring tight regulation. TOR regulates cell growth and differentiation largely by controlling the rates of protein synthesis, ribosome production and polysome integrity (Li et al., 2006; Wullschleger et al., 2006). In Caenorhabditis elegans, manipulations to protein synthesis decrease rates of aging and lengthen life (Hansen et al., 2007; Pan et al., 2007; Syntichaki et al., 2007; Chiocchetti et al., 2007). TOR serves a central role in regulating the translation machinery of the eukaryotic cell, although select downstream effectors and regulatory mechanisms appear unique in plants. As in other eukaryotes, TAP46, E2Fs and S6Ks are phosphorylation targets of plant TOR kinase and affect a number of processes including rRNA transcription and protein translation. In Arabidopsis, there are two S6Ks, AtS6K1 and AtS6K2 (Zhang et al., 1994; Mizoguchi et al., 1995) and both are phosphorylation targets of TOR (Xiong and Sheen, 2012), but exhibit distinct subcellular localization patterns (Mahfouz et al., 2006). The predominantly cytosolic AtS6K1 appears to function as the mammalian (p70) S6K1 counterpart, and its phosphorylation by TOR is likely aided by Raptor (Schalm and Blenis, 2002; Mahfouz et al., 2006; Schepetilnikov et al., 2011; Xiong and Sheen, 2012). AtS6K2 exhibits nuclear localization and has been implicated in ribosome biogenesis (Mahfouz et al., 2006). Downstream phosphorylation targets of S6Ks have been identified in plants, including ribosomal

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protein S6 (RPS6) and BIN2 (Mahfouz et al., 2006; Xiong et al., 2017). In TOR-S6K1 signaling, Arabidopsis TORC1 can interact with eukaryotic initiation factor 3 (eIF3), a central complex involved in translation initiation and polysomes. This recruitment of TORC1 is thought to facilitate S6K1 phosphorylation under activating conditions (Schepetilnikov et al., 2011; 2013). AtS6K2 directly phosphorylates BIN2, regulating photoautotrophic growth and chloroplast development in seedlings (Xiong et al., 2017). Protein synthesis (and photosynthesis) also appears to be negatively regulated by Sucrose non-fermenting related kinase 1 (SnRK1) (Nukarinen et al., 2016). RPS6 hyperphosphorylation in SnRK-deficient Arabidopsis, along with in vivo interaction between Raptor and SnRK1, revealed links between the antagonistic TOR and SnRK1 pathways in regulating protein translation (Nukarinen et al., 2016). Perhaps of most interest to our understanding of longevity determination in plants, rps6 (a and b) mutants and RPS6 overexpression in Arabidopsis produce opposing phenotypes, extending or reducing lifespan, respectively (Ren et al., 2012). In addition to longer lifespan, rps6 mutant lines exhibited reduced vegetative growth, and delayed development including delayed phase transitions and late flowering. These alterations to RPS6 gene expression mirrored the phenotypes observed in equivalent TOR modulated lines, emphasizing the critical roles of TOR and RPS6 in mRNA translational control and their intimate connection with lifespan determination. These studies emphasized the key role of protein synthesis and its regulation in plant vegetative growth and longevity.

In many eukaryotes, translational regulation by TOR involves the key phosphorylation targets, eIF4E-binding proteins (4E-BPs). In response to select inputs, phosphorylation of 4E-BP translational initiation repressors by TOR results in their release from eIF4E and enables the assembly of the eIF4F complex for cap-dependent initiation of translation (Jackson et al., 2010). A number of proteins with the signature eIF4E -binding motif have been identified in plants, but whether these proteins are targeted by TOR remains to be determined. For comprehensive discussions on the regulation of plant protein synthesis, ribosome biogenesis and/or polysome integrity by TOR, the reader is directed to (Schepetilnikov and Ryabova, 2018; Shi et al., 2018).

The rate of general protein synthesis declines with age in a number of organisms, while the accumulation of macromolecule modifications and damage naturally increase over development (Hipkiss, 2006; Tavernarakis, 2008). TOR functions as a regulator of mitochondrial activity, managing the formation of reactive oxygen species (ROS) and their detrimental effects on lifespan (Schieke and Finkel, 2006; Cunningham et al., 2007). Oxidative stress levels associated with mitochondrial ROS influence properties of plant cell wall extensibility and cellular growth (Gapper and Dolan, 2006; Rhoads et al., 2006). In addition to the intracellular processes supporting and regulating cellular growth, plant cell size is governed by the properties of the encompassing cell wall and requires synthesis and remodeling of cell wall components to accommodate expansion (Carpita and Gibeaut, 1993). Not surprisingly, TOR s role as a master regulator of growth and development includes coordinating cell expansion through changes to cell wall structure (Leiber et al., 2010). Consistent with this, comparative transcriptomes of rapamycin-treated BP12-2 and wild type identified a large number of differentially expressed genes

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involved in Arabidopsis cell wall biogenesis (Ren et al., 2012). Verification of the gene expression changes in BP12-2 lines supported the downregulation of genes encoding cell wall structural proteins, including extensins and expansins, in accordance with the reduced cell expansion, growth and lifespan phenotypes observed. In estradiol-inducible tor RNAi mutants, artificial microRNA tor lines and Lethal with Sec Thirteen8 mutants, transcriptome and metabolome analyses revealed broad changes in pathways modulating membrane and storage lipids, cell walls and sucrose synthesis (Moreau et al., 2012; Caldana et al., 2013; Xiong et al., 2013). Mutant analysis in Arabidopsis identified a role played by the LST8-1 component of the TOR complex in cell wall formation and vegetative growth (Moreau et al., 2012). Although a role for TOR in regulating components of the cytoskeleton has been discovered in yeast and animal systems and involves the rapamycin insensitive TORC2 RICTOR complex, a role for TOR in regulating cytoskeletal organization in plant cells has not been identified (Loewith et al., 2002; Sarbassov et al., 2004; Jacinto et al., 2004).

Metabolism plays a central role in the growth, development and performance of an organism throughout its life cycle. TOR has emerged as an indispensable player in the regulation of plant metabolism, mediating cross talk between multiple metabolic pathways (Moreau et al., 2012; Ren et al., 2012; Caldana et al., 2013; Dobrenel et al., 2013; Xiong et al., 2013; Shi et al., 2018; Salem et al., 2018). The role of metabolism in TOR mediated aging processes, although well-known in animals (Johnson et al., 2013; Weichhart, 2018) is in relatively early stages of study in plants. Metabolic and transcriptomic profiling of the Arabidopsis BP12-2 line identified a role for the TOR signaling pathway in plant lifespan regulation (Ren et al., 2012). Similar sustained increases in biomass and growth over prolonged periods were reported in photosynthetic algae Chlamydomonas reinhardtii (Jüppner et al., 2018), and support conservation among the metabolic pathways that function in photoautotrophic organisms.

Carbon metabolism is closely linked to the growth and lifespan of plants (van Doorn, 2008; Wingler et al., 2009) and is impacted dramatically by the manipulation of TOR expression (Ren et al., 2012; Caldana et al., 2013; Xiong et al., 2013; Dobrenel et al., 2013; Dong et al., 2015; Dobrenel et al., 2016a; Shi et al., 2018; Zhang et al., 2018). TOR and SnRK1 operate antagonistically at the centre of sugar/carbon metabolic regulation (Ghillebert et al., 2011; Cho et al., 2012; Xiong and Sheen, 2012; Xiong et al., 2013). TORC1 is activated by nutrient availability and inactivated by stresses that cause energy depletion in the cell (Xiong and Sheen, 2015; Dobrenel et al., 2016a; González and Hall, 2017; Saxton and Sabatini, 2017). Conversely, mutations in TORC1 members and inducible silencing of TOR in Arabidopsis lead to decreased photosynthesis, accumulation of soluble sugars, starch and amino acids (Deprost et al., 2007; Ren et al., 2012; Salem et al., 2018). Similarly, rapamycin treated rice cells showed transcriptional reprogramming of genes involved in primary and secondary metabolic pathways (De Vleesschauwer et al., 2017). Indeed, higher accumulation of storage compounds and induction of autophagy make raptor plants more tolerant to carbon starvation conditions. In addition to SnRK1, RPS6 could serve as a link between carbon catabolism and the TOR pathway in plants (Williams et al., 2003; Mahfouz et al., 2006).

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In addition to carbon metabolism, the TOR pathway senses and integrates signals from nitrogen, sulphur, and phosphorus to coordinate the synthesis of cellular building blocks, such as nucleotides, amino acids and membrane lipids (Xiong and Sheen, 2014; Dong et al., 2017; Zhang et al., 2018; Salem et al., 2018). Activation of TOR kinase results in the upregulation of genes involved in sulphur-assimilation, glutathione and nucleotide synthesis (Xiong et al., 2013). On the other hand, TORC1 inhibition significantly affects expression of nitrogen and amino acid metabolism pathway genes (Dong et al., 2015) and the accumulation of nitrate, ammonium, amino acids and storage compounds such as triacylglycerol in Arabidopsis (Caldana et al., 2013; Salem et al., 2018). Amino acids can directly activate TORC1 on the lysosome surface in animals and yeast (Kim et al., 2008; Zoncu et al., 2011; Bar-Peled et al., 2012; Cornu et al., 2013). Rather than direct activation by amino acids, sulfide reductase, a cysteine anabolism pathway precursor that catalyzes the entry point for sulphur into the amino acid backbone, activates the TOR pathway in Arabidopsis (Dong et al., 2017). These data strengthen the role of TOR signaling in the modulation of plant metabolism, which ultimately contribute to plant growth and lifespan potential (Salem et al., 2018).

TOR plays a pivotal role in a diverse array of plant processes that impact lifespan, yet, how TOR regulates lifespan at the molecular level remains poorly understood. Plant lifespan is partly determined by the coordinated arrest and/or terminal differentiation of all meristems, or global proliferative arrest (GPA) (Balanzà et al., 2018) at the end of the life cycle. A genetic pathway regulating GPA in Arabidopsis, FRUITFULL-APETALA2 (FUL/AP2), responds to age-dependent factors and acts in parallel to seed-derived signals. The ectopic expression of TOR and resulting changes to the expression of genes in this novel pathway could provide a new approach to characterize the molecular mechanism of lifespan determination by TOR. This hypothesis gains support from studies in white beech mushroom (basidiomycete Hypsizygus marmoreus), where transcriptome analysis suggested that nitrogen starvation might be one of the most important factors in promoting fruiting body maturation, and the TOR signaling pathway was associated with this process (Zhang et al., 2015).

Over the course of an orga is s life, parti ularl i lo g li i g spe ies su h as trees, there is pote tial for the actively dividing cells of meristematic zones to undergo imperfect mitosis and accumulate somatic mutations. Unlike animals, somatic mutations in plants can be inherited and provide a mechanism for increasing genetic diversity and the potential for long-lived species to gain an adaptive advantage. Although studies on long living tree species and the mechanisms enabling their lifespan achievements are limited, sequencing of an oak tree species (Quercus robur L.) capable of living several hundred years revealed high nucleotide diversity (as a measure of genetic diversity) and elevated deleterious mutation rates relative to over thirty other species (Plomion et al., 2018). This work demonstrates that significant genetic diversity exists in association with plant longevity. Although further work is required to clarify if somatic mutations contribute to oak longevity and adaptation, this work reveals the importance of tackling genetic analyses in such organisms.

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Examining the role of TOR in tree species in our search for mechanistic understanding of longevity will be of value in future studies.

4. Transitioning from vegetative growth to reproductive growth and senescence

The transitions into reproductively competent and senescent life phases are genetically regulated and influenced by hormones such as ethylene and gibberellins (Thomas, 2012). Following reproduction, many plants progressively transition directly into a final senescent stage of life, where photosynthesis is reduced and nutrients are remobilized (Thomas, 2012). Intriguing discoveries surrounding the function of TOR in flowering and senescence timing have been made using the overexpression of TOR and different combinations of TOR domains in the monocarpic species, Arabidopsis (Ren et al., 2011), however, there is still much uncertainty regarding the role of TOR in late stages of plant development. While pleiotropic developmental changes were observed in plants overexpressing full length TOR, or truncated versions of TOR with or without the kinase domain, phenotypes revealed the importance of TOR in coordinating the timing of flowering and senescence. Specifically, when TOR was overexpressed, plants showed early transition to flowering and onset of senescence (Figure 1B). Furthermore, in Arabidopsis mutants of TORC1 members, including lst8-1 and raptor1b, flowering onset was delayed and abnormal flower development was observed (Moreau et al., 2012; Salem et al., 2018). This is consistent with the assertion that compromising TOR signaling activity influences growth and the transition to flowering. Under conditionally induced silencing at bolting, strong TOR downregulation produced Arabidopsis plants with early yellowing and elevated soluble sugars, supporting early senescence and perturbed nutrient recycling (Deprost et al., 2007). While delayed senescence was observed in BP12-2 lines treated with rapamycin, early senescence in Arabidopsis plants with strong TOR downregulation suggests a balance in the level of TOR expression may be required to regulate the onset of senescence and nutrient remobilization (Deprost et al., 2007; Ren et al., 2012).

The developmental programs regulating meristem determinancy are fundamental in differentiating annual, biennial or perennial life spans, and monocarpic or polycarpic reproductive strategies (Melzer et al., 2008). The role played by TOR in reproductive cycle regulation, senescence and longevity has largely focused on monocarpic species, leaving TO‘ s role i go er i g multiple plant reproductive cycles and different lifespan strategies unknown. For perennial species that live for more than two years and are often polycarpic, the onset of flowering and senescence must additionally be timed with the seasons. The circadian clock plays a role in timing this seasonal response with reproductive onset (Brachi et al., 2010; Johansson and Köster, 2018). Through multiple feedback loops, the circadian clock is regulated at the transcriptional and post-transcriptional levels and has been proposed to control TOR activity (Khapre et al., 2014; Schepetilnikov and Ryabova, 2018). Based on evidence that TOR contributes to the regulation of flowering and senescence timing in monocarpic Arabidopsis and to light perception, it will be interesting to determine what role TOR plays in the meristem indeterminancy and flowering time of

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polycarpic species, whether this master regulator limits energy expenditure during reproductive phases to enable subsequent vegetative periods of growth, and if TOR coordinately regulates these processes with input from the circadian clock. Co sideri g TO‘ s role i regulati g e tr i to the reprodu ti e phase, examining TO‘ s contribution to the mechanistic regulation of meristem determinancy by known key regulators such as SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and FRUITFULL (FUL) will be a compelling path for future work (Melzer et al., 2008).

The established link between caloric intake and lifespan in a broad range of eukaryotes has also been demonstrated in photoautotrophic species, where plant caloric restriction can be modeled by reducing light intensity (Minina et al., 2013). In Arabidopsis, this light-based caloric restriction lengthened each stage of the life cycle, producing an overall lifespan extension of approximately 25%. Autophagy has been established as a key mechanistic link between caloric restriction and lifespan extension in heterotrophs (Rubinsztein, 2006; Madeo et al., 2010) and photoautotrophs (Minina et al., 2013), and may be a common degradative mechanism enabling longevity extension (Madeo et al., 2010). In accordance with the lifespan extension observed in calorie restrictive conditions, Arabidopsis autophagy-deficient mutants exhibit early senescence, with shortened duration spent in each stage of the life cycle and attenuated lifespan decreases under low light/calorie restrictive conditions (Thompson et al., 2005; Minina et al., 2013). While the role that autophagy plays in removing cellular damage has connected this process to lifespan extension, the mechanisms triggering plant autophagy are less clear (Liu and Bassham, 2012). TOR has been identified as a negative regulator of autophagy in photosynthetic organisms, where TOR inhibition by rapamycin treatment in Chlamydomonas reinhardtii, or TOR gene RNAi-based knock-down in Arabidopsis activated autophagy and growth reductions (Pérez-Pérez et al., 2010; Liu and Bassham, 2010). Further, TORs role as a negative regulator of autophagy was found to occur only under specific stressors, such as nutrient or osmotic stress, and was mediated by upstream auxin signaling (Pu et al., 2017). In yeast, negative regulation of autophagy occurs through TO‘C s direct phosphorylation of autophagy-related gene 13 (ATG13), which prevents autophagy, while rapamycin treatment induces autophagy and extends life (Alvers et al., 2009; Kamada et al., 2010). In Arabidopsis, double mutation of the two ATG13 homologs produces autophagy defects, however phosphorylation of ATG13 or other autophagy-related gene product has not been demonstrated (Suttangkakul et al., 2011). Additionally, Arabidopsis SnRK1 acts as an upstream regulator of autophagy by phosphorylating ATG1 under abiotic stress conditions (Soto-Burgos and Bassham, 2017). Thus, despite breakthrough discoveries linking TOR to autophagy in plants, the upstream regulation of TOR and the downstream phosphorylation of autophagy-related gene(s) by TOR are not well understood (Yoshimoto and Ohsumi, 2018).

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Advancing the u dersta di g TO‘ s fu tio holds promise for lifespan research and extension efforts (Kaeberlein and Kennedy, 2008). While severe reduction of TOR produces dire consequences for plant life, as in tor null mutants that exhibit early embryo arrest, less severe reductions in TOR expression slow plant growth and appear to enable lifespan extension. TOR signaling orchestrates a far-reaching network of effectors that influence all stages of plant life, but also exhibits unique regulatory features from other eukaryotes. Aided by the development of high-throughput next generation sequencing, studies employing global transcriptome analyses of TOR have facilitated the systematic investigation of the TOR regulatory network, particularly in response to TOR repression, under different biotic or abiotic conditions, and at critical junctures in the plant life cycle. Altogether, a central role is emerging for TOR signaling and its influence on the rate of development, the transition between growth phases, aging and ultimately, the span of a plants life.

The vast array of responses driven by TOR highlight the importance of integrating this master regulator for improved plant productivity and performance. Targeting TOR to improve crop traits has been applied successfully in genetically engineered rice, where broadly expressed Arabidopsis full-length TOR increased plant biomass and photosynthetic efficiency under drought conditions (Bakshi et al., 2017). Similarly, a wheat line with elevated levels of TOR expression exhibited leaf epidermal cell expansion and enlarged architecture (Shin et al., 2015). The manipulation of crop lifespan to reduce the duration of the growing season, without affecting yields, is an agricultural priority, particularly in regions with short growing seasons. Conversely, many horticultural and grocery produce suppliers desire plant lifespan extension to optimize shelf life. TOR’s role in regulating the vegetative to reproductive transition could also be targeted for a number of applications. For example, biomass required for biofuel production is based on vegetative growth yields and could be improved by hindering entry into the reproductive phase. Manipulating TOR to affect the balance of anabolic and catabolic processes could be used to favor the accumulation of desirable plant products, such as seed oils or proteins. Although our understanding of TOR’s role in plant longevity is in its infancy, expanding this knowledge to target TOR in plant lifespan engineering has the potential to create transformative improvements in agriculture crops to support global food security.

Acknowledgements

This work was supported by the National Research Council of Canada Aquatic and Crop Resource Development Research Centre (ACRD manuscript #56422) in partnership with the Government of Saskatchewan Agriculture Development Fund. Conceptual framework was devised by TDQ and RD. Written contributions and advice were provided by TDQ, PG, PP, DX, and MR.

Conclusions and perspectives

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Figure 1. Plant lifespan varies significantly and is regulated, in part, by TOR. A) Long-lived coast redwoods, Sequoia sempervirens. B) A composite image showing the influence of TOR expression on the growth and development of Arabidopsis thaliana. Representative plants for conditional TOR suppression derived from the FKBP12-2 line (left) and WT (middle) control were grown in jars and treated with rapamycin. The TOR overexpressing plant (right) is derived from the 35S:TOR OE1 line described in Ren et al., 2011 The three images in this panel represent plants of comparable age at 30 days after germination. The image in panel A was taken by TDQ.

Figure 2. A schematic representation of TOR signaling in the circle of plant life. Highlighted in this figure are elements of TOR signaling, selected for their relevance to discussions in this review. TOR kinase research in plant life has revealed a complex network of regulatory controls governing embryogenesis, heterotrophic seedling germination, photoautotrophic seedling establishment, vegetative growth, reproduction and senescence (see respective illustrations) and the transitions between many of these stages, including from heterotrophic growth in the dark (represented by a purple background) to photoautotrophic growth in the light (represented by a yellow background). TOR regulates a broad range of anabolic and catabolic processes, including but not limited to polysome loading, ribosome biogenesis, protein translation, cell cycle, meristem activation, chloroplast biogenesis, photosynthesis, carbon/sugar metabolism and autophagy. Links between regulation by TOR and additional processes, including modulation of the cytoskeleton, cell wall structure and expansion, somatic mutations, epigenetic modifications, circadian clock and meristem arrest have also been supported or hypothesized. In Arabidopsis, TORC1, composed of TOR, Raptor and LST8, phosphorylates several effectors (as indicated by arrows) including, but not restricted to S6K1, S6K2, E2Fa, TAP46, while phosphorylation of WUS, GA-DELLA, FUL/AP2 or other mediators remains to be studied. Repression activity of TORC1 has also been identified for select effectors, including ATGs, and the antagonistic regulation by SnRK1. For the purposes of illustration, some effectors and TOR-regulated pathways are shown in one life stage, but are relevant throughout the plant life cycle. ATGs, autophagy-related genes; BIN2, brassinosteroid insensitive 2; E2Fa, transcription factor involved in eukaryotic cell cycle regulation; eIF3, eukaryotic initiation factor 3; FUL/AP2, FRUITFULL-APETALA2; GA-DELLA, gibberellin-DELLA; LST8, lethal with sec thirteen 8; PP2A, protein phosphatase 2A; Raptor, regulatory-associated protein of mTOR; RPS6, ribosome protein 6; S6K1/2, small ribosome protein 6 kinase 1/2; SnRK1, sucrose non-fermenting related kinase 1; TAP46, regulatory subunit of PP2A; TOR, target of rapamycin kinase; TORC1, TOR complex 1; WUS, WUSCHEL.

Figure Legends Downloaded from ht tps: //academic. oup. com/ jxb/ advance-art icle-abst ract /doi/ 10. 1093/ jxb/ erz125/ 5418542 by Nat ional Research Council Canada user on 09 A pril 2019

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