Dynamic Nutrient Signaling Networks in Plants.

Nutrients are vital to life through intertwined sensing, signaling, and metabolic processes. Emerging research focuses on how distinct nutrient signaling networks integrate and coordinate gene expression, metabolism, growth, and survival. We review the multifaceted roles of sugars, nitrate, and phosphate as essential plant nutrients in controlling complex molecular and cellular mechanisms of dynamic signaling networks. Key advances in central sugar and energy signaling mechanisms mediated by the evolutionarily conserved master regulators HEXOKINASE1 (HXK1), TARGET OF RAPAMYCIN (TOR), and SNF1-RELATED PROTEIN KINASE1 (SNRK1) are discussed. Significant progress in primary nitrate sensing, calcium signaling, transcriptome analysis, and root-shoot communication to shape plant biomass and architecture are elaborated. Discoveries on intracellular and extracellular phosphate signaling and the intimate connections with nitrate and sugar signaling are examined. This review highlights the dynamic nutrient, energy, growth, and stress signaling networks that orchestrate systemwide transcriptional, translational, and metabolic reprogramming, modulate growth and developmental programs, and respond to environmental cues.

eIF4F complex dynamics are important for the activation of the integrated stress response.

In response to stress, eukaryotes activate the integrated stress response (ISR) via phosphorylation of eIF2α to promote the translation of pro-survival effector genes, such as GCN4 in yeast. Complementing the ISR is the target of rapamycin (TOR) pathway, which regulates eIF4E function. Here, we probe translational control in the absence of eIF4E in Saccharomyces cerevisiae. Intriguingly, we find that loss of eIF4E leads to de-repression of GCN4 translation. In addition, we find that de-repression of GCN4 translation is accompanied by neither eIF2α phosphorylation nor reduction in initiator ternary complex (TC). Our data suggest that when eIF4E levels are depleted, GCN4 translation is de-repressed via a unique mechanism that may involve faster scanning by the small ribosome subunit due to increased local concentration of eIF4A. Overall, our findings suggest that relative levels of eIF4F components are key to ribosome dynamics and may play important roles in translational control of gene expression.

Functional Metabolomics Describes the Yeast Biosynthetic Regulome.

Genome-metabolism interactions enable cell growth. To probe the extent of these interactions and delineate their functional contributions, we quantified the Saccharomyces amino acid metabolome and its response to systematic gene deletion. Over one-third of coding genes, in particular those important for chromatin dynamics, translation, and transport, contribute to biosynthetic metabolism. Specific amino acid signatures characterize genes of similar function. This enabled us to exploit functional metabolomics to connect metabolic regulators to their effectors, as exemplified by TORC1, whose inhibition in exponentially growing cells is shown to match an interruption in endomembrane transport. Providing orthogonal information compared to physical and genetic interaction networks, metabolomic signatures cluster more than half of the so far uncharacterized yeast genes and provide functional annotation for them. A major part of coding genes is therefore participating in gene-metabolism interactions that expose the metabolism regulatory network and enable access to an underexplored space in gene function.

TOR regulates variability of protein synthesis rates.

Cellular processes are subject to inherent variability, but the extent to which cells can regulate this variability has received little investigation. Here, we explore the characteristics of the rate of cellular protein synthesis in single cells of the eukaryote fission yeast. Strikingly, this rate is highly variable despite protein synthesis being dependent on hundreds of reactions which might be expected to average out at the overall cellular level. The rate is variable over short time scales, and exhibits homoeostatic behaviour at the population level. Cells can regulate the level of variability through processes involving the TOR pathway, suggesting there is an optimal level of variability conferring a selective advantage. While this could be an example of bet-hedging, but we propose an alternative explanation: regulated 'loose' control of complex processes of overall cellular metabolism such as protein synthesis, may lead to this variability. This could ensure cells are fluid in control and agile in response to changing conditions, and may constitute a novel organisational principle of complex metabolic cellular systems.

AMPK, a Regulator of Metabolism and Autophagy, Is Activated by Lysosomal Damage via a Novel Galectin-Directed Ubiquitin Signal Transduction System.

AMPK is a central regulator of metabolism and autophagy. Here we show how lysosomal damage activates AMPK. This occurs via a hitherto unrecognized signal transduction system whereby cytoplasmic sentinel lectins detect membrane damage leading to ubiquitination responses. Absence of Galectin 9 (Gal9) or loss of its capacity to recognize lumenal glycans exposed during lysosomal membrane damage abrogate such ubiquitination responses. Proteomic analyses with APEX2-Gal9 have revealed global changes within the Gal9 interactome during lysosomal damage. Gal9 association with lysosomal glycoproteins increases whereas interactions with a newly identified Gal9 partner, deubiquitinase USP9X, diminishes upon lysosomal injury. In response to damage, Gal9 displaces USP9X from complexes with TAK1 and promotes K63 ubiquitination of TAK1 thus activating AMPK on damaged lysosomes. This triggers autophagy and contributes to autophagic control of membrane-damaging microbe Mycobacterium tuberculosis. Thus, galectin and ubiquitin systems converge to activate AMPK and autophagy during endomembrane homeostasis.

TOR acts as a metabolic gatekeeper for auxin-dependent lateral root initiation in Arabidopsis thaliana.

Plant organogenesis requires matching the available metabolic resources to developmental programs. In Arabidopsis, the root system is determined by primary root-derived lateral roots (LRs), and adventitious roots (ARs) formed from non-root organs. Lateral root formation entails the auxin-dependent activation of transcription factors ARF7, ARF19, and LBD16. Adventitious root formation relies on LBD16 activation by auxin and WOX11. The allocation of shoot-derived sugar to the roots influences branching, but how its availability is sensed for LRs formation remains unknown. We combine metabolic profiling with cell-specific interference to show that LRs switch to glycolysis and consume carbohydrates. The target-of-rapamycin (TOR) kinase is activated in the lateral root domain. Interfering with TOR kinase blocks LR initiation while promoting AR formation. The target-of-rapamycin inhibition marginally affects the auxin-induced transcriptional response of the pericycle but attenuates the translation of ARF19, ARF7, and LBD16. TOR inhibition induces WOX11 transcription in these cells, yet no root branching occurs as TOR controls LBD16 translation. TOR is a central gatekeeper for root branching that integrates local auxin-dependent pathways with systemic metabolic signals, modulating the translation of auxin-induced genes.

TOR-mediated Ypt1 phosphorylation regulates autophagy initiation complex assembly.

The regulation of autophagy initiation is a key step in autophagosome biogenesis. However, our understanding of the molecular mechanisms underlying the stepwise assembly of ATG proteins during this process remains incomplete. The Rab GTPase Ypt1/Rab1 is recognized as an essential autophagy regulator. Here, we identify Atg23 and Atg17 as binding partners of Ypt1, with their direct interaction proving crucial for the stepwise assembly of autophagy initiation complexes. Disruption of Ypt1-Atg23 binding results in significantly reduced Atg9 interactions with Atg11, Atg13, and Atg17, thus preventing the recruitment of Atg9 vesicles to the phagophore assembly site (PAS). Likewise, Ypt1-Atg17 binding contributes to the PAS recruitment of Ypt1 and Atg1. Importantly, we found that Ypt1 is phosphorylated by TOR at the Ser174 residue. Converting this residue to alanine blocks Ypt1 phosphorylation by TOR and enhances autophagy. Conversely, the Ypt1S174D phosphorylation mimic impairs both PAS recruitment and activation of Atg1, thus inhibiting subsequent autophagy. Thus, we propose TOR-mediated Ypt1 as a multifunctional assembly factor that controls autophagy initiation via its regulation of the stepwise assembly of ATG proteins.

Nitrogen availability is important for preventing catastrophic mitosis in fission yeast.

Mitosis is a crucial stage in the cell cycle, controlled by a vast network of regulators responding to multiple internal and external factors. The fission yeast Schizosaccharomyces pombe demonstrates catastrophic mitotic phenotypes due to mutations or drug treatments. One of the factors provoking catastrophic mitosis is a disturbed lipid metabolism, resulting from, for example, mutations in the acetyl-CoA/biotin carboxylase (cut6), fatty acid synthase (fas2, also known as lsd1) or transcriptional regulator of lipid metabolism (cbf11) genes, as well as treatment with inhibitors of fatty acid synthesis. It has been previously shown that mitotic fidelity in lipid metabolism mutants can be partially rescued by ammonium chloride supplementation. In this study, we demonstrate that mitotic fidelity can be improved by multiple nitrogen sources. Moreover, this improvement is not limited to lipid metabolism disturbances but also applies to a number of unrelated mitotic mutants. Interestingly, the partial rescue is not achieved by restoring the lipid metabolism state, but rather indirectly. Our results highlight a novel role for nitrogen availability in mitotic fidelity.

GRD-1/PTR-11, the C. elegans hedgehog/patched-like morphogen-receptor pair, modulates developmental rate.

Both hedgehog (Hh) and target of rapamycin complex 2 (TORC2) are central, evolutionarily conserved signaling pathways that regulate development and metabolism. In C. elegans, loss of the essential TORC2 component RICTOR (rict-1) causes delayed development, shortened lifespan, reduced brood, small size and increased fat. Here, we report that knockdown of both the hedgehog-related morphogen grd-1 and its patched-related receptor ptr-11 rescues delayed development in TORC2 loss-of-function mutants, and grd-1 and ptr-11 overexpression delays wild-type development to a similar level to that in TORC2 loss-of-function animals. These findings potentially indicate an unexpected role for grd-1 and ptr-11 in slowing developmental rate downstream of a nutrient-sensing pathway. Furthermore, we implicate the chronic stress transcription factor pqm-1 as a key transcriptional effector in this slowing of whole-organism growth by grd-1 and ptr-11. We propose that TORC2, grd-1 and ptr-11 may act linearly or converge on pqm-1 to delay organismal development.

Parallels between oncogene-driven cardiac hyperplasia and heart regeneration in zebrafish.

The human heart is poorly regenerative and cardiac tumors are extremely rare. Whether the adult zebrafish myocardium is responsive to oncogene overexpression and how this condition affects its intrinsic regenerative capacity remains unknown. Here, we have established a strategy of inducible and reversible expression of HRASG12V in zebrafish cardiomyocytes. This approach stimulated a hyperplastic cardiac enlargement within 16 days. The phenotype was suppressed by rapamycin-mediated inhibition of TOR signaling. As TOR signaling is also required for heart restoration after cryoinjury, we compared transcriptomes of hyperplastic and regenerating ventricles. Both conditions were associated with upregulation of cardiomyocyte dedifferentiation and proliferation factors, as well as with similar microenvironmental responses, such as deposition of nonfibrillar Collagen XII and recruitment of immune cells. Among the differentially expressed genes, many proteasome and cell-cycle regulators were upregulated only in oncogene-expressing hearts. Preconditioning of the heart with short-term oncogene expression accelerated cardiac regeneration after cryoinjury, revealing a beneficial synergism between both programs. Identification of the molecular bases underlying the interplay between detrimental hyperplasia and advantageous regeneration provides new insights into cardiac plasticity in adult zebrafish.

mTORC1 Signaling in Brain Endothelial Progenitors Contributes to CCM Pathogenesis.

BACKGROUND: Cerebral vascular malformations (CCMs) are primarily found within the brain, where they result in increased risk for stroke, seizures, and focal neurological deficits. The unique feature of the brain vasculature is the blood-brain barrier formed by the brain neurovascular unit. Recent studies suggest that loss of CCM genes causes disruptions of blood-brain barrier integrity as the inciting events for CCM development. CCM lesions are proposed to be initially derived from a single clonal expansion of a subset of angiogenic venous capillary endothelial cells (ECs) and respective resident endothelial progenitor cells (EPCs). However, the critical signaling events in the subclass of brain ECs/EPCs for CCM lesion initiation and progression are unclear. METHODS: Brain EC-specific CCM3-deficient (Pdcd10BECKO) mice were generated by crossing Pdcd10fl/fl mice with Mfsd2a-CreERT2 mice. Single-cell RNA-sequencing analyses were performed by the chromium single-cell platform (10× genomics). Cell clusters were annotated into EC subtypes based on visual inspection and GO analyses. Cerebral vessels were visualized by 2-photon in vivo imaging and tissue immunofluorescence analyses. Regulation of mTOR (mechanistic target of rapamycin) signaling by CCM3 and Cav1 (caveolin-1) was performed by cell biology and biochemical approaches. RESULTS: Single-cell RNA-sequencing analyses from P10 Pdcd10BECKO mice harboring visible CCM lesions identified upregulated CCM lesion signature and mitotic EC clusters but decreased blood-brain barrier-associated EC clusters. However, a unique EPC cluster with high expression levels of stem cell markers enriched with mTOR signaling was identified from early stages of the P6 Pdcd10BECKO brain. Indeed, mTOR signaling was upregulated in both mouse and human CCM lesions. Genetic deficiency of Raptor (regulatory-associated protein of mTOR), but not of Rictor (rapamycin-insensitive companion of mTOR), prevented CCM lesion formation in the Pdcd10BECKO model. Importantly, the mTORC1 (mTOR complex 1) pharmacological inhibitor rapamycin suppressed EPC proliferation and ameliorated CCM pathogenesis in Pdcd10BECKO mice. Mechanistic studies suggested that Cav1/caveolae increased in CCM3-depleted EPC-mediated intracellular trafficking and complex formation of the mTORC1 signaling proteins. CONCLUSIONS: CCM3 is critical for maintaining blood-brain barrier integrity and CCM3 loss-induced mTORC1 signaling in brain EPCs initiates and facilitates CCM pathogenesis.

Target of rapamycin signaling couples energy to oxygen sensing to modulate hypoxic gene expression in Arabidopsis.

Plants respond to oxygen deprivation by activating the expression of a set of hypoxia-responsive genes (HRGs). The master regulator of this process is a small group of transcription factors belonging to group VII of the ethylene response factors (ERF-VIIs). ERF-VIIs are highly unstable under aerobic conditions due to the continuous oxidation of their characteristic Cys residue at the N terminus by plant cysteine oxidases (PCOs). Under hypoxia, PCOs are inactive and the ERF-VIIs activate transcription of the HRGs required for surviving hypoxia. However, if the plant exposed to hypoxia has limited sugar reserves, the activity of ERF-VIIs is severely dampened. This suggests that oxygen sensing by PCO/ERF-VII is fine-tuned by another sensing pathway, related to sugar or energy availability. Here, we show that oxygen sensing by PCO/ERF-VII is controlled by the energy sensor target of rapamycin (TOR). Inhibition of TOR by genetic or pharmacological approaches leads to a much lower induction of HRGs. We show that two serine residues at the C terminus of RAP2.12, a major ERF-VII, are phosphorylated by TOR and are needed for TOR-dependent activation of transcriptional activity of RAP2.12. Our results demonstrate that oxygen and energy sensing converge in plants to ensure an appropriate transcription of genes, which is essential for surviving hypoxia. When carbohydrate metabolism is inefficient in producing ATP because of hypoxia, the lower ATP content reduces TOR activity, thus attenuating the efficiency of induction of HRGs by the ERF-VIIs. This homeostatic control of the hypoxia-response is required for the plant to survive submergence.

TORC pathway intersects with a calcium sensor kinase network to regulate potassium sensing in Arabidopsis.

Potassium (K) is an essential macronutrient for plant growth, and its availability in the soil varies widely, requiring plants to respond and adapt to the changing K nutrient status. We show here that plant growth rate is closely correlated with K status in the medium, and this K-dependent growth is mediated by the highly conserved nutrient sensor, target of rapamycin (TOR). Further study connected the TOR complex (TORC) pathway with a low-K response signaling network consisting of calcineurin B-like proteins (CBL) and CBL-interacting kinases (CIPK). Under high K conditions, TORC is rapidly activated and shut down the CBL-CIPK low-K response pathway through regulatory-associated protein of TOR (RAPTOR)-CIPK interaction. In contrast, low-K status activates CBL-CIPK modules that in turn inhibit TORC by phosphorylating RAPTOR, leading to dissociation and thus inactivation of the TORC. The reciprocal regulation of the TORC and CBL-CIPK modules orchestrates plant response and adaptation to K nutrient status in the environment.

Amino acid-dependent regulation of insulin-like peptide signaling is mediated by TOR and GATA factors in the disease vector mosquito Aedes aegypti.

Aedes aegypti female mosquitoes require vertebrate blood for their egg production and consequently they become vectors of devastating human diseases. Amino acids (AAs) and nutrients originating from a blood meal activate vitellogenesis and fuel embryo development of anautogenous mosquitoes. Insulin-like peptides (ILPs) are indispensable in reproducing female mosquitoes, regulating glycogen and lipid metabolism, and other essential functions. However, how ILPs coordinate their action in response to the AA influx in mosquito reproduction was unknown. We report here that the AA/Target of Rapamycin (TOR) signaling pathway regulates ILPs through GATA transcription factors (TFs). AA infusion combined with RNA-interference TOR silencing of revealed their differential action on ILPs, elevating circulating levels of several ILPs but inhibiting others, in the female mosquito. Experiments involving isoform-specific CRISPR-Cas9 genomic editing and chromatin immunoprecipitation assays showed that the expression of ilp4, ilp6, and ilp7 genes was inhibited by the GATA repressor (GATAr) isoform in response to low AA-TOR signaling, while the expression of ilp1, ilp2, ilp3, ilp5, and ilp8 genes was activated by the GATA activator isoform after a blood meal in response to the increased AA-TOR signaling. FoxO, a downstream TF in the insulin pathway, was involved in the TOR-GATAr-mediated repression of ilp4, ilp6, and ilp7 genes. This work uncovered how AA/TOR signaling controls the ILP pathway in modulation of metabolic requirements of reproducing female mosquitoes.

Trehalose-6-phosphate signaling regulates lateral root formation in Arabidopsis thaliana.

Plant roots explore the soil for water and nutrients, thereby determining plant fitness and agricultural yield, as well as determining ground substructure, water levels, and global carbon sequestration. The colonization of the soil requires investment of carbon and energy, but how sugar and energy signaling are integrated with root branching is unknown. Here, we show through combined genetic and chemical modulation of signaling pathways that the sugar small-molecule signal, trehalose-6-phosphate (T6P) regulates root branching through master kinases SNF1-related kinase-1 (SnRK1) and Target of Rapamycin (TOR) and with the involvement of the plant hormone auxin. Increase of T6P levels both via genetic targeting in lateral root (LR) founder cells and through light-activated release of the presignaling T6P-precursor reveals that T6P increases root branching through coordinated inhibition of SnRK1 and activation of TOR. Auxin, the master regulator of LR formation, impacts this T6P function by transcriptionally down-regulating the T6P-degrader trehalose phosphate phosphatase B in LR cells. Our results reveal a regulatory energy-balance network for LR formation that links the 'sugar signal' T6P to both SnRK1 and TOR downstream of auxin.

Mechanical stress regulates insulin sensitivity through integrin-dependent control of insulin receptor localization.

Insulin resistance, the failure to activate insulin signaling in the presence of ligand, leads to metabolic diseases, including type 2 diabetes. Physical activity and mechanical stress have been shown to protect against insulin resistance, but the molecular mechanisms remain unclear. Here, we address this relationship in the Drosophila larval fat body, an insulin-sensitive organ analogous to vertebrate adipose tissue and livers. We found that insulin signaling in Drosophila fat body cells is abolished in the absence of physical activity and mechanical stress even when excess insulin is present. Physical movement is required for insulin sensitivity in both intact larvae and fat bodies cultured ex vivo. Interestingly, the insulin receptor and other downstream components are recruited to the plasma membrane in response to mechanical stress, and this membrane localization is rapidly lost upon disruption of larval or tissue movement. Sensing of mechanical stimuli is mediated in part by integrins, whose activation is necessary and sufficient for mechanical stress-dependent insulin signaling. Insulin resistance develops naturally during the transition from the active larval stage to the immotile pupal stage, suggesting that regulation of insulin sensitivity by mechanical stress may help coordinate developmental programming with metabolism.

TORC2 is required for the accumulation of γH2A in response to DNA damage.

TOR protein kinases serve as the catalytic subunit of the TORC1 and TORC2 complexes, which regulate cellular growth, proliferation, and survival. In the fission yeast, Schizosaccharomyces pombe, cells lacking TORC2 or its downstream kinase Gad8 (AKT or SGK1 in human cells) exhibit sensitivity to a wide range of stress conditions, including DNA damage stress. One of the first responses to DNA damage is the phosphorylation of C-terminal serine residues within histone H2AX in human cells (γH2AX), or histone H2A in yeast cells (γH2A). The kinases responsible for γH2A in S. pombe are the two DNA damage checkpoint kinases Rad3 and Tel1 (ATR and ATM, respectively, in human cells). Here we report that TORC2-Gad8 signaling is required for accumulation of γH2A in response to DNA damage and during quiescence. Using the TOR-specific inhibitor, Torin1, we demonstrate that the effect of TORC2 on γH2A in response to DNA damage is immediate, rather than adaptive. The lack of γH2A is restored by deletion mutations of transcription and chromatin modification factors, including loss of components of Paf1C, SAGA, Mediator, and the bromo-domain proteins Bdf1/Bdf2. Thus, we suggest that TORC2-Gad8 may affect the accumulation of γH2A by regulating chromatin structure and function.

A drug-resistant mutation in plant target of rapamycin validates the specificity of ATP-competitive TOR inhibitors in vivo.

Kinases are major components of cellular signaling pathways, regulating key cellular activities through phosphorylation. Kinase inhibitors are efficient tools for studying kinase targets and functions, however assessing their kinase specificity in vivo is essential. The identification of resistant kinase mutants has been proposed to be the most convincing approach to achieve this goal. Here, we address this issue in plants via a pharmacogenetic screen for mutants resistant to the ATP-competitive TOR inhibitor AZD-8055. The eukaryotic TOR (Target of Rapamycin) kinase is emerging as a major hub controlling growth responses in plants largely thanks to the use of ATP-competitive inhibitors. We identified a dominant mutation in the DFG motif of the Arabidopsis TOR kinase domain that leads to very strong resistance to AZD-8055. This resistance was characterized by measuring root growth, photosystem II (PSII) activity in leaves and phosphorylation of YAK1 (Yet Another Kinase 1) and RPS6 (Ribosomal protein S6), a direct and an indirect target of TOR respectively. Using other ATP-competitive TOR inhibitors, we also show that the dominant mutation is particularly efficient for resistance to drugs structurally related to AZD-8055. Altogether, this proof-of-concept study demonstrates that a pharmacogenetic screen in Arabidopsis can be used to successfully identify the target of a kinase inhibitor in vivo and therefore to demonstrate inhibitor specificity. Thanks to the conservation of kinase families in eukaryotes, and the possibility of creating amino acid substitutions by genome editing, this work has great potential for extending studies on the evolution of signaling pathways in eukaryotes.

The TOR-dependent phosphoproteome and regulation of cellular protein synthesis.

Cell growth is orchestrated by a number of interlinking cellular processes. Components of the TOR pathway have been proposed as potential regulators of cell growth, but little is known about their immediate effects on protein synthesis in response to TOR-dependent growth inhibition. Here, we present a resource providing an in-depth characterisation of Schizosaccharomyces pombe phosphoproteome in relation to changes observed in global cellular protein synthesis upon TOR inhibition. We find that after TOR inhibition, the rate of protein synthesis is rapidly reduced and that notable phosphorylation changes are observed in proteins involved in a range of cellular processes. We show that this reduction in protein synthesis rates upon TOR inhibition is not dependent on S6K activity, but is partially dependent on the S. pombe homologue of eIF4G, Tif471. Our study demonstrates the impact of TOR-dependent phospho-regulation on the rate of protein synthesis and establishes a foundational resource for further investigation of additional TOR-regulated targets both in fission yeast and other eukaryotes.

Cellular responses to compound stress induced by atmospheric-pressure plasma in fission yeast.

The stress response is one of the most fundamental cellular processes. Although the molecular mechanisms underlying responses to a single stressor have been extensively studied, cellular responses to multiple stresses remain largely unknown. Here, we characterized fission yeast cellular responses to a novel stress inducer, non-thermal atmospheric-pressure plasma. Plasma irradiation generates ultraviolet radiation, electromagnetic fields and a variety of chemically reactive species simultaneously, and thus can impose multiple stresses on cells. We applied direct plasma irradiation to fission yeast and showed that strong plasma irradiation inhibited fission yeast growth. We demonstrated that mutants lacking sep1 and ace2, both of which encode transcription factors required for proper cell separation, were resistant to plasma irradiation. Sep1-target transcripts were downregulated by mild plasma irradiation. We also demonstrated that plasma irradiation inhibited the target of rapamycin kinase complex 1 (TORC1). These observations indicate that two pathways, namely the Sep1-Ace2 cell separation pathway and TORC1 pathway, operate when fission yeast cope with multiple stresses induced by plasma irradiation.