Diverse nitrogen signals activate convergent ROP2-TOR signaling in Arabidopsis

•Nitrogen is essential for TOR-promoted true leaf growth•NO3?, NH4+, and Gln function as separate upstream signals to activate TOR•AAs generated from plant-specific pathways have the highest potency TOR•NO3?, all stimulate ROP2 TOR The evolutionarily conserved target-of-rapamycin (TOR) kinase coordinates cellular organismal growth in eukaryotes. Amino acids (AAs) are key mammalian activation, but how nitrogen-related nutrients regulate signaling plants poorly understood. Here, we discovered that, independent of nitrogen assimilation, nitrate ammonium primary Arabidopsis primordium. We further identified that a total 15 proteinogenic AAs also able TOR, first plant specific assimilation (glutamine), sulfur (cysteine), glycolate cycle (glycine), exhibit potency. Interestingly, nitrate, ammonium, glutamine small GTPase Rho-related protein 2 (ROP2), constitutively active restores activation under nitrogen-starvation conditions. Our findings suggest evolutionary adaptations nitrogen-TOR pathway occurred lineages, can integrate diverse hormone activation. Plants assimilate inorganic (nitrate ammonium) environment into organic compounds, such amino (AAs). Beyond acting macronutrients structural components macromolecules, these nitrogen-containing molecules could act orchestrate gene expression, control root architecture, seed germination, developmental phase transitions, flowering (Gaudinier et al., 2018Gaudinier A. Rodriguez-Medina J. Zhang L. Olson Liseron-Monfils C. Bågman A.M. Foret Abbitt S. Tang M. Li B. al.Transcriptional regulation nitrogen-associated metabolism growth.Nature. 2018; 563: 259-264Crossref PubMed Scopus (98) Google Scholar; 2016Li P. Zhuang Z. Cai H. Cheng Soomro A.A. Liu Gu R. Mi G. Yuan Chen F. Use genotype-environment interactions elucidate pattern maize plasticity deficiency.J. Integr. Plant Biol. 2016; 58: 242-253Crossref (25) Wang 2018Wang Y.Y. Y.H. K.E. Tsay Y.F. Nitrate transport, signaling, use efficiency.Annu. Rev. 69: 85-122Crossref (202) Xuan 2017Xuan W. Beeckman T. Xu nutrition: sensing signaling.Curr. Opin. 2017; 39: 57-65Crossref (118) 2020Zhang Hu Chu Towards understanding hierarchical signalling network plants.Curr. 2020; 55: 60-65Crossref (24) Scholar). Recent breakthrough studies implicated nitrate-transporter-1.1 (NRT1.1, called CHL1) transporter 1.1 (AMT1.1) potential sensors respectively (Ho 2009Ho C.H. Lin S.H. H.C. CHL1 functions sensor plants.Cell. 2009; 138: 1184-1194Abstract Full Text PDF (804) Lanquar 2009Lanquar V. Loqué D. Hörmann Bohner Engelsberger W.R. Lalonde Schulze W.X. von Wirén N. Frommer W.B. Feedback inhibition uptake by phospho-dependent allosteric mechanism Arabidopsis.Plant Cell. 21: 3610-3622Crossref (132) Scholar), Ca2+-sensor (CPK)-signaling transcription factors mediating response (Armijo Gutiérrez, 2017Armijo Gutiérrez R.A. Emerging players pathway.Mol. Plant. 10: 1019-1022Abstract (12) Guan 2017Guan Ripoll J.J. Vuong Bailey-Steinitz L.J. Ye Crawford N.M. Interacting TCP NLP responses availability.Proc. Natl. Acad. Sci. USA. 114: 2419-2424Crossref (113) Krouk, 2017Krouk signalling: calcium bridges gap.Nat. Plants. 3: 17095Crossref (22) Lam 2006Lam H.-M. Chiao Y.A. M.-W. Yung Y.-K. Ji Putative systems higher plants.J. Integrative 2006; 48: 873-888Crossref (17) 2017Liu K.H. Niu Y. Konishi Wu Du Sun Chung Boudsocq McCormack Maekawa al.Discovery nitrate–CPK–NLP central nutrient–growth networks.Nature. 545: 311-316Crossref (230) Marchive 2013Marchive Roudier Castaings Bréhaut Blondet E. Colot Meyer Krapp Nuclear retention factor NLP7 orchestrates early plants.Nat. Commun. 2013; 4: 1713Crossref (275) Para 2014Para Marshall-Colón Varala K. Francoeur N.J. Moran T.M. Edwards M.B. Hackley Bargmann B.O.R. Birnbaum K.D. al.Hit-and-run transcriptional bZIP1 mediates rapid nutrient Arabidopsis.Proc. 2014; 111: 10371-10376Crossref (105) Qin 2020Qin D.B. M.Y. Zhu X.D. L.M. W.H. CALCIUM-DEPENDENT PROTEIN KINASE 32-mediated phosphorylation transport activity AMT1;1 roots.J. Exp. Bot. 71: 5087-5097Crossref (6) Rubin 2009Rubin Tohge Matsuda Saito Scheible Members LBD family repress anthocyanin synthesis affect additional 3567-3584Crossref (369) 2016Xu Zhao Lei NRG2 interacts with regulates regulators.Plant 28: 485-504Crossref (96) However, little known about AA plants, mainly hindered innate capacity cells produce themselves via nitrogen-assimilation complexity biosynthesis metabolisms. In mammals yeasts, pathway, which acts regulator integrating status promote cell proliferation (Fu 2020Fu Xiong Target rapamycin stress responses.Plant Physiol. 182: 1613-1623Crossref Sabatini, 2020Liu G.Y. Sabatini D.M. mTOR at nexus nutrition, growth, ageing disease.Nat. Mol. Cell 183-203Crossref (523) Strikingly, despite distinct body plans, strategies, survival behaviors between animals, coordinator networks has been (Brunkard, 2020Brunkard J.O. Exaptive evolution target multicellular eukaryotes.Dev. 54: 142-155Abstract (19) Fu autotrophic photosynthesis-derived glucose or auxin directly phosphorylates E2FA E2FB, activates S-phase genes meristems primordium, resulting supply new (Li 2017Li X. Differential shoot apexes.Proc. 2765-2770Crossref (133) 2013Xiong Hall Q. Xiang Sheen Glucose–TOR reprograms transcriptome meristems.Nature. 496: 181-186Crossref (405) Very recently, linked branched-chain AAs, e.g., Val, actin cytoskeleton organization characterizing loss-of-function mutant IPMS1, encodes committed enzyme Leu (Cao 2019Cao Kim S.J. Xing Schenck C.A. Jiang Last R.L. Brandizzi Homeostasis critical Arabidopsis.eLife. 2019; 8: e50747Crossref (30) Schaufelberger 2019Schaufelberger Galbier Herger de Brito Francisco Roffler Clement Diet Hörtensteiner Wicker Ringli Mutations ROL17/isopropylmalate synthase 1 locus alter acid content, modify network, suppress hair development lrx1.J. 70: 2313-2323Crossref (18) AA-derived metabolic influence respiratory rate mature leaves (O'Leary 2020O'Leary B.M. Oh G.G.K. Lee C.P. Millar A.H. Metabolite regulatory activation.Plant 32: 666-682Crossref (33) well-established (e.g., Arg SLC38A9 sestrin) transducers Ras-related GTP-binding (RAG) GTPase, RAGULATOR, GATOR) yeasts absent (Kim Guan, 2019Kim K.L. hub growth.Nat. 63-71Crossref (386) Saxton 2017Saxton MTOR metabolism, disease.Cell. 169: 361-371Abstract (916) 2019Wu Shi Integration nutrient, energy, light, 2227-2238Crossref (51) Nevertheless, underlying whereby still primordium apices production strictly controlled internal cues, nutrients, hormones, external environmental (Cai 2017Cai Zhou COP1 integrates light Signal. Behav. 12: e1363946Crossref previously showed sense coordinate energy (glucose), (auxin), (light) depletion inorganic-nitrogen medium strongly inhibited abolished Arabidopsis. both development, uncoupled NRT1.1. Furthermore, revealed significantly when exogenously supplied inorganic-nitrogen-starved seedlings different capacities, Cys Gly pathway) exhibited although lack RAG GTPases, found forms “Rho-like” (ROP2) Together, our work convergent ROP2-TOR axis, Arabidopsis, indicating might lineages. nature, absorb soil convert them pathway. How mixed largely unknown. To address fundamental questions, germinated inorganic-nitrogen-free liquid supplemented (Figures S1A S1B; Table S1). roots shoots grew differently starvation conditions: rapidly, was impaired, completely 1A, 1B , S1D, S1E). Similar phenotypes were seen grown on inorganic-nitrogen- glucose-free solid medium, supporting idea this caused starvation, instead putative high C/N ratio S1F S1G). better define physiological state conditions, measured vivo levels separately (Figure S1C) concentration decreased an undetectable level 4 days after germination (DAG) conditions S1H). remained compared 9 DAG S1I). Given division organogenesis assessed mitotic seedlings. Specifically, examined expression reporter, pCYCB1;1::?-glucuronidase (GUS). pCYCB1;1::GUS progressively 1A 1B). contrast, apex relatively 1C 1D). Exogenously mix (Table S2) triggered reactivation formation induced various including ORC2/6 (ORIGIN RECOGNITION COMPLEX), MCM3/5/7 (MINOCHROMOSOME MAINTENANCE), ETG1 (E2F TARGET GENE), PCNA1 (PROLIFERATING CELL NUCLEAR ANTIGEN) 1E–1I). data indicate blocked and, therefore, next investigated molecular inorganic-nitrogen-mediated growth. previous (glucose) hormones (auxin) investigate whether affects endogenous TOR-kinase activity, monitored based T449 TOR-conserved substrate ribosomal S6 (S6K1) (Xiong Sheen, 2012Xiong Rapamycin glucose-target Chem. 2012; 287: 2836-2842Abstract (169) apices, activities maintained inorganic-nitrogen-supplied S2A S2B), gradually whereas mRNA not affected 2A, 2B S2G–S2I). nitrogen-supplied nitrogen-free S2C–S2F). then determined required development. avoid embryonic lethality tor null mutants, utilized Torin2, chemical inhibitor estradiol-inducible RNAi (tor-es) Torin2 treatment wild-type (WT) estradiol tor-es fully rescue exogenous 2C 2D). estradiol-treated 2E–2G). characterized kinetics only 100 nM (standard 1XMS contains 59.3 mM mix) 5 min time efficiently 2H–2K).Together, sensitive rapid. Glucose through glycolysis-mitochondria electron chain (ETC) relay 2016Zhang J.Y. Roh S.K. Z.Y. promotes accumulation BZR1 balance carbon availability Arabidopsis.Curr. 26: 1854-1860Abstract (115) Sulfate reported glucose-mediated (Dong 2017Dong Silbermann Speiser Forieri I. Linster Poschet Allboje Samami Wanatabe Sticht Teleman al.Sulfur glucose-TOR signaling.Nat. 1174Crossref (100) Pretreatment mitochondrial antimycin A (AMA) mitochondria uncoupler 2,4-dinitrophenol (DNP), shown block glucose- sulfite-starved inorganic-nitrogen-starvation be sufficiently reactivated even presence AMA DNP 3A B), suggesting nitrate-ammonium does dependent signaling. Nitrogen cross-talks multiple phytophormone-signaling modulate (Vega 2019Vega O’Brien J.A. hormonal crosstalk development.Curr. 52: 155-163Crossref (35) apical meristem (SAM) maintenance cytokinin (Landrein 2018Landrein Formosa-Jordan Malivert Schuster Melnyk C.W. Yang Turnbull Meyerowitz E.M. Locke J.C.W. Jönsson modulates stem dynamics cytokinins.Proc. 115: 1382-1387Crossref (79) glucose, Pfeiffer 2016Pfeiffer Janocha Dong Medzihradszky Schöne Daum Suzaki Forner Langenecker Rempel al.Integration meristem.eLife. 5: e17023Crossref (101) effect auxin, cytokinin, gibberellins, brassinosteroids, none replace reactivate 3C 3D) 3E–3G) These insufficient media used above included (Tables S1 S2), converted (Krapp, 2015Krapp its regulation: complex puzzle missing pieces.Curr. 2015; 25: 115-122Crossref (224) 4A 4B ), induce S3A–3D) addition Gln, recently other Glu, Leu, Ile O'Leary Therefore, conducted unbiased examination role 20 quickly capacities 4C 4D). Among them, Ala, Gly, (Group 1) (80%–100% nitrate/ammonium mix); Ser, Asp, 2) (60%–80% activation); His, Ile, Thr, Met, Asn, Lys 3) moderate (35%–60% activation) By Tyr, Pro, Arg, Phe, Trp 4) 4D) unable starts skeleton, made intermediates glycolysis, TCA cycle, there clearly intrinsic correlation AAs’ TOR-activation potencies their carbon-skeleton donors 4E). originating glycolysis-derived pyruvate Ala Leu) had activation; most TCA-cycle-derived oxaloacetate modest potency; phosphoenolpyruvate no More strikingly, originated (Gly), (Gln), sulfur-assimilation (Cys) performed metabolomics analyses many downregulated S4A; S3). 10 recovery, effectively absorbed reached than those continuously condition Importantly, lowest Trp) S3), low AA’s rates. retained S4B; S4). During process, nitrite reductases (NRs), reductase, synthetase/glutamine-2-oxoglutarate aminotransferase converts Gln. genetic pharmacological approaches determine responsible form prevented conversion treating tungstate, NR (Deng 1989Deng Moureaux Caboc