BACKGROUND: Many regulatory circuits in plants contain steps of targeted proteolysis, with the ubiquitin proteasome system (UPS) as the mediator of these proteolytic events. In order to decrease ubiquitin-dependent proteolysis, we inducibly expressed a ubiquitin variant with Arg at position 48 instead of Lys (ubK48R). This variant acts as an inhibitor of proteolysis via the UPS, and allowed us to uncover processes that are particularly sensitive to UPS perturbation. RESULTS: Expression of ubK48R during germination leads to seedling death. We analyzed the seedling transcriptome, proteome and metabolome 24 h post ubK48R induction and confirmed defects in chloroplast development. We found that mutations in single genes can suppress seedling lethality, indicating that a single process in seedlings is critically sensitive to decreased performance of the UPS. Suppressor mutations in phototropin 2 (PHOT2) suggest that a contribution of PHOT2 to chloroplast protection is compromised by proteolysis inhibition. CONCLUSIONS: Overall, the results reveal protein turnover as an integral part of a signal transduction chain that protects chloroplasts during development.

CEITEC Central European Institute of Technology Mendel University in Brno CZ 61300 Brno Czech Republic

Department of Biochemistry and Cell Biology Max Perutz Labs Center for Molecular Biology University of Vienna A 1030 Vienna Austria

Department of Molecular Biology and Radiobiology Faculty of AgriSciences Mendel University in Brno CZ 613 00 Brno Czech Republic

Institute of Plant Physiology RWTH Aachen 52056 Aachen Germany

Max Planck Genome Centre Cologne Max Planck Institute for Plant Breeding Research 50829 Cologne Germany

Present address Department of Integrative Biology Oregon State University 3029 Cordley Hall Corvallis OR 97331 USA

Vienna Biocenter Core Facilities Electron Microscopy A 1030 Vienna Austria

Zobrazit více v PubMed

Miricescu A, Goslin K, Graciet E. Ubiquitylation in plants: signaling hub for the integration of environmental signals. J Exp Bot. 2018;69:4511–4527. doi: 10.1093/jxb/ery165. PubMed DOI

Millar AH, Heazlewood JL, Giglione C, Holdsworth MJ, Bachmair A, Schulze WX. The scope, functions, and dynamics of posttranslational modifications. Annu Rev Plant Biol. 2019;70:119–151. doi: 10.1146/annurev-arplant-050718-100211. PubMed DOI

Dreher K, Callis J. Ubiquitin, hormones and biotic stress in plants. Ann Bot (Lond) 2007;99:787–822. doi: 10.1093/aob/mcl255. PubMed DOI PMC

Adams EHG, Spoel SH. The ubiquitin–proteasome system as a transcriptional regulator of plant immunity. J Exp Bot. 2018;69:4529–4537. doi: 10.1093/jxb/ery216. PubMed DOI

Tal L, Anleu Gil MX, Guercio AM, Shabek N. Structural aspects of plant hormone signal perception and regulation by ubiquitin ligases. Plant Physiol. 2020;182:1537–1544. doi: 10.1104/pp.19.01282. PubMed DOI PMC

Dikic I. Proteasomal and autophagic degradation systems. Annu Rev Biochem. 2017;86:193–224. doi: 10.1146/annurev-biochem-061516-044908. PubMed DOI

Zheng N, Shabek N. Ubiquitin ligases: structure, function and regulation. Annu Rev Biochem. 2017;86:129–157. doi: 10.1146/annurev-biochem-060815-014922. PubMed DOI

Hua Z, Vierstra RD. The cullin-RING ubiquitin-protein ligases. Annu Rev Plant Biol. 2011;62:299–334. doi: 10.1146/annurev-arplant-042809-112256. PubMed DOI

Genschik P, Sumara I, Lechner E. The emerging family of Cullin3-RING ubiquitin ligases (CRL3s): cellular functions and disease implications. EMBO J. 2013;32:2307–2320. doi: 10.1038/emboj.2013.173. PubMed DOI PMC

Gingerich DJ, Gagne JM, Salter DW, Hellmann H, Estelle M, Ma L, Vierstra RD. Cullins 3a and 3b assemble with members of the broad complex/tramtrack/bric-a-brac (BTB) protein family to form essential ubiquitin-protein ligases (E3s) in Arabidopsis. J Biol Chem. 2005;280:18810–18821. doi: 10.1074/jbc.M413247200. PubMed DOI

Roberts D, Pedmale UV, Morrow J, Sachdev S, Lechner E, Tang X, Zheng N, Hannink M, Genschik P, Liscum E. Modulation of phototropic responsiveness in Arabidopsis through ubiquitination of phototropin 1 by the CUL3-Ring E3 ubiquitin ligase CRL3(NPH3) Plant Cell. 2011;23:3627–3640. doi: 10.1105/tpc.111.087999. PubMed DOI PMC

Inada S, Ohgishi M, Mayama T, Okada K, Sakai T. RPT2 is a signal transducer involved in phototropic response and stomatal opening by association with phototropin 1 in Arabidopsis thaliana. Plant Cell. 2004;16:887–896. doi: 10.1105/tpc.019901. PubMed DOI PMC

Haga K, Tsuchida-Mayama T, Yamada M, Sakai T. Arabidopsis ROOT PHOTOTROPISM2 contributes to the adaptation to high-intensity light in phototropic responses. Plant Cell. 2015;27:1098–1112. doi: 10.1105/tpc.15.00178. PubMed DOI PMC

Mena EL, Jevtić P, Greber BJ, Gee CL, Lew BG, Akopian D, Nogales E, Kuriyan J, Rape M. Structural basis for dimerization quality control. Nature. 2020;586:452–456. doi: 10.1038/s41586-020-2636-7. PubMed DOI PMC

Morrow J, Willenburg KT, Liscum E. Phototropism in land plants: molecules and mechanism from light perception to response. Front Biol. 2018;13:342–357. doi: 10.1007/s11515-018-1518-y. DOI

Christie JM, Suetsugu N, Sullivan S, Wada M. Shining light on the function of NPH3/RPT2-like proteins in phototropin signaling. Plant Physiol. 2018;176:1015–1024. doi: 10.1104/pp.17.00835. PubMed DOI PMC

Demarsy E, Fankhauser C. Higher plants use LOV to perceive blue light. Curr Opin Plant Biol. 2009;12:69–74. doi: 10.1016/j.pbi.2008.09.002. PubMed DOI

Banas AK, Aggarwal C, Labuz J, Sztatelman O, Gabrys H. Blue light signalling in chloroplast movements. J Exp Bot. 2012;63:1559–1574. doi: 10.1093/jxb/err429. PubMed DOI

Petroutsos D, Tokutsu R, Maruyama S, Flori S, Greiner A, Magneschi L, Cusant L, Kottke T, Mittag M, Hegemann P, Finazzi G, Minagawa J. A blue-light photoreceptor mediates the feedback regulation of photosynthesis. Nature. 2016;537:563–566. doi: 10.1038/nature19358. PubMed DOI

Ishishita K, Higa T, Tanaka H, Inoue S-i, Chung A, Ushijima T, Matsushita T, Kinoshita T, Nakai M, Wada M, Suetsugu N, Gotoh E. Phototropin 2 contributes to the chloroplast avoidance response at the chloroplast-plasma membrane interface. Plant Physiol. 2020;183:304–316. doi: 10.1104/pp.20.00059. PubMed DOI PMC

Finley D, Sadis S, Monia BP, Boucher P, Ecker DJ, Crooke ST, Chau V. Inhibition of proteolysis and cell cycle progression in a multiubiquitination-deficient yeast mutant. Mol Cell Biol. 1994;14:5501–5509. PubMed PMC

Spence J, Sadis S, Haas AL, Finley D. A ubiquitin mutant with specific defects in DNA repair and multiubiquitination. Mol Cell Biol. 1995;15:1265–1273. doi: 10.1128/MCB.15.3.1265. PubMed DOI PMC

Kim DY, Scalf M, Smith LM, Vierstra RD. Advanced proteomic analyses yield a deep catalog of ubiquitylation targets in Arabidopsis. Plant Cell. 2013;25:1523–1540. doi: 10.1105/tpc.112.108613. PubMed DOI PMC

Schlögelhofer P, Garzon M, Kerzendorfer C, Nizhynska V, Bachmair A. Expression of the ubiquitin variant ubR48 decreases proteolytic activity in Arabidopsis and induces cell death. Planta. 2006;223:684–697. doi: 10.1007/s00425-005-0121-z. PubMed DOI

Debrieux D, Trevisan M, Fankhauser C. Conditional involvement of COP1 in the degradation of phytochrome A. Plant Physiol. 2013;161:2136–2145. doi: 10.1104/pp.112.213280. PubMed DOI PMC

Mitsiades N, Mitsiades CS, Puolaki V, Chauhan D, Fanourakis G, Gu X, Bailey C, Joseph M, Liberman TA, Treon SP, Munshi NC, Richardson PG, Hideshima T, Anderson KC. Molecular sequelae of proteasome inhibition in human multiple myeloma cells. Proc Natl Acad Sci U S A. 2002;99:14374–14379. doi: 10.1073/pnas.202445099. PubMed DOI PMC

Fleming JA, Lightcap ES, Sadis S, Thoroddsen V, Bulawa CE, Blackman RK. Complementary whole-genome technologies reveal the cellular response to proteasome inhibition by PS-341. Proc Natl Acad Sci U S A. 2002;99:1461–1466. doi: 10.1073/pnas.032516399. PubMed DOI PMC

Kim M, Ahn J-W, Jin U-H, Choi D, Paek K-H, Pai H-S. Activation of programmed cell death pathway by inhibition of proteasome function in plants. J Biol Chem. 2003;278:19406–19415. doi: 10.1074/jbc.M210539200. PubMed DOI

Van Hoewyk D. Use of the non-radioactive SUnSET method to detect decreased protein synthesis in proteasome inhibited Arabidopsis roots. Plant Methods. 2016;12:20. doi: 10.1186/s13007-016-0120-z. PubMed DOI PMC

Pereksta D, King D, Saki F, Maroli A, Leonard E, Suseela V, May S, Castellanos Uribe M, Tharayil N, Van Hoewyk D. Proteasome inhibition in Brassica napus roots increases amino acid synthesis to offset reduced proteolysis. Plant Cell Physiol. 2020;61:1028–1040. doi: 10.1093/pcp/pcaa047. PubMed DOI

Mendoza F, Berry C, Prestigiacomo L, Van Hoewyk D. Proteasome inhibition rapidly exacerbates photoinhibition and impedes recovery during high light stress in Chlamydomonas reinhardtii. BMC Plant Biol. 2020;20:22. doi: 10.1186/s12870-020-2236-6. PubMed DOI PMC

Smalle J, Kurepa J, Yang P, Emborg TJ, Babiychuk E, Kushnir S, Vierstra RD. The pleiotropic role of the 26S proteasome subunit RPN10 in Arabidopsis growth and development supports a substrate-specific function in abscisic acid signaling. Plant Cell. 2003;15:965–980. doi: 10.1105/tpc.009217. PubMed DOI PMC

Ling Q, Huang W, Baldwin A, Jarvis P. Chloroplast biogenesis is regulated by direct action of the ubiquitin-proteasome system. Science. 2012;338:655–659. doi: 10.1126/science.1225053. PubMed DOI

Woodson JD, Joens MS, Sinson AB, Gilkerson J, Salomé PA, Weigel D, Fitzpatrick JA, Chory J. Ubiquitin facilitates a quality-control pathway that removes damaged chloroplasts. Science. 2015;350:450–454. doi: 10.1126/science.aac7444. PubMed DOI PMC

Haakonsen DL, Rape M. Branching out: improved signaling by heterotypic ubiquitin chains. Trends Cell Biol. 2019;29:704–716. doi: 10.1016/j.tcb.2019.06.003. PubMed DOI

Lee BH, Lu Y, Prado MA, Shi Y, Tian G, Sun S, Elsasser S, Gygi SP, King RW, Finley D. USP14 deubiquitinates proteasome-bound substrates that are ubiquitinated at multiple sites. Nature. 2016;532:398–401. doi: 10.1038/nature17433. PubMed DOI PMC

Lu Y, Lee BH, King RW, Finley D, Kirschner MW. Substrate degradation by the proteasome: a single-molecule kinetic analysis. Science. 2015;348:1250834. doi: 10.1126/science.1250834. PubMed DOI PMC

Kami C, Lorrain S, Hornitschek P, Fankhauser C. Light-regulated plant growth and development. Curr Top Dev Biol. 2010;91:29–66. doi: 10.1016/S0070-2153(10)91002-8. PubMed DOI

Ruckle ME, Burgoon LD, Lawrence LA, Sinkler CA, Larkin RM. Plastids are major regulators of light signaling in Arabidopsis. Plant Physiol. 2012;159:366–390. doi: 10.1104/pp.112.193599. PubMed DOI PMC

Seluzicki A, Burko Y, Chory J. Dancing in the dark: darkness as a signal in plants. Plant Cell Environ. 2017;40:2487–2501. doi: 10.1111/pce.12900. PubMed DOI PMC

Suraweera A, Munch C, Hanssum A, Bertolotti A. Failure of amino acid homeostasis causes cell death following proteasome inhibition. Mol Cell. 2012;48:242–253. doi: 10.1016/j.molcel.2012.08.003. PubMed DOI PMC

Ding Q, Dimayuga E, Markesbery WR, Keller JN. Proteasome inhibition induces reversible impairments in protein synthesis. FEBS J. 2006;20:1055–1063. PubMed

Hu W, Franklin KA, Sharrock RA, Jones MA, Harmer SL, Lagarias JC. Unanticipated regulatory roles for Arabidopsis phytochromes revealed by null mutant analysis. Proc Natl Acad Sci U S A. 2013;110:1542–1547. doi: 10.1073/pnas.1221738110. PubMed DOI PMC

Leivar P, Monte E, Oka Y, Liu T, Carle C, Castillon A, et al. Multiple Phytochrome-interacting bHLH transcription factors repress premature seedling photomorphogenesis in darkness. Curr Biol. 2008;18:1815–23. PubMed PMC

Hornitschek P, Kohnen MV, Lorrain S, Rougemont J, Ljung K, López-Vidriero I, Franco-Zorrilla JM, Solano R, Trevisan M, Pradervand S, Xenarios I, Fankhauser C. Phytochrome interacting factors 4 and 5 control seedling growth in changing light conditions by directly controlling auxin signaling. Plant J. 2012;71:699–711. doi: 10.1111/j.1365-313X.2012.05033.x. PubMed DOI

Stokes ME, Chattopadhyay A, Wilkins O, Nambara E, Campbell MM. Interplay between sucrose and folate modulates auxin signaling in Arabidopsis. Plant Physiol. 2013;162:1552–65. PubMed PMC

Schreiber KJ, Austin RS, Gong Y, Zhang J, Fung P, Wang PW, Guttman DS, Desveaux D. Forward chemical genetic screens in Arabidopsis identify genes that influence sensitivity to the phytotoxic compound sulfamethoxazole. BMC Plant Biol. 2012;12:226. doi: 10.1186/1471-2229-12-226. PubMed DOI PMC

Mitchoulski A, Liscum E. Arabidopsis NPH3: A NPH1 photoreceptor-interacting protein essential for phototropism. Science. 1999;286:961–964. doi: 10.1126/science.286.5441.961. PubMed DOI

Baubec T, Pecinka A, Rozhon W, Mittelsten Scheid O. Effective, homogeneous and transient interference with cytosine methylation in plant genomic DNA by zebularine. Plant J. 2009;57:542–54. PubMed PMC

Nakasako M, Zikihara K, Matsuoka D, Katsura H, Tokutomi S. Structural basis of the LOV1 dimerization of Arabidopsis phototropins 1 and 2. J Mol Biol. 2008;381:718–733. doi: 10.1016/j.jmb.2008.06.033. PubMed DOI

Oide M, Okajima K, Nakagami H, Kato T, Sekiguchi Y, Oroguchi T, Hikima T, Yamamoto M, Nakasako M. Blue light-excited LOV1 and LOV2 domains cooperatively regulate the kinase activity of full-length phototropin 2 from Arabidopsis. J Biol Chem. 2018;293:963–972. doi: 10.1074/jbc.RA117.000324. PubMed DOI PMC

Aoyama T, Chua N-H. A glucocorticoid-mediated transcriptional induction system in transgenic plants. Plant J. 1997;11:605–612. doi: 10.1046/j.1365-313X.1997.11030605.x. PubMed DOI

Kang H-G, Fang Y, Singh KB. A glucocorticoid-inducible transcription system causes severe growth defects in Arabidopsis and induces defense-related genes. Plant J. 1999;20:127–133. doi: 10.1046/j.1365-313X.1999.00575.x. PubMed DOI

Marquès-Bueno MM, Moreno-Romero J, Abas L, De Michele R, Martinez MC. A dominant negative mutant of protein kinase CK2 exhibits altered auxin responses in Arabidopsis. Plant J. 2011;67:169–180. doi: 10.1111/j.1365-313X.2011.04585.x. PubMed DOI

Shanklin J, Jabben M, Vierstra RD. Red light-induced formation of ubiquitin-phytochrome conjugates: identification of possible intermediates in phytochrome degradation. Proc Natl Acad Sci U S A. 1987;84:359–363. doi: 10.1073/pnas.84.2.359. PubMed DOI PMC

Lin C, Yang H, Guo H, Mockler T, Chen J, Cashmore AR. Enhancement of blue-light sensitivity of Arabidopsis seedlings by a blue light receptor cryptochrome 2. Proc Natl Acad Sci U S A. 1998;95:2686–90. PubMed PMC

Wang Q, Zuo Z, Wang X, Liu Q, Gu L, Oka Y, Lin C. Beyond the photocycle — how cryptochromes regulate photoresponses in plants. Curr Opin Plant Biol. 2018;45:120–126. doi: 10.1016/j.pbi.2018.05.014. PubMed DOI PMC

Sakamoto K, Briggs WR. Cellular and subcellular localization of phototropin 1. Plant Cell. 2002;14:1723–1735. doi: 10.1105/tpc.003293. PubMed DOI PMC

D’Alessandro S, Beaugelin I, Havaux M. Tanned or sunburned: how excessive light triggers plant cell death. Mol Plant. 2020;13:1545–1555. doi: 10.1016/j.molp.2020.09.023. PubMed DOI

Roeber VM, Bajaj I, Rohde M, Schmülling T, Cortleven A. Light acts as a stressor and influences abiotic and biotic stress responses in plants. Plant Cell Environ. 2021;44:645–664. doi: 10.1111/pce.13948. PubMed DOI

Kagaya Y, Toyoshima R, Okuda R, Usui H, Yamamoto A, Hattori T. LEAFY COTYLEDON1 controls seed storage protein genes through its regulation of FUSCA3 and ABSCISIC ACID INSENSITIVE3. Plant Cell Physiol. 2005;46:399–406. doi: 10.1093/pcp/pci048. PubMed DOI

Ge X, Dietrich C, Matsuno M, Li G, Berg H, Xia Y. An Arabidopsis aspartic protease functions as an anti-cell-death component in reproduction and embryogenesis. EMBO Rep. 2005;6:282–288. doi: 10.1038/sj.embor.7400357. PubMed DOI PMC

Gao H, Li R, Guo Y. Arabidopsis asp proteases A36 and A39 play roles in plant reproduction. Plant Signal Behav. 2017;12:e1304343. doi: 10.1080/15592324.2017.1304343. PubMed DOI PMC

Alomrani S, Kunert KJ, Foyer CH. Papain-like cysteine proteases are required for the regulation of photosynthetic gene expression and acclimation to high light stress. J Exp Bot. 2021;72:3441–3454. doi: 10.1093/jxb/erab101. PubMed DOI PMC

Suetsugu N, Takemiya A, Kong SG, Higa T, Komatsu A, Shimazaki K, Kohchi T, Wada M. RPT2/NCH1 subfamily of NPH3-like proteins is essential for the chloroplast accumulation response in land plants. Proc Natl Acad Sci U S A. 2016;113:10424–10429. doi: 10.1073/pnas.1602151113. PubMed DOI PMC

Kimura T, Tsuchida-Mayama T, Imai H, Okajima K, Ito K, Sakai T. Arabidopsis ROOT PHOTOTROPISM2 is a light-dependent dynamic modulator of Phototropin1. Plant Cell. 2020;32:2004–2019. doi: 10.1105/tpc.19.00926. PubMed DOI PMC

Neff MM, Chory J. Genetic interactions between phytochrome A, phytochrome B, and cryptochrome 1 during Arabidopsis development. Plant Physiol. 1998;118:27–36. doi: 10.1104/pp.118.1.27. PubMed DOI PMC

Kikuchi Y, Nakamura S, Woodson JD, Ishida H, Ling Q, Hidema J, Jarvis P, Hagihara S, Izumi M. Chloroplast autophagy and ubiquitination combine to manage oxidative damage and starvation responses. Plant Physiol. 2020;183:1531–1544. doi: 10.1104/pp.20.00237. PubMed DOI PMC

Vizcaíno JA, Csordas A, Del-Toro N, Dianes JA, Griss J, Lavidas I, Mayer G, Perez.-Riverol Y, Resinger F, Ternent T, Xu QW, Wang R, Hermjakob H. Update of the PRIDE database and its related tools. Nucleic Acids Res. 2016;2016(44):11033. doi: 10.1093/nar/gkw880. PubMed DOI PMC

Cerna H, Černý M, Hábanová H, Šafářová D, Abushamsiya K, Nevrátil M, et al. Proteomics offers insight to the mechanism behind Pisum sativum L. response to pea seed-bourne mosaic virus (PSbMV). J Proteome. 2017;153:78–88. PubMed

Fauser F, Schiml S, Puchta H. Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana. Plant J. 2014;79:348–359. doi: 10.1111/tpj.12554. PubMed DOI

Seki M, Carninci P, Nishiyama Y, Hayashizaki Y, Shinozaki K. High-efficiency cloning of Arabidopsis full-length cDNA by biotinylated CAP trapper. Plant J. 1998;15:707–720. doi: 10.1046/j.1365-313x.1998.00237.x. PubMed DOI

Seki M, Narusaka M, Kamiya A, Ishida J, Satou M, Sakurai T, Nakajima M, Enju A, Akiyama K, Oono Y, Muramatsu M, Hayashizaki Y, Kawai J, Carninci P, Itoh M, Ishii Y, Arakawa T, Shibata K, Shinagawa A, Shinozaki K. Functional annotation of a full-length Arabidopsis cDNA collection. Science. 2002;296:141–145. doi: 10.1126/science.1071006. PubMed DOI

Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen H, Shinn P, et al. Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science. 2003;301:653–7. PubMed

Mollier P, Montoro P, Delarue M, Bechtold N, Bellini C, Pelletier G. Promoterless gusA expression in a large number of Arabidopsis thaliana transformants obtained by the in planta infiltration method. C R Acad Sci Paris Sci Vie Life Sci. 1995;318:465–474.