Xylem sap proteomics provides crucial insights into plant defense and root-to-shoot communication. This study highlights the sensitivity and reproducibility of xylem sap proteome analyses, using a single plant per sample to track over 3000 proteins in two model crop plants, Solanum tuberosum and Hordeum vulgare. By analyzing the flg22 response, we identified immune response components not detectable through root or shoot analyses. Notably, we discovered previously unknown elements of the plant immune system, including calcium/calmodulin-dependent kinases and G-type lectin receptor kinases. Despite similarities in the metabolic pathways identified in the xylem sap of both plants, the flg22 response differed significantly: S. tuberosum exhibited 78 differentially abundant proteins, whereas H. vulgare had over 450. However, an evolutionarily conserved overlap in the flg22 response proteins was evident, particularly in the CAZymes and lipid metabolism pathways, where lipid transfer proteins and lipases showed a similar response to flg22. Additionally, many proteins without conserved signal sequences for extracellular targeting were found, such as members of the HSP70 family. Interestingly, the HSP70 response to flg22 was specific to the xylem sap proteome, suggesting a unique regulatory role in the extracellular space similar to that reported in mammalians.

Department of Molecular Biology and Radiobiology Faculty of AgriSciences Mendel University in Brno 61300 Brno Czech Republic

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Koenig A.M., Hoffmann-Benning S. The interplay of phloem-mobile signals in plant development and stress response. Biosci. Rep. 2020;40:BSR20193329. doi: 10.1042/BSR20193329. PubMed DOI PMC

Lucas W.J., Groover A., Lichtenberger R., Furuta K., Yadav S., Helariutta Y., He X., Fukuda H., Kang J., Brady S.M., et al. The Plant Vascular System: Evolution, Development and Functions. J. Integr. Plant Biol. 2013;55:294–388. doi: 10.1111/jipb.12041. PubMed DOI

Taleski M., Jin M., Chapman K., Taylor K., Winning C., Frank M., Imin N., Djordjevic M.A. CEP hormones at the nexus of nutrient acquisition and allocation, root development, and plant–microbe interactions. J. Exp. Bot. 2024;75:538–552. doi: 10.1093/jxb/erad444. PubMed DOI PMC

Ladeynova M., Kuznetsova D., Mudrilov M., Vodeneev V. Integration of Electrical Signals and Phytohormones in the Control of Systemic Response. Int. J. Mol. Sci. 2023;24:847. doi: 10.3390/ijms24010847. PubMed DOI PMC

Houmani H., Corpas F.J. Can nutrients act as signals under abiotic stress? Plant Physiol. Biochem. 2024;206:108313. doi: 10.1016/j.plaphy.2023.108313. PubMed DOI

Gao Y.-Q., Morin H., Marcourt L., Yang T.-H., Wolfender J.-L., Farmer E.E. Chloride, glutathiones, and insect-derived elicitors introduced into the xylem trigger electrical signaling. Plant Physiol. 2024;194:1091–1103. doi: 10.1093/plphys/kiad584. PubMed DOI PMC

Rüscher D., Vasina V.V., Knoblauch J., Bellin L., Pommerrenig B., Alseekh S., Fernie A.R., Neuhaus H.E., Knoblauch M., Sonnewald U., et al. Symplasmic phloem loading and subcellular transport in storage roots are key factors for carbon allocation in cassava. Plant Physiol. 2024 doi: 10.1093/plphys/kiae298. in press . PubMed DOI

Akhiyarova G., Finkina E.I., Zhang K., Veselov D., Vafina G., Ovchinnikova T.V., Kudoyarova G. The Long-Distance Transport of Some Plant Hormones and Possible Involvement of Lipid-Binding and Transfer Proteins in Hormonal Transport. Cells. 2024;13:364. doi: 10.3390/cells13050364. PubMed DOI PMC

Shabala S., White R.G., Djordjevic M.A., Ruan Y.-L., Mathesius U. Root-to-shoot signalling: Integration of diverse molecules, pathways and functions. Funct. Plant Biol. 2016;43:87. doi: 10.1071/FP15252. PubMed DOI

De Schepper V., De Swaef T., Bauweraerts I., Steppe K. Phloem transport: A review of mechanisms and controls. J. Exp. Bot. 2013;64:4839–4850. doi: 10.1093/jxb/ert302. PubMed DOI

Garg V., Kühn C. What determines the composition of the phloem sap? Is there any selectivity filter for macromolecules entering the phloem sieve elements? Plant Physiol. Biochem. 2020;151:284–291. doi: 10.1016/j.plaphy.2020.03.023. PubMed DOI

Tolstyko E.A., Lezzhov A.A., Morozov S.Y., Solovyev A.G. Phloem transport of structured RNAs: A widening repertoire of trafficking signals and protein factors. Plant Sci. 2020;299:110602. doi: 10.1016/j.plantsci.2020.110602. PubMed DOI

Hu C., Ham B., El-shabrawi H.M., Alexander D., Zhang D., Ryals J., Lucas W.J. Proteomics and metabolomics analyses reveal the cucurbit sieve tube system as a complex metabolic space. Plant J. 2016;87:442–454. doi: 10.1111/tpj.13209. PubMed DOI

Brodersen C.R., Roddy A.B., Wason J.W., McElrone A.J. Functional Status of Xylem Through Time. Annu. Rev. Plant Biol. 2019;70:407–433. doi: 10.1146/annurev-arplant-050718-100455. PubMed DOI

Rodríguez-Celma J., Ceballos-Laita L., Grusak M.A., Abadía J., López-Millán A.-F. Plant fluid proteomics: Delving into the xylem sap, phloem sap and apoplastic fluid proteomes. BBA—Proteins Proteom. 2016;1864:991–1002. doi: 10.1016/j.bbapap.2016.03.014. PubMed DOI

Wheeldon C.D., Bennett T. There and back again: An evolutionary perspective on long-distance coordination of plant growth and development. Semin. Cell Dev. Biol. 2021;109:55–67. doi: 10.1016/j.semcdb.2020.06.011. PubMed DOI

Sakakibara H. Cytokinin biosynthesis and transport for systemic nitrogen signaling. Plant J. 2021;105:421–430. doi: 10.1111/tpj.15011. PubMed DOI

Mashiguchi K., Seto Y., Yamaguchi S. Strigolactone biosynthesis, transport and perception. Plant J. 2021;105:335–350. doi: 10.1111/tpj.15059. PubMed DOI

Pérez-Pérez J.G., Puertolas J., Albacete A., Dodd I.C. Alternation of wet and dry sides during partial rootzone drying irrigation enhances leaf ethylene evolution. Environ. Exp. Bot. 2020;176:104095. doi: 10.1016/j.envexpbot.2020.104095. DOI

Regnault T., Davière J.-M., Wild M., Sakvarelidze-Achard L., Heintz D., Carrera Bergua E., Lopez Diaz I., Gong F., Hedden P., Achard P. The gibberellin precursor GA12 acts as a long-distance growth signal in Arabidopsis. Nat. Plants. 2015;1:15073. doi: 10.1038/nplants.2015.73. PubMed DOI

Thorpe M.R., Ferrieri A.P., Herth M.M., Ferrieri R.A. 11C-imaging: Methyl jasmonate moves in both phloem and xylem, promotes transport of jasmonate, and of photoassimilate even after proton transport is decoupled. Planta. 2007;226:541. doi: 10.1007/s00425-007-0503-5. PubMed DOI

Park S.-W., Kaimoyo E., Kumar D., Mosher S., Klessig D.F. Methyl Salicylate Is a Critical Mobile Signal for Plant Systemic Acquired Resistance. Science. 2007;318:113–116. doi: 10.1126/science.1147113. PubMed DOI

Broussard L., Abadie C., Lalande J., Limami A.M., Lothier J., Tcherkez G. Phloem Sap Composition: What Have We Learnt from Metabolomics? Int. J. Mol. Sci. 2023;24:6917. doi: 10.3390/ijms24086917. PubMed DOI PMC

Aoki K., Suzui N., Fujimaki S., Dohmae N., Yonekura-Sakakibara K., Fujiwara T., Hayashi H., Yamaya T., Sakakibara H. Destination-Selective Long-Distance Movement of Phloem Proteins. Plant Cell. 2005;17:1801–1814. doi: 10.1105/tpc.105.031419. PubMed DOI PMC

Djordjevic M.A., Oakes M., Li D.X., Hwang C.H., Hocart C.H., Gresshoff P.M. The Glycine max Xylem Sap and Apoplast Proteome. J. Proteome Res. 2007;6:3771–3779. doi: 10.1021/pr0606833. PubMed DOI

Carella P., Merl-Pham J., Wilson D.C., Dey S., Hauck S.M., Vlot C., Cameron R.K. Comparative Proteomics Analysis of Arabidopsis Phloem Exudates Collected During the Induction of Systemic Acquired Resistance. Plant Physiol. 2016;171:1495–1510. doi: 10.1104/pp.16.00269. PubMed DOI PMC

Rep M., Dekker H.L., Vossen J.H., de Boer A.D., Houterman P.M., Speijer D., Back J.W., de Koster C.G., Cornelissen B.J.C. Mass Spectrometric Identification of Isoforms of PR Proteins in Xylem Sap of Fungus-Infected Tomato. Plant Physiol. 2002;130:904–917. doi: 10.1104/pp.007427. PubMed DOI PMC

Pu Z., Ino Y., Kimura Y., Tago A., Shimizu M., Natsume S., Sano Y., Fujimoto R., Kaneko K., Shea D.J., et al. Changes in the Proteome of Xylem Sap in Brassica oleracea in Response to Fusarium oxysporum Stress. Front. Plant Sci. 2016;7:31. doi: 10.3389/fpls.2016.00031. PubMed DOI PMC

Abeysekara N.S., Bhattacharyya M.K. Analyses of the Xylem Sap Proteomes Identified Candidate Fusarium virguliforme Proteinacious Toxins. PLoS ONE. 2014;9:e93667. doi: 10.1371/journal.pone.0093667. PubMed DOI PMC

Floerl S., Druebert C., Majcherczyk A., Karlovsky P., Kües U., Polle A. Defence reactions in the apoplastic proteome of oilseed rape (Brassica napus var. napus) attenuate Verticillium longisporum growth but not disease symptoms. BMC Plant Biol. 2008;8:129. doi: 10.1186/1471-2229-8-129. PubMed DOI PMC

Zheng T., Haider M.S., Zhang K., Jia H., Fang J. Biological and functional properties of xylem sap extracted from grapevine (cv. Rosario bianco) Sci. Hortic. 2020;272:109563. doi: 10.1016/j.scienta.2020.109563. DOI

Notaguchi M., Okamoto S. Dynamics of long-distance signaling via plant vascular tissues. Front. Plant Sci. 2015;6:161. doi: 10.3389/fpls.2015.00161. PubMed DOI PMC

Kehr J., Buhtz A., Giavalisco P. Analysis of xylem sap proteins from Brassica napus. BMC Plant Biol. 2005;5:11. doi: 10.1186/1471-2229-5-11. PubMed DOI PMC

Buhtz A., Kolasa A., Arlt K., Walz C., Kehr J. Xylem sap protein composition is conserved among different plant species. Planta. 2004;219:610–618. doi: 10.1007/s00425-004-1259-9. PubMed DOI

Yadeta K.A.J., Thomma B.P.H. The xylem as battleground for plant hosts and vascular wilt pathogens. Front. Plant Sci. 2013;4:97. doi: 10.3389/fpls.2013.00097. PubMed DOI PMC

Jelenska J., Davern S.M., Standaert R.F., Mirzadeh S., Greenberg J.T. Flagellin peptide flg22 gains access to long-distance trafficking in Arabidopsis via its receptor, FLS2. J. Exp. Bot. 2017;68:1769–1783. doi: 10.1093/jxb/erx060. PubMed DOI PMC

Moroz N., Tanaka K. FlgII-28 Is a Major Flagellin-Derived Defense Elicitor in Potato. Mol. Plant Microbe Interact. 2020;33:247–255. doi: 10.1094/MPMI-06-19-0164-R. PubMed DOI

Colaianni N.R., Parys K., Lee H.-S., Conway J.M., Kim N.H., Edelbacher N., Mucyn T.S., Madalinski M., Law T.F., Jones C.D., et al. A complex immune response to flagellin epitope variation in commensal communities. Cell Host Microbe. 2021;29:635–649.e9. doi: 10.1016/j.chom.2021.02.006. PubMed DOI

Buscaill P., Chandrasekar B., Sanguankiattichai N., Kourelis J., Kaschani F., Thomas E.L., Morimoto K., Kaiser M., Preston G.M., Ichinose Y., et al. Glycosidase and glycan polymorphism control hydrolytic release of immunogenic flagellin peptides. Science. 2019;364:eaav0748. doi: 10.1126/science.aav0748. PubMed DOI

Hooper C.M., Castleden I.R., Tanz S.K., Aryamanesh N., Millar A.H. SUBA4: The interactive data analysis centre for Arabidopsis subcellular protein locations. Nucleic Acids Res. 2017;45:D1064–D1074. doi: 10.1093/nar/gkw1041. PubMed DOI PMC

Hooper C.M., Castleden I.R., Aryamanesh N., Jacoby R.P., Millar A.H. Finding the Subcellular Location of Barley, Wheat, Rice and Maize Proteins: The Compendium of Crop Proteins with Annotated Locations (CropPAL) Plant Cell Physiol. 2016;57:e9. doi: 10.1093/pcp/pcv170. PubMed DOI

Ge S.X., Jung D., Yao R. ShinyGO: A graphical gene-set enrichment tool for animals and plants. Bioinformatics. 2020;36:2628–2629. doi: 10.1093/bioinformatics/btz931. PubMed DOI PMC

Simpson C., Thomas C., Findlay K., Bayer E., Maule A.J. An Arabidopsis GPI-Anchor Plasmodesmal Neck Protein with Callose Binding Activity and Potential to Regulate Cell-to-Cell Trafficking. Plant Cell. 2009;21:581–594. doi: 10.1105/tpc.108.060145. PubMed DOI PMC

Kim C., Park J., Choi G., Kim S., Vo K.T.X., Jeon J., Kang S., Lee Y. A rice gene encoding glycosyl hydrolase plays contrasting roles in immunity depending on the type of pathogens. Mol. Plant Pathol. 2022;23:400–416. doi: 10.1111/mpp.13167. PubMed DOI PMC

Schimoler-O’Rourke R., Richardson M., Selitrennikoff C.P. Zeamatin Inhibits Trypsin and α-Amylase Activities. Appl. Environ. Microbiol. 2001;67:2365–2366. doi: 10.1128/AEM.67.5.2365-2366.2001. PubMed DOI PMC

Jia R., Yu L., Chen J., Hu L., Cao S., Wang Y. Characterization of the Fasciclin-like arabinogalactan gene family in Brassica napus and the negative regulatory role of BnFLA39 in response to clubroot disease stress. Ind. Crop Prod. 2023;196:116400. doi: 10.1016/j.indcrop.2023.116400. DOI

Sun L., Dong S., Ge Y., Fonseca J.P., Robinson Z.T., Mysore K.S., Mehta P. DiVenn: An Interactive and Integrated Web-Based Visualization Tool for Comparing Gene Lists. Front. Genet. 2019;10:421. doi: 10.3389/fgene.2019.00421. PubMed DOI PMC

Pan L., Berka M., Černý M., Novák J., Luklová M., Brzobohatý B., Saiz-Fernández I. Cytokinin Deficiency Alters Leaf Proteome and Metabolome during Effector-Triggered Immunity in Arabidopsis thaliana Plants. Plants. 2022;11:2123. doi: 10.3390/plants11162123. PubMed DOI PMC

Narváez-Barragán D.A., Tovar-Herrera O.E., Guevara-García A., Serrano M., Martinez-Anaya C. Mechanisms of plant cell wall surveillance in response to pathogens, cell wall-derived ligands and the effect of expansins to infection resistance or susceptibility. Front. Plant Sci. 2022;13:969343. doi: 10.3389/fpls.2022.969343. PubMed DOI PMC

Raiola A., Lionetti V., Elmaghraby I., Immerzeel P., Mellerowicz E.J., Salvi G., Cervone F., Bellincampi D. Pectin Methylesterase Is Induced in Arabidopsis upon Infection and Is Necessary for a Successful Colonization by Necrotrophic Pathogens. Mol. Plant Microbe Interact. 2011;24:432–440. doi: 10.1094/MPMI-07-10-0157. PubMed DOI

Zhou X., Gao H., Zhang X., Khashi u Rahman M., Mazzoleni S., Du M., Wu F. Plant extracellular self-DNA inhibits growth and induces immunity via the jasmonate signaling pathway. Plant Physiol. 2023;192:2475–2491. doi: 10.1093/plphys/kiad195. PubMed DOI PMC

Pernis M., Salaj T., Bellová J., Danchenko M., Baráth P., Klubicová K. Secretome analysis revealed that cell wall remodeling and starch catabolism underlie the early stages of somatic embryogenesis in Pinus nigra. Front. Plant Sci. 2023;14:1225424. doi: 10.3389/fpls.2023.1225424. PubMed DOI PMC

Batailler B., Lemaître T., Vilaine F., Sanchez C., Renard D., Cayla T., Beneteau J., Dinant S. Soluble and filamentous proteins in Arabidopsis sieve elements. Plant Cell Environ. 2012;35:1258–1273. doi: 10.1111/j.1365-3040.2012.02487.x. PubMed DOI

Zimmermann M.R., Knauer T., Furch A.C.U. Phytoplasmas. Methods in Molecular Biology. Humana Press; New York, NY, USA: 2019. Collection of Phloem Sap in Phytoplasma-Infected Plants; pp. 291–299. PubMed DOI

Liu Y., Lin T., Valencia M.V., Zhang C., Lv Z. Unraveling the Roles of Vascular Proteins Using Proteomics. Molecules. 2021;26:667. doi: 10.3390/molecules26030667. PubMed DOI PMC

Maricchiolo E., Panfili E., Pompa A., De Marchis F., Bellucci M., Pallotta M.T. Unconventional Pathways of Protein Secretion: Mammals vs. Plants. Front. Cell Dev. Biol. 2022;10:895853. doi: 10.3389/fcell.2022.895853. PubMed DOI PMC

Zipfel C., Robatzek S., Navarro L., Oakeley E.J., Jones J.D.G., Felix G., Boller T. Bacterial disease resistance in Arabidopsis through flagellin perception. Nature. 2004;428:764–767. doi: 10.1038/nature02485. PubMed DOI

Takai R., Isogai A., Takayama S., Che F.-S. Analysis of Flagellin Perception Mediated by Flg22 Receptor OsFLS2 in Rice. Mol. Plant Microbe Interact. 2008;21:1635–1642. doi: 10.1094/MPMI-21-12-1635. PubMed DOI

Wei Y., Balaceanu A., Rufian J.S., Segonzac C., Zhao A., Morcillo R.J.L., Macho A.P. An immune receptor complex evolved in soybean to perceive a polymorphic bacterial flagellin. Nat. Commun. 2020;11:3763. doi: 10.1038/s41467-020-17573-y. PubMed DOI PMC

Hind S.R., Strickler S.R., Boyle P.C., Dunham D.M., Bao Z., O’Doherty I.M., Baccile J.A., Hoki J.S., Viox E.G., Clarke C.R., et al. Tomato receptor FLAGELLIN-SENSING 3 binds flgII-28 and activates the plant immune system. Nat. Plants. 2016;2:16128. doi: 10.1038/nplants.2016.128. PubMed DOI

Murakami T., Katsuragi Y., Hirai H., Wataya K., Kondo M., Che F.-S. Distribution of flagellin CD2-1, flg22, and flgii-28 recognition systems in plant species and regulation of plant immune responses through these recognition systems. Biosci. Biotechnol. Biochem. 2022;86:490–501. doi: 10.1093/bbb/zbac007. PubMed DOI

Sun Y., Qiao Z., Muchero W., Chen J.-G. Lectin Receptor-like Kinases: The Sensor and Mediator at the Plant Cell Surface. Front. Plant Sci. 2020;11:596301. doi: 10.3389/fpls.2020.596301. PubMed DOI PMC

Yip Delormel T., Boudsocq M. Properties and functions of calcium-dependent protein kinases and their relatives in Arabidopsis thaliana. New Phytol. 2019;224:585–604. doi: 10.1111/nph.16088. PubMed DOI

Lampl N., Alkan N., Davydov O., Fluhr R. Set-point control of RD21 protease activity by AtSerpin1 controls cell death in Arabidopsis. Plant J. 2013;74:498–510. doi: 10.1111/tpj.12141. PubMed DOI

Tunc-Ozdemir M., Jones A.M. BRL3 and AtRGS1 cooperate to fine tune growth inhibition and ROS activation. PLoS ONE. 2017;12:e0177400. doi: 10.1371/journal.pone.0177400. PubMed DOI PMC

Matsui S., Noda S., Kuwata K., Nomoto M., Tada Y., Shinohara H., Matsubayashi Y. Arabidopsis SBT5.2 and SBT1.7 subtilases mediate C-Terminal Cleavage of flg22 epitope from bacterial flagellin. Nat. Commun. 2024;15:3762. doi: 10.1038/s41467-024-48108-4. PubMed DOI PMC

Gao H., Ma K., Ji G., Pan L., Zhou Q. Lipid transfer proteins involved in plant–pathogen interactions and their molecular mechanisms. Mol. Plant Pathol. 2022;23:1815–1829. doi: 10.1111/mpp.13264. PubMed DOI PMC

Berka M., Kopecká R., Berková V., Brzobohatý B., Černý M. Regulation of heat shock proteins 70 and their role in plant immunity. J. Exp. Bot. 2022;73:1894–1909. doi: 10.1093/jxb/erab549. PubMed DOI PMC

Lopez V., Cauvi D.M., Arispe N., De Maio A. Bacterial Hsp70 (DnaK) and mammalian Hsp70 interact differently with lipid membranes. Cell Stress. Chaperones. 2016;21:609–616. doi: 10.1007/s12192-016-0685-5. PubMed DOI PMC

Dufková H., Berka M., Psota V., Brzobohatý B., Černý M. Environmental impacts on barley grain composition and longevity. J. Exp. Bot. 2023;74:1609–1628. doi: 10.1093/jxb/erac498. PubMed DOI

Berková V., Berka M., Kameniarová M., Kopecká R., Kuzmenko M., Shejbalová Š., Abramov D., Čičmanec P., Frejlichová L., Jan N., et al. Salicylic Acid Treatment and Its Effect on Seed Yield and Seed Molecular Composition of Pisum sativum under Abiotic Stress. Int. J. Mol. Sci. 2023;24:5454. doi: 10.3390/ijms24065454. PubMed DOI PMC

Dorfer V., Pichler P., Stranzl T., Stadlmann J., Taus T., Winkler S., Mechtler K. MS Amanda, a Universal Identification Algorithm Optimized for High Accuracy Tandem Mass Spectra. J. Proteome Res. 2014;13:3679–3684. doi: 10.1021/pr500202e. PubMed DOI PMC

Kong A.T., Leprevost F.V., Avtonomov D.M., Mellacheruvu D., Nesvizhskii A.I. MSFragger: Ultrafast and comprehensive peptide identification in mass spectrometry-based proteomics. Nat. Methods. 2017;14:513–520. doi: 10.1038/nmeth.4256. PubMed DOI PMC

Dufková H., Berka M., Greplová M., Shejbalová Š., Hampejsová R., Luklová M., Domkářová J., Novák J., Kopačka V., Brzobohatý B., et al. The Omics Hunt for Novel Molecular Markers of Resistance to Phytophthora infestans. Plants. 2022;11:61. doi: 10.3390/plants11010061. PubMed DOI PMC

Perez-Riverol Y., Bai J., Bandla C., García-Seisdedos D., Hewapathirana S., Kamatchinathan S., Kundu D.J., Prakash A., Frericks-Zipper A., Eisenacher M., et al. The PRIDE database resources in 2022: A hub for mass spectrometry-based proteomics evidences. Nucleic Acids Res. 2022;50:D543–D552. doi: 10.1093/nar/gkab1038. PubMed DOI PMC

Pang Z., Lu Y., Zhou G., Hui F., Xu L., Viau C., Spigelman A.F., MacDonald P.E., Wishart D.S., Li S., et al. MetaboAnalyst 6.0: Towards a unified platform for metabolomics data processing, analysis and interpretation. Nucleic Acids Res. 2024;52:W398–W406. doi: 10.1093/nar/gkae253. PubMed DOI PMC

Liebermeister W., Noor E., Flamholz A., Davidi D., Bernhardt J., Milo R. Visual account of protein investment in cellular functions. Proc. Natl. Acad. Sci. USA. 2014;111:8488–8493. doi: 10.1073/pnas.1314810111. PubMed DOI PMC

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