KEY MESSAGE: In Physcomitrium patens, PpRH1/PpRH2 are GUCT-domain-containing DEAD-BOX RNA helicases localize to the nucleus. They are implicated in cell and tissue development in all stages of the moss life cycle. ABSTRACT: The DEAD-box-containing RNA helicase family encompasses a large and functionally important group of enzymes involved in cellular processes committed to the metabolism of RNA, including its transcription, processing, transport, translation and decay. Studies indicate this protein family has implied roles in plant vegetative and reproductive developmental processes as well as response to environmental stresses such has cold and high salinity. We focus here on a small conserved sub-group of GUCT domain-containing RNA helicase in the moss Physcomitrium patens. Phylogenetic analysis shows that RNA helicases containing the GUCT domain form a distinct conserved clade across the green lineage. In this clade, the P. patens genome possesses two closely related paralogues RNA helicases predicted to be nuclear, PpRH1 and PpRH2. Using in-locus gene fluorescent tagging we show that PpRH1 is localized to the nucleus in protonema. Analysis of PpRH1 and PpRH2 deletions, individually and together, indicates their potential roles in protonema, gametophore and sporophyte cellular and tissue development in P. patens. Additionally, the ultrastructural analysis of phyllid chloroplasts in Δrh2 and Δrh1/2 shows distinct starch granule accumulation under standard growth conditions associated with changes in photosynthetic activity parameters. We could not detect effects of either temperature or stress on protonema growth or PpRH1 and PpRH2 expression. Together, these results suggest that nuclear GUCT-containing RNA helicases play a role primarily in developmental processes directly or indirectly linked to photosynthesis activity in the moss P. patens. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11103-021-01152-w.

Comenius University in Bratislava Science Park Ilkovicova 8 84215 Bratislava Slovakia

Department of Biophysics Centre of the Region Haná for Biotechnological and Agricultural Research Faculty of Science Palacký University Šlechtitelů 27 78371 Olomouc Czech Republic

Department of Biotechnology Inland Norway University of Applied Sciences Holsetgata 31 2318 Hamar Norway

Department of Gastroenterology Hepatology and Endocrinology Hannover Medical School Carl Neuberg Str 1 30625 Hannover Germany

Department of Plant Physiology Faculty of Natural Sciences Comenius University in Bratislava Ilkovicova 6 84215 Bratislava Slovakia

Institut Jean Pierre Bourgin INRAE AgroParisTech Université Paris Saclay 78000 Versailles France

Plant Cell Biology Faculty of Biology University of Marburg Karl von Frisch Str 8 35043 Marburg Germany

Plant Science and Biodiversity Center Slovak Academy of Sciences Dúbravská cesta 9 84523 Bratislava Slovakia

School of Animal Rural and Environmental Sciences Nottingham Trent University Brackenhurst Campus Southwell NG25 0QF Nottinghamshire UK

See more in PubMed

Andersen JS, Lyon CE, Fox AH, Leung AKL, Lam YW, Steen H, Mann M, Lamond AI. Directed proteomic analysis of the human nucleolus. Curr Biol. 2002;12:1–11. doi: 10.1016/s0960-9822(01)00650-9. PubMed DOI

Andreou AZ, Klostermeier D. eIF4B and eIF4G jointly stimulate eIF4A ATPase and unwinding activities by modulation of the eIF4A conformational cycle. J Mol Biol. 2014;426:51–61. doi: 10.1016/j.jmb.2013.09.027. PubMed DOI

Asakura Y, Galarneau E, Watkins KP, Barkan A, van Wijk KJ. Chloroplast RH3 DEAD box RNA helicases in maize and Arabidopsis function in splicing of specific group II introns and affect chloroplast ribosome biogenesis. Plant Physiol. 2012;159:961–974. doi: 10.1104/pp.112.197525. PubMed DOI PMC

Bush MS, Hutchins AP, Jones AME, Naldrett MJ, Jarmolowski A, Lloyd CW, Doonan JH. Selective recruitment of proteins to 5′ cap complexes during the growth cycle in Arabidopsis. Plant J. 2009;59:400–412. doi: 10.1111/j.1365-313X.2009.03882.x. PubMed DOI

Bush MS, Crowe N, Zheng T, Doonan JH. The RNA helicase, eIF4A-1, is required for ovule development and cell size homeostasis in Arabidopsis. Plant J. 2015;84:989–1004. doi: 10.1111/tpj.13062. PubMed DOI PMC

Bush MS, Pierrat O, Nibau C, Mikitova V, Zheng T, Corke FMK, Vlachonasios K, Mayberry LK, Browning KS, Doonan JH. eIF4A RNA Helicase associates with cyclin-dependent protein Kinase A in proliferating cells and is modulated by phosphorylation. Plant Physiol. 2016;172:128–140. doi: 10.1104/pp.16.00435. PubMed DOI PMC

Cai J, Meng X, Li G, Dong T, Sun J, Xu T, Li Z, Han Y, Zhu M. Identification, expression analysis, and function evaluation of 42 tomato DEAD-box RNA helicase genes in growth development and stress response. Acta Physiol Plant. 2018;40:94. doi: 10.1007/s11738-018-2665-0. DOI

Chou KC, Shen HB. Plant-mPLoc: A top-down strategy to augment the power for predicting plant protein subcellular localization. PLoS ONE. 2010 doi: 10.1371/journal.pone.0011335. PubMed DOI PMC

Cove DJ, Perroud P-F, Charron AJ, McDaniel SF, Khandelwal A, Quatrano RS. The moss Physcomitrella patens: a novel model system for plant development and genomic studies. Cold Spring Harb Protoc. 2009;4:69–104. doi: 10.1101/pdb.emo115. PubMed DOI

Henning D, Rolando B. So, Runyan J, Lester F. Lau, Benigno C. Valdez (2003) Silencing of RNA helicase II/Guα inhibits mammalian ribosomal RNA production. J Biol Chem 278(52):52307–52314 PubMed

Elzanati O, Mouzeyar S, Roche J. Dynamics of the transcriptome response to heat in the moss, Physcomitrella patens. Int J Mol Sci. 2020 doi: 10.3390/ijms21041512. PubMed DOI PMC

Engel PP. The induction of biochemical and morphological mutants inthe moss Physcomitrella patens. Am J Bot. 1968;55:438–446. doi: 10.1002/j.1537-2197.1968.tb07397.x. DOI

Fairman-Williams ME, Guenther U-P, Jankowsky E. SF1 and SF2 helicases: family matters. Curr Opin Struct Biol. 2010;20:313–324. doi: 10.1016/j.sbi.2010.03.011. PubMed DOI PMC

Fernandez-Pozo N, Haas FB, Meyberg R, Ullrich KK, Hiss M, Perroud P-F, Hanke S, Kratz V, Powell AF, Vesty EF, Daum CG, Zane M, Lipzen A, Sreedasyam A, Grimwood J, Coates JC, Barry K, Schmutz J, Mueller LA, Rensing SA. PEATmoss (Physcomitrella Expression Atlas Tool): a unified gene expression atlas for the model plant Physcomitrella patens. Plant J. 2020;1:165–177. doi: 10.1111/tpj.14607. PubMed DOI

Haas FB, Fernandez-Pozo N, Meyberg R, Perroud P-F, Göttig M, Stingl N, Saint-Marcoux D, Langdale J, Rensing SR. Single nucleotide polymorphism charting of P. patens reveals accumulation of somatic mutations during in vitro culture on the scale of natural variation by selfing. Front Plant Sci. 2020;11:813. doi: 10.3389/fpls.2020.00813. PubMed DOI PMC

Hájek T, Tuittila E-S, Ilomets M, Laiho R. Light responses of mire mosses – a key to survival after water-level drawdown? Oikos. 2009;118:240–250. doi: 10.1111/j.1600-0706.2008.16528.x. DOI

Hiss M, Meyberg R, Westermann J, Haas FB, Schneider L, Schallenberg-Rüdinger M, Ullrich KK, Rensing SA. Sexual reproduction, sporophyte development and molecular variation in the model moss Physcomitrella patens: introducing the ecotype Reute. Plant J. 2017;90:606–620. doi: 10.1111/tpj.13501. PubMed DOI

Ho J, Adeolu M, Khadka B, Gupta RS. Identification of distinctive molecular traits that are characteristic of the phylum “Deinococcus-Thermus” and distinguish its main constituent groups. Syst Appl Microbiol. 2016;39:453–463. doi: 10.1016/j.syapm.2016.07.003. PubMed DOI

Hohe A, Rensing SA, Mildner M, Lang D, Reski R. Day length and temperature strongly influence sexual reproduction and expression of a novel MADS-box gene in the moss Physcomitrella patens. Plant Biol. 2002;4:595–602. doi: 10.1055/s-2002-35440. DOI

Huang C-K, Shen Y-L, Huang L-F, Wu S-J, Yeh C-H, Lu C-A. The DEAD-Box RNA Helicase AtRH7/PRH75 participates in pre-rRNA processing, plant development and cold tolerance in Arabidopsis. Plant Cell Physiol. 2016;57:174–191. doi: 10.1093/pcp/pcv188. PubMed DOI

Hutchins AP, Roberts GR, Lloyd CW, Doonan JH. In vivo interaction between CDKA and eIF4A: a possible mechanism linking translation and cell proliferation. FEBS Lett. 2004;556:91–94. doi: 10.1016/S0014-5793(03)01382-6. PubMed DOI

Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013;30:772–780. doi: 10.1093/molbev/mst010. PubMed DOI PMC

Khraiwesh B, Qudeimat E, Thimma M, Chaiboonchoe A, Jijakli K, Alzahmi A, Arnoux M, Salehi-Ashtiani K. Genome-wide expression analysis offers new insights into the origin and evolution of Physcomitrella patens stress response. Sci Rep. 2015;5:17434. doi: 10.1038/srep17434. PubMed DOI PMC

Kim M-H, Sonoda Y, Sasaki K, Kaminaka H, Imai R. Interactome analysis reveals versatile functions of Arabidopsis COLD SHOCK DOMAIN PROTEIN 3 in RNA processing within the nucleus and cytoplasm. Cell Stress Chaperones. 2013;18:517–525. doi: 10.1007/s12192-012-0398-3. PubMed DOI PMC

Liang Z, Geng Y, Ji C, Du H, Wong CE, Zhang Q, Zhang Y, Zhang PP, Riaz A, Chachar S, Ding Y, Wen J, Wu YY, Wang M, Zheng H, Wu YY, Demko V, Shen L, Han X, Zhang PP, Gu X, Yu H. Mesostigma viride genome and transcriptome provide insights into the origin and evolution of Streptophyta. Adv Sci. 2020 doi: 10.1002/advs.201901850. PubMed DOI PMC

Linder P, Owttrim GW. Plant RNA helicases: linking aberrant and silencing RNA. Trends Plant Sci. 2009;14:344–352. doi: 10.1016/j.tplants.2009.03.007. PubMed DOI

Liu Y, Tabata D, Imai R. A cold-inducible DEAD-box RNA helicase from Arabidopsis thaliana regulates plant growth and development under low temperature. PLoS ONE. 2016 doi: 10.1371/journal.pone.0154040. PubMed DOI PMC

Lorković ZJ, Herrmann RG, Oelmüller R. PRH75, a new nucleus-localized member of the DEAD-box protein family from higher plants. Mol Cell Biol. 1997;17:2257–2265. doi: 10.1128/mcb.17.4.2257. PubMed DOI PMC

Lu C-A, Huang C-K, Huang W-S, Huang T-S, Liu H-Y, Chen Y-F. DEAD-Box RNA Helicase 42 plays a critical role in pre-mRNA splicing under cold stress. Plant Physiol. 2020;182:255–271. doi: 10.1104/pp.19.00832. PubMed DOI PMC

Maxwell K, Johnson GN. Chlorophyll fluorescence—a practical guide. J Exp Bot. 2000;51:659–668. doi: 10.1093/jexbot/51.345.659. PubMed DOI

Mdodana NT, Jewell JF, Phiri EE, Smith ML, Oberlander K, Mahmoodi S, Kossmann J, Lloyd JR. Mutations in glucan, water dikinase affect starch degradation and gametophore development in the moss Physcomitrella patens. Sci Rep. 2019;9:15114. doi: 10.1038/s41598-019-51632-9. PubMed DOI PMC

Medina R, Johnson MG, Liu Y, Wickett NJ, Shaw AJ, Goffinet B. Phylogenomic delineation of Physcomitrium (Bryophyta: Funariaceae) based on targeted sequencing of nuclear exons and their flanking regions rejects the retention of Physcomitrella, Physcomitridium and Aphanorrhegma. J Syst Evol. 2019;57:404–417. doi: 10.1111/jse.12516. DOI

Meyberg R, Perroud P-F, Haas FB, Schneider L, Heimerl T, Renzaglia KS, Rensing SA. Characterisation of evolutionarily conserved key players affecting eukaryotic flagellar motility and fertility using a moss model. New Phytol. 2020;227:440–454. doi: 10.1111/nph.16486. PubMed DOI PMC

Nayak NR, Putnam AA, Addepalli B, Lowenson JD, Chen T, Jankowsky E, Perry SE, Dinkins RD, Limbach PA, Clarke SG, Downie AB. An Arabidopsis ATP-Dependent, DEAD-Box RNA helicase loses activity upon isoAsp formation but Is restored by PROTEIN ISOASPARTYL METHYLTRANSFERASE. Plant Cell. 2013;25:2573–2586. doi: 10.1105/tpc.113.113456. PubMed DOI PMC

Ohnishi S, Pääkkönen K, Koshiba S, Tochio N, Sato M, Kobayashi N, Harada T, Watanabe S, Muto Y, Güntert P, Tanaka A, Kigawa T, Yokoyama S. Solution structure of the GUCT domain from human RNA helicase II/Guβ reveals the RRM fold, but implausible RNA interactions. Proteins Struct Funct Bioinforma. 2009;74:133–144. doi: 10.1002/prot.22138. PubMed DOI

Ortiz-Ramírez C, Hernandez-Coronado M, Thamm A, Catarino B, Wang M, Dolan L, Feijó JA, Becker JD. A transcriptome atlas of Physcomitrella patens provides insights into the evolution and development of land plants. Mol Plant. 2016;9:205–220. doi: 10.1016/j.molp.2015.12.002. PubMed DOI

Ou Y, Fritzler MJ, Valdez BC, Rattner JB. Mapping and characterization of the functional domains of the nucleolar protein RNA Helicase II/Gu. Exp Cell Res. 1999;247:389–398. doi: 10.1006/excr.1998.4365. PubMed DOI

Pandey S, Muthamilarasan M, Sharma N, Chaudhry V, Dulani P, Shweta S, Jha S, Mathur S, Prasad M. Characterization of DEAD-box family of RNA helicases in tomato provides insights into their roles in biotic and abiotic stresses. Environ Exp Bot. 2019;158:107–116. doi: 10.1016/j.envexpbot.2018.11.018. DOI

Peltier J-BB, Yang C, Qi S, Zabrouskov V, Giacomelli LL, Rudella A, Ytterberg AJ, Rutschow H, van Wijk KJ, Cai Y, Sun Q, Zabrouskov V, Giacomelli LL, Rudella A, Ytterberg AJ, Rutschow H, van Wijk KJ. The oligomeric stromal proteome of Arabidopsis thaliana chloroplasts. Mol Cell Proteomics. 2006;5:114–133. doi: 10.1074/mcp.M500180-MCP200. PubMed DOI

Perroud PF, Quatrano RS. The role of ARPC4 in tip growth and alignment of the polar axis in filaments of of Physcomitrella patens. Cell Motil cytoskelet. 2006;63(3):162–171. doi: 10.1002/cm.20114. PubMed DOI

Perroud P-F, Cove DJ, Quatrano RS, Mcdaniel SF. An experimental method to facilitate the identification of hybrid sporophytes in the moss Physcomitrella patens using fluorescent tagged lines. New Phytol. 2011;191:301–306. doi: 10.1111/j.1469-8137.2011.03668.x. PubMed DOI PMC

Perroud P-F, Meyberg R, Rensing SA. Physcomitrella patens Reute mCherry as a tool for efficient crossing within and between ecotypes. Plant Biol. 2019;21:143–149. doi: 10.1111/plb.12840. PubMed DOI

Perroud P-F, Meyberg R, Demko V, Quatrano RS, Olsen OA, Rensing SA. DEK1 displays a strong subcellular polarity during Physcomitrella patens 3D growth. New Phytol. 2020;226:1029–1041. doi: 10.1111/nph.16417. PubMed DOI

Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001;29:e45. doi: 10.1093/nar/29.9.e45. PubMed DOI PMC

Putnam AA, Jankowsky E. DEAD-box helicases as integrators of RNA, nucleotide and protein binding. Biochim Biophys Acta. 2013;1829:884–893. doi: 10.1016/j.bbagrm.2013.02.002. PubMed DOI PMC

Rasband W. ImageJ 1997–2018. Bethesda, Maryland, USA: U. S. Natl. Institutes Heal; 2018.

Reynolds ES. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol. 1963;17:208–212. doi: 10.1083/jcb.17.1.208. PubMed DOI PMC

Rogers GWJ, Richter NJ, Merrick WC. Biochemical and kinetic characterization of the RNA helicase activity of eukaryotic initiation factor 4A. J Biol Chem. 1999;274:12236–12244. doi: 10.1074/jbc.274.18.12236. PubMed DOI

Rosado A, Li R, van de Ven W, Hsu E, Raikhel NV. Arabidopsis ribosomal proteins control developmental programs through translational regulation of auxin response factors. Proc Natl Acad Sci U S A. 2012;109:19537–19544. doi: 10.1073/pnas.1214774109. PubMed DOI PMC

Ruijter JM, Ramakers C, Hoogaars WMH, Karlen Y, Bakker O, van den Hoff MJB, Moorman AFM. Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data. Nucleic Acids Res. 2009;37:e45–e45. doi: 10.1093/nar/gkp045. PubMed DOI PMC

Schaefer DG, Zrÿd J-P. Efficient gene targeting in the moss Physcomitrella patens. Plant J. 1997;11:1195–1206. doi: 10.1046/j.1365-313X.1997.11061195.x. PubMed DOI

Schaefer DG, Delacote F, Charlot F, Vrielynck N, Guyon-Debast A, Le Guin S, Neuhaus J-M, Doutriaux MP, Nogué F. RAD51 loss of function abolishes gene targeting and de-represses illegitimate integration in the moss Physcomitrella patens. DNA Repair (Amst) 2010;9:526–533. doi: 10.1016/j.dnarep.2010.02.001. PubMed DOI

Singleton MR, Dillingham MS, Wigley DB. Structure and mechanism of helicases and nucleic acid translocases. Annu Rev Biochem. 2007;76:23–50. doi: 10.1146/annurev.biochem.76.052305.115300. PubMed DOI

Spurr AR. A low-viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res. 1969;26:31–43. doi: 10.1016/s0022-5320(69)90033-1. PubMed DOI

Staub E, Fiziev P, Rosenthal A, Hinzmann B. Insights into the evolution of the nucleolus by an analysis of its protein domain repertoire. BioEssays. 2004;26:567–581. doi: 10.1002/bies.20032. PubMed DOI

Tanner NKK, Linder P. DExD/H Box RNA Helicases: from generic motors to specific dissociation functions. Mol Cell. 2001;8:251–262. doi: 10.1016/S1097-2765(01)00329-X. PubMed DOI

Thelander M, Landberg K, Sundberg E. Auxin-mediated developmental control in the moss Physcomitrella patens. Bot: J. Exp; 2018. PubMed

Trouiller B, Schaefer DG, Charlot F, Nogué F. MSH2 is essential for the preservation of genome integrity and prevents homeologous recombination in the moss Physcomitrella patens. Nucleic Acids Res. 2006;34:232–242. doi: 10.1093/nar/gkj423. PubMed DOI PMC

Tyagi V, Parihar V, Malik G, Kalra V, Kapoor S, Kapoor M. The DEAD-box RNA helicase eIF4A regulates plant development and interacts with the hnRNP LIF2L1 in Physcomitrella patens. Mol Genet Genomics. 2020;295:373–389. doi: 10.1007/s00438-019-01628-x. PubMed DOI

Valdez BC. Structural domains involved in the RNA folding activity of RNA helicase II/Gu protein. Eur J Biochem. 2000;267:6395–6402. doi: 10.1046/j.1432-1327.2000.01727.x. PubMed DOI

Valdez BC, Henning D, Perlaky L, Busch RK, Busch H. Cloning and characterization of Gu/RH-II binding protein. Biochem Biophys Res Commun. 1997;234:335–340. doi: 10.1006/bbrc.1997.6642. PubMed DOI

Vitha S, Baluška F, Volkmann D, Barlow P. Steedman’s wax for F-actin visualization. In: Baluška F, Barlow PW, Staiger CJ, Volkmann D, editors. Actin: a dynamic framework for multiple plant cell functions. Dordrecht: Kluwer; 2000. pp. 619–636.

Wang D, Qin B, Li X, Tang D, Zhang Y, Cheng Z, Xue Y. Nucleolar DEAD-Box RNA Helicase TOGR1 regulates thermotolerant growth as a pre-rRNA chaperone in rice. PLOS Genet. 2016;12:e1005844. doi: 10.1371/journal.pgen.1005844. PubMed DOI PMC

Xiao L, Wang H, Wan P, Kuang T, He Y. Genome-wide transcriptome analysis of gametophyte development in Physcomitrella patens. BMC Plant Biol. 2011;11:177. doi: 10.1186/1471-2229-11-177. PubMed DOI PMC

Xu R, Zhang S, Huang J, Zheng C. Genome-wide comparative in silico analysis of the RNA helicase gene family in Zea mays and Glycine max: A comparison with Arabidopsis and Oryza sativa. PLoS ONE. 2013 doi: 10.1371/journal.pone.0078982. PubMed DOI PMC