The absence of state transitions in a Nt(Hn) cybrid is due to a cleavage of the threonine residue from the misprocessed N-terminus of the LHCII polypeptides. The cooperation between the nucleus and chloroplast genomes is essential for plant photosynthetic fitness. The rapid and specific interactions between nucleus-encoded and chloroplast-encoded proteins are under intense investigation with potential for applications in agriculture and renewable energy technology. Here, we present a novel model for photosynthesis research in which alien henbane (Hyoscyamus niger) chloroplasts function on the nuclear background of a tobacco (Nicotiana tabacum). The result of this coupling is a cytoplasmic hybrid (cybrid) with inhibited state transitions-a mechanism responsible for balancing energy absorption between photosystems. Protein analysis showed differences in the LHCII composition of the cybrid plants. SDS-PAGE analysis revealed a novel banding pattern in the cybrids with at least one additional 'LHCII' band compared to the wild-type parental species. Proteomic work suggested that the N-terminus of at least some of the cybrid Lhcb proteins was missing. These findings provide a mechanistic explanation for the lack of state transitions-the N-terminal truncation of the Lhcb proteins in the cybrid included the threonine residue that is phosphorylated/dephosphorylated in order to trigger state transitions and therefore crucial energy balancing mechanism in plants.

Faculty of Science and Engineering Manchester Metropolitan University John Dalton Building Chester St Manchester M1 5GD UK

Mikrobiologický Institute Novohradská 237 Opatovický Mlýn 37901 Třeboň Czech Republic

School of Biological and Chemical Sciences Queen Mary University of London Mile End Road London E1 4NS UK

Zobrazit více v PubMed

Adam Z. Chloroplast proteases: possible regulators of gene expression? Biochimie. 2000;82:647–654. doi: 10.1016/S0300-9084(00)00612-X. PubMed DOI

Adam Z, Adamska I, Nakabayashi K, Ostersetzer O, Haussuhl K, Manuell A, Clarke AK. Chloroplast and mitochondrial proteases in Arabidopsis. A proposed nomenclature. Plant Physiol. 2001;125:1912–1918. doi: 10.1104/pp.125.4.1912. PubMed DOI PMC

Allen JF. State transitions—a question of balance. Science. 2009;299:1530–1532. doi: 10.1126/science.1082833. PubMed DOI

Allen JF, de Paula WBM, Puthiyaveetil S, Niel J. A structural phylogenetic map for chloroplast photosynthesis. Trends Plant Sci. 2011;16:645–655. doi: 10.1016/j.tplants.2011.10.004. PubMed DOI

Archibald JM. The puzzle of plastid evolution. Curr Biol. 2009;19:81–88. doi: 10.1016/j.cub.2008.11.067. PubMed DOI

Aro E, Suorsa M, Rokka A, Allahverdiyeva Y, Paakkarinen V, Saleem A, Battchikova N, Rintamäki E. Dynamics of photosystem II: a proteomic approach to thylakoid protein complexes. J Exp Bot. 2005;56:347–356. doi: 10.1093/jxb/eri041. PubMed DOI

Artimo P, Jonnalagedda M, Arnold K, Baratin D, Csardi G, de Castro E, Duvaud S, Flegel V, Fortier A, Gasteiger E, Grosdidier A, Hernandez C, Ioannidis V, Kuznetsov D, Liechti R, Moretti S, Mostaguir K, Redaschi N, Rossier G, Xenarios I, Stockinger H. ExPASy: SIB bioinformatics resource portal. Nucleic Acids Res. 2012;40:597–603. doi: 10.1093/nar/gks400. PubMed DOI PMC

Babiychuk E, Schantz R, Cherep N, Weil JH, Gleba Y, Kushnir S. Alterations in chlorophyll a/b binding proteins in Solanaceae cybrids. Mol Gen Genet. 1995;249:648–654. doi: 10.1007/BF00418034. PubMed DOI

Bassi R, Silvestri M, Dainese P, Moya I, Giacometti GM. Effects of a nonionic detergent on the spectral properties and aggregation state of the light-harvesting chlorophyll a/b protein complex (LHCII) J Photochem Photobiol, B. 1991;9:335–354. doi: 10.1016/1011-1344(91)80170-M. DOI

Belliard G, Vedel F, Pelletier G. Mitochondrial recombination in cytoplasmic hybrid of Nicotiana tabacum by protoplast fusion. Nature. 1979;281:401–403. doi: 10.1038/281401a0. DOI

Blankenship RE. Molecular mechanisms of photosynthesis. Oxford: Blackwell Science; 2002.

Bouchnak I, Brugière S, Moyet L, Le Gall S, Salvi D, Kuntz M, Tardif M, Rolland N (2019) Unravelling hidden components of the chloroplast envelope proteome: opportunities and limits of better MS sensitivity. Mol Cell Proteomics, mcp-RA118 PubMed PMC

Caffarri S, Kouřil R, Kereïche S, Boekema EJ, Croce R. Functional architecture of higher plant photosystem II supercomplexes. The EMBO J. 2009;28:3052–3063. doi: 10.1038/emboj.2009.232. PubMed DOI PMC

Carlson PS, Smith HH, Dearing RD. Parasexual interspecific plant hybridisation. Proc Natl Acad Sci USA. 1972;69:2292–2294. doi: 10.1073/pnas.69.8.2292. PubMed DOI PMC

Chen YL, Chen LJ, Chu CC, Huang PK, Wen JR, Li HM. TIC236 links the outer and inner membrane translocons of the chloroplast. Nature. 2018;564:125–144. doi: 10.1038/s41586-018-0713-y. PubMed DOI

Chotewutmontri P, Holbrook K, Bruce BD. Plastid protein targeting: preprotein recognition and translocation. Int Rev Cell Mol Biol. 2017;330:227–239. doi: 10.1016/bs.ircmb.2016.09.006. PubMed DOI

Cline K, Henry R. Import and routing of nucleus-encoded chloroplast proteins. Annu Rev Cell Dev Biol. 1996;12:1–26. doi: 10.1146/annurev.cellbio.12.1.1. PubMed DOI

Dainese P, Hoyer-Hansen G, Bassi R. The resolution of chlorophyll a/b binding proteins by a preparative method based on flat bed isoelectric focusing. Photochem Photobiol. 1990;51:693–703. doi: 10.1111/php.1990.51.6.693. PubMed DOI

Dix PJ, Gray JC, Kavanagh TA, Lerbs-Mache S, Medgyesy P, Mordhorst G, Peltier G, Schäfer C, Uijtewaal B. Developments in plastid transformation. Dev Plant Genet Breed. 2000;6:59–66.

Dodds JH, Roberts LW. Experiments in plant tissue culture. Cambridge: Cambridge University Press; 1985.

Eberhard S, Finazzi G, Wollman FA. The dynamics of photosynthesis. Annu Rev Genet. 2008;42:463–515. doi: 10.1146/annurev.genet.42.110807.091452. PubMed DOI

Forsberg J, Allen JF. Protein tyrosine phosphorylation in the transition to light state 2 of chloroplast thylakoids. Photosynth Res. 2001;68:71–79. doi: 10.1023/A:1011891017067. PubMed DOI

Fuks B, Schnell DJ. Mechanism of protein transport across the chloroplast envelope. Plant Physiol. 1997;114:405–420. doi: 10.1104/pp.114.2.405. PubMed DOI PMC

Gao F, Kight AD, Henderson R, Jayanthi S, Patel P, Murchison M, Heyes CD. Regulation of structural dynamics within a signal recognition particle promotes binding of protein targeting substrates. J Biol Chem. 2015;290:15462–15474. doi: 10.1074/jbc.M114.624346. PubMed DOI PMC

Grosser JW, Cmitter FC. Applications of somatic hybridization and cybridization in crop improvement, with citrus as a model. Vitro Cell Dev Biol Plant. 2004;41:220–225. doi: 10.1079/IVP2004634. DOI

Hasegawa K, Yukawa Y, Sugita M, Sugiura M. Organization and transcription of the gene family encoding chlorophyll a/b binding proteins in Nicotiana sylvestris. Gene. 2002;289:161–168. doi: 10.1016/S0378-1119(02)00539-5. PubMed DOI

Kochevenko A, Ratushnyak Y, Kornyeyev D, Stasik O, Porublyova L, Kochubey S, Suprunova T, Gleba Y. Functional cybrid plants of Lycopersicon peruvianum var ‘dentatum’ with chloroplasts of Lycopersicon esculentum. Plant Cell Rep. 2000;19:588–597. doi: 10.1007/s002990050778. PubMed DOI

Kumar A, Cocking EC. Protoplast fusion—a novel approach to organelle genetics in higher plants. Am J Bot. 1987;74:289–1303. doi: 10.1002/j.1537-2197.1987.tb08742.x. DOI

Kuroda H, Maliga P. The plastid clpP1 protease gene is essential for plant development. Nature. 2003;425(6953):86. doi: 10.1038/nature01909. PubMed DOI

Kushnir SG, Shlumukov LR, Pogrebnyak NJ, Berger J, Gleba Y. Functional cybrid plants possessing a Nicotiana genome and an Atropa plastome. Mol Gen Genet. 1987;209:159–163. doi: 10.1007/BF00329852. PubMed DOI

Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680–685. doi: 10.1038/227680a0. PubMed DOI

Liu JH, Xu XY, Deng XX. Intergeneric somatic hybridization and its application to crop genetic improvement. Plant Cell Tissue Organ Cult. 2005;82:19–44. doi: 10.1007/s11240-004-6015-0. DOI

Martin W, Herrmann RG. Gene transfer from organelles to the nucleus: how much, what happens, and why? Plant Physiol. 1998;118:9–17. doi: 10.1104/pp.118.1.9. PubMed DOI PMC

Michel H, Griffin PR, Shabanowitz J, Hunts DF, Bennett J. Tandem mass spectrometry identifies sites of three post-translational modifications of spinach light-harvesting chlorophyll protein II. Proteolytic cleavage, acetylation, and phosphorylation. J Biol Chem. 1991;266:17584–17591. PubMed

Negrutiu I, Hinnisdaels S, Mouras A, Gill BS, Ghartichhetri GB, Davey MR, Gleba YY, Sidorov V, Jacobs M. Somatic versus sexual hybridization—features, facts and future. Acta Bot Neerl. 1989;38:253–272. doi: 10.1111/j.1438-8677.1989.tb01350.x. DOI

Nishimura K, Kato Y, Sakamoto W. Chloroplast proteases: updates on proteolysis within and across suborganellar compartments. Plant Physiol. 2016;171:2280–2293. PubMed PMC

Oblong JE, Lamppa GK. Identification of two structurally related proteins involved in proteolytic processing of precursors targeted to the chloroplast. EMBO J. 1992;11:4401–4409. doi: 10.1002/j.1460-2075.1992.tb05540.x. PubMed DOI PMC

Paila YD, Richardson LG, Schnell DJ. New insights into the mechanism of chloroplast protein import and its integration with protein quality control, organelle biogenesis and development. J Mol Biol. 2015;427:1038–1060. doi: 10.1016/j.jmb.2014.08.016. PubMed DOI PMC

Peter S, Spang O, Medgyesy P, Schafer C. Consequences of intergeneric chloroplast transfers on photosynthesis and sensitivity to high light. Aust J Plant Physiol. 1999;26:171–177.

Porra RJ, Thompson WA, Kriedemann PE. Determination of accurate extinction coefficients and simultaneous-equations for assaying chlorophyll-a and chlorophyll-b with 4 different solvents—verification of the concentration of chlorophyll standards by atomic-absorption spectroscopy. Biochim Biophys Acta. 1989;975:384–394. doi: 10.1016/S0005-2728(89)80347-0. DOI

Puthiyaveetil S, Ibrahim IM, Allen JF. Oxidation–reduction signalling components in regulatory pathways of state transitions and photosystem stoichiometry adjustment in chloroplasts. Plant Cell Environ. 2012;35:347–359. doi: 10.1111/j.1365-3040.2011.02349.x. PubMed DOI

Richter CV, Träger C, Schünemann D. Evolutionary substitution of two amino acids in chloroplast SRP54 of higher plants cause its inability to bind SRP RNA. FEBS Lett. 2008;582:3223–3229. doi: 10.1016/j.febslet.2008.08.014. PubMed DOI

Ruban AV. Plants in light. Commun Integr Biol. 2009;2:50–55. doi: 10.4161/cib.2.1.7504. PubMed DOI PMC

Ruban AV, Johnson MP. Dynamics of higher plant photosystem cross-section associated with state transitions. Photosynth Res. 2009;99:173–183. doi: 10.1007/s11120-008-9387-x. PubMed DOI

Ruban AV, Young AJ, Pascal AA, Horton P. The effects of illumination on the Xanthophyll composition of the photosystem-II light-harvesting complexes of spinach thylakoid membranes. Plant Physiol. 1994;104:227–234. doi: 10.1104/pp.104.1.227. PubMed DOI PMC

Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9:676–682. doi: 10.1038/nmeth.2019. PubMed DOI PMC

Schmitz-Linneweber C, Kushnir S, Babiychuk E, Poltnigg P, Herrmann RG, Maier RM. Pigment deficiency in nightshade/tobacco cybrids is caused by the failure to edit the plastid ATPase alpha-subunit mRNA. Plant Cell. 2005;17:1815–1828. doi: 10.1105/tpc.105.032474. PubMed DOI PMC

Soll J, Schleiff E. Protein import into chloroplasts. Nat Rev Mol Cell Biol. 2004;5:198–218. doi: 10.1038/nrm1333. PubMed DOI

Standfuss J, van Scheltinga ACT, Lamborghini M, Kühlbrandt W. Mechanisms of photoprotection and nonphotochemical quenching in pea light-harvesting complex at 2.5 Å resolution. EMBO J. 2005;24:919–928. doi: 10.1038/sj.emboj.7600585. PubMed DOI PMC

Stengel KF, Holdermann I, Cain P, Robinson C, Wild K, Sinning I. Structural basis for specific substrate recognition by the chloroplast signal recognition particle protein cpSRP43. Science. 2008;321:253–256. doi: 10.1126/science.1158640. PubMed DOI

van Amerongen H, Croce R. Primary processes of photosynthesis, part 1. London: Royal Society of Chemistry; 2007. Structure and function of photosystem II light-harvesting proteins (Lhcb) of higher plants; pp. 329–367.

Yukawa M, Tsudzuki T, Sugiura M. The 2005 version of the chloroplast DNA sequence from tobacco (Nicotiana tabacum) Plant Mol Biol Report. 2005;23:359–365. doi: 10.1007/BF02788884. DOI

Zubko MK, Zubko EI, Patskovsky YV, Khvedynich OA, Fisahn J, Gleba YY, Schieder O. Novel ‘homeotic’ CMS patterns generated in Nicotiana via cybridization with Hyoscyamus and Scopolia. J Exp Bot. 1996;47:1101–1110. doi: 10.1093/jxb/47.8.1101. DOI

Zubko MK, Zubko EI, Gleba YY. Self-fertile cybrids Nicotiana tabacum (+Hyoscyamus aureus) with a nucleo-plastome incompatibility. Theor Appl Genet. 2002;105:822–828. doi: 10.1007/s00122-002-1037-7. PubMed DOI