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Neither the Availability of D2 Nor CP43 Limits the Biogenesis of PSII in Tobacco

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Neither the Availability of D2 Nor CP43 Limits the Biogenesis of PSII in Tobacco bioRxiv preprint doi: https://doi.org/10.1101/2020.08.31.272526; this version posted September 1, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Short title: Regulation of PSII biogenesis 2 Author for Contact details: Mark Aurel Schöttler 3 Title: Neither the availability of D2 nor CP43 limits the biogenesis of PSII in tobacco 4 5 All author names: Han-Yi Fu, Rabea Ghandour, Stephanie Ruf, Reimo Zoschke, Ralph 6 Bock, and Mark Aurel Schöttler 7 Affiliation: Max-Planck-Institut für Molekulare Pflanzenphysiologie, Am Mühlenberg 1, D- 8 14476 Potsdam-Golm, Germany 9 10 One sentence summary: PSII biogenesis in tobacco is neither limited by transcript 11 accumulation nor translation of psbD and psbC. 12 13 Author contributions: M.A.S. and R.B. conceived the project. H.Y.F. performed most 14 experiments. R.G. and R.Z. performed ribosomal footprint analysis. S.R. supervised plant 15 transformation and transformant regeneration. M.A.S. wrote the article with input from all co- 16 authors. All authors contributed to the editing and review of the manuscript. M.A.S. serves as 17 the author responsible for contact and ensures communication. 18 Funding information: This research was supported by the Max Planck Society, the 19 International Max-Planck Research School “Primary Metabolism and Plant Growth”, and by a 20 grants from the Deutsche Forschungsgemeinschaft (DFG) to R.B. / R.Z. (SFB-TRR 175, A04) 21 and R.Z. (ZO 302/5-1). 22 Present addresses: Han-Yi Fu, Department of Biological Sciences, National Sun Yat-sen 23 University, Kaohsiung 80424, Taiwan 24 Email address of author for contact: [email protected] 25 26 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.31.272526; this version posted September 1, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 27 Abstract: The pathway of photosystem II assembly is well understood and multiple auxiliary 28 proteins supporting it have been identified. By contrast, little is known about rate-limiting steps 29 controlling PSII biogenesis. In the green alga Chlamydomonas reinhardtii, biosynthesis of the 30 chloroplast-encoded D2 reaction center subunit (PsbD) limits PSII accumulation. To determine 31 the importance of D2 synthesis for PSII accumulation in vascular plants and elucidate the 32 contributions of transcriptional and translational regulation, the 5´-untranslated region of psbD 33 was modified via chloroplast transformation in tobacco. A drastic reduction in psbD mRNA 34 abundance resulted in a strong decrease of PSII content, impaired photosynthetic electron 35 transport, and retarded growth under autotrophic conditions. Overexpression of the psbD 36 mRNA also increased transcript abundance of psbC (the CP43 inner antenna protein), which 37 is co-transcribed with psbD. Because translation efficiency remained unaltered, translation 38 output of pbsD and psbC increased with mRNA abundance. However, this did not result in 39 increased PSII accumulation. The introduction of point mutations into the Shine-Dalgarno-like 40 sequence or start codon of psbD decreased translation efficiency without causing pronounced 41 effects on PSII accumulation and function. These data show that neither transcription nor 42 translation of psbD and psbC are rate-limiting for PSII biogenesis in vascular plants, and that 43 PSII assembly and accumulation in tobacco are controlled by different mechanisms than in 44 Chlamydomonas. 45 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.31.272526; this version posted September 1, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 46 Introduction 47 Photosystem II (PSII), the water-plastoquinone oxidoreductase protein supercomplex of 48 oxygenic photosynthesis, catalyzes the first step of linear electron flux in the thylakoid 49 membranes of cyanobacteria and photosynthetic eukaryotes (Shen, 2015). Electron transfer 50 within PSII is initiated by a light-induced charge separation at the reaction center (RC) 51 chlorophyll-a dimer P680, which then transfers one electron to the first quinone acceptor QA. 52 From QA, the electron is transferred to the plastoquinone-binding site (QB-site) and reduces 53 plastoquinone to plastosemiquinone. Following a second charge separation, the subsequent 54 reduction of the plastosemiquinone to plastoquinol is coupled to the uptake of two protons from 55 the stroma. Plastoquinol is released into the thylakoid membrane and re-oxidized at the + 56 cytochrome b6f complex (cyt b6f). After each charge separation, P680 is reduced by the oxygen- 57 evolving Mn4O5Ca complex (OEC) near the lumenal surface of PSII. After four oxidation 58 steps, two water molecules are oxidized to molecular oxygen, and the Mn4O5Ca cluster is 59 reduced again by the four electrons abstracted from the two water molecules (Dau et al., 2012; 60 Vinyard and Brudvig, 2017). 61 PSII functions as a dimer, and each monomer is composed of more than 20 core subunits, and 62 additional peripheral antenna proteins of the light-harvesting complex II (LHCII) type. With a 63 molecular mass of up to 1300 kDa, the PSII-LHCII supercomplexes are the largest complexes 64 of the photosynthetic apparatus (Dekker and Boekema, 2005; Kouril et al., 2012; Shen, 2015). 65 The PSII RC core is formed by the D1 and D2 heterodimer that binds all redox-active cofactors 66 necessary for rapid electron transfer from water to plastoquinone. D1 and D2 are encoded in 67 the chloroplast genome (plastome) by the psbA and psbD genes, respectively. An additional 68 redox-active cofactor, the heme of cytochrome b559 (cyt b559), is bound by PsbE and PsbF, 69 which are also plastome-encoded. Cyt b559 is an essential structural component and required 70 for PSII assembly, but its physiological function is enigmatic. It has been suggested to mediate 71 a cyclic electron flux within PSII when the PSII donor side is inactive (Shinopoulos and 72 Brudvig, 2012; Takagi et al., 2019), and may function as a plastoquinol oxidase (Bondarava et 73 al., 2003; Bondarava et al., 2010). 74 The PSII RC is surrounded by the inner antenna proteins CP47 (PsbB) and CP43 (PsbC) also 75 encoded in the plastome, and multiple membrane-intrinsic low molecular mass subunits 76 encoded either in the plastome or the nucleus. Some of these subunits are essential for PSII 77 accumulation or function, while others are not (reviewed by Shi et al., 2012; Plöchinger et al., 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.31.272526; this version posted September 1, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 78 2016). Additionally, the three nuclear-encoded extrinsic subunits PSBO, PSBP, and PSBQ are 79 associated with the lumenal side of PSII and stabilize the OEC (Bricker et al., 2012). 80 The process of de novo PSII assembly is largely conserved from cyanobacteria to vascular 81 plants, except that the subcellular localization of some steps varies (Komenda et al., 2012; 82 Nickelsen and Rengstl, 2013). Assembly proceeds in a modular fashion and starts with cyt b559, 83 which stably accumulates even in the absence of the other PSII RC subunits (Müller and 84 Eichacker, 1999; Kanervo et al., 2008; Plöscher et al., 2009; Schmitz et al., 2012). 85 Subsequently, D2 is co-translationally inserted into the thylakoid membrane (Zoschke and 86 Barkan, 2015) and binds to cyt b559, forming the D2-cyt b559 subcomplex (Komenda et al., 87 2004). The addition of a complex consisting of pre-D1, a D1 protein precursor with a short C- 88 terminal extension, and PsbI leads to the formation of the “RC-like complex” (Dobakova et al., 89 2007), which is stabilized by multiple auxiliary proteins (Li et al., 2019). After maturation of 90 the D1 protein by the lumenal C-terminal processing protease CTPA (Che et al., 2013), the 91 “RC47 subcomplex” is formed by binding of CP47 (PsbB) and the rapid addition of PsbH, 92 PsbR, and PsbTc (Rokka et al., 2005). Finally, CP43 (PsbC), PsbK, and PsbZ bind to the RC 93 (Rokka et al., 2005; Boehm et al., 2011). This complex migrates into the grana stacks, where 94 it is photoactivated by binding the extrinsic OEC subunits (Mamedov et al., 2000; Mamedov 95 et al., 2008). Ultimately, two PSII monomers form a PSII dimer and bind additional LHCIIs 96 (Shevela et al., 2016). During the assembly process, more than 20 auxiliary proteins located in 97 the stroma, the thylakoids, and the lumen transiently bind to PSII. They protect and stabilize 98 all assembly intermediates except for the early D2-cyt b559 subcomplex, which appears to 99 accumulate without the support of auxiliary proteins (Shi et al., 2012; Nickelsen and Rengstl, 100 2013; Plöchinger et al., 2016). Some auxiliary proteins mediate chlorophyll and cofactor 101 insertion into the nascent complex (Hey and Grimm, 2020). 102 In vascular plants, PSII contents are highly variable (reviewed by Schöttler and Toth, 2014). 103 In Arabidopsis thaliana, PSII contents increase almost fourfold from 2.5 to 9 mmol PSII per 104 mol chlorophyll, at the expense of the peripheral LHCII, when the actinic light intensity is 105 increased from 35 to 600 µE m-2 s-1 (Bailey et al., 2001). Similar changes have been observed 106 in tobacco (Nicotiana tabacum; Petersen et al., 2011; Schöttler et al., 2017) and in Chamerion 107 angustifolium (Murchie and Horton, 1998). The mechanisms, by which these adjustments are 108 achieved, are largely unknown.
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