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Page 1 of 37 Physiologia Plantarum 1 2 3 The heterologous expression of a plastocyanin in the diatom 4 5 Phaeodactylum tricornutum improves cell growth under iron-deficient 6 7 conditions 8 9 10 11 12 Carmen Castell1, Pilar Bernal-Bayard1, José M. Ortega1, Mercedes Roncel1, Manuel Hervás1, José 13 14 A. Navarro1* 15 16 17 18 19 20 21 1Instituto de Bioquímica VegetalFor y PeerFotosíntesis, Review cicCartuja, Universidad de Sevilla and CSIC, 22 23 Seville, Spain. 24 25 26 27 28 29 30 31 32 *Corresponding author: José A. Navarro, Instituto de Bioquímica Vegetal y Fotosíntesis, Centro de 33 34 Investigaciones Científicas Isla de la Cartuja, Universidad de Sevilla and CSIC, Américo Vespucio 35 36 49, 41092-Sevilla, Spain; E-mail: [email protected] 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 1 Physiologia Plantarum Page 2 of 37 1 2 3 ABSTRACT 4 5 6 We have investigated if the heterologous expression in the diatom Phaeodactylum tricornutum 7 8 of a functional green alga plastocyanin, as an alternative to cytochrome c6 under iron-limiting 9 conditions, can improve photosynthetic activity and cell growth. Transformed P. tricornutum strains 10 11 were obtained expressing a single-mutant of the plastocyanin from the green algae Chlamydomonas 12 13 reinhardtii that had previously shown to be the more effective in reducing P. tricornutum 14 photosystem I in vitro. Our results indicate that even the relatively low intracellular concentrations of 15 16 holo-plastocyanin detected (≈4 µM) are enough to promote an increased growth (up to 60%) under 17 iron-deficient conditions as compared with the WT strain, measured as higher cell densities, content 18 19 in pigments and active photosystem I, global photosynthetic rates per cell and even cell volume. In 20 addition, the presence of plastocyanin as an additional photosynthetic electron carrier seems to 21 For Peer Review 22 decrease the over-reduction of the plastoquinone pool, and consequently promotes an improvement 23 24 in the maximum quantum yield of both photosystem II and I, together with a decrease in the acceptor 25 side photoinhibition of photosystem II –also associated to a reduced oxidative stress–, a decrease in 26 27 the peroxidation of membrane lipids in the choroplast, and a lower degree of limitation on the donor 28 side of photosystem I. 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 Abbreviations: 45 46 Cb6f, cytochrome b6f; Cc550, cytochrome c550; Cc6, cytochrome c6; Cf, cytochrome f; Chl, 47 chlorophyll; ET, electron transfer; Fd, ferredoxin; Fld, flavodoxin; Pc, plastocyanin; PQ, 48 49 plastoquinone; PSI, photosystem I; PSII, photosystem II; P700, photosystem I primary donor; TL, 50 51 STL and HTL, thermoluminescence, standard thermoluminescence and high temperature 52 thermoluminescence, respectively; tmax, temperature of the maximum of a TL band; t1/2, half-life 53 54 time. 55 56 57 58 59 60 2 Page 3 of 37 Physiologia Plantarum 1 2 3 INTRODUCTION 4 5 6 7 Photosynthetic productivity, which is equally shared between terrestrial and oceanic systems, is due 8 to a wide variety of organisms, from cyanobacteria to eukaryotic microalgae and plants (Hall 1976, 9 10 Field et al. 1998). Within the group of eukaryotic microalgae, diatoms constitute the dominant life 11 form in oceanic phytoplankton, probably representing the largest group of biomass producers on 12 13 earth (Bowler et al. 2010). 14 The oxygenic photosynthetic electron transfer (ET) chain is composed by three large 15 16 multisubunit membrane-embedded complexes, all of which contain iron cofactors: photosystem II 17 18 (PSII), cytochrome b6f (Cb6f) and photosystem I (PSI), that are connected by mobile electron carriers 19 (Blankenship 2014). In terrestrial plants, electrons from the Cb6f complex are transferred to PSI 20 21 inside the thylakoidal lumenFor by means Peer of plastocyanin Review (Pc), a small (ca. 100 amino acids) soluble 22 Type-1 blue copper-protein, whereas electrons from PSI are delivered to ferredoxin (Fd), an iron- 23 24 sulphur protein. In aquatic systems however, many photosynthetic microorganisms maintain 25 26 alternative electron transporters couples (Hervás et al. 2003). Thus, in most cyanobacteria and 27 unicellular green algae, Pc is produced when copper is available, but under low-copper conditions Pc 28 29 is replaced by cytochrome c6 (Cc6), a typical Class I c-type cytochrome (ca. 90 amino acids). By its 30 turn, the flavoprotein flavodoxin (Fld) replaces Fd under iron-limiting conditions (Hervás et al. 2003, 31 32 De la Rosa et al. 2006, Sancho 2006, Sétif 2006). The functional and structural equivalence of both 33 couples, Pc/Cc and Fd/Fld, is well established (Hervás et al. 1995, 2003, Hippler and Drepper 2006, 34 6 35 Bendall and Howe 2016). 36 37 The existence of redox alternative couples in photosynthesis can be understood as an 38 adaptation to fluctuations in the bioavailability of iron in the aquatic environments. Many studies 39 40 have revealed that iron limitation is the major factor controlling the primary productivity and growth 41 of phytoplankton in vast oceanic areas (Boyd et al. 2007, Moore and Braucher 2008). Massive 42 43 oceanic iron fertilization experiments have also shown the appearance of algal blooms, in many 44 45 cases dominated by diatoms, suggesting that these eukaryotic algae, in particular, are very sensitive 46 to iron limitations (Morrissey and Bowler 2012). Among other effects, iron starvation causes a 47 48 severe decrease in the chlorophyll content and drastic alterations in the photosynthetic efficiency in 49 diatoms, promoting a decrease in the content of both the iron-rich PSI and Cb6f complexes and 50 51 resulting in an increased PSII:PSI ratio (Allen et al. 2008, Lommer et al. 2012, Marchetti et al. 2012). 52 However, the occurrence of alternative couples is only partially maintained in the red lineage 53 54 of photosynthetic organisms, that diverged along evolution from the green lineage, and that 55 56 comprises, among others, red algae, cryptophytes, stramenopiles (that include diatoms) and 57 haptophytes (Falkowski et al. 2004). Thus, whereas Fld is widely produced in the red lineage as an 58 59 alternative to Fd under low iron conditions (Pierella Karlusich et al. 2014), Cc6 is the only electron 60 carrier from cytochrome f (Cf) –in the Cb6f complex– to PSI in red algae and in the majority of 3 Physiologia Plantarum Page 4 of 37 1 2 3 organisms belonging to the red-plastid lineage, including diatoms, as confirmed by protein 4 5 characterization, genome sequencing and transcriptomic analyses (Akazaki et al. 2009, Bowler et al. 6 2010, Blaby-Haas and Merchant 2012, Bernal-Bayard et al. 2013, Groussman et al. 2015). The 7 8 absence of Pc as an alternative to Cc6 can thus represent an extra requirement of iron that affects the 9 adaption of these organisms to iron limitations, as Cc replacement has been suggested to reduce iron 10 6 11 requirements by up to 10% in open-ocean waters (Boyd et al. 2007, Guo et al. 2010). However, 12 13 although petJ genes encoding Cc6 have been found in all the sequenced stramenopiles genomes, petE 14 genes encoding Pc, closely related to those from green algae, have been also found in the genomes of 15 16 several strains of oceanic diatom species. These include Thalassiosira oceanica and Fragilariopsis 17 cylindrus, but also the haptophyte Emiliania huxleyi and the dinoflagellate Karenia brevis (Nosenko 18 19 et al. 2006, Peers and Price 2006, Blaby-Haas and Merchant 2012). In addition, analyses of 20 microbial eukaryotic transcriptomes detected transcripts encoding for putative Pcs in at least one 21 For Peer Review 22 species from each of the major diatom classes (Groussman et al. 2015). All of this has been 23 24 attributed to the acquisition of petE Pc genes from green alga by horizontal gene transfer, as an 25 adaptation to iron limitation (Peers and Price 2006, Marchetti et al. 2012, Groussman et al. 2015, 26 27 Hippmann et al. 2017). A particular strain of the open ocean diatom T. oceanica has been even 28 described to express constitutively Pc as the main electron carrier from Cf to PSI, as the two Cc 29 6 30 genes of its genome are weakly expressed (Peers and Price 2006, Lommer et al. 2012, Kong and 31 32 Price 2020). The presence of Pc in diatoms leaves, however, some open questions, such as the 33 mechanism regulating either its expression or a putative Cc6/Pc alternation in function of the 34 35 copper/iron levels (Lommer et al. 2012, Marchetti et al. 2012, Roncel et al. 2016, Hippmann et al. 36 2017). In addition, the existence of a still not found specific transporting system carrying copper into 37 38 the lumen, to be incorporated into the Pc, is another unresolved question (Levy et al. 2008, Guo et al. 39 2010, 2015, Kong and Price 2019). 40 41 Clear evidences for the production of a functional Pc protein have been only presented in the 42 43 case of T. oceanica, where relatively small amounts per cell of the holoprotein were detected, 44 purified and partially sequenced (Peers and Price 2006, Kong and Price 2020). However, there is a 45 46 discordance between the theoretical molecular mass of the T. oceanica mature Pc (10,300 Da) and 47 the apparent experimental mass found for the purified protein (18,200 Da) (Peers and Price 2006). In 48 49 any case, the Pc-translated sequence in diatoms is closely related to that from green algae, and thus 50 51 the "red-type" acquired Pc would show the features of a "green-type" Pc.

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