Regulation of light harvesting in Chlamydomonas: two protein phosphatases are involved in state transitions

Protein phosphorylation plays important roles in short-term regulation of photosynthetic electron transfer. In a mechanism known as state transitions, the kinase STATE TRANSITION 7 (STT7) of Chlamydomonas reinhardtii phosphorylates components of light-harvesting antenna complex II (LHCII). This reversible phosphorylation governs the dynamic allocation of a part of LHCII to photosystem I or photosystem II, depending on light conditions and metabolic demands. Little is however known in the green alga on the counteracting phosphatase(s). In Arabidopsis, the homologous kinase STN7 is specifically antagonized by PROTEIN PHOSPHATASE 1/THYLAKOID-ASSOCIATED PHOSPHATASE 38 (PPH1/TAP38). Furthermore, the paralogous kinase STN8 and the countering phosphatase PHOTOSYSTEM II PHOSPHATASE (PBCP), which count subunits of PSII amongst their major targets, influence thylakoid architecture and high-light tolerance. Here we analyze state transitions in C. reinhardtii mutants of the two homologous phosphatases, CrPPH1 and CrPBCP. The transition from state 2 to state 1 is retarded in pph1, and surprisingly also in pbcp. However both mutants can eventually return to state 1. In contrast, the double mutant pph1;pbcp appears strongly locked in state 2. The complex phosphorylation patterns of the LHCII trimers and of the monomeric subunits are affected in the phosphatase mutants. Their analysis indicates that the two phosphatases have different yet overlapping sets of protein targets. The dual control of thylakoid protein de-phosphorylation and the more complex antenna phosphorylation patterns in Chlamydomonas compared to Arabidopsis are discussed in the context of the stronger amplitude of state transitions and the more diverse LHCII isoforms in the alga.


INTRODUCTION 45
To fulfill their energy requirements, photoautotrophic plants and algae rely on a photosynthetic 46 electron transfer chain embedded in the thylakoid membrane of the chloroplast. Two 47 immunoblotting of total proteins separated by SDS PAGE, the protein was detected in the wild 292 type but not in the pbcp mutant (Fig. 3B). The chlorophyll content and the maximum quantum 293 yield of PSII were not significantly different in pbcp and in the wild type (Table S1). 294 Furthermore, the pbcp mutant showed normal growth under a variety of conditions (Fig. S2). 295 We analyzed state transitions in the pbcp mutant by determining its fluorescence emission 296 spectra at 77 K in St 2 and after a subsequent transition to conditions promoting St 1 for 20 297 minutes. Compared to the wild type, the transition to St 1 was significantly impaired in pbcp 298 (Fig. 3C). Using PAM chlorophyll fluorescence spectroscopy, we observed that anaerobiosis 299 in the dark promoted a transition to St 2 in the pbcp mutant as in the wild type. However, upon 300 aeration and re-oxidation of the PQ pool, the transition from St 2 to St 1 was strongly delayed 301 in pbcp (Fig. 3D). Similar observations were made using the protocol where this transition was 302 induced by light in the presence of DCMU (Fig. S4). Thus, CrPBCP seems to play a major role 303 in state transitions, unlike its homologue in Arabidopsis. 304 To confirm that the state transition phenotype was due to the pbcp mutation, we transformed 305 the pbcp mutant with a plasmid carrying a wild-type copy of PBCP tagged with a sequence 306 encoding a triple HA epitope (PBCP-HA) and a selectable marker (aph7''). Four rescued lines 307 (pbcp;PBCP-HA) that expressed the tagged CrPBCP-HA protein were retained for further 308 analysis. Immunoblotting with the anti-PBCP antibodies (Fig. 4A) showed that the different 309 pbcp;PBCP-HA lines expressed the protein at levels similar or somewhat reduced compared 310 to the wild type. State transitions were restored in the pbcp;PBCP-HA lines, as monitored by 311 PAM fluorescence spectroscopy (Fig. 4B). 312 To investigate the alteration of thylakoid protein phosphorylation in the pbcp mutant, we used 313 SDS-PAGE and immunoblotting (Fig. 3E). With an anti-P-Thr antibody we observed that 314 bands which migrate as components of LHCII (Fig. S5) were over-phosphorylated in pbcp, but 315 not in the complemented pbcp;PBCP-HA lines. Unfortunately, these commercial anti-P-Thr 316 antibodies (now discontinued) did not clearly identify the phosphorylated forms of PSII 317 subunits D2 (PsbD) or CP43 (PsbC) (Fig. S5). High phosphorylation of LHCII constituents was 318 confirmed with the Arabidopsis anti-P-Lhcb2 antibodies, which showed over-phosphorylation 319 in pbcp of the bands that are under-phosphorylated in the stt7 mutant. Thus, CrPBCP has a 320 different range of targets in Chlamydomonas than its homologue in Arabidopsis, where the 321 major targets of PBCP are the subunits of the PSII core. It was striking that excess 322 phosphorylation of thylakoid proteins in pbcp appeared not only after a transition from St 2 to 323 St 1, but also under normal growth conditions (GL, 80 μmol photons m -2 s -1 ), as well as after 324 growth under high light (HL, 300 μmol photons m -2 s -1 ) or in the dark (Fig. 3F). the two phosphatases play partly redundant roles in the regulation of state transitions, we 332 generated double pph1;pbcp mutants by crossing pph1 and pbcp and genotyping the progeny 333 by PCR (Fig. S7). The pph1;pbcp mutants also carry the cw15 mutation like both their parents. 334 The double mutants were, as expected, deficient for both CrPPH1 and CrPBCP (Fig. 5A). The 335 maximum quantum yield of PSII and the chlorophyll content of pph1;pbcp were not 336 significantly different from the wild type (Table S1), and the double mutant showed normal 337 growth under a set of different conditions that were tested (Fig. S2). 338 When state transitions were monitored in pph1;pbcp double mutants, it was remarkable that, 339 in the psal mutant, which is deficient in the docking of LHCII to PSI and thus incapable of 12 completing state transitions (Rantala et al., 2016). These observations prompted us to 362 determine whether in Chlamydomonas the amounts of STT7, CrPPH1 or CrPBCP are altered 363 in the single kinase or phosphatase mutants as well as in the pph1;pbcp double mutant, using 364 SDS PAGE and immunoblotting of protein extracts from cells grown under normal conditions 365 (Fig. 5A). However, no significant differences were observed in the accumulation of the three 366 regulatory proteins. 367 We also investigated the possibility that the phosphatase mutations might be compensated in 368 the long term by changes in the stoichiometry of the photosystems or other major 369 photosynthetic complexes. Proteins extracts of the wild type, the single mutants pph1 and 370 pbcp as well as the double mutant pph1;pbcp grown under normal conditions were compared 371 by SDS PAGE and immunoblotting (Fig. S8). Antisera against representative subunits of the 372 major complexes were used for this analysis: AtpB (ATP synthase), D1 (PSII), PsaA (PSI), 373 Cytf (cytochrome b6f complex) or COXIIb (mitochondrial cytochrome oxidase). However, no 374 significant differences were apparent in the relative amounts of the photosynthetic complexes 375 in the mutant lines. 376 377

CrPPH1 and CrPBCP have overlapping but distinct de-phosphorylation targets 378
In Chlamydomonas, LHCII is composed of monomeric LHCB4 (CP29) and LHCB5 (CP26) as 379 well as trimers of isoforms LHCBM1 through LHCBM9. Based on their primary sequences, the 380 trimer subunits belong to four types ( Fig. S9): type I in which three subgroups can be 381 distinguished (LHCBM3; LHCBM4/LHCBM6/LHCBM8; LHCBM9), type II (LHCBM5), type III 382 (LHCBM2/LHCBM7, which are identical in mature sequence) and type IV (LHCBM1). While 383 the P-Thr antiserum and the Arabidopsis P-Lhcb2 antiserum showed clear differences in the 384 patterns of LHCII phosphorylation in St 1 in both phosphatase mutants (Fig. 6A), the ill-defined 385 specificity of these antisera did not allow the discrimination of the different components of the 386 antenna, or reveal any target specificity of the respective phosphatases. To address these 387 questions, we used Phos-tag polyacrylamide gel electrophoresis (Phos-tag PAGE) followed 388 by immunoblotting with specific antibodies. We obtained previously described antisera against 389 LHCB4, LHCB5 and LHCBM5 (type II) (Takahashi et al., 2006), and also generated antisera 390 against peptides that are characteristic for the other types of LHCBM subunits. Because these 391 isoforms share a high degree of sequence similarity, the antigenic peptides were selected in 392 the N-terminal region of the proteins, which is the most divergent between LHCII types (Fig.  393   S9). The specificity of the affinity-purified antibodies was tested against the recombinant LHCII 394 subunits expressed in E. coli (Fig. S10). The antisera against LHCBM1 (type IV), LHCBM3 395 (type I), LHCB4 and LHCB5 (minor antenna) proved to be very specific. The antiserum against 396 13 LHCBM2/7 (type III) showed minor cross-reactions towards type I isoforms. Finally the 397 antiserum against LHCBM4/6/8 (type I, which share the same sequence in the N-terminal 398 region) also reacted towards LHCBM3 (also type I) but unexpectedly very strongly decorated 399 LHCBM9 (type I). It should be noted however, that LHCBM9 is only expressed under 400 To avoid any bias due to differential recognition by these antibodies of the phosphorylated and  The antibodies against LHCBM1 (type IV) labelled two bands in the wild type in St 2 (Fig. 6B). 419 The lower one co-migrated with the single band in the λ-phosphatase-treated sample,  The antibodies against LHCBM3 (type I) decorated two bands after Phos-tag gel 430 electrophoresis of the λ-phosphatase-treated sample (Fig. 6B), as well as after conventional 431 gel electrophoresis of an untreated sample (Fig. S8B), even though these antibodies were 432 specific for LHCBM3 amongst the recombinant proteins expressed in E. coli (Fig. S10). The 433 lower band (marked with an asterisk), which was partially resolved as a doublet, is unlikely to 434 represent processed forms of LHCBM3 lacking amino-acid residues at the N-terminus 435 (Stauber et al., 2003) since the LHCBM3 antibodies were raised against this region and would 436 not recognize a truncated protein. Thus the lower band may reflect non-specific binding to 437 another protein. There were two additional slower-migrating bands in the wild type in St 2, 438 largely absent from the λ-phosphatase-treated sample, suggesting that LHCBM3 may 439 undergo phosphorylation at more than one site, or that the non-specific protein is 440 phosphorylated as well. One of these bands were also clearly present in stt7, suggesting the 441 involvement of another protein kinase in LHCBM3 phosphorylation. The relative intensity of 442 the top-most of the phosphorylated bands to the non-phosphorylated ones clearly decreased 443 after transition to St 1 in the wild type. Some de-phosphorylation was still apparent in the pph1 444 mutant, but the overall phosphorylation level appeared to be higher. In contrast, the pbcp 445 mutant showed a much higher ratio of phosphorylated bands in both St 2 and St 1, as did the 446 pph1;pbcp double mutant. Thus, CrPBCP appears to play the major role for de-447 With the antibodies against LHCBM4/6/8 (type I), a single major band was observed in the λ- where the most phosphorylated form was prevalent. We tentatively infer that CrPBCP is 458 involved in de-phosphorylation of LHCBM4/6/8 and, since it also affects LHCBM3, more 459 generally in the de-phosphorylation of type I isoforms. 460 The antibodies against LHCBM2/7 (type III) decorated a strong band in the λ-phosphatase-461 treated sample, with a second minor band above it. Likewise, after conventional gel 462 electrophoresis, these antibodies labelled a major and a minor band (Fig. S8B). Amongst the 463 recombinant proteins expressed in E. coli, the LHCBM2/7 antibodies strongly recognized 464 LHCBM2/7, but also weakly cross-reacted with the type I isoforms LHCBM3, LHCBM4/6/8 and 465 LHCBM9 (Fig. S10). The patterns of the phosphorylated bands together with the upper non-466 phosphorylated band (marked with a triangle), but excluding the strong non-phosphorylated 467 band below (marked with a square), were similar to the pattern obtained with anti-468 LHCBM4/6/8, and may mostly represent the cross-reaction to these isoforms. Compared to 469 LHCBM4/6/8, the strong additional non-phosphorylated band (marked with a square) can 470 tentatively be ascribed to LHCBM2/7, which is thus apparently not subject to phosphorylation 471 under these conditions, or only to a small degree. 472 With antiserum against LHCBM5 (type II), two bands were detected in the λ-phosphatase- Unexpectedly, we found that in Chlamydomonas CrPBCP is also involved in state transitions. 518 This is in contrast to PBCP from Arabidopsis, which is required for the de-phosphorylation of 519 several PSII core subunits but not of the LHCII antenna (Samol et al., 2012). In Arabidopsis, 520 lack of PBCP does not affect state transitions and it is only when this phosphatase is strongly to St 1. In particular we found that LHCBM1 was phosphorylated in St 2, that its 588 phosphorylation depends on STT7, and that it was de-phosphorylated in St 1, mainly reliant 589 on CrPPH1. However mutants which lack LHCBM1 were previously found to be able to Compared to Arabidopsis, Chlamydomonas lacks LHCB6 (CP24), but has a more complex 606 complement of the LHCBM subunits composing the LHCII trimers (reviewed by (Crepin and 607 Caffarri, 2018)). Another difference is that in Chlamydomonas, some PSI-LHCI-LHCII 608 complexes contain not only LHCII trimers but also the "minor" antennae LHCB4 and LHCB5 609 pre-acclimated in dim light (~10 µmol m -2 s -1 ) for 2 h with shaking. 642

Mapping of the insertion in pbcp 643
DNA was isolated using the CTAB method and quantified using a NanoDrop 644 spectrophotometer (ThermoScientific). The Resda-PCR protocol was used to identify the left 645 border, the technique was adapted from (Gonzalez-Ballester et al., 2005). The first PCR is 646 performed with Taq polymerase and 10% DMSO. The primers and the program used are 647 described in Table X and Y. A nested PCR was performed on the product of the first PCR 648 diluted to 1/1000, 1/500 or 1/50 using KOD polymerase Xtreme™ (MerckMillipore). The 649 product of the second PCR was then loaded onto an agarose gel 2% and fragments with a 650 molecular weight greater than 800 bp were isolated. PCR products were extracted from the 651 gel and purified with NucleoSpin® Gel and PCR Clean-up Kit (Macherey Nagel) and eluted in 652 water. The Genome Walker technique (Clonetech) was used to identify the right border of the 653 flanking sequence of the cassette insertion site. Genomic DNA was successively digested 654 using the enzyme PvuII followed by the ligation of a specific adapter. The primary PCR uses 655 a primer specific to the insert (AphVIII) and an adapter specific primer. This was followed by

21
The CrPPH1 coding sequence (CDS) was amplified from a Chlamydomonas cDNA library with 677 primers pFC_216 and pFC_217 and cloned by Gibson assembly into pFC18 linearized with 678 EcoRV / BglII, resulting in pFC18_CrPPH1_HA. The CrPBCP CDS was amplified from a cDNA 679 library with primers pFC_218 and pFC_219 and cloned as above into pFC18 resulting in 680 pFC18_CrPBCP_HA. The CrPPH1 CDS without the predicted chloroplast transit peptide 681 (cTP) was amplified with primer pFC_265 and primer pFC_266 from pFC18_CrPPH1_HA, 682 then cloned into vector pet28a digested with NdeI and SalI to obtain pet28a_CrPPH1_∆cTP. 683 The CrPBCP CDS without the predicted cTP was amplified with primer pFC_267 and primer 684 pFC_268 from pFC18_CrPBCP_HA, and cloned into pet28a as above to obtain 685 pET28a_CrPBCP_∆cTP. All vectors used were verified by sequencing. 686

Transformation 687
Nuclear transformation by electroporation was modified from (Shimogawara et al., 1998). The data are normalized on the PSII peak at 680 nm. immunoblotting with antisera against CrPPH1, the HA epitope and D1 (loading control). 795 B) State transitions in the pph1 mutant and two complemented lines were monitored by PAM 796 chlorophyll fluorescence spectroscopy as in Figure 1D. 797 C) Phospho-immunoblot analysis. Total protein extracts (10 µg) of the pph1 mutant and of 798 complemented lines were subjected to SDS-PAGE and immunoblotting with antisera against 799 P-Thr or AtpB (loading control).  chlorophyll fluorescence spectroscopy as in Figure 1D. chlorophyll fluorescence spectroscopy at room temperature as in Figure 1D. PAM chlorophyll fluorescence spectroscopy at room temperature, as in Figure 1D. The data 835 were not normalized to the first Fm' peak. were subjected to SDS-PAGE and immunoblotting with antisera against P-Thr, P-Lhcb2 or 839 AtpB (loading control).

Fig. 2. Complementation of the pph1 mutant
A) Immunoblot analysis. Total protein extracts (50 g) of the wild type, the pph1 mutant and four complemented lines (pph1:PPH1-HA) were subjected to SDS-PAGE and immunoblotting with antisera against CrPPH1, the HA epitope and D1 (loading control).
B) State transitions in the pph1 mutant and two complemented lines were monitored by PAM chlorophyll fluorescence spectroscopy as in Figure 1D.
C) Phospho-immunoblot analysis. Total protein extracts (10 g) of the pph1 mutant and of complemented lines were subjected to SDS-PAGE and immunoblotting with antisera against P-Thr or AtpB (loading control). D) State transitions of the wild type (WT) and the pbcp mutant were monitored by PAM chlorophyll fluorescence spectroscopy as in Figure 1D.
E) Phospho-immunoblot analysis of state transitions. Total protein extracts of wild type and pbcp cells in St 2 and St1 (10 g, treated as in panel 2 C)) were subjected to SDS-PAGE and immunoblotting with antisera against P-Thr, P-Lhcb2 or AtpB (loading control). F) Phospho-immunoblot analysis of light acclimation. The cells were grown in low light and then transferred to the dark (D), growth light (GL, 80 µE m -2 s -1 ) or high light (HL, 300 µE m -2 s -1 ) for 2 hours and analyzed like in panel E).

Fig. 4. Complementation of the pbcp mutant
A) Immunoblot analysis. Total protein extracts (50 g) of the wild type, the pbcp mutant and four complemented lines (pbcp:PBCP-HA) were subjected to SDS-PAGE and immunoblotting with antisera against CrPBCP, the HA epitope or AtpB (loading control).
B) State transitions in the pbcp mutant and two complemented lines were monitored by PAM chlorophyll fluorescence spectroscopy at room temperature as in Figure 1D.
B) State transitions of the wild type (WT) and two pph1;pbcp mutants were monitored by PAM chlorophyll fluorescence spectroscopy at room temperature, as in Figure 1D. The data were not normalized to the first Fm' peak.   B) Phos-tag PAGE and immunoblot analysis. Total protein extracts (10 g) of stt7, wild type, pph1, pbcp, or pph1;pbcp in St 2 and then St 1 were subjected to Phos-tag PAGE and immunoblotting with antisera against LHCBM1, LHCBM3, LHCBM4/6/8, LHCBM2/7, LHCBM5, LHCB4, LHCB5, PsbH or PETO. A sample of the wild type in state 2 was treated with lambda phosphatase (+ ) and used as a reference for the migration of the non-phosphorylated form (NP). The migration of the phosphorylated forms (P) is retarded by the Phos-tag immobilized in the polyacrylamide gel. Bands marked with an asterisk, a triangle and a square are discussed in the main text.