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Article Exploring the Link between Photosystem II Assembly and of the psbA mRNA

Prakitchai Chotewutmontri †, Rosalind Williams-Carrier † and Alice Barkan * Institute of , University of Oregon, Eugene, OR 97403, USA; [email protected] (P.C.); [email protected] (R.W.-C.) * Correspondence: [email protected] These authors contributed equally to this work. †  Received: 20 December 2019; Accepted: 21 January 2020; Published: 25 January 2020 

Abstract: Photosystem II (PSII) in and cyanobacteria contains approximately fifteen core proteins, which organize numerous pigments and prosthetic groups that mediate the light-driven water-splitting activity that drives oxygenic photosynthesis. The PSII reaction center protein D1 is subject to photodamage, whose repair requires degradation of damaged D1 and its replacement with nascent D1. Mechanisms that couple D1 synthesis with PSII assembly and repair are poorly understood. We address this question by using ribosome profiling to analyze the translation of chloroplast mRNAs in maize and Arabidopsis mutants with defects in PSII assembly. We found that OHP1, OHP2, and HCF244, which comprise a recently elucidated complex involved in PSII assembly and repair, are each required for the recruitment of ribosomes to psbA mRNA, which encodes D1. By contrast, HCF136, which acts upstream of the OHP1/OHP2/HCF244 complex during PSII assembly, does not have this effect. The fact that the OHP1/OHP2/HCF244 complex brings D1 into proximity with three proteins with dual roles in PSII assembly and psbA ribosome recruitment suggests that this complex is the hub of a translational autoregulatory mechanism that coordinates D1 synthesis with need for nascent D1 during PSII biogenesis and repair.

Keywords: photosystem II; psbA; translation; chloroplast; plastid; ribosome profiling; Arabidopsis; maize

1. Introduction Photosystem II (PSII) is a large protein–pigment complex whose light-driven water splitting activity is the foundation of oxygenic photosynthesis (reviewed in [1,2]). PSII localizes to thylakoid membranes of cyanobacteria and chloroplasts, where it consists of a highly conserved core complex surrounded by distinct light harvesting complexes. The PSII core contains approximately fifteen proteins in 1:1 stoichiometry, which serve as scaffolds to organize the numerous pigments and prosthetic groups that mediate light-absorption, charge separation and electron transport. This structure is assembled in an ordered pathway with the assistance of accessory factors that are, in many cases, conserved between cyanobacteria and chloroplasts [3]. Furthermore, PSII is a highly dynamic structure due to the damaging effects of light on its D1 reaction center protein. PSII repair involves an elaborate process that includes partial disassembly of the complex, proteolysis of damaged D1, new D1 synthesis, and re-assembly of the complex [4–6]. PSII biogenesis in plants and algae is further complicated by the fact that genes encoding PSII subunits are distributed between the nuclear and chloroplast genomes. During the biogenesis of chloroplasts, chloroplast-encoded PSII subunits are synthesized at rates that are roughly proportional to their stoichiometry in PSII [7,8]. However, in mature chloroplasts, exposure to light induces an excess of D1 synthesis [9–11], which provides D1 for PSII repair. Thus, mechanisms must exist to program

Plants 2020, 9, 152; doi:10.3390/plants9020152 www.mdpi.com/journal/plants Plants 2020, 9, 152 2 of 13 either stoichiometric or excess D1 synthesis in accord with specific developmental and environmental conditions. Seminal experiments with the green alga Chlamydomonas reinhardtii (Chlamydomonas) revealed mechanisms that couple the synthesis of some PSII subunits with their assembly status [12]. However, the underlying mechanisms and the degree to which similar themes apply in plants remain unclear. In this study we explored these issues by examining how mutations that disrupt PSII assembly impact the association of ribosomes with chloroplast mRNAs encoding PSII subunits. We focus on mutants lacking four nucleus-encoded proteins: OHP1, OHP2, HCF244, and HCF136. These proteins are conserved in cyanobacteria, where the OHP orthologs are named HliD and HliC, the HCF244 ortholog is named Ycf39, and the HCF136 ortholog is named Ycf48 (reviewed in [3]). Recent work has provided evidence that OHP1, OHP2, and HCF244 (and their cyanobacterial counterparts) comprise a complex required for the addition of nascent D1 to a D2/PsbE/PsbF-containing assembly intermediate [13–17]. Ycf48/HCF136 is believed to act upstream of this step by promoting incorporation of nascent D1 into the OHP1/OHP2/HCF244 complex [14,18–21]. In addition, there is evidence that these proteins are involved not only in the de novo biogenesis of PSII, but also in PSII repair [14,20]. HCF244 was originally discovered in a genetic screen in Arabidopsis [22], at which time it was shown to be essential for PSII accumulation. The PSII defect in the hcf244 mutant was ascribed to the failure to translate the chloroplast psbA mRNA, which encodes D1. Thus, there is evidence that HCF244 functions in both PSII assembly and D1 synthesis, evoking the intriguing possibility that it plays a role in coupling these two processes. The accumulation of HCF244, OHP1, and OHP2 is mutually inter-dependent [14,16], leading to the prediction that psbA translation would be similarly affected in ohp1, ohp2, and hcf244 mutants. However, polysome analyses led to the conclusion that OHP1 and OHP2 do not affect the recruitment of ribosomes to psbA mRNA [14], contrasting with the polysome data for HCF244 [22]. To clarify the role of the HCF244/OHP1/OHP2 complex in coordinating D1 synthesis with PSII assembly, we used ribosome profiling to analyze chloroplast translation in a maize hcf244 mutant and in Arabidopsis ohp1 and ohp2 mutants. These experiments used deep-sequencing to map and quantify ribosome protected fragments, a method that can be useful for clarifying the interpretation of polysome and pulse-labeling experiments (see, e.g., [23]). Our results show that maize HCF244 is required for psbA translation initiation, consistent with the prior conclusions from polysome data in Arabidopsis [22]. However, in contrast with previous conclusions [14], our results indicate that OHP1 and OHP2 are likewise required for the recruitment of ribosomes to the psbA mRNA. This observation is consistent with the loss of HCF244 when OHP1 and OHP2 expression is reduced [14,16]. Importantly, the absence of HCF136, which also functions very early in PSII assembly, does not decrease ribosome occupancy on psbA mRNA in maize or Arabidopsis. These results reveal an intimate connection between psbA translation and a specific PSII assembly complex, and suggest models for the coupling of D1 synthesis to D1 assembly during PSII biogenesis and repair.

2. Results

2.1. Maize HCF244 Is Required for the Recruitment of Ribosomes Specifically to psbA mRNA Polysome analysis of an Arabidopsis hcf244 mutant provided evidence that HCF244 promotes psbA ribosome association [22]. However, interpretation of psbA polysome data can be challenging due to the large pool of untranslated psbA RNA (see, for example [23]). In addition, effects of HCF244 on translation of other chloroplast mRNAs had not been examined. To address these issues, we recovered transposon insertion alleles of maize hcf244 (Zm-hcf244) from the Photosynthetic Mutant Library [24], and analyzed chloroplast translation in the mutants by ribosome profiling. We recovered two independent alleles, which had Mu transposon insertions at the identical position in the 5’-untranslated region (Figure1a). Homozygous mutant seedlings appear very similar to their wild-type siblings when grown in a growth chamber under moderate light conditions, and they grow normally in soil until Plants 2020, 9, x FOR PEER REVIEW 3 of 13 Plants 2020, 9, 152 3 of 13 type siblings when grown in a growth chamber under moderate light conditions, and they grow normally in soil until seed reserves are exhausted (Figure 1b). Immunoblot analysis showed that this seedinsertion reserves conditions are exhausted a severe (Figure loss1 b).of HCF244 Immunoblot protein analysis (Figure showed 1c). As that expected this insertion based on conditions the hcf244 a severemutant loss phenotype of HCF244 in Arabidopsis, protein (Figure Zm-1c).hcf244 As expected mutants basedlack the on theD1 reactionhcf244 mutant center phenotypeprotein of inPSII Arabidopsis,(Figure 1c). Zm- Corehcf244 subunitsmutants of lackthe thechloroplast D1 reaction ATP center synthase, protein Ph ofotosystem PSII (Figure I (PSI),1c). Core cytochrome subunits b6f ofcomplex, the chloroplast and Rubisco ATP synthase, accumulate Photosystem normally I (PSI), (Figure cytochrome 1c), consistentb6f complex, with and the Rubisco established accumulate role for normallyHCF244 (Figure specifically1c), consistent in PSII biogenesis. with the established role for HCF244 specifically in PSII biogenesis.

FigureFigure 1. 1.Maize Maize mutants mutants usedused inin thisthis study.study. ( (aa)) Insertion Insertion sites sites of of MuMu transposonstransposons in in Zm- Zm-hcf244hcf244 and andZm- Zm-hcf136hcf136 mutants.mutants. The The Zm- Zm-hcf244-1hcf244-1 andand Zm- hcf244-3hcf244-3 alleles arosearose independentlyindependently but but have have an an insertioninsertion at theat the same same position. position. The The sequences sequences flanking flanking each each insertion insertion are are shown shown below, below, with with the the target target sitesite duplications duplications underlined. underlined. (b ()b Mutant) Mutant seedlings seedlings and and their their phenotypically phenotypically normal normal siblings siblings at at the the developmentaldevelopmental stage stage used used for for experiments experiments reported reported here. here. Plants Plants were were grown grown in in soil soil for for eight eight days days as as describeddescribed in in Materials Materials and and Methods. Methods. Zm- Zm-hcf244-1hcf244-1/-3/-3 is theis the progeny progeny of of across a cross between between heterozygous heterozygous plantsplants harboring harboring each each allele. allele. (c )(c Immunoblot) Immunoblot analysis analysis of of leaf leaf proteins proteins in in Zm- Zm-hcf24hcf244 and4 and Zm- Zm-hcf136hcf136 mutants.mutants. Replicate Replicate blots blots were were probed probed to detect to detect HCF244, HCF244, HCF136, HCF136, AtpB AtpB (subunit (sub ofunit the chloroplastof the chloroplast ATP synthase), D1, PsaD (subunit of PSI), and PetD (subunit of the cytochrome b f complex). An excerpt of ATP synthase), D1, PsaD (subunit of PSI), and PetD (subunit of the cytochrome6 b6f complex). An oneexcerpt of the of Ponceau one of the S-stained Ponceau blots S-stained illustrates blots relative illustra sampletes relative loading sample and loading the abundance and the ofabundance the large of subunitthe large of Rubisco subunit (RbcL).of Rubisco (RbcL). We compared chloroplast translation in Zm-hcf244 mutants to that in their phenotypically normal We compared chloroplast translation in Zm-hcf244 mutants to that in their phenotypically siblings by sequencing ribosome-protected mRNA fragments (Ribo-seq) recovered from seedling leaf normal siblings by sequencing ribosome-protected mRNA fragments (Ribo-seq) recovered from tissue. Two replicate experiments showed a specific loss of ribosomes from psbA mRNA in the mutant seedling leaf tissue. Two replicate experiments showed a specific loss of ribosomes from psbA mRNA (Figure2a,b and Figure S1a). RNA-seq analysis of the same leaf homogenate used for Ribo-seq showed in the mutant (Figure 2a,b and Figure S1a). RNA-seq analysis of the same leaf homogenate used for no significant changes in the abundance of chloroplast RNAs (Figure2a), but slot-blot hybridization Ribo-seq showed no significant changes in the abundance of chloroplast RNAs (Figure 2a), but slot- analysis suggested a modest (~30%) decrease in psbA mRNA abundance in the mutant (Figure S1a). blot hybridization analysis suggested a modest (~30%) decrease in psbA mRNA abundance in the These results indicate that the loss of psbA ribosome footprints in the mutant is due almost entirely to mutant (Figure S1a). These results indicate that the loss of psbA ribosome footprints in the mutant is reduced translation. To more clearly visualize effects on translation of other chloroplast open reading due almost entirely to reduced translation. To more clearly visualize effects on translation of other frames (ORFs) encoding PSII subunits, the relative abundance of ribosome footprints in the wild-type chloroplast open reading frames (ORFs) encoding PSII subunits, the relative abundance of ribosome relative to the mutant for these genes is plotted in Figure2c. This display suggests a slight increase footprints in the wild-type relative to the mutant for these genes is plotted in Figure 2c. This display in ribosome footprints on many PSII genes in the mutant; however, this is likely due, at least in suggests a slight increase in ribosome footprints on many PSII genes in the mutant; however, this is part, to the fact that psbA ribosome footprints make up such a high fraction of the total footprints likely due, at least in part, to the fact that psbA ribosome footprints make up such a high fraction of

Plants 2020, 9, 152 4 of 13 Plants 2020, 9, x FOR PEER REVIEW 4 of 13 thein thetotal wild-type; footprints the in the sharp wild-type; drop in thepsbA sharpreads drop in the in psbA mutant reads therefore in the mutant increases therefore cpRPKM increases values cpRPKM(calculated values by normalizing (calculated to by total normalizing chloroplast to reads)total chloroplast for other genes. reads) The for residualother genes. ribosomes The residual bound ribosomesto psbA mRNA bound in to the psbA mutant mRNA were in the distributed mutant were along distributed the ORF similarlyalong the toORF those similarly in the to wild-type those in the(Figure wild-type2d), indicating (Figure 2d), that indicating the loss of that ribosomes the loss of is notribosomes due to is ribosome not due stallingto ribosome early stalling in the ORF.early inThese the resultsORF. These show thatresults HCF244 show activatesthat HCF244psbA translationactivates psbA initiation, translation validating initiation, the conclusion validating in the the conclusionoriginal report in the on Arabidopsisoriginal report HCF244 on Arabidopsis [22]. These HCF244 results show [22]. furtherThese results that HCF244’s show further function that in HCF244’spsbA translation function is conservedin psbA translation in monocots, is conserved and that in HCF244 monocots, has littleand that or no HCF244 effect on has translation little or no of effectother on chloroplast translation mRNAs. of other chloroplast mRNAs.

FigureFigure 2. 2. AnalysisAnalysis of of chloroplast chloroplast gene gene expression expression in in the the Zm- Zm-hcf244hcf244 mutantmutant by by Ribo-seq Ribo-seq and and RNA-seq. RNA-seq. ResultsResults from from a replicate experiment are shown in Figure S1a. ( a) Comparison of ribosome footprint abundanceabundance (Ribo-seq)(Ribo-seq) and and RNA RNA abundance abundance (RNA-seq) (RNA-seq) for allfor chloroplast all chloroplast genes genes in the Zm-in thehcf244 Zm-mutanthcf244 mutantand its phenotypically-normaland its phenotypically-normal sibling (WT). sibling Each (WT) symbol. Each represents symbol onerepresents gene. Values one gene. are expressed Values are as expressedreads per kilobaseas reads per in the kilobase ORF perin the million ORF per reads million mapped reads to mapped chloroplast to chloroplast ORFs (cpRPKM). ORFs (cpRPKM). (b) Screen (captureb) Screen from capture the Integrated from the Integrated Genome Viewer Genome (IGV) View showinger (IGV) the showing distribution the distribution of ribosome of footprintsribosome footprintsalong psbA alongand adjacentpsbA and ORFs adjacent in the ORFs wild-type in the andwild-type Zm-hcf244 and mutant.Zm-hcf244 The mutant. Y-axis showsThe Y-axis the number shows theofreads number at eachof reads position at each (not position normalized); (not norma the maximumlized); the Y-axismaximum values Y-axis are shownvalues are in the shown upper in left,the upperand were left, chosen and were such chosen that peak such heights that peak in psbD heightsand inpsbC psbDwere and similarpsbC were in the similar two samples.in the two (c )samples. Ratio of (Ribo-seqc) Ratio of reads Ribo-seq in the reads wild-type in the relativewild-type to relative the mutant to the for mutant chloroplast for chloroplast genes encoding genes encoding PSII subunits. PSII subunits.Several atp Severalgenes areatp genes shown are for shown comparison. for comparison. The average The of averag the twoe of replicates the two (+/replicates– SD) is shown.(+/– SD) (dis) shown.Distribution (d) Distribution of ribosome of footprints ribosome alongfootprints the psbA alongORF. the ThepsbA Y-axis ORF. maximaThe Y-axis were maxima adjusted were to adjusted facilitate tocomparison facilitate comparison of ribosome of distributions ribosome distributions in the two genotypes. in the two genotypes.

2.2.2.2. OHP1 OHP1 and and OHP2 OHP2 Are Are Required Required to to Recr Recruituit Ribosomes Specifically Specifically to psbA mRNA. TheThe abundance of of HCF244 is is strongly reduced in OHP1 and OHP2 insertion mutants mutants and and knockdownknockdown lines lines [14,16]. [14,16]. Therefor Therefore,e, it it might might be be expected expected that that ohp1ohp1 and ohp2 mutants would exhibit psbApsbA translation defects defects similar to those in hcf244hcf244 mutants.mutants. In In fact, pulse-labeling data suggested reducedreduced D1 D1 synthesis in Arabidopsis ohp1 and ohp2 mutant and knockdown lines [[14,16].14,16]. Polysome analysesanalyses of ohp1ohp1 andand ohp2ohp2 mutants,mutants, however, however, led to the the conclusion conclusion that that OHP1 OHP1 and and OHP2 OHP2 do do not not affect affect psbApsbA translationtranslation initiation initiation [14]. [14]. ToTo clarify the role of OHP1 and OHP2 in psbA translation, we analyzed Arabidopsis ohp1 and ohp2ohp2 mutant leaf tissue by Ribo-seq. Both Both mutant mutantss exhibited exhibited a a strong strong and and specific specific decrease decrease in in psbApsbA ribosomeribosome footprints footprints (Figures (Figure3 a,b3a,b and and Figure 4a,b). 4Replicatea,b). Replicate experiments experiments gave similar gave similar results results (Figure (Figure S1b,c). ItS1b,c). was shown It was shownpreviously previously that psbA that mRNApsbA mRNA accumulates accumulates normally normally in ohp1 in andohp1 ohp2and mutantsohp2 mutants [14], and [14], we confirmed this to be true by slot-blot or RNA-gel blot hybridization analysis of the same leaf homogenates used for Ribo-seq (Figure S1b,c). The ohp1 and ohp2 mutations had little effect on

Plants 2020, 9, 152 5 of 13 and we confirmed this to be true by slot-blot or RNA-gel blot hybridization analysis of the same Plants 2020, 9, x FOR PEER REVIEW 5 of 13 leaf homogenates used for Ribo-seq (Figure S1b,c). The ohp1 and ohp2 mutations had little effect on ribosomeribosome occupancyoccupancy onon otherother chloroplastchloroplast genesgenes encodingencoding PSIIPSII subunitssubunits (Figures(Figures3 3cc and and4 c)4c) and and did did notnot significantlysignificantly alteralter thethe distributiondistribution ofof the residual ribosomes on psbA mRNA (Figures3 3dd andand4 d).4d). Therefore,Therefore, OHP1OHP1 andand OHP2,OHP2, likelike HCF244,HCF244, promotepromotepsbA psbA translationtranslation initiationinitiation andand dodo notnot havehave strongstrong eeffectsffects onon thethe recruitmentrecruitment ofof ribosomesribosomes toto otherother chloroplastchloroplast ORFs.ORFs. TheThe similar translation phenotypesphenotypes ofof thethe ohp1,ohp1, ohp2,ohp2, andand hcf244hcf244 mutantsmutants isis consistentconsistent withwith thethe interdependentinterdependent accumulationaccumulation of OHP1, OHP2,OHP2, andand HCF244HCF244 [[14,16].14,16].

FigureFigure 3.3. Ribo-seqRibo-seq analysisanalysis ofof ArabidopsisArabidopsis ohp1ohp1 mutants.mutants. DataData forfor aa replicatereplicate experimentexperiment areare shownshown inin FigureFigure S1b.S1b. SymbolsSymbols andand nomenclaturenomenclature areare asas describeddescribed inin FigureFigure2 .(2. (aa)) ComparisonComparison ofof ribosomeribosome footprintfootprint abundanceabundance for for all all chloroplast chloroplast genes genes in ohp1 in mutantsohp1 mutants to that to in phenotypically-normalthat in phenotypically-normal siblings. (siblings.b) Screen (b capture) Screen from capture the Integratedfrom the Integrated Genome Viewer Genome (IGV) Viewer showing (IGV) the showing distribution the distribution of ribosome of footprintsribosome footprints along psbA alongand adjacentpsbA and ORFs. adjacent Diff ORFs.erent genesDifferent are displayedgenes are displa here andyed in here Figure and2 bin due Figure to the2b due different to the gene different order ingene the maizeorder in and the Arabidopsis maize and chloroplast Arabidopsis genomes. chloroplast (c) Ratio genomes. of Ribo-seq (c) Ratio reads of inRibo-seq the wild-type reads in relative the wild-type to the mutant relative for to chloroplast the mutant genes for chloroplast encoding PSII genes subunits. encoding Several PSII atpsubunits.genes areSeveral shown atp forgenes comparison. are shown The for averagecomparison. of the The two average replicates of the (+/ two– SD) replicates is shown. (+/– (d SD)) Distribution is shown. ( ofd) ribosomeDistribution footprints of ribosome along footprints the psbA ORF.along The the Y-axispsbA ORF. maxima The wereY-axis adjusted maxima to were facilitate adjusted comparison to facilitate of ribosomecomparison distributions of ribosome in distributions the two genotypes. in the two genotypes.

2.3. Ribosome Occupancy on psbA mRNA Is Not Reduced in hcf136 Mutants Results presented above are consistent with the possibility that any defect in assembly of the PSII core reduces psbA ribosome recruitment as a secondary effect. However, hcf107 and lpe1 mutants, which lack PSII due to defects in expression of the psbH and psbN/psbJ genes, respectively, show only minor reductions in psbA ribosome occupancy [23]. Still, this leaves open the possibility that defects very early in PSII assembly (as in hcf244, ohp1, and ohp2 mutants) secondarily cause a defect in psbA translation. To address this possibility, we performed similar experiments with mutants lacking HCF136. HCF136 acts upstream of the OHP1/OHP2/HCF244 complex, possibly to facilitate the insertion of D1 into that complex or the insertion of chlorophyll into D1 [14,18–21]. Furthermore, the HCF136 ortholog in cyanobacteria, Ycf48, associates with the Ycf39/HliD/HliC complex [13], and analysis of ycf48/ycf39 double mutants suggested that Ycf48 cooperates with the Ycf39 complex to deliver chlorophyll to the assembling PSII reaction center [25].

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(a) (b) 20 26000 WT psbA 15 psbB psbC psbD cpRPKM) 10 2 22000 ohp2 (log

5 coverage Read WT

0 0 5 10 15 20 psbA psbK psbI atpA atpF atpH (log cpRPKM) ohp2 2 (c) (d) 21795 15 WT

10 ohp2

/ 1818 ohp2

WT WT 5 Read coverage Ribo-seq cpRPKM 1 0 psbI sbM atpA atpB atpH psbJ psbL psbZ psbF psbA psbB psbT psbE psbD psbC psbK psbN psbH psbA p FigureFigure 4. Ribo-seq analysis analysis of of Arabidopsis ohp2ohp2 mutants. Data Data for for a a replicate experiment are are shown inin Figure Figure S1c. Symbols Symbols and and nomenclature nomenclature are are as as described described in in Figure 22..( (a)) Comparison of of ribosome footprintfootprint abundanceabundance for for all all chloroplast chloroplast genes genes in ohp2 in ohp2mutants mutants to that to in that phenotypically-normal in phenotypically-normal siblings. siblings.(b) Screen (b capture) Screen fromcapture the from Integrated the Integrated Genome ViewerGenome (IGV) Viewer showing (IGV) theshowing distribution the distribution of ribosome of ribosomefootprints footprints along psbA alongand psbA adjacent and adjacent ORFs. (c ORFs.) Ratio (c of) Ratio Ribo-seq of Ribo-seq reads in reads the wild-typein the wild-type relative relative to the tomutant the mutant for chloroplast for chloroplast genes encoding genes encoding PSII subunits. PSII subunits. Several atp Severalgenes areatp showngenes forare comparison.shown for comparison.The average ofThe the average two replicates of the (+/two– SD) replicates is shown. (+/– (d )SD) Distribution is shown. of ( ribosomed) Distribution footprints of ribosome along the footprintspsbA ORF. along The Y-axis the psbA maxima ORF. wereThe Y-axis adjusted maxima to facilitate were adjusted comparison to facilitate of ribosome comparison distributions of ribosome in the distributionstwo genotypes. in the two genotypes.

2.3. RibosomeTo address Occupancy whether on HCF136 psbA mRNA impacts Is Not the Reduced recruitment in hcf136 of ribosomes Mutants to psbA mRNA, we analyzed hcf136 mutants in maize (Zm-hcf136) and Arabidopsis by Ribo-seq. The Zm-hcf136 mutant grew Results presented above are consistent with the possibility that any defect in assembly of the similarly to the wild-type and Zm-hcf244 mutant to the three-leaf stage, but showed a more pronounced PSII core reduces psbA ribosome recruitment as a secondary effect. However, hcf107 and lpe1 mutants, chlorophyll deficiency than the Zm-hcf244 mutant (Figure1b). D1 abundance is severely reduced in the which lack PSII due to defects in expression of the psbH and psbN/psbJ genes, respectively, show only mutant, whereas core subunits of other photosynthetic complexes were found at near normal levels minor reductions in psbA ribosome occupancy [23]. Still, this leaves open the possibility that defects (Figure1c). These results are as expected based on the phenotype of Arabidopsis hcf136 [19] and a very early in PSII assembly (as in hcf244, ohp1, and ohp2 mutants) secondarily cause a defect in psbA previously characterized Zm-hcf136 allele [26]. Ribo-seq analysis showed no apparent difference in translation. To address this possibility, we performed similar experiments with mutants lacking psbA ribosome occupancy in the mutant and wild-type in two replicate experiments (Figure5a and HCF136. HCF136 acts upstream of the OHP1/OHP2/HCF244 complex, possibly to facilitate the Figure S1d). We obtained similar results with the Arabidopsis hcf136 allele reported previously [19] insertion of D1 into that complex or the insertion of chlorophyll into D1 [14,18–21]. Furthermore, the (Figure5b). The psbA mRNA was previously shown to accumulate normally in Arabidopsis hcf136 HCF136 ortholog in cyanobacteria, Ycf48, associates with the Ycf39/HliD/HliC complex [13], and mutants [19]; we confirmed the same to be true for maize by slot-blot hybridization analysis of RNA analysis of ycf48/ycf39 double mutants suggested that Ycf48 cooperates with the Ycf39 complex to from the same extracts used for Ribo-seq (Figure S1d). These results show that an early defect in PSII deliver chlorophyll to the assembling PSII reaction center [25]. assembly does not, in and of itself, prevent the recruitment of ribosomes to psbA RNA, highlighting To address whether HCF136 impacts the recruitment of ribosomes to psbA mRNA, we analyzed the HCF244/OHP1/OHP2 complex as playing a special role in the coordination of PSII assembly with hcf136 mutants in maize (Zm-hcf136) and Arabidopsis by Ribo-seq. The Zm-hcf136 mutant grew D1 synthesis. similarly to the wild-type and Zm-hcf244 mutant to the three-leaf stage, but showed a more pronounced chlorophyll deficiency than the Zm-hcf244 mutant (Figure 1b). D1 abundance is severely reduced in the mutant, whereas core subunits of other photosynthetic complexes were found at near normal levels (Figure 1c). These results are as expected based on the phenotype of Arabidopsis hcf136 [19] and a previously characterized Zm-hcf136 allele [26]. Ribo-seq analysis showed no apparent difference in psbA ribosome occupancy in the mutant and wild-type in two replicate experiments (Figure 5a and Figure S1d). We obtained similar results with the Arabidopsis hcf136 allele reported previously [19] (Figure 5b). The psbA mRNA was previously shown to accumulate normally in Arabidopsis hcf136 mutants [19]; we confirmed the same to be true for maize by slot-blot hybridization analysis of RNA from the same extracts used for Ribo-seq (Figure S1d). These results show that an early defect in PSII assembly does not, in and of itself, prevent the recruitment of

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Plantsribosomes2020, 9 ,to 152 psbA RNA, highlighting the HCF244/OHP1/OHP2 complex as playing a special role7 of in 13 the coordination of PSII assembly with D1 synthesis.

(a) 20 psbA 2

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10 psbC 1 2 (log WT / Zm- WT

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0 0 psbI atpA atpB psbJ psbL atpH psbZ psbA psbB psbF psbD psbE psbT psbC psbK psbH 0 5 10 15 20 psbN psbM

Zm-hcf136 (log2 cpRPKM) (b) 20 psbA 2 psbC 15 psbB psbD cpRPKM) 10 hcf136 2 1 (log WT / WT

5 Ribo-seq cpRPKM WT WT

0 0 psbI atpA atpB psbJ psbL atpH psbZ psbA psbB psbF psbD psbE 0 5 10 15 20 psbT psbC psbK psbH psbN psbM hcf136 (log cpRPKM) 2 Figure 5. Ribo-seq analysis of hcf136 mutants in maize and Arabidopsis. Symbols and nomenclature are as describeddescribed inin FigureFigure2 .(2. a(a)) Comparison Comparison of of ribosome ribosome footprint footprin abundancet abundance for for chloroplast chloroplast genes genes in inZm- Zm-hcf136hcf136mutants mutants to thatto that in theirin their phenotypically phenotypically normal normal siblings. siblings. Results Results for for a biological a biological replicate replicate are areshown shown in Figure in Figure S1d. TheS1d. bar The graph bar showsgraph theshows average the ofaverage the two of replicates the two (+/replicates– SD). (b )(+/– Comparison SD). (b) Comparisonof ribosome footprintof ribosome abundance footprint for abundance chloroplast for genes chloroplast in Arabidopsis genes in Arabidopsishcf136 mutants hcf136 to that mutants in their to phenotypicallythat in their phenotypically normal siblings. normal siblings.

3. Discussion Discussion The OHP1OHP1/OHP2/HCF244/OHP2/HCF244 complex and its ortholog in cyanobacteria facilitate assembly of an early PSII assembly intermediate, and are believed to do so by delivering chlorophyll chlorophyll to to nascent nascent D1, D1, scavenging chlorophyll from damaged D1, and/or and/or pr protectingotecting the assembling reaction center from photooxidative damage [13,14,16,17,27]. [13,14,16,17,27]. HCF244 HCF244 had, had, in in addition, addition, been been reported reported to promote psbA translation initiation [22], [22], whereas polysome analyses of ohp1 and ohp2 mutants led to the conclusion that OHP1 and OHP2 do not have this function [14]. [14]. These These apparently apparently contrasting contrasting effects effects of HCF244 and OHP1OHP1/OHP2/OHP2 onon psbApsbAtranslation translation were were puzzling puzzling in in light light of of the the decreased decreased accumulation accumulation of of each each of ofthese these proteins proteins in mutantin mutant and and knockdown knockdown lines lines for for each each of theof the others others [14 [14,16].,16]. Data presentedpresented here here resolve resolve this this issue issue by demonstratingby demonstrating that psbA thattranslation psbA translation initiation initiation is strongly is stronglyand specifically and specifically reduced inreducedohp1, ohp2, in ohp1,and ohp2, Zm-hcf244 and Zm-mutants.hcf244 Inmutants. fact, inspection In fact, inspection of the polysome of the polysomedata reported data for reportedohp1 and forohp2 ohp1[14] suggestsand ohp2 a considerable[14] suggests decrease a considerable in ribosome decrease occupancy in ribosome on psbA occupancymRNA. Interpretation on psbA mRNA. of polysome Interpretation data for psbA of polysomeis challenging data duefor topsbA the unusuallyis challenging small due fraction to the of unusuallythe mRNA small pool thatfraction is polysome-associated of the mRNA pool even that in theis polysome-associated wild-type. Similarly, even inference in the about wild-type. rates of Similarly,D1 and D2 inference synthesis about from pulse-labelingrates of D1 and assays D2 synt in PSIIhesis assembly from pulse-labeling mutants is complicated assays in PSII by their assembly rapid mutantsdegradation is complicated when their assembly by their is disrupted.rapid degradat Theseion complications when their in assembly interpreting is pulse-labelingdisrupted. These and polysomecomplications data in for interpretingpsbA led to pulse-labeling confusion about and apolysome role for the data pentatricoptide for psbA led to repeat confusion protein about LPE1 a role in forpsbA thetranslation pentatricoptide [23,28]. repeat Ribosome protein profiling LPE1 in or psbA the use translation of 5’-UTR: [23,28]. reporter Ribosome gene fusions profiling (e.g., or [ 12the]) use are ofbetter 5’-UTR: suited reporter to providing gene fusions unambiguous (e.g., [12]) information are better aboutsuitedpsbA to providingtranslation unambiguous initiation. information about psbA translation initiation. Elegant experiments with Chlamydomonas revealed feedback loops that couple the synthesis of certain chloroplast-encoded subunits of the photosynthetic apparatus with the availability of partner subunits. These phenomena are collectively referred to as Control by Epistasy of Synthesis (CES) [29]. The degree to which CES interactions are conserved in plant chloroplasts remains unclear. For example, CES interactions among ATP synthase subunits that were observed in Chlamydomonas [30]

Plants 2020, 9, 152 8 of 13

Elegant experiments with Chlamydomonas revealed feedback loops that couple the synthesis of certain chloroplast-encoded subunits of the photosynthetic apparatus with the availability of partner subunits. These phenomena are collectively referred to as Control by Epistasy of Synthesis (CES) [29]. The degree to which CES interactions are conserved in plant chloroplasts remains unclear. For example, CES interactions among ATP synthase subunits that were observed in Chlamydomonas [30] do not occur in maize [31]. Results presented here add to this body of information by addressing whether the D1-dependent translation of the chloroplast psbB ORF observed in Chlamydomonas [12] also occurs in plants. Our data show that the severe defect in psbA translation initiation in hcf244, ohp1, and ohp2 mutants is not accompanied by a strong decrease in psbB ribosome occupancy, suggesting that this CES interaction is not conserved. That said, our data hint at a related phenomenon: The ratio of psbB ribosome footprints in the wild-type relative to that in each of these mutants is slightly higher than that for most other chloroplast PSII genes (see Figures2c,3c and4c, and Figure S1); this small effect is not apparent in hcf136 mutants. We observed a similar weak effect on psbB ribosome occupancy in an Arabidopsis hcf173 mutant, which likewise has a strong defect in psbA translation initiation [23,32]. The consistency of this correlation suggests that D1 synthesis mildly enhances PsbB synthesis in plants. It seems possible that this is mechanistically related to the D1-psbB CES interaction observed in Chlamydomonas. Experiments in Chlamydomonas also revealed that unassembled D1 autoregulates D1 synthesis by repressing psbA translation initiation. For example, deletion or truncation of the psbA ORF caused a dramatic increase in synthesis of reporter proteins whose expression was driven by the psbA 5’ UTR [12,33]. In a similar vein, deletion of all psbA genes in Synechocystis increased the recovery of ribosomes in Ycf39 affinity purifications [12]. These observations, in conjunction with our results and the recent elucidation of the role of the HCF244/OHP1/OHP2 complex in PSII assembly, suggests that this complex is the hub of an autoregulatory mechanism that couples D1 synthesis with demand for D1. First, the dual effects of the HCF244/OHP1/OHP2 complex on psbA translation and PSII assembly are unique to this step in PSII biogenesis: disruption of upstream or downstream steps in PSII biogenesis (e.g., in hcf136 or hcf107 mutants) has little or no effect on psbA translation initiation (Figure5 and Reference [ 23]). Second, there is strong biochemical evidence that nascent D1 is found in a complex together with OHP1/OHP2/HCF244 in plants, and in the corresponding complex in cyanobacteria [13,14,17,27]. That this complex brings D1 into proximity with proteins that are required for the recruitment of ribosomes to psbA mRNA suggests a model in which D1 autoregulates psbA translation through direct inhibitory interactions with HCF244/OHP1/OHP2 (Figure6). To elicit e ffects specifically on psbA translation, this complex must influence the activity of one or more proteins that bind directly to psbA mRNA. Translation initiates on psbA mRNA in the stroma or in loose association with the thylakoid membrane [31]. An exhaustive search for candidate regulators that bind psbA RNA in the stroma detected the known translational activator HCF173, but did not detect any other proteins that have strong effects on psbA ribosome occupancy [34,35]. As such, we propose that the membrane-associated HCF244 complex communicates in some manner with HCF173 to trigger psbA translation (Figure6). Future experiments will be directed toward testing and elaborating on this model. Plants 2020, 9, 152 9 of 13 Plants 2020, 9, x FOR PEER REVIEW 9 of 13

Figure 6. ModelModel for for negative negative autoregulation autoregulation of of psbApsbA translationtranslation initiation initiation in in the the context context of of the HCF244/OHP1/OHP2HCF244/OHP1/OHP2 complex. OHP1 OHP1 and and OHP2 OHP2 are are integral integral thylakoid thylakoid proteins, proteins, and and HCF244 is

tetheredtethered to to OHP2 OHP2 on on the the stromal stromal face face of of the the membrane membrane [13,14,16,17] [13,14,16,17].. HCF173HCF173 bindsbinds thethe 550′-UTR of psbA mRNAmRNA andand is is required required for forpsbA psbAtranslation translation initiation initiation [23,32 [23,32,34],34]. We propose. We propose that stroma-exposed that stroma- exposedfeatures offeatures the complex of the (HCF244complex and(HCF244/or OHP2’s and/or stromal OHP2’s tail) stromal collaborate tail) withcollaborate HCF173 with to activate HCF173psbA to activatetranslation psbA initiation, translation and initiation, that the presenceand that the of D1presence in the of complex D1 in the inhibits complex communication inhibits communication to HCF173. toThis HCF173. is a simplified This iscartoon a simplified that does cartoon not incorporate that does evidencenot incorporate that other evidence PSII core that subunits other PSII (PsbI, core D2, subunitsPsbE, PsbF) (PsbI, also D2, associate PsbE, PsbF) with al theso associate HCF244/ Ycf39with the complex HCF244/Ycf under39 some complex conditions under [some13,14 conditions,17]. Ycf48 [13,14,17](the HCF136. Ycf48 homolog (the in HCF136 cyanobacteria) homolog associates in cyanobacteri with thisa) complex associates when with downstream this complex steps inwhen PSII downstreamassembly are steps disrupted, in PSII but assembly there is conflicting are disrupted, evidence but aboutthere theis conflicti associationng evidence of HCF136 about with the associationHCF244 complex of HCF136 in plants with [ 13the,14 HCF244,17]. complex in plants [13,14,17]. 4. Materials and Methods 4. Materials and Methods 4.1. Mutant Lines and Plant Growth. 4.1. Mutant Lines and Plant Growth. OHP1 is encoded by Arabidopsis gene AT5G02120 and OHP2 is encoded by AT1G34000. The ohp1OHP1and ohp2 is encodedmutants by were Arabidopsis T-DNA insertion gene AT5G021 lines GABI_362D0220 and OHP2 andis encoded GABI_071E10, by AT1G34000. respectively. The ohp1These and were ohp2 the mutants same alleles were asT-DNA those usedinsertion in [36 lines], where GABI_362D02 they were and demonstrated GABI_071E10, to be respectively. null alleles. TheseBoth lineswere werethe same obtained alleles from as those the Arabidopsisused in [36], Biologicalwhere they Resource were demonstrated Center. HCF136 to be null is encoded alleles. Bothby Arabidopsis lines were obtained gene AT5G23120. from the TheArabidopsishcf136 mutant Biological allele Resource was reported Center. in HCF136 [19] and is the encoded seed was by Arabidopsisa generous giftgene of AT5G23120. Joerg Meurer The and hcf136 Peter mutant Westho ffallele. Maize was HCF136reported (Zm-HCF136) in [19] and the is encodedseed was by a generousmaize gene gift GRMZM2G102838 of Joerg Meurer and (B73 Peter RefGen Westhoff v3). orMaize Zm00001d036340 HCF136 (Zm-HCF136) (B73 RefGen is encoded v4) and by ismaize also genecalled GRMZM2G102838 PSB1. Maize HCF244 (B73 (Zm-HCF244) RefGen v3) or isZm00001d036340 encoded by gene (B73 GRMZM2G143917 RefGen v4) and (B73is also RefGen called PSB1. v3) or MaizeZm00001d031997 HCF244 (Zm-HCF244) (B73 RefGen v4).is Weencoded recovered by Mugenetransposon GRMZM2G143917 insertion alleles (B73 inRefGen Zm-hcf244 v3) andor Zm00001d031997Zm-hcf136 from the (B73 Photosynthetic RefGen v4). MutantWe recovered Library Mu [24 transposon]. The positions insertion of the alleles insertions in Zm- andhcf244 evidence and Zm-thathcf136 they are from null the alleles Photosynthetic are shown inMutant Figure Library1. [24]. The positions of the insertions and evidence that they are null alleles are shown in Figure 1. 2 Maize was grown in a growth chamber in diurnal cycles of 16 h light (~200 µE/m /s at 28 ◦C)/ Maize was grown in a growth chamber in diurnal cycles of 16 h light (~200 µE/m2/s at 28 °C)/ 8 8 h dark (26 ◦C). RNA and ribosome footprints were extracted from the second and third leaves of h7-to-8-day-old dark (26 °C). seedlings,RNA and ribosome at which footprints point the thirdwere leafextracted was beginning from the second to emerge. and third Arabidopsis leaves of was 7- to-8-day-oldgerminated and seedlings, grown onat sterilewhich Murashige point the andthird Skoog leaf was medium beginning (4.33 g /toL Murashigeemerge. Arabidopsis and Skoog Basalwas germinatedMedium (Sigma, and grown St. Louis, on sterile MO, USA), Murashige 2% sucrose, and Sk 0.3%oog medium Phytagel (4.33 (Sigma), g/L Murashige pH 5.7) in and diurnal Skoog cycles Basal of Medium (Sigma, St.2 Louis, MO, USA), 2% sucrose, 0.3% Phytagel (Sigma), pH 5.7) in diurnal cycles 10 h light (80 µE/m /s)/14 h dark at 22 ◦C. of 10 h light (80 µE/m2/s)/14 h dark at 22 °C. 4.2. Ribosome Profiling and RNA-seq. 4.2. Ribosome Profiling and RNA-seq. Ribosome footprint preparation and library construction were performed as described previouslyRibosome [9]. footprint Each replicate preparation experiment and performedlibrary construction with the ohp1 weremutant performed used a as pool described of three previouslymutant plants. [9]. Each Leaves replicate were harvested experiment at midday performed on the with 18th the day ohp1 post mutant vernalization. used a pool The replicateof three mutant plants. Leaves were harvested at midday on the 18th day post vernalization. The replicate experiments performed with the ohp2 mutant used pools of four and five mutant plants, respectively.

Plants 2020, 9, 152 10 of 13 experiments performed with the ohp2 mutant used pools of four and five mutant plants, respectively. Leaves were harvested at midday on the 15th day post vernalization. The Arabidopsis hcf136 experiment used a pool of four mutant plants. Leaves were harvested at midday on the 14th day post vernalization. The Zm-hcf136 replicates and the first replicate of the Zm-hcf244 experiment used leaves from one mutant seedling, whereas the second Zm-hcf244 replicate used leaves pooled from two seedlings. Phenotypically normal siblings were used as the wild-type control for all of the experiments except the second replicate of ohp2 (Figure S1c), for which we used Col-0 grown in parallel. RNA for RNA-seq was extracted from an aliquot of the same leaf homogenate used for ribosome profiling. Libraries were prepared using Bioo Scientific NEXTflex qRNA-seq kit V2. Data analyses were performed largely as described previously [9], but with the following modifications: (i) Sequence reads were aligned to genomes using STAR instead of Bowtie2; (ii) Read counts excluded the first 10 or 25 nucleotides of each organellar or cytosolic ORF, respectively. Sequencing of Ribo-seq and RNA-seq libraries was performed at the University of Oregon Genomics Core Facility. Read counts for chloroplast genes in Ribo-seq and RNA-seq experiments are summarized in Table S1.

4.3. RNA Blot Hybridizations RNA gel blot hybridizations were performed as described previously [37]. The psbA mRNA was detected on the RNA gel blot with an end-labeled antisense DNA oligonucleotide corresponding to Arabidopsis chloroplast genome (TAIR10) nucleotide positions 1410–1444. Slot-blot hybridizations were performed as described previously [38], using 500 ng of total RNA in each slot. Probes were generated by PCR and radiolabeled with random hexamer priming. The psbA probe for both maize and Arabidopsis corresponded to maize chloroplast genome (Accession X86563) nucleotide positions 295–1074. The probe for maize atpB probe corresponded to maize chloroplast genome nucleotide positions 54590–55790. The probe for Arabidopsis atpB corresponded to Arabidopsis chloroplast genome nucleotide positions 52660–54156.

4.4. Immunoblot Analysis and Antibodies. Immunoblots were performed as described previously [37]. Proteins were extracted from the apical 2-cm of the second leaf of 8 day old maize seedlings. Antibodies to AtpB, D1, PsaD and PetD were generated by our group and were described previously [39]. Polyclonal antibody to Zm-HCF244 was raised in rabbits (Alpha Diagnostic Intl., San Antonio, TX, USA) using a recombinant antigen corresponding to amino acids 146–385 of Zm-HCF244. The HCF136 antibody was a generous gift of Peter Westhoff and Joerg Meurer.

Supplementary Materials: The following are available online at http://www.mdpi.com/2223-7747/9/2/152/s1, Figure S1: Supporting data for Ribo-seq assays. Ribo-seq graphs are as described in Figure2. Slot blots were imaged and quantified with a Storm phosphorimager. (a–c) Replicate Ribo-seq data and psbA RNA quantification for Zm-hcf244, ohp1, and ohp2 mutants. The abundance of psbA RNA relative to the chloroplast atpB mRNA in the extracts used for Ribo-seq was analyzed by slot blot hybridization. The psbA RNA in ohp2 mutants was, in addition, quantified by RNA gel blot hybridization (c, right panel). (d) Replicate Ribo-seq data for Zm-hcf136 (left panel) and quantification of psbA RNA in Zm-hcf136 extracts used for Ribo-seq (first replicate) (right panel). The abundance of psbA RNA relative to atpB RNA was analyzed by slot blot hybridization. Table S1: Ribo-seq and RNA-seq read counts for all chloroplast genes. Author Contributions: All authors contributed to the conceptualization of this study, development of the methodology, interpretation of the data, and validation of the findings. A.B. wrote the original draft of the manuscript. P.C. and R.W.-C. edited the manuscript and prepared figures. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the US National Science Foundation, grant number MCB-1616016. Acknowledgments: We are grateful to Margarita Rojas and Susan Belcher (University of Oregon) for expert technical assistance, Carolyn Brewster for making the recombinant HCF244 antigen, and Joerg Meurer and Peter Westhoff for providing Arabidopsis hcf136 seed and HCF136 antiserum. Conflicts of Interest: The authors declare no conflict of interest. Plants 2020, 9, 152 11 of 13

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