REVIEW

Chloroplast Translation: Structural and FunctionalOrganization, Operational Control, and Regulation[OPEN]

Reimo Zoschke1 and Ralph Bock1

Max Planck Institute of Molecular Plant Physiology, 14476 Potsdam, Germany

ORCID IDs: 0000-0002-6898-6836 (R.Z.); 0000-0001-7502-6940 (R.B.)

Chloroplast translation is essential for cellular viability and plant development. Its positioning at the intersection of organellarRNA and protein metabolism makes it a unique point for the regulation of gene expression in response to internal and externalcues. Recently obtained high-resolution structures of plastid ribosomes, the development of approaches allowing genome-wideanalyses of chloroplast translation (i.e., ribosome profiling), and the discovery of RNA binding proteins involved in the control oftranslational activity have greatly increased our understanding of the chloroplast translation process and its regulation. In thisreview, we provide an overview of the current knowledge of the chloroplast translation machinery, its structure, organization,and function. In addition, we summarize the techniques that are currently available to study chloroplast translation and describehow translational activity is controlled and which cis-elements and trans-factors are involved. Finally, we discuss howtranslational control contributes to the regulation of chloroplast gene expression in response to developmental, environmental,and physiological cues. We also illustrate the commonalities and the differences between the chloroplast and bacterialtranslation machineries and the mechanisms of protein biosynthesis in these two prokaryotic systems.

INTRODUCTION

Chloroplasts are the characteristic organelle of plant cells. Theyhost numerous essential metabolic pathways including photo-synthesis, whichmakes chloroplasts the primary source of chemicalenergy on earth. All chloroplasts are likely derived from a singleancient photosynthetic cyanobacterium that was engulfed byamitochondriateeukaryoticcellmore thanabillionyearsago.Duringsubsequent host-endosymbiont coevolution, the genome of theendosymbiont shrank significantly (Timmis et al., 2004). While somegenes were lost, many others were transferred to the host genome(Martinetal.,2002;BockandTimmis,2008).Theproteomeof today’schloroplasts consists of;3000proteins,most ofwhichare nucleus-encoded and posttranslationally imported into the organelle.

Present-day chloroplasts still harbor a genome, which com-prises ;120 genes in green plants. Most plastid genes are es-sential for plant viability because they encodecrucial componentsof thephotosynthesismachinery (the large subunit ofRubiscoandapproximately half of the subunits of the thylakoidal proteincomplexes involved in the light reactions:photosystem Iand II [PSIand PSII], cytochrome b6f complex [Cyt b6f], and ATP synthase)and the gene expression system of the plastid (including a com-plete set of bacterial-type RNA polymerase core subunits, rRNAsand tRNAs, and approximately one-third of the ribosomal pro-teins; Allen et al., 2011; Green, 2011). The reasons for retention ofthis particular set of genes in the plastid genome are not fullyunderstood. Several hypotheses that are not necessarily mutu-ally exclusive have been put forward, including constraints on

importabilityofproteins (andRNAs) intoplastidsand requirementsfor efficient and organelle-specific redox regulation of geneexpression (Allen, 2015). An intriguing consequence of endosym-biont-hostcoevolution ismultimericchloroplastproteincomplexes,whose subunits are encoded in different compartments (i.e., theplastid and thenucleus). This necessitates the tight orchestrationofnuclear and chloroplast gene expression (Jarvis and López-Juez,2013; Kleine and Leister, 2016).The bacterial origin of chloroplast gene expression is evident,

forexample, fromtheoperon-likestructureofplastidgeneclustersand the highly similar composition of the translation machinery.However, several features clearly distinguish chloroplast geneexpression from that of bacteria. For example, chloroplasts pos-sess an astoundingly complex RNAmetabolism that includes theusage of different RNA polymerases and extensive posttran-scriptional RNA processing by splicing, editing, end processing,and intercistronic processing of polycistronic RNAs (Barkan, 2011;Lyska et al., 2013; Börner et al., 2015). These processes are nearlyexclusively conducted by nucleus-encoded protein factors, mostof which were likely established during host-endosymbiont co-evolution (Barkan,2011;Lyskaetal.,2013;PfalzandPfannschmidt,2013). Also different from bacteria, the regulatory influence oftranscription is limited and posttranscriptional and translationalevents represent key points in controlling chloroplast gene ex-pression (Barkan, 2011; Sun and Zerges, 2015).In recent years, numerous specific features of chloroplast trans-

lation were uncovered, including its interconnection with cotrans-lational processes in RNA and protein metabolism, its regulation inresponse to internal and external triggers, and the presence ofunusual components of the translation machinery. Genome-wideanalyses have unraveled the suborganellar localization of trans-lationand itsparticipation incontrolling thedevelopmental programof chloroplast gene expression (Zoschke and Barkan, 2015;

1 Address correspondence to zoschke@mpimp-golm.mpg.de or rbock@mpimp-golm.mpg.de.[OPEN]Articles can be viewed without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.18.00016

The Plant Cell, Vol. 30: 745–770, April 2018, www.plantcell.org ã 2018 ASPB.

Chotewutmontri and Barkan, 2016). Studies of sequence-specificchloroplast RNA binding proteins that comprise helical repeatdomains, especially the pentatricopeptide repeat (PPR) proteins,revealed their concerted function inRNAmetabolismandpromotionof translation (Barkan and Small, 2014; Hammani et al., 2014). Lastbut not least, high-resolution structural analysesprovidedadetailedthree-dimensional picture of the plastid ribosome (Graf et al., 2016;Bieri et al., 2017). These discoveries were largely enabled by novelapproaches toward the quantitative transcriptome-wide analysis oftranslational activity, the identificationof specific factors that controlprotein synthesis, and the structural elucidation of the translationalapparatus in plastids (e.g., by ribosome profiling techniques, RNAcoimmunoprecipitation assays, and refined methods for 3D struc-tural analysis). In the light of these and other findings, we now canreevaluate classical models of chloroplast translation and reassesscontroversially discussed hypotheses.

Chloroplast translation has been mainly studied in the unicel-lular green alga Chlamydomonas reinhardtii and in model seedplants such as Arabidopsis thaliana, maize (Zea mays), and to-bacco (Nicotianatabacum). Inthisreview,wefocusonembryophytesand, wherever appropriate, refer to breakthrough discoveriesmade in Chlamydomonas. For a broader overview of chloroplasttranslation inChlamydomonas, the interested reader is referred tocomprehensive review articles (Stern et al., 2009; Nickelsen et al.,2014; Sun and Zerges, 2015).

METHODS TO ANALYZE CHLOROPLAST TRANSLATION

Methods todetermine translational activityeither indirectlyexaminethe ribosome coverage of the translation template (i.e., the mRNA)ordirectlymeasure theaccumulationofnewlysynthesizedproteins.

Classical Methods to Analyze Translation

Pulse labeling is the method of choice to directly measuretranslational activity in vivo. In this approach, isolated chlor-oplasts, cells, or intact plant tissues are fed with the 35S-radio-labeled amino acids methionine and/or cysteine (Barkan, 1998).The isotopes are incorporated into newly synthesized proteins toan extent that mirrors their synthesis rate. Subsequently, the ra-diolabeled proteins can be separated by gel electrophoresis, vi-sualized, and quantified (Figure 1A). An advantage of pulse labelingis that it has the potential to measure protein synthesis rates in-dependent of the dynamics of ribosome movement along themRNA. By pulse labeling, the synthesis rate of especially the largeplastid-encoded core subunits of the photosynthesis machinerycan be readily quantified. However, the method has several limi-tations: (1) Small subunits and subunits of similar molecular weightare difficult to resolve in protein gels and may require selectivepurification by immunoprecipitation (Barkan, 1998). (2) The syn-thesis rates of many plastid-encoded proteins cannot be de-termined by pulse labeling approaches due to their low expressionlevels. (3) The measured quantities of labeled proteins are de-termined by their synthesis and degradation rates. Consequently,for proteins with high turnover rates (e.g., PsbA, the D1 protein ofPSII), results of pulse-labeling experiments are often difficult tointerpret (even if followed by a chase with unlabeled amino acids

to examine the stability of the labeled protein). Also, (4) in multi-cellular organisms, neither the pulsing nor the chasing occurshomogenously in all cells, thus making quantitative comparisonsvery challenging.Polysome analysis is a widely used method that indirectly

measures translational activity using the association of mRNAswith ribosomes as a proxy. Polysomes are high molecularweight assemblies of actively translating ribosomes held to-gether by the strands of mRNA being translated. They can beseparated from free mRNAs and ribosomes (monosomes) byultracentrifugation in sucrose density gradients (Figure 1B). Fol-lowing RNA extraction from gradient fractions and RNA gel blotanalysis, the distribution of specificmRNAs across the gradient isvisualized and provides a qualitativemeasure of their translationalactivity (Barkan, 1998). For genome-wide analysis (translatomics),mRNAs recovered fromdifferent density fractionscanbeexaminedby microarray hybridization (Kahlau and Bock, 2008). Polysomeanalyses are often complicated by the operon-like organization ofgenes in the plastid genome. The processing of transcripts pro-duced from polycistronic transcription units frequently gives rise toa multitude of mono-, oligo-, and polycistronic RNA species, all ofwhich representpotential translation templates (Barkan,1988).Dueto the physical linkage of reading frames located on the sametranscript, their individual translation rates cannot be resolved. Inaddition, only translational regulation at the level of initiation can bedetected because the molecular weight of mRNAs loaded withactively elongating ribosomes is indistinguishable from those withpaused or stalled ribosomes.Other elegant though labor intensive methods have been used

to examine the regulatory capacity of cis-elements in chloroplasttranslation: (1) Chloroplast in vitro translation systems have beenestablished and used to analyze the regulatory influence of pu-tative cis-elements residing in the 59 untranslated region (59UTR)ontranslation (Hirose and Sugiura, 1996; Yukawa et al., 2007). (2) Re-porter genes (e.g., GFP or GUS) have been fused to different pre-sumed cis-elements and inserted into the plastid genome bychloroplast transformation to examine the translational activity con-ferred by these sequences (Staub andMaliga, 1994; Eibl et al., 1999;Drechsel and Bock, 2011).

Ribosome Profiling: Genome-Wide Analysis of Translation atHigh Resolution

The above described classical methods have been informative,but they are labor intensive and limited in resolution, and none ofthem is suited to genome-wide and/or high-throughput analyses.Thesedeficitswere addressedby ribosomeprofiling, anapproachthat enables the quantitative genome-wide analysis of translationin unprecedented depth and resolution (Ingolia et al., 2009). Ri-bosome profiling takes advantage of the remarkable stability oftranslating ribosomes, which protect the mRNA sequence theyphysically cover from attack by nucleases, thereby producingprotected fragments, so-called ribosome footprints (Wolin andWalter, 1988; Figure 1C). Next-generation sequencing analysis ofthese footprints determines the in vivo positions and abundancesof translating ribosomes. Considering that each elongating ri-bosome produces one protein, ribosome footprint abundancesreflect the protein synthesis rate for each reading frame (Ingolia

746 The Plant Cell

et al., 2009). Footprint abundance is typically normalized tomRNAabundance (assayed by RNA sequencing), so that relativetranslation efficiencies can be inferred (Ingolia et al., 2009). Con-sequently, the approach measures the two determinants of geneexpression that define the final protein output: transcript amountand translational activity. In recent years, ribosome profiling hasbeen extensively used to study translation in prokaryotes andeukaryotes (Ingolia, 2016).

Inchloroplasts, ribosomeprofilingwasfirst applied inamodifiedapproach, exchanging the next-generation sequencing analysisof footprints by microarray hybridization (Zoschke et al., 2013a;Figure 1C). More recently, deep sequencing was used to studychloroplast translational dynamics in maize and Arabidopsis(Chotewutmontri and Barkan, 2016; Lukoszek et al., 2016;Gawronski et al., 2018).

Despite the compelling attractions of ribosome profiling, itshould be noted that the method cannot distinguish activelytranslating from paused ribosomes. This may be problematic if

translation is regulated at the level of elongation, as described forsome chloroplast genes (see below). Application of inhibitors ofinitiation or early elongation (e.g., lincomycin) and examination ofthe run-off kinetics of ribosomes over time should allow dis-tinguishing pausing from elongating ribosomes.

THE PLASTID TRANSLATION MACHINERY: VARIATIONSON A BACTERIAL THEME

A Bacterial-Like Translation Machinery Whose ComponentsAre Encoded by Two Genomes

Chloroplast translation is performed by prokaryotic-type 70Sribosomes that are composed of a small 30S and a large 50Ssubunit and contain orthologs of most proteins and all rRNAs ofthe Escherichia coli reference ribosome (Kössel et al., 1985;Yamaguchi and Subramanian, 2000; Yamaguchi et al., 2000). All

Figure 1. Common Methods to Analyze Chloroplast Translation.

(A) Pulse labeling. Plant cells (chloroplast, large green oval; nucleus, white circle) are fed with radiolabeled cysteine and/or methionine (red dots), which isincorporated together with unlabeled amino acids (black dots) into nascent peptides by translation (for simplicity, only chloroplast ribosomes are shown).Proteins are then isolated, separated by gel electrophoresis, and visualized/quantified by radio-detection methods. (Gel picture kindly provided by KarinMeierhoff.)(B) Polysome analysis. Plant cell lysates are loaded on sucrose gradients (white to black: low to high concentration) to separate RNPs according to theirmolecularweight byultracentrifugation.RNA is isolated fromgradient fractions andexaminedbyRNAgel blot analysis todetermine the ribosome loadingofspecific mRNAs.(C) Ribosome profiling. Plant cell lysates are treated with nuclease to degrade ribosome-free mRNA sequences. This generates monosomes, whoseprotected mRNA fragments (ribosome footprints) are subsequently purified. The positions and abundances of the ribosome footprints are determined bynext-generation sequencing or microarray hybridization and reflect protein synthesis rates.

Chloroplast Translation 747

rRNAs, a complete set of ;30 tRNAs, approximately half of theribosomal proteins of the 30S subunit, and one-quarter of theproteins of the 50S subunit are encoded in the plastid genome(Sugiura, 1995). The remaining ribosomal proteins are nucleus-encoded (Tiller and Bock, 2014; http://www.bangroup.ethz.ch/research/nomenclature-of-ribosomal-proteins.html). Interestingly,the genomic distribution of genes for organellar ribosomal pro-teins is, to some degree, evolutionarily conserved, suggestingconstraints in ribosome assembly that require on-site coex-pression of rRNAs and organelle-encoded core ribosomal pro-teins (Maier et al., 2013). Most other components of the plastidtranslation machinery are nucleus-encoded (e.g., initiation/elongation/termination/ribosome recycling factors and aminoacyl-tRNA synthetases), except for initiation factor 1 (IF1), which isplastid-encoded in many plants (Millen et al., 2001).

Plastid Deviations from the Bacterial Ribosome and TheirStructural and Functional Consequences

Despite the generally bacterial structure of the chloroplast ribo-some, there are some features that clearly distinguish plastidribosomes from the E. coli reference ribosome. Chloroplast ri-bosomes contain the full set of bacterial rRNAs (23S, 16S, and 5SrRNAs), which comprise the peptidyl transferase activity (23SrRNA) and the decoding center (16S rRNA) and serve as scaffoldfor ribosomal proteins during ribosome assembly (Shajani et al.,2011; Maier et al., 2013). However, the 23S rRNA gene was splitinto twogenes in theplastidgenome:a large59portionencodedbythe 23S rRNA gene and a small 39 fragment encoded by the 4.5SrRNA gene (Whitfeld et al., 1978). In addition, the 23S rRNA isposttranscriptionally processed at two so-called “hidden breaks”into three fragments, whose abundances and precise sizes varyamong species (e.g., ;0.5, ;1.2, and ;1.1 kb from 59 to 39 inArabidopsis plastids). These fragments are found in the mature70S ribosome and held together by intermolecular base pairing(Kössel et al., 1985; Bieri et al., 2017). Despite their general ho-mology, some structural elements of the E. coli 16S and 23SrRNAs are absent from chloroplasts and, conversely, the chloro-plast 23S rRNA contains additional secondary structures (Kösseletal., 1985).Particularlywell conservedare thecatalyticdomainV inthe 23S rRNA, which carries the peptidyl transferase activity, andthe anti-Shine-Dalgarnosequence in the16S rRNA,which is crucialfor translation initiation (Scharff et al., 2017).

In the course of evolution, significant changes also occurred intheproteinaceouspart of thechloroplast ribosome.TheRpl25andRpl30 proteins were completely lost, and, in some species, thebacterial Rpl23 was replaced by its counterpart from the cyto-solic 80S ribosome (Bubunenko et al., 1994; Yamaguchi andSubramanian, 2000). Furthermore, the first complete inventory ofplastid ribosomal proteins identified six proteins that were as-sumed to lack bacterial orthologs and, consequently, were des-ignated as plastid-specific ribosomal proteins (PSRP) 1-6(Yamaguchi and Subramanian, 2000; Yamaguchi et al., 2000).However, later it was shown that PSRP1 is not a genuine ribosomalprotein, but the ortholog of the bacterial cold-shock protein pY,which is associatedwith the small subunit of the ribosome but nota structural part of it (Sharma et al., 2007, 2010). PSRP4 alsoshows homology to a bacterial protein: THX, an intrinsic part of

the 30S ribosomal subunit in Thermus thermophilus, which,so far, has not been found in other bacteria (Yamaguchi andSubramanian, 2003). At present, PSRP2 and PSRP3 in the 30Sribosomal subunit, and PSRP5 and PSRP6 in the 50S subunit areconsidered genuine plastid-specific ribosomal proteins; conse-quently, their renaming to RPS22/23 and RPL37/38 was sug-gested (Bieri et al., 2017). After their discovery, PSRPs werehypothesized to act in light regulation of translation (YamaguchiandSubramanian, 2003;Manuell et al., 2007).WhereasplastidpYmay indeed perform this function (see below), a number of studieshave suggested that the major function of PSRPs lies in thestructural compensation of evolutionarily modified rRNA domains(Sharmaetal., 2007;Tiller etal., 2012;Ahmedetal., 2016;Grafet al.,2016; Bieri et al., 2017). However, this role does not necessarilyexclude additional functions in translational regulation.Some of the conserved plastid ribosomal proteins also exhibit

N- or C-terminal extensions (or internal expansions) comparedwith their E. coli orthologs (Yamaguchi and Subramanian, 2000;Yamaguchi et al., 2000). Many of these extensions mediate newinteractionswith rRNAsor ribosomalproteinsandmaystructurallycompensate for missing ormodified rRNA domains (Ahmed et al.,2016; Graf et al., 2016; Bieri et al., 2017). However, the extensionsof some ribosomal proteins (e.g., S2, S18, and S21) representpotential new contact sites with the mRNA and therefore werehypothesized to be involved in translational regulation (Manuellet al., 2007; Sharma et al., 2007;Graf et al., 2016). Notably, severalalterations (e.g., extensions of RPS5 andRPS1 and an insertion inRPS4) narrow the mRNA entry site of the chloroplast ribosomecompared with the E. coli reference ribosome (Bieri et al., 2017).Furthermore, structural changes in thepolypeptide exit tunnel andthe tunnel exit site were hypothesized to support the cotransla-tional binding of the chloroplast signal recognition particle (SRP),which diverges substantially from that of bacteria (Ahmed et al.,2016; Graf et al., 2016; Bieri et al., 2017).Altogether, the chloroplast ribosome has a substantially higher

protein mass (by ;170 kD) and a slightly lower RNA content (by;0.4 kD) than the E. coli ribosome, resulting in a considerablyincreased protein to RNA ratio (;2:3 comparedwith 1:3 in E. coli).The partial replacement of rRNA domains by protein elements inchloroplast ribosomes follows the general evolutionary trend ofreducing RNA components in enzymatically active chloroplastribonucleoprotein particles (RNPs) and substituting them byprotein constituents (Barbrook et al., 2006). Other examples in-clude the chloroplast tRNA processing enzyme RNase P that lostits RNA component in the Viridiplantae lineage (Pinker et al., 2013)and the chloroplast SRP of seed plants that lacks the SRP RNA(Ziehe et al., 2017). The tendency to lose RNA functions inchloroplastsmay be driven by the evolutionary genome reductionand the massive transfer of protein-coding genes to the nucleus.Whereas proteins can be reimported posttranslationally intoplastids, this route seems to be blocked for RNA components,which may have enforced their evolutionary loss, replacement byproteins, or retention in the chloroplast genome (in the case ofrRNAs and tRNAs; Barbrook et al., 2006). Also, it was predictedthat ribosomecomposition (including its relative protein and rRNAcontents) is optimized for the production of the translation ma-chinery itself, a process that strongly limits cell division rates inprokaryotes (Reuveni et al., 2017). However, in plastids, the

748 The Plant Cell

synthesis of ribosomal proteins and rRNAs is partially uncoupledfrom translation due to the transfer of genes for ribosomal proteinsto the nucleus and the presence of nucleus-encoded RNA poly-merases, both of which are produced by cytosolic rather than or-ganellar ribosomes. Consequently, the evolutionary constraints onribosome composition are somewhat relaxed in organelles, whichmay have facilitated the observed shifts in protein-to-rRNA ratios inplastid and mitochondrial ribosomes (Reuveni et al., 2017).

Some Ribosomal Proteins Are Dispensable under StandardGrowth Conditions

Most constituents of the plastid translation machinery are es-sential for chloroplast biogenesis and, consequently, for plantviability (Tiller and Bock, 2014). In many species, plastid trans-lation is essential even under heterotrophic growth conditions(Ahlert et al., 2003; Sosso et al., 2012), presumably due to thenecessity to express a few essential plastid genes such as accD,clpP, ycf1, and ycf2 (Bock, 2007). However, some ribosomalproteins and tRNAs are nonessential, at least under standardgreenhouse conditions.

Nonessential ribosomal proteins have been identified in bothE. coli and plastids. Surprisingly, despite the shared ancestry ofbacterial and plastid ribosomes, the essentiality of ribosomalproteins is not fully conservedbetween the twosystems (Tiller andBock, 2014). The nucleus-encoded chloroplast ribosomal pro-teins RPL11, RPL24, RPS17, RPS21, PSRP3, andPSRP6 and theplastid-encoded Rps15, Rpl33, and Rpl36 are nonessential (Tillerand Bock, 2014). The phenotypes of viable ribosomal proteinmutants range from wild-type appearance to very strong phe-notypes with altered leaf morphology, variegated leaves, cold-induced bleaching, and retarded growth. Nonessential plastidribosomal proteins have been speculated to facilitate ribosomeassembly and structural integrity, or act in regulation, optimiza-tion, or localizationof translation (Pesaresi et al., 2001;Yamaguchiand Subramanian, 2003; Tiller et al., 2012; Tiller and Bock, 2014).However, their distinct molecular functions remain to be eluci-dated. Inbacteria, ribosomescandiffer in theirproteincomposition,for instance, under different growth conditions. Moreover, there isgrowing evidence that this ribosome heterogeneity (additionallyinvolving posttranscriptional and posttranslationalmodifications ofrRNAs and ribosomal proteins) creates regulatory capacity byconferring translational selectivity of ribosomal subpools for spe-cificmRNAs (Sauert et al., 2015; Shi et al., 2017). However,whethernonessential plastid ribosomal proteins can act as modulators ofribosome affinity to specific sets of mRNAs is currently unknown.

Translation with a Minimal Set of tRNAs

Similar to some bacterial species, plastids do not encode the fullset of 32 tRNAs that are required to serve the 61 codons bystandard andwobble basepairing betweencodon andanticodon.For example, only 30 tRNA genes are present in the plastid ge-nome of most seed plants. Since there is no evidence for tRNAimport from the cytosol, superwobble base pairing has beenconsidered as amechanismenabling translationwith a reduced setof tRNAs. A systematic reverse genetic screen revealed that thechloroplast tRNAs trnG-GCC, trnL-CAA, trnS-GGA, trnT-GGU, and

trnV-GACarenotessential forplantviability (Alkatib etal., 2012, andreferences therein). Thesenonessential tRNAsdecodecodonswithapyrimidine in the thirdpositionand, for all of them,essentialplastidisoacceptor tRNAs with a uracil (U) in the first anticodon positionexist in plastids. These isoacceptor tRNAscan serve the respectivecodons by superwobble base pairing (i.e., the U in the wobbleposition of the anticodon can pair with all four nucleotides in thirdcodon position; Alkatib et al., 2012). The diverse phenotypes ofthese mutants suggest that superwobble base pairing causesdistinct molecular constraints on translation (Alkatib et al., 2012),possibly explaining the evolutionary conservation of some non-essential tRNAs.

CONSERVATION AND MODIFICATION OF BACTERIALTRANSLATION MECHANISMS IN CHLOROPLASTS

The overall structural conservation of essential functional ele-ments of bacterial ribosomes in chloroplasts is generally assumedto reflect the functional preservation of the bacterial translationmechanisms in plastids (Peled-Zehavi and Danon, 2007).

Initiation

The initiation process starts with the contact of a preinitiationcomplex consisting of the 30S subunit and the initiator tRNA (forN-formylmethionine [fMet]) to the initiation site in the mRNA.Bacterial initiation depends on the initiation factors IF1, 2, and 3,which facilitate initiator tRNA binding and ribosome subunit as-sembly. Functional chloroplast orthologs were identified for allthree IFs (Sijben-Müller et al., 1986; Campos et al., 2001; Miuraet al., 2007; Zheng et al., 2016). Interestingly, many plants containtwo or more paralogous genes for plastid IF3, whose differentialexpression was proposed to regulate chloroplast translationinitiation (Nesbit et al., 2015). Also similar to the standard bacterialtranslation initiation, approximately two-thirds of the chloroplastreading frames are preceded by the purine-rich Shine-Dalgarnosequence (SD) (Shine and Dalgarno, 1974; Scharff et al., 2011). Inbacteria, the SD interacts by base pairing with a pyrimidine-richsequence in the 16S rRNA (the anti-Shine-Dalgarno sequence[aSD]) to ensure proper positioning of the initiation complex at thestart codon. The aSD is fully conserved in plant plastids, andevidence for its functionality has been provided for many geneswith SD (Kim and Mullet, 1994; Hirose and Sugiura, 2004). Never-theless, the significance of SD-dependent initiation in chloroplastshas been questioned (Fargo et al., 1998). To provide ultimate clar-ification, mutations were introduced into the aSD in the tobaccochloroplast genome and shown to cause reduced translation formany reading frames with upstream SDs, thus confirming thefunctionality of SD-aSD interactions (Scharff et al., 2017). On theother hand, roughly a third of the chloroplast genes and also manybacterial genes do not contain SD sequences (or contain a putativeSD but display SD-independent translation initiation). It was shownthat low amounts of mRNA secondary structure around the startcodon facilitate SD-independent translation initiation (Scharff et al.,2011, 2017; Nakagawa et al., 2017). Furthermore, in bacteria, theribosomal protein S1 preferentially binds polypyrimidine tracts andacts as an RNA chaperone that unfolds structured regions inmRNAs, thereby enabling efficient SD-independent translation

Chloroplast Translation 749

initiation (Qu et al., 2012). Chloroplast S1 binds RNA with a prefer-ence for adenineor uracil-rich sequences,butwhether this supportsSD-independent translation initiation is unknown (Franzetti et al.,1992; Shteiman-Kotler and Schuster, 2000).

Studies in both bacteria and chloroplasts have pointed toa 59-to-39 ribosome scanning mechanism and the preferentialutilization of the 59-most start codon (Drechsel and Bock, 2011;Yamamoto et al., 2016). Moreover, extended interactions ofmRNA sequences upstream of the start codon and adjacent tothe SD with bases downstream of the anticodon of the initiatortRNA-fMet and bases next to the aSD, respectively, have beensuggested to facilitate chloroplast translation initiation (Ruf andKössel, 1988; Esposito et al., 2003; Kuroda et al., 2007). How-ever, the exact mechanism and the quantitative contribution ofthese interactions to the efficiency of translation initiation remainto be elucidated. Similar to bacteria, the triplets AUG, GUG, andUUGcan be utilized as start codons in chloroplasts (Hirose et al.,1999; Kuroda et al., 2007; Rott et al., 2011;Moreno et al., 2017), withthe recognition efficiency of non-AUG start codons presumablydepending on the sequence context (Boeck and Kolakofsky,1994).

Elongation

After bindingof the 50S subunit to thepreinitiation complex, thefunctional 70S ribosome is completed and starts moving alongthe coding sequence of the mRNA to translate it into a poly-peptide chain. Bacterial translation elongation depends on thefactors EF-Tu, EF-G, and EF-Ts for which conserved chloro-plast orthologs were identified (Breitenberger et al., 1979; Foxet al., 1980; Sreedharan et al., 1985). Notably, the expression ofchloroplast elongation factors is regulated by light and otherstimuli (temperature, phytohormones, anddevelopmental cues),suggesting involvement of elongation factors in the regulation oftranslation (Akkaya and Breitenberger, 1992; Bhadula et al.,2001; Singh et al., 2004; Albrecht et al., 2006; Liu et al., 2010;Schröter et al., 2010).

Analogous to bacterial gene expression, many chloroplastgenes are cotranscribed from operon-like gene clusters. Theresulting polycistronic transcripts contain reading frames thatare separated by (often short) spacer sequences or evenoverlapbya fewnucleotides. Inbacteria, translationof adjacentand overlapping reading frames is frequently coupled in thattranslation of the second coding sequence depends on that ofthe first one (Jackson et al., 2007). In some cases, the strongRNA helicase activity of the ribosome translating the upstreamreading frame is needed to unfold RNA secondary structuresthat mask initiation elements (SD and/or start codon) of thedownstream reading frame (Jackson et al., 2007). Recently,direct coupling of termination on the upstream reading frame(without ribosomedisassembly) with subsequent scanning andreinitiation on the downstream reading frame also has beendemonstrated (Yamamoto et al., 2016). In chloroplasts, casesof coupled (ndhC/K and psbD/C ) and uncoupled (atpB/E )translation were identified by in vitro and in vivo analyses,respectively, but the detailed mechanisms of translationalcoupling are unknown (Yukawa and Sugiura, 2008; Adachiet al., 2012; Zoschke et al., 2013a). A recently designed in vivo

expression system exploits coupled translation in chloroplastsand suggests that the RNA helicase function of the ribosomemay also mediate translational coupling in chloroplasts (MartinAvila et al., 2016).

Termination and Ribosome Recycling

When one of the three stop codons is reached, the orthologousrelease factors RF1/PrfA (serving UAA and UAG) and RF2/PrfB1 (serving UAA and UGA) set the synthesized protein freeby hydrolysis of the ester bond (Buckingham et al., 1997;Meurer et al., 2002; Motohashi et al., 2007). Additionally, PrfB1is involved in the stabilization of plastid mRNAs containingreading frames with UGA stop codons (Meurer et al., 2002).PrfB3, a nonfunctional chloroplast-targeted paralog of PrfB1,lacks domains that are essential for stop codon recognition andhydrolytic activity of release factors. Remarkably, PrfB3 gaineda new function in transcript stabilization of the petB mRNA(Stoppel et al., 2011). Another release factor, RF3, facilitatesdissociation of RF1 and RF2 from the ribosome in bacteria(Buckinghamet al., 1997) and likely also in chloroplasts (Beligniet al., 2004). In the final step, ribosome recycling factor (Rollandet al., 1999), EF-G, and IF3 facilitate the release of mRNA andtRNA, and the disassembly of the small and large ribosomalsubunits, thereby recycling them for the next round of trans-lation initiation (Kiel et al., 2007).

PLASTID TRANSLATION IS INTERCONNECTED WITH RNAAND PROTEIN METABOLISM

Relaxed Coupling of Translation and Transcription

In bacteria, translation initiates and elongates cotranscriptionally,thus ensuring efficient transcription (e.g., by preventing RNApolymerase backtracking), conferringRNAstability (by translatingribosomes protecting the mRNA from ribonucleolytic attack),enabling timely translation, and maintaining genome integrity(e.g., by avoiding extended hybridization of RNA and DNA thatwould cause collision of the transcription and replication ma-chineries; McGary and Nudler, 2013). A similar coupling of tran-scription and translation was proposed for chloroplast geneexpression based on early electron micrographs that were in-terpreted as evidence for ribosomes being associated with na-scent transcripts (Rose and Lindbeck, 1982; Figure 2). Additionalevidence for coupling of transcription and translation has comefrom the findings that (1) ribosomal proteins are associated withthe transcription machinery (Pfalz et al., 2006), and (2) translationfactors and other proteins involved in translation are enriched inplastid nucleoids in a ribonuclease-sensitive manner, suggestingtethering by nascent transcripts (Majeran et al., 2012). Moreover,orthologs of the Nus proteins that couple transcription andtranslation in bacteria have been identified in chloroplast nucle-oids (Majeran et al., 2012). However, chloroplast transcripts havea longer half-life than bacterial transcripts and are stablewhen notcovered by ribosomes, and many translated RNA species aregenerated by RNA processing (see below). Together, this impliesthat, simply due to the kinetics of mRNAprocessing and turnover,

750 The Plant Cell

there may be a quantitative shift toward posttranscriptionaltranslation in chloroplasts.

The chloroplast genome is regularly transcribed by bacterial-type and phage-type RNA polymerases that have very differentproperties (Börner et al., 2015). For example, the twopolymerases

transcribe with different speeds and recognize different pro-moters, thusproducingprimary transcriptswithdivergent 59ends.Notably, in phage-infected E. coli cells, the speedy transcriptionby the RNA polymerase of bacteriophage T7 is not coupled withtranslationand thusproduces initially “naked” (i.e., ribosome-free)

Figure 2. Overview of Internal and External Triggers That Cause Regulatory Adjustments of Translation in Chloroplasts, the Mechanisms That ControlTranslation, the Coupling of RNA and Protein Metabolism to Chloroplast Translation, and the Localization of the Chloroplast Translation Machinery.

Chloroplast translation is regulated in response to internal and external triggers (listed in the upper part). Nucleus-encoded factors are translated in thecytosol (shown in the upper left part) and imported into the chloroplast, where they control and/or regulate chloroplast protein synthesis directly (by alteringchloroplast translation activity) or indirectly (by controlling cotranslational chloroplast RNA or protein metabolisms). Chloroplast translation occurscotranscriptionally (left); however, due to the slow mRNA turnover, the majority of ribosomes act posttranscriptionally (right). RNA binding proteins assistcotranslational RNA processing and/or facilitate translation initiation. Ribosomes initiate and elongate regularly on both processed and unprocessedtranscripts, the extent of which seems to mainly depend on the kinetics of the processing events (see text and Figure 3). Many of the factors involved inprotein processing, folding, targeting, and assembly act cotranslationally on the nascent polypeptide. See text for details.

Chloroplast Translation 751

transcripts with a higher decay rate (Makarova et al., 1995). Howtranscription by the different plastid RNA polymerases is co-ordinated with translation and whether or not the utilized RNApolymerase influences the kinetics of protein synthesis are cur-rently unknown.

Unprocessed Transcripts Can Be Translated

Primary chloroplast transcripts undergo extensive RNA pro-cessing, including splicing of group I and II introns, RNAediting(changing cytosine to uracil residues to restore codons forconserved amino acids or start or stop codons), 59 and 39 endtrimming, and intercistronic processing that generates diverse

transcript isoforms from polycistronic primary transcripts(Barkan, 2011; Lyska et al., 2013). There is no obvious spatialseparation that would compartmentalize RNAmetabolism andtranslation. Interestingly, many of the factors known to beinvolved in transcript processing (e.g., RNA binding proteins),stabilization, and translation colocalize with processed andunprocessed transcripts in nucleoids and transcriptioncomplexes (Pfalz et al., 2006;Majeranetal., 2012;Lehnigeretal.,2017). This raises the question whether unprocessed mRNAs,often referred to as “precursors” or “immature transcripts,” areutilized as templates for translation or whether any partitioning(e.g., temporal separation) exists between RNA processing andtranslation in plastids.

Figure 3. Ribosomes Translate Unprocessed Chloroplast Transcripts (See Text for Details).

(A)Several chloroplast reading frames are interrupted by group II introns. Left: Translating ribosomes cover exon 1 of unspliced atpF, ndhA, ndhB, and ycf3transcripts (Zoschke et al., 2013a; Alice Barkan, personal communication). Middle and right: Splicing releases the intron and ligates the exons. Con-sequently, both exons of the spliced transcript are occupied by ribosomes, producing full-length proteins (chain of black dots: nascent polypeptide).(B)Chloroplast transcripts are edited at specific sitesbymodificationof cytosine (C) to uracil (U) residues, often restoringcodons for conservedamino acids(change fromyellow towhitedot in thenascentpeptide). Actively translatedmRNAshave thesameeditingstatusas the total transcriptome (Chotewutmontriand Barkan, 2016), indicating that, in a partially edited transcript pool, unedited transcripts also are translated.(C) Polycistronic chloroplast transcripts often undergo posttranscriptional processing that generates smaller transcript isoforms (represented by the threemonocistronic transcripts on the right; RF, reading frame). Often all transcript isoforms are used as translation templates. The extent to which transcriptprocessing may enhance translation efficiency needs to be determined on a case-by-case basis.

752 The Plant Cell

Up to 12 reading frames are interrupted by group II introns inchloroplast genomes of seed plants (Sugiura, 1995). In all of them,a substantial fraction of the coding region is located downstreamof the intron; consequently, splicing is essential to producefunctional proteins (Barkan, 2011). Surprisingly, recent ribosomeprofiling studies in maize chloroplasts have demonstrated thattranslation initiates on unspliced atpF, ndhA, ndhB, and ycf3transcripts and also elongates (Zoschke et al., 2013a; AliceBarkan, personal communication; Figure 3A).Whether translationelongationpausesat the robust intronstructureor, alternatively, theRNAhelicase functionof the ribosomeallows translation toproceedinto the intronuntil it terminatesat thefirst in-framestopcodon, thusproducing nonfunctional proteins, has not yet been possible toresolve.Nevertheless, it isclear that ribosomes initiateandelongateon unspliced transcripts, strongly arguing against a spatial ortemporal separation of splicing and translation processes.

High-resolution ribosomeprofilinganalysisofmaizechloroplasttranslation also demonstrated that the degree of editing in mRNAfootprints of actively translating ribosomes is not substantiallydifferent from that in the general transcript pool (Chotewutmontriand Barkan, 2016). Specific editing sites that are only partiallyedited in the transcript pool showed a similar degree of partialediting in ribosome footprints. This is in line with the earlier findingthat editing of tobacco rps14 is not a requirement for efficientin vitro translation (Hirose et al., 1998). Together, these data implythat ribosomes cannot distinguish between edited and uneditedtranscripts (Figure3B).However, twoexceptionswere identified inmaize: the transcriptsof rpl2andndhA,whichareonly translated intheir edited form (Chotewutmontri and Barkan, 2016). In rpl2,ACG-to-AUG editing restores the start codon (Hoch et al., 1991),therebyactivating translation (Chotewutmontri andBarkan,2016).This is consistent with the earlier finding that restoration of thetobacco ndhD start codon by editing is essential for efficienttranslation initiation in vitro (Hirose and Sugiura, 1997). In someinstances, start codon restoration by editing is subject to tissue-specific or developmental variation, thus raising the intriguingpossibility that editing may control translational activity (IchinoseandSugita, 2016). In thecaseofndhA, splicing of its group II intronis required to enable editing of the first editing site in exon II(Schmitz-Linneweber et al., 2001, and references therein). Con-sequently, ndhA transcripts that are unedited at this particular siteare unspliced, and elongating ribosomes cannot get through tothe editing site in the second exon (Chotewutmontri and Barkan,2016). Leaving aside these special cases, it can be assumed that,normally, unedited and unspliced transcripts are translated(Figure 3) and potentially give rise to the synthesis of low amountsof nonfunctional and potentially deleterious proteins. In accor-dance with this assumption, failure to restore conserved aminoacids in subunits of PSII, the Cyt b6f complex, and the ATPsynthase by mRNA editing strongly impairs the function of therespectivecomplexes (Bocketal., 1994;Zitoet al., 1997;Schmitz-Linneweber et al., 2005b). Hence, rapid proteolytic removal of thedysfunctional proteins synthesized from unedited and unsplicedtranscripts has to be assumed to prevent deleterious effects onchloroplast function.

End trimming and intercistronic processing of plastid tran-scripts have been proposed to enhance their translational activity(Drechsel and Bock, 2011). However, internal reading frames on

polycistronic transcripts derived from the maize psbB-psbT-psbH-petB-petD transcription unit are actively translated, despitethe fact that they exist also as 59 reading frames on processedtranscripts (Barkan, 1988). Likewise, unprocessed tobacco atpHand rbcL mRNAs were translated as efficiently as processedmRNAs in vitro (Yukawa et al., 2007). Furthermore, synthetictranscription units that were engineered into plastids gave rise topolycistronic transcripts that were efficiently translated in theabsenceofprocessing (StaubandMaliga,1995).Finally, agenome-wide ribosome profiling study revealed that several polycis-tronic mRNAs are efficiently used as translation template,despite the known coexistence of monocistronic transcriptisoforms (Zoschke andBarkan, 2015). In all of these examples, thetranslation of downstream reading frames in polycistronicmRNAswas not dependent on transcript processing into monocistronicunits, indicating that internal start codons can be efficiently rec-ognized. Also, a number of plastid reading frames are only presentin di- or polycistronic transcripts, indicating that these must un-dergo translation. Altogether, these data provide compellingevidence that transcript end processing and intercistronic pro-cessing are not general requirements for efficient translation(Figure 3C). However, in a few cases, there is good evidence thattranscript processing stimulates translation. For instance, in to-bacco, abase-pairing interactionbetween thepsaCcoding regionand the ndhD 59UTR in the dicistronic transcript was shown toprevent efficient ndhD translation in vitro, whereas processedmonocistronic transcripts were translationally active, suggestingthat, in this operon, processing is required to activate translation(Hirose and Sugiura, 1997). Similarly, in vitro translation providedevidence thatunprocessedatpB,psbB, andpsbD transcripts fromtobacco chloroplasts are less efficiently translated than theirprocessed isoforms (Yukawa et al., 2007; Adachi et al., 2012).Moreover, in tobacco chloroplasts, heterologous expression ofGFP from engineered polycistronic mRNAs was more efficientwhenGFPwasplacedat the59endof thesyntheticoperon (Drechseland Bock, 2011). Finally, in maize, the monocistronic forms of psaIand rps14 show the highest accumulation in those developmentalstages where the highest translation rates of these reading framesoccur, a finding that would be consistent with the monocistronicRNAspeciesbeingbetter translatable (Chotewutmontri andBarkan,2016).In sum, although in somecases, translation is indeedstimulated

byRNAprocessing, there is no general dependence of translationonprocessing. Unprocessed, unspliced, andunedited transcriptshave been shown to be used as translation templates (Figure 3);therefore, these transcript isoforms are not necessarily “immature”or “precursors.”

Several RNA Binding Proteins Act Dually in TranscriptProcessing/Stabilization and Promotion of Translation

In recent years, plastid RNA binding proteins, many of them withhelical repeat domains,were shown tobe involved in specificRNAend trimming and intercistronic processing events (Barkan andSmall, 2014; Hammani et al., 2014). Mutants of some of thesefactors (Table 1) displayed defects in RNA processing that wereaccompanied by translation deficiencies (Barkan et al., 1994;Felder et al., 2001; Hashimoto et al., 2003). Initially, these were

Chloroplast Translation 753

interpreted as RNA processing-dependent translation defects (inthat processing was required for efficient translation). However,later, it wasobserved that the knockout of PPR10, anRNAbindingprotein involved in the processing and stabilization of specificatpH transcript isoforms, caused much stronger translation andprotein accumulation defects than expected from its RNA pro-cessing defect, suggesting a more direct role of PPR10 in trans-lation of atpH (Pfalz et al., 2009). In vitro assays showed thatPPR10 binds to the atpH 59UTR and protects transcripts from 59-to-39exonucleolyticdegradation.Consequently, thePPR10bindingsite defines the 59 end of these transcripts (Prikryl et al., 2011). Inaddition, PPR10 binding remodels the atpH 59UTR such that an RNAstem-loopstructure thatoccludestheputativeSDsequenceofatpH isdissolvedandtheribosomebindingsitebecomesexposed(Prikryletal.,2011). This suggests a dual function of PPR10 in RNA stabilizationand stimulation of atpH translation. A similar mode of action wasshown, or is discussed, for other chloroplast RNA binding proteinssuch as HCF107, PGR3, CRR2, and CRP1 (see Table 1 and refer-ences therein). Theadditional translation-promoting function shouldbe independent of processing in that it should also occur in poly-cistronic transcripts where the target reading frame is locateddownstream of other reading frames. In fact, reanalysis of ppr10,crp1, pgr3, and hcf107maize mutants by ribosome profiling revealedsubstantial translation defects in vivo (and less severe transcriptaccumulation defects) for the reading frames downstream of theRNAbinding sites of the respective protein (in atpH, petD, petL, andpsbH expression, respectively; Zoschke et al., 2013a; Alice Barkan,personal communication). In line with a dual function of some RNAbinding proteins, PPR53, a member of the small PPR-SMR family,was recently described to be involved in both promotion of ndhAtranslation and processing/stabilization of transcript isoforms withndhA as the 59 reading frame (Zoschke et al., 2016).

These examples support the idea that RNA binding proteinsacting in processing/stabilization of specific 59 transcript endscan also directly promote translation of the reading framedownstream of their binding site (Figure 2). Such proteins pro-vide a physical link between RNA metabolism and transla-tion; therefore, co-occurrence of intercistronic processing and

stimulation of translation does not necessarily imply a strict re-quirement of RNA processing to facilitate translation.Notably, a widely employed sequence element in chloroplast

biotechnology (IEE, for intercistronic expression element) thatenhances the heterologous expression of reading frames locatedin polycistronic transcription units includes the HCF107 bindingsite (Zhou et al., 2007; Hammani et al., 2012). The insertion of theIEE between reading frames enhances the accumulation of mon-ocistronic transcripts. This could be related to the RNA secondarystructure formedby the IEE (Zhou et al., 2007), the action of RNase E(Walter et al., 2010), and/or thebindingofHCF107 (Hammani et al.,2012; Legen et al., 2018).

The Turnover of Plastid Transcripts Is Not Determined byTheir Translation Status

In E. coli, mRNAs are stabilized by translating ribosomes, pre-sumably by ribosome coverage providing physical protectionfrom ribonucleases (Laalami et al., 2014). By contrast, the study ofmaize and Arabidopsis mutants with transcript-specific trans-lation defects has revealed that atpB, petA, psaC, and psbAtranscripts are stable although their translation and, consequently,their ribosome occupancy was dramatically reduced (Barkan et al.,1994;McCormacandBarkan,1999;Linketal., 2012;Zoschkeetal.,2012, 2013b). Furthermore, exchange of the canonical AUG startcodonby thenonstandard initiationcodonGUGorUUGin theatpB,clpP, and psbD reading frames in tobacco chloroplasts diminishedtranslation initiationandproteinsynthesisbutdidnotdestabilize thetranscripts (Rott et al., 2011;Moreno et al., 2017;MarkA. Schöttler,personal communication). Also, mutants with general impairmentsin chloroplast translation do not exhibit general transcript accu-mulation defects (Barkan, 1993; Scharff et al., 2017). Similarly, thetreatment of wild-type Arabidopsis plants with lincomycin, an an-tibiotic that disturbs 70S translation elongation only at the earlieststeps, thus causing runoff of ribosomes (Kallia-Raftopoulos andKalpaxis, 1999), did not cause a substantial decrease in the ac-cumulation of any of the analyzed chloroplast transcripts (Meureret al., 2002; Stoppel et al., 2011). Accumulation of chloroplast

Table 1. Factors Demonstrated or Suggested to Facilitate Translation of Specific Transcripts in Seed Plant Plastids

Factor Protein Domain(s)Reading Frames withTranslation Promoted Species References for Translational Function

ATP1 Unknown atpB Maize McCormac and Barkan (1999); Zoschke et al. (2013a)ATP4/SVR7 PPR, SMR atpB Maize, Arabidopsis Zoschke et al. (2012)CRP1 PPR petA, petD*, psaC Maize Barkan et al. (1994); Zoschke et al. (2013a)CRR2 PPR ndhB* Arabidopsis Hashimoto et al. (2003)HCF107 HAT psbH* Arabidopsis Felder et al. (2001); Hammani et al. (2012)HCF152 PPR petB*? Arabidopsis Meierhoff et al. (2003)HCF173 Atypical SDR psbA* Arabidopsis Schult et al. (2007)HCF244 Atypical SDR psbA Arabidopsis Link et al. (2012)PGR3 PPR petL*, ndhA? Arabidopsis Yamazaki et al. (2004); Cai et al. (2011)PPR10 PPR atpH* Maize Pfalz et al. (2009); Prikryl et al. (2011); Zoschke et al.

(2013a)PPR53 PPR, SMR ndhA* Maize, Arabidopsis Zoschke et al. (2016)

Asterisks indicate an additional function in stabilization of the transcript 59 end upstream of the reading frame whose translation is stimulated. Questionmarks denote proposed but experimentally unconfirmed functions in stimulation of translation. SDR, short-chain dehydrogenase/reductase; HAT, halfa tetratricopeptide repeat; SMR, small MutS-related.

754 The Plant Cell

transcripts was also not increased after treatment with chloram-phenicol, a 70S elongation inhibitor that arrests ribosomes andinhibits their release, thus resulting in densely ribosome-coveredtranscripts (Nierhaus and Wittmann, 1980).

Altogether, the available data demonstrate that chloroplastmRNAs are stable in the absence of translation and do not requirephysical protection by ribosomes. This may not be surprisinggiven the evolutionary switch from a largely transcriptional reg-ulation of gene expression, as found in bacteria, to predominantlyposttranscriptional regulation in chloroplasts (which stronglydepends on transcripts with long half-lives). Chloroplasts harbormany endo- and exoribonucleases that potentially could degrade“naked” transcripts (Germain et al., 2013). This implies that non-translated chloroplast mRNAs must be somehow protectedagainst nuclease attack. A small family of RNA recognition motifdomain-containing proteins, the chloroplast RNPs (cpRNPs),were shown tobe involved indifferent stepsofmRNAmetabolism,including transcript stabilization (Ruwe et al., 2011). Taking intoaccount the high abundance of these proteins in the chloroplast,their broad RNAbinding activity in vivo (in that they associate withvirtually allmRNAs), and the fact that theyare specifically bound tononpolysomal mRNAs (Nakamura et al., 2001; Kupsch et al., 2012;Teubner et al., 2017), cpRNPs are strong candidates for providingstability to untranslatedmRNAs. Notably, mutants of the cpRNPsCP29A and CP31A show a conditional cold-sensitive phenotype,presumably caused by a reduction in the stability ofmanymRNAs(Kupsch et al., 2012). A possible explanation is a runoff of trans-lating ribosomes in the cold and subsequent transcript degra-dation in the absence of stabilizing cpRNPs. A recent structuralanalysis revealed a narrowed mRNA entry site of the chloroplastribosome comparedwith that of E. coli (Bieri et al., 2017). It seemstempting to speculate that this is because chloroplast ribosomes,different from their bacterial counterparts, need to strip offabundantRNAbinding proteins such as cpRNPswhen translationreinitiates andelongates onpreviously “stored” (i.e., untranslated)mRNAs or even on normally translated mRNAs with low initiationrates (resulting in larger ribosome spacing).

In sum, untranslated chloroplastmRNAsare stable, and cpRNPbinding may protect them against ribonucleolytic attack, thuscausing the observed uncoupling of mRNA stability from trans-lation. However, a direct functional connection between trans-lational activity, cpRNP (un)binding, and mRNA stability remainsto be demonstrated.

Cotranslational Folding, Maturation, Targeting, andAssembly of Proteins

In bacteria, several steps in protein metabolism, including pro-teolytic processing, chemical modification, cofactor binding,folding, targeting, and assembly, can occur cotranslationally(Gloge et al., 2014). It seems clear that this is also the case inchloroplasts, although the knowledge about the intersectionsbetween plastid translation and protein metabolism is still scarce(Giglione et al., 2015; Breiman et al., 2016; Figure 2). Removal ofthe N-terminal N-formylmethionine often represents the first stepof nascent peptide chain processing in bacteria and chloroplasts.It occurs cotranslationally by the consecutive reactions of peptidedeformylase and methionine aminopeptidase (Breiman et al.,

2016). Likewise, the N-terminal signal peptide for thylakoid tar-geting of PetA (cytochrome f) is cotranslationally cleaved (seebelow). Another widespread N-terminal modification of the na-scent peptide is theN-a-acetylationof thepenultimate aminoacid(Zybailov et al., 2008; Breiman et al., 2016). A complete list ofidentified N-terminal processing events in plastid-encoded pro-teins is provided at http://www.i2bc.paris-saclay.fr/spip.php?article1261&lang=fr (Breiman et al., 2016).In bacteria and eukaryotes, folding of the nascent peptide chain

has been shown to start already in the ribosome exit tunnel(Bhushan et al., 2010; Gloge et al., 2014). With dimensions of10nm in lengthand1 to2nm inwidth, the70S ribosomeexit tunnelhas a sufficient size to shelter 30 to 60 amino acids (depending onthe folding status) and allows the formation of small protein do-mains consisting of a-helices (Holtkamp et al., 2015). Proteinfolding in the exit tunnel is assisted by the ribosome itself throughinteractions of the nascent peptide with the 23S rRNA and ribo-somal proteins (e.g., L4, L22, and L23; Gloge et al., 2014). Giventhe high conservation of the peptide exit tunnel, cotranslationalfolding is expected to occur also during chloroplast translation.The ribosome-associated chaperone trigger factor binds thenascent peptideuponexit from the ribosomeandstabilizes it, thuspreventing protein aggregation and assisting cotranslationalprotein folding in bacteria and, most likely, also in chloroplasts(Breiman et al., 2016; Ries et al., 2017). Subsequently, otherchaperones are recruited and take over (Trösch et al., 2015).In parallel to folding, cofactors such as chlorophylls, hemes,

carotenoids, quinones, and metal ions can associate cotransla-tionally with chloroplast apoproteins (Schöttler et al., 2011, 2015;Nickelsen and Rengstl, 2013). Several studies suggest that theplastid-encoded apoproteins of PSI and PSII must bind chloro-phylls cotranslationally to ensure faithful complex biogenesis,most likely, because chlorophyll binding is required for correctprotein folding and assembly (Nickelsen and Rengstl, 2013). Thisis supported by evidence that chlorophyll stabilizes nascentchlorophyll binding proteins (Mullet et al., 1990; Kim et al., 1994b;Eichacker et al., 1996). Specific pausing sites during psbA, psaA,psaB, and psaC translation elongation were suggested to facili-tate the cotranslational binding of chlorophyll and other cofactorssuch as pheophytin, quinone, iron sulfur, andmanganese clusters(Kim et al., 1991, 1994a;Gawronski et al., 2018). In cyanobacteria,unassembled PsbB and PsbC apoproteins contain chlorophyll aandb-carotene, suggesting their early cotranslational association(Boehm et al., 2011).Chloroplasts comprise different suborganellar compartments:

stroma, thylakoid membrane, thylakoid lumen, inner and outerenvelope membranes, and the intermembrane space. The tar-geting of some chloroplast-encoded proteins to the thylakoidmembrane has long been recognized to occur cotranslationally(reviewed in Celedon and Cline, 2013; Figure 2). Early on, it wasshown that puromycin treatment (which causes prematuretranslation terminationand thereby releaseof thenascentpeptide)also releases ribosomes from the thylakoid membrane in chloro-plasts, suggesting cotranslational protein targeting mechanisms(Yamamoto et al., 1981). In later studies, chloroplast subfrac-tionation coupled with polysome analysis and pulse labelingstudies revealed that polytopic proteins of PSI (PsaA and PsaB)and PSII (PsbA/D1, PsbB/CP47, PsbC/CP43, and PsbD/D2),

Chloroplast Translation 755

and the bitopic cytochrome f subunit (PetA) of the Cyt b6f com-plex associate with the thylakoid membrane cotranslationally(Margulies et al., 1987; Friemann and Hachtel, 1988; Kim et al.,1994b; van Wijk et al., 1996). However, the interpretation of theresults from these experiments was sometimes controversial(Ibhaya and Jagendorf, 1984) and complicated by the fact that inchloroplasts, polycistronic transcripts can be used as translationtemplates. Consequently, one cotranslationally inserted poly-peptide produced from a polycistronic transcript is sufficient totether all cotranscribed cistrons to the thylakoid membrane. Thisdifficultywasovercome in a recent studyusing ribosomeprofiling,a method that employs nucleases to degrade mRNAs in poly-somes down to the footprints protected bymonosomes (ZoschkeandBarkan, 2015). By coupling this approachwith fractionation ofchloroplasts into thylakoid membranes and stroma, cotransla-tionalmembrane insertion couldbecomprehensively examinedatagenome-widescale. Thestudy revealed that 19of the37plastid-encoded intrinsic transmembrane domain-containing thylakoidproteins in maize insert cotranslationally into the membrane andsupplied evidence that exposure of the first transmembrane do-main provides the signal and/or the anchor for stable membraneassociation (with the sole exception of PetA, as described below).The data suggest a model for ribosome-mediated mRNA tar-geting, in which the nascent polypeptide exposed by the first“pioneer” ribosome anchors the translation machinery togetherwith the translated mRNA at the thylakoid membrane. Continuedtranslation by the following ribosomes keeps the mRNA tetheredto the thylakoidmembrane. A similarmodelwas suggested for thecytosolic ribosomes that are associated with mitochondria andthe endoplasmic reticulum (Jan et al., 2014; Williams et al., 2014).In addition, a recent ribosome profiling study in Arabidopsiscorrelatedplastid ribosomepausingeventswith thesynthesis andcorrect integration of transmembrane domains (Gawronski et al.,2018). Electron microscopic evidence indicates that membrane-associated chloroplast polysomes are connected with all un-stacked thylakoid membrane regions (i.e., stroma lamellae andgrana margins), but not with internal membranes in grana stacks,which are inaccessible due to their tight packing (Yamamoto et al.,1981). Consequently, once grana stacks are assembled duringchloroplast biogenesis, plastid-encoded grana proteins (mainlyPSII subunits) need to be transported posttranslationally fromunstacked membrane regions into grana stacks, for example,during photosystem repair (Puthiyaveetil et al., 2014; Pribil et al.,2014).

The mechanisms involved in suborganellar protein targetinghave been best studied for nucleus-encoded chloroplastproteins that are posttranslationally distributed (Celedon andCline, 2013). Five major pathways with partially overlappingfunctions (and some shared components) have been described(Schünemann, 2007; Celedon and Cline, 2013). The secretory(Sec) pathway and the twin-arginine translocase (Tat) transportproteins across the thylakoid membrane into the lumen (re-viewed in Schünemann, 2007). The chloroplast SRP (cpSRP)interacts with the cpSRP receptor cpFtsY and the insertaseALB3 to insert nucleus-encoded light-harvesting complexproteins into the thylakoid membrane (reviewed in Ziehe et al.,2017). Some proteins apparently insert spontaneously into thethylakoid membrane; finally, a recently discovered parallel Sec

pathway targets proteins to the inner envelope membrane (Liet al., 2017b).Much less is known about the mechanisms involved in

cotranslational suborganellar targeting of chloroplast-encodedproteins. Most plastid-encoded proteins are found in either thestroma or the thylakoid membrane. Plastid-encoded membraneproteins likely utilize one of the above-mentioned targetingpathways, either co- or posttranslationally. So far, the cotrans-lational targeting mechanisms were elucidated in some detail foronly two plastid-encoded proteins: PetA and PsbA. PetA is theonly plastid-encoded protein containing a cleavable signal pep-tide at its N terminus, which is recognized by cpSecA. In vitro andgenetic data suggest that PetA targeting to the thylakoid mem-brane occurs cotranslationally (Voelker et al., 1997; Röhl and vanWijk, 2001, and references therein). A recent ribosome profilingstudy showed that nascent PetA engages the thylakoid mem-brane long before its single transmembrane domain becomesexposed outside the ribosome exit tunnel, thus confirmingcotranslational action of cpSecA-dependent targeting in vivo(Zoschke and Barkan, 2015). Only recently, SecA-mediated tar-geting in bacteria was demonstrated to also occur cotransla-tionally (Huber et al., 2016).Cotranslational interaction of PsbA and cpSRP54 has been

suggested based on in vitro cross-linking experiments (NilssonandvanWijk, 2002,andreferences therein).However,Arabidopsismutants lacking cpSRP54 have very mild phenotypes, arguingagainst an essential role of cpSRP54 in membrane targetingof PsbA or any other core subunit of the photosynthesis ma-chinery (Amin et al., 1999; Tzvetkova-Chevolleau et al., 2007).Nonetheless, cpFtsY, the essential chloroplast SRP receptor ho-mologthathadpreviouslybeen implicated inPSII repair (Tzvetkova-Chevolleau et al., 2007; Asakura et al., 2008), was shown to beassociated with nascent PsbA in vitro suggesting a role in co-translational targeting (Walter et al., 2015). Furthermore, thetranslocons cpSecY and ALB3 and the multifunctional proteinVipp1, all of which are essential for thylakoid biogenesis (Sundberget al., 1997; Roy and Barkan, 1998; Kroll et al., 2001), appear tointeractwithnascentPsbA invitro, suggesting their involvement incotranslational membrane targeting of PsbA (Zhang et al., 2001;Walter et al., 2015). Recent in vitro data suggest that the co-translational targeting of PetB (cytochrome b6) also involves theinsertase ALB3 (Króliczewski et al., 2016).The assembly of proteins into functional complexes often ini-

tiates cotranslationally (Natan et al., 2017). Biogenesis of themultiprotein complexes in the thylakoid membrane requires thetightly coordinated action of multiple assembly factors that guidethe association of plastid-encoded and nucleus-encoded sub-units (Schöttler et al., 2011; Nickelsen and Rengstl, 2013). Manyplastid-encoded subunits of these complexes are believed tobe assembled cotranslationally (Figure 2) because unassem-bled free subunits are usually condemned to rapid degradation.However, only in few cases, direct evidence for cotranslationalassembly has been obtained. The PSII assembly process is bestunderstood (Nickelsen and Rengstl, 2013), and several plastid-encoded core subunits of PSII have been suggested to assemblecotranslationally into thecomplex.Duringbothdenovoassemblyand repair of PSII, nascent PsbA subunits are integrated intoan early assembly intermediate that contains PsbD (Müller and

756 The Plant Cell

Eichacker, 1999; Zhang et al., 1999).Whereas the first and secondtransmembrane domains of PsbA only weakly interact with PsbD,a robust association is established after synthesis of the fourthtransmembrane domain (Zhang et al., 1999; Zhang and Aro,2002). It is tempting to speculate that the operon-like organizationof chloroplast genes enhances the efficiency of cotranslationaltargeting and assembly (e.g., in the dicistronic psaA/B andpsbD/C transcripts), as this was suggested for bacteria (Natanet al., 2017).

In summary, a multitude of factors act cotranslationally as“welcoming committee” for nascent polypeptides and assist withthe amazing metamorphosis of linear amino acid chains intofunctional proteins and protein complexes in specific sub-organellar locations (Figure 2). We are just beginning to un-derstand the complex interconnections of the diverse processesinvolved in cotranslational protein maturation, targeting, andassembly, but it is becoming increasingly evident that the ribo-some acts as a central hub in the coordination of these processes.

OPERATIONAL CONTROL AND REGULATION OFCHLOROPLAST TRANSLATION

Upon stress and under changing environmental conditions, thethylakoid membrane system is adjusted to achieve optimumphotosynthetic performance and prevent photooxidative dam-age. These acclimation responses require integration of multipleexternal and internal signals (Figure 2) and involve extensiveregulation of chloroplast translation (Nickelsen et al., 2014; Sunand Zerges, 2015).

Translational Control Versus Regulation

The terms “translational control” and “translational regulation” aresometimes used synonymously. However, factors controllingtranslation (e.g., the strength of a ribosome binding site in the59UTR) do not necessarily also regulate translation (i.e., dynam-ically changeprotein synthesis rates in response to environmentalstimuli or developmental programs).

The interplay of trans-acting protein factors and cis-actingRNA elements determines the translation output of chloroplastgenes. The functional involvement of these elements in trans-lational control typically is demonstrated by their genetic ma-nipulation causing altered translational activity. However, thisdoes not necessarily imply a regulatory function in translation,resulting in a change in the protein synthesis rate during ad-aptation processes. In other words, a given factor would havea regulatory function if it were to become limiting for translationunder specific conditions, thus altering protein synthesis output.According to this definition, a true regulatory function of chlo-roplast translation factors has been established only in very fewcases in Chlamydomonas (see below). It was proposed thatmany of the nucleus-encoded factors involved in chloroplastRNA metabolism simply suppress mutations that accumulate inthe (asexually reproducing) plastid genome over time (Maieret al., 2008; Lefebvre-Legendre et al., 2014). Similarly, nucleus-encoded translation factors could be needed constitutively to fixchloroplast mutations at the RNA level (e.g., by resolving sec-ondary structures around ribosome binding sites to facilitate

translation initiation). However, on an evolutionary time scale,factors that control translation may also be recruited as trueregulators that modulate translation in response to internal andexternal triggers.

Translation Plays a Major Role in the Control and Regulationof Chloroplast Gene Expression

Translation is an extremely resource-consuming process due tothe energy and nutrient demands involved in the assembly ofribosomes, the synthesis of amino acids, the expression, pro-cessing, and charging of tRNAs, and the GTP-dependent reac-tions during initiation and elongation. Dividing bacterial cells use;50% of their energy for protein synthesis (Russell and Cook,1995).Millar andcoworkers calculated thecellular energybudgetsused for protein synthesis in Arabidopsis leaves (Li et al., 2017a).Their estimate is that, dependent on the developmental stage,13 to 38% of the cellular ATP is used for protein synthesis, withplastid translation accounting for ;70% of the costs of cellularprotein synthesis.SynthesisofRbcLaloneaccounts formore than15% of the cellular ATP equivalents used for protein synthesis (Liet al., 2017a). In view of the high costs of translation, the rate ofprotein synthesis is tightly coordinated to thecellular demands inalldomains of life. In addition, translational regulation has severaladvantages. As translation is the final synthesis step in gene ex-pression, its regulation (1)canmediaterapid responses to internalorexternal stimuli, (2) most directly affects the protein accumulationlevels, and (3) can be readily exploited to enhance or attenuatechanges in upstream steps of gene expression (i.e., transcriptionand transcript accumulation). Importantly, translation can also bedynamically localized to the site where the synthesized protein isneeded,whereas transcription is largelybound to thepositionof thegenomicDNA. Finally, although in bacterial systems transcriptionalcoregulation is easily achievedbycombininggenes inoperons, thisgenomic organization is rather static and, in contrast to transla-tionally coregulated mRNAs (qualifying as “regulons”), cannotreadilymediatedynamic responses todifferent cues (Keene, 2007).It is generally accepted that chloroplast gene expression is

largely controlled and regulated at the posttranscriptional and,especially, the translational levels, contrasting gene expression incyanobacteria,which ismainly transcriptionally regulated.Severallines of evidence led to this conclusion. First, bacterial transcriptstend to be unstable (with half-lives in the range of minutes), en-abling transcriptional regulation (Pedersen and Reeh, 1978; Klug,1993). By contrast, the half-lives of chloroplast transcripts are inthe range of hours or even days, thus disabling fast transcriptionalresponses (Mullet and Klein, 1987; Klaff and Gruissem, 1991).Second, although chloroplast gene clusters are reminiscent ofbacterial operons, they often are transcribed from different pro-moters (including operon-internal promoters) and, frequently,the resulting primary transcripts are further processed intosmaller units (Barkan, 2011; Lyska et al., 2013;Börner et al., 2015).Also, chloroplast polycistronic transcription units often com-prise functionally unrelated genes (Sugiura, 1995), unlike bacterialoperons,wherecotranscription serves tocouple theexpressionoffunctionally related genes. Third, the translation of some chlo-roplast mRNAs coding for core components of the photosyn-thesis machinery is induced by light, whereas their mRNA levels

Chloroplast Translation 757

remain virtually unaffected (Berry et al., 1988; Mühlbauer andEichacker, 1998). Fourth, translation of many chloroplast mRNAswas shown to be the rate-limiting step in gene expression inChlamydomonas (Eberhard et al., 2002), although the situationmay be different in angiosperms (Udy et al., 2012). Moreover, ina regulatory mechanism termed control by epistasy of synthesis(CES), the translation rate of some subunits of photosyntheticprotein complexes is regulatedby thepresenceorabsenceof theirassembly partners (Choquet andWollman, 2009; see also below).Fifth, in recent years, a number of factors required for the trans-lation of specific chloroplast reading frames were discovered,which may be suggestive of extensive translational regulation(Table 1).

RNA cis-Elements Controlling Translation

RNA sequence or structural elements that are located in cis,typically upstream of the reading frame, play crucial roles intranslational control. A major cis-acting sequence element fortranslation initiation in chloroplasts is the SD sequence (Scharffet al., 2017; see above). Recent ribosome profiling studies havesuggested that, in bacteria and chloroplasts, SD-like sequencesthatare locatedwithin reading framescauseprogrammedpausingof elongating ribosomes (Li et al., 2012; Zoschke et al., 2013a;Gawronski et al., 2018). It has been proposed that SD-dependentpausing facilitates cotranslational foldingand targetingof nascentpolypeptides (Li et al., 2012; Fluman et al., 2014). Additionalchloroplast cis-acting sequence elements for translational controlare the binding sites of trans-acting factors that stimulate trans-lation of specific reading frames (Table 1).

The degeneracy of the genetic code offers another elegantmeans of influencing the translation rate in cis. Since an organism’scodon usage is usually adapted to the relative abundances ofisoaccepting tRNAs, the choice of synonymous codons canpotentially control translation efficiency (Supek, 2016).Whether ornot this also applies to chloroplasts is currently controversiallydiscussed (Sugiura, 2014; Suzuki and Morton, 2016; Gawronskiet al., 2018). Although initial ribosome profiling studies could notconfirm a robust correlation between codon adaptation andtranslational speed/efficiency, recent methodological and bio-informatic refinements uncovered the suspected relationship inseveral organisms (DanaandTuller, 2014;Nakahigashi et al., 2014).

In addition to primary sequence elements, features ofmRNA2Dor 3D structure (or lack of structure) can represent cis-elementsthat influence the translation process (Mauger et al., 2013). Forexample, the lack of secondary structures at the start codon fa-cilitates efficient SD-independent translation initiation in bacteriaand chloroplasts (Scharff et al., 2011, 2017). Furthermore, trans-lation initiation in the chloroplast atpH and psbH mRNAs is stim-ulated by resolving secondary structures that mask ribosomebinding site and start codon, respectively (Prikryl et al., 2011;Hammani et al., 2012). A recent ribosome profiling study also re-vealed transcriptome-widecorrelationsbetween ribosomepausingand secondary structures in chloroplast mRNAs (Gawronski et al.,2018).

Riboswitches are structured RNA elements that act as sensorsfor small molecules (metabolites). Metabolite binding triggersa conformational switch that regulates transcription (typically by

inducing termination or antitermination) or translation (by ex-posing or sequestering the ribosome binding site; Serganov andNudler, 2013). Although riboswitches have not yet been identifiedin plastids, they have been exploited as tools to control thetranslation of transgenes in chloroplast biotechnology (Verhouniget al., 2010). Theuseof improvedalgorithms for the computationalprediction of riboswitches (Philips et al., 2013) should help toclarify whether endogenous riboswitches exist in chloroplastgenomes.Internal RNA structures within reading frames can adjust the

translational speed by decelerating elongating ribosomes inbacteria and chloroplasts (Mauger et al., 2013; Gawronski et al.,2018), a mechanism that has been suggested to aid cotransla-tional protein-complex maturation steps. Specific translationalpausing sites have been identified in the chloroplast psbA andatpA mRNAs (Kim et al., 1991; Kim and Hollingsworth, 1992).However, although these pausing events appear to be influencedby light or temperature (Kim et al., 1994a; Grennan andOrt, 2007),they could not be assigned to particular sequences or structuralelements of either themRNAor the nascent polypeptide chain norcouldanyobviousmolecular functiondirectlybeascribed to them.In bacteria, structured RNA elements are also involved in pro-grammed frameshifting and translational coupling of neighboringreading frames on the same transcript (Jackson et al., 2007;Mauger et al., 2013).

cis-Acting Elements in the Nascent Peptide

Translational cis-elements can also be located in the nascentpeptide. For example, translation of consecutive proline codons iscomplicated due to the physicochemical properties of the sec-ondary amino acid proline, which make it a weak peptidyl donorand acceptor. Therefore, reading of consecutive proline tripletscauses translational pausing (Artieri and Fraser, 2014, and ref-erences therein). Specific elongation factors such as EF-P inbacteria facilitate the translation of consecutive proline codons(Doerfel et al., 2013; Ude et al., 2013). EF-P is conserved inchloroplasts suggesting a similar function (Manuell et al., 2007).More than twoconsecutive proline codonsare usually avoided inproteins of all organisms. For example, the entire tobaccochloroplast genome does not encode a single stretch of threeprolines.A recent study alsoprovided evidence for other specificpeptide

motifs (e.g., domains comprised of small polar residues) inhibitingpeptidyl transfer during elongation or peptide release duringtermination in bacteria (Woolstenhulme et al., 2013). Furthermore,specific translational pausing events that are induced by inter-actions of theprokaryotic ribosomewith the nascent peptide haveemerged as programmed switches that regulate translation inresponse to small metabolites or the availability of protein trans-location factors (Itoet al., 2010). Inchloroplasts, ribosomepausingevents were correlated with positively charged amino acids(Gawronski et al., 2018).

Protein Factors That Control Translation in cis or trans

Proteins that bind the mRNA template, the ribosome, or the na-scent peptide chain can influence translation. These proteins act

758 The Plant Cell

either in anmRNA-specificmanner or asmore general factors thatcontrol translation of many transcripts.

All mRNA-specific chloroplast translation factors character-ized to date act in trans by binding the 59UTRs of one (or a few)mRNAs and promoting translation of the downstream readingframe (see above; Table 1). More generally acting trans-factorscontrol the translation of several reading frames. These factorsareeithergenuineorauxiliaryconstituentsof the ribosomeorbindthe nascent peptide. Hence, these factors act in cis or trans. cis-regulatory functions have been suggested for several ribosomalproteins or ribosome-associated proteins such as PSRP2-6, pY,and RPS1 (Yamaguchi and Subramanian, 2003; Manuell et al.,2007; Sharma et al., 2010). In bacteria, Rps1 is involved intranslation initiation of specific transcripts and a similar functionhas been suggested for chloroplast RPS1 (see above). Chloro-plasts also contain an ortholog of the bacterial ribosome-associated protein pY that binds 70S ribosomes or 30S subunitsunder cold-shock conditions, thus inactivating translation andstabilizing monomeric 70S ribosomes (Vila-Sanjurjo et al., 2004,and references therein). Similarly, it has been proposed thatplastid pY could be involved in the deactivation of translation andthe stabilization of chloroplast ribosomes in the dark (Sharmaet al., 2010, and references therein). Also, the expression ofchloroplast translation elongation factors is regulated by light,suggesting their contribution to translational regulation. How-ever, direct evidence for a regulatory function of chloroplastelongation factors RPS1 or pY is lacking.

In both bacteria and eukaryotes, the binding of specificprotein factors can create subpools of specialized ribosomesthat selectively translate a specific set of mRNAs (Sauert et al.,2015). More systematic and quantitative proteomic studies ofchloroplast ribosomes will be required to determine whetherspecialized ribosomes exist also in plastids.

Translation Is Regulated at the Initiation andElongation Levels

In theory, translation can be regulated at any of its three stages:initiation, elongation, or termination (Hershey et al., 2012). How-ever, by far most common is the regulation of initiation. All chlo-roplast factors characterized to date that promote translation ofspecific reading frames act at the level of initiation (Table 1).However, in somecases, elongation also appears to be regulated.For example, elongationofpsaA,psaB,psbA, and rbcL translationis regulated by light, and the synthesis of PsbA was suggestedadditionally to depend on the availability of cofactors and as-sembly partners (Berry et al., 1988; Klein et al., 1988b;Mullet et al.,1990; Kim et al., 1994a; Mühlbauer and Eichacker, 1998; Kim andMullet, 2003).

Light- and Redox-Dependent Regulation of Translation

Light regulation of translation ensures coordination of the ma-jor energy-producing process (photosynthesis) with the majorenergy-consuming process (protein synthesis) in the chloro-plast. In addition, components of the photosynthetic apparatus(especially PsbA) are damaged by light, necessitating specificrepair synthesis.

The synthesis of several chloroplast proteins was shown to beactivated at the translational level in response to increased lightintensity. Upon illumination, translation of rbcL and psbA is ac-tivated at the level of elongation, amechanism that was proposedto be regulated by the light-dependent generation of a protongradient across the thylakoid membrane, the ATP status of thechloroplast and/or redox signal(s) generated by photosyntheticelectron transfer (Taniguchi et al., 1993;MühlbauerandEichacker,1998; Zhang et al., 1999). In line with regulation of elongation, thetransfer of amaranth plants into the dark caused a decline in RbcLprotein synthesis that was not accompanied by the loss of rbcLmRNA association with ribosomes (in polysomes), suggestingthat elongating ribosomes paused (Berry et al., 1988). In addition,psaA and psaBwere shown to be light-regulated at the elongationlevel during deetiolation (Klein et al., 1988b). A potential mecha-nism for the general light-dependent activation of chloroplasttranslation elongation could rely on pY (see above) or elongationfactor Tu, whichwas identified as redox-regulated (Schröter et al.,2010).During deetiolation, psbA translation is also stimulated at the

level of initiation (Klein et al., 1988b; Kim and Mullet, 1994). Evi-dence currently available suggests that psbA translation for PSIIbiogenesis is regulated at the initiation level, whereas PsbAsynthesis for PSII repair is regulated at the (presumably fasterresponding) elongation level, amodel supportedalsobydata fromChlamydomonas (reviewed in Nickelsen et al., 2014). In seedplants, psbA translation initiation is controlled by specific cis-elements in the 59UTR (Staub and Maliga, 1994; Hirose andSugiura, 1996; Eibl et al., 1999) and by the trans-factors HCF173and HCF244, two putative oxidoreductases that promote psbAtranslation cooperatively (Schult et al., 2007; Link et al., 2012). Allof these elements may also play a role in the light-dependenttranslation initiation of psbA.Variations in light quantity and quality can cause imbalances in

the activities of PSI and PSII, changing the redox state of thechloroplast and potentially generating harmful reactive oxygenspecies (Pfalz et al., 2012). The chloroplast redox state controlstranscriptional and posttranscriptional adaptation responses inthe chloroplast (Allen and Pfannschmidt, 2000; Rochaix, 2007). InChlamydomonas, a redox-dependent regulatory mechanism hasrecentlybeensuggested for the light-activated translationofpsbD(Schwarz et al., 2012). In this model, the RNA binding proteinsNac2 and RBP40 form a disulfide bridge-connected complex inthe light that stabilizes psbD mRNA and activates its translation(Schwarz et al., 2012, and references therein). In the dark, thecomplex is reduced and disassembles, and, as a result, PsbDsynthesis is inactivated. The electrons are likely provided by theNADPH-dependent thioredoxin reductaseC,anenzymeshown toreducedisulfidebonds in thedarkbyutilizingNADPHgenerated inthe oxidative pentose phosphate pathway (Schwarz et al., 2012).Plants perceive light independently of photosynthesis by

photoreceptors that are locatedoutsideof chloroplasts.However,light-induced regulation of translation was also observed in iso-latedchloroplasts, arguingagainstacrucial roleofphotoreceptors(Sun and Zerges, 2015).Altogether, our current knowledge about light-induced trans-

lational regulation in seed plant chloroplasts is restricted to veryfew photosynthesis-related genes, with psbA translation being

Chloroplast Translation 759

best studied. However, even for psbA, the underlying mecha-nisms and themode of action of the factors involved are not wellunderstood.

Developmental Regulation of Translation

Developmental regulation of plastid gene expression is crucialfor the differentiation and interconversion of plastid types. Forexample, conversion of proplastids to chloroplasts requiresestablishment of the thylakoid system and must be tightly co-ordinated with cell division and the emergence of photosyn-thetic tissues frommeristems (Jarvis and López-Juez, 2013). Themechanisms involved in chloroplast differentiationwere identifiedin classical experiments that analyzed the process of deetiolation.During deetiolation, a rapid rise in translational activity was ob-served by pulse labeling experiments for several plastid mRNAsencoding subunits of PSI (psaA/B), PSII (psbA/B/C/D), andRubisco(rbcL; Klein and Mullet, 1986, 1987; Kim and Mullet, 2003;Kleffmann et al., 2007). The initial steep increase in translation ofthese mRNAs was followed by a slow decline as chloroplastdifferentiation continued in the light. Polysome analyses dem-onstrated that psaA/B, rbcL, and psbA transcripts are found inpolysomes throughout the chloroplast differentiation process,suggesting that the regulation of their translation occurs, at leastin part, at the elongation level (Klein et al., 1988b). This con-clusion was further substantiated by the observation that psaA,psbA, and rbcL translation initiation remains unaltered duringlight-dependent chloroplast differentiation (Kim et al., 1994b;Kim and Mullet, 2003).

There is also evidence for developmentally activated translationinitiationofplastidmRNAs. InitiationofpsbA translation is inducedduring deetiolation (Eichacker et al., 1992; Kim and Mullet, 1994),and this induction is likely controlled by cis-elements in the psbA59UTR (Staub and Maliga, 1994, and references therein). It wasfurther proposed that the light-induced synthesis of chlorophyllcontrols both the accumulation and the translation of chlorophyllbinding apoproteins ofPSI andPSII duringdeetiolation (Eichackeret al., 1992). However, this conclusion was mainly based on theobserved lack of accumulation of chlorophyll binding apoproteinsin the absence of chlorophyll, as determined by protein immu-noblotting andpulse labelingexperiments in vivoand in vitro (Kleinet al., 1988a, 1988b; Eichacker et al., 1990, 1992). Although pulselabeling can reveal protein synthesis rates, it cannot unambigu-ously distinguish between the absence of synthesis and rapiddegradation of newly synthesized proteins (especially not forproteins with high turnover rates, such as PsbA). A recent ribo-someprofilinganalysisofplastid translation inamaizemutantwithknocked-out chlorophyll synthesis showed that the synthesis ofplastid-encoded chlorophyll binding apoproteins is virtually un-altered in the absence of chlorophyll (Zoschke et al., 2017). Eventhe cotranslational thylakoid membrane engagement of nascentapoproteins was shown to be independent of chlorophyll syn-thesis (Zoschkeetal., 2017).However, apoproteinswereshown toundergo rapid degradation in the absence of chlorophyll (Mulletet al., 1990; Kim et al., 1994b; Eichacker et al., 1996).

The above described deetiolation of seedlings representsadramatic change inplant development and reflects thesituationduring germination, when light-dependent and developmental

programs operate simultaneously. Another useful experimentalsystem to study translational regulation during chloroplast dif-ferentiation is provided by the longitudinal developmental gra-dient of young leaves in grasses such as maize, rice (Oryzasativa), or barley (Hordeum vulgare). At the leaf base, meriste-matic, proplastid-containing tissue is found, followed by a de-velopmental gradient toward the leaf tip that includes etioplasts,differentiating chloroplasts (etiochloroplasts), and fully dif-ferentiated chloroplasts (Leech et al., 1973). This natural de-velopmental gradient has been exploited to systematically studythe dynamic changes in gene expression at the level of transcriptand protein accumulation (Barkan, 1989; Baumgartner et al.,1989; Cahoon et al., 2004; Li et al., 2010; Majeran et al., 2010).Recently, ribosome profiling enabled the transcriptome-wide

examination of plastid translation in the longitudinal develop-mental gradient of maize leaves (Chotewutmontri and Barkan,2016). This pioneering study comprehensively determined therelative contributions of changing transcript levels and trans-lational activity to the protein synthesis output (Chotewutmontriand Barkan, 2016). The data revealed that the synthesis rates ofmost plastid-encoded proteins increases early in developmentand drops later, once the photosynthetic machinery is set up.Strikingly, PsbA and PetD, two proteins that do not follow thisgeneral rule and instead display increasing synthesis levels thatpeak in the latest examined developmental stage, represent thesubunitswith thehighest turnover rates in their complexes (Li etal.,2017a), likely explaining their continued production.In general, the regulation of plastid gene expression during

leaf development in maize is achieved by changes in tran-script levels that are often tuned by changes in translationefficiency (Chotewutmontri and Barkan, 2016). Remarkably,the developmental dynamics in protein synthesis that was dis-covered revealed twomajor regulons that are defined by changesin RNA accumulation and translation efficiency: Early in devel-opment, proteins needed for chloroplast gene expression havethe highest synthesis output (e.g., RNA polymerase subunitsand ribosomal proteins), whereas in later developmental stages,proteins required for photosynthesis are extensively synthesized(i.e., subunits of PSII, Cyt b6f, PSI, ATP synthase, and NDHcomplexes). To achieve this developmental pattern in plastidprotein synthesis, translational regulation is especially importantfor polycistronic transcription units that encode proteins of bothregulons. Indeed, in these transcripts (e.g., the psaA/psaB/rps14transcription unit encoding two PSI subunits and a ribosomalprotein), strong differential translational regulation was observed(Chotewutmontri and Barkan, 2016).Unfortunately, none of the factors involved in the complex de-

velopmental regulation of chloroplast translation has been iden-tified so far. It would be particularly exciting to reveal themechanismsunderlying the translational switchbetween these twomajor regulons during leaf development. A possible explanationcould lie in the known developmental switch in the usageof the twodistinct typesofRNApolymerasesduringchloroplastdifferentiation(Börner et al., 2015). In theory, the two polymerase activities couldproduce transcriptscoding for identicalproteinsbutdiffering in theirtranslational competence. Alternatively, RNA binding proteins thatfacilitate translation (see above and Table 1) could mediate thedevelopmental regulation of translation.

760 The Plant Cell

Translational Autoregulation and Feedback Regulation

Control of protein synthesis by translational autoregulation orfeedback regulation represents an elegantmeans of robustly fine-tuning protein synthesis levels to changing cellular requirements.In the chloroplast of Chlamydomonas, the translation of specificsubunits of the four photosynthetic complexes (PSII, Cyt b6f, PSI,and ATP synthase) was shown to be feedback regulated bythe assembly state of the respective complex, a process termedcontrol by epistasy of synthesis (CES; Choquet and Wollman,2009). CES regulation ensures the stoichiometric production ofchloroplast subunits that reside in oligomeric complexes ac-cording to the requirementsof their sequential assembly (ChoquetandWollman, 2009). The best-studied casemechanistically is theCES involved inpetAgene expression. PetA is a subunit of theCytb6f complex, whose synthesis is strongly reduced in the absenceof its assembly partners PetB (cytochrome b6) and PetD (subunitIV; Kuras and Wollman, 1994). The protein factors MCA1 andTCA1 were shown to be cooperatively associated with the 59UTRof the petA mRNA, where they stabilize the transcript (MCA1)and promote translation initiation (TCA1; Wostrikoff et al., 2001;Loiselay et al., 2008).Boulouis et al. (2011) discovereda regulationmechanism in which a C-terminal domain of PetA that is exposedonly in the unassembled PetA protein binds to MCA1 and causesits proteolytic degradation. In turn, this leads to downregulation ofpetA gene expression at both the level of transcript accumulationand the translational level. Conversely, if the assembly partnersPetB and PetD are available, PetA is assembled into the Cyt b6fcomplex, the C-terminal domain is inaccessible, and, hence,MCA1 remains stable and facilitates petA expression in complexwith TCA1. In thisway, theCESmechanismensures the adequatesynthesis of PetA according to the availability of its assemblypartners in the Cyt b6f complex.

In chloroplasts of seed plants, evidence for a similar feedbackregulationmechanismhas been obtained in only a single case. Thesynthesis of the plastid-encoded large subunit of Rubisco (RbcL)in tobacco is adjusted to that of its nucleus-encoded assem-bly partner, the small subunit RBCS, by a mechanism that issimilar to the CES-regulated rbcL expression in Chlamydomonas(Rodermel et al., 1988, 1996; Khrebtukova and Spreitzer, 1996).Furthermore, evidence was provided that unassembled RbcLrepresses itsown translation, possibly throughdirectRNAbinding(Yosef et al., 2004; Wostrikoff and Stern, 2007). In a systematicgenetic approach, the essential plastid-encoded Cyt b6f sub-units PetA, PetB, and PetD were knocked out to disrupt complexassembly and test for potentially conserved CES regulatorymechanisms in tobacco (Monde et al., 2000). In the mutants,only amild effect on translation of thepolycistronicpetA transcriptwas detected by polysome analyses, suggesting that the CESmechanism is not conserved in seed plants (Monde et al., 2000).Thismay not be entirely surprising, given that the regulatory trans-factors involved, MCA1 and TCA1, are not found in seed plants.Moreover, a ribosomeprofiling analysis of twomaizemutantswithdefectiveAtpBsynthesis did not showasubstantial effect on atpAtranslation (Zoschke et al., 2013a). This suggests that also theAtpB-dependent translationofatpA, theonly trans-activatingCESmechanism observed in Chlamydomonas (Drapier et al., 2007), isnot conserved in seed plants. However, in Arabidopsis, it was

observed that mutation of the chloroplast RNA binding proteinHCF107 that facilitatespsbH translationalsocausesa reduction inPsbB (CP47) synthesis (Felder et al., 2001). Remarkably, whenpsbH expression was rescued by introducing a psbH gene copyinto the nuclear genome of an hcf107 mutant, accumulation ofthe PsbB protein was rescued as well, despite the absence offunctional HCF107 protein (Levey et al., 2014). A possible expla-nation for this observation is thatPsbHaccumulation isneeded forPsbB synthesis, pointing to a potential CES mechanism in PSIIbiogenesis (Levey et al., 2014). Interestingly, recent transcriptome-wide analyses of translation inmaize and Arabidopsis chloroplastsrevealed that most proteins are synthesized in the amounts thatcorrespond to (1) their steady state stoichiometry as subunits ofprotein complexes and (2) the abundance of the respective proteincomplex (Chotewutmontri andBarkan,2016;Lukoszeketal., 2016).This suggests that precise regulation of gene expression is moreimportant than posttranslational proteolytic adjustments of subunitstoichiometry.However,severalalternative regulatorymechanismscan explain this behavior, and it remains to be examinedwhether ornot CES is involved.Inmanyorganisms,autoregulatedproteinshavebeenobserved

to comprise an intrinsic RNA binding activity. Prominent exam-ples include several ribosomal proteins, the initiation factor IF3and the b-subunit of the bacterial RNA polymerase (Choquet andWollman, 2009). The RNA binding motifs allow these proteins tobind the 59UTRof their ownmRNAand inhibit translation initiationwhenever their assembly partners are not available, thus gen-erating a negative regulatory feedback loop. The chloroplastgenome codes for numerous RNA binding proteins (e.g., or-thologs of bacterial RNA polymerase subunits and ribosomalproteins). Hence, it is tempting to speculate that some of themmay also autoregulate their own synthesis. A plastid-encodedprotein that has been shown to associatewith its ownRNA in vivois the putative splicing factor MatK (Zoschke et al., 2010). Thematurase MatK is encoded in the intron of the trnK gene. MatKbinds to the trnK transcript, most likely assisting in splicing(Zoschke et al., 2010, and references therein). Notably, a MatK-related bacterial maturase is translationally autoregulated (Singhet al., 2002), and an autoregulatory mechanism was also pro-posed for the expression of plastid MatK (Hertel et al., 2013).Insummary,whetherautoregulationand/or feedback regulation

of translation are common in seed plant chloroplasts or whetherthese regulatory mechanisms are specific to Chlamydomonasremains to be investigated. Pronounced differences betweenmicroalgae and seedplantsmaynot be surprising if one considersthe dissimilar evolutionary forces that act on regulatory circuits ina unicellular organismwith onechloroplast per cell comparedwithmulticellular organisms with numerous chloroplasts per cell.

Translational Regulation in Response to Other Internal andExternal Triggers

Temperature is a major abiotic signal influencing translationalregulation. In bacteria, cold-stress conditions induce a generalblock of translation that is mediated by the pY protein. However,the synthesis of a small subsetof proteins, suchasRNAhelicases,IF1-3, and trigger factor, is induced under these conditions (Barriaet al., 2013). In chloroplasts, changes in temperature alter the

Chloroplast Translation 761

speed of all enzymatic reactions (including the Calvin-Benson-Basshamcycle), but, unlessextreme, theyhavevirtually no impacton the light reactions of photosynthesis. Consequently, temper-ature changes can cause dramatic imbalances in the redox ho-moeostasis of photosynthesis (Crosatti et al., 2013). It has longbeen known that the chloroplast translation capacity is crucial toplant adaptation to chilling stress. This became evident by thediscovery of numerous mutants with impaired chloroplast trans-lation that exhibit cold-sensitive phenotypes (Barkan, 1993;Rogalski et al., 2008; Liu et al., 2010).

A recent ribosome profiling study comprehensively examinedchloroplast translational regulation upon temperature stress(Lukoszek et al., 2016). Ignatova and coworkers observed inArabidopsis that elevated temperature causes dramatic changesin the protein synthesis output of subunits of photosyntheticcomplexes (Lukoszek et al., 2016). This regulation was driven byboth changes in transcript levels and translational regulation.Interestingly, in several cases, unbalanced changes in proteinsynthesis rates were observed for different subunits withina complex, in that the altered synthesis rates did not reflect thesubunit stoichiometry. This finding may suggest an increasedrelevanceof control at the level of protein turnover of at least somesubunits during heat stress (Lukoszek et al., 2016). In sum, al-though it is well established that plastid translation is critical foracclimation to changing temperature conditions, the molecularmechanisms and the factors involved are currently unknown.

A special case of internal specialization of gene expression isthe chloroplast dimorphism in C4 plants. In C4 species such asmaize, light reactions and carbon reactions of photosynthesis arepartitioned betweenmesophyll cells and bundle sheath cells. Thedivision of labor between the two cell types is reflected by spe-cialized chloroplast types, referred to as chloroplast dimorphism.At the molecular level, bundle sheath chloroplasts have a highcontent of Rubisco and NDH complex, whereas mesophyllchloroplasts are enriched in PSII (Majeran et al., 2010). Thisspecialization is achieved by differential gene expression (Sharpeet al., 2011, and references therein). Recently, transcript accu-mulation and translation have been comprehensively analyzed inbundle sheath and mesophyll chloroplasts by ribosome profiling(Chotewutmontri and Barkan, 2016). This study confirmed thatmost of the observed differences in protein accumulation can beexplained by differential protein synthesis, which in turn is pre-dominantly achieved by differential expression at the RNA level.However, the synthesis of some proteins such as the PSII coresubunits PsbA/B/C/D is additionally tuned by translational reg-ulation. The underlying regulatory mechanisms are unknown. Thestudy showed that the synthesis output of Rubisco, PSII, PSI, andNDH complex subunits reflects well their protein accumulation inthedifferent chloroplast types. This is not the case for the subunitsof the ATP synthase and the Cyt b6f complex, suggesting moreextensive posttranslational adjustments in these complexes.

Internal metabolic signals can also control translation (e.g., viariboswitches in bacteria; Serganov and Nudler, 2013). Severalrecent discoveries inChlamydomonashaveestablished interestinglinks between chloroplast metabolism and gene expression(Bohne and Nickelsen, 2017). DLA2, a subunit of the chloroplastpyruvate dehydrogenase complex, was shown to have RNAbinding activity which enables its moonlighting function in

translational regulation of the psbA mRNA (Bohne et al., 2013).Considering its functions in metabolism and translational regu-lation, DLA2 is anticipated to coordinate fatty acid and proteinsyntheses during thylakoid biogenesis (Bohne et al., 2013). Thephylogenetic conservation of the DLA2 amino acid sequence andRNA binding properties suggests that it performs a similar dualfunction in seed plants (Bohne et al., 2013). A moonlightingfunction was also suggested for RbcL, which was shown to bindRNA (Yosef et al., 2004; Cohen et al., 2006) and recently has beenproposed tobe involved in the redoxstress-induced localizationofoxidized chloroplastRNAs inChlamydomonas, independent of itsfunction in the Rubisco complex (Zhan et al., 2015). Moreover,recent in vitro data suggest that PsbD protein synthesis inChlamydomonas ismetabolically controlled (Schwarz et al., 2012;see above). Whether similar regulatory connections between me-tabolism and chloroplast translation exist in seed plants remains tobe investigated.In bacteria, stress-induced regulation of gene expression is

triggered by the so-called stringent response. Upon nutrientdeprivation and other stresses, bacteria produce the effectormolecule (p)ppGpp (guanosine tetraphosphate/pentaphosphate;also known as alarmone) that coordinates numerous cellularresponses, including broad changes in transcription and trans-lation (Gaca et al., 2015). Chloroplasts also harbor the enzymesneeded for ppGpp synthesis (and degradation). Plastids producealarmone not only in response to stress, but also to orchestratechloroplast and nuclear gene expression (reviewed in Field,2017). ppGpp has been shown to influence transcription,translation, and many other metabolic and physiological pro-cesses in plants (Field, 2017). In an in vitro chloroplast translationsystem from pea (Pisum sativum), ppGpp inhibited protein syn-thesis, suggesting conservation of the bacterial mode of ppGpp-mediated translational repression by interaction with IF2 and/orEF-G (Nomura et al., 2012). Furthermore, overaccumulationof ppGpp in vivo caused a dramatic reduction in the accumulationof many plastid-encoded proteins, mRNAs, tRNAs, and rRNAs,suggesting an overall reduced translation capacity (Sugliani et al.,2016). Whether the translational activity of specific chloroplastmRNAs is regulated by ppGpp and how this contributes toppGpp-induced changes in chloroplast gene expression stillneeds to be determined.In summary, considerableprogress hasbeenmade indescribing

changes in translation in response to light and developmentalsignals, but comparably little is known about the mechanisms andfactors that adjust plastid protein synthesis to other internal andexternal cues that affect protein homeostasis.

OUTLOOK

Over the last decades, extensive research on the structure andfunctionof the translational apparatusofbacteriaandchloroplastshas revealed many similarities but also substantial differences.Unfortunately, our knowledgeabout genuine regulatory processes,the underlyingmolecular mechanisms and the factors involved, isstill limited. For example, the influence of internal and externaltriggers such as light, temperature, nutrient availability, osmoticstatus, redox state, phytohormones, alarmone, and diurnal and

762 The Plant Cell

circadian rhythms on chloroplast gene expression has so far notbeen comprehensively examined at the translational level. Like-wise, investigation of the interconnection of chloroplast trans-lationwith protein synthesis in the cytosol and themitochondria islikely to provide fresh insights into intracellular signaling andcrosstalk in response to changing environmental conditions anddevelopmental programs. Last but not least, the detailedmolecularfunction of RNA cis-elements andproteinaceous trans-factors inthe regulation of chloroplast translation is mostly unknown. Theexciting advent of chloroplast ribosome profiling and otherin vivo ribonomic methods, such as the comprehensive exam-ination of RNA secondary structures or the mapping of bindingsites for RNA binding proteins, can be anticipated to addressmanyof theseopenquestions in thebiologyof plant organelles inthe future.

ACKNOWLEDGMENTS

We apologize to the authors of numerous articles we were unable to citedue to space constraints. We thank Alice Barkan (University of Oregon) forhelpful suggestions on the manuscript. We acknowledge the gener-ous sharing of unpublished data by Alice Barkan, Christian Schmitz-Linneweber (Humboldt University of Berlin), Karin Meierhoff (HeinrichHeine University Düsseldorf), and Mark Aurel Schöttler (Max Planck In-stituteofMolecularPlantPhysiology).Researchonplastid translation in theauthors’ laboratories is funded by the Max Planck Society, the DeutscheForschungsgemeinschaft (Grant ZO 302/4-1 to R.Z. and SFB-TRR 175 toR.Z. andR.B.), theGermanAcademic ExchangeService (DAAD; Project ID57387429 to R.Z.), and the European Research Council under the Euro-pean Union’s Horizon 2020 research and innovation program (ERC-ADG-2014; grant agreement 669982 to R.B.).

AUTHOR CONTRIBUTIONS

R.Z. and R.B. wrote the article.

Received January 8, 2018; revisedMarch 26, 2018; acceptedApril 1, 2018;published April 2, 2018.

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770 The Plant Cell

DOI 10.1105/tpc.18.00016; originally published online April 2, 2018; 2018;30;745-770Plant Cell

Reimo Zoschke and Ralph BockRegulation

Chloroplast Translation: Structural and Functional Organization, Operational Control, and

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