REVIEW

Chloroplast Translation: Structural and Functional Organization,

Operational Control, and Regulation

Reimo Zoschke* and Ralph Bock*

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

* To whom correspondence should be addressed. Email: zoschke@mpimp-golm.mpg.de or

rbock@mpimp-golm.mpg.de

Short title: Chloroplast Translation

One-sentence summary:

We review the structure of the plastid ribosome, the mechanisms of chloroplast protein biosynthesis, the control and regulation of plastid translation, and the methods available to study translation.

ABSTRACT

Chloroplast translation is essential for cellular viability and plant development. Its positioning at the intersection of organellar RNA and protein metabolism makes it a unique point for the regulation of gene expression in response to internal and external cues. Recently obtained high-resolution structures of plastid ribosomes, the development of approaches allowing genome-wide analyses of chloroplast translation (i.e., ribosome profiling), and the discovery of RNA-binding proteins involved in the control of translational activity have greatly increased our understanding of the chloroplast translation process and its regulation. In this review, 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 describe how translational activity is controlled and which cis-elements and trans-factors are involved. Finally, we discuss how translational 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 bacterial translation machineries and the mechanisms of protein biosynthesis in these two prokaryotic systems.

Keywords: ribosome, chloroplast, plastid, gene expression, translational regulation, translational control, translation initiation, translation elongation, translation termination

Plant Cell Advance Publication. Published on April 2, 2018, doi:10.1105/tpc.18.00016

©2018 American Society of Plant Biologists. All Rights Reserved

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INTRODUCTION

Chloroplasts are the characteristic organelle of plant cells. They host numerous essential metabolic

pathways including photosynthesis, which makes chloroplasts the primary source of chemical energy on

earth. All chloroplasts are likely derived from a single ancient photosynthetic cyanobacterium that was

engulfed by a mitochondriate eukaryotic cell more than a billion years ago. During subsequent host-

endosymbiont co-evolution, the genome of the endosymbiont shrank significantly (e.g., Timmis et al.,

2004). While some genes were lost, many others were transferred to the host genome (Martin et al., 2002;

Bock and Timmis, 2008). The proteome of today’s chloroplasts consists of ~3000 proteins, most of which

are nucleus-encoded and post-translationally imported into the organelle.

Present-day chloroplasts still harbor a genome, which comprises ~120 genes in green plants. Most plastid

genes are essential for plant viability because they encode crucial components of the photosynthesis

machinery (the large subunit of Rubisco and approximately half of the subunits of the thylakoidal protein

complexes involved in the light reactions: photosystem I and II [PSI and PSII], cytochrome b6f complex

[Cyt b6f] and ATP synthase) and the gene expression system of the plastid (including a complete set of

bacterial-type RNA polymerase core subunits, rRNAs and tRNAs, and approximately one third of the

ribosomal proteins; Allen et al., 2011; Green, 2011). The reasons for retention of this particular set of

genes in the plastid genome are not fully understood. Several hypotheses that are not necessarily mutually

exclusive have been put forward, including constraints on importability of proteins (and RNAs) into

plastids and requirements for efficient and organelle-specific redox-regulation of gene expression (Allen,

2015). An intriguing consequence of endosymbiont-host co-evolution are multimeric chloroplast protein

complexes, whose subunits are encoded in different compartments (i.e., the plastid and the nucleus). This

necessitates the tight orchestration of nuclear and chloroplast gene expression (e.g., Jarvis and Lopez-

Juez, 2013; Kleine and Leister, 2016).

The bacterial origin of chloroplast gene expression is evident, for example, from the operon-like structure

of plastid gene clusters and the highly similar composition of the translation machinery. However, several

features clearly distinguish chloroplast gene expression from that of bacteria. For example, chloroplasts

possess an astoundingly complex RNA metabolism that includes the usage of different RNA polymerases

and extensive post-transcriptional 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 nearly exclusively conducted by nucleus-encoded protein factors, most of which were likely

established during host-endosymbiont co-evolution (Barkan, 2011; Lyska et al., 2013; Pfalz and

Pfannschmidt, 2013). Also different from bacteria, the regulatory influence of transcription is limited and

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post-transcriptional and translational events represent key points in controlling chloroplast gene

expression (e.g., Barkan, 2011; Sun and Zerges, 2015).

In recent years, numerous specific features of chloroplast translation were uncovered, including its

interconnection with co-translational processes in RNA and protein metabolism, its regulation in response

to internal and external triggers and the presence of unusual components of the translation machinery.

Genome-wide analyses have unraveled the suborganellar localization of translation and its participation in

controlling the developmental program of chloroplast gene expression (Zoschke and Barkan, 2015;

Chotewutmontri and Barkan, 2016). Studies of sequence-specific chloroplast RNA-binding proteins that

comprise helical repeat domains, especially the pentatricopeptide repeat (PPR) proteins, revealed their

concerted function in RNA metabolism and promotion of translation (Barkan and Small, 2014; Hammani

et al., 2014). Last but not least, high-resolution structural analyses provided a detailed three-dimensional

picture of the plastid ribosome (e.g., Graf et al., 2016; Bieri et al., 2017). These discoveries were largely

enabled by novel approaches towards the quantitative transcriptome-wide analysis of translational

activity, the identification of specific factors that control protein synthesis, and the structural elucidation

of the translational apparatus in plastids (e.g., by ribosome profiling techniques, RNA co-

immunoprecipitation assays, and refined methods for 3D structural analysis). In the light of these and

other findings, we now can reevaluate classical models of chloroplast translation and reassess

controversially discussed hypotheses.

Chloroplast translation has been mainly studied in the unicellular green alga Chlamydomonas reinhardtii

and in model seed plants such as Arabidopsis, maize, and tobacco. In this review, we focus on

embryophytes and, wherever appropriate, refer to breakthrough discoveries made in Chlamydomonas. For

a broader overview of chloroplast translation in Chlamydomonas, the interested reader is referred to

comprehensive review articles (Stern et al., 2009; Nickelsen et al., 2014; Sun and Zerges, 2015).

METHODS TO ANALYZE CHLOROPLAST TRANSLATION

Methods to determine translational activity either indirectly examine the ribosome coverage of the

translation template (i.e., the mRNA), or directly measure the accumulation of newly synthesized

proteins.

Classical methods to analyze translation

Pulse labeling is the method of choice to directly measure translational activity in vivo. In this approach,

isolated chloroplasts, cells or intact plant tissues are fed with the 35S radiolabeled amino acids methionine

and/or cysteine (e.g., Barkan, 1998). The isotopes are incorporated into newly synthesized proteins to an

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extent that mirrors their synthesis rate. Subsequently, the radiolabeled proteins can be separated by gel

electrophoresis, visualized and quantified (Figure 1A). An advantage of pulse labeling is that it has the

potential to measure protein synthesis rates independent of the dynamics of ribosome movement along the

mRNA. By pulse labeling, the synthesis rate of especially the large plastid-encoded core subunits of the

photosynthesis machinery can be readily quantified. However, the method has several limitations: (i)

Small subunits and subunits of similar molecular weight are difficult to resolve in protein gels and may

require selective purification by immunoprecipitation (Barkan, 1998). (ii) The synthesis rates of many

plastid-encoded proteins cannot be determined by pulse labeling approaches due to their low expression

levels. (iii) The measured quantities of labeled proteins are determined by their synthesis and degradation

rates. Consequently, for proteins with high turnover rates (e.g., PsbA, the D1 protein of PSII), results of

pulse-labeling experiments are often difficult to interpret (even if followed by a chase with unlabeled

amino acids to examine the stability of the labeled protein). Also, (iv) in multicellular organisms, neither

the pulsing nor the chasing occur homogenously in all cells, thus making quantitative comparisons very

challenging.

Polysome analysis is a widely used method that indirectly measures translational activity using the

association of mRNAs with ribosomes as a proxy. Polysomes are high molecular weight assemblies of

actively translating ribosomes held together by the strands of mRNA being translated. They can be

separated from free mRNAs and ribosomes (monosomes) by ultracentrifugation in sucrose density

gradients (Figure 1B). Following RNA extraction from gradient fractions and RNA gel blot analysis, the

distribution of specific mRNAs across the gradient is visualized and provides a qualitative measure of

their translational activity (Barkan, 1998). For genome-wide analysis (translatomics), mRNAs recovered

from different density fractions can be examined by microarray hybridization (e.g., Kahlau and Bock,

2008). Polysome analyses are often complicated by the operon-like organization of genes in the plastid

genome. The processing of transcripts produced from polycistronic transcription units frequently gives

rise to a multitude of mono-, oligo- and polycistronic RNA species, all of which represent potential

translation templates (e.g., Barkan, 1988). Due to the physical linkage of reading frames located on the

same transcript, their individual translation rates cannot be resolved. In addition, only translational

regulation at the level of initiation can be detected, because the molecular weight of mRNAs loaded with

actively elongating ribosomes is indistinguishable from those with paused or stalled ribosomes.

Other elegant though labor intensive methods have been used to examine the regulatory capacity of cis-

elements in chloroplast translation: (i) Chloroplast in vitro translation systems have been established and

used to analyze the regulatory influence of putative cis-elements residing in the 5’ untranslated region

(5’UTR) on translation (e.g., Hirose and Sugiura, 1996; Yukawa et al., 2007). (ii) Reporter genes (e.g.,

A Pulse labeling

3s5_1abeledcysteine/methionine

• + ••

Cell lysis

+

Proteins \\ � J'-� 1D or 2D gel electrophoresis

Labeled Proteins

+

1- PsaA/B-AtpA/B ( PsbB (CP47)

1

_ PsbC (CP43)

1- PsbD (D2)- PsbA (D1)

B Polysome analysis

Polysomes, monosomes, free mRNA

Cell lysis

+ ;-e5

�_rJ

Ultracentrifugation

Separated RNPs

Sucrose gradien

Free mRNA

+ Polysomes

RNA purification and detection

RNA gel blot

+

---�-1

Figure 1. Common methods to analyze chloroplast translation.

C Ribosome profiling

Polysomes, monosomes, free mRNA

Cell lysis

+ ;-e5

�_rJ

Nuclease treatment

Monosomes

Ribosome footprints

+ B B

B B

RNA purification

+

NGS or microarray analysis

Ribosome distribution

(A) Pulse labeling. Plant cells (chloroplast: large green oval, nucleus: white circle) are fed with radiolabeled cysteine and/ormethionine (red dots) which is incorporated together with unlabeled amino acids (black dots) into nascent peptides bytranslation (for simplicity, only chloroplast ribosomes are shown). Proteins are then isolated, separated by gel electrophoresisand visualized/quantified by radio-detection methods (gel picture kindly provided by Karin Meierhoff).

(B) Polysome analysis. Plant cell lysates are loaded on sucrose gradients (white to black: low to high concentration) toseparate ribonucleoprotein particles (RNPs) according to their molecular weight by ultracentrifugation. RNA is isolated fromgradient fractions and examined by RNA gel blot analysis to determine the ribosome loading of specific mRNAs.

(C) Ribosome profiling. Plant cell lysates are treated with nuclease to degrade ribosome-free mRNA sequences. Thisgenerates monosomes, whose protected mRNA fragments (ribosome footprints) are subsequently purified. The positions andabundances of the ribosome footprints are determined by next-generation sequencing or microarray hybridization and reflectprotein synthesis rates.

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GFP or GUS) have been fused to different presumed cis-elements and inserted into the plastid genome by

chloroplast transformation to examine the translational activity conferred by these sequences (e.g., Staub

and Maliga, 1994; Eibl et al., 1999; Drechsel and Bock, 2011).

Ribosome profiling: genome-wide analysis of translation at high resolution

The above described classical methods have been informative, but they are labor intensive, limited in

resolution, and none of them is suited to genome-wide and/or high-throughput analyses. These deficits

were addressed by ribosome profiling, an approach that enables the quantitative genome-wide analysis of

translation in unprecedented depth and resolution (Ingolia et al., 2009). Ribosome profiling takes

advantage of the remarkable stability of translating ribosomes, which protect the mRNA sequence they

physically cover from attack by nucleases, thereby producing protected fragments, so-called ribosome

footprints (Wolin and Walter, 1988; Figure 1C). Next-generation sequencing analysis of these footprints

determines the in vivo positions and abundances of translating ribosomes. Considering that each

elongating ribosome produces one protein, ribosome footprint abundances reflect the protein synthesis

rate for each reading frame (Ingolia et al., 2009). Footprint abundance is typically normalized to mRNA

abundance (assayed by RNA sequencing), so that relative translation efficiencies can be inferred (Ingolia

et al., 2009). Consequently, the approach measures the two determinants of gene expression that define

the final protein output: transcript amount and translational activity. In recent years, ribosome profiling

has been extensively used to study translation in prokaryotes and eukaryotes (e.g., Ingolia, 2016).

In chloroplasts, ribosome profiling was first applied in a modified approach, exchanging the next-

generation sequencing analysis of footprints by microarray hybridization (Zoschke et al., 2013a; Figure

1C). More recently, deep sequencing was used to study chloroplast 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, it should be noted that the method cannot

distinguish actively translating from paused ribosomes. This may be problematic if translation is

regulated at the level of elongation, as described for some chloroplast genes (see below). Application of

inhibitors of initiation or early elongation (e.g., lincomycin) and examination of the run-off kinetics of

ribosomes over time should allow to distinguish pausing from elongating ribosomes.

THE PLASTID TRANSLATION MACHINERY – VARIATIONS ON A BACTERIAL THEME

A bacterial-like translation machinery whose components are encoded by two genomes

Chloroplast translation is carried out by prokaryotic-type 70S ribosomes that are composed of a small 30S

and a large 50S subunit and contain orthologues of most proteins and all rRNAs of the Escherichia coli

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(E. coli) reference ribosome (Kössel et al., 1985; Yamaguchi and Subramanian, 2000; Yamaguchi et al.,

2000). All rRNAs, a complete set of ~30 tRNAs, approximately half of the ribosomal proteins of the 30S

subunit and one quarter of the proteins 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 proteins is, to some degree, evolutionarily

conserved, suggesting constraints in ribosome assembly that require on-site co-expression of rRNAs and

organelle-encoded core ribosomal proteins (Maier et al., 2013). Most other components of the plastid

translation machinery are nucleus-encoded (e.g., initiation/elongation/termination/ribosome recycling

factors, aminoacyl-tRNA synthetases), except for initiation factor 1 (IF1) which is plastid-encoded in

many plants (Millen et al., 2001).

Plastid deviations from the bacterial ribosome and their structural and functional consequences

Despite the generally bacterial structure of the chloroplast ribosome, there are some features that clearly

distinguish plastid ribosomes from the E. coli reference ribosome. Chloroplast ribosomes contain the full

set of bacterial rRNAs (23S, 16S, and 5S rRNAs), which comprise the peptidyl transferase activity (23S

rRNA) and the decoding center (16S rRNA) and serve as scaffold for ribosomal proteins during ribosome

assembly (Shajani et al., 2011; Maier et al., 2013). However, the 23S rRNA gene was split into two genes

in the plastid genome: a large 5’ portion encoded by the 23S rRNA gene and a small 3’ fragment encoded

by the 4.5S rRNA gene (Whitfeld et al., 1978). In addition, the 23S rRNA is post-transcriptionally

processed at two so-called “hidden breaks” into three fragments, whose abundances and precise sizes vary

among species (e.g., ~0.5, ~1.2 and ~1.1 kb from 5’ to 3’ in Arabidopsis plastids). These fragments are

found in the mature 70S ribosome and held together by intermolecular base pairing (Kössel et al., 1985;

Bieri et al., 2017). Despite their general homology, some structural elements of the E. coli 16S and 23S

rRNAs are absent from chloroplasts and, conversely, the chloroplast 23S rRNA contains additional

secondary structures (Kössel et al., 1985). Particularly well conserved are the catalytic domain V in the

23S rRNA, which carries the peptidyl transferase activity, and the anti-Shine-Dalgarno sequence in the

16S rRNA, which is crucial for translation initiation (Scharff et al., 2017).

In the course of evolution, significant changes also occurred in the proteinaceous part of the chloroplast

ribosome. The Rpl25 and Rpl30 proteins were completely lost and, in some species, the bacterial Rpl23

was replaced by its counterpart from the cytosolic 80S ribosome (Bubunenko et al., 1994; Yamaguchi and

Subramanian, 2000). Furthermore, the first complete inventory of plastid ribosomal proteins identified six

proteins that were assumed to lack bacterial orthologues and, consequently, were designated as plastid-

specific ribosomal proteins (PSRP) 1-6 (Yamaguchi and Subramanian, 2000; Yamaguchi et al., 2000).

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However, later it was shown that PSRP1 is not a genuine ribosomal protein, but the orthologue of the

bacterial cold-shock protein pY, which is associated with the small subunit of the ribosome but not a

structural part of it (Sharma et al., 2007; Sharma et al., 2010). PSRP4 also shows 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 and Subramanian, 2003). At present, PSRP2 and PSRP3 in

the 30S ribosomal subunit, and PSRP5 and PSRP6 in the 50S subunit are considered genuine plastid-

specific ribosomal proteins and, consequently, their renaming to Rps22/23 and Rpl37/38 was suggested

(Bieri et al., 2017). After their discovery, PSRPs were hypothesized to act in light-regulation of

translation (Yamaguchi and Subramanian, 2003; Manuell et al., 2007). Whereas plastid pY may indeed

perform this function (see below), a number of studies have suggested that the major function of PSRPs

lies in the structural compensation of evolutionarily modified rRNA domains (Sharma et al., 2007; Tiller

et al., 2012; Ahmed et al., 2016; Graf et al., 2016; Bieri et al., 2017). However, this role does not

necessarily exclude additional functions in translational regulation.

Some of the conserved plastid ribosomal proteins also exhibit N- or C-terminal extensions (or internal

expansions) compared to their E. coli orthologues (Yamaguchi and Subramanian, 2000; Yamaguchi et al.,

2000). Many of these extensions mediate new interactions with rRNAs or ribosomal proteins and may

structurally compensate for missing or modified rRNA domains (Ahmed et al., 2016; Graf et al., 2016;

Bieri et al., 2017). However, the extensions of some ribosomal proteins (e.g., S2, S18, S21) represent

potential new contact sites with the mRNA and, therefore, were hypothesized to be involved in

translational regulation (Manuell et al., 2007; Sharma et al., 2007; Graf et al., 2016). Notably, several

alterations (e.g., extensions of Rps5 and Rps1 and an insertion in Rps4) narrow the mRNA entry site of

the chloroplast ribosome compared to the E. coli reference ribosome (Bieri et al., 2017). Furthermore,

structural changes in the polypeptide exit tunnel and the tunnel exit site were hypothesized to support the

co-translational 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 kDa) and a slightly

lower RNA content (by ~0.4 kDa) than the E. coli ribosome, resulting in a considerably increased protein

to RNA ratio (~2:3 compared to 1:3 in E. coli). The partial replacement of rRNA domains by protein

elements in chloroplast ribosomes follows the general evolutionary trend of reducing RNA components in

enzymatically active chloroplast ribonucleoprotein particles (RNPs) and substituting them by protein

constituents (Barbrook et al., 2006). Other examples include the chloroplast tRNA processing enzyme

RNase P that lost its 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 in

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chloroplasts may be driven by the evolutionary genome reduction and the massive transfer of protein-

coding genes to the nucleus. Whereas proteins can be reimported post-translationally into plastids, this

route seems to be blocked for RNA components, which may have enforced their evolutionary loss,

replacement by proteins or retention in the chloroplast genome (in the case of rRNAs and tRNAs;

Barbrook et al., 2006). Also, it was predicted that ribosome composition (including its relative protein and

rRNA contents) is optimized for the production of the translation machinery itself, a process that strongly

limits cell division rates in prokaryotes (Reuveni et al., 2017). However, in plastids, the synthesis of

ribosomal proteins and rRNAs is partially uncoupled from translation, due to the transfer of genes for

ribosomal proteins to the nucleus and the presence of nucleus-encoded RNA polymerases, both of which

are produced by cytosolic rather than organellar ribosomes. Consequently, the evolutionary constraints on

ribosome composition are somewhat relaxed in organelles, which may have facilitated the observed shifts

in protein-to-rRNA ratios in plastid and mitochondrial ribosomes (Reuveni et al., 2017).

Some ribosomal proteins are dispensable under standard growth conditions

Most constituents of the plastid translation machinery are essential for chloroplast biogenesis and

consequently, for plant viability (Tiller and Bock, 2014). In many species, plastid translation is essential

even under heterotrophic growth conditions (Ahlert et al., 2003; Sosso et al., 2012), presumably due to

the necessity to express a few essential plastid genes such as accD, clpP, ycf1 and ycf2 (Bock, 2007).

However, some ribosomal proteins and tRNAs are non-essential, at least under standard greenhouse

conditions.

Non-essential ribosomal proteins have been identified in both E. coli and plastids. Surprisingly, despite

the shared ancestry of bacterial and plastid ribosomes, the essentiality of ribosomal proteins is not fully

conserved between the two systems (Tiller and Bock, 2014). The nucleus-encoded chloroplast ribosomal

proteins RPL11, RPL24, RPS17, RPS21, PSRP3, PSRP6 and the plastid-encoded Rps15, Rpl33 and

Rpl36 are non-essential (Tiller and Bock, 2014). The phenotypes of viable ribosomal protein mutants

range from wild-type appearance to very strong phenotypes with altered leaf morphology, variegated

leaves, cold-induced bleaching and retarded growth. Non-essential plastid ribosomal proteins have been

speculated to facilitate ribosome assembly and structural integrity, or act in regulation, optimization or

localization of translation (Pesaresi et al., 2001; Yamaguchi and Subramanian, 2003; Tiller et al., 2012;

Tiller and Bock, 2014). However, their distinct molecular functions remain to be elucidated. In bacteria,

ribosomes can differ in their protein composition, for instance, under different growth conditions.

Moreover, there is growing evidence that this ribosome heterogeneity (additionally involving post-

transcriptional and post-translational modifications of rRNAs and ribosomal proteins) creates regulatory

capacity by conferring translational selectivity of ribosomal sub-pools for specific mRNAs (e.g., Sauert et

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al., 2015; Shi et al., 2017). However, whether non-essential plastid ribosomal proteins can act as

modulators of ribosome 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 full set of 32 tRNAs that are required to

serve the 61 codons by standard and wobble base pairing between codon and anticodon. For example,

only 30 tRNA genes are present in the plastid genome of most seed plants. Since there is no evidence for

tRNA import from the cytosol, superwobble base pairing has been considered as a mechanism enabling

translation with a reduced set of tRNAs. A systematic reverse genetic screen revealed that the chloroplast

tRNAs trnG-GCC, trnL-CAA, trnS-GGA, trnT-GGU, and trnV-GAC are not essential for plant viability

(Alkatib et al., 2012; and references therein). These non-essential tRNAs decode codons with a

pyrimidine in the third position and, for all of them, essential plastid isoacceptor tRNAs with a uracil (U)

in the first anticodon position exist in plastids. These isoacceptor tRNAs can serve the respective codons

by superwobble base pairing (i.e., the U in the wobble position of the anticodon can pair with all four

nucleotides in third codon position; Alkatib et al., 2012). The diverse phenotypes of these mutants suggest

that superwobble base pairing causes distinct molecular constraints on translation (Alkatib et al., 2012),

possibly explaining the evolutionary conservation of some non-essential tRNAs.

CONSERVATION AND MODIFICATION OF BACTERIAL TRANSLATION MECHANISMS

IN CHLOROPLASTS

The overall structural conservation of essential functional elements of bacterial ribosomes in chloroplasts

is generally assumed to reflect the functional preservation of the bacterial translation mechanisms in

plastids (e.g., Peled-Zehavi and Danon, 2007).

Initiation

The initiation process starts with the contact of a pre-initiation complex consisting of the 30S subunit and

the initiator tRNA (for N-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 assembly. Functional chloroplast orthologues were identified for all three IFs (Sijben-Müller et

al., 1986; Campos et al., 2001; Miura et al., 2007; Zheng et al., 2016). Interestingly, many plants contain

two or more paralogous genes for plastid IF3, whose differential expression was proposed to regulate

chloroplast translation initiation (Nesbit et al., 2015). Also similar to the standard bacterial translation

initiation, approximately two thirds of the chloroplast reading frames are preceded by the purine-rich

Shine-Dalgarno sequence (SD) (Shine and Dalgarno, 1974; Scharff et al., 2011). In bacteria, the SD

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interacts by base pairing with a pyrimidine-rich sequence in the 16S rRNA (the anti-Shine-Dalgarno

sequence, aSD) to ensure proper positioning of the initiation complex at the start codon. The aSD is fully

conserved in plant plastids, and evidence for its functionality has been provided for many genes with SD

(e.g., Kim and Mullet, 1994; Hirose and Sugiura, 2004). Nevertheless, the significance of SD-dependent

initiation in chloroplasts has been questioned (e.g., Fargo et al., 1998). To provide ultimate clarification,

mutations were introduced into the aSD in the tobacco chloroplast genome and shown to cause reduced

translation for many reading frames with upstream SDs, thus confirming the functionality of SD-aSD

interactions (Scharff et al., 2017). On the other hand, roughly a third of the chloroplast genes and also

many bacterial genes do not contain SD sequences (or contain a putative SD but display SD-independent

translation initiation). It was shown that low amounts of mRNA secondary structure around the start

codon facilitate SD-independent translation initiation (e.g., Scharff et al., 2011; Nakagawa et al., 2017;

Scharff et al., 2017). Furthermore, in bacteria, the ribosomal protein S1 preferentially binds

polypyrimidine tracts and acts as an RNA chaperone that unfolds structured regions in mRNAs, thereby

enabling efficient SD-independent translation initiation (Qu et al., 2012). Chloroplast S1 binds RNA with

a preference for adenine or uracil-rich sequences, but whether this supports SD-independent translation

initiation is unknown (e.g., Franzetti et al., 1992; Shteiman-Kotler and Schuster, 2000).

Studies in both bacteria and chloroplasts have pointed to a 5’-to-3’ ribosome scanning mechanism and the

preferential utilization of the 5’-most start codon (e.g., Drechsel and Bock, 2011; Yamamoto et al., 2016).

Moreover, extended interactions of mRNA sequences upstream of the start codon and adjacent to the SD

with bases downstream of the anticodon of the initiator tRNA-fMet and bases next to the aSD,

respectively, have been suggested to facilitate chloroplast translation initiation (e.g., Ruf and Kössel,

1988; Esposito et al., 2003; Kuroda et al., 2007). However, the exact mechanism and the quantitative

contribution of these interactions to the efficiency of translation initiation remain to be elucidated. Similar

to bacteria, the triplets AUG, GUG and UUG can be utilized as start codons in chloroplasts (e.g., Hirose

et al., 1999; Kuroda et al., 2007; Rott et al., 2011; Moreno et al., 2017), with the recognition efficiency of

non-AUG start codons presumably depending on the sequence context (Boeck and Kolakofsky, 1994).

Elongation

After binding of the 50S subunit to the pre-initiation complex, the functional 70S ribosome is completed

and starts moving along the coding sequence of the mRNA to translate it into a polypeptide chain.

Bacterial translation elongation depends on the factors EF-Tu, EF-G and EF-Ts for which conserved

chloroplast orthologues were identified (Breitenberger et al., 1979; Fox et al., 1980; Sreedharan et al.,

1985). Notably, the expression of chloroplast elongation factors is regulated by light and other stimuli

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(temperature, phytohormones, developmental cues), suggesting involvement of elongation factors in the

regulation of translation (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 chloroplast genes are co-transcribed from operon-like gene

clusters. The resulting polycistronic transcripts contain reading frames that are separated by (often short)

spacer sequences or even overlap by a few nucleotides. In bacteria, translation of adjacent and

overlapping reading frames is frequently coupled in that translation of the second coding sequence

depends on that of the first one (Jackson et al., 2007). In some cases, the strong RNA helicase activity of

the ribosome translating the upstream reading frame is needed to unfold RNA secondary structures that

mask initiation elements (SD and/or start codon) of the downstream reading frame (Jackson et al., 2007).

Recently, also direct coupling of termination on the upstream reading frame (without ribosome

disassembly) with subsequent scanning and re-initiation on the downstream reading frame has been

demonstrated (Yamamoto et al., 2016). In chloroplasts, cases of coupled (ndhC/K, psbD/C) and

uncoupled (atpB/E) translation were identified by in vitro and in vivo analyses, respectively, but the

detailed mechanisms of translational coupling are unknown (Yukawa and Sugiura, 2008; Adachi et al.,

2012; Zoschke et al., 2013a). A recently designed in vivo expression system exploits coupled translation

in chloroplasts and suggests that the RNA helicase function of the ribosome may also mediate

translational coupling in chloroplasts (Martin Avila et al., 2016).

Termination and ribosome recycling

When one of the three stop codons is reached, the orthologous release factors RF1/PrfA (serving UAA

and UAG) and RF2/PrfB1 (serving UAA and UGA) set the synthesized protein free by hydrolysis of the

ester bond (Buckingham et al., 1997; Meurer et al., 2002; Motohashi et al., 2007). Additionally, PrfB1 is

involved in the stabilization of plastid mRNAs containing reading frames with UGA stop codons (Meurer

et al., 2002). PrfB3, a non-functional chloroplast-targeted paralog of PrfB1, lacks domains that are

essential for stop codon recognition and hydrolytic activity of release factors. Remarkably, PrfB3 gained a

new function in transcript stabilization of the petB mRNA (Stoppel et al., 2011). Another release factor,

RF3, facilitates dissociation of RF1 and RF2 from the ribosome in bacteria (Buckingham et al., 1997) and

likely also in chloroplasts (Beligni et al., 2004). In the final step, ribosome recycling factor (RRF; Rolland

et al., 1999), EF-G and IF3 facilitate the release of mRNA and tRNA, and the disassembly of the small

and large ribosomal subunits, thereby recycling them for the next round of translation initiation.

13

PLASTID TRANSLATION IS INTERCONNECTED WITH RNA AND PROTEIN

METABOLISM

Relaxed coupling of translation and transcription

In bacteria, translation initiates and elongates co-transcriptionally, thus ensuring efficient transcription

(e.g., by preventing RNA polymerase backtracking), conferring RNA stability (by translating ribosomes

protecting the mRNA from ribonucleolytic attack), enabling timely translation, and maintaining genome

integrity (e.g., by avoiding extended hybridization of RNA and DNA that would cause collision of the

transcription and replication machineries; e.g., McGary and Nudler, 2013). A similar coupling of

transcription and translation was proposed for chloroplast gene expression based on early electron

micrographs that were interpreted as evidence for ribosomes being associated with nascent transcripts

(e.g., Rose and Lindbeck, 1982; Figure 2). Additional evidence for coupling of transcription and

translation has come from the findings that (i) ribosomal proteins are associated with the transcription

machinery (Pfalz et al., 2006), and (ii) translation factors and other proteins involved in translation are

enriched in plastid nucleoids in a ribonuclease-sensitive manner, suggesting tethering by nascent

transcripts (Majeran et al., 2011). Moreover, orthologues of the Nus proteins that couple transcription and

translation in bacteria have been identified in chloroplast nucleoids (Majeran et al., 2011). However,

chloroplast transcripts have a longer half-life than bacterial transcripts, are stable when not covered by

ribosomes, and many translated RNA species are generated by RNA processing (see below). Together,

this implies that, simply due to the kinetics of mRNA processing and turnover, there may be a

quantitative shift towards post-transcriptional translation in chloroplasts.

The chloroplast genome is regularly transcribed by bacterial-type and phage-type RNA polymerases that

have very different properties (Börner et al., 2015). For example, the two polymerases transcribe with

different speeds and recognize different promoters thus producing primary transcripts with divergent 5’

ends. Notably, in phage-infected E. coli cells, the speedy transcription by the RNA polymerase of

bacteriophage T7 is not coupled with translation and thus produces initially “naked” (i.e., ribosome-free)

transcripts with a higher decay rate (e.g., Makarova et al., 1995). How transcription by the different

plastid RNA polymerases is coordinated with translation and whether or not the utilized RNA polymerase

influences the kinetics of protein synthesis, is currently unknown.

Unprocessed transcripts can be translated

Primary chloroplast transcripts undergo extensive RNA processing, including splicing of group I and II

introns, RNA editing (changing cytosine to uracil residues to restore codons for conserved amino acids or

start or stop codons), 5’ and 3’ end trimming, and intercistronic processing that generates diverse

-

Thylakoids

Internal triggers:

- developmental stage

- cell type

- phytohormones- redox state

- circadian clock- alarmone - (p)ppGpp- metabolites

Unprocessed RNA

Transcription

External triggers:

- light (quality/quantity)- temperature

- osmotic status- nutrient availability

Processed RNA

R:licing,� Protein proce�sing,e�iting,_ intra: & �����11;

1targeting,

1nterc1stronic Y processing

Chloroplast ----i Protein

___ _, complex

DNA

Thylakoids

RNA polymerase

RNA with reading frame, --�-- and translating ribosome

with nascent peptide

Group II intron

Transcript-specific RNA-binding proteins (or complexes) involved in RNA processing and/or translational activation

Factors involved in protein processing, folding, targeting and assembly

Membrane-embedded protein import

apparatus (TOC/TIC)

Figure 2. Overview of internal and external triggers that cause regulatory adjustments of translation in chloroplasts, the mechanisms that control translation, the coupling of RNA and protein metabolisms 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 the cytosol (shown in the upper left part) and imported into the chloroplast, where they control and/or regulate chloroplast protein synthesis directly (by altering chloroplast translation activity) or indirectly (by controlling co-translational chloroplast RNA or protein metabolisms). Chloroplast translation occurs co-transcriptionally (left), however, due to the slow mRNA turnover, the majority of ribosomes act post-transcriptionally (right). RNA-binding proteins assist co-translational RNA processing and/or facilitate translation initiation. Ribosomes initiate and elongate regularly on both processed and unprocessed transcripts, 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 in protein processing, folding, targeting and assembly act co-translationally on the nascent polypeptide. See text for details.

14

15

transcript isoforms from polycistronic primary transcripts (e.g., Barkan, 2011; Lyska et al., 2013). There

is no obvious spatial separation that would compartmentalize RNA metabolism and translation.

Interestingly, many of the factors known to be involved in transcript processing (e.g., RNA-binding

proteins), stabilization and translation co-localize with processed and unprocessed transcripts in nucleoids

and transcription complexes (e.g., Pfalz et al., 2006; Majeran et al., 2011; Lehniger et al., 2017). This

raises the question whether unprocessed mRNAs, often referred to as “precursors” or “immature

transcripts”, are utilized as templates for translation, or whether any partitioning (e.g., temporal

separation) exists between RNA processing and translation in plastids.

Up to twelve reading frames are interrupted by group II introns in chloroplast genomes of seed plants

(e.g., Sugiura, 1995). In all of them, a substantial fraction of the coding region is located downstream of

the intron and, consequently, splicing is essential to produce functional proteins (Barkan, 2011).

Surprisingly, recent ribosome profiling studies in maize chloroplasts have demonstrated that translation

initiates on unspliced atpF, ndhA, ndhB and ycf3 transcripts and also elongates (Zoschke et al., 2013a;

Alice Barkan, personal communication; Figure 3A). Whether translation elongation pauses at the robust

intron structure or, alternatively, the RNA helicase function of the ribosome allows translation to proceed

into the intron until it terminates at the first in-frame stop codon, thus producing non-functional proteins,

has not yet been possible to resolve. Nevertheless, it is clear that ribosomes initiate and elongate on

unspliced transcripts, strongly arguing against a spatial or temporal separation of splicing and translation

processes.

High-resolution ribosome profiling analysis of maize chloroplast translation also demonstrated that the

degree of editing in mRNA footprints of actively translating ribosomes is not substantially different from

that in the general transcript pool (Chotewutmontri and Barkan, 2016). Specific editing sites that are only

partially edited in the transcript pool showed a similar degree of partial editing in ribosome footprints.

This is in line with the earlier finding that editing of tobacco rps14 is not a requirement for efficient in

vitro translation (Hirose et al., 1998). Together, these data imply that ribosomes cannot distinguish

between edited and unedited transcripts (Figure 3B). However, two exceptions were identified in maize:

the transcripts of rpl2 and ndhA which are only translated in their edited form (Chotewutmontri and

Barkan, 2016). In rpl2, ACG-to-AUG editing restores the start codon (Hoch et al., 1991), thereby

activating translation (Chotewutmontri and Barkan, 2016). This is consistent with the earlier finding that

restoration of the tobacco ndhD start codon by editing is essential for efficient translation initiation in

vitro (Hirose and Sugiura, 1997). In some instances, start codon restoration by editing is subject to tissue-

specific or developmental variation, thus raising the intriguing possibility that editing may control

translational activity (Ichinose and Sugita, 2016). In the case of ndhA, splicing of its group II intron is

A Unspliced

5' 3'

Exon 2

Unedited

5' 3'

C

Polycistronic

RF 1 RF 2 RF 3

Processing

\ •

w

Spliced

5'

�

3'

Edited

5' 3'

Monocistronic

5'�3' 5'�3' RF 1 RF 3

5'�3 RF 2

Figure 3. Ribosomes translate unprocessed chloroplast transcripts (see text for details).

(A) Several chloroplast reading frames are interrupted by group II intrans. Left: Translating ribosomes cover exon 1 ofunspliced atpF, ndhA, ndhB, and ycf3 transcripts (Zoschke et al., 2013a; Alice Barkan, personal communication). Middle andright: Splicing releases the intron and ligates the exons. Consequently, both exons of the spliced transcript are occupied byribosomes, producing full-length proteins (chain of black dots: nascent polypeptide).

(B) Chloroplast transcripts are edited at specific sites by modification of cytosine (C) to uracil (U) residues, often restoringcodons for conserved amino acids (change from yellow to white dot in the nascent peptide). Actively translated mRNAs havethe same editing status as the total transcriptome (Chotewutmontri and Barkan, 2016), indicating that, in a partially editedtranscript pool, also unedited transcripts are translated.

(C) Polycistronic chloroplast transcripts often undergo post-transcriptional processing that generates smaller transcriptisoforms (represented by the three monocistronic transcripts on the right; RF : reading frame). Often all transcript isoforms areused as translation templates. The extent to which transcript processing may enhance translation efficiency needs to bedetermined on a case-by-case basis.

16

17

required to enable editing of the first editing site in exon II (which is separated by the intron; Schmitz-

Linneweber et al., 2001; and references therein). Consequently, ndhA transcripts that are unedited at this

particular site are unspliced, and elongating ribosomes cannot get through to the 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 amounts of non-functional and potentially deleterious proteins. In accordance with this

assumption, failure to restore conserved amino acids in subunits of PSII, the cytb6f complex and the ATP

synthase by mRNA editing strongly impairs the function of the respective complexes (Bock et al., 1994;

Zito et al., 1997; Schmitz-Linneweber et al., 2005b). Hence, rapid proteolytic removal of the

dysfunctional proteins synthesized from unedited and unspliced transcripts has to be assumed to prevent

deleterious effects on chloroplast function.

End trimming and intercistronic processing of plastid transcripts have been proposed to enhance their

translational activity (e.g., 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,

despite the fact that they exist also as 5’ reading frames on processed transcripts (Barkan, 1988).

Likewise, unprocessed tobacco atpH and rbcL mRNAs were translated as efficiently as processed

mRNAs in vitro (Yukawa et al., 2007). Furthermore, synthetic transcription units that were engineered

into plastids gave rise to polycistronic transcripts that were efficiently translated in the absence of

processing (e.g., Staub and Maliga, 1995). Finally, a genome-wide ribosome profiling study revealed that

several polycistronic mRNAs are efficiently used as translation template, despite the known co-existence

of monocistronic transcript isoforms (Zoschke and Barkan, 2015). In all of these examples, the translation

of downstream reading frames in polycistronic mRNAs was not dependent on transcript processing into

monocistronic units, indicating that internal start codons can be efficiently recognized. Also, a number of

plastid reading frames are only present in di- or polycistronic transcripts, indicating that these must

undergo translation. Altogether, these data provide compelling evidence that transcript end processing and

intercistronic processing are not general requirements for efficient translation (Figure 3C). However, in a

few cases there is good evidence that transcript processing stimulates translation. For instance, in tobacco,

a base-pairing interaction between the psaC coding region and the ndhD 5’UTR in the dicistronic

transcript was shown to prevent efficient ndhD translation in vitro, whereas processed monocistronic

transcripts were translationally active, suggesting that, in this operon, processing is required to activate

translation (Hirose and Sugiura, 1997). Similarly, in vitro translation provided evidence that unprocessed

atpB, psbB and psbD transcripts from tobacco chloroplasts are less efficiently translated than their

processed isoforms (Yukawa et al., 2007; Adachi et al., 2012). Moreover, in tobacco chloroplasts,

18

heterologous expression of GFP from engineered polycistronic mRNAs was more efficient when GFP

was placed at the 5’ end of the synthetic operon (Drechsel and Bock, 2011). Finally, in maize, the

monocistronic forms of psaI and rps14 show the highest accumulation in those developmental stages

where the highest translation rates of these reading frames occur, a finding that would be consistent with

the monocistronic RNA species being better translatable (Chotewutmontri and Barkan, 2016).

In sum, although in some cases, translation is indeed stimulated by RNA processing, there is no general

dependence of translation on processing. Unprocessed, unspliced and unedited transcripts have been

shown to be used as translation templates (Figure 3), and, therefore, these transcript isoforms are not

necessarily “immature” or “precursors”.

Several RNA-binding proteins act dually in transcript processing/stabilization and promotion of

translation

In recent years, plastid RNA-binding proteins, many of them with helical repeat domains, were shown to

be involved in specific RNA end trimming and intercistronic processing events (Barkan and Small, 2014;

Hammani et al., 2014). Mutants of some of these factors (Table 1) displayed defects in RNA processing

that were accompanied by translation deficiencies (e.g., Barkan et al., 1994; Felder et al., 2001;

Hashimoto et al., 2003). Initially, these were interpreted as RNA processing-dependent translation defects

(in that processing was required for efficient translation). However, later, it was observed that the

knockout of PPR10, an RNA-binding protein involved in the processing and stabilization of specific atpH

transcript isoforms, caused much stronger translation and protein accumulation defects than expected

from its RNA processing defect, suggesting a more direct role of PPR10 in translation of atpH (Pfalz et

al., 2009). In vitro assays showed that PPR10 binds to the atpH 5’UTR and protects transcripts from 5’-

to-3’exonucleolytic degradation. Consequently, the PPR10-binding site defines the 5’ end of these

transcripts (Prikryl et al., 2011). In addition, PPR10 binding remodels the atpH 5’UTR such that an RNA

stem-loop structure that occludes the putative SD sequence of atpH is dissolved and the ribosome-binding

site becomes exposed (Prikryl et al., 2011). This suggests a dual function of PPR10 in RNA stabilization

and stimulation of atpH translation. A similar mode of action was shown, or is discussed, for other

chloroplast RNA-binding proteins such as HCF107, PGR3, CRR2 and CRP1 (see Table 1 and references

therein). The additional translation-promoting function should be independent of processing in that it

should also occur in polycistronic transcripts where the target reading frame is located downstream of

other reading frames. In fact, reanalysis of ppr10, crp1, pgr3 and hcf107 maize mutants by ribosome

profiling revealed substantial translation defects in vivo (and less severe transcript accumulation defects)

for the reading frames downstream of the RNA-binding sites of the respective protein (in atpH, petD,

petL, and psbH expression, respectively; Zoschke et al., 2013a; Alice Barkan, personal communication).

19

In line with a dual function of some RNA-binding proteins, PPR53, a member of the small PPR-SMR

family, was recently described to be involved in both promotion of ndhA translation and

processing/stabilization of transcript isoforms with ndhA as the 5’ reading frame (Zoschke et al., 2016).

These examples support the idea that RNA-binding proteins acting in processing/stabilization of specific

5’ transcript ends can also directly promote translation of the reading frame downstream of their binding

site (Figure 2). Such proteins provide a physical link between RNA metabolism and translation, and

therefore, co-occurrence of intercistronic processing and stimulation of translation does not necessarily

imply a strict requirement of RNA processing to facilitate translation.

Table 1. Factors demonstrated or suggested to facilitate translation of specific transcripts in seed plant plastids. Asterisks (*) indicate an additional function in stabilization of the transcript 5’ end upstream of the reading frame whose translation is stimulated. Question marks (?) denote proposed but experimentally unconfirmed functions in stimulation of translation. SDR: short-chain dehydrogenase/reductase, HAT: half a tetratricopeptide repeat, PPR: pentatricopeptide repeat, SMR: small MutS-related.

Factor Protein domain(s)

Reading frames with translation 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; Zoschke et al., 2013b; Zoschke et al., 2013a

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 possibly 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

20

Notably, a widely employed sequence element in chloroplast biotechnology (IEE, for intercistronic

expression element) that enhances the heterologous expression of reading frames located in polycistronic

transcription units includes the HCF107-binding site (Zhou et al., 2007; Hammani et al., 2012). The

insertion of the IEE between reading frames enhances the accumulation of monocistronic transcripts. This

could be related to the RNA secondary structure formed by the IEE (Zhou et al., 2007), the action of

RNase E (Walter et al., 2010) and/or the binding of HCF107 (Hammani et al., 2012; Legen et al., 2018).

The turnover of plastid transcripts is not determined by their translation status

In E. coli, mRNAs are stabilized by translating ribosomes, presumably by ribosome coverage providing

physical protection from ribonucleases (e.g., Laalami et al., 2014). By contrast, the study of maize and

Arabidopsis mutants with transcript-specific translation defects has revealed that atpB, petA, psaC and

psbA transcripts are stable although their translation and, consequently, their ribosome occupancy was

dramatically reduced (Barkan et al., 1994; McCormac and Barkan, 1999; Link et al., 2012; Zoschke et al.,

2012; Zoschke et al., 2013b). Furthermore, exchange of the canonical AUG start codon by the non-

standard initiation codon GUG or UUG in the atpB, clpP and psbD reading frames in tobacco

chloroplasts diminished translation initiation and protein synthesis but did not destabilize the transcripts

(Rott et al., 2011; Moreno et al., 2017; Mark A. Schöttler, personal communication). Also, mutants with

general impairments in chloroplast translation do not exhibit general transcript accumulation defects (e.g.,

Barkan, 1993; Scharff et al., 2017). Similarly, the treatment of wild-type Arabidopsis plants with

lincomycin, an antibiotic that disturbs 70S translation elongation only at the earliest steps, thus causing

run-off of ribosomes (Kallia-Raftopoulos and Kalpaxis, 1999), did not cause a substantial decrease in the

accumulation of any of the analyzed chloroplast transcripts (e.g., Meurer et al., 2002; Stoppel et al.,

2011). Accumulation of chloroplast transcripts was also not increased after treatment with

chloramphenicol, a 70S elongation inhibitor that arrests ribosomes and inhibits their release, thus

resulting in densely ribosome-covered transcripts (Nierhaus and Wittmann, 1980).

Altogether, the available data demonstrate that chloroplast mRNAs are stable in the absence of translation

and do not require physical protection by ribosomes. This may not be surprising given the evolutionary

switch from a largely transcriptional regulation of gene expression, as found in bacteria, to predominantly

post-transcriptional regulation in chloroplasts (which strongly depends on transcripts with long half-

lives). Chloroplasts harbor many endo- and exoribonucleases that potentially could degrade “naked”

transcripts (e.g., Germain et al., 2013). This implies that non-translated chloroplast mRNAs must be

somehow protected against nuclease attack. A small family of RNA recognition motif (RRM) domain-

21

containing proteins, the chloroplast ribonucleoproteins (cpRNPs), were shown to be involved in different

steps of mRNA metabolism, including transcript stabilization (Ruwe et al., 2011). Taking into account the

high abundancy of these proteins in the chloroplast, their broad RNA-binding activity in vivo (in that they

associate with virtually all mRNAs), and the fact that they are specifically bound to non-polysomal

mRNAs (Nakamura et al., 2001; Kupsch et al., 2012; Teubner et al., 2017), cpRNPs are strong candidates

for providing stability to untranslated mRNAs. Notably, mutants of the cpRNPs CP29A and CP31A show

a conditional cold-sensitive phenotype, presumably caused by a reduction in the stability of many

mRNAs (Kupsch et al., 2012). A possible explanation is a run-off of translating ribosomes in the cold and

subsequent transcript degradation in the absence of stabilizing cpRNPs. A recent structural analysis

revealed a narrowed mRNA entry site of the chloroplast ribosome compared to that of E. coli (Bieri et al.,

2017). It seems tempting to speculate that this is because chloroplast ribosomes, different from their

bacterial counterparts, need to strip off abundant RNA-binding proteins such as cpRNPs when translation

reinitiates and elongates on previously “stored” (i.e., untranslated) mRNAs, or even on normally

translated mRNAs with low initiation rates (resulting in larger ribosome spacing).

In sum, untranslated chloroplast mRNAs are stable and cpRNP binding may protect them against

ribonucleolytic attack, thus causing the observed uncoupling of mRNA stability from translation.

However, a direct functional connection between translational activity, cpRNP (un)binding and mRNA

stability remains to be demonstrated.

Co-translational folding, maturation, targeting and assembly of proteins

In bacteria, several steps in protein metabolism, including proteolytic processing, chemical modification,

co-factor binding, folding, targeting and assembly, can occur co-translationally (e.g., Gloge et al., 2014).

It seems clear that this is also the case in chloroplasts, although the knowledge about the intersections

between plastid translation and protein metabolism is still scarce (e.g., Giglione et al., 2015; Breiman et

al., 2016; Figure 2). Removal of the N-terminal N-formylmethionine often represents the first step of

nascent peptide chain processing in bacteria and chloroplasts. It occurs co-translationally by the

consecutive reactions of peptide deformylase and methionine aminopeptidase (Breiman et al., 2016).

Likewise, the N-terminal signal peptide for thylakoid targeting of PetA (cytochrome f) is co-

translationally cleaved (see below). Another widespread N-terminal modification of the nascent peptide is

the N-α-acetylation of the penultimate amino acid (e.g., Zybailov et al., 2008; Breiman et al., 2016). A

complete list of identified N-terminal processing events in plastid-encoded proteins is provided at

http://www.i2bc.paris-saclay.fr/spip.php?article1261&lang=fr (Breiman et al., 2016).

22

In bacteria and eukaryotes, folding of the nascent peptide chain has been shown to start already in the

ribosome exit tunnel (e.g., Bhushan et al., 2010; Gloge et al., 2014). With dimensions of 10 nm in length

and 1 - 2 nm in width, the 70S ribosome exit tunnel has a sufficient size to shelter 30 - 60 amino acids

(depending on the folding status) and allows the formation of small protein domains consisting of α-

helices (e.g., Holtkamp et al., 2015). Protein folding in the exit tunnel is assisted by the ribosome itself

through interactions of the nascent peptide with the 23S rRNA and ribosomal proteins (e.g., L4, L22 and

L23; Gloge et al., 2014). Given the high conservation of the peptide exit tunnel, co-translational folding is

expected to occur also during chloroplast translation. The ribosome-associated chaperone trigger factor

binds the nascent peptide upon exit from the ribosome and stabilizes it, thus preventing protein

aggregation and assisting co-translational protein folding in bacteria and, most likely, also in chloroplasts

(Breiman et al., 2016; Ries et al., 2017). Subsequently, other chaperones are recruited and take over

(Trösch et al., 2015).

In parallel to folding, co-factors such as chlorophylls, hemes, carotenoids, quinones and metal ions can

associate co-translationally with chloroplast apoproteins (Schöttler et al., 2011; Nickelsen and Rengstl,

2013; Schöttler et al., 2015). Several studies suggest that the plastid-encoded apoproteins of PSI and PSII

must bind chlorophylls co-translationally to ensure faithful complex biogenesis, most likely, because

chlorophyll binding is required for correct protein folding and assembly (e.g., Nickelsen and Rengstl,

2013). This is supported by evidence that chlorophyll stabilizes nascent chlorophyll-binding proteins

(e.g., 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 facilitate the co-translational binding of

chlorophyll and other co-factors such as pheophytin, quinone, iron sulfur and manganese clusters (Kim et

al., 1991, 1994a; Gawronski et al., 2018). In cyanobacteria, unassembled PsbB and PsbC apoproteins

contain chlorophyll a and β-carotene, suggesting their early co-translational association (Boehm et al.,

2011).

Chloroplasts comprise different suborganellar compartments: stroma, thylakoid membrane, thylakoid

lumen, inner and outer envelope membranes and the intermembrane space. The targeting of some

chloroplast-encoded proteins to the thylakoid membrane has long been recognized to occur co-

translationally (reviewed in Celedon and Cline, 2013; Figure 2). Early on, it was shown that puromycin

treatment (which causes premature translation termination and thereby release of the nascent peptide) also

releases ribosomes from the thylakoid membrane in chloroplasts, suggesting co-translational protein

targeting mechanisms (e.g., Yamamoto et al., 1981). In later studies, chloroplast sub-fractionation

coupled with polysome analysis and pulse labeling studies revealed that polytopic proteins of PSI (PsaA,

PsaB) and PSII (PsbA/D1, PsbB/CP47, PsbC/CP43, PsbD/D2), and the bitopic cytochrome f subunit

23

(PetA) of the cyt b6f complex associate with the thylakoid membrane co-translationally (e.g., Margulies et

al., 1987; Friemann and Hachtel, 1988; Kim et al., 1994b; van Wijk et al., 1996). However, the

interpretation of the results from these experiments was sometimes controversial (e.g., Ibhaya and

Jagendorf, 1984) and complicated by the fact that in chloroplasts, polycistronic transcripts can be used as

translation templates. Consequently, one co-translationally inserted polypeptide produced from a

polycistronic transcript is sufficient to tether all co-transcribed cistrons to the thylakoid membrane. This

difficulty was overcome in a recent study by using ribosome profiling, a method that employs nucleases

to degrade mRNAs in polysomes down to the footprints protected by monosomes (Zoschke and Barkan,

2015). By coupling this approach with fractionation of chloroplasts into thylakoid membranes and stroma,

co-translational membrane insertion could be comprehensively examined at a genome-wide scale. The

study revealed that 19 of the 37 plastid-encoded intrinsic transmembrane domain-containing thylakoid

proteins in maize insert co-translationally into the membrane, and supplied evidence that exposure of the

first transmembrane domain provides the signal and/or the anchor for stable membrane association (with

the sole exception of PetA, as described below). The data suggest a model for ribosome-mediated mRNA

targeting, in which the nascent polypeptide exposed by the first “pioneer” ribosome anchors the

translation machinery together with the translated mRNA at the thylakoid membrane. Continued

translation by the following ribosomes keeps the mRNA tethered to the thylakoid membrane. A similar

model was suggested for the cytosolic ribosomes that are associated with mitochondria and the

endoplasmic reticulum (Jan et al., 2014; Williams et al., 2014). In addition, a recent ribosome profiling

study in Arabidopsis correlated plastid ribosome pausing events with the synthesis and correct integration

of transmembrane domains (Gawronski et al., 2018). Electron microscopic evidence indicates that

membrane-associated chloroplast polysomes are connected with all unstacked thylakoid membrane

regions (i.e., stroma lamellae, grana margins), but not with internal membranes in grana stacks which are

inaccessible due to their tight packing (e.g., Yamamoto et al., 1981). Consequently, once grana stacks are

assembled during chloroplast biogenesis, plastid-encoded grana proteins (mainly PSII subunits) need to

be transported post-translationally from unstacked membrane regions into grana stacks, for example,

during photosystem repair (e.g., Pribil et al., 2014).

The mechanisms involved in suborganellar protein targeting have been best studied for nucleus-encoded

chloroplast proteins that are post-translationally distributed (Celedon and Cline, 2013). Five major

pathways with partially overlapping functions (and some shared components) have been described

(Schünemann, 2007; Celedon and Cline, 2013). The secretory (Sec) pathway and the twin-arginine

translocase (Tat) transport proteins across the thylakoid membrane into the lumen (reviewed in

Schünemann, 2007). The chloroplast signal recognition particle (cpSRP) interacts with the cpSRP

24

receptor cpFtsY and the insertase ALB3 to insert nucleus-encoded light-harvesting complex proteins into

the thylakoid membrane (reviewed in Ziehe et al., 2017). Some proteins apparently insert spontaneously

into the thylakoid membrane, and finally, a recently discovered parallel Sec pathway targets proteins to

the inner envelope membrane (Li et al., 2017b).

Much less is known about the mechanisms involved in co-translational suborganellar targeting of

chloroplast-encoded proteins. Most plastid-encoded proteins are found in either the stroma or the

thylakoid membrane. Plastid-encoded membrane proteins likely utilize one of the above-mentioned

targeting pathways, either co- or post-translationally. So far, the co-translational targeting mechanisms

were elucidated in some detail for only two plastid-encoded proteins: PetA and PsbA. PetA is the only

plastid-encoded protein containing a cleavable signal peptide at its N-terminus which is recognized by

cpSecA. In vitro and genetic data suggest that PetA targeting to the thylakoid membrane occurs co-

translationally (Voelker et al., 1997; Röhl and van Wijk, 2001; and references therein). A recent ribosome

profiling study showed that nascent PetA engages the thylakoid membrane long before its single

transmembrane domain becomes exposed outside the ribosome exit tunnel, thus confirming co-

translational action of cpSecA-dependent targeting in vivo (Zoschke and Barkan, 2015). Only recently,

SecA-mediated targeting in bacteria was demonstrated to also occur co-translationally (Huber et al.,

2017).

Co-translational interaction of PsbA and cpSRP54 has been suggested based on in vitro cross-linking

experiments (Nilsson and van Wijk, 2002; and references therein). However, Arabidopsis mutants lacking

cpSRP54 have very mild phenotypes, arguing against an essential role of cpSRP54 in membrane targeting

of PsbA or any other core subunit of the photosynthesis machinery (Amin et al., 1999; Tzvetkova-

Chevolleau et al., 2007). Nonetheless, cpFtsY, the essential chloroplast SRP receptor homologue that had

previously been implicated in PSII repair (Tzvetkova-Chevolleau et al., 2007; Asakura et al., 2008), was

shown to be associated with nascent PsbA in vitro suggesting a role in co-translational targeting (Walter

et al., 2015). Furthermore, the translocons cpSecY and ALB3 and the multifunctional protein Vipp1, all

of which are essential for thylakoid biogenesis (Sundberg et al., 1997; Roy and Barkan, 1998; Kroll et al.,

2001), appear to interact with nascent PsbA in vitro, suggesting their involvement in co-translational

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 the insertase ALB3 (Kroliczewski et al.,

2016).

The assembly of proteins into functional complexes often initiates co-translationally (e.g., Natan et al.,

2017). Biogenesis of the multiprotein complexes in the thylakoid membrane requires the tightly

25

coordinated action of multiple assembly factors that guide the association of plastid-encoded and nucleus-

encoded subunits (e.g., Schöttler et al., 2011; Nickelsen and Rengstl, 2013). Many plastid-encoded

subunits of these complexes are believed to be assembled co-translationally (Figure 2), because

unassembled free subunits are usually condemned to rapid degradation. However, only in few cases,

direct evidence for co-translational assembly has been obtained. The PSII assembly process is best

understood (e.g., Nickelsen and Rengstl, 2013), and several plastid-encoded core subunits of PSII have

been suggested to assemble co-translationally into the complex. During both de novo assembly and repair

of PSII, nascent PsbA subunits are integrated into an early assembly intermediate that contains PsbD

(Müller and Eichacker, 1999; Zhang et al., 1999). Whereas the first and second transmembrane domains

of PsbA only weakly interact with PsbD, a robust association is established after synthesis of the fourth

transmembrane domain (Zhang et al., 1999; Zhang and Aro, 2002). It is tempting to speculate that the

operon-like organization of chloroplast genes enhances the efficiency of co-translational targeting and

assembly (e.g., in the dicistronic psaA/B and psbD/C transcripts), as this was suggested for bacteria

(Natan et al., 2017).

In summary, a multitude of factors act co-translationally as “welcoming committee” for nascent

polypeptides and assist with the amazing metamorphosis of linear amino acid chains into functional

proteins and protein complexes in specific suborganellar locations (Figure 2). We are just beginning to

understand the complex interconnections of the diverse processes involved in co-translational protein

maturation, targeting and assembly, but it is becoming increasingly evident that the ribosome acts as a

central hub in the coordination of these processes.

OPERATIONAL CONTROL AND REGULATION OF CHLOROPLAST TRANSLATION

Upon stress and under changing environmental conditions, the thylakoid membrane system is adjusted to

achieve optimum photosynthetic performance and prevent photo-oxidative damage. These acclimation

responses require integration of multiple external and internal signals (Figure 2), and involve extensive

regulation of chloroplast translation (e.g., Nickelsen et al., 2014; Sun and Zerges, 2015).

Translational control versus regulation

The terms ‘translational control’ and ‘translational regulation’ are sometimes used synonymously.

However, factors controlling translation (e.g., the strength of a ribosome-binding site in the 5’UTR) do

not necessarily also regulate translation (i.e., dynamically change protein synthesis rates in response to

environmental stimuli or developmental programs).

26

The interplay of trans-acting protein factors and cis-acting RNA elements determines the translation

output of chloroplast genes. The functional involvement of these elements in translational control

typically is demonstrated by their genetic manipulation causing altered translational activity. However,

this does not necessarily imply a regulatory function in translation, resulting in a change in the protein

synthesis rate during adaptation processes. In other words, a given factor would have a regulatory

function if it were to become limiting for translation under specific conditions, thus altering protein

synthesis output. According to this definition, a true regulatory function of chloroplast translation factors

has been established only in very few cases in Chlamydomonas (see below). It was proposed that many of

the nucleus-encoded factors involved in chloroplast RNA metabolism simply suppress mutations that

accumulate in the (asexually reproducing) plastid genome over time (Maier et al., 2008; Lefebvre-

Legendre et al., 2014). Similarly, nucleus-encoded translation factors could be needed constitutively to fix

chloroplast mutations at the RNA level (e.g., by resolving secondary structures around ribosome-binding

sites to facilitate translation initiation). However, on an evolutionary time scale, factors that control

translation may also be recruited as true regulators that modulate translation in response to internal and

external triggers.

Translation plays a major role in the control and regulation of chloroplast gene expression

Translation is an extremely resource-consuming process due to the energy and nutrient demands involved

in the assembly of ribosomes, the synthesis of amino acids, the expression, processing and charging of

tRNAs, and the GTP-dependent reactions during initiation and elongation. Dividing bacterial cells use

~50 % of their energy for protein synthesis (Russell and Cook, 1995). Millar and co-workers calculated

the cellular energy budgets used for protein synthesis in Arabidopsis leaves (Li et al., 2017a). Their

estimate is that, dependent on the developmental stage, 13 – 38 % of the cellular ATP is used for protein

synthesis, with plastid translation accounting for ~70 % of the costs of cellular protein synthesis.

Synthesis of RbcL alone accounts for more than 15 % of the cellular ATP equivalents used for protein

synthesis (Li et al., 2017a). In view of the high costs of translation, the rate of protein synthesis is tightly

coordinated to the cellular demands in all domains of life. In addition, translational regulation has several

advantages. As translation is the final synthesis step in gene expression, its regulation (i) can mediate

rapid responses to internal or external stimuli, (ii) most directly affects the protein accumulation levels,

and (iii) can be readily exploited to enhance or attenuate changes in upstream steps of gene expression

(i.e., transcription and transcript accumulation). Importantly, translation can also be dynamically localized

to the site where the synthesized protein is needed, whereas transcription is largely bound to the position

of the genomic DNA. Finally, although in bacterial systems transcriptional co-regulation is easily

achieved by combining genes in operons, this genomic organization is rather static and, in contrast to

27

translationally co-regulated mRNAs (qualifying as ‘regulons’), cannot readily mediate dynamic responses

to different cues (Keene, 2007).

It is generally accepted that chloroplast gene expression is largely controlled and regulated at the post-

transcriptional and, especially, the translational levels, contrasting gene expression in cyanobacteria,

which is mainly transcriptionally regulated. Several lines of evidence led to this conclusion. First,

bacterial transcripts tend to be unstable (with half-lives in the range of minutes), enabling transcriptional

regulation (e.g., Pedersen and Reeh, 1978; Klug, 1993). By contrast, the half-lives of chloroplast

transcripts are in the range of hours or even days, thus disabling fast transcriptional responses (e.g.,

Mullet and Klein, 1987; Klaff and Gruissem, 1991). Second, although chloroplast gene clusters are

reminiscent of bacterial operons, they often are transcribed from different promoters (including operon-

internal promoters) and, frequently, the resulting primary transcripts are further processed into smaller

units (e.g., Barkan, 2011; Lyska et al., 2013; Börner et al., 2015). Also, chloroplast polycistronic

transcription units often comprise functionally unrelated genes (Sugiura, 1995), unlike bacterial operons,

where co-transcription serves to couple the expression of functionally related genes. Third, the translation

of some chloroplast mRNAs coding for core components of the photosynthesis machinery is induced by

light, whereas their mRNA levels remain virtually unaffected (e.g., Berry et al., 1988; Mühlbauer and

Eichacker, 1998). Fourth, translation of many chloroplast mRNAs was shown to be the rate-limiting step

in gene expression in Chlamydomonas (Eberhard et al., 2002), although the situation may be different in

angiosperms (Udy et al., 2012). Moreover, in a regulatory mechanism termed control by epistasy of

synthesis (CES), the translation rate of some subunits of photosynthetic protein complexes is regulated by

the presence or absence of their assembly partners (Choquet and Wollman, 2009; see also below). Fifth,

in recent years, a number of factors required for the translation 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 in translational control. A major cis-acting sequence element for translation initiation in

chloroplasts is the SD sequence (Scharff et al., 2017; see above). Recent ribosome profiling studies have

suggested that, in bacteria and chloroplasts, SD-like sequences that are located within reading frames

cause programmed pausing of elongating ribosomes (Li et al., 2012; Zoschke et al., 2013a; Gawronski et

al., 2018). It has been proposed that SD-dependent pausing facilitates co-translational folding and

targeting of nascent polypeptides (Li et al., 2012; Fluman et al., 2014). Additional chloroplast cis-acting

sequence elements for translational control are the binding sites of trans-acting factors that stimulate

translation of specific reading frames (Table 1).

28

The degeneracy of the genetic code offers another elegant means of influencing the translation rate in cis.

Since an organism’s codon usage is usually adapted to the relative abundances of isoaccepting tRNAs, the

choice of synonymous codons can potentially control translation efficiency (e.g., Supek, 2016). Whether

or not this also applies to chloroplasts, is currently controversially discussed (e.g., Sugiura, 2014; Suzuki

and Morton, 2016; Gawronski et al., 2018). Although initial ribosome profiling studies could not confirm

a robust correlation between codon adaptation and translational speed/efficiency, recent methodological

and bioinformatic refinements uncovered the suspected relationship in several organisms (e.g., Dana and

Tuller, 2014; Nakahigashi et al., 2014).

In addition to primary sequence elements, features of mRNA 2D or 3D structure (or lack of structure) can

represent cis-elements that influence the translation process (e.g., Mauger et al., 2013). For example, the

lack of secondary structures at the start codon facilitates efficient SD-independent translation initiation in

bacteria and chloroplasts (Scharff et al., 2011; Scharff et al., 2017). Furthermore, translation initiation in

the chloroplast atpH and psbH mRNAs is stimulated by resolving secondary structures that mask

ribosome-binding site and start codon, respectively (Prikryl et al., 2011; Hammani et al., 2012). A recent

ribosome profiling study also revealed transcriptome-wide correlations between ribosome pausing and

secondary structures in chloroplast mRNAs (Gawronski et al., 2018).

Riboswitches are structured RNA elements that act as sensors for small molecules (metabolites).

Metabolite binding triggers a conformational switch that regulates transcription (typically by inducing

termination or antitermination) or translation (by exposing or sequestering the ribosome-binding site; e.g.,

Serganov and Nudler, 2013). Although riboswitches have not yet been identified in plastids, they have

been exploited as tools to control the translation of transgenes in chloroplast biotechnology (e.g.,

Verhounig et al., 2010). The use of improved algorithms for the computational prediction of riboswitches

(e.g., Philips et al., 2013) should help to clarify whether endogenous riboswitches exist in chloroplast

genomes.

Internal RNA structures within reading frames can adjust the translational speed by decelerating

elongating ribosomes in bacteria and chloroplasts (Mauger et al., 2013; Gawronski et al., 2018), a

mechanism that has been suggested to aid co-translational protein-complex maturation steps. Specific

translational pausing sites have been identified in the chloroplast psbA and atpA mRNAs (Kim et al.,

1991; Kim and Hollingsworth, 1992). However, although these pausing events appear to be influenced by

light or temperature (Kim et al., 1994a; Grennan and Ort, 2007), they could not be assigned to particular

sequences or structural elements of either the mRNA or the nascent polypeptide chain, nor could any

obvious molecular function directly be ascribed to them. In bacteria, structured RNA elements are also

29

involved in programmed frameshifting and translational coupling of neighboring reading 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 nascent peptide. For example, translation of

consecutive proline codons is complicated due to the physicochemical properties of the secondary amino

acid proline, which make it a weak peptidyl donor and acceptor. Reading of consecutive proline triplets,

therefore, causes translational pausing (Artieri and Fraser, 2014; and references therein). Specific

elongation factors such as EF-P in bacteria facilitate the translation of consecutive proline codons

(Doerfel et al., 2013; Ude et al., 2013). EF-P is conserved in chloroplasts suggesting a similar function

(e.g., Manuell et al., 2007). More than two consecutive proline codons are usually avoided in proteins of

all organisms. For example, the entire tobacco chloroplast genome does not encode a single stretch of

three prolines.

A recent study also provided evidence for other specific peptide motifs (e.g., domains comprised of small

polar residues) inhibiting peptidyl transfer during elongation or peptide release during termination in

bacteria (Woolstenhulme et al., 2013). Furthermore, specific translational pausing events that are induced

by interactions of the prokaryotic ribosome with the nascent peptide have emerged as programmed

switches that regulate translation in response to small metabolites or the availability of protein

translocation factors (Ito et al., 2010). In chloroplasts, ribosome pausing events 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 nascent peptide chain can influence

translation. These proteins act either in an mRNA-specific manner or as more general factors that control

translation of many transcripts.

All mRNA-specific chloroplast translation factors characterized to date act in trans by binding the

5’UTRs of one (or a few) mRNAs and promoting translation of the downstream reading frame (see

above; Table 1). More generally acting trans-factors control the translation of several reading frames.

These factors are either genuine or auxiliary constituents of the ribosome, or bind the nascent peptide.

Hence these factors act in cis or trans. Cis-regulatory functions have been suggested for several ribosomal

proteins or ribosome-associated proteins such as PSRP2-6, pY and Rps1 (e.g., Yamaguchi and

Subramanian, 2003; Manuell et al., 2007; Sharma et al., 2010). In bacteria, Rps1 is involved in translation

initiation of specific transcripts and a similar function has been suggested for chloroplast Rps1 (see

30

above). Chloroplasts also contain an orthologue of the bacterial ribosome-associated protein pY that binds

70S ribosomes or 30S subunits under cold-shock conditions, thus inactivating translation and stabilizing

monomeric 70S ribosomes (Vila-Sanjurjo et al., 2004; and references therein). Similarly, it has been

proposed that plastid pY could be involved in the deactivation of translation and the stabilization of

chloroplast ribosomes in the dark (Sharma et al., 2010; and references therein). Also, the expression of

chloroplast translation elongation factors is regulated by light, suggesting their contribution to

translational regulation. However, direct evidence for a regulatory function of chloroplast elongation

factors Rps1 or pY is lacking.

In both bacteria and eukaryotes, the binding of specific protein factors can create subpools of specialized

ribosomes that selectively translate a specific set of mRNAs (e.g., Sauert et al., 2015). More systematic

and quantitative proteomic studies of chloroplast ribosomes will be required to determine whether

specialized ribosomes exist also in plastids.

Translation is regulated at the initiation and elongation levels

In theory, translation can be regulated at any of its three stages: initiation, elongation, or termination (e.g.,

Hershey et al., 2012). However, by far most common is the regulation of initiation. All chloroplast factors

characterized to date that promote translation of specific reading frames act at the level of initiation

(Table 1). However, in some cases, elongation also appears to be regulated. For example, elongation of

psaA, psaB, psbA and rbcL translation is regulated by light, and the synthesis of PsbA was suggested

additionally to depend on the availability of co-factors and assembly partners (e.g., Berry et al., 1988;

Klein et al., 1988b; Mullet et al., 1990; Kim et al., 1994a; Mühlbauer and Eichacker, 1998; Kim and

Mullet, 2003).

Light- and redox-dependent regulation of translation

Light regulation of translation ensures coordination of the major energy-producing process

(photosynthesis) with the major energy-consuming process (protein synthesis) in the chloroplast. In

addition, components of the photosynthetic apparatus (especially PsbA) are damaged by light,

necessitating specific repair synthesis.

The synthesis of several chloroplast proteins was shown to be activated at the translational level in

response to increased light intensity. Upon illumination, translation of rbcL and psbA is activated at the

level of elongation, a mechanism that was proposed to be regulated by the light-dependent generation of a

proton gradient across the thylakoid membrane, the ATP status of the chloroplast and/or redox signal(s)

generated by photosynthetic electron transfer (e.g., Taniguchi et al., 1993; Mühlbauer and Eichacker,

31

1998; Zhang et al., 1999). In line with regulation of elongation, the transfer of amaranth plants into the

dark caused a decline in RbcL protein synthesis that was not accompanied by the loss of rbcL mRNA

association with ribosomes (in polysomes), suggesting that elongating ribosomes paused (Berry et al.,

1988). In addition, psaA and psaB were shown to be light-regulated at the elongation level during de-

etiolation (Klein et al., 1988b). A potential mechanism for the general light-dependent activation of

chloroplast translation elongation could rely on pY (see above) or elongation factor Tu, which was

identified as redox-regulated (Schröter et al., 2010).

During de-etiolation, psbA translation is also stimulated at the level of initiation (e.g., Klein et al., 1988b;

Kim and Mullet, 1994). Evidence currently available suggests that psbA translation for PSII biogenesis is

regulated at the initiation level, whereas PsbA synthesis for PSII repair is regulated at the (presumably

faster responding) elongation level, a model supported also by data from Chlamydomonas (reviewed in

Nickelsen et al., 2014). In seed plants, psbA translation initiation is controlled by specific cis-element(s)

in the 5’UTR (Staub and Maliga, 1994; Hirose and Sugiura, 1996; Eibl et al., 1999) and by the trans-

factors HCF173 and HCF244, two putative oxidoreductases that promote psbA translation cooperatively

(Schult et al., 2007; Link et al., 2012). All of these elements may also play a role in the light-dependent

translation initiation of psbA.

Variations in light quantity and quality can cause imbalances in the activities of PSI and PSII, changing

the redox state of the chloroplast and potentially generating harmful reactive oxygen species (Pfalz et al.,

2012). The chloroplast redox state controls transcriptional and post-transcriptional adaptation responses in

the chloroplast (e.g., Allen and Pfannschmidt, 2000; Rochaix, 2007). In Chlamydomonas, a redox-

dependent regulatory mechanism has recently been suggested for the light-activated translation of psbD

(Schwarz et al., 2012). In this model, the RNA-binding proteins Nac2 and RBP40 form a disulfide bridge-

connected complex in the light which stabilizes psbD mRNA and activates its translation (Schwarz et al.,

2012; and references therein). In the dark, the complex is reduced and disassembles, and, as a result, PsbD

synthesis is inactivated. The electrons are likely provided by the NADPH-dependent thioredoxin

reductase C, an enzyme shown to reduce disulfide bonds in the dark by utilizing NADPH generated in the

oxidative pentose phosphate pathway (Schwarz et al., 2012).

Plants perceive light independently of photosynthesis by photoreceptors that are located outside of

chloroplasts. However, light-induced regulation of translation was also observed in isolated chloroplasts,

arguing against a crucial role of photoreceptors (Sun and Zerges, 2015).

Altogether, our current knowledge about light-induced translational regulation in seed plant chloroplasts

is restricted to very few photosynthesis-related genes, with psbA translation being best studied. However,

32

even for psbA, the underlying mechanisms and the mode of action of the factors involved are not well

understood.

Developmental regulation of translation

Developmental regulation of plastid gene expression is crucial for the differentiation and interconversion

of plastid types. For example, conversion of proplastids to chloroplasts requires establishment of the

thylakoid system and must be tightly coordinated with cell division and the emergence of photosynthetic

tissues from meristems (Jarvis and Lopez-Juez, 2013). The mechanisms involved in chloroplast

differentiation were identified in classical experiments that analyzed the process of de-etiolation. During

de-etiolation, a rapid rise in translational activity was observed by pulse labeling experiments for several

plastid mRNAs encoding subunits of PSI (psaA/B), PSII (psbA/B/C/D) and Rubisco (rbcL; e.g., Klein and

Mullet, 1986, 1987; Kim and Mullet, 2003; Kleffmann et al., 2007). The initial steep increase in

translation of these mRNAs was followed by a slow decline as chloroplast differentiation continued in the

light. Polysome analyses demonstrated that psaA/B, rbcL and psbA transcripts are found in polysomes

throughout the chloroplast differentiation process, suggesting that the regulation of their translation

occurs, at least in part, at the elongation level (Klein et al., 1988b). This conclusion was further

substantiated by the observation that psaA, psbA and rbcL translation initiation remains unaltered during

light-dependent chloroplast differentiation (Kim et al., 1994b; Kim and Mullet, 2003).

There is also evidence for developmentally activated translation initiation of plastid mRNAs. Initiation of

psbA translation is induced during de-etiolation (Eichacker et al., 1992; Kim and Mullet, 1994), and this

induction is likely controlled by cis-elements in the psbA 5’UTR (Staub and Maliga, 1994; and references

therein). It was further proposed that the light-induced synthesis of chlorophyll controls both the

accumulation and the translation of chlorophyll-binding apoproteins of PSI and PSII during de-etiolation

(e.g., Eichacker et al., 1992). However, this conclusion was mainly based on the observed lack of

accumulation of chlorophyll-binding apoproteins in the absence of chlorophyll, as determined by protein

immunoblotting and pulse labeling experiments in vivo and in vitro (e.g., Klein et al., 1988a; Klein et al.,

1988b; Eichacker et al., 1990; Eichacker et al., 1992). Although pulse labeling can reveal protein

synthesis rates, it cannot unambiguously distinguish between the absence of synthesis and rapid

degradation of newly synthesized proteins (especially not for proteins with high turnover rates, such as

PsbA). A recent ribosome profiling analysis of plastid translation in a maize mutant with knocked-out

chlorophyll synthesis showed that the synthesis of plastid-encoded chlorophyll-binding apoproteins is

virtually unaltered in the absence of chlorophyll (Zoschke et al., 2017). Even the co-translational

thylakoid membrane engagement of nascent apoproteins was shown to be independent of chlorophyll

33

synthesis (Zoschke et al., 2017). However, apoproteins were shown to undergo rapid degradation in the

absence of chlorophyll (e.g., Mullet et al., 1990; Kim et al., 1994b; Eichacker et al., 1996).

The above described de-etiolation of seedlings represents a dramatic change in plant development and

reflects the situation during germination, when light-dependent and developmental programs operate

simultaneously. Another useful experimental system to study translational regulation during chloroplast

differentiation is provided by the longitudinal developmental gradient of young leaves in grasses such as

maize, rice or barley. At the leaf base, meristematic, proplastid-containing tissue is found, followed by a

developmental gradient towards the leaf tip which includes etioplasts, differentiating chloroplasts

(etiochloroplasts) and fully differentiated chloroplasts (e.g., Leech et al., 1973). This natural

developmental gradient has been exploited to systematically study the dynamic changes in gene

expression at the level of transcript and protein accumulation (e.g., 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 developmental gradient of maize leaves (Chotewutmontri and Barkan, 2016). This

pioneering study comprehensively determined the relative contributions of changing transcript levels and

translational activity to the protein synthesis output (Chotewutmontri and Barkan, 2016). The data

revealed that the synthesis rates of most plastid-encoded proteins increases early in development and

drops later, once the photosynthetic machinery is set up. Strikingly, PsbA and PetD, two proteins that do

not follow this general rule and, instead, display increasing synthesis levels that peak in the latest

examined developmental stage, represent the subunits with the highest turnover rates in their complexes

(Li et al., 2017a), likely explaining their continued production.

In general, the regulation of plastid gene expression during leaf development in maize is achieved by

changes in transcript levels that are often tuned by changes in translation efficiency (Chotewutmontri and

Barkan, 2016). Remarkably, the developmental dynamics in protein synthesis that was discovered

revealed two major regulons that are defined by changes in RNA accumulation and translation efficiency:

early in development, proteins needed for chloroplast gene expression have the highest synthesis output

(e.g., RNA polymerase subunits and 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 NDH complexes). To achieve this developmental pattern in plastid protein synthesis, translational

regulation is especially important for polycistronic transcription units that encode proteins of both

regulons. Indeed, in these transcripts (e.g., the psaA/psaB/rps14 transcription unit encoding two PSI

34

subunits and a ribosomal protein), strong differential translational regulation was observed

(Chotewutmontri and Barkan, 2016).

Unfortunately, none of the factors involved in the complex developmental regulation of chloroplast

translation has been identified so far. It would be particularly exciting to reveal the mechanisms

underlying the translational switch between these two major regulons during leaf development. A possible

explanation could lie in the known developmental switch in the usage of the two distinct types of RNA

polymerases during chloroplast differentiation (Börner et al., 2015). In theory, the two polymerase

activities could produce transcripts coding for identical proteins but differing in their translational

competence. Alternatively, RNA-binding proteins that facilitate translation (see above and Table 1) could

mediate the developmental regulation of translation.

Translational autoregulation and feedback regulation

Control of protein synthesis by translational autoregulation or feedback regulation represents an elegant

means of robustly fine-tuning protein synthesis levels to changing cellular requirements. In the

chloroplast of Chlamydomonas, the translation of specific subunits of the four photosynthetic complexes

(PSII, Cyt b6f, PSI, ATP synthase) was shown to be feedback-regulated by the assembly state of the

respective complex, a process termed “control by epistasy of synthesis” (CES; Choquet and Wollman,

2009). CES regulation ensures the stoichiometric production of chloroplast subunits that reside in

oligomeric complexes according to the requirements of their sequential assembly (Choquet and Wollman,

2009). The best-studied case mechanistically is the CES involved in petA gene expression. PetA is a

subunit of the cytochrome b6f complex, whose synthesis is strongly reduced in the absence of its assembly

partners PetB (cytochrome b6) and PetD (subunit IV; Kuras and Wollman, 1994). The protein factors

MCA1 and TCA1 were shown to be cooperatively associated with the 5’UTR of 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. discovered a regulation mechanism in which a C-terminal domain of

PetA that is exposed only in the unassembled PetA protein binds to MCA1 and causes its proteolytic

degradation (Boulouis et al., 2011). This leads, in turn, to down-regulation of petA gene expression at

both the level of transcript accumulation and the translational level. Conversely, if the assembly partners

PetB and PetD are available, PetA is assembled into the Cyt b6f complex, the C-terminal domain is

inaccessible, and hence, MCA1 remains stable and facilitates petA expression in complex with TCA1. In

this way, the CES mechanism ensures the adequate synthesis of PetA according to the availability of its

assembly partners in the Cyt b6f complex.

35

In chloroplasts of seed plants, evidence for a similar feedback regulation mechanism has been obtained in

only a single case. The synthesis of the plastid-encoded large subunit of Rubisco (RbcL) in tobacco is

adjusted to that of its nucleus-encoded assembly partner, the small subunit RBCS, by a mechanism that is

similar to the CES-regulated rbcL expression in Chlamydomonas (Rodermel et al., 1988; Khrebtukova

and Spreitzer, 1996; Rodermel et al., 1996). Furthermore, evidence was provided that unassembled RbcL

represses its own translation, possibly through direct RNA binding (Yosef et al., 2004; Wostrikoff and

Stern, 2007). In a systematic genetic approach, the essential plastid-encoded Cyt b6f subunits PetA, PetB

and PetD were knocked out to disrupt complex assembly and test for potentially conserved CES

regulatory mechanisms in tobacco (Monde et al., 2000). In the mutants, only a mild effect on translation

of the polycistronic petA transcript was detected by polysome analyses, suggesting that the CES

mechanism is not conserved in seed plants (Monde et al., 2000). This may not be entirely surprising,

given that the regulatory trans-factors involved, MCA1 and TCA1, are not found in seed plants.

Moreover, a ribosome profiling analysis of two maize mutants with defective AtpB synthesis did not

show a substantial effect on atpA translation (Zoschke et al., 2013a). This suggests that also the AtpB-

dependent translation of atpA, the only trans-activating CES mechanism observed in Chlamydomonas

(Drapier et al., 2007), is not conserved in seed plants. However, in Arabidopsis, it was observed that

mutation of the chloroplast RNA-binding protein HCF107 that facilitates psbH translation also causes a

reduction in PsbB (CP47) synthesis (Felder et al., 2001). Remarkably, when psbH expression was rescued

by introducing a psbH gene copy into the nuclear genome of an hcf107 mutant, accumulation of the PsbB

protein was rescued as well, despite the absence of functional HCF107 protein (Levey et al., 2014). A

possible explanation for this observation is that PsbH accumulation is needed for PsbB synthesis, pointing

to a potential CES mechanism in PSII biogenesis (Levey et al., 2014). Interestingly, recent transcriptome-

wide analyses of translation in maize and Arabidopsis chloroplasts revealed that most proteins are

synthesized in the amounts that correspond to (i) their steady state stoichiometry as subunits of protein

complexes, and (ii) the abundancy of the respective protein complex (Chotewutmontri and Barkan, 2016;

Lukoszek et al., 2016). This suggests that precise regulation of gene expression is more important than

post-translational proteolytic adjustments of subunit stoichiometry. However, several alternative

regulatory mechanisms can explain this behavior, and it remains to be examined whether or not CES is

involved.

In many organisms, auto-regulated proteins have been observed to comprise an intrinsic RNA-binding

activity. Prominent examples include several ribosomal proteins, the initiation factor IF3 and the β

subunit of the bacterial RNA polymerase (Choquet and Wollman, 2009). The RNA-binding motifs allow

these proteins to bind the 5’UTR of their own mRNA and inhibit translation initiation whenever their

36

assembly partners are not available, thus generating a negative regulatory feedback loop. The chloroplast

genome codes for numerous RNA-binding proteins (e.g., orthologues of bacterial RNA polymerase

subunits and ribosomal proteins). Hence, it is tempting to speculate that some of them may also auto-

regulate their own synthesis. A plastid-encoded protein that has been shown to associate with its own

RNA in vivo is the putative splicing factor MatK (Zoschke et al., 2010). The maturase MatK is encoded in

the intron of the trnK gene. MatK binds to the trnK transcript, most likely assisting in splicing (Zoschke

et al., 2010; and references therein). Notably, a MatK-related bacterial maturase is translationally auto-

regulated (Singh et al., 2002), and an auto-regulatory mechanism was also proposed for the expression of

plastid MatK (Hertel et al., 2013).

In summary, whether autoregulation and/or feedback regulation of translation are common in seed plant

chloroplasts, or whether these regulatory mechanisms are specific to Chlamydomonas, remains to be

investigated. Pronounced differences between microalgae and seed plants may not be surprising, if one

considers the dissimilar evolutionary forces that act on regulatory circuits in a unicellular organism with

one chloroplast per cell compared to multicellular organisms with numerous chloroplasts per cell.

Translational regulation in response to other internal and external triggers

Temperature is a major abiotic signal influencing translational regulation. In bacteria, cold-stress

conditions induce a general block of translation that is mediated by the pY protein. However, the

synthesis of a small subset of proteins, such as RNA helicases, IF1-3 and trigger factor, is induced under

these conditions (Barria et al., 2013). In chloroplasts, changes in temperature alter the speed of all

enzymatic reactions (including the Calvin-Benson-Bassham cycle) but, unless extreme, they have

virtually no impact on the light reactions of photosynthesis. Consequently, temperature changes can cause

dramatic imbalances in the redox homoeostasis of photosynthesis (e.g., Crosatti et al., 2013). It has long

been known that the chloroplast translation capacity is crucial to plant adaptation to chilling stress. This

became evident by the discovery of numerous mutants with impaired chloroplast translation that exhibit

cold-sensitive phenotypes (e.g., Barkan, 1993; Rogalski et al., 2008; Liu et al., 2010).

A recent ribosome profiling study comprehensively examined chloroplast translational regulation upon

temperature stress (Lukoszek et al., 2016). Ignatova and co-workers observed in Arabidopsis that elevated

temperature causes dramatic changes in the protein synthesis output of subunits of photosynthetic

complexes (Lukoszek et al., 2016). This regulation was driven by both changes in transcript levels and

translational regulation. Interestingly, in several cases, unbalanced changes in protein synthesis rates were

observed for different subunits within a complex, in that the altered synthesis rates did not reflect the

subunit stoichiometry. This finding may suggest an increased relevance of control at the level of protein

37

turnover of at least some subunits during heat stress (Lukoszek et al., 2016). In sum, although it is well

established that plastid translation is critical for acclimation to changing temperature conditions, the

molecular mechanisms and the factors involved are currently unknown.

A special case of internal specialization of gene expression is the chloroplast dimorphism in C4 plants. In

C4 species such as maize, light reactions and carbon reactions of photosynthesis are partitioned between

mesophyll cells and bundle sheath cells. The division of labor between the two cell types is reflected by

specialized chloroplast types, referred to as chloroplast dimorphism. At the molecular level, bundle sheath

chloroplasts have a high content of Rubisco and NDH complex, whereas mesophyll chloroplasts are

enriched in PSII (e.g., Majeran et al., 2010). This specialization is achieved by differential gene

expression (e.g., Sharpe et al., 2011; and references therein). Recently, transcript accumulation and

translation have been comprehensively analyzed in bundle sheath and mesophyll chloroplasts by

ribosome profiling (Chotewutmontri and Barkan, 2016). This study confirmed that most of the observed

differences in protein accumulation can be explained by differential protein synthesis which in turn is

predominantly achieved by differential expression at the RNA level. However, the synthesis of some

proteins such as the PSII core subunits PsbA/B/C/D is additionally tuned by translational regulation. The

underlying regulatory mechanisms are unknown. The study showed that the synthesis output of Rubisco,

PSII, PSI and NDH complex subunits reflects well their protein accumulation in the different chloroplast

types. This is not the case for the subunits of the ATP synthase and the Cyt b6f complex, suggesting more

extensive post-translational adjustments in these complexes.

Internal metabolic signals can also control translation (e.g. via riboswitches in bacteria; Serganov and

Nudler, 2013). Several recent discoveries in Chlamydomonas have established interesting links between

chloroplast metabolism and gene expression (Bohne and Nickelsen, 2017). DLA2, a subunit of the

chloroplast pyruvate dehydrogenase complex, was shown to have RNA-binding activity which enables its

moonlighting function in translational regulation of the psbA mRNA (Bohne et al., 2013). Considering its

functions in metabolism and translational regulation, DLA2 is anticipated to coordinate fatty acid and

protein syntheses during thylakoid biogenesis (Bohne et al., 2013). The phylogenetic conservation of the

DLA2 amino acid sequence and RNA-binding properties suggests that it performs a similar dual function

in seed plants (Bohne et al., 2013). A moonlighting function was also suggested for RbcL, which was

shown to bind RNA (Yosef et al., 2004; Cohen et al., 2006), and recently has been proposed to be

involved in the redox stress-induced localization of oxidized chloroplast RNAs in Chlamydomonas,

independent of its function in the Rubisco complex (Zhan et al., 2015). Moreover, recent in vitro data

suggest that PsbD protein synthesis in Chlamydomonas is metabolically controlled (Schwarz et al., 2012;

38

see above). Whether similar regulatory connections between metabolism and chloroplast translation exist

in seed plants remains to be investigated.

In bacteria, stress-induced regulation of gene expression is triggered by the so-called stringent response.

Upon nutrient deprivation and other stresses, bacteria produce the effector molecule (p)ppGpp (guanosine

tetraphosphate/pentaphosphate, also known as alarmone) that coordinates numerous cellular responses,

including broad changes in transcription and translation (Gaca et al., 2015). Chloroplasts also harbor the

enzymes needed for ppGpp synthesis (and degradation). Plastids produce alarmone not only in response

to stress, but also to orchestrate chloroplast and nuclear gene expression (reviewed, e.g., in Field, 2017).

ppGpp has been shown to influence transcription, translation, and many other metabolic and

physiological processes in plants (e.g., Field, 2017). In an in vitro chloroplast translation system from pea,

ppGpp inhibited protein synthesis, suggesting conservation of the bacterial mode of ppGpp-mediated

translational repression by interaction with IF2 and/or EF-G (Nomura et al., 2012). Furthermore,

overaccumulation of ppGpp in vivo caused a dramatic reduction in the accumulation of many plastid-

encoded proteins, mRNAs, tRNA and rRNAs, suggesting an overall reduced translation capacity (e.g.,

Sugliani et al., 2016). Whether the translational activity of specific chloroplast mRNAs is regulated by

ppGpp and how this contributes to ppGpp-induced changes in chloroplast gene expression still needs to

be determined.

In summary, considerable progress has been made in describing changes in translation in response to light

and developmental signals, but comparably little is known about the mechanisms and factors that adjust

plastid protein synthesis to other internal and external cues that affect protein homeostasis.

OUTLOOK

Over the last decades, extensive research on the structure and function of the translational apparatus of

bacteria and chloroplasts has revealed many similarities but also substantial differences. Unfortunately,

our knowledge about genuine regulatory processes, the underlying molecular mechanisms and the factors

involved is still limited. For example, the influence of internal and external triggers such as light,

temperature, nutrient availability, osmotic status, redox state, phytohormones, alarmone, and diurnal and

circadian rhythms on chloroplast gene expression has so far not been comprehensively examined at the

translational level. Likewise, investigation of the interconnection of chloroplast translation with protein

synthesis in the cytosol and the mitochondria is likely to provide fresh insights into intracellular signaling

and crosstalk in response to changing environmental conditions and developmental programs. Last but

not least, the detailed molecular function of RNA cis-elements and proteinaceous trans-factors in the

regulation of chloroplast translation is mostly unknown. The exciting advent of chloroplast ribosome

39

profiling and other in vivo ribonomic methods, such as the comprehensive examination of RNA

secondary structures or the mapping of binding sites for RNA-binding proteins, can be anticipated to

address many of these open questions in the biology of plant organelles in the future.

ACKNOWLEDGEMENTS

We apologize to the authors of numerous papers we were unable to cite due to space constraints. We

thank Alice Barkan (University of Oregon) for helpful suggestions on the manuscript. We acknowledge

the generous sharing of unpublished data by Alice Barkan, Christian Schmitz-Linneweber (Humboldt

University of Berlin), Karin Meierhoff (Heinrich Heine University Düsseldorf), and Mark Aurel Schöttler

(Max Planck Institute of Molecular Plant Physiology). Research on plastid translation in the authors’

laboratories is funded by the Max Planck Society, the Deutsche Forschungsgemeinschaft (DFG; grant ZO

302/4-1 to R.Z., SFB-TRR 175 to R.Z. and R.B.), the German Academic Exchange Service (DAAD;

project ID 57387429 to R.Z.), and the European Research Council (ERC) under the European Union’s

Horizon 2020 research and innovation programme (ERC-ADG-2014; grant agreement No 669982 to

R.B.).

AUTHOR CONTRIBUTIONS

RZ and RB wrote the paper.

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DOI 10.1105/tpc.18.00016; originally published online April 2, 2018;Plant CellReimo Zoschke and Ralph Bock

RegulationChloroplast Translation: Structural and Functional Organization, Operational Control and

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