Expression of Plastid Genes: Organelle-Specific Elaborations on a Prokaryotic Scaffold

Chloroplasts and their nonphotosynthetic relatives in the plastid organelle family evolved from a cyanobacterial endosymbiont (for review, see Timmis et al., 2004). The subsequent coevolution of the chloroplast and nuclear genomes produced an organelle that is eubacterial at its core but with extensive chloroplast-specific embellishments. Of the thousands of genes in the cyanobacterial ancestor, only approximately 100 are retained in chloroplast genomes. These genes fall into three categories: those encoding (1) components of the chloroplast gene expression machinery (RNA polymerase, ribosomal proteins, tRNAs, and rRNAs), (2) subunits of photosynthetic enzymes (Rubisco, PSII, the cytochrome b 6 f complex, PSI, the ATP synthase, and the NADH dehydrogenase), and (3) proteins involved in other metabolic processes (e.g. ClpP, AccD, Ycf1, and Ycf2). The chloroplast proteome has a complexity of several thousand proteins and is dominated by nuclear gene products that are synthesized in the cytosol and imported into the organelle. Many of these are encoded by genes of cyanobacterial ancestry that were transferred to the nucleus and that have retained their ancestral functions. As a result, the chloroplast gene expression and photosynthesis machineries consist of proteins that are derived from two physically separate genetic systems. Detailed knowledge of chloroplast gene expression and the nucleus-encoded proteins that influence it are prerequisites for understanding nuclear-organellar cross talk and chloroplast evolution, and will aid in optimizing transgene expression in the plastid compartment. The use of genetic and biochemical approaches, together with the ability to manipulate the chloroplast genome in several species, have brought most aspects of chloroplast gene expression out of the “black box” and into the realm of concrete, mechanistic hypotheses. The intent of this contribution is to highlight new perspectives that have resulted from recent observations and instances in which current data warrant the revision of previous paradigms. For more comprehensive information, I refer the reader to recent reviews of chloroplast RNA metabolism (Stern et al., 2010), transcription (Liere and Börner, 2007; Lerbs-Mache, 2010), and translation (Peled-Zehavi and Danon, 2007). Mechanisms of chloroplast gene expression have been studied primarily in land plants and in the green alga Chlamydomonas reinhardtii. Here, I emphasize findings with land plants, as detailed reviews of chloroplast gene expression in Chlamydomonas have been published in a recent volume (Stern and Harris, 2009). The bacterial ancestry of chloroplasts is readily apparent in the organization of chloroplast genomes and in the machineries for chloroplast transcription, translation, and RNA turnover. Polycistronic transcription units that resemble bacterial operons predominate in land plant chloroplasts (Bock, 2007). Chloroplast ribosomes are similar in protein content and antibiotic sensitivities to bacterial ribosomes (Peled-Zehavi and Danon, 2007). A bacterial-type RNA polymerase contributes to chloroplast transcription (Liere and Börner, 2007; Lerbs-Mache, 2010), and chloroplast RNA turnover employs ribonucleases that are derived from those in bacteria (Stern et al., 2010). Superimposed on this bacterial infrastructure are features that were acquired only after the chloroplast became incorporated into a eukaryotic cell. Examples include a plethora of introns, a phage-type RNA polymerase, the modification of mRNA sequences by RNA editing, and the processing of polycistronic primary transcripts to generate complex transcript populations. The analysis of nonphotosynthetic mutants in maize (Zea mays), Arabidopsis (Arabidopsis thaliana), and Chlamydomonas has revealed numerous nucleus-encoded RNA-binding proteins that participate in the expression of chloroplast genes. Two major themes emerged from this large body of work: (1) the repertoire of nucleus-encoded chloroplast RNA-binding proteins is remarkably complex given the small coding capacity of the chloroplast genome; and (2) most such proteins belong to protein families that function almost exclusively in organellar gene expression. Both of these points are exemplified by the pentatricopeptide repeat (PPR) family, whose members are defined by degenerate 35-amino acid helical repeats (for review, see Schmitz-Linneweber and Small, 2008). PPR proteins are not represented in bacteria, and PPR proteins in eukaryotes function almost exclusively in organellar gene expression. The PPR family consists of over 400 members in angiosperms, approximately half of which are predicted to localize to chloroplasts and half to mitochondria. Several other predicted helical repeat protein classes have also been implicated in chloroplast RNA metabolism (Stern et al., 2010); it is likely that these share mechanistic similarities with PPR proteins, so these and PPR proteins are referred to together below as “PPR-like” proteins. Current data support the view that PPR tracts are sequence-specific RNA-binding motifs that bind single-stranded RNA along a surface formed by stacked, helical repeating units (Schmitz-Linneweber and Small, 2008; Williams-Carrier et al., 2008; Prikryl et al., 2011). Genetic data have implicated PPR proteins in many aspects of organellar RNA metabolism, and it is often suggested that they mediate their multifarious effects by recruiting different effector proteins to specific RNA sites. Indeed, there is good evidence that an appended domain found in a subset of PPR proteins functions in this manner during the process of plant organellar RNA editing (see below). However, recent data also support an alternative view: that many of the functions attributed to PPR proteins may result directly from the unusual nature of the PPR-RNA interface, which sequesters an extended RNA segment such that it cannot interact with other proteins or et al., of a segment of single-stranded RNA by a PPR may for many functions attributed to PPR proteins. is exemplified by which specific RNA and translation by the RNA segment et al., Prikryl et al., 2011). influence RNA and translation in other of RNA The RNA derived from polycistronic transcripts and are at the this and the of by from The and transcripts from different of The is in a with a of the the for translation et al., Prikryl et al., 2011). classes of organellar RNA-binding proteins a similar on a The et al., et al., and et al., et al., are represented in small gene members of these families are predicted to localize to chloroplasts or and that have been studied bind RNA and in plant and proteins are to in RNA metabolism a process which these protein families were in with the processes they The of proteins in to and The is in plants, members of the plastid organelle family different in different For the in and the in chloroplast gene products involved in photosynthesis but the expression of chloroplast genes involved in other aspects of metabolism and 2008; et al., 2009). The effects of and are on to influence the of chloroplast gene A chloroplasts and bacteria the at which gene expression is transcription is the most of in bacteria, in the of features et al., in the of nuclear genes for in chloroplast gene expression has many for Examples of gene and are in the of of gene expression The of gene expression may in land plant chloroplasts in bacteria, the use of is likely to have a on the of subunits photosynthetic complex is in by of subunits 2007). The of protein has been most in a of as by of the of subunits of photosynthetic complex that are by specific subunits and 2009). The to which in land plants is Several proteins that in Chlamydomonas have been not to so in land plants and et al., the other a the of the and subunits of in et al., and 2007). alternative for the expression of chloroplast genes nucleus-encoded proteins that the expression of of chloroplast genes (e.g. RNA-binding proteins or as many such proteins have been (Schmitz-Linneweber et al., et al., et al., Lerbs-Mache, 2010). in chloroplast transcription in to and (for review, see and Börner, 2007). for chloroplast transcription during the of et al., and and to the transcription of a subset of chloroplast genes in chloroplasts et al., 2008). The is by and and the of and transcription in to different of to the of to et al., in the chloroplast transcription machinery has a for the the of chloroplast transcription (for review, see and Börner, 2007). to a polymerase chloroplasts in and in the or two nucleus-encoded RNA are to those in and in mitochondria. data suggested a of in which genes (e.g. genes for tRNAs, ribosomal proteins, ClpP, and and genes involved in photosynthesis et al., The organization of chloroplast genes into two such to a in which the of in chloroplast the of chloroplast ribosomes and of which photosynthetic genes at in its has not the of is that most chloroplast genes by or from (for review, see and Börner, 2007). the of the of transcripts those involved in gene and transcription predominate in chloroplast is for the of chloroplasts et al., et al., that chloroplast genes or the other polymerase for expression. are by sequences that resemble by in Indeed, are by nucleus-encoded proteins that are to et al., Chloroplast are encoded by a small gene family in land The of the to chloroplast gene at the there is evidence that different members of the are and different chloroplast the of this of to as in Arabidopsis the different for functions of chloroplast in Arabidopsis is by A is for the use of the and transcript are in to the of transcription may to for of the of by and in Arabidopsis are for in et al., and in et al., in to the of the and of the the of to transcription et al., 2010). Several different protein have been to to the of et al., et al., but the that to in chloroplast transcription are other proteins have been in chloroplasts that bind or that are with the chloroplast (for review, see and Börner, 2007; Lerbs-Mache, 2010). in several of the genes chloroplast gene expression et al., However, more detailed will to which of these proteins are transcription and which function in other processes or in processes that are to The of transcription in chloroplasts has Many RNA are not products of transcription but result from the processing of transcripts (for review, see et al., 2010). However, and RNA in at bacterial which consist of RNA by several and et al., et al., 2007). it likely that and to sequences with these features in As only a of have been for transcription more comprehensive may transcription in A of approximately I and approximately acquired during the of land plants and is by most land plants et al., chloroplast were acquired of those in land plants, so the chloroplast content in Chlamydomonas is chloroplast were derived from I or However, has not been for in land plant and it is that their is protein I and have and Both are found in bacteria, in small they with the aid of proteins and Chloroplast introns, are degenerate and with proteins of A to the that is to the of nuclear in the in two of which are found in Chlamydomonas and in land proteins in land plants and three in Chlamydomonas have been that are to chloroplast and that with in et al., et al., 2010). but of these proteins are encoded by the nuclear the the encoded in the in land is that many such proteins by into a biochemical data support this view for et al., which is for the of the chloroplast in to from the of chloroplast are of I and in bacteria and revealed or two proteins and an that is and chloroplast are more protein For different proteins have been to and to for the the maize is of the other in land plant for expression of the chloroplast gene The gene genes and approximately transcripts processing and the of in the and genes and The a subset of the for the and are with their RNA-binding in (for review, see et al., et al., 2011). The of a protein the of RNA in by genetic data and in the et al., et al., et al., but these have not been The and are to at to to RNA A of this on the effects of on two other transcription units et al., 2009). A of the of most nuclear different chloroplast to the of different the proteins that participate in chloroplast are to those that participate in nuclear The and were to RNA-binding the analysis of chloroplast RNA and members of these protein families participate in in the of in plant may have been the for the of these protein the other several chloroplast a to bacterial proteins. Examples include maize which is to bacterial and Chlamydomonas which is to bacterial to these proteins. The of in to the of gene expression in As for other in chloroplast gene it is not to the of gene However, of several maize chloroplast a et al., that these proteins and the they are for gene expression. The to which these and other a to of gene expression in land plant chloroplasts is the modification of mRNA sequences by RNA editing (for review, see and Small, 2010). mRNA editing has not been in bacteria or in However, this is with plant and RNA editing in plant and chloroplasts is to have a angiosperms, chloroplast mRNA editing is to the of specific to and at approximately is a as more half of the and Chloroplast genomes that a a at the editing in chloroplasts are for gene and the coding such that a acid is to a and The of the editing is to (for review, see and Small, 2010). the editing machinery has been to consist of two of (1) that a for editing, and (2) a of the with the that the PPR protein is to a in the mRNA et al., with to a RNA the et al., The of the with in and in of for a of approximately is to most with the found the of this to a of PPR proteins that include appended motifs at their and et al., 2004). the of genetic many more in chloroplasts and also in plant mitochondria. of these are or proteins. These the and motifs in the editing have the function to in the PPR and have the domain to for editing et al., 2009). The from several editing of et al., 2009). has been that the the editing has et al., 2007; et al., et al., 2009). These a in which the subset of PPR proteins or are the primary for RNA editing in the PPR approximately of the and the editing in an as such proteins editing at a but many and are to a degenerate et al., 2008; et al., 2009). the approximately PPR proteins of this in land plant chloroplasts are more to for editing of the chloroplast family of RNA-binding proteins have also been to several editing in and and in et al., 2009). bind RNA so the by which they influence RNA editing is likely to from that of PPR editing the of RNA editing in chloroplast gene The of a comprehensive analysis and suggested that the editing in maize are to a in gene expression. the that produced plant organellar RNA et al., that the of new for RNA editing the of in the chloroplast The of chloroplast and in to and during and et al., et al., The below support the view that the influence of chloroplast RNA-binding proteins, proteins, is an RNA turnover machinery from bacteria, to RNA in Chloroplast for many and et al., and are more are in the chloroplast ribonucleases that are implicated most in RNA are to those in bacteria (Stern et al., 2010). The most studied for RNA in chloroplasts the (for review, see and 2009). As in bacteria, chloroplast is by of its RNA and it is by RNA that chloroplasts also have a for that has apparent in a PPR protein in and in and et al., Prikryl et al., 2011). is genetic evidence for a in chloroplasts whose by a RNA or protein (for review, see et al., 2010). The has not been but it is likely to in chloroplast whose in bacteria has and 2007). The in RNA in as in bacteria, is which products that are to by features at the RNA (for review, see et al., 2010). However, the of the a A chloroplast protein in but evidence that RNA in has not and this function in bacteria, at sequences 2007). but and plant chloroplasts Arabidopsis mutants chloroplast not a in mRNA et al., 2010), that cannot the that chloroplast mRNA the for chloroplast in et al., and the that bacterial and have similar it may that and in chloroplasts to mRNA The that the of the for chloroplast RNA are not These are likely to include the and of the its of and the of other proteins that or sites. it that of the ribonucleases and proteins the of chloroplast RNA and that the of these will in the A of gene expression in land plant chloroplasts is the complexity of the RNA from most genes. is exemplified by the gene and mRNA from the processing of polycistronic transcripts coding in the and by the of The and of these in chloroplasts have been findings have to these and have that have these were more been that mRNA processing in chloroplasts from that generate and on data that and in The that this view may with the of the RNA the and in maize et al., the of the RNA from the approximately of the of the RNA from the that this of not result from a data that this and from the is and evidence for a of processing that not a of the maize protein et al., in the and were the by approximately as been for the and are of only in which and an have been not to in this and these to from processing et al., is evidence that the in the and in the manner et al., Prikryl et al., 2011). to these two at that have similar and the of from the or in the of whose or is defined by the or of by that is to and in et al., 2011). in with a is to generate a that to the found in that proteins are is likely to the the as genetic data three other PPR proteins and to the of transcripts with or in three other et al., et al., and more as from a to the involved in RNA alternative for processing has also been a PPR protein an with RNA et al., 2009). The and are not current data the to the for RNA processing a for to at transcript a the that mRNA et al., is by the of Arabidopsis mutants chloroplast which have in the processing of several polycistronic transcripts et al., 2010). However, many processing were not in these that other as Genetic data proteins not only in RNA processing but also in the of specific in the chloroplasts of land plants and Chlamydomonas (Stern et al., 2010). processing and processing to but it that they the the of at specific by a processing from from a the transcription processing from from the to that the machineries for and mRNA processing in chloroplasts by the of evolved proteins a for RNA turnover that from Chloroplasts proteins that are to nuclear and that have also been implicated in mRNA in chloroplast and et al., and a for a in the of several chloroplast in an in analysis et al., 2009). genetic data to proteins as the primary protein involved in chloroplast RNA (Stern et al., 2010), that the by a PPR et al., a to Several observations have translation as an in chloroplast gene expression (Peled-Zehavi and Danon, 2007). (1) The translation of chloroplast is by (2) has been to a in the expression of many chloroplast genes in Chlamydomonas et al., and by the of the plastid gene products and 2009). (3) Genetic have numerous nucleus-encoded proteins that are for the translation of specific chloroplast a large of the genome in chloroplast gene expression at the to the are that chloroplast translation may that are from those in are not in many chloroplast to in chloroplasts are of chloroplast translation, in bacteria is by Chloroplast ribosomes are formed from and proteins that a to those in bacteria, and they function in with bacterial-type and (Peled-Zehavi and Danon, 2007). this as the below from the that translation and its in chloroplasts and bacteria similar of chloroplast translation that at with this are in this Chloroplast ribosomes include several ribosomal and et al., which have been as for of chloroplast However, a of the chloroplast suggested that several for the of specific et al., 2007). and a in Chlamydomonas et al., highlight of ribosomal proteins as for in chloroplast-specific For chloroplast-specific on ribosomal protein in et al., and ribosomal protein in Chlamydomonas et al., are to the mRNA during translation has to a ribosomal protein plastid specific et al., 2010). and its bacterial translation by sites. the gene encoding the cyanobacterial is after a and cyanobacterial the to the that or may in the and that this the of plastid translation after a from to The most of in bacteria the the of the of the with sequences of the The bacterial has the and is approximately from the of chloroplast genes in land plants are by predicted at the and several of these have been to translation (Peled-Zehavi and Danon, 2007). However, the of chloroplast genes to alternative for Two are often that for and that ribosomal proteins a in is to is bacterial translation to to to observations in is that are from in bacterial genes et al., for only approximately of genes in have apparent from bacteria is that ribosomal subunits bind to single-stranded RNA and that a approximately on the is for translation and et al., 2009). Current data support the that translation in bacteria an RNA from which the to the and similar for observations in A recent that in chloroplasts and bacteria, translation a are are those a that is in the of a and the chloroplast whose are to function by a for the (see below). The that contributes to in chloroplasts in two which use of in and and 2010). that observations have been in and ribosomes and polycistronic are not to these observations in chloroplasts may not chloroplast-specific but may of in RNA from in and of nonphotosynthetic mutants in plants and Chlamydomonas have revealed numerous nucleus-encoded proteins that influence chloroplast translation (for review, see and Danon, 2007). These proteins are and they on or several chloroplast Many of are proteins they the of the many proteins that translation also the Two in Chlamydomonas on the et al., and et al., genetic data evidence that a protein the mRNA and the RNA These proteins interact with a protein that the RNA segment and the translation of the proteins also the translation of specific in the chloroplasts of land plants with specific et al., et al., Schmitz-Linneweber et al., et al., 2009). Two for the of ribosomes or translation or the of an RNA of that is to and the mechanistic data for chloroplast support the revealed the likely by which translation of the et al., to the it the RNA such that the is from a Genetic data support an for two in Chlamydomonas chloroplasts et al., et al., 2007). Detailed of will to other of are also at The of translation has The gene the protein of PSII, which is to that new for the of translation the (for review, see and Danon, 2007). Genetic the Arabidopsis protein et al., and the Chlamydomonas protein et al., as for and are they are not proteins, and their of are A biochemical in Chlamydomonas to a for the of translation a of RNA-binding proteins whose is by (for review, see and Danon, 2007). However, there is evidence for a in land plants, and recent are at with several aspects of that (for review, see and 2009). The of is often as the for the mRNA processing in is an but the body of evidence to not evidence in of this many chloroplast genes are represented by polycistronic for several genes that are represented by processing has been not to for to the in for maize and to and were to in and transcripts or sequences were of the or sequences A similar for on from a chloroplast in translation et al., 2007). in polycistronic transcription units in chloroplasts processing and is also that mRNA processing A from an analysis of the transcription and in the chloroplast in translation the and were more as as to to an a in the coding and its in the A that has been to support the that processing translation from the genetic analysis of the proteins and in and the of specific with et al., et al., et al., However, recent with warrant of an alternative for those The those of and in that specific transcripts are and the translation is also Both of those effects result from the of with the RNA and the translation to the et al., Prikryl et al., These findings the that the of in and mutants is the of it may that it is the of and proteins on their that translation, and that the translation and of in the mutants are effects of the of these proteins. the current evidence as a for the of chloroplast mRNA processing likely in it that a acquired mRNA processing over for translation to on RNA an from sequences by it will from that have with sequences found on the RNA it is that chloroplast-specific are at during translation and it is to of the view that chloroplasts and bacterial translation in the of RNA-binding proteins that are to RNA and processing (e.g. in the translation process transcription and translation are to in that ribosomes translation after the RNA The of chloroplast and the that they after from RNA sequences that translation is not to transcription in translation in chloroplasts may on transcripts during the process of Chloroplast is in with the which is the of many RNA-binding proteins and it that RNA-binding proteins and ribosomes to with transcripts in a view is by the that the chloroplast et al., with the et al., 2004). The to which RNA processing and translation are may the that more will those that will is evidence for the of proteins into the a machinery et al., has been to the of into the et al., et al., this is a of the of the with the machinery or as a to to is to the and to the that has been in understanding the of chloroplast gene expression. processes that to complex to have For the complexity of RNA processing to to a of proteins that bind specific RNA and the process of RNA editing to a of PPR proteins that a of but that a aspects of chloroplast gene expression that been to have out to A is chloroplast RNA which whose a of different proteins. complexity also in the and the that chloroplast The of chloroplast gene expression to a of of bacterial ancestry in core gene expression processes translation, and RNA aspects of chloroplast gene expression (RNA editing, and RNA were by the of evolved proteins these core The of plant to have been for the of RNA-binding the of PPR proteins. it likely that the complex RNA metabolism and complex RNA-binding protein in plant an as of the of chloroplast gene expression. is in gene expression that is the primary to different of chloroplast gene expression different that the and of chloroplast gene expression are in the is to chloroplast gene expression to and and A current is to the subset of nucleus-encoded whose is for gene expression and the that these to I to the of the many I to to I to Börner, and for and for on the I also for and for data and Williams-Carrier and Prikryl for in the