Plastid Proteostasis and Heterologous Protein Accumulation in Transplastomic Plants

The insertion of transgenes into the plastid genome (plastome) has proved to be an effective alternative to nuclear transformation for producing stable transformants expressing heterologous proteins. Recently, methodology, characteristics, and possible fields of application of plastome transformation have been extensively reviewed (Lössl and Waheed, 2011; Scotti et al., 2012). From these reports, plastid transformation turns out to be a very promising tool for the production of recombinant proteins in plants, yet some limitations must still be overcome. Although the plastome of many species has been successfully transformed, an efficient and reproducible transformation procedure easily adoptable by any plant laboratory has been established only in tobacco (Nicotiana tabacum) and to a minor extent in two other solanaceous species (potato [Solanum tuberosum] and tomato [Solanum lycopersicum]) as well as in a few other species (Maliga and Bock, 2011). Moreover, up to now, the steady-state level of a recombinant protein coded by a gene inserted into the plastome has failed to be predictable. In spite of a huge number of reports on successful foreign protein production in plastids, above all proteins with a pharmaceutical interest, in many cases the expression level of other recombinant proteins in the transplastomic plants appears to be very low or even undetectable (Birch-Machin et al., 2004; Bellucci et al., 2005; Wirth et al., 2006). We believe that this is mainly due to our limited knowledge of the mechanisms in plastids influencing the maintenance of protein homeostasis, or proteostasis. This term refers to a complex network of biological pathways capable of maintaining the dynamic equilibrium of the protein pool, thus allowing the cell to get used to environmental changes (Balch et al., 2008). This concept can also be extended to the subcellular level, especially for those organelles with an endosymbiotic origin like the plastids and mitochondria (Waller, 2012). These organelles retain a whole series of mechanisms for the preservation of their protein balance, including specific proteases, transcriptional and translational control, as well as molecular chaperones and enzymes useful in protein folding. Therefore, it is important to develop basic studies in factors regulating protein synthesis, stability, folding, targeting, and accumulation in plastids, including the protein quality control that contributes to the functional integrity of proteins. Our intention here is not to provide a comprehensive review of the mechanisms that regulate plastid proteostasis but to discuss some recent insights into this field that might bring beneficial applications in plastid biotechnology for transgene expression and foreign protein accumulation. In spite of the prokaryotic origin and the similarity to its ancestors of the gene expression machinery, the presence of introns, RNA editing, and processing of mRNA polycistronic precursors make the chloroplast a unique organelle that combines eukaryotic and prokaryotic features (Stern et al., 2010). This uniqueness can be noticed in at least two cases: (1) the dual genetic origin of the plastid proteome, made up of proteins encoded either by the plastome or the nucleus; (2) the presence of unique ribosomal proteins, named plastid-specific ribosomal proteins, together with ribosomal proteins of prokaryotic origin (Tiller et al., 2012). As an endosymbiont, chloroplast has almost totally replaced the control of gene expression at the transcriptional level, switching to a predominantly posttranscriptional control (Marìn-Navarro et al., 2007). However, some transcriptional regulation may also take place, because plastid transcription is performed by three RNA polymerases, two monomeric, nucleus-encoded, phage-type enzymes (dubbed RPOTp and RPOTmp) and one multimeric, plastid-encoded, eubacteria-type enzyme (dubbed PEP), which need to interact with up to six nucleus-encoded transcription initiation factors in order to perform their activity (Liere and Börner, 2007; Lerbs-Mache, 2011). Hence, transcription is considered an important step for transgene expression, and it can be controlled by using appropriate promoters in the plastid transformation vectors. Most of the chloroplast transformation vectors utilize the strong promoter of the plastid ribosomal RNA operon (Prrn), but also other promoters like the plastidial psbA and clpP or the bacterial trc are sometimes used. The technology of plastid transformation, from the engineering of plastid transgenes in Escherichia coli cloning vectors to the obtainment of stable transplastomic plants, is clearly described in a recent review (Maliga and Bock, 2011), but here we intend to underline the importance of some specific motifs present in the chloroplast transformation vectors for transgene expression. In addition to the promoters, the transgene expression cassettes commonly employed for plastid vector construction harbor other regulatory elements like 5′-untranslated regions (UTRs) and 3′-UTRs. These regions are both involved in the regulation of RNA stability and the translation efficiency of plastid transcripts (Zou et al., 2003; Pfalz et al., 2009). The exact modality for the correct positioning of the chloroplast ribosome on the translation initiation codon of mRNAs is still beyond our understanding, but most of the plastid mRNAs possess a Shine-Dalgarno (SD) sequence in the 5′-UTR. In prokaryotes, the SD sequence (GGAGG) is located between four and 12 nucleotides upstream from the start codon and, being complementary to the 3′ terminus of the 16S ribosomal RNA, mediates the binding of the ribosome to an mRNA. In order to find out the mechanism of SD sequence recognition, Drechsel and Bock (2011) analyzed translation initiation from mRNAs containing multiple SD sequences, demonstrating that the plastid ribosomes displayed a bias toward preferentially utilizing the 5′-most SD sequence. The facts that the location of plastidial SD elements is much more variable than in E. coli and that for some plastid genes SD-like sequences have turned out to be less important for translation initiation lead to the possibility that other mechanisms for translation initiation may exist (Marìn-Navarro et al., 2007; Scharff et al., 2011). Indeed, detailed analyses of the plastid 5′-UTRs have detected the presence of cis-elements that facilitate the interaction with nucleus-encoded RNA-binding proteins (Merhige et al., 2005). These associations between protein factors and the respective RNA sequences influence the translation initiation process. There is a little concern about the use of homologous regulatory elements in chloroplast transformation vectors due to the risk of potential recombination with the endogenous ones (Rogalski et al., 2006). Therefore, for example, to transform the tobacco plastome, Elghabi et al. (2011) utilized promoter and 3′-UTR sequences from Chlamydomonas reinhardtii in order to avoid unwanted recombination with the resident copies in the plastome. Conversely, in other laboratories, the use of homologous regulatory sequences is preferred, as shown in the case of the psbA 5′-UTR. It has been demonstrated that, despite the possible accumulation of foreign proteins using a heterologous psbA 5′-UTR, it occurs with a reduced efficiency in comparison with the results achieved when the homologous regulatory elements are used (Ruhlman et al., 2010). The reason for these results is probably the preferential association to stromal RNA-binding proteins with the endogenous psbA 5′-UTR, whereas the heterologous one is an ineffective competitor. On the basis of the results obtained and after a taxonomic analysis of the region 200 bases upstream of the translation start codon, the authors suggested the use of species-specific regulatory elements for significant accumulation of foreign protein in transplastomic plants. The 5′-UTR of gene 10 of bacteriophage T7 (T7g10) has been successfully used in plastids achieving high protein expression (Kuroda and Maliga 2001) as well as intact or truncated 5′-UTR from highly expressed genes like rbcL, psbA, and atbP (Herz et al., 2005; Valkov et al., 2011). The inverted repeat sequence, typically present in the 3′-UTR of many plastid genes, can potentially form a stem-loop structure, thus becoming important in the stabilization and the accumulation of the transcript coded by the upstream sequence. This concept has been recently highlighted in transplastomic tobacco plants expressing several gfp gene constructs with the same 5′ regulatory sequence but different 3′-UTRs et al., 2011). 3′ regulatory elements from plastid or bacterial genes are used in plastid transformation vectors. The E. coli 3′ region to be the most both in tobacco and transplastomic plants, a mRNA stability and not to high heterologous protein expression et al., 2011; Valkov et al., 2011). Therefore, a of the promoters, and 3′-UTR utilized in different to the transcript accumulation and stability can be made et al., 2005; Maliga and Bock, 2011; et al., 2011; Valkov et al., 2011), but it is that the of transgene even with stability, not to an foreign protein and accumulation plastids et al., This is because the translational control is much more than transcriptional regulation for transgene expression and this in many cases the present in mRNA the sequences of the translation initiation codon transcript stability or The in both cases is results obtained by of highly expressed proteins in plastids to the transgene region that it is possible to the mRNA by sequence of nucleotides or between the 5′-UTR and the region et al., 2011). a it is also possible to that encoded in the plastid gene are important of translation efficiency (Kuroda and These results many to the sequence of the region located of the start codon et al., 2011). processing of polycistronic transcripts into mRNAs is to the efficiency of a expression can be used to foreign genes of the same operon et al., 2007). heterologous protein expression plastids is that, almost all plastid transformation vectors have been to expression of the However, expression of a transgene may in a or due to the of the expressed or due to its interaction with or the plastid (Lössl et al., 2003; et al., 2007). expression of a highly stable protein can also lead to the of chloroplast protein in et al., 2009). avoid by transgene expression, different have been as the one on a nucleus-encoded, T7 RNA that plastid transgene transcription from a T7 promoter (Lössl et al., or that on the control of transgene expression using the bacterial and 2005). However, the use of these is limited by the transformation of two cell and or by the of a regulation of gene expression in the Recently, the use of engineering et al., has been as a tool to control chloroplast transgene expression the use of nuclear or plastid The use of a regulatory because the undetectable expression in the transplastomic tobacco plants after application of the coded by genes located in the plastome are on plastid and the expression of these genes is mainly translation of the mRNAs and at the level as Indeed, after synthesis, these proteins must into stable that to perform their biological and with the of molecular chaperones et al., 2011). The of molecular chaperones and the quality control protein and et al., quality control of plastids are very to of or and and several for example, proteins, and 2004; et al., 2008). also of bacterial which are involved in of the and the maintenance of these These the of stromal and their and several other in recent et al., 2006). of the psbA for the one of the proteins of is an of the complex regulatory utilized in the of plants. is by and is and the In plant a of psbA mRNA with the but the translation of can be only when a complex is to the of the protein et al., 2012). This to the that translation is the mechanism for the regulation of psbA gene expression. The of a quality control on is the which of the protein to regulate its and of the and the from the to the the protein is and by the and et al., 2011). of protein also the of molecular chaperones that are proteins and or a protein et al., 2012). on transplastomic plants have that the expression of genes into the plastome is mainly after the translation of the mRNAs into the stability is the that foreign protein accumulation et al., 2005; et al., et al., 2009). proteins are to be more et al., but on plastid biotechnology are very in of protein stability in the example, which are the on the protein that regulate the of the proteins or the proteins of the recent reports that in plastids the stability in the terminus of the protein et al., et al., 2011). This may to foreign proteins are in plastids after the addition of et al., et al., 2008). It is that the of the of a protein is to its in a as the are as or and in the eukaryotic proteins a are by the are by the or must be also a the protein can be by to the The is being present in all et al., including even the prokaryotic of the in the order of the et al., and in the by the et al., 2009). and by and sequences from plastid proteins to that the terminus important the of the whereas the terminus to have influence on protein also in one of the constructs all in the after the for plastids the of an that is not homologous to the bacterial However, the in protein accumulation by the different sequences be only by the of the of plastid protein stability must in the region of the In an to in transplastomic tobacco plants, the accumulation and stability of the even et al. the importance of the for protein The to at the 5′ of the region the expression of the foreign gene in the transplastomic plants and the protein in However, the and are not the only ones to influence protein stability, because in other for in E. the of protein turns out to be a of protein stability et al., 2009). The to almost all proteins is in and the by the enzyme the of the the is and or for the protein can by which to of the proteins in a et al., and of by that is involved in the regulation of protein in the chloroplast et al., In of the in and proteins, which can in of their et al., and proteins are of a of has been on the involved in their especially for et al., 2012). In to regulate the accumulation of and proteins, in with chloroplast et al., 2011). The authors that of and are for both quality control of the proteins and of and Moreover, of the of the and by other than Therefore, the terminus of both or and proteins, and the is can the of these proteins et al., 2007). to the in and proteins with an are very On the the in and proteins, due to of may as a for Therefore, that the stability of other chloroplast proteins by the of their might be but not to the of a other protein on or to the are to the protein et al., 2006). Indeed, other in regulating the stability of a than different of protein or have been described and which the can be as (1) of one or more as or (2) of the intact or proteins as or or the of the et al., 2011). These can influence protein or to stability, some can in proteins for example, by in the or of in the or Indeed, of the in the from the and the of the to the transcription for by the and 2011). Moreover, in and are of the and proteins for et al., 2010). can be a very useful tool to a comprehensive of plastid protein recent review has of the of plastid the importance of a plastid that to the in on protein and and 2011). an can provide useful for functional studies and and with on the protein it can also to the between protein stability and in with and it has been shown in reinhardtii that the most stromal proteins of less than possess an in to the most proteins et al., 2011). This that, in to in the to protein stabilization in the stromal In analysis of has recently been out using chloroplast et al., and as well as whole et al., 2012). These studies have of a of and plastid proteins, on the protein the processing for of plastid proteins, and the of many in which is considered an mainly in the of appears to be a protein also in the because a high of nucleus-encoded chloroplast proteins of the protein after of the et al., 2012). The that for nucleus-encoded proteins the have a of and whereas for proteins the are or the of two different et al., 2008). and accumulation of a recombinant protein in it is to this protein to an appropriate cell The plant and the accumulation of foreign protein in the is the This is because a of on protein quality control is on the and and this knowledge to to retain and heterologous proteins of in this subcellular for by of as the et al., or the protein of with a protein capable of et al., 2008). the of the plastids the of of foreign proteins. inserted into the at the 5′ a sequence for a that the of the from the the transgene mRNA is to the the to plastids, the present in the and 2008). Recently, the by expression of heterologous genes, above all those to the high level of protein expression and transgene have many to use the plastome transformation technology for the accumulation of in the of reinhardtii and plants (Lössl and Waheed, 2011; et al., 2011). proteins expressed in the chloroplast can be in the of and or in the by and The of these proteins the chloroplast insertion in different with a more or less Moreover, its proteome, which a specific of enzymes and other involved in protein et al., et al., 2011). to the to be possible only for nucleus-encoded genes utilizing the or a insertion of the protein and its is very different to the of tobacco from tobacco plants analyzed by analysis using and expressed from plants to the it is to to can be in the of nuclear plants, either to the a not or expressing to a of the protein is and The accumulation of in the much than that in the both as not and as which due to the of et al., In the chloroplast of transplastomic plants expressing the protein the recombinant protein of its in the and when to the and it is clearly et al., 2011). in at is The are the pathways chloroplast proteins, either encoded or plastome the same in the a protein can and perform its biological or can be to other it specific The most mechanisms the chloroplast concern the of proteins to the even other pathways have been to proteins from the to the and the between the two et al., 2011). least four different homologous to bacterial have been for the of proteins into and the of these one a and the other one appears to of the factors et al., 2011). The other two pathways proteins the and proteins in an by a The other one is the that proteins into the using a et al., 2012). The chloroplast has been used to recombinant proteins in the but few reports have analyzed other alternative like the et al., or the et al., 2008). The correct of a protein to its or and one of the of is that many have been described in this the possibility to a of functional heterologous proteins. of the chloroplast have been here in the and but have also been as and et al., et al., 2011). However, we need more detailed on the of in different plastidial in order to the recombinant protein of has to be to its specific this an case of is the of by many proteins for their activity as Moreover, is also important for the of recombinant proteins to be used as it has been demonstrated that recombinant proteins can be successfully expressed in both in the and in the of but it is still not which of these two is more to this of protein with et al., or a protein with et al., 2001) is when expressed in the of plant a and functional can be expressed in the chloroplast of the reinhardtii et al., However, other recombinant proteins containing form in the chloroplast and order that are in the as et al., 2003; et al., 2007; et al., 2007; and 2011; et al., 2011). the which is a by when expressed in the chloroplast in it is to the heterologous protein to its biological activity et al., 2011). can be easily and of the recombinant protein the of the production the of the protein et al., In an to the of and thus avoid the of et al. (2011) have tobacco together with the of recombinant proteins that form in expression but this has failed to the of protein of the possible for the of chloroplast in the is that the high translation and the high stability of the recombinant proteins the organelle protein homeostasis, the of as that accumulation in et al., et al., 2011). Recently, it has been that the be a more than the for the and activity of proteins that the of the enzyme a with two for for stability and activity of the expressed both in the and in the the results that to the to and more enzyme et al., 2008). In the of recombinant capable of can be to the et al., 2011). Conversely, the same proteins when into the are to form this of has been for a with expressed both in the and in the et al., 2012). This is not as the as well as the of is to the of and pathways are present in these in order to protein and regulate protein et al., 2009). are used for regulation in both the and the and 2011), but in the be a with proteins in a reduced by that the activity of molecular whereas enzymes are and 2005). Moreover, the for functional as the protein which the and of has been in reinhardtii but not in the of plant et al., several chloroplast are described in up to only for one has a chloroplast location been but its its has been shown et al., 2009). Conversely, functional have been in the like which has a to the capable of in the a of for its stability the of a et al., 2011). Recently, other reports described two proteins activity located in the and involved in the maintenance of et al., or et al., 2012). This together that a in the of the with a the of and a to and 2010). protein in the chloroplast to be of importance for and functional a of the mechanisms the the as well as the of a chloroplast complex are in order to the have many in chloroplast for example, protein is by that perform an in protein and and in proteins from unwanted et al., 2010). The of the chloroplast is a molecular that the of its proteins et al., 2010). This that, to their proteins by the are from Recently, it has been shown that a plant is capable of a protein gene is inserted into the to the the recombinant protein as In the the of the same protein the is as the protein at low in a form et al., 2011). we can that is the mechanism that to the it probably also has a on the recombinant protein of the possible of accumulation in the chloroplast of recombinant proteins containing proteins for by a protein with an when expressed in transplastomic plants have been in most cases in the In this only a few studies the obtainment of whereas the other recombinant proteins described probably in with by the proteins are for by stromal proteases, in a low level of protein expression. Recently, recombinant proteins the of also by to the or The of the is to the number of the expression of a recombinant protein in a chloroplast more The with a pathways that in protein and different can be to transform a and plastid biotechnology is one of the most promising The of plastid genetic engineering have been clearly described by et al. and here we to that a has to be made in the factors that regulate plastid proteostasis studies on are to the between protein stability and the of its Moreover, the of from studies can be utilized to to for and their with protein as for the tool which and of either prokaryotic or eukaryotic proteins from or nuclear The of chaperones in different plastid also has to be clearly in order to these protein in plastids and this can be used in plastid genetic example, several chaperones have been together with recombinant proteins in the chloroplast to the accumulation of the The results are only for the bacterial et al., 2001) but not for the binding protein and the chloroplast tobacco and et al., 2007; et al., 2011). to be into is that the of the plastid are to their the cell and that more than 10 different can be like and gene expression in plastids are not well but some results that these are from those of and Bock, Valkov et al., et al., 2012). for the be the of for the chloroplast in order to the of protein Indeed, it which the most plastid proteins. for possible is the of et al. to potential for in plants. and involved in the maintenance of plastid which foreign protein accumulation. and translational control, proteases, molecular protein folding, and are The with a the with a The with the transgene 5′-UTRs and 3′-UTRs are as well as the translation initiation codon and the sequences of it that transcript stability translation region RNA-binding proteins. In a of basic and studies on plastid proteostasis is in the to foreign proteins from the plastid We Bock for of the We to all not be because of region Shine-Dalgarno protein