Non-green Plastids Research Articles

Design of chimeric expression elements that confer high‐level gene activity in chromoplasts

Non-green plastids, such as chromoplasts, generally have much lower activity of gene expression than chloroplasts in photosynthetically active tissues. Suppression of plastid genes in non-green tissues occurs through a complex interplay of transcriptional and translational control, with the contribution of regulation of transcript abundance versus translational activity being highly variable between genes. Here, we have investigated whether the low expression of the plastid genome in chromoplasts results from inherent limitations in gene expression capacity, or can be overcome by designing appropriate combinations of promoters and translation initiation signals in the 5' untranslated region (5'-UTR). We constructed chimeric expression elements that combine promoters and 5'-UTRs from plastid genes, which are suppressed during chloroplast-to-chromoplast conversion in Solanum lycopersicum (tomato) fruit ripening, either just at the translational level or just at the level of mRNA accumulation. These chimeric expression elements were introduced into the tomato plastid genome by stable chloroplast transformation. We report the identification of promoter-UTR combinations that confer high-level gene expression in chromoplasts of ripe tomato fruits, resulting in the accumulation of reporter protein GFP to up to 1% of total cellular protein. Our work demonstrates that non-green plastids are capable of expressing genes to high levels. Moreover, the chimeric cis-elements for chromoplasts developed here are widely applicable in basic and applied research using transplastomic methods.

Unsolved problems in plastid transformation

Plants have been proved as a novel production platform for a wide range of biologically important compounds such as enzymes, therapeutic proteins, antibiotics, and proteins with immunological properties. In this context, plastid genetic engineering can be potentially used to produce recombinant proteins. However, several challenges still remain to be overcome if the full potential of plastid transformation technology is to be realized. They include the development of plastid transformation systems for species other than tobacco, the expression of transgenes in non-green plastids, the increase of protein accumulation and the appearance of pleiotropic effects. In this paper, we discuss the novel tools recently developed to overcome some limitations of chloroplast transformation.

Chloroplast redox homeostasis is essential for lateral root formation in Arabidopsis

Redox regulation based on dithiol-disulphide interchange is an essential component of the control of chloroplast metabolism. In contrast to heterotrophic organisms, and non-photosynthetic plant tissues, chloroplast redox regulation relies on ferredoxin (Fd) reduced by the photosynthetic electron transport chain, thus being highly dependent on light. The finding of the NADPH-dependent thioredoxin reductase C (NTRC), a chloroplast-localized NTR with a joint thioredoxin domain, showed that NADPH is also used as source of reducing power for chloroplast redox homeostasis. Recently we have found that NTRC is also in plastids of non-photosynthetic tissues. Because these non-green plastids lack photochemical reactions, their redox homeostasis depends exclusively on NADPH produced from sugars and, thus, NTRC may play an essential role maintaining the redox homeostasis in these plastids. The fact that redox regulation occurs in any type of plastids raises the possibility that the functions of chloroplasts and non-green plastids, such as amyloplasts, are integrated to harmonize the growth of the different organs of the plant. To address this question, we generated Arabidopsis plants the redox homeostasis of which is recovered exclusively in chloroplasts, by leaf-specific expression of NTRC in the ntrc mutant, or exclusively in amyloplasts, by root-specific expression of NTRC. The analysis of these plants suggests that chloroplasts exert a pivotal role on plant growth, as expected because chloroplasts constitute the major source of nutrients and energy, derived from photosynthesis, for growth of heterotrophic tissues. However, NTRC deficiency causes impairment of auxin synthesis and lateral root formation. Interestingly, recovery of redox homeostasis of chloroplasts, but not of amyloplasts, was sufficient to restore wild type levels of lateral roots, showing the important signaling function of chloroplasts for the development of heterotrophic organs.

Identification of cis‐elements conferring high levels of gene expression in non‐green plastids

Although our knowledge about the mechanisms of gene expression in chloroplasts has increased substantially over the past decades, next to nothing is known about the signals and factors that govern expression of the plastid genome in non-green tissues. Here we report the development of a quantitative method suitable for determining the activity of cis-acting elements for gene expression in non-green plastids. The in vivo assay is based on stable transformation of the plastid genome and the discovery that root length upon seedling growth in the presence of the plastid translational inhibitor kanamycin is directly proportional to the expression strength of the resistance gene nptII in transgenic tobacco plastids. By testing various combinations of promoters and translation initiation signals, we have used this experimental system to identify cis-elements that are highly active in non-green plastids. Surprisingly, heterologous expression elements from maize plastids were significantly more efficient in conferring high expression levels in root plastids than homologous expression elements from tobacco. Our work has established a quantitative method for characterization of gene expression in non-green plastid types, and has led to identification of cis-elements for efficient plastid transgene expression in non-green tissues, which are valuable tools for future transplastomic studies in basic and applied research.

CLUMPED CHLOROPLASTS 1 is required for plastid separation in Arabidopsis

We identified an Arabidopsis thaliana mutant, clumped chloroplasts 1 (clmp1), in which disruption of a gene of unknown function causes chloroplasts to cluster instead of being distributed throughout the cytoplasm. The phenotype affects chloroplasts and nongreen plastids in multiple organs and cell types, but is detectable only at certain developmental stages. In young leaf petioles of clmp1, where clustering is prevalent, cells lacking chloroplasts are detected, suggesting impaired chloroplast partitioning during mitosis. Although organelle distribution and partitioning are actin-dependent in plants, the actin cytoskeleton in clmp1 is indistinguishable from that in WT, and peroxisomes and mitochondria are distributed normally. A CLMP1-YFP fusion protein that complements clmp1 localizes to discrete foci in the cytoplasm, most of which colocalize with the cell periphery or with chloroplasts. Ultrastructural analysis revealed that chloroplasts within clmp1 clusters are held together by membranous connections, including thin isthmi characteristic of late-stage chloroplast division. This finding suggests that constriction of dividing chloroplasts proceeds normally in clmp1, but separation is impaired. Consistently, chloroplast size and number, as well as positioning of the plastid division proteins FtsZ and ARC5/DRP5B, are unaffected in clmp1, indicating that loss of CLMP1-mediated chloroplast separation does not prevent otherwise normal division. CLMP1-like sequences are unique to green algae and land plants, and the CLMP1 sequence suggests that it functions through protein-protein interactions. Our studies identify a unique class of proteins required for plastid separation after the constriction stage of plastid division and indicate that CLMP1 activity is also required for plastid distribution and partitioning during cell division.

The role of transporters in supplying energy to plant plastids

The energy status of plant cells strongly depends on the energy metabolism in chloroplasts and mitochondria, which are capable of generating ATP either by photosynthetic or oxidative phosphorylation, respectively. Another energy-rich metabolite inside plastids is the glycolytic intermediate phosphoenolpyruvate (PEP). However, chloroplasts and most non-green plastids lack the ability to generate PEP via a complete glycolytic pathway. Hence, PEP import mediated by the plastidic PEP/phosphate translocator or PEP provided by the plastidic enolase are vital for plant growth and development. In contrast to chloroplasts, metabolism in non-green plastids (amyloplasts) of starch-storing tissues strongly depends on both the import of ATP mediated by the plastidic nucleotide transporter NTT and of carbon (glucose 6-phosphate, Glc6P) mediated by the plastidic Glc6P/phosphate translocator (GPT). Both transporters have been shown to co-limit starch biosynthesis in potato plants. In addition, non-photosynthetic plastids as well as chloroplasts during the night rely on the import of energy in the form of ATP via the NTT. During energy starvation such as prolonged darkness, chloroplasts strongly depend on the supply of ATP which can be provided by lipid respiration, a process involving chloroplasts, peroxisomes, and mitochondria and the transport of intermediates, i.e. fatty acids, ATP, citrate, and oxaloacetate across their membranes. The role of transporters involved in the provision of energy-rich metabolites and in pathways supplying plastids with metabolic energy is summarized here.

High efficiency plastid transformation in potato and regulation of transgene expression in leaves and tubers by alternative 5′ and 3′ regulatory sequences

Transformation of potato plastids is limited by low transformation frequencies and low transgene expression in tubers. In order to improve the transformation efficiency, we modified the regeneration procedure and prepared novel vectors containing potato flanking sequences for transgene integration by homologous recombination in the Large Single Copy region of the plastome. Vector delivery was performed by the biolistic approach. By using the improved regeneration procedure and the potato flanking sequences, we regenerated about one shoot every bombardment. This efficiency corresponds to 15-18-fold improvement compared to previous results with potato and is comparable to that usually achieved with tobacco. Further, we tested five promoters and terminators, and four 5'-UTRs, to increase the expression of the gfp transgene in tubers. In leaves, accumulation of GFP to about 4% of total soluble protein (TSP) was obtained with the strong promoter of the rrn operon, a synthetic rbcL-derived 5'-UTR and the bacterial rrnB terminator. GFP protein was detected in tubers of plants transformed with only four constructs out of eleven. Best results (up to approximately 0.02% TSP) were achieved with the rrn promoter and rbcL 5'-UTR construct, described above, and another containing the same terminator, but with the promoter and 5'-UTR from the plastid clpP gene. The results obtained suggest the potential use of clpP as source of novel regulatory sequences in constructs aiming to express transgenes in amyloplasts and other non-green plastids. Furthermore, they represent a significant advancement of the plastid transformation technology in potato, of relevance to its implementation in potato breeding and biotechnology.

Carotenoid Crystal Formation in Arabidopsis and Carrot Roots Caused by Increased Phytoene Synthase Protein Levels

BackgroundAs the first pathway-specific enzyme in carotenoid biosynthesis, phytoene synthase (PSY) is a prime regulatory target. This includes a number of biotechnological approaches that have successfully increased the carotenoid content in agronomically relevant non-green plant tissues through tissue-specific PSY overexpression. We investigated the differential effects of constitutive AtPSY overexpression in green and non-green cells of transgenic Arabidopsis lines. This revealed striking similarities to the situation found in orange carrot roots with respect to carotenoid amounts and sequestration mechanism.Methology/Principal FindingsIn Arabidopsis seedlings, carotenoid content remained unaffected by increased AtPSY levels although the protein was almost quantitatively imported into plastids, as shown by western blot analyses. In contrast, non-photosynthetic calli and roots overexpressing AtPSY accumulated carotenoids 10 and 100-fold above the corresponding wild-type tissues and contained 1800 and 500 µg carotenoids per g dry weight, respectively. This increase coincided with a change of the pattern of accumulated carotenoids, as xanthophylls decreased relative to β-carotene and carotene intermediates accumulated. As shown by polarization microscopy, carotenoids were found deposited in crystals, similar to crystalline-type chromoplasts of non-green tissues present in several other taxa. In fact, orange-colored carrots showed a similar situation with increased PSY protein as well as carotenoid levels and accumulation patterns whereas wild white-rooted carrots were similar to Arabidopsis wild type roots in this respect. Initiation of carotenoid crystal formation by increased PSY protein amounts was further confirmed by overexpressing crtB, a bacterial PSY gene, in white carrots, resulting in increased carotenoid amounts deposited in crystals.ConclusionsThe sequestration of carotenoids into crystals can be driven by the functional overexpression of one biosynthetic enzyme in non-green plastids not requiring a chromoplast developmental program as this does not exist in Arabidopsis. Thus, PSY expression plays a major, rate-limiting role in the transition from white to orange-colored carrots.

Genome-Wide Analysis of Plastid Gene Expression in Potato Leaf Chloroplasts and Tuber Amyloplasts: Transcriptional and Posttranscriptional Control

Gene expression in nongreen plastids is largely uncharacterized. To compare gene expression in potato (Solanum tuberosum) tuber amyloplasts and leaf chloroplasts, amounts of transcripts of all plastid genes were determined by hybridization to plastome arrays. Except for a few genes, transcript accumulation was much lower in tubers compared with leaves. Transcripts of photosynthesis-related genes showed a greater reduction in tubers compared with leaves than transcripts of genes for the genetic system. Plastid genome copy number in tubers was 2- to 3-fold lower than in leaves and thus cannot account for the observed reduction of transcript accumulation in amyloplasts. Both the plastid-encoded and the nucleus-encoded RNA polymerases were active in potato amyloplasts. Transcription initiation sites were identical in chloroplasts and amyloplasts, although some differences in promoter utilization between the two organelles were evident. For some intron-containing genes, RNA splicing was less efficient in tubers than in leaves. Furthermore, tissue-specific differences in editing of ndh transcripts were detected. Hybridization of the plastome arrays with RNA extracted from polysomes indicated that, in tubers, ribosome association of transcripts was generally low. Nevertheless, some mRNAs, such as the transcript of the fatty acid biosynthesis gene accD, displayed relatively high ribosome association. Selected nuclear genes involved in plastid gene expression were generally significantly less expressed in tubers than in leaves. Hence, compared with leaf chloroplasts, gene expression in tuber amyloplasts is much lower, with control occurring at the transcriptional, posttranscriptional, and translational levels. Candidate regulatory sequences that potentially can improve plastid (trans)gene expression in amyloplasts have been identified.

Control of Arabidopsis meristem development by thioredoxin-dependent regulation of intercellular transport

Cell-to-cell transport in plants occurs through cytoplasmic channels called “plasmodesmata” and is regulated by developmental and environmental factors. Callose deposition modulates plasmodesmal transport in vivo, but little is known about the mechanisms that regulate this process. Here we report a genetic approach to identify mutants affecting plasmodesmal transport. We isolated 5 mutants, named gfp arrested trafficking ( gat ), affected in GFP unloading from the phloem into the meristem. gat1 mutants were seedling lethal and carried lesions in an m -type thioredoxin that is expressed in non-green plastids of meristems and organ primordia. Callose and hydrogen peroxide accumulated in gat1 mutants, and WT plants subjected to oxidative conditions phenocopied the gat1 trafficking defects. Ectopic expression of GAT1 in mature leaves increased plasmodesmal permeability and led to a delay in senescence and flowering time. We propose a role for the GAT1 thioredoxin in the redox regulation of callose deposition and symplastic permeability that is essential for meristem maintenance in Arabidopsis .

Chloroplast-derived vaccine antigens and biopharmaceuticals: expression, folding, assembly and functionality.

Chloroplast genetic engineering offers several advantages, including high levels of transgene expression, transgene containment via maternal inheritance, and multi-gene expression in a single transformation event. Oral delivery is facilitated by hyperexpression of vaccine antigens against cholera, tetanus, anthrax, plague, or canine parvovirus (4%–31% of total soluble protein, TSP) in transgenic chloroplasts (leaves) or non-green plastids (carrots, tomato) as well as the availability of antibiotic free selectable markers or the ability to excise selectable marker genes. Hyperexpression of several therapeutic proteins, including human serum albumin (11.1% TSP), somatotropin (7% TSP), interferon-alpha (19% TSP), interferon-gamma (6% TSP), and antimicrobial peptide (21.5% TSP), facilitates efficient and economic purification. Also, the presence of chap-erones and enzymes in chloroplasts facilitates assembly of complex multisubunit proteins and correct folding of human blood proteins with proper disulfide bonds. Functionality of chloroplast-derived vaccine antigens and therapeutic proteins has been demonstrated by several assays, including the macrophage lysis assay, GM1-ganglioside binding assay, protection of HeLA cells or human lung carcinoma cells against encephalomyocarditis virus, systemic immune response, protection against pathogen challenge, and growth or inhibition of cell cultures. Purification of human proinsulin has been achieved using novel purification strategies (inverse temperature transition property) that do not require expensive column chromatography techniques. Thus, transgenic chloroplasts are ideal bio-reactors for production of functional human and animal therapeutic proteins in an environmentally friendly manner.

Effects of arc3, arc5 and arc6 Mutations on Plastid Morphology and Stromule Formation in Green and Nongreen Tissues of Arabidopsis thaliana†

Mutations in the ARC3, ARC5 and ARC6 genes of Arabidopsis thaliana affect chloroplast division. We investigated whether ARC3, ARC5 and ARC6 are also involved in determining plastid morphology in nongreen tissues, where stromules, stroma-filled tubular extensions of the plastid envelope membrane, are more abundant than in mesophyll cells. Using plastid-targeted green fluorescent protein to observe plastids throughout the organs of these mutants, we have discovered a number of new mutant phenotypes. The size of arc3 plastids was heterogeneous in various tissues. arc5 plastids appeared wild-type in the majority of nongreen tissues examined. However, in cells of stamen filaments, the arc5 mutant showed an increase in the frequency of stromules. Increased stromule frequency was observed for a number of organs in the arc6 mutant. Some arc6 cells contained heterogeneous mixtures of plastids; epidermal cells of hypocotyls, stamen filaments and the bases of petals possessed both very large chloroplasts as well as much smaller nongreen plastids. Quantitative analysis in hypocotyl cells revealed that the alteration in stromule length in arc3 and arc6 mutants occurred despite wild-type plastid densities. Thus, in hypocotyls, the effects of the arc3 and arc6 mutations on stromule length and frequency are independent of changes in plastid division. Although electron micrographs of stromules emanating from chloroplasts have rarely been reported, within the arc3 mutant, narrow, 40-50 nm diameter, recoiled stromules could be followed for about 10 microm in electron micrographs of leaf tissue.

Plastid Transcriptomics and Translatomics of Tomato Fruit Development and Chloroplast-to-Chromoplast Differentiation: Chromoplast Gene Expression Largely Serves the Production of a Single Protein

Plastid genes are expressed at high levels in photosynthetically active chloroplasts but are generally believed to be drastically downregulated in nongreen plastids. The genome-wide changes in the expression patterns of plastid genes during the development of nongreen plastid types as well as the contributions of transcriptional versus translational regulation are largely unknown. We report here a systematic transcriptomics and translatomics analysis of the tomato (Solanum lycopersicum) plastid genome during fruit development and chloroplast-to-chromoplast conversion. At the level of RNA accumulation, most but not all plastid genes are strongly downregulated in fruits compared with leaves. By contrast, chloroplast-to-chromoplast differentiation during fruit ripening is surprisingly not accompanied by large changes in plastid RNA accumulation. However, most plastid genes are translationally downregulated during chromoplast development. Both transcriptional and translational downregulation are more pronounced for photosynthesis-related genes than for genes involved in gene expression, indicating that some low-level plastid gene expression must be sustained in chromoplasts. High-level expression during chromoplast development identifies accD, the only plastid-encoded gene involved in fatty acid biosynthesis, as the target gene for which gene expression activity in chromoplasts is maintained. In addition, we have determined the developmental patterns of plastid RNA polymerase activities, intron splicing, and RNA editing and report specific developmental changes in the splicing and editing patterns of plastid transcripts.

Visualisation of plastids in endosperm, pollen and roots of transgenic wheat expressing modified GFP fused to transit peptides from wheat SSU RubisCO, rice FtsZ and maize ferredoxin III proteins

The ability to target marker proteins to specific subcellular compartments is a powerful research tool to study the structure and development of organelles. Here transit sequences from nuclear-encoded, plastid proteins, namely rice FtsZ, maize non-photosynthetic ferredoxin III (FdIII) and the small subunit of RubisCO were used to target a modified synthetic GFP (S65G, S72A) to plastids. The localisations of the fusion proteins expressed in transgenic wheat plants and under the control of the rice actin promoter were compared to an untargeted GFP control. GFP fluorescence was localised to non-green plastids in pollen, roots and seed endosperm and detected in isolated leaf chloroplasts using a GFP-specific antibody. Transit peptides appeared to influence the relative fluorescence intensities of plastids in different tissues. This is consistent with differential targeting and/or turnover of GFP fusion proteins in different plastid types. Replacement of GFP sequences with alternative coding regions enables immediate applications of our vectors for academic research and commercial applications.

Carbohydrate Metabolism in theToxoplasma gondiiApicoplast: Localization of Three Glycolytic Isoenzymes, the Single Pyruvate Dehydrogenase Complex, and a Plastid Phosphate Translocator

Many apicomplexan parasites, such as Toxoplasma gondii and Plasmodium species, possess a nonphotosynthetic plastid, referred to as the apicoplast, which is essential for the parasites' viability and displays characteristics similar to those of nongreen plastids in plants. In this study, we localized several key enzymes of the carbohydrate metabolism of T. gondii to either the apicoplast or the cytosol by engineering parasites which express epitope-tagged fusion proteins. The cytosol contains a complete set of enzymes for glycolysis, which should enable the parasite to metabolize imported glucose into pyruvate. All the glycolytic enzymes, from phosphofructokinase up to pyruvate kinase, are present in the T. gondii genome, as duplicates and isoforms of triose phosphate isomerase, phosphoglycerate kinase, and pyruvate kinase were found to localize to the apicoplast. The mRNA expression levels of all genes with glycolytic products were compared between tachyzoites and bradyzoites; however, a strict bradyzoite-specific expression pattern was observed only for enolase I. The T. gondii genome encodes a single pyruvate dehydrogenase complex, which was located in the apicoplast and absent in the mitochondrion, as shown by targeting of epitope-tagged fusion proteins and by immunolocalization of the native pyruvate dehydrogenase complex. The exchange of metabolites between the cytosol and the apicoplast is likely to be mediated by a phosphate translocator which was localized to the apicoplast. Based on these localization studies, a model is proposed that explains the supply of the apicoplast with ATP and the reduction power, as well as the exchange of metabolites between the cytosol and the apicoplast.

Optical Manipulation Reveals Strong Attracting Forces at Membrane Contact Sites between Endoplasmic Reticulum and Chloroplasts

Eukaryote cells depend on membrane lipid trafficking from biogenic membranes, like the endoplasmic reticulum (ER), to other membranes in the cell. Two major routes for membrane lipid transport are recognized: vesicular trafficking and lipid transfer at zones of close contact between membranes. Specific ER regions involved in such membrane contact sites (MCSs) have been isolated, and lipid transfer at MCSs as well as protein-protein interactions between the partaking membranes have been demonstrated (reviewed by Holthuis, J. C. M., and Levine, T. P. (2005) Nat. Rev. 6, 209-220). Here we present the first demonstration of the physical association between membranes involved in MCSs: by using optical imaging and manipulation, strong attracting forces between ER and chloroplasts are revealed. We used Arabidopsis thaliana expressing green fluorescent protein in the ER lumen and observed leaf protoplasts by confocal microscopy. The ER network was evident, with ER branch end points apparently localized at chloroplast surfaces. After rupture of a protoplast using a laser scalpel, the cell content was released. ER fragments remained attached to the released chloroplasts and could be stretched out by optical tweezers. The applied force, 400 pN, could not drag a chloroplast free from its attached ER, which could reflect protein-protein interactions at the ER-chloroplast MCSs. As chloroplasts rely on import of ER-synthesized lipids, we propose that lipid transfer occurs at these MCSs. We suggest that lipid transfer at the MCSs also occurs in the opposite direction, for example to channel plastid-synthesized acyl groups to supply substrates for ER-localized synthesis of membrane and storage lipids.

Contained metabolic engineering in tomatoes by expression of carotenoid biosynthesis genes from the plastid genome

Applications of chloroplast engineering in agriculture and biotechnology will depend critically on success in extending the crop range of chloroplast transformation, and on the feasibility of expressing transgenes in edible organs (such as tubers and fruits), which often are not green and thus are much less active in chloroplast gene expression. We have improved a recently developed chloroplast-transformation system for tomato plants and applied it to engineering one of the central metabolic pathways in fruits: carotenoid biosynthesis. We report that plastid expression of a bacterial lycopene beta-cyclase gene results in herbicide resistance and triggers conversion of lycopene, the main storage carotenoid of tomatoes, to beta-carotene, resulting in fourfold enhanced pro-vitamin A content of the fruits. Our results demonstrate the feasibility of engineering nutritionally important biochemical pathways in non-green plastids by transformation of the chloroplast genome.

Production of biopharmaceuticals and vaccines in plants via the chloroplast genome

Transgenic plants offer many advantages, including low cost of production (by elimination of fermenters), storage and transportation; heat stability; and absence of human pathogens. When therapeutic proteins are orally delivered, plant cells protect antigens in the stomach through bioencapsulation and eliminate the need for expensive purification and sterile injections, in addition to development of both systemic and mucosal immunity. Chloroplast genetic engineering offers several advantages, including high levels of transgene expression, transgene containment via maternal inheritance and multi-gene expression in a single transformation event. Hyper-expression of vaccine antigens against cholera, tetanus, anthrax, plague or canine parvovirus (4-31% of total soluble protein, tsp) in transgenic chloroplasts (leaves) or non-green plastids (carrots, tomato), as well as the availability of antibiotic-free selectable markers or the ability to excise selectable marker genes, facilitate oral delivery. Hyper-expression of several therapeutic proteins, including human serum albumin (11.1% tsp), somatotropin (7% tsp), interferon-gamma (6% tsp), anti-microbial peptide (21.5% tsp), facilitates efficient and economic purification. Also, the presence of chaperones and enzymes in chloroplasts facilitate assembly of complex multi-subunit proteins and correct folding of human blood proteins with proper disulfide bonds. Functionality of chloroplast-derived vaccine antigens and therapeutic proteins has been demonstrated by several assays, including the macrophage lysis assay, GM1-ganglioside binding assay, protection of HeLa cells or human lung carcinoma cells against encephalomyocarditis virus, systemic immune response, protection against pathogen challenge, and growth or inhibition of cell cultures. Thus, transgenic chloroplasts are ideal bioreactors for production of functional human and animal therapeutic proteins in an environmentally friendly manner.

Recent advances in the study of Clp, FtsH and other proteases located in chloroplasts

Several chloroplast proteases have been characterized in recent years. The ATP-dependent chloroplast proteases Clp and FtsH stand out because they form multi-subunit complexes consisting of different gene products. Surprisingly, both green and non-green plastids appear to contain a similar soluble Clp core proteolytic complex, consisting of five ClpP proteases, their non-catalytic ClpR homologs, and two ClpS homologs that have unknown function. Analyses of single and double FtsH1, FtsH2, FtsH5 and FtsH8 mutants, and overexpression of FtsH proteins in these Arabidopsis thaliana mutants show partial redundancies within pairs of closely related FtsH thylakoid proteins. The presence of at least one member of each pair is essential for functional accumulation. Other chloroplast proteases have also been identified recently. Future challenges include the identification of substrate recognition mechanisms and elucidating the role of proteases in chloroplast biogenesis and function.

Carotenoid accumulation and function in seeds and non‐green tissues

Carotenoids are plant pigments that function as antioxidants, hormone precursors, colourants and essential components of the photosynthetic apparatus. Carotenoids accumulate in nearly all types of plastids, not just the chloroplast, and are thus found in most plant organs and tissues, albeit at trace levels in some tissues. In this review we summarise the current knowledge of the carotenoid content of non-green plastids and discuss what is known about the regulation of their biosynthesis in roots, fruits, flowers, tubers and seeds. The emphasis is on food crops as carotenoids are essential components of human diets, primarily as some are precursors of vitamin A. The low carotenoid content of many staple foods, such as cereals, can exacerbate dietary deficiencies. The World Health Organisation has estimated that more than 100 million children are vitamin A-deficient and up to 500,000 of these children become blind each year. Many of these children die within 12 months of going blind. Thus, understanding the regulation of carotenoid accumulation in food crops, especially tubers and cereals, should facilitate improvements to nutritional value with potentially significant health benefits.