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The high-value carotenoid astaxanthin (3,3'-dihydroxy-β,β-carotene-4,4'-dione) is one of the most potent antioxidants in nature. In addition to its large-scale use in fish farming, the pigment has applications as a food supplement and an active ingredient in cosmetics and in pharmaceuticals for the treatment of diseases linked to reactive oxygen species. The biochemical pathway for astaxanthin synthesis has been introduced into seed plants, which do not naturally synthesize this pigment, by nuclear and plastid engineering. The highest accumulation rates have been achieved in transplastomic plants, but massive production of astaxanthin has resulted in severe growth retardation. What limits astaxanthin accumulation levels and what causes the mutant phenotype is unknown. Here, we addressed these questions by making astaxanthin synthesis in tobacco (Nicotiana tabacum) plastids inducible by a synthetic riboswitch. We show that, already in the uninduced state, astaxanthin accumulates to similarly high levels as in transplastomic plants expressing the pathway constitutively. Importantly, the inducible plants displayed wild-type-like growth properties and riboswitch induction resulted in a further increase in astaxanthin accumulation. Our data suggest that the mutant phenotype associated with constitutive astaxanthin synthesis is due to massive metabolite turnover, and indicate that astaxanthin accumulation is limited by the sequestration capacity of the plastid., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

De novo fatty acid biosynthesis in plants relies on a prokaryotic-type acetyl-CoA carboxylase (ACCase) that resides in the plastid compartment. The enzyme is composed of four subunits, one of which is encoded in the plastid genome, whereas the other three subunits are encoded by nuclear genes. The plastid gene (accD) encodes the β-carboxyltransferase subunit of ACCase and is essential for cell viability. To facilitate the functional analysis of accD, we pursued a transplastomic knockdown strategy in tobacco (Nicotiana tabacum). By introducing point mutations into the translational start codon of accD, we obtained stable transplastomic lines with altered ACCase activity. Replacement of the standard initiator codon AUG with UUG strongly reduced AccD expression, whereas replacement with GUG had no detectable effects. AccD knockdown mutants displayed reduced ACCase activity, which resulted in changes in the levels of many but not all species of cellular lipids. Limiting fatty acid availability caused a wide range of macroscopic, microscopic, and biochemical phenotypes, including impaired chloroplast division, reduced seed set, and altered storage metabolism. Finally, while the mutants displayed reduced growth under photoautotrophic conditions, they showed exaggerated growth under heterotrophic conditions, thus uncovering an unexpected antagonistic role of AccD activity in autotrophic and heterotrophic growth., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The Clp protease in the chloroplasts of plant cells is a large complex composed of at least 13 nucleus-encoded subunits and one plastid-encoded subunit, which are arranged in several ring-like structures. The proteolytic P-ring and the structurally similar R-ring form the core complex that contains the proteolytic chamber. Chaperones of the HSP100 family help with substrate unfolding, and additional accessory proteins are believed to assist with Clp complex assembly and/or to promote complex stability. Although the structure and function of the Clp protease have been studied in great detail in both bacteria and chloroplasts, the identification of bona fide protease substrates has been very challenging. Knockout mutants of genes for protease subunits are of limited value, due to their often pleiotropic phenotypes and the difficulties with distinguishing primary effects (i.e. overaccumulation of proteins that represent genuine protease substrates) from secondary effects (proteins overaccumulating for other reasons). Here, we have developed a new strategy for the identification of candidate substrates of plant proteases. By combining ethanol-inducible knockdown of protease subunits with time-resolved analysis of changes in the proteome, proteins that respond immediately to reduced protease activity can be identified. In this way, secondary effects are minimized and putative protease substrates can be identified. We have applied this strategy to the Clp protease complex of tobacco ( Nicotiana tabacum ) and identified a set of chloroplast proteins that are likely degraded by Clp. These include several metabolic enzymes but also a small number of proteins involved in photosynthesis., (© 2018 American Society of Plant Biologists. All Rights Reserved.)

To avoid photodamage plants regulate the amount of excitation energy in the membrane at the level of the light-harvesting complexes (LHCs). It has been proposed that the energy absorbed in excess is dissipated via protein conformational changes of individual LHCs. However, the exact quenching mechanism remains unclear. Here we study the mechanism of quenching in LHCs that bind a single carotenoid species and are constitutively in a dissipative conformation. Via femtosecond spectroscopy we resolve a number of carotenoid dark states, demonstrating that the carotenoid is bound to the complex in different conformations. Some of those states act as excitation energy donors for the chlorophylls, whereas others act as quenchers. Via in silico analysis we show that structural changes of carotenoids are expected in the LHC protein domains exposed to the chloroplast lumen, where acidification triggers photoprotection in vivo. We propose that structural changes of LHCs control the conformation of the carotenoids, thus permitting access to different dark states responsible for either light harvesting or photoprotection.

Transgene expression from the plastid (chloroplast) genome provides unique advantages, including high levels of foreign protein accumulation, convenient transgene stacking in operons, and increased biosafety due to exclusion of plastids from pollen transmission [1, 2]. However, applications in biotechnology and synthetic biology are severely restricted by the very small number of plant species whose plastid genomes currently can be transformed [3, 4]. Here we report a simple method for the introduction of useful plastid transgenes into non-transformable species. The transgenes tested comprised a synthetic operon encoding three components of a biosynthetic pathway for producing the high-value ketocarotenoid astaxanthin in the plastids of the cigarette tobacco, Nicotiana tabacum. Transplastomic N. tabacum plants accumulated astaxanthin to up to 1% of the plants' dry weight. We then used grafting, a procedure recently shown to facilitate horizontal genome transfer between plants [5-7], to let the transgenic chloroplast genome move across the graft junction from N. tabacum plants into plants of the nicotine-free tree species Nicotiana glauca. Transplastomic N. glauca trees expressing the synthetic pathway were recovered at high frequency, thus providing a straightforward method for extension of the transplastomic technology to new species., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Protein degradation in chloroplasts is carried out by a set of proteases that eliminate misfolded, damaged, or superfluous proteins. The ATP-dependent caseinolytic protease (Clp) is the most complex protease in plastids and has been implicated mainly in stromal protein degradation. In contrast, FtsH, a thylakoid membrane-associated metalloprotease, is believed to participate mainly in the degradation of thylakoidal proteins. To determine the role of specific Clp and FtsH subunits in plant growth and development, RNAi lines targeting at least one subunit of each Clp ring and FtsH were generated in tobacco. In addition, mutation of the translation initiation codon was employed to down-regulate expression of the plastid-encoded ClpP1 subunit. These protease lines cover a broad range of reductions at the transcript and protein levels of the targeted genes. A wide spectrum of phenotypes was obtained, including pigment deficiency, alterations in leaf development, leaf variegations, and impaired photosynthesis. When knock-down lines for the different protease subunits were compared, both common and specific phenotypes were observed, suggesting distinct functions of at least some subunits. Our work provides a well-characterized collection of knock-down lines for plastid proteases in tobacco and reveals the importance of the Clp protease in physiology and plant development., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Artemisinin-based therapies are the only effective treatment for malaria, the most devastating disease in human history. To meet the growing demand for artemisinin and make it accessible to the poorest, an inexpensive and rapidly scalable production platform is urgently needed. Here we have developed a new synthetic biology approach, combinatorial supertransformation of transplastomic recipient lines (COSTREL), and applied it to introduce the complete pathway for artemisinic acid, the precursor of artemisinin, into the high-biomass crop tobacco. We first introduced the core pathway of artemisinic acid biosynthesis into the chloroplast genome. The transplastomic plants were then combinatorially supertransformed with cassettes for all additional enzymes known to affect flux through the artemisinin pathway. By screening large populations of COSTREL lines, we isolated plants that produce more than 120 milligram artemisinic acid per kilogram biomass. Our work provides an efficient strategy for engineering complex biochemical pathways into plants and optimizing the metabolic output.

Plant chloroplasts are light-driven cell factories that have great potential to act as a chassis for metabolic engineering applications. Using plant chloroplasts, we demonstrate how photosynthetic reducing power can drive a metabolic pathway to synthesise a bio-active natural product. For this purpose, we stably engineered the dhurrin pathway from Sorghum bicolor into the chloroplasts of Nicotiana tabacum (tobacco). Dhurrin is a cyanogenic glucoside and its synthesis from the amino acid tyrosine is catalysed by two membrane-bound cytochrome P450 enzymes (CYP79A1 and CYP71E1) and a soluble glucosyltransferase (UGT85B1), and is dependent on electron transfer from a P450 oxidoreductase. The entire pathway was introduced into the chloroplast by integrating CYP79A1, CYP71E1, and UGT85B1 into a neutral site of the N. tabacum chloroplast genome. The two P450s and the UGT85B1 were functional when expressed in the chloroplasts and converted endogenous tyrosine into dhurrin using electrons derived directly from the photosynthetic electron transport chain, without the need for the presence of an NADPH-dependent P450 oxidoreductase. The dhurrin produced in the engineered plants amounted to 0.1-0.2% of leaf dry weight compared to 6% in sorghum. The results obtained pave the way for plant P450s involved in the synthesis of economically important compounds to be engineered into the thylakoid membrane of chloroplasts, and demonstrate that their full catalytic cycle can be driven directly by photosynthesis-derived electrons., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Allopolyploidization, the combination of the genomes from two different species, has been a major source of evolutionary innovation and a driver of speciation and environmental adaptation. In plants, it has also contributed greatly to crop domestication, as the superior properties of many modern crop plants were conferred by ancient allopolyploidization events. It is generally thought that allopolyploidization occurred through hybridization events between species, accompanied or followed by genome duplication. Although many allopolyploids arose from closely related species (congeners), there are also allopolyploid species that were formed from more distantly related progenitor species belonging to different genera or even different tribes. Here we have examined the possibility that allopolyploidization can also occur by asexual mechanisms. We show that upon grafting--a mechanism of plant-plant interaction that is widespread in nature--entire nuclear genomes can be transferred between plant cells. We provide direct evidence for this process resulting in speciation by creating a new allopolyploid plant species from a herbaceous species and a woody species in the nightshade family. The new species is fertile and produces fertile progeny. Our data highlight natural grafting as a potential asexual mechanism of speciation and also provide a method for the generation of novel allopolyploid crop species.

The engineering of complex metabolic pathways requires the concerted expression of multiple genes. In plastids (chloroplasts) of plant cells, genes are organized in operons that are coexpressed as polycistronic transcripts and then often are processed further into monocistronic mRNAs. Here we have used the tocochromanol pathway (providing tocopherols and tocotrienols, collectively also referred to as "vitamin E") as an example to establish principles of successful multigene engineering by stable transformation of the chloroplast genome, a technology not afflicted with epigenetic variation and/or instability of transgene expression. Testing a series of single-gene constructs (encoding homogentisate phytyltransferase, tocopherol cyclase, and γ-tocopherol methyltransferase) and rationally designed synthetic operons in tobacco and tomato plants, we (i) confirmed previous results suggesting homogentisate phytyltransferase as the limiting enzymatic step in the pathway, (ii) comparatively characterized the bottlenecks in tocopherol biosynthesis in transplastomic leaves and tomato fruits, and (iii) achieved an up to tenfold increase in total tocochromanol accumulation. In addition, our results uncovered an unexpected light-dependent regulatory link between tocochromanol metabolism and the pathways of photosynthetic pigment biosynthesis. The synthetic operon design developed here will facilitate future synthetic biology applications in plastids, especially the design of artificial operons that introduce novel biochemical pathways into plants.

Plants with transgenic plastid (chloroplast) genomes represent a promising production platform in molecular farming, mainly because of the plastids' potential to accumulate foreign proteins to very high levels and the increased biosafety conferred by the maternal mode of plastid inheritance. Although some transgenes can be expressed to extraordinarily high levels, the expression of others has been unsuccessful. Lack of detectable transgene expression is usually attributable to either RNA instability or protein instability. Here, we have investigated the possibilities to improve the production of a pharmaceutical protein that is difficult to express in chloroplasts: the HIV-1 fusion inhibitor cyanovirin-N (CV-N). Testing various N-terminal and C-terminal fusions to peptide sequences from two proteins known to accumulate to high levels in transgenic plastids (GFP and the protein antibiotic PlyGBS), we show that both low mRNA stability and low protein stability contribute to the lack of detectable CV-N expression in chloroplasts. Both problems can be alleviated by N-terminal fusions to the CV-N coding region, thus highlighting a suitable strategy for optimization of plastid transgene expression., (© 2011 The Authors. Plant Biotechnology Journal © 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.)

Chloroplast transformation of the high-biomass tobacco variety Maryland Mammoth has been assessed as a production platform for the human immunodeficiency virus type 1 (HIV-1) p24 antigen. Maryland Mammoth offers the prospect of higher yields of intact functional protein per unit floor area of contained glasshouse per unit time prior to flowering. Two different transformation constructs, pZSJH1p24 (for the insertion of a native p24 cDNA between the rbcL and accD genes) and pZF5 (for the insertion of a chloroplast-codon-optimized p24 gene between trnfM and trnG) were examined for the production of p24. Plants generated with construct pZSJH1p24 exhibited a normal green phenotype, but p24 protein accumulated only in the youngest leaves (up to approximately 350 microg/g fresh weight or approximately 2.5% total soluble protein) and was undetectable in mature leaves. In contrast, some of the plants generated with pZF5 exhibited a yellow phenotype (pZF5-yellow) with detectable p24 accumulation (up to approximately 450 microg/g fresh weight or approximately 4.5% total soluble protein) in all leaves, regardless of age. Total protein in pZF5-yellow leaves was reduced by approximately 40%. The pZF5-yellow phenotype was associated with recombination between native and introduced direct repeat sequences of the rbcL 3' untransformed region in the plastid genome. Chloroplast-expressed p24 was recognized by a conformation-dependent monoclonal antibody to p24, and p24 protein could be purified from pZF5-yellow leaves using a simple procedure, involving ammonium sulphate precipitation and ion-exchange chromatography, without the use of an affinity tag. The purified p24 was shown to be full length with no modifications, such as glycosylation or phosphorylation, using N-terminal sequencing and mass spectrometry.

Transgene expression from the plant's plastid genome represents a promising strategy in molecular farming because of the plastid's potential to accumulate foreign proteins to high levels and the increased biosafety provided by the maternal mode of organelle inheritance. In this article, we explore the potential of transplastomic plants to produce human immunodeficiency virus (HIV) antigens as potential components of an acquired immunodeficiency syndrome (AIDS) vaccine. It is shown that the HIV antigens p24 (the major target of T-cell-mediated immune responses in HIV-positive individuals) and Nef can be expressed to high levels in plastids of tobacco, a non-food crop, and tomato, a food crop with an edible fruit. Optimized p24-Nef fusion gene cassettes trigger antigen protein accumulation to up to approximately 40% of the plant's total protein, demonstrating the great potential of transgenic plastids to produce AIDS vaccine components at low cost and high yield.

RNA editing sites and their site-specific trans-acting recognition factors are thought to have coevolved. Hence, evolutionary loss of an editing site by a genomic mutation is normally followed by the loss of the specific recognition factor for this site, due to the absence of selective pressure for its maintenance. Here, we have tested this scenario for the only tomato-specific plastid RNA editing site. A single C-to-U editing site in the tomato rps12 gene is absent from the tobacco and nightshade plastid genomes, where the presence of a genomic T nucleotide obviates the need for editing of the rps12 mRNA. We have introduced the tomato editing site into the tobacco rps12 gene by plastid transformation and find that, surprisingly, this heterologous site is efficiently edited in the transplastomic plants. This suggests that the trans-acting recognition factor for the rps12 editing site has been maintained, presumably because it serves another function in tobacco plastids. Bioinformatics analyses identified an editing site in the rpoB gene of tobacco and tomato whose sequence context exhibits striking similarity to that of the tomato rps12 editing site. This may suggest that requirement for rpoB editing resulted in maintenance of the rps12 editing activity or, alternatively, the pre-existing rpoB editing activity facilitated the evolution of a novel editing site in rps12.

Most plastid genes are part of operons and expressed as polycistronic mRNAs. Many primary polycistronic transcripts undergo post-transcriptional processing in monocistronic or oligocistronic units. At least some polycistronic transcripts are not translatable, and endonucleolytic processing may therefore be a prerequisite for translation to occur. As the requirements for intercistronic mRNA processing into stable monocistronic transcript are not well understood, we have sought to define minimum sequence elements that trigger processing and thus are capable of generating stable translatable monocistronic mRNAs. We describe here the in vivo identification of a small intercistronic expression element that mediates intercistronic cleavage into stable monocistronic transcripts. Separation of foreign genes by this element facilitates transgene stacking in operons, and thus will help to expand the range of applications of transplastomic technology.

Plastids (chloroplasts) are maternally inherited in most crops. Maternal inheritance excludes plastid genes and transgenes from pollen transmission. Therefore, plastid transformation is considered a superb tool for ensuring transgene containment and improving the biosafety of transgenic plants. Here, we have assessed the strictness of maternal inheritance and the extent to which plastid transformation technology confers an increase in transgene confinement. We describe an experimental system facilitating stringent selection for occasional paternal plastid transmission. In a large screen, we detected low-level paternal inheritance of transgenic plastids in tobacco. Whereas the frequency of transmission into the cotyledons of F(1) seedlings was approximately 1.58 x 10(-5) (on 100% cross-fertilization), transmission into the shoot apical meristem was significantly lower (2.86 x 10(-6)). Our data demonstrate that plastid transformation provides an effective tool to increase the biosafety of transgenic plants. However, in cases where pollen transmission must be prevented altogether, stacking with other containment methods will be necessary to eliminate the residual outcrossing risk.

Primary transcripts in higher plant cell organelles undergo a series of essential RNA processing steps, including 5'- and 3'-terminal maturation, intron splicing and RNA editing. Most of the protein factors involved in these RNA processing mechanisms are still unknown. Also, little is known about the environmental and developmental factors regulating RNA processing events in plastids. Here we have tested the influence of the environmental factor temperature on RNA processing in the ndhB mRNA from tobacco plastids. We find that, whereas temperature increase to 37 degrees C fully inhibits RNA editing at only one out of nine sites in the tobacco ndhB transcript, further temperature increase to 42 degrees C selectively blocks editing at two additional sites. As these temperature effects are strictly site-specific, our findings provide evidence for the existence of site-specific editing factors which exhibit differential temperature sensitivity. Furthermore, splicing of the group II intron present in the ndhB pre-mRNA is largely blocked at 42 degrees C, suggesting that chloroplast RNA processing steps are more sensitive to high temperature than chloroplast transcription. Our findings may also suggest that impaired plastid RNA processing contributes to the loss of chloroplast function upon plant growth at high temperature.