Removal of the large inverted repeat from the plastid genome reveals gene dosage effects and leads to increased genome copy number.

The chloroplast genomes of most plants and algae contain a large inverted repeat (IR) region that separates two single-copy regions and harbours the ribosomal RNA operon. We have addressed the functional importance of the IR region by removing an entire copy of the 25.3-kb IR from the tobacco plastid genome. Using plastid transformation and subsequent selectable marker gene elimination, we precisely excised the IR, thus generating plants with a substantially reduced plastid genome size. We show that the lack of the IR results in a mildly reduced plastid ribosome number, suggesting a gene dosage benefit from the duplicated presence of the ribosomal RNA operon. Moreover, the IR deletion plants contain an increased number of plastid genomes, suggesting that genome copy number is regulated by measuring total plastid DNA content rather than by counting genomes. Together, our findings (1) demonstrate that the IR can enhance the translation capacity of the plastid, (2) reveal the relationship between genome size and genome copy number, and (3) provide a simplified plastid genome structure that will facilitate future synthetic biology applications.

Characterization of mutants deficient in N-terminal phosphorylation of the chloroplast ATP synthase subunit ß.

Understanding the regulation of photosynthetic light harvesting and electron transfer is of great importance to efforts to improve the ability of the electron transport chain to supply downstream metabolism. A central regulator of the electron transport chain is ATP synthase, the molecular motor that harnesses the chemiosmotic potential generated from proton-coupled electron transport to synthesize ATP. ATP synthase is regulated both thermodynamically and post-translationally, with proposed phosphorylation sites on multiple subunits. In this study we focused on two N-terminal serines on the catalytic subunit ß in tobacco (Nicotiana tabacum), previously proposed to be important for dark inactivation of the complex to avoid ATP hydrolysis at night. Here we show that there is no clear role for phosphorylation in the dark inactivation of ATP synthase. Instead, mutation of one of the two phosphorylated serine residues to aspartate to mimic constitutive phosphorylation strongly decreased ATP synthase abundance. We propose that the loss of N-terminal phosphorylation of ATPß may be involved in proper ATP synthase accumulation during complex assembly.

Control of plastid inheritance by environmental and genetic factors.

The genomes of cytoplasmic organelles (mitochondria and plastids) are maternally inherited in most eukaryotes, thus excluding organellar genomes from the benefits of sexual reproduction and recombination. The mechanisms underlying maternal inheritance are largely unknown. Here we demonstrate that two independently acting mechanisms ensure maternal inheritance of the plastid (chloroplast) genome. Conducting large-scale genetic screens for paternal plastid transmission, we discovered that mild chilling stress during male gametogenesis leads to increased entry of paternal plastids into sperm cells and strongly increased paternal plastid transmission. We further show that the inheritance of paternal plastid genomes is controlled by the activity of a genome-degrading exonuclease during pollen maturation. Our data reveal that (1) maternal inheritance breaks down under specific environmental conditions, (2) an organelle exclusion mechanism and a genome degradation mechanism act in concert to prevent paternal transmission of plastid genes and (3) plastid inheritance is determined by complex gene-environment interactions.

Transgene insertion into the plastid genome alters expression of adjacent native chloroplast genes at the transcriptional and translational levels.

In plant biotechnology and basic research, chloroplasts have been used as chassis for the expression of various transgenes. However, potential unintended side effects of transgene insertion and high-level transgene expression on the expression of native chloroplast genes are often ignored and have not been studied comprehensively. Here, we examined expression of the chloroplast genome at both the transcriptional and translational levels in five transplastomic tobacco (Nicotiana tabacum) lines carrying the identical aadA resistance marker cassette in diverse genomic positions. Although none of the lines exhibits a pronounced visible phenotype, the analysis of three lines that contain the aadA insertion in different locations within the petL-petG-psaJ-rpl33-rps18 transcription unit demonstrates that transcriptional read-through from the aadA resistance marker is unavoidable, and regularly causes overexpression of downstream sense-oriented chloroplast genes at the transcriptional and translational levels. Investigation of additional lines that harbour the aadA intergenically and outside of chloroplast transcription units revealed that expression of the resistance marker can also cause antisense effects by interference with transcription/transcript accumulation and/or translation of downstream antisense-oriented genes. In addition, we provide evidence for a previously suggested role of genomically encoded tRNAs in chloroplast transcription termination and/or transcript processing. Together, our data uncover principles of neighbouring effects of chloroplast transgenes and suggest general strategies for the choice of transgene insertion sites and expression elements to minimize unintended consequences of transgene expression on the transcription and translation of native chloroplast genes.

Transplastomic tomatoes expressing double-stranded RNA against a conserved gene are efficiently protected from multiple spider mites.

Spider mites are serious pests and have evolved significant resistance to many chemical pesticides, thus making their control challenging. Several insect pests can be combated by plastid-mediated RNA interference (PM-RNAi), but whether PM-RNAi can be utilized to control noninsect pests is unknown. Here, we show that three species of spider mites (Tetranychus evansi, Tetranychus truncatus, and Tetranychus cinnabarinus) take up plastid RNA upon feeding. We generated transplastomic tomato plants expressing double-stranded RNA (dsRNA) targeted against a conserved region of the spider mite ß-Actin mRNA. Transplastomic plants exhibited high levels of resistance to all three spider mite species, as evidenced by increased mortality and suppression of target gene expression. Notably, transplastomic plants induced a more robust RNAi response, caused higher mortality, and were overall better protected from spider mites than dsRNA-expressing nuclear transgenic plants. Our data demonstrate the potential of PM-RNAi as an efficient pest control measure for spider mites and extend the application range of the technology to noninsect pests.

Efficient control of western flower thrips by plastid-mediated RNA interference.

Plastid-mediated RNA interference (PM-RNAi) has emerged as a promising strategy for pest control. Expression from the plastid genome of stable double-stranded RNAs (dsRNAs) targeted against essential insect genes can effectively control some herbivorous beetles, but little is known about the efficacy of the transplastomic approach in other groups of pest insects, especially nonchewing insects that do not consume large amounts of leaf material. Here we have investigated the susceptibility of the western flower thrip (WFT, Frankliniella occidentalis), a notorious pest in greenhouses and open fields, to PM-RNAi. We show that WFTs ingest chloroplasts and take up plastid-expressed dsRNAs. We generated a series of transplastomic tobacco plants expressing dsRNAs and hairpin RNAs (hpRNAs) targeted against four essential WFT genes. Unexpectedly, we discovered plastid genome instability in transplastomic plants expressing hpRNAs, suggesting that dsRNA cassettes are preferable over hpRNA cassettes when designing PM-RNAi strategies. Feeding studies revealed that, unlike nuclear transgenic plants, transplastomic plants induced a potent RNAi response in WFTs, causing efficient suppression of the targeted genes and high insect mortality. Our study extends the application range of PM-RNAi technology to an important group of nonchewing insects, reveals design principles for the construction of dsRNA-expressing transplastomic plants, and provides an efficient approach to control one of the toughest insect pests in agriculture and horticulture.

Riboswitch-mediated inducible expression of an astaxanthin biosynthetic operon in plastids.

The high-value carotenoid astaxanthin (3,3'-dihydroxy-ß,ß-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.

Contributions of the international plant science community to the fight against human infectious diseases - part 1: epidemic and pandemic diseases.

Infectious diseases, also known as transmissible or communicable diseases, are caused by pathogens or parasites that spread in communities by direct contact with infected individuals or contaminated materials, through droplets and aerosols, or via vectors such as insects. Such diseases cause ˜17% of all human deaths and their management and control places an immense burden on healthcare systems worldwide. Traditional approaches for the prevention and control of infectious diseases include vaccination programmes, hygiene measures and drugs that suppress the pathogen, treat the disease symptoms or attenuate aggressive reactions of the host immune system. The provision of vaccines and biologic drugs such as antibodies is hampered by the high cost and limited scalability of traditional manufacturing platforms based on microbial and animal cells, particularly in developing countries where infectious diseases are prevalent and poorly controlled. Molecular farming, which uses plants for protein expression, is a promising strategy to address the drawbacks of current manufacturing platforms. In this review article, we consider the potential of molecular farming to address healthcare demands for the most prevalent and important epidemic and pandemic diseases, focussing on recent outbreaks of high-mortality coronavirus infections and diseases that disproportionately affect the developing world.

Contributions of the international plant science community to the fight against infectious diseases in humans-part 2: Affordable drugs in edible plants for endemic and re-emerging diseases.

The fight against infectious diseases often focuses on epidemics and pandemics, which demand urgent resources and command attention from the health authorities and media. However, the vast majority of deaths caused by infectious diseases occur in endemic zones, particularly in developing countries, placing a disproportionate burden on underfunded health systems and often requiring international interventions. The provision of vaccines and other biologics is hampered not only by the high cost and limited scalability of traditional manufacturing platforms based on microbial and animal cells, but also by challenges caused by distribution and storage, particularly in regions without a complete cold chain. In this review article, we consider the potential of molecular farming to address the challenges of endemic and re-emerging diseases, focusing on edible plants for the development of oral drugs. Key recent developments in this field include successful clinical trials based on orally delivered dried leaves of Artemisia annua against malarial parasite strains resistant to artemisinin combination therapy, the ability to produce clinical-grade protein drugs in leaves to treat infectious diseases and the long-term storage of protein drugs in dried leaves at ambient temperatures. Recent FDA approval of the first orally delivered protein drug encapsulated in plant cells to treat peanut allergy has opened the door for the development of affordable oral drugs that can be manufactured and distributed in remote areas without cold storage infrastructure and that eliminate the need for expensive purification steps and sterile delivery by injection.

Plastid Transformation in Tomato: A Vegetable Crop and Model Species.

Tomato (Solanum lycopersicum L.), a member of the nightshade family (Solanaceae), is one of the most important vegetable crops and has long been an important model species in plant biology. Plastid biology in tomato is especially interesting due to the chloroplast-to-chromoplast conversion occurring during fruit ripening. Moreover, as tomato represents a major food crop with a fleshy fruit that can be eaten raw, the development of a plastid transformation protocol for tomato was of particular interest to plant biotechnologists. Recent methodological improvements have made tomato plastid transformation more efficient, and facilitated applications in metabolic engineering and molecular farming. This chapter describes the basic methods involved in the generation and analysis of tomato plants with transgenic chloroplast genomes and summarizes recent applications of tomato plastid transformation in plant biotechnology.

Chloroplast Transformation in Arabidopsis.

Plastids (chloroplasts) are the defining organelles of plants and eukaryotic algae. In addition to performing photosynthesis, plastids harbor numerous other metabolic pathways and therefore are often referred to as the biosynthetic center of the plant cell. The chloroplasts of seed plants possess dozens of copies of a circular genome of ∼150 kb that contains a conserved set of 120 to 130 genes. The engineering of this genome by genetic transformation is technically challenging and currently only possible in a small number of species. In this article, we describe the methods involved in generating stable chloroplast-transformed (transplastomic) plants in the model species Arabidopsis (Arabidopsis thaliana). The protocols presented here can be applied to (1) target genes in the Arabidopsis chloroplast genome by reverse genetics and (2) express reporter genes or other foreign genes of interest in plastids of Arabidopsis plants. © 2021 The Authors. Basic Protocol 1: Generation of root-derived microcallus material for biolistic transformation Basic Protocol 2: Chloroplast transformation by biolistic bombardment of root-derived microcalli Basic Protocol 3: Regeneration of transplastomic lines and seed production.

The availability of neither D2 nor CP43 limits the biogenesis of photosystem II in tobacco.

The pathway of photosystem II (PSII) assembly is well understood, and multiple auxiliary proteins supporting it have been identified, but little is known about rate-limiting steps controlling PSII biogenesis. In the cyanobacterium Synechocystis PCC6803 and the green alga Chlamydomonas reinhardtii, indications exist that the biosynthesis of the chloroplast-encoded D2 reaction center subunit (PsbD) limits PSII accumulation. To determine the importance of D2 synthesis for PSII accumulation in vascular plants and elucidate the contributions of transcriptional and translational regulation, we modified the 5'-untranslated region of psbD via chloroplast transformation in tobacco (Nicotiana tabacum). A drastic reduction in psbD mRNA abundance resulted in a strong decrease in PSII content, impaired photosynthetic electron transport, and retarded growth under autotrophic conditions. Overexpression of the psbD mRNA also increased transcript abundance of psbC (the CP43 inner antenna protein), which is co-transcribed with psbD. Because translation efficiency remained unaltered, translation output of pbsD and psbC increased with mRNA abundance. However, this did not result in increased PSII accumulation. The introduction of point mutations into the Shine-Dalgarno-like sequence or start codon of psbD decreased translation efficiency without causing pronounced effects on PSII accumulation and function. These data show that neither transcription nor translation of psbD and psbC are rate-limiting for PSII biogenesis in vascular plants and that PSII assembly and accumulation in tobacco are controlled by different mechanisms than in cyanobacteria or in C. reinhardtii.

Knockdown of the plastid-encoded acetyl-CoA carboxylase gene uncovers functions in metabolism and development.

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 ß-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.

Correction of frameshift mutations in the atpB gene by translational recoding in chloroplasts of Oenothera and tobacco.

Translational recoding, also known as ribosomal frameshifting, is a process that causes ribosome slippage along the messenger RNA, thereby changing the amino acid sequence of the synthesized protein. Whether the chloroplast employs recoding is unknown. I-iota, a plastome mutant of Oenothera (evening primrose), carries a single adenine insertion in an oligoA stretch [11A] of the atpB coding region (encoding the ß-subunit of the ATP synthase). The mutation is expected to cause synthesis of a truncated, nonfunctional protein. We report that a full-length AtpB protein is detectable in I-iota leaves, suggesting operation of a recoding mechanism. To characterize the phenomenon, we generated transplastomic tobacco lines in which the atpB reading frame was altered by insertions or deletions in the oligoA motif. We observed that insertion of two adenines was more efficiently corrected than insertion of a single adenine, or deletion of one or two adenines. We further show that homopolymeric composition of the oligoA stretch is essential for recoding, as an additional replacement of AAA lysine codon by AAG resulted in an albino phenotype. Our work provides evidence for the operation of translational recoding in chloroplasts. Recoding enables correction of frameshift mutations and can restore photoautotrophic growth in the presence of a mutation that otherwise would be lethal.

Lycopene ß-cyclase expression influences plant physiology, development, and metabolism in tobacco plants.

Carotenoids are important isoprenoids produced in the plastids of photosynthetic organisms that play key roles in photoprotection and antioxidative processes. ß-Carotene is generated from lycopene by lycopene ß-cyclase (LCYB). Previously, we demonstrated that the introduction of the Daucus carota (carrot) DcLCYB1 gene into tobacco (cv. Xanthi) resulted in increased levels of abscisic acid (ABA) and especially gibberellins (GAs), resulting in increased plant yield. In order to understand this phenomenon prior to exporting this genetic strategy to crops, we generated tobacco (Nicotiana tabacum cv. Petit Havana) mutants that exhibited a wide range of LCYB expression. Transplastomic plants expressing DcLCYB1 at high levels showed a wild-type-like growth, even though their pigment content was increased and their leaf GA1 content was reduced. RNA interference (RNAi) NtLCYB lines showed different reductions in NtLCYB transcript abundance, correlating with reduced pigment content and plant variegation. Photosynthesis (leaf absorptance, Fv/Fm, and light-saturated capacity of linear electron transport) and plant growth were impaired. Remarkably, drastic changes in phytohormone content also occurred in the RNAi lines. However, external application of phytohormones was not sufficient to rescue these phenotypes, suggesting that altered photosynthetic efficiency might be another important factor explaining their reduced biomass. These results show that LCYB expression influences plant biomass by different mechanisms and suggests thresholds for LCYB expression levels that might be beneficial or detrimental for plant growth.

Improving plant drought tolerance and growth under water limitation through combinatorial engineering of signalling networks.

Agriculture is by far the biggest water consumer on our planet, accounting for 70 per cent of all freshwater withdrawals. Climate change and a growing world population increase pressure on agriculture to use water more efficiently ('more crop per drop'). Water-use efficiency (WUE) and drought tolerance of crops are complex traits that are determined by many physiological processes whose interplay is not well understood. Here, we describe a combinatorial engineering approach to optimize signalling networks involved in the control of stress tolerance. Screening a large population of combinatorially transformed plant lines, we identified a combination of calcium-dependent protein kinase genes that confers enhanced drought stress tolerance and improved growth under water-limiting conditions. Targeted introduction of this gene combination into plants increased plant survival under drought and enhanced growth under water-limited conditions. Our work provides an efficient strategy for engineering complex signalling networks to improve plant performance under adverse environmental conditions, which does not depend on prior understanding of network function.

Multi-gene metabolic engineering of tomato plants results in increased fruit yield up to 23%.

The capacity to assimilate carbon and nitrogen, to transport the resultant sugars and amino acids to sink tissues, and to convert the incoming sugars and amino acids into storage compounds in the sink tissues, are key determinants of crop yield. Given that all of these processes have the potential to co-limit growth, multiple genetic interventions in source and sink tissues, plus transport processes may be necessary to reach the full yield potential of a crop. We used biolistic combinatorial co-transformation (up to 20 transgenes) for increasing C and N flows with the purpose of increasing tomato fruit yield. We observed an increased fruit yield of up to 23%. To better explore the reconfiguration of metabolic networks in these transformants, we generated a dataset encompassing physiological parameters, gene expression and metabolite profiling on plants grown under glasshouse or polytunnel conditions. A Sparse Partial Least Squares regression model was able to explain the combination of genes that contributed to increased fruit yield. This combinatorial study of multiple transgenes targeting primary metabolism thus offers opportunities to probe the genetic basis of metabolic and phenotypic variation, providing insight into the difficulties in choosing the correct combination of targets for engineering increased fruit yield.

Stem Cell Transplantation and Vinblastine Monotherapy for Relapsed Pediatric Anaplastic Large Cell Lymphoma: Results of the International, Prospective ALCL-Relapse Trial.

PURPOSE: To analyze the efficacy of a risk-stratified treatment of children with relapsed anaplastic large cell lymphoma (ALCL). The ALCL-Relapse trial (ClinicalTrials.gov identifier: NCT00317408) stratified patients according to the time of relapse and CD3 expression to prospectively test reinduction approaches combined with consolidation by allogeneic or autologous hematopoietic stem cell transplantation (SCT) and vinblastine monotherapy. PATIENTS AND METHODS: Patients with progression during frontline therapy (very high risk) or a CD3-positive relapse (high risk) were scheduled for allogeneic SCT after reinduction chemotherapy. Patients with a CD3-negative relapse within 1 year after initial diagnosis or prior exposure to vinblastine (intermediate risk) received autologous SCT after carmustine-etoposide-cytarabine-melphalan. This arm was terminated prematurely, and subsequent patients received vinblastine monotherapy instead. Patients with a CD3-negative relapse > 1 year after initial diagnosis (low risk) received vinblastine monotherapy. RESULTS: One hundred sixteen patients met the inclusion criteria; 105 evaluable patients with CNS-negative disease had a 5-year event-free survival (EFS) of 53% ± 5% and a 5-year overall survival (OS) of 78% ± 4%. Before termination of autologous SCT, EFS rates of patients in the very-high- (n = 17), high- (n = 26), intermediate- (n = 32), and low- (n = 21) risk groups were 41% ± 12%, 62% ± 10%, 44% ± 9%, and 81% ± 9%; the respective OS rates were 59% ± 12%, 73% ± 9%, 78% ± 7%, and 90% ± 6%. Analyzing only the patients in the intermediate-risk group consolidated per protocol by autologous SCT, EFS and OS of 23 patients were 30% ± 10% and 78% ± 9%, respectively. All 5 patients with intermediate risk receiving vinblastine monotherapy after the amendment experienced relapse again. CONCLUSION: Shorter time to relapse was the strongest predictor of subsequent relapse. Allogeneic SCT offers a chance for cure in patients with high-risk ALCL relapse. For early relapsed ALCL autologous SCT was not effective. Vinblastine monotherapy achieved cure in patients with late relapse; however, it was not effective for early relapses.

The Functions of Chloroplast Glutamyl-tRNA in Translation and Tetrapyrrole Biosynthesis.

The chloroplast glutamyl-tRNA (tRNAGlu) is unique in that it has two entirely different functions. In addition to acting in translation, it serves as the substrate of glutamyl-tRNA reductase (GluTR), the enzyme catalyzing the committed step in the tetrapyrrole biosynthetic pathway. How the tRNAGlu pool is distributed between the two pathways and whether tRNAGlu allocation limits tetrapyrrole biosynthesis and/or protein biosynthesis remains poorly understood. We generated a series of transplastomic tobacco (Nicotiana tabacum) plants to alter tRNAGlu expression levels and introduced a point mutation into the plastid trnE gene, which has been reported to uncouple protein biosynthesis from tetrapyrrole biosynthesis in chloroplasts of the protist Euglena gracilis We show that, rather than comparable uncoupling of the two pathways, the trnE mutation is lethal in tobacco because it inhibits tRNA processing, thus preventing translation of Glu codons. Ectopic expression of the mutated trnE gene uncovered an unexpected inhibition of glutamyl-tRNA reductase by immature tRNAGlu We further demonstrate that whereas overexpression of tRNAGlu does not affect tetrapyrrole biosynthesis, reduction of GluTR activity through inhibition by tRNAGlu precursors causes tetrapyrrole synthesis to become limiting in early plant development when active photosystem biogenesis provokes a high demand for de novo chlorophyll biosynthesis. Taken together, our findings provide insight into the roles of tRNAGlu at the intersection of protein biosynthesis and tetrapyrrole biosynthesis.