Transplastomic tobacco plants expressing a fatty acid desaturase gene exhibit altered fatty acid profiles and improved cold tolerance.

The possibility of altering the unsaturation level of fatty acids in plant lipids by genetic transformation has implications for the stress tolerance of higher plants as well as for their nutritional value and industrial utilisation. While the integration and expression of transgenes in the plastome has several potential advantages over nuclear transformation, very few attempts have been made to manipulate fatty acid biosynthesis using plastid transformation. We produced transplastomic tobacco plants that express a Delta(9) desaturase gene from either the wild potato species Solanum commersonii or the cyanobacterium Anacystis nidulans, using PEG-mediated DNA uptake by protoplasts. Incorporation of chloroplast antibiotic-insensitive point mutations in the transforming DNA was used to select transformants. The presence of the transcript and the Delta(9) desaturase protein in transplastomic plants was confirmed by northern and western blot analyses. In comparison with control plants, transplastomic plants showed altered fatty acid profiles and an increase in their unsaturation level both in leaves and seeds. The two transgenes produced comparable results. The results obtained demonstrate the feasibility of using plastid transformation to engineer lipid metabolic pathways in both vegetative and reproductive tissues and suggest an increase of cold tolerance in transplastomic plants showing altered leaf fatty acid profiles. This is the first example of transplastomic plants expressing an agronomically relevant gene produced with the "binding-type" vectors, which do not contain a heterologous marker gene. In fact, the transplastomic plants expressing the S. commersonii gene contain only plant-derived sequences, a clear attraction from a public acceptability perspective.

Hybrid transcription system for controlled plastid transgene expression.

Plastid transformation technologies have developed rapidly over the last few years, reflecting their value in the study of the principal mechanisms of plastid gene expression and commercial interest in using plastids as bioreactors. Application of this technology is still limited by the difficulty of obtaining regulated, selective expression of plastid transgenes. The plastid genome is transcribed by two different types of RNA polymerase. One of them is of the eubacterial type of polymerase, and its subunits are encoded in the plastid genome [plastid-encoded RNA polymerase (PEP)]. The other one is of the phage type and nucleus-encoded [nucleus-encoded RNA polymerase (NEP)]. To obtain selective transgene expression, we have made use of the similarities and differences between the eubacterial and the plastid eubacterial type transcription systems. We created a hybrid transcription system in which the transgene is placed under the control of a eubacterial promoter which does not exist in the plastid genome and which is not recognized by the plastid endogenous transcriptional machinery. Selective transcription of the transgene is achieved by the supply of a chimeric transcription factor that interacts with PEP and directs it specifically to the foreign eubacterial-type transgene promoter. This hybrid transcription system could be used for biotechnological and fundamental research applications as well as in the characterization of the evolutionary differences between the eubacterial and the plastid eubacterial-type transcription systems.

T7 RNA polymerase-directed expression of an antibody fragment transgene in plastids causes a semi-lethal pale-green seedling phenotype.

A T7 promoter-controlled transgene, AbL, encoding a camel single-domain antibody fragment that binds to the model antigen chicken egg-white lysozyme was introduced into the plastid genome of tobacco. AbL expression was activated in the transplastomic line by introducing a nuclear transgene, ST7, encoding a light-regulated plastid-targeted T7RNAP by cross-pollination. The resulting AbL x ST7 progeny seedlings developed a pale-green phenotype and ceased growth soon after germination. High levels of AbL transcripts accumulated in AbL x ST7 seedlings and expression of functional AbL antibody was detected by ELISA. Transplastomic AbL plants were also crossed with nuclear-transformed tobacco plants containing a salicylic acid-inducible transgene encoding a plastid-targeted T7RNAP (PR-T7 transgene). The resulting AbL x PR-T7 progeny were wild-type in appearance but were slow growing and prone to wilting even when provided with adequate water. Although AbL transcription was inducible by treating AbL x PR-T7 leaves with salicylic acid, high levels of T7RNAP-dependent AbL transcripts also accumulated in the absence of induction. However, AbL antibody did not accumulate at levels detectable by immunoblotting or ELISA in AbL x PR-T7 plants despite the fact that total leaf RNA containing AbL transcripts was capable of directing AbL antibody synthesis in an E. coli-derived in vitro translation system.

Increased zinc content in transplastomic tobacco plants expressing a polyhistidine-tagged Rubisco large subunit.

Rubisco is a hexadecameric enzyme composed of two subunits: a small subunit (SSU) encoded by a nuclear gene (rbcS), and a large subunit (LSU) encoded by a plastid gene (rbcL). Due to its high abundance, Rubisco represents an interesting target to express peptides or small proteins as fusion products at high levels. In an attempt to modify the plant metal content, a polyhistidine sequence was fused to Rubisco, the most abundant protein of plants. Plastid transformation was used to express a polyhistidine (6x) fused to the C-terminal extremity of the tobacco LSU. Transplastomic tobacco plants were generated by cotransformation of polyethylene glycol-treated protoplasts using two vectors: one containing the 16SrDNA marker gene, conferring spectinomycin resistance, and the other the polyhistidine-tagged rbcL gene. Homoplasmic plants containing L8-(His)6S8 as a single enzyme species were obtained. These plants contained normal Rubisco amounts and activity and displayed normal photosynthetic properties and growth. Interestingly, transplastomic plants accumulated higher zinc amounts than the wild-type when grown on zinc-enriched media. The highest zinc increase observed exceeded the estimated chelating ability of the polyhistidine sequence, indicating a perturbation in intracellular zinc homeostasis. We discuss the possibility of using Rubisco to express foreign peptides as fusion products and to confer new properties to higher plants.