Arabidopsis COP9 is a component of a large protein complex that is essential for the light control of a developmental switch and whose conformation or size is modulated by light. The complex is acidic, binds heparin, and is localized within the nucleus. Biochemical purification of the complex to near homogeneity revealed that it contains 12 distinct subunits. One of the other subunits is COP11, mutations in which result in a phenotype identical to cop9 mutants. The COP9 complex may act to regulate the nuclear abundance of COP1, an established repressor of photomorphogenic development. During the biogenesis of the COP9 complex, a certain degree of prior subunit association is a prerequisite for proper nuclear translocation. Since both COP9 and COP11 have closely related human counterparts, the COP9 complex probably represents a conserved developmental regulator in higher eukaryotes.

Transgenic plants have become attractive systems for production of human therapeutic proteins because of the reduced risk of mammalian viral contaminants, the ability to do large scale-up at low cost, and the low maintenance requirements. Here we report a feasibility study for production of a human therapeutic protein through transplastomic transformation technology, which has the additional advantage of increased biological containment by apparent elimination of the transmission of transgenes through pollen. We show that chloroplasts can express a secretory protein, human somatotropin, in a soluble, biologically active, disulfide-bonded form. High concentrations of recombinant protein accumulation are observed (>7% total soluble protein), more than 300-fold higher than a similar gene expressed using a nuclear transgenic approach. The plastid-expressed somatotropin is nearly devoid of complex post-translational modifications, effectively increasing the amount of usable recombinant protein. We also describe approaches to obtain a somatotropin with a non-methionine N terminus, similar to the native human protein. The results indicate that chloroplasts are a highly efficient vehicle for the potential production of pharmaceutical proteins in plants.

SummaryWe describe here the development of a reproducible plastid transformation system for potato and regeneration of plants with uniformly transformed plastids. Two distinct tobacco-speci®c plastid vectors, pZS197 (Prrn/aadA/TpsbA) and pMON30125 (Prrn/GFP/ Trps16::PpsbA/aadA/TpsbA), designed for integration into the large single copy and inverted repeat regions of the plastid genome, respectively, were bombarded into leaf explants of potato line FL1607. A total of three transgenic lines were selected out of 46 plates bombarded with pZS197 and three transgenic lines out of 104 plates were obtained with pMON30125. Development of a high frequency leaf-based regeneration system, a stringent selection scheme and optimization of biolistic transformation protocol were critical for recovery of plastid transformants. Plastidexpressed green¯uorescent protein was used as a visual marker for identi®cation of plastid transformants at the early stage of selection and shoot regeneration. The establishment of a plastid transformation system in potato, which has several advantages over routinely used nuclear transformation, offers new possibilities for genetic improvement of this crop.

). ² These authors contributed equally to this work. SummaryPlastid transformation (transplastomic) technology has several potential advantages for biotechnological applications including the use of unmodi®ed prokaryotic genes for engineering, potential high-level gene expression and gene containment due to maternal inheritance in most crop plants. However, the ef®cacy of a plastid-encoded trait may change depending on plastid number and tissue type. We report a feasibility study in tobacco plastids to achieve high-level herbicide resistance in both vegetative tissues and reproductive organs. We chose to test glyphosate resistance via over-expression in plastids of tolerant forms of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Immunological, enzymatic and whole-plant assays were used to prove the ef®cacy of three different prokaryotic (Achromobacter, Agrobacterium and Bacillus) EPSPS genes. Using the Agrobacterium strain CP4 EPSPS as a model we identi®ed translational control sequences that direct a 10 000-fold range of protein accumulation (to >10% total soluble protein in leaves). Plastid-expressed EPSPS could provide very high levels of glyphosate resistance, although levels of resistance in vegetative and reproductive tissues differed depending on EPSPS accumulation levels, and correlated to the plastid abundance in these tissues. Paradoxically, higher levels of plastid-expressed EPSPS protein accumulation were apparently required for ef®cacy than from a similar nuclear-encoded gene. Nevertheless, the demonstration of high-level glyphosate tolerance in vegetative and reproductive organs using transplastomic technology provides a necessary step for transfer of this technology to other crop species.

The plastid psbA gene encodes the 32 kDa D1 polypeptide of photosystem II. It has previously been shown that the initiation of psbA mRNA translation in tobacco is regulated by sequences outside of the coding region. To identify the cis-acting regulatory sequences involved in the translational control, a series of chimeric uidA genes, encoding the beta-glucuronidase (GUS) reporter enzyme, were introduced into the plastid genome. GUS accumulation in response to the light (135- to 200-fold), and the arrest of uidA mRNA translation in light-grown seedlings following transfer for 2 h to the dark, was observed only if the transgenes contained the psbA 5'-untranslated leader region (UTR). Changes in GUS accumulation were accompanied by little or no changes in the uidA mRNA levels. The data indicate that the initiation of D1 translation in tobacco plastids is controlled via the psbA 5'-UTR.