The new pPZP Agrobacterium binary vectors are versatile, relatively small, stable and are fully sequenced. The vectors utilize the pTiT37 T-DNA border regions, the pBR322 bom site for mobilization from Escherichia coli to Agrobacterium, and the ColE1 and pVS1 plasmid origins for replication in E. coli and in Agrobacterium, respectively. Bacterial marker genes in the vectors confer resistance to chloramphenicol (pPZP100 series) or spectinomycin (pPZP200 series), allowing their use in Agrobacterium strains with different drug resistance markers. Plant marker genes in the binary vectors confer resistance to kanamycin or to gentamycin, and are adjacent to the left border (LB) of the transferred region. A lacZ alpha-peptide, with the pUC18 multiple cloning site (MCS), lies between the plant marker gene and the right border (RB). Since the RB is transferred first, drug resistance is obtained only if the passenger gene is present in the transgenic plants.

We report here a 100-fold increased frequency of plastid transformation in tobacco by selection for a chimeric aadA gene encoding aminoglycoside 3"-adenylyltransferase, as compared with that obtained with mutant 16S rRNA genes. Expression of aad4 confers resistance to spectinomycin and streptomycin. In transforming plasmid pZS197, a chimeric aadA is cloned between rbcL and open reading frame ORF512 plastid gene sequences. Selection was for spectinomycin resistance after biolistic delivery of pZS197 DNA into leaf cells.

deleting the rpoB gene encoding the essential β subunit 1 Present address: University of Nebraska, Lincoln, of the tobacco PEP, we established the existence of a N209 Beadle Center, Lincoln, NE 68588-0664, USA second nuclear-encoded plastid RNA polymerase (NEP) in photosynthetic higher plants (Allison et al., 1996). Corresponding authorDeletion of rpoB yielded photosynthetically defective, pigment-deficient plants. An examination of ΔrpoB plants The plastid genome in photosynthetic higher plants encodes subunits of an Escherichia coli-like RNA polyrevealed proplastid-like structures containing low levels of mRNAs for the photosynthetic genes rbcL, psbA and merase (PEP) which initiates transcription from E.coli σ 70 -type promoters. We have previously established psbD due to the lack of PEP promoter activity. In wildtype tobacco leaves, the ribosomal RNA operon (rrn) is the existence of a second nuclear-encoded plastid RNA polymerase (NEP) in photosynthetic higher plants. We transcribed by PEP. Interestingly, in the ΔrpoB plants the rrn mRNA accumulated close to wild-type levels due to report here that many plastid genes and operons have at least one promoter each for PEP and NEP (Class II transcription by NEP acting at a downstream non-σ 70 -type promoter. The rRNA operon is the first plastid transcription unit). However, a subset of plastid genes, including photosystem I and II genes, are transcribed transcription unit for which a promoter for both PEP and NEP was identified. from PEP promoters only (Class I genes), while in some instances (e.g. accD) genes are transcribed excluWe report here that the rRNA operon is not unique, but represents a class of plastid transcription units which have sively by NEP (Class III genes). Sequence alignment identified a 10 nucleotide NEP promoter consensus at least one promoter each for PEP and NEP. These genes or operons have a potential for expression by either of around the transcription initiation site. Distinct NEP and PEP promoters reported here provide a general the two plastid RNA polymerases. Furthermore, some genes are transcribed by only one of the two RNA mechanism for group-specific gene expression through recognition by the two RNA polymerases.polymerases. We propose that transcription by NEP and PEP, through recognition of distinct promoters, is a general Keywords: Nicotiana tabacum/nuclear-encoded plastid RNA polymerase (NEP)/plastid-encoded RNA mechanism of group-specific gene regulation during chloroplast development. A tentative NEP promoter conpolymerase (PEP)/plastid gene expression/rpoB deficient mutant sensus is derived by the alignment of the transcription initiation sites.

Stable genetic transformation of the plastid genome is reported in a higher plant, Nicotiana tabacum. Plastid transformation was obtained after bombardment of leaves with tungsten particles coated with pZS148 plasmid DNA. Plasmid pZS148 (9.6 kilobases) contains a 3.7-kilobase plastid DNA fragment encoding the 16S rRNA. In the 16S rRNA-encoding DNA (rDNA) a spectinomycin resistance mutation is flanked on the 5' side by a streptomycin resistance mutation and on the 3' side by a Pst I site generated by ligating an oligonucleotide in the intergenic region. Transgenic lines were selected by spectinomycin resistance and distinguished from spontaneous mutants by the flanking, cotransformed streptomycin resistance and Pst I markers. Regenerated plants are homoplasmic for the spectinomycin resistance and the Pst I markers and heteroplasmic for the unselected streptomycin resistance trait. Transgenic plastid traits are transmitted to the seed progeny. The transgenic plastid genomes are products of a multistep process, involving DNA recombination, copy correction, and sorting out of plastid DNA copies.Transgenic plants are widely used to study nuclear gene function and regulation and to improve agronomically important crop plants (1, 2). Production of transgenic plants is achieved routinely by several alternative methods developed specifically for transformation of the nuclear genome of higher plants. Transgenic technology has not, however, been applied yet to genomes of plastids and mitochondria of higher plants.Introduction and stable integration of exogenous DNA have been reported recently in the plastid genome of a unicellular alga, Chlamydomonas reinhardtii (3-5). In this paper, we report stable genetic transformation of the plastid genome in a higher plant, Nicotiana tabacum. We propose the term transplastomic for the lines carrying a transgenic plastome. The transplastomic lines were selected by a nonlethal marker, resistance to the antibiotic spectinomycin that allows differentiation by color. On selective media the resistant clones are green and the sensitive clones are white (6). When cells are grown in a selective medium, plastids carrying the resistance genes are preferentially maintained (7). Selection, aided by an active system of recombination (8), was expected to facilitate the recovery of transplastomic clones.The antibiotic resistance markers were derived from N. tabacum SPC2, a line that exhibits a high level of resistance to spectinomycin and streptomycin due to mutations 278 base pairs (bp) apart in the 16S rRNA-encoding DNA (rDNA) (Z.S. and P.M., unpublished results). The 16S rDNA gene is in the repeated region and therefore is present in two copies per plastid genome. The SPC2 16S rDNA clone was further marked by a silent mutation resulting in a new restriction site to facilitate identification of transplastomic clones. Although the efficiency of transformation is low, the results reported in this paper demonstrate the requirements of plastid transformation in higher plants. MATERIALS AND MET...

The plastid genomes of several plants contain homologues, termed ndh genes, of genes encoding subunits of the NADH:ubiquinone oxidoreductase or complex I of mitochondria and eubacteria. The functional significance of the Ndh proteins in higher plants is uncertain. We show here that tobacco chloroplasts contain a protein complex of 550 kDa consisting of at least three of the ndh gene products: NdhI, NdhJ and NdhK. We have constructed mutant tobacco plants with disrupted ndhC, ndhK and ndhJ plastid genes, indicating that the Ndh complex is dispensible for plant growth under optimal growth conditions. Chlorophyll fluorescence analysis shows that in vivo the Ndh complex catalyses the post-illumination reduction of the plastoquinone pool and in the light optimizes the induction of photosynthesis under conditions of water stress. We conclude that the Ndh complex catalyses the reduction of the plastoquinone pool using stromal reductant and so acts as a respiratory complex. Overall, our data are compatible with the participation of the Ndh complex in cyclic electron flow around the photosystem I complex in the light and possibly in a chloroplast respiratory chain in the dark.

Plastids of higher plants are semi-autonomous organelles with a small, highly polyploid genome and their own transcription-translation machinery. This review provides an overview of the technology for the genetic modification of the plastid genome including: vectors, marker genes and gene design, the use of gene knockouts and over-expression to probe plastid function and the application of site-specific recombinases for excision of target DNA. Examples for applications in basic science include the study of plastid gene transcription, mRNA editing, photosynthesis and evolution. Examples for biotechnological applications are incorporation of transgenes in the plastid genome for containment and high-level expression of recombinant proteins for pharmaceutical and industrial applications. Plastid transformation is routine only in tobacco. Progress in implementing the technology in other crops is discussed.

The plastid genome in higher plants encodes subunits of an Escherichia coli‐like RNA polymerase which initiates transcription of plastid genes from sequences resembling E.coli sigma70‐type promoters. By deleting the gene for the essential beta subunit of the tobacco E.coli‐like RNA polymerase, we have established the existence of a second plastid transcription system which does not utilize E.coli‐like promoters. In contrast to the E.coli‐like RNA polymerase, the novel transcription machinery preferentially transcribes genetic system genes rather than photosynthetic genes. Although the mutant plants are photosynthetically defective, transcription by this polymerase is sufficient for plastid maintenance and plant development.

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