Val missense mutation, visualized by the development of anthocyanin pigment. Missense suppression is blocked by mutation of tRNA(ala)(GAC) at a site that prevents aminoacylation by the dicot alanyl-tRNA synthetase, indicating that features identified for expression and utilization of dicot tRNAs also function in monocots. This assay of the expression and utilization of tRNA(ala)(GAC) also can be used to study a variety of tRNAs and their genes, most of which can be relatively easily altered to be charged by alanyl tRNA synthetase.

Identification of a genetically stable Nicotiana tabacum (tobacco) plant with a uniform population of transformed plastid genomes (ptDNA) takes two cycles of plant regeneration from chimeric leaves and analysis of multiple shoots by Southern probing in each cycle. Visual detection of transgenic sectors facilitates identification of transformed shoots in the greenhouse, complementing repeated cycles of blind purification in culture. In addition, it provides a tool to monitor the maintenance of transplastomic state. Our current visual marker system requires two genes: the aurea bar (bar(au)) gene that confers a golden leaf phenotype and a spectinomycin resistance (aadA) gene that is necessary for the introduction of the bar(au) gene in the plastid genome. We developed a novel aadA gene that fulfills both functions: it is a conventional selectable aadA gene in culture, and allows detection of transplastomic sectors in the greenhouse by leaf color. Common causes of pigment deficiency in leaves are mutations in photosynthetic genes, which affect chlorophyll accumulation. We use a different approach to achieve pigment deficiency: post-transcriptional interference with the expression of the clpP1 plastid gene by aurea aadA(au) transgene. This interference produces plants with reduced growth and a distinct color, but maintains a wild-type gene set and the capacity for photosynthesis. Importantly, when the aurea gene is removed, green pigmentation and normal growth rate are restored. Because the aurea plants are viable, the new aadA(au) genes are useful to query rare events in large populations and for in planta manipulation of the plastid genome.

Successful manipulation of the plastid genome (ptDNA) has been carried out so far only in tissue-culture cells, a limitation that prevents plastid transformation being applied in major agronomic crops. Our objective is to develop a tissue-culture independent protocol that enables manipulation of plastid genomes directly in plants to yield genetically stable seed progeny. We report that in planta excision of a plastid aurea bar gene (bar(au) ) is detectable in greenhouse-grown plants by restoration of the green pigmentation in tobacco leaves. The P1 phage Cre or PhiC31 phage Int site-specific recombinase was delivered on the Agrobacterium T-DNA injected at the axillary bud site, resulting in the excision of the target-site flanked marker gene. Differentiation of new apical meristems was forced by decapitating the plants above the injection site. The new shoot apex that differentiated at the injection site contained bar(au)-free plastids in 30-40% of the injected plants, of which 7% transmitted the bar(au)-free plastids to the seed progeny. The success of obtaining seed with bar(au)-free plastids depended on repeatedly forcing shoot development from axillary buds, a process that was guided by the size and position of green sectors in the leaves. The success of in planta plastid marker excision proved that manipulation of the plastid genomes is feasible within an intact plant. Extension of the protocol to in planta plastid transformation depends on the development of new protocols for the delivery of transforming DNA encoding visual markers.

A primer for DNA sequencing by the chain-termination method in the M13mp2 cloning system was constructed and amplified. The primer was isolated as an EcoRI/AluI restriction fragment. After conversion of the AluI end into an EcoRI end the fragment was cloned in pBR325 from which it can be recovered by cleavage with EcoRI. The primer hybridizes to the single-stranded DNA of the mature M13mp2 phage next to the site of insertion thereby directing DNA synthesis along the inserted DNA.

Two instances of genetic transmission of spontaneous epimutation of the maize P-rr gene were identified. Transmission gave rise to two similar, moderately stable alleles, designated P-pr-1 and P-pr-2, that exhibited Mendelian behavior. Both isolates of P-pr conditioned a variable and variegated phenotype, unlike the uniform pigmentation conditioned by P-rr. Extensive genomic analysis failed to reveal insertions, deletions or restriction site polymorphisms between the new allele and its progenitor. However, methylation of the P gene was increased in P-pr relative to P-rr, and was greatly reduced (though not lost) in a revertant to uniform pigmentation. Variability in pigmentation conditioned by P-pr correlated with variability in transcript levels of the P gene, and both correlated inversely with variability in its methylation. Part of the variability in methylation could be accounted for by a developmental decrease in methylation in all tissues of plants carrying P-pr. We hypothesize that the variegated phenotype results from a general epigenetic pathway which causes a progressive decrease in methylation and increase in expression potential of the P gene as a function of cell divisions in each meristem of the plant. This renders all tissues chimeric for a functional gene; chimerism is visualized as variegation only in pericarp due to the tissue specificity of P gene expression. Therefore, this allele that originates from epimutation may exemplify an epigenetic mechanism for variegation in maize.