Genetic and Biochemical Analysis of Plant Mitochondrial Transcription

Boyce Thompson Institute Plant Research, Ithaca NY

Investigators

Abstract

Mitochondrial function is essential for plant metabolism and male fertility, yet little is known of the mechanisms regulating mitochondrial gene expression, in particular the role of the nucleus. Because mitochondria contain few genes, the vast majority of its proteins are nucleus encoded, including all factors required for the first committed step in gene expression, transcription initiation. This project addresses the role of nuclear gene products in mitochondrial transcription and by extension, the role of mitochondrial gene expression during vegetative and reproductive stages. The organism chosen for this work is maize, an important crop species for which appropriate genetic and molecular resources are available. The project was initiated using in vitro transcription to define promoter regulatory elements. Subsequently, a gene encoding the core subunit of the RNA polymerase was identified and the product, RpoTm, was found to be related to those of phages such as T3 and T7. Because RpoTm alone cannot recognize mitochondrial promoters, a search for possible transcription specificity factors was carried out, and four candidates were obtained. These include three genes that encode DNA binding proteins, and an ortholog of bacterial sigma-70. One possibility is that each of these proteins lends a particular specificity to the polymerase and if so, this would allow a fine-tuning of mitochondrial function during development and gametogenesis. This need for flexibility could explain why plant mitochondrial transcription would be more complex that the cognate systems in fungi and animals. To determine the form(s) and composition of mitochondrial RNA polymerase, and the role of each of these in plant function, experiments will be conducted that use reverse genetic and biochemical approaches. Reverse genetics will entail the phenotypic and biochemical characterization of Mutator transposon insertion loss-of-function alleles of rpoTm and one or more of the possible transcription factors. If technology permits, an antisense approach will be used to obtain results more rapidly. Mutant plants have already been obtained for RpoTm and will be analyzed first. In addition to direct measurements of transcription products, there may be specific developmental responses to the mutations. For example, the loss-of-function allele of rpoTm may reduce pollen vigor and the frequency of embryo development, but has no apparent effect on the viability of female gametophytes. Therefore, pollen morphology and competitiveness will be examined in segregating plants, as well as embryo structure. To analyze effects in vegetative tissues, mosaic plants will be constructed that will develop mutant sectors in an otherwise wild-type background. A second and parallel objective is to reconstitute the RNA polymerase using the proteins described above, following expression in bacterial or insect cells. This will lead to a greater understanding of the mechanism of promoter recognition, and aid in interpreting the mutant phenotypes. The concomitant use of genetic and biochemical techniques will provide broad training to those involved in the project. In addition, undergraduate students will be involved in the genetic aspects, giving them early exposure to plant biology that may encourage them in this career path, or at least dramatically improve their scientific literacy.