The nuclear impact on plastid-deficient plants

Androgenesis, the production of plants from microspores, which are the precursors of the male sex cells in plants, is a method of an increasing commercial importance in plant breeding programmes. At present, the list of embryos or plants which have been regenerated by this method includes a large number of plant species, among them many species important in agriculture. However, the practical application of microspore culture in wheat and other cereals is limited by the occurrence of pigment-deficient (albino) plants among the regenerants. Earlier studies demonstrated that in such plants the plastids show alterations ("albino plastids") compared to the chloroplasts of green plants. In our previous study, we examined plastid DNA and plastid gene expression in microspore-derived albino plants of wheat (Triticum aestivum L.). We could verify that the majority of albino plants had deletions in their plastid genomes. However, in a group of albino plants no plastid DNA deletions could be detected. Instead, all albino plants showed an altered plastid RNA pattern and a deficiency in plastid protein synthesis irrespective of the presence or absence of deletions. Our results indicate that the absence of plastid protein synthesis is a primary and fundamental defect in microspore-derived albino plants. We hypothesise that the deficiencies found in albino plants are caused by a defective interaction between plastid and nuclear-encoded factors. The proposed study includes experiments to find (1) molecular reasons for the deficiency in plastid protein synthesis, (2) the role of interactions between the plastid and the nuclear genome in albino plant formation and (3) correlations to plastid DNA deletions. The latter part will be supplemented by a FWF project at the Vienna Biocenter (P14983), which is investigating the formation of plastid DNA deletions. However, the main focus of the proposed study is on albino plants without plastid DNA deletions. The experiments presented here should, for the first time, reveal the important roles of plastid translation deficiencies and of the nuclear genome in the formation of albino plants during microspore culture. Answers to these questions can help to better understand regulatory feedback mechanisms on plastid gene expression and to improve the application of doubled haploid plant production methods in wheat and other cereals.

In the course of this project, we carried out a detailed molecular characterization of albino plants of wheat obtained from microspore culture, which is a widely used method for plant breeding. The occurrence of albino plants is a frequently observed phenomenon when this culture method is applied to cereal plant species, like barley, rice and wheat. We found extensive alterations of gene expression in plastids as well as in the nuclear genome of such plants. Plastid-encoded proteins were completely absent in albino plants, while nuclear-encoded plastid proteins were present. As a consequence of the plastid translation-deficiency, an RNA polymerase, which is encoded in the plastid genome, could not be produced and lead to a completely altered plastid RNA pattern in albino plants compared to green plants. Since most plastid functions are encoded in the nuclear genome, we examined the expression of selected nuclear genes encoding plastid proteins, but most of the plastid translation-related genes examined, showed a similar expression pattern as in wild-type plants. To get more information about the expression of the nuclear genome in albino plants in general, we carried out differential display experiments, which showed that a relatively high proportion of about 0.4% - 0.8% of all nuclear genes are differentially expressed in green and in albino wheat plants. This indicates that the nuclear genome plays an important role in albino plant formation during microspore culture. About one half of the differentially expressed genes showed a reduced expression in albino plants, while the other half showed an increased expression. The determination of possible functions of such differentially expressed nuclear genes showed that they are involved in a variety of plant functions and metabolic pathways. As expected from the chlorophyll-deficient phenotype, several of the isolated genes with reduced expression in albino plants were photosynthesis-related genes. Furthermore, genes of the energy household of plants have an altered expression, for example, of glycolysis and of the mitochondrial respiratory chain. An interesting observation was the reduced transcript levels of several genes involved in amino acid metabolism. Since amino acid metabolism takes place to a great extent in plastids, the reduction of this pathway could be an indication for additional defects in plastids of albino plants. Several of the differentially expressed genes identified played a role in nuclear gene expression at different levels, e. g. DNA methylation, transcription regulation, RNA splicing, protein synthesis, protein folding and protein degradation indicating extensive modulations of nuclear gene expression. An interesting observation was that many of the genes with an increased expression in albino plants are involved in stress responses of plants. Our results could help to improve microspore culture in cereals, since we detected several of the deficiencies and interactions, which are responsible for the formation of albino plants during microspore culture. Since our results indicate that regulatory mechanisms could be responsible for acquiring the deficiencies leading to albinism, this could open the way to develop strategies and approaches to prevent formation of albino plants, for example by reducing stress situations during microspore culture. Our results can be a starting point for further research on chlorophyll-deficiency, chloroplast gene expression, interactions between plastids and the nucleus and for the isolation of previously unidentified nuclear genes of wheat.