Plants and animals use oxygen from the atmosphere to produce the energy needed for cell function. These organisms respond in an exquisite manner when oxygen levels are diminished. This can occur in plant roots when a field is flooded or in a brain cell during a heart attack or stroke. The goal is to understand the process by which cells endure transient periods of inadequate oxygen. The focus is on the conservation of cellular energy that occurs through a change in the manner in which proteins are synthesized from gene transcripts - the intermediate between gene and protein. In plants, as well as in animals, cells that lack oxygen become more selective in protein synthesis. This occurs by storing most gene transcripts. However, some gene transcripts escape this repression; these generally encode the proteins that the cell requires to cope with the stress. This project will study the regulation of gene transcript use during oxygen deprivation. This will include evaluation of the management in the root and the shoot, as well as in specific layers of cells. This analysis will provide a level of resolution that has previously not been obtained - yielding information on the response to the stress that occurs in organs and specialized cells. The research will also address the mechanism by which cells store gene transcripts, in order to spare energy. A broader impact will be cross-disciplinary training of scientists: a biologist, will be trained in computer science and a chemist will be trained in plant physiology. Undergraduate students, some of whom will be from small colleges, will gain hands-on research experience. Another broader impact will be the provision of plant lines to the research community that can be used to monitor gene transcripts associated with the protein synthesis machinery in specialized cell types.
" investigated how plants respond to their dynamic environment. When plants are exposed to different levels of light or stressed by a flood, heat wave or drought, a consequence is altered synthesis of some but not all proteins. Because plants are multicelluar organisms, it has been a challenge to determine if stress responses are distinct in the different kinds of cells in a root, leaf or other organ. To better understand how individual types of cells respond to hypoxia (i.e. oxygen deprivation caused by a flood) we developed a widely applicable method to evaluate protein synthesis in targeted cells. More specifically, tools and protocols were established for the isolation of the subset of cellular gene transcripts undergoing translation into protein in specific cell types of an organ. This enabled the a detailed evaluation of the response to hypoxia in a variety of cell types in the roots, stems and leaves of the model plant Arabidopsis thaliana (thale cress). A major intellectual merit of the project was the finding that hypoxia triggers a similar core stress response in all cell types tested; this core response was accompanied by responses that were specific to different types of cells. As a broader impact, Arabidopsis varieties that can be used by other scientists to study the gene transcripts undergoing translation into protein in specific cell types were donated to the Arabidopsis Biological Resource Center repository. Also, an atlas of cell-type specific gene expression data was generated. This Arabidopsis translatome dataset is freely available on-line (www.efp.ucr.edu). This atlas was used to compare the response to hypoxia in plants, animals and fungi. From the data we generated new hypotheses on how plants respond to hypoxia to enable survival. This led to the identification of a mechanism by which plant cells sense a decline in oxygen availability and alter gene regulation. By this mechanism, proteins needed to switch on genes for hypoxia survival are destroyed when oxygen is present but accumulate when oxygen is diminished. Thus, the major intellectual merit of the project is new and detailed information on environmental regulation of gene transcription and mRNA translation that control survival of a bout of stress, such as a short-term flood. We also gained new insights into the dynamic regulation of translation of individual mRNAs in response to environmental stimuli. An additional broader impact was that postdoctoral researchers, graduate students and undergraduate students received cross-disciplinary training in biology, computer science and chemistry.
