Broken chromosomes are exceptionally dangerous to the cell, as a single break can result in the simultaneous loss of hundreds of genes. For this reason, all organisms can detect even a single double-strand break (DSB), initiating a response that includes the induction of repair, the arrest of cell division, changes in developmental fate, and in some cases programmed cell death (PCD). These responses vary from organism to organism, and with cell type. The cell-type specificity of damage response has not been well characterized in any system, in spite of its importance. Recent advances make it feasible to simultaneously measure changes in the transcription levels of every gene. Plants exhibit a robust transcriptional response to DSBs, involving the induction or suppression of thousands of genes. New technologies also allow purification of nuclei from specific cell types. Combining "transcriptomic" and phenotypic analysis will increase understanding of the role of transcriptional regulation in the repair and tolerance of DSBs. The transcriptional response in a small and specialized population of cells within the plant which undergo PCD in response to breaks will be compared to that of surrounding cells that do not die in spite of experiencing the same DNA-damaging treatment. This approach will allow identification of genes required for break-induced programmed cell death, providing insights into the mechanism of the process and markers for early and late steps in the induction of PCD in plants. Several transcription factors are also induced by chromosomal breaks. Using mutants defective in these regulatory genes, the subroutines within the response and their biological significance will be investigated. Finally, the natural source of chromosomal breaks that makes this response selectively advantageous in plants is unknown. Recent work indicates that both the desiccation experienced during seed storage and damage induced by growth in acidic, metal-rich soils provoke the transcriptional response described above, and this hypothesis will be investigated further.
Broader impacts: This project will contribute to the understanding of cell cycle regulation and the mechanisms of programmed cell death in plants. Novel assays for genomic stability in plants and exploration of potential natural sources of DSBs in plants should result in findings of relevance to agriculture. Undergraduates, a graduate student, and a postdoctoral scholar will be employed and trained through this grant, and will be recruited from a very diverse population.
