EAGER: Methods for chromatin profiling of plant gametes and zygotes

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Plant reproduction requires the fusion of a male gamete, a sperm cell, delivered by the pollen, and a female gamete, an egg cell within the ovule, to form a unique cell called the zygote that ultimately regenerates a new plant. The instructions for making functional gametes and zygotes are contained within their genome sequences, as well as modifications of the proteins constituting the chromatin packaging of their DNA that will determine which genes will be switched on or off. These modifications of the chromatin proteins throughout the genome can be revealed by a technique called chromatin profiling. However, chromatin profiling requires large numbers of cells, and currently is out of reach for highly specialized cells such as female gametes and zygotes, whose isolation is both difficult and laborious. As a result, we know almost nothing about the chromatin modifications that mark these critical stages of the plant life cycle. The project will develop new methods to isolate gametes and zygotes, and adapt methods of chromatin profiling that require lower numbers of cells. Successful development of such methods will enable detailed characterization of genome-wide modifications to the chromatin of these important cells, and more generally, they could be used for chromatin studies of any rare cell types in plants. Understanding how gametes form embryos has agricultural applications for increased seed yields, for regeneration of plants from tissue culture, for haploid plants for rapid breeding, and for hybrid seed production through clonal propagation to reduce costs of hybrid seeds.Methods to study egg cells and zygotes currently rely on laborious dissection that is performed one cell from one ovule at a time. We will address this needle in a haystack problem through a novel strategy using an affinity-tag based protocol, initially in Arabidopsis, to specifically fish out egg cell or zygote nuclei from the thousands of somatic cells in the ovule. Because of the relative ease of harvesting large numbers of ovules, the protocol should be scalable for bulk isolation of egg cells or zygotes from rice and other plants. A second hurdle for chromatin profiling is that methods currently used in plants require tens of thousands of cells. Recently, a method called CUT&Tag, which requires one to two orders of magnitude fewer cells, has been used for low-input chromatin profiling in animals. However, this method is not tested for profiling of plant chromatin at very low-inputs. We will attempt to adapt the new CUT&Tag method for low-input chromatin profiling of plant cells to enable chromatin studies of purified egg and zygote nuclei at the scales that will be isolated by the proposed affinity tag approach. More generally, successful development of the above proposed methods will have an impact beyond these reproductive cells, as they will open up the wider possibility of chromatin studies in plants at the single cell type level.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.