EAGER: Methods for chromatin profiling of plant gametes and zygotes

植物繁殖需要将雄配子(由花粉传递的精子细胞)和雌配子(胚珠内的卵细胞)融合，形成一种独特的细胞，称为受精卵，最终再生出新的植物。制造功能性配子和受精卵的指令包含在它们的基因组序列中，以及组成它们DNA染色质包装的蛋白质的修改，这将决定哪些基因将被打开或关闭。整个基因组中染色质蛋白质的这些修饰可以通过一种称为染色质图谱的技术来揭示。然而，染色质图谱需要大量的细胞，目前还无法获得高度专业化的细胞，如雌配子和受精卵，这些细胞的分离既困难又费力。因此，我们对标志着植物生命周期这些关键阶段的染色质修饰几乎一无所知。该项目将开发分离配子和受精卵的新方法，并采用需要较少细胞数量的染色质图谱方法。这种方法的成功开发将使对这些重要细胞染色质的全基因组修饰的详细表征成为可能，更广泛地说，它们可以用于植物中任何稀有细胞类型的染色质研究。了解配子如何形成胚胎对于提高种子产量、从组织培养中再生植物、用于快速育种的单倍体植物以及通过克隆繁殖生产杂交种子以降低杂交种子成本具有重要的农业应用价值。目前研究卵细胞和受精卵的方法依赖于费力的解剖，每次从一个胚珠中分离一个细胞。我们将通过一种新的策略来解决这个大海捞针的问题，该策略最初在拟南芥中使用基于亲和力标签的协议，专门从胚珠中数千个体细胞中提取卵细胞或合子核。由于采集大量胚珠相对容易，该方案应该可以扩展到从水稻和其他植物中大量分离卵细胞或受精卵。染色质图谱的第二个障碍是，目前在植物中使用的方法需要数万个细胞。最近，一种名为Cut&amp；Tag的方法被用于动物的低输入染色质分析，这种方法需要减少一到两个数量级的细胞。然而，这种方法没有在非常低的输入条件下对植物染色质进行测试。我们将尝试将新的切割和标记方法应用于植物细胞的低输入染色质分析，以便能够对纯化的卵子和受精卵的细胞核进行染色质研究，这些研究将通过拟议的亲和标记方法分离出来。更广泛地说，上述拟议方法的成功开发将产生超出这些生殖细胞的影响，因为它们将打开在单细胞类型水平上对植物进行染色质研究的更广泛的可能性。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.