EAGER: Methods for chromatin profiling of plant gametes and zygotes

植物的繁殖需要一个雄性配子（由花粉传递的精子细胞）和一个雌性配子（胚珠内的卵细胞）的融合，形成一个称为受精卵的独特细胞，最终再生出一株新植物。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，以及构成它们DNA染色质包装的蛋白质的修饰，这些蛋白质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的这些修饰可以通过一种称为染色质谱分析的技术来揭示。然而，染色质谱分析需要大量的细胞，目前还无法实现高度特化的细胞，如雌性配子和受精卵，它们的分离既困难又费力。因此，我们对标记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，并调整需要较少细胞数量的染色质分析方法。这些方法的成功发展将使这些重要细胞的染色质全基因组修饰的详细特征，更一般地说，它们可以用于植物中任何罕见细胞类型的染色质研究。了解配子如何形成胚胎在提高种子产量、组织培养植物再生、单倍体植物快速育种以及通过无性繁殖生产杂交种子以降低杂交种子成本等方面具有农业应用价值。研究卵细胞和受精卵的方法目前依赖于一次从一个胚珠中进行一个细胞的艰苦解剖。我们将通过一种基于亲和标签的新策略来解决这个大海捞针的问题，首先在拟南芥中，从胚珠中的数千个体细胞中特异性地捞出卵细胞或受精卵核。由于相对容易收获大量的胚珠，该方案应该可扩展到从水稻和其他植物中大量分离卵细胞或受精卵。染色质分析的第二个障碍是，目前在植物中使用的方法需要数万个细胞。最近，一种被称为CUT&amp；Tag的方法被用于动物的低输入染色质分析，这种方法需要的细胞数量要少一到两个数量级。然而，这种方法并没有在非常低的投入下测试植物染色质谱。我们将尝试将新的CUT&amp；Tag方法应用于植物细胞的低输入染色质分析，使纯化的卵子和受精卵核的染色质研究能够在通过提议的亲和标签方法分离的尺度上进行。更广泛地说，上述方法的成功发展将对这些生殖细胞以外的细胞产生影响，因为它们将在单细胞类型水平上开辟更广泛的植物染色质研究的可能性。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值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.