Madrid Gabriela, Raimundo Gabriel Angelo Saraiva, Reyes Fabian Andres, Picoli Edgard Augusto de Toledo, Resende Marcio F R, Balmant Kelly M
Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, Florida, United States of America.
Horticultural Sciences Department, University of Florida, Gainesville, Florida, United States of America.
PLoS One. 2025 Sep 10;20(9):e0302118. doi: 10.1371/journal.pone.0302118. eCollection 2025.
The study of plant biology has traditionally focused on investigations conducted at the tissue, organ, or whole plant level. However, single-cell transcriptomics has recently emerged as an important tool for plant biology, enabling researchers to uncover the expression profiles of individual cell types within a tissue. The application of this tool has revealed new insights into cell-to-cell gene expression heterogeneity and has opened new avenues for research in plant biology. A critical step in the successful application of single-cell and single-nuclei RNA-seq (scRNA-seq and snRNA-seq) is the isolation of individual cells or nuclei, respectively, from tissue to recover their transcriptional profile. A critical step during nuclei isolation for snRNA-seq studies is Fluorescent-Activated Cell Sorting (FACS). During this step, nuclei stained with DAPI (4',6-diamidino-2-phenylindole) can be sorted and separated from cell debris and organelles. Leaf tissue presents a unique challenge due to its high content of chloroplasts, which can interfere with obtaining high-quality results. Because DAPI can also bind to the plastid genome, these organelles will be sorted as nuclei. Thus, in tissues with a high content of chloroplasts, we have a high contamination of these organelles and an overestimation of the number of nuclei. In this study, we introduce a straightforward alternative method for isolating nuclei from Zea mays leaves with reduced chloroplast contamination. By effectively removing chloroplasts during the FACS step of our protocol, using the autofluorescence from the chloroplasts, we achieved improved alignment of reads to the genome and transcriptome. Our enhanced protocol offers a valuable solution for applying snRNA-seq in tissues with a high content of chloroplasts.
传统上，植物生物学的研究主要集中在组织、器官或整株植物水平上进行的调查。然而，单细胞转录组学最近已成为植物生物学的一种重要工具，使研究人员能够揭示组织内单个细胞类型的表达谱。该工具的应用揭示了细胞间基因表达异质性的新见解，并为植物生物学研究开辟了新途径。成功应用单细胞和单细胞核RNA测序（scRNA-seq和snRNA-seq）的关键步骤分别是从组织中分离单个细胞或细胞核，以恢复其转录谱。snRNA-seq研究中细胞核分离的一个关键步骤是荧光激活细胞分选（FACS）。在这一步骤中，用DAPI（4',6-二脒基-2-苯基吲哚）染色的细胞核可以与细胞碎片和细胞器分选并分离。叶片组织由于其叶绿体含量高而带来了独特的挑战，叶绿体含量高会干扰获得高质量的结果。因为DAPI也可以与质体基因组结合，这些细胞器会被分选成细胞核。因此，在叶绿体含量高的组织中，我们会有这些细胞器的高污染以及对细胞核数量的高估。在本研究中，我们引入了一种简单的替代方法，用于从玉米叶片中分离细胞核，减少叶绿体污染。通过在我们方案的FACS步骤中利用叶绿体的自发荧光有效去除叶绿体，我们实现了读取与基因组和转录组的更好比对。我们改进后的方案为在叶绿体含量高的组织中应用snRNA-seq提供了一个有价值的解决方案。
