单细胞 RNA-seq (scRNA-seq)作为描述人类细胞转录组的有力工具已经受到重视，但该技术在植物细胞中的应用还不广泛。在此，我们描述了在花生叶原生质体细胞分离系统的成功发展。根据已报道的标记基因，应用 scRNA-seq 技术将6,815个单细胞分为8个细胞群。此外，还利用伪时间分析方法描述了不同细胞类型转录因子在叶片生长过程中的发育轨迹和相互作用网络。这一轨迹使人们能够重新研究叶肉和表皮原基驱动的发育过程。这些结果提示栅栏细胞可能分化为海绵细胞，而表皮细胞起源早于原基。随后，该方法结合多种技术，有效地验证了 scRNA-seq 方法在同质细胞群中的有效性。几个转录因子的表达水平与表皮个体发育密切相关，符合获得的 scRNA-seq 值。此外，花生 AHL23(AT-HOOK 核定位蛋白23)定位于细胞核内，通过调节植物激素途径促进 Arabidopsis 叶片的生长。
总之，我们的研究表明，应用 scRNA-seq 可以提供有关细胞分化在花生叶片的新假说。我们相信，这种方法将使异源四倍体花生等植物叶片细胞的功能研究取得重大进展。
Single cell RNA-seq (scRNA-seq) has been highlighted as a powerful tool for the description of the human cell transcriptome, but the technology has not been broadly applied in plant cells. Herein, we describe the successful development of a robust protoplast cell isolation system in the peanut leaf. A total of 6,815 single-cells were divided into eight cell-clusters based on reported marker genes by applying scRNA-seq. Further, a pseudo-time analysis was used to describe the developmental trajectory and interaction network of transcription factors (TFs) of distinct cell types during leaf growth. The trajectory enabled re-investigation of the primordium-driven development processes of the mesophyll and epidermis. These results suggest that palisade cells likely differentiate into spongy cells, while the epidermal cells originated earlier than the primordium. Subsequently, the developed method integrated multiple technologies to efficiently validate the scRNA-seq result in a homogenous cell population. The expression levels of several TFs were strongly correlated with epidermal ontogeny in accordance with obtained scRNA-seq values. Additionally, peanut AHL23 (AT-HOOK MOTIF NUCLEAR LOCALIZED PROTEIN 23), which is localized in nucleus, promoted leaf growth when ectopically expressed in Arabidopsis by modulating the phytohormone pathway. Together, our study displays that application of scRNA-seq can provide new hypotheses regarding cell differentiation in the leaf blade of Arachis hypogaea. We believe that this approach will enable significant advances in the functional study of leaf blade cells in the allotetraploid peanut and other plant species.
Hao Liu, Dongxiu Hu, Puxuan Du, et al. Single-cell RNA-seq Describes the Transcriptome Landscape and Identifies Critical Transcription Factors in the Leaf Blade of the Allotetraploid Peanut (Arachis hypogaea L.). Plant Biotechnology Journal, 2021
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