Mesophyll cells are the main site of photosynthesis and the largest cell population in leaves, with tightly packed cylinder palisade mesophyll cells (PMCs) on the adaxial side and loosely arranged rounded spongy mesophyll cells (SMCs) on the abaxial side. Loss of dorsoventral differentiation of PMCs and SMCs causes alterations in leaf phenotypes (Yu et al., 2020). Brassica rapa encompasses many leafy vegetables with extreme morphological diversity, such as Chinese cabbage with leafy head and pak choi with flat leaves. Exploring the differentiation between PMCs and SMCs and identifying key regulatory genes are important for unravelling the mechanisms underlying leaf development and heading in vegetable crops. However, little is known about them.

Here, we prepared protoplasts from young leaves of Chinese cabbage at the rosette stage for single-cell RNA-seq (scRNA-seq) (Figure 1a). After removing low-quality cells and genes, we obtained 16 055 high-quality cells and 30 214 genes. Our scRNA-seq data showed high reproducibility and a strong correlation with the bulk RNA-seq data (Figure 1b,c). These cells were classified into 17 clusters (Figure 1d). Using the orthologs of marker genes in Arabidopsis, we identified eight cell types, namely mesophyll cells (MCs), epidermis, vasculature, bundle sheath, guard cells, proliferating cells, phloem, and xylem (Figure 1d,e). The expression of Bra001929, a guard cell marker gene, was found to be consistent with the result of in situ RT-PCR (Song et al., 2021). To further verify the annotation result, we compared our results to an Arabidopsis leaf scRNA-seq dataset (Zhang et al., 2021). Pairwise comparisons of cell types and integration analysis of scRNA-seq data between two species both supported our annotation (Figure 1f–h).

Because MCs were not divided into PMCs and SMCs, we developed an optimized tape-sandwich method to specifically enrich PMCs and SMCs to identify specific marker genes for distinguishing them (Figure 1i). SMCs were enriched according to Uemoto et al. (2018). To collect PMCs, the upper epidermis was removed using tweezers. Then, digestion solution was added to the PMCs and incubated for 30 min to allow the PMCs to be released. We confirmed the successful enrichment of PMCs and SMCs by the specific expression of adaxial and abaxial marker genes (Figure 1j). We identified 6731 differentially expressed genes (DEGs) between them using RNA-seq. Based on many reliable DEGs between adaxial and abaxial domains in Arabidopsis leaves identified by Tian et al. (2019), we selected 433 and 510 potential PMC and SMC marker genes (Table S1), respectively, from overlapping orthologous DEGs shared by B. rapa and Arabidopsis.

To identify PMCs and SMCs, the MC population was re-clustered into 9 subclusters (M0-M8) (Figure 1k). Combining the expression of photosynthetic genes (BrLHCB4.3, BrLHCA6, and Bra018819) and the top 100 potential PMC marker genes, we assigned M0 and M2 to PMC (Figure 1l,m,p). The other 7 subclusters were assigned to SMC by combining the expression of abaxial genes (BrFIL.1, BrYAB3 and BrYAB5) and the top 100 potential SMC marker genes (Figure 1l,n,p). In situ hybridization assays of BrFIL.1 supported the correct definition of SMCs and PMCs (Figure 1o,p).

Using “FindMarkers” in Seurat (v4.0.3), we identified 944 and 1387 genes preferentially expressed in the PMCs and SMCs, respectively (Table S1). Surprisingly, besides identifying many photosynthesis-related genes, a large number of ribosomal protein-encoding genes (RPEGs) (239/944) were enriched in PMCs (Figure 1q,r). Out of the 239 RPEGs found, 27 were orthologous genes of 15 Arabidopsis RPEGs involved in the development of adaxial cells or PMCs (Table S1). Interestingly, protein–protein interaction analysis showed that the top 10 genes with the most connections in PMCs were RPEGs (Figure 1s). These results strongly suggested that RPEGs were involved in PMCs development. Unlike PMCs, SMCs were mainly enriched in the response to abiotic and biotic stimulus, signalling responsive genes (temperature, jasmonic acid, salicylic acid and calcium), and protein modification (Figure 1t). Additionally, almost all the top 10 genes with the most connections in SMCs responded to biotic or abiotic stress (Figure u). Overall, these results indicated that PMCs are the major machine for photosynthesis, while SMCs may be involved in tuning the machine to adapt to external environment.

Surprisingly, we found most of the reported adaxial–abaxial polarity genes to be barely detectable in MCs. To determine why this might be, we collected a series of samples representing the shoot apical meristem (SAM) and different regions of inner and outer leaves at the seedling and rosette stages for RNA-seq (Figure S1a,b). Results showed that most adaxial–abaxial polarity genes were preferentially expressed in the SAM, while they were sharply and continuously downregulated from inner to outer leaves at both stages (Figure S1c). This may be why few adaxial–abaxial polarity genes were detected in MCs, as the middle rosette leaves were collected for scRNA-seq.

Overall, we generated a transcriptome atlas of Chinese cabbage rosette leaves at single-cell resolution. A key finding was the identification of adaxial PMCs and abaxial SMCs from MCs. We identified functional differences between PMCs and SMCs, and the potential role of RPEG in PMC development. Our study also provided many cell type-specific marker genes, which will facilitate the application of scRNA-seq in B. rapa. Comparing the leaf single-cell transcriptome by each cell type, focusing on SMCs and PMCs, across different developmental stages and different subspecies will expand our knowledge of the differentiation of PMCs and SMCs in leaf adaxial–abaxial patterning, and in the processes underlying leaf development and morphogenesis in B. rapa leafy vegetables.

Accession numbers

All sequencing data are available from the NGDC (https://ngdc.cncb.ac.cn/gsa/) under BioProject accession number PRJCA009630.

中文翻译：

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单细胞转录组揭示了白菜近轴和远轴叶肉细胞的分化

叶肉细胞是光合作用的主要场所，也是叶片中最大的细胞群，近轴侧为紧密排列的圆柱栅栏叶肉细胞 (PMC)，远轴侧为松散排列的圆形海绵状叶肉细胞 (SMC)。PMC 和 SMC 背腹分化的丧失导致叶片表型的改变（Yu 等人，  2020 年）。白菜包括许多具有极端形态多样性的叶菜类蔬菜，例如带叶菜头的大白菜和带平叶的白菜。探索 PMC 和 SMC 之间的差异并确定关键调控基因对于揭示蔬菜作物叶片发育和抽穗的潜在机制非常重要。然而，人们对他们知之甚少。

在这里，我们从莲座期的大白菜幼叶中制备了原生质体，用于单细胞 RNA-seq (scRNA-seq)（图 1a）。去除低质量细胞和基因后，我们得到了16 055个高质量细胞和30 214个基因。我们的 scRNA-seq 数据显示出高重现性和与大量 RNA-seq 数据的强相关性（图 1b，c）。这些细胞被分为 17 个簇（图 1d）。使用拟南芥中标记基因的直系同源物，我们鉴定了八种细胞类型，即叶肉细胞 (MC)、表皮、脉管系统、束鞘、保卫细胞、增殖细胞、韧皮部和木质部（图 1d、e）。发现保卫细胞标记基因Bra001929的表达与原位RT-PCR结果一致(Song et al.,  2021 )). 为了进一步验证注释结果，我们将我们的结果与拟南芥叶 scRNA-seq 数据集进行了比较 (Zhang et al.,  2021 )。两个物种之间细胞类型的成对比较和 scRNA-seq 数据的整合分析都支持我们的注释（图 1f-h）。

因为 MC 没有分为 PMC 和 SMC，我们开发了一种优化的胶带夹心方法来专门富集 PMC 和 SMC，以识别用于区分它们的特定标记基因（图 1i）。根据 Uemoto 等人的说法，SMC 得到了丰富。（2018 年）。为了收集 PMC，使用镊子去除上表皮。然后，将消化溶液加入到 PMC 中并孵育 30 分钟以释放 PMC。我们通过近轴和远轴标记基因的特异性表达证实了 PMC 和 SMC 的成功富集（图 1j）。我们使用 RNA-seq 鉴定了它们之间的 6731 个差异表达基因 (DEG)。基于 Tian 等人确定的拟南芥叶片近轴和远轴域之间的许多可靠 DEG。( 2019)，我们分别从白菜和拟南芥共有的重叠直系同源 DEG 中选择了 433 和 510 个潜在的 PMC 和 SMC 标记基因（表 S1 ）。

为了识别 PMC 和 SMC，将 MC 群体重新聚类为 9 个亚群 (M0-M8)（图 1k）。结合光合基因（BrLHCB4.3、BrLHCA6和Bra018819）的表达和前 100 个潜在 PMC 标记基因，我们将 M0 和 M2 分配给 PMC（图 1l、m、p）。通过组合远轴基因（BrFIL.1、BrYAB3和BrYAB5）的表达和前 100 个潜在的 SMC 标记基因（图 1l、n、p），将其他 7 个亚群分配给 SMC。BrFIL.1的原位杂交分析支持 SMC 和 PMC 的正确定义（图 1o，p）。

使用 Seurat (v4.0.3) 中的“FindMarkers”，我们分别鉴定了优先在 PMC 和 SMC 中表达的 944 和 1387 个基因（表 S1）。令人惊讶的是，除了识别许多光合作用相关基因外，大量核糖体蛋白编码基因 (RPEG) (239/944) 在 PMC 中富集（图 1q，r）。在发现的 239 个 RPEG 中，27 个是参与近轴细胞或 PMC 发育的 15 个拟南芥 RPEG 的直系同源基因（表 S1）。有趣的是，蛋白质-蛋白质相互作用分析表明，PMC 中连接最多的前 10 个基因是 RPEG（图 1s）。这些结果强烈表明 RPEG 参与了 PMC 的发展。与 PMC 不同，SMC 主要富集对非生物和生物刺激的反应，信号响应基因（温度、茉莉酸、水杨酸和钙），和蛋白质修饰（图 1t）。此外，几乎所有在 SMC 中连接最多的前 10 个基因都对生物或非生物胁迫做出反应（图 u）。总的来说，这些结果表明 PMC 是光合作用的主要机器，而 SMC 可能参与调整机器以适应外部环境。

令人惊讶的是，我们发现大多数报道的近轴-远轴极性基因在 MCs 中几乎检测不到。为了确定为什么会这样，我们收集了一系列代表茎尖分生组织 (SAM) 以及幼苗和莲座丛阶段内叶和外叶不同区域的样本，用于 RNA-seq（图 S1a、b）。结果表明，大多数近轴-远轴极性基因优先在 SAM 中表达，而在两个阶段它们从内叶到外叶都急剧且持续下调（图 S1c）。这可能是为什么在 MCs 中几乎没有检测到近轴-远轴极性基因，因为收集了中间的莲座叶用于 scRNA-seq。

总的来说，我们以单细胞分辨率生成了大白菜莲座叶的转录组图谱。一个关键发现是从 MC 中识别出近轴 PMC 和远轴 SMC。我们确定了 PMC 和 SMC 之间的功能差异，以及 RPEG 在 PMC 发展中的潜在作用。我们的研究还提供了许多细胞类型特异性标记基因，这将有助于scRNA-seq在白菜中的应用。比较每种细胞类型的叶单细胞转录组，重点关注不同发育阶段和不同亚种的 SMC 和 PMC，将扩展我们对 PMC 和 SMC 在叶近轴-远轴模式以及叶发育过程中的分化的认识和B. rapa叶类蔬菜的形态发生。

登录号

所有测序数据均可从 NGDC (https://ngdc.cncb.ac.cn/gsa/) 获得，BioProject 登录号为 PRJCA009630。