Single cell-type transcriptome profiling reveals genes that promote nitrogen fixation in the infected and uninfected cells of legume nodules.
Single cell-type transcriptome profiling reveals genes that promote nitrogen fixation in the infected and uninfected cells of legume nodules.
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单细胞型转录组分析揭示了豆科植物根瘤感染和未感染细胞中促进固氮的基因
DOI：
10.1111/pbi.13778
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发表时间：
2022-04
影响因子：
13.8
通讯作者：
Duanmu D
中科院分区：
文献类型：
--
作者：
Wang L;Zhou Y;Li R;Liang J;Tian T;Ji J;Chen R;Zhou Y;Fan Q;Ning G;Larkin RM;Becana M;Duanmu D
过量施用氮肥不可避免地造成了环境问题。豆科植物根瘤中的共生固氮（SNF）为农业生态系统提供了可持续的氮源。已经报道了超过200种基因调节SNF，包括根瘤菌感染、根瘤器官发生和衰老（Roy et al.，2020年）。成熟的根瘤主要由两种细胞类型组成：含有固氮类杆菌的感染细胞（IC）和介导活性代谢和营养运输的未感染细胞（UC）。尽管众所周知SNF需要功能特化，但负责IC和UC中转录调控和碳/氮代谢和运输的特定基因仍然在很大程度上未被探索。单细胞转录组学已成为一种强大的技术，研究基因表达的时空模式。这些基于液滴的方法可接受的最大细胞直径约为40 μ m。来自豆科植物根瘤的IC的平均尺寸通常为50 - 100 μ m。因此，我们在用百脉根中慢生根瘤菌MAFF 303099接种后4周（wpi）从百脉根成熟根瘤中手动分离红棕色IC和透明UC（这两种细胞类型各50 - 100个细胞）（图1a，B;附录S1）。由于无法克服的技术困难，我们无法将IC分离成具有不同水平的核DNA核内复制的细胞，或将UC分离成外皮层细胞和内皮层细胞、维管束细胞和间质细胞，这些细胞通常存在于有限的结节中。构建Smart-Seq2文库（Picelli等人，2014），并且具有> 50%映射率的样品用于转录组分析，包括UC的四个重复和IC的两个重复（图Ic）。第939章我的天（|Log2-倍数变化（IC vs. UC）|> 3，FDR <0.05;表S1）。在检测到的基因中，有925个先前未被表征，因此，我们的转录组学分析提供了研究成熟结节中SNF的资源（Roy等人，2020年）。值得注意的是，我们发现了55个编码假定转录因子的基因和73个编码转运蛋白的基因（图1d）。我们发现，先前与SNF相关的几个基因，如豆血红蛋白基因（LjLbs）和硫酸盐转运蛋白基因（LjSST1），在IC中高水平表达。有趣的是，在UC中检测到约2%的总Lb 2 mRNA，这与最近的研究显示该基因在L.结节（Wang等人，2019年）。相反，我们发现氨转运蛋白（LjAMT1. 1)和ERF转录因子（LjERF1）仅在UC中表达（图1e; Roy等，2020年）。接下来，我们确定了参与碳和氮代谢的基因的表达谱。蔗糖合成酶和转化酶是蔗糖代谢的关键酶。我们发现六个蔗糖合成酶基因中的两个（Lj4g3v2215210. 1和Lj6g3v1162830。1)和7个转化酶基因之一（Lj3g3v0323220. 1)在结节中以更高水平表达，主要在UC中相对于IC（图1f）。这些结果支持蔗糖被转运至UC并在那里分解代谢为二羧酸盐的提议（白色等人，2007年）。固氮酶复合物在IC中将N2还原为氨。谷氨酰胺合成酶（GS）和天冬酰胺合成酶（AS）是将氨同化为谷氨酰胺和天冬酰胺的两个关键酶，是L. Kai（Miao et al.，1991年）。其中两个基因编码GS...
Excessive application of nitrogen fertilizers has inevitably resulted in environmental problems. The symbiotic nitrogen fixation (SNF) that occurs in the root nodules of leguminous plants provides a sustainable source of reduced nitrogen in agricultural ecosystems. More than 200 genes have been reported to regulate SNF, including rhizobial infection, nodule organogenesis and senescence (Roy et al., 2020). Mature nodules consist mainly of two cell types: infected cells (IC) that contain nitrogen-fixing bacteroids and uninfected cells (UC) that mediate active metabolism and nutrient transport. Although it is well known that SNF requires functional specialization, the specific genes responsible for transcriptional regulation and carbon/nitrogen metabolism and transport in IC and UC remain largely unexplored. Single-cell transcriptomics has emerged as a powerful technique for investigating spatiotemporal patterns of gene expression. The maximum cell diameter acceptable for these dropletbased methods is~ 40 μm. An average size of IC from a legume nodule is typically 50–100 μm. We therefore manually separated reddish-brown IC and transparent UC (50–100 cells for each of these two cell types) from the mature nodules of Lotus japonicus at 4 weeks post-inoculation (wpi) with Mesorhizobium loti MAFF 303099 (Figure 1a, b; Appendix S1). Due to insurmountable technical difficulties, we were unable to separate IC into cells having various levels of nuclear DNA endoreduplication, or UC into outer and inner cortical cells, vascular bundle cells and interstitial cells, typically found in determinate nodules. Smart-Seq2 libraries were constructed (Picelli et al., 2014), and samples with> 50% mapping rates were used for the transcriptome analysis, including four replicates for UC and two replicates for IC (Figure 1c). We finally obtained 939 DEGs (| Log2-fold change (IC vs. UC)|> 3, FDR< 0.05; Table S1). Of the detected genes, 925 had not been previously characterized, and therefore, our transcriptomic analysis provides a resource to study SNF in mature nodules (Roy et al., 2020). Notably, we found 55 genes that encode putative transcription factors and 73 genes that encode transporters (Figure 1d). We found that several genes previously linked to SNF, such as the leghaemoglobin genes (LjLbs) and a sulphate transporter gene (LjSST1), are expressed at high levels in IC. Interestingly,~ 2% of the total Lb2 mRNA was detected in UC, which is consistent with a recent study showing promoter activity of that gene in the interstitial cells of L. japonicus nodules (Wang et al., 2019). In contrast, we found that an ammonia transporter (LjAMT1. 1) and an ERF transcription factor (LjERF1) are exclusively expressed in UC (Figure 1e; Roy et al., 2020). Next, we determined the expression profiles of genes involved in carbon and nitrogen metabolism. Sucrose synthase and invertase are key enzymes involved in sucrose metabolism. We found that two of the six sucrose synthase genes (Lj4g3v2215210. 1 and Lj6g3v1162830. 1) and one of the seven invertase genes (Lj3g3v0323220. 1) are expressed at higher levels in nodules, predominantly in UC relative to IC (Figure 1f). These results support the proposal that sucrose is transported to UC and catabolized there to dicarboxylates (White et al., 2007). The nitrogenase enzymatic complex reduces N2 to ammonia in IC. Glutamine synthetase (GS) and asparagine synthetase (AS) are two key enzymes for the assimilation of ammonia into glutamine and asparagine, which serve as the two major forms of fixed nitrogen being translocated to the shoot in L. japonicus (Miao et al., 1991). Two of the five genes encoding GS …
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影响因子：
5.1
作者：
Rogato, Alessandra;D'Apuzzo, Enrica;Chiurazzi, Maurizio
通讯作者：
Chiurazzi, Maurizio
影响因子：
7.2
作者：
Hou, Zhiwei;Yang, Yanyu;Grimm, Bernhard
通讯作者：
Grimm, Bernhard
CRISPR/Cas9敲除百脉根豆血红蛋白基因揭示了它们在共生固氮中的协同作用
影响因子：
9.4
作者：
Wang, Longlong;Rubio, Maria Carmen;Duanmu, Deqiang
通讯作者：
Duanmu, Deqiang
影响因子：
11.6
作者：
MIAO, GH;HIREL, B;VERMA, DPS
通讯作者：
VERMA, DPS
影响因子：
14.8
作者：
Picelli, Simone;Faridani, Omid R.;Sandberg, Rickard
通讯作者：
Sandberg, Rickard