Effects of oxidative stress on growth and development of suspension cells of Populus tomentosa by transcriptome analysis
目的毛白杨是多年生木本植物，由于其固着的生长模式，毛白杨存在长期承受胁迫的可能性。氧化胁迫是常见的非生物胁迫方式，在多个物种中均有不同程度的研究，然而以毛白杨为材料在转录水平的研究目前鲜有报道。通过转录组数据探明活性氧平衡的打破对毛白杨悬浮细胞生长发育的影响。 方法本研究对毛白杨悬浮细胞系进行H2O2胁迫处理，通过激光共聚焦显微镜和转录组测序技术（RNA-Seq），观察、分析了线粒体的形态学和基因表达量的差异。 结果通过对转录组数据的分析得到了806个显著差异基因（P < 0.001且 |log2ratio| > 1），其中449个差异基因下调，357个差异基因上调。这些差异基因涉及细胞分裂、细胞分裂素的激活、赤霉素调控以及磷酸化途径。通过线粒体特异性染料并使用激光共聚焦显微镜观察氧化胁迫条件下毛白杨线粒体的形态学，结果表明氧化胁迫下线粒体以蠕虫状为主。 结论通过分析氧化胁迫条件下细胞转录组数据，在RNA水平揭示了氧化胁迫对植物细胞生长发育的影响，为植物的应激反应和生长发育研究提供了理论基础。Abstract: Objective Populus tomentosa is a perennial woody plant. Because of its fixed growth pattern, P. tomentosa may under long-term stress. Oxidative stress is a common way of abiotic stress, which has been studied in many researches. However, there are few reports on the transcriptional level of P. tomentosa as a material. The transcriptome data were used to investigate the effects of breaking the balance of reactive oxygen species (ROS) on the growth and development of suspension cells of P. tomentosa. Method In this study, the suspension cell lines of P. tomentosa were treated with hydrogen peroxide. The morphological and gene expression differences of mitochondria were observed and analyzed by confocal laser microscopy and transcriptome sequencing (RNA-Seq). Result Through the analysis of transcriptome data, 806 differentially expressed genes (P < 0.001 and |log2ratio| > 1) were identified, of which 449 differentially expressed genes were down-regulated and 357 differentially expressed genes were up-regulated. These differential genes involve cell division, cytokinin activation, gibberellin regulation and phosphorylation pathways. Mitochondrial morphology of P. tomentosa under oxidative stress was observed using mitochondrial specific dyes and laser confocal microscopy. The results showed that the mitochondria were mostly vermiform under oxidative stress. Conclusion By analyzing cell transcriptome data under oxidative stress, the effects of oxidative stress on plant growth and development were revealed at RNA level, which provided a theoretical basis for the study of plant stress response and growth and development.
图 1 差异表达基因火山图
x轴为差异倍数。y轴为校正后的P值。红点表示显著差异基因中上调的基因，绿点表示显著差异基因中下调的基因，蓝点表示不显著差异的基因。x, fold change. y, P-adjusted value. Red dots indicate significantly up-regulated differentially expressed genes, green dots indicate significantly down-regulated differentially expressed genes. Blue dots indicate non-significantly differentially expressed genes.
Figure 1. Volcano map of differentially expressed genes
图 5 线粒体形态学
A、B、C分别是野生型细胞在未处理条件下线粒体形态（WT）、野生型细胞在AsA处理条件（1 mmol/L，3 h）下线粒体形态（WTA）、野生型细胞在H2O2处理条件（10 mmol/L，3 h）下线粒体形态（WTH）；D是基于200个不同细胞的形态学定量统计。A, mitochondrial morphology of wild type cells under untreated conditions (WT). B, mitochondrial morphology of wild type cells under AsA treatment (1 mmol/L, 3 h) (WTA). C, mitochondrial morphology of wild type cells under H2O2 treatment (10 mmol/L, 3 h) (WTH). D, quantitative statistics based on 200 cells.
Figure 5. Mitochondrial morphology
表 1 转录组测序数据的