拟南芥根毛衰老死亡过程的PCD检测

doi:10.11983/CBB15081

摘要/Abstract

摘要： 根毛是植物体吸收养分的重要器官, 自然条件下根毛的寿命很短, 仅能存活2-3周, 随即脱落死亡。以模式植物拟南芥(Arabidopsis thaliana)根毛为材料, 对根毛死亡的细胞学特征进行了报道。结果发现,根毛衰老死亡后细胞内的原生质体发生了收缩, 并在胞质中观察到凝集物的出现; 通过原位末端标记(TUNEL)检测, 发现幼根上的根毛细胞核DNA发生了片段化。上述结果表明, 拟南芥根毛的衰老死亡很可能是植物体自主调控的程序性细胞死亡(PCD)。另外, 当根毛衰老死亡后, 细胞核大多会迁移到靠近根毛基部的位置, 且正常的长管状根毛发生旋转扭曲。

关键词: 细胞程序性死亡, 根毛

Abstract: The root hair is an essential organ for the uptake of nutrients in plants; root hair cells can only survive for 2 to 3 weeks and then die off, but the mechanisms underlying root hair death is not clear. In this study, we investigated the cellular characteristics of root hair death. Arabidopsis dying root hairs underwent protoplast retraction from the cell wall, and some unknown aggregates were observed in the cell. On detecting DNA fragmentation by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL), we found TUNEL-positive nuclei in root hairs. Protoplast retraction and nuclear DNA fragmentation are both considered hallmark features of apoptotic-like programmed cell death in plants. Therefore, we propose that the death of Arabidopsis root hairs belongs to programmed cell death, which is mediated by an intracellular program. As well, fluorescence staining with DAPI demonstrated that in dying root hairs, the nucleus would migrate to a position close to the basement of the root hair. Moreover, the tubular-shaped root hairs finally twist.

Key words: programmed cell death, root hairs

李林, 谭康, 唐秀光, 晁晓婷, 汶晨曦, 白壮东, 丰华玲, 刘文哲, 苏慧. 拟南芥根毛衰老死亡过程的PCD检测. 植物学报, 2016, 51(2): 194-201.

Lin Li, Kang Tan, Xiuguang Tang, Xiaoting Chao, Chenxi Wen, Zhuangdong Bai, Hualing Feng, Wenzhe Liu, Hui Su. Characterization of Programmed Cell Death During the Senescence of Root Hairs in Arabidopsis. Chinese Bulletin of Botany, 2016, 51(2): 194-201.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB15081

https://www.chinbullbotany.com/CN/Y2016/V51/I2/194

图/表 5

图1 模式植物拟南芥根毛的死亡过程 (A)-(E) 连续跟踪观察1、2、3、4和5天的根毛(箭头所示); (F) 在观察的第8天, 根毛形态发生变化(箭头所示); (G) 在观察的第10天, 根毛发生明显的扭曲(箭头所示), 此时已经死亡 ; (H) 观察第13天的扭曲的根毛(箭头所示)。Bar=10 μm

Figure 1 Time-lapse of root hairs in Arabidopsis (A)-(E) The morphology of the root hair indicated by the arrow head was observed for 1, 2, 3, 4 and 5 d, respectively; (F) The vary of the root hair was observed in the 8th day (indicating by the arrow head) ; (G) The root hair obviously twisted and curved in the 10th day (indicating by the arrow head), and at that time, root hair has been died); (H) Twisted and curved root hair in the 13th day (indicating by the arrow head). Bar=10 μm

图2 拟南芥根毛细胞的TUNEL原位检测 (A)-(A3) 阳性对照。DNA酶I处理后, 根毛及主根细胞的细胞核均为TUNEL阳性。(B)-(B3) 发生PCD的根毛细胞。图中所示的根毛细胞TUNEL原位检测为阳性。(C)-(C3) 阴性对照。图中所示的根毛细胞TUNEL原位检测为阴性。Bar=50 μm

Figure 2 The TUNEL assay in roots and root hairs of Arabidopsis (A)-(A3) DNase I treated seedlings were fixed and subjected to TUNEL assay. The root and root hairs were TUNEL-positive; (B)-(B3) PCD root hair. A representative image shows TUNEL-positive root hair. (C)-(C3) Negative control. A representative image shows TUNEL-negative root hair. Bar=50 μm

图3 自然死亡的拟南芥根毛细胞发生质壁分离 (A)-(A1) 正常的根毛细胞。FDA染色可以观察到明显的绿色荧光(A); 明场下细胞形态正常(A1)。(B)-(B1) 自然死亡的根毛细胞。FDA染色显示根毛已死亡(B); 明场下可以观察到明显的质壁分离(B1)。(C)和(D) 自然死亡的根毛细胞基部发生了质壁分离; 死亡后期的根毛细胞内部原生质体断裂。(E) 自然生长1周的拟南芥幼苗的热激处理(阳性对照)。Bar=10 μm

Figure 3 The plasmolysis of cell in Arabidopsis naturally dead root hair (A)-(A1) Normal root hairs stained with FDA and viewed under white (A1) or fluorescent light (A); (B)-(B1) Naturally dead root hair. The root hairs with no fluorescence after FDA staining shows obvious retraction of the cytoplasm; (C) and (D) The extent of protoplast shrinking varies depending on the sequential process of programmed cell death. (E) Root hair of Arabidopsis thaliana 6 h after a 10 minutes heat shock at 55°C. The root hair shows retraction of the cytoplasm indicating it has undergone AL-PCD (the positive control). Bar=10 μm

图4 不同生长天数的拟南芥幼苗根毛的FDA和DAPI着色比率(n≥10)

Figure 4 The FDA and DAPI stain of root hairs in different stage of Arabidopsis seedlings (n≥10)

图5 自然死亡的根毛细胞的细胞核大多接近根毛基部 (A) 活着的根毛细胞核中心距根毛基部的距离与根毛全长比率的统计。横坐标为比值, 纵坐标为根毛数目(n=57)。(B) 自然死亡的根毛细胞核中心距根毛基部的距离与根毛全长比率的统计。横坐标为比值, 纵坐标为根毛数目(n=75)。(C)-(C2) FDA (C1)和DAPI (C2)染色表明死亡的根毛细胞核的位置靠近根毛基部。C: 明场。箭头指示细胞核的位置。Bar=50 μm

Figure 5 The position of nuclei in natural death root hairs is close to the basement of root hairs (A) Quantitative analysis of the position of nuclei in living root hairs; Abscissa represents ratio between the distance of the nucleus center from the basement of the root hair and root hair length; Ordinate represents the number of root hairs (n=57); (B) Quantitative analysis of the position of nuclei in dead root hairs; Abscissa represents ratio; Ordinate represents the number of root hairs (n=75); (C)-(C2) A representative image stained by FDA and DAPI shows that the nucleus in the dead root hair can be colored by DAPI (indicating by the arrow head) and the location of the nucleus is near the basement of the root hair. C: Bright field. Arrow head: Nucleus. Bar=50 μm

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