Al3+对大豆根边缘细胞程序性死亡诱导的生理生态作用

doi:10.3773/j.issn.1005-264x.2008.03.019

摘要/Abstract

摘要：

设置不同的Al³⁺浓度(0、25、50、100、200、400 μmol·L ^-1)和培养时间(12、24 h),研究了边缘细胞活性和大豆(Glycine max)根中过氧化氢酶(CAT)、过氧化物酶(POD)、超氧化物歧化酶(SOD)随Al³⁺浓度及处理时间变化的规律,并通过Hoechst33342-PI双重荧光染色、梯状DNA(即DNA ladder)分析和末端脱氧核糖核酸转移酶介导的dUTP切口末端标记(即TUNEL原位标记)检测,研究了Al³⁺对大豆根边缘细胞程序性死亡诱导的生理生态作用。结果表明,Al³⁺胁迫能诱导边缘细胞的死亡,随着Al³⁺浓度的升高和处理时间的延长,细胞死亡率增加。通过Hoechst33342-PI双重荧光染色、DNA ladder分析和TUNEL原位标记,检测到Al³⁺胁迫下发生程序性死亡的边缘细胞。其表现为:在400 μmol·L ^-1 Al³⁺诱导大豆根24 h时, 核酸电泳显示细胞DNA发生特异性降解并形成阶梯状电泳条带(DNA ladder),用TUNEL原位标记检测200和400 μmol·L ^-1 Al³⁺ 处理12 h后的大豆根边缘细胞,发现DNA的3'-OH端被原位特异标记,二氨基联苯胺(DAB)显色后,细胞核为阳性或强阳性。同时,高浓度Al³⁺(>100 μmol·L^-1)处理下,CAT、POD和SOD活性均有不同程度的下降,CAT和SOD的活性也随处理时间的延长而降低。说明在Al³⁺胁迫下边缘细胞的死亡可能是一种程序性死亡形式,高浓度Al³⁺胁迫下,通过诱导活性氧在细胞体内的产生和累积而导致细胞凋亡,此过程是其对逆境胁迫所作出的生理生态防御性应答方式之一。

关键词: 铝毒, 大豆, 边缘细胞, 细胞程序性死亡, 抗氧化酶, 生理生态

Abstract:

Aims Programmed cell death (PCD) plays an important role in plant growth and development, which correlates with adaptation of the plant to environment stress. Root border cells have many important biological functions in protecting the plant root tip from biotic and abiotic stress, which is a popular research topic recently. However, few studies have focused on PCD of root border cells and the phytoecological effect of aluminum on it. Our objective was to study the phytoecological and molecular ecological mechanism of root border cells in resisting aluminum (Al) toxicity.

Methods We investigated change in viability of border cells, activities of catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD), and PCD of root border cells by Al³⁺ induced in soybean (Glycine max) root tips with different Al³⁺ concentrations (0, 25, 50, 100, 200 and 400 μmol·L ^-1 Al³⁺) and treatment times (12 and 24 h). PCD was observed through Hoechst33342-PI fluorescent staining, DNA ladder and TdT-mediated dUTP nick end labeling (TUNEL) analysis.

Important findings Aluminum can induce the death of root border cells. The viability of border cells decreases with increased Al³⁺ concentration and treatment time. The progressive delineation of fragmented DNA was coincident with the appearance of DNA ladder after being exposed to 400 μmol·L ^-1 Al³⁺ for 24 h. TUNEL analysis of border cells revealed that the nuclear DNA strand breaks can be identified by labeling free 3'-OH termini after treatment with 200 and 400 μmol·L ^-1 Al³⁺ for 12 h. Diaminobenzidine (DBA) staining indicated that the nucleus was positive and strong positive. Otherwise, CAT and SOD activities declined with increasing Al³⁺ concentration and treatment time under higher Al³⁺ concentration (>100 μmol·L^-1), and we observed no significant differences in POD activity among different Al³⁺ concentrations and treatment times. These results indicated that border cell death may be a PCD under Al³⁺ stress. High Al³⁺ concentration induced PCD through enhancing reactive oxygen species (ROS), which is one of the ways of resisting adversity in plants.

Key words: aluminum toxicity, soybean, border cells (BC), programmed cell death (PCD), antioxidase, phytoecology

李荣峰, 蔡妙珍, 刘鹏, 徐根娣, 陈敏燕, 梁和. Al³⁺对大豆根边缘细胞程序性死亡诱导的生理生态作用. 植物生态学报, 2008, 32(3): 690-697. DOI: 10.3773/j.issn.1005-264x.2008.03.019

LI Rong-Feng, CAI Miao-Zhen, LIU Peng, XU Gen-Di, CHEN Min-Yan, LIANG He. PHYTOECOLOGICAL EFFECT OF Al³⁺ ON THE INDUCTIVITY OF PROGRAMMED CELL DEATH OF BORDER CELLS IN SOYBEAN ROOT. Chinese Journal of Plant Ecology, 2008, 32(3): 690-697. DOI: 10.3773/j.issn.1005-264x.2008.03.019

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https://www.plant-ecology.com/CN/Y2008/V32/I3/690

图/表 5

表1 大豆边缘细胞存活率随Al3+浓度和处理时间的变化(平均值±标准误差)

Table 1 Viabilities (%) of soybean root border cells treated with different Al3+ concentrations and treatment time (Mean±SE)

处理时间 Treatment time (h)	Al³⁺ 浓度 Al³⁺ concentration (μmol·L^-1)
处理时间 Treatment time (h)	0	50	100	200	400
12	84.39±3.68^ab	79.64±6.52^abc	72.68±7.36^bc	71.26±6.41^c	67.49±15.30^cd
24	85.78±2.72^a	72.00±5.38^bc	66.87±0.29^cd	57.33±4.08^de	49.54±5.79^e

图1 大豆边缘细胞Hoechst33342-PI双重染色荧光效果 A:活的边缘细胞 Live root border cells B:程序性死亡的大豆边缘细胞 Soybean root border cells of programmed cell death C:坏死细胞或晚期的凋亡细胞 Dead cells or later-phase apoptosis cells in soybean root border cells

Fig.1 Hoechst33342-PI double staining results in soybean root border cells

图2 Al3+处理12和24 h后大豆边缘细胞DNA的凝胶电泳图谱 1~6:分别表示0、25、50、100、200、400 μmol·L -1 Al3+处理12 h Treated with 0, 25, 50, 100, 200 and 400 μmol·L -1 Al3+ for 12 h, respectively 7~12:分别表示0、25、50、100、200、400 μmol·L -1 Al3+处理24 h Treated with 0, 25, 50, 100, 200 and 400 μmol·L -1 Al3+ for 24 h, respectively

Fig.2 DNA band patterns are visualized in soybean root border cells treated with different Al3+ concentrations for 12 h and 24 h, respectively

图3 Al3+诱导大豆边缘细胞程序性死亡的TUNEL检测结果 A、B、C、D、E、F:分别表示0、25、50、100、200和400 μmol·L -1 Al3+处理12 h后的边缘细胞 Showing the root border cells treated with 0, 25, 50, 100, 200 and 400 μmol·L -1 Al3+ for 12 h, respectively

Fig.3 In situ detection with the TUNEL reaction for soybean root border cells exposed to Al3+

表2 Al3+对大豆根尖CAT、POD和SOD酶活性的影响(平均值±标准误差)

Table 2 Effects of different Al3+ concentrations on the activities of CAT, POD and SOD in soybean root tips (Mean±SE)

处理时间 Treatment time (h)	Al³⁺ 浓度 Al³⁺ concentration (μmol·L^-1)	CAT活性 CAT activity (U·mg^-1 Pr)	POD活性 POD activity (Δ470·mg^-1Pr·min^-1)	SOD 活性 SOD activity (U·mg^-1Pr)(×10³)
12	0	265.9±76.52^a	144.5±87.65^a	27.7±2.68^ab
	25	291.1±41.92^a	148.1±32.58^a	30.5±11.04^ab
	50	283.3±4.45^a	151.9±12.84^a	31.2±4.09^ab
	100	272.0±113.6^a	139.5±34.01^a	36.2±13.70^a
	200	255.2±30.23^a	118.2±8.03^a	30.7±1.35^ab
	400	159.0±43.93^b	114.5±12.59^a	28.0±4.90^ab
24	0	67.68±18.65^d	152.8±7.92^a	20.5±9.87^bc
	25	106.2±13.08^bcd	158.2±30.98^a	21.4±0.51^bc
	50	147.7±45.46^bc	160.5±23.4^a	31.1±8.47^ab
	100	113.2±7.96^bcd	157.4±12.73^a	23.3±0.55^abc
	200	77.10±0.17^cd	163.5±2.15^a	21.4±3.45^bc
	400	138.5±21.28^bcd	151.9±8.25^a	14.3±3.08^c

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Al³⁺对大豆根边缘细胞程序性死亡诱导的生理生态作用

PHYTOECOLOGICAL EFFECT OF Al³⁺ ON THE INDUCTIVITY OF PROGRAMMED CELL DEATH OF BORDER CELLS IN SOYBEAN ROOT

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