Ethylene Improves Root System Development under Cadmium Stress by Modulating Superoxide Anion Concentration in Arabidopsis thaliana

Abstract

This work aims at identifying the effects of ethylene on the response of Arabidopsis thaliana root system to cadmium chloride (CdCl₂) stress. Two ethylene-insensitive mutants, ein2-5 and ein3-1eil1-1, were subjected to (25, 50, 75, and 100 μM) CdCl₂ concentrations, from which 75 μM concentration decreased root growth by 40% compared with wild type Col-0 as a control. Ethylene biosynthesis increased in response to CdCl₂ treatment. The length of primary root and root tip in ein2-5 and ein3-1eil1-1 decreased compared with wild type after CdCl₂ treatment, suggesting that ethylene play a role in root system response to Cd stress. The superoxide concentration in roots of ein2-5 and ein3-1eil1-1 was greater than in wild type seedlings under Cd stress. Application of exogenous 1-aminocyclopropane-1-carboxylic acid (ACC) (a precursor of ethylene biosynthesis) in different concentrations (0.01, 0.05 and 0.5 μM) decreased superoxide accumulation in Col-0 root tips and increased the activities of superoxide dismutase (SOD) isoenzymes under Cd stress. This result was reversed with 5 μM of aminoisobutyric acid AIB (an inhibitor of ethylene biosynthesis). Moreover, it was accompanied by increase in lateral roots number and root hairs length, indicating the essential role of ethylene in modulating root system development by controlling superoxide accumulation through SOD isoenzymes activities. The suppressed Cd-induced superoxide accumulation in wild type plants decreased the occurrence of cells death while programmed cell death (PCD) was initiated in the root tip zone, altering root morphogenesis (decreased primary root length, more lateral roots and root hairs) to minimize the damage caused by Cd stress, whereas this response was absent in the ein2-5 and ein3-1eil1-1 seedlings. Hence, ethylene has a role in modulating root morphogenesis during CdCl₂ stress in A. thaliana by increasing the activity of SOD isoenzymes to control superoxide accumulation.

Keywords: Arabidopsis thaliana; Cd stress; ethylene; programmed cell death; root system; superoxide.

FIGURE 1

Ethylene signaling involved in plant root response to Cd stress. (A) QRT-PCR analysis of relative gene expression of ACS2, ACS6, EIN3 and ERF2 in Col-0 plants under Cd stress. (B) Increased ethylene response after Cd treatment visualized by EBS::GUS staining. (C) Frequency of stained root tip as (B) indicated. Seeds were germinated and grown on MS agar plates for 4 days and then transferred to CdCl₂ (μM) pretreated plates, as indicated, for another 4 days.

FIGURE 2

The inhibitory effects of increased duration of exposure to Cd on the elongation of the primary roots and root tips of the Col-0, ein2-5, and ein3-1eil1-1 plants. Four-day-old seedlings were transferred to CdCl₂ (75 μM)-treated agar plates for 2 and 4 days, as indicated. (A) The primary root lengths of the Col-0, ein2-5, and ein3-1eil1-1 plants after being transferred to the Cd-treated agar plates for different periods, (B) and (C) the root tip length. Values represent the mean ± SE of 20 individual plants, and the letters indicate significant differences (P < 0.05). Scale bars = 50 μm.

FIGURE 3

Effects of Cd stress on the root system architecture of the Col-0, ein2-5, and ein3-1eil1-1 plants. (A) Lateral root number and (B) Root hair density of the 4-days-old seedlings transferred to agar plates with or without CdCl₂ (75 μM) for 7 days. Values represent the mean ± SE of more than 20 individual seedlings, and the bars indicate the standard error. The letters indicate significant differences (P < 0.05).

FIGURE 4

The Cd content of Arabidopsis seedlings roots. Three-week-old Arabidopsis thaliana seedlings were exposed to 0, 10, 20, and 40 μM CdCl2 for 8 days. At the 20 μM CdCl2 treatment, the Cd contents of ein2-5 and ein3-1eil1-1 were even higher than the Cd content of Col-0. Furthermore, the Bfs of ein2-5 and ein3-1eil1-1 were even bigger than Col-0 under 20 μM CdCl2 treatment.

FIGURE 5

The accumulation of superoxide in the root tips of the Col-0, ein2-5, and ein3-1eil1-1 plants. Four-day-old seedlings were transferred to CdCl₂ (75 μM) -treated agar plates for different periods, as indicated. 2 days treatment significantly increased the production of superoxide in all plants. In addition, the accumulation of superoxide in both the ein2-5 and ein3-1eil1-1 mutants was significantly greater than in the wild-type plants even after 4 days of Cd treatment. Values (mean ± SE) show the staining intensity, and the letters indicate significant differences (P < 0.05). At least 20 individual roots were analyzed for each genotype and treatment, and one representative image was selected for the figure.

FIGURE 6

Effects of exogenous ACC on the accumulation of superoxide in the root tips of the Col-0 plants under Cd stress. Four-day-old seedlings were transferred to various pretreated agar plates with CdCl₂ (75 μM) or CdCl₂ (75 μM) plus various concentrations of ACC (0.01, 0.05, and 0.5 μM, a precursor of ethylene biosynthesis) with or without 5 μM AIB (an inhibitor of ethylene biosynthesis), as indicated, for 4 days. Values (mean ± SE) show the staining intensity, and the letters indicate significant differences (P < 0.05). At least 20 individual roots were analyzed for each genotype and treatment, and one representative image was selected for the figure.

FIGURE 7

Effects of various pretreatments (ACC, 0.01 μM; AIB, 5 μM) on the production of O₂^- in roots of Arabidopsis Col-0 seedlings under Cd (75 μM) stress. Four-day-old seedlings were transferred to various pretreated agar plates for 4 days. Values represent the mean ± SE, and the bars indicate the standard error.

FIGURE 8

Activities of SOD and its Cu/Zn-SOD, Fe-SOD, and Mn-SOD isoenzymes in the roots of the Col-0, ein2-5, and ein3-1eil1-1 plants under Cd stress. Four-day-old seedlings were transferred for 4 days to agar plates, or agar plates with CdCl₂ (75 μM), or with ACC (0.01 μM, a precursor of ethylene biosynthesis), or with CdCl₂ (75 μM) plus ACC (0.01 μM), as indicated. Values represent the mean ± SE, and the bars indicate the standard error. The letters indicate significant differences (P < 0.05).

FIGURE 9

Progression of cell death/necrosis in the root tips of the Col-0, ein2-5, and ein3-1eil1-1 plants during Cd stress. Four-day-old seedlings were transferred to CdCl₂ (75 μM)-treated agar plates for different periods, as indicated. The seedlings were then collected and incubated in propidium iodide (PI) or Trypan blue. At least 20 individual roots were analyzed for each genotype and treatment, and one representative image was selected for the figure.

FIGURE 10

Progression of programmed cell death, indicated by DAPI and TUNEL staining in the root tips of the Col-0, ein2-5, and ein3-1eil1-1 plants during Cd stress. Four-day-old seedlings were transferred to CdCl₂ (75 μM)-treated agar plates for different time points, as indicated. Insets: close up observations enlargement of chromatin condensation. At least 20 individual roots were analyzed for each genotype and treatment, and one representative image was selected for the figure.

FIGURE 11

A schematic model elucidating the interaction of ethylene, SOD, superoxide and PCD in the root meristematic and elongation zones under Cd stress.