SNF1-related protein kinases type 2 are involved in plant responses to cadmium stress

Abstract

Cadmium ions are notorious environmental pollutants. To adapt to cadmium-induced deleterious effects plants have developed sophisticated defense mechanisms. However, the signaling pathways underlying the plant response to cadmium are still elusive. Our data demonstrate that SnRK2s (for SNF1-related protein kinase2) are transiently activated during cadmium exposure and are involved in the regulation of plant response to this stress. Analysis of tobacco (Nicotiana tabacum) Osmotic Stress-Activated Protein Kinase activity in tobacco Bright Yellow 2 cells indicates that reactive oxygen species (ROS) and nitric oxide, produced mainly via an l-arginine-dependent process, contribute to the kinase activation in response to cadmium. SnRK2.4 is the closest homolog of tobacco Osmotic Stress-Activated Protein Kinase in Arabidopsis (Arabidopsis thaliana). Comparative analysis of seedling growth of snrk2.4 knockout mutants versus wild-type Arabidopsis suggests that SnRK2.4 is involved in the inhibition of root growth triggered by cadmium; the mutants were more tolerant to the stress. Measurements of the level of three major species of phytochelatins (PCs) in roots of plants exposed to Cd(2+) showed a similar (PC2, PC4) or lower (PC3) concentration in snrk2.4 mutants in comparison to wild-type plants. These results indicate that the enhanced tolerance of the mutants does not result from a difference in the PCs level. Additionally, we have analyzed ROS accumulation in roots subjected to Cd(2+) treatment. Our data show significantly lower Cd(2+)-induced ROS accumulation in the mutants' roots. Concluding, the obtained results indicate that SnRK2s play a role in the regulation of plant tolerance to cadmium, most probably by controlling ROS accumulation triggered by cadmium ions.

Figure 1.

MAPK and SnRK2 are activated in plant cells in response to cadmium stress. Six-day-old BY-2 suspension cell cultures were treated with various concentrations of CdCl₂ (0–1,000 μm; A) or CdSO₄ (0–200 μm; G) for 30 min, or with 100 μm CdCl₂ for various times (0–90 min; B–F). Protein kinase activity in cells untreated and treated with the stressor was monitored by in-gel kinase activity assay with MBP as a substrate (A, B, D, and G), or by western blotting (C and E). D and G, Activity of NtOSAK analyzed by immunocomplex kinase activity assay using specific anti-C-terminal NtOSAK antibodies. C, Immunoblot probed with antiphospho-p44/42 MAPK antibodies. E, Immunoblot probed with anti-Ser-158(P) antibodies. F, NtOSAK protein level determined by western blotting with specific anti-C-terminal NtOSAK antibodies.

Figure 2.

Cadmium induces NtOSAK activation in NO- and ROS-dependent manner. BY-2 cells were treated with 100 μm CdCl₂ without (A) or with pretreatment with: 500 μm cPTIO for 1 h (B), 1,000 units/mL catalase for 30 min (C), 300 μm l-NAME for 10 min (D), 1 mm sodium tungstate for 30 min or 24 h (E). NtOSAK activity in BY-2 cells was detected by immunocomplex in-gel kinase activity assay using specific anti-C-terminal NtOSAK antibodies and MBP as a substrate. Additionally, NtOSAK activity was monitored by immunocomplex kinase activity assay in BY-2 cells treated with 2 mm H₂O₂ for various times (F). NtOSAK protein level determined by western blotting with specific anti-C-terminal NtOSAK antibodies in protein extracts before immunoprecipitation (black diamond).

Figure 3.

Effect of cadmium ions on SnRK2.4 and SnRK2.10 activity and on SnRK2.4 and SnRK2.10 transcript level. Ten-day-old seedlings of Arabidopsis wild-type Col-0, as well as insertion mutants nia1nia2 and atnoa1 were treated with 750 mm sorbitol or with different concentration of CdCl₂: 150, 300, or 500 μm Col-0 (A) and 300 μm nia1nia2 and atnoa1 (B). Activity of SnRK2.4/SnRK2.10 in seedling extracts was monitored by immunocomplex in-gel kinase activity assay using specific antibodies against N-terminal peptide of both kinases and MBP as a substrate. C, EGFP-SnRK2.4 and EGFP-SnRK2.10 were transiently expressed in Arabidopsis T87 protoplasts. Protoplasts were treated with 200 μm CdCl₂ or 300 mm NaCl for 30 min and activity of EGFP-SnRK2.4 and EGFP-SnRK2.10 was analyzed by in-gel kinase activity assay with MBP as a substrate. D, Ten-day-old Col-0 Arabidopsis seedlings were treated with 100 μm CdCl₂ for various time (0–12 h), total RNA was isolated and transcript level of SnRK2.4 and SnRK2.10 was analyzed by semiquantitative RT-PCR. As a control Actin2 transcript level was monitored. E, Four-week-old Col-0 Arabidopsis plants grown in hydroponic culture were treated with 20 μm cadmium chloride for 2 d and transcript level of SnRK2.4 and SnRK2.10 was monitored in plant roots by RT-PCR. The results were confirmed by qRT-PCR (F).

Figure 4.

Localization of NtOSAK, SnRK2.10, and SnRK2.4 before and after cadmium treatment. Tobacco protoplast were prepared from BY-2 cells and transformed with EGFP-NtOSAK construct. The kinase localization was monitored before and after 30-min treatment with 50 μm CdCl₂. In both conditions NtOSAK localized to cytoplasm and nucleus (A). Arabidopsis protoplasts were prepared from T87 cells and transformed with EGFP-SnRK2.4, EGFP-SnRK2.10, or EGFP constructs. Localization of the fluorescent proteins was observed before or after 30-min treatment with 50 μm CdCl₂. EGFP-SnRK2.10 protein was observed only in cytoplasm in both conditions, whereas EGFP-SnRK2.4 was localized simultaneously to cytoplasm and nucleus in control and in cadmium-containing medium. n indicates position of the nucleus. Data represent one of several independent experiments showing similar results.

Figure 5.

SnRK2.4 regulates root growth in response to cadmium ions. Sites of T-DNA insertions in SnRK2.4 gene (A). SnRK2.4 transcript levels in leaves of snrk2.4-1 (SALK_080588) and snrk2.4-2 (SALK_146522), and Col-0 determined by semiquantitative RT-PCR (B). Seeds of wild-type Arabidopsis and both mutants were sterilized and sown on square petri plates on control media or media supplemented with 30 or 100 μm CdCl₂. Seeds were vernalized for 3 d at 4°C in darkness and germinated in standard germination condition. After 14 d of growth, root length was measured by ImageJ free software (C). Values correspond to means ± sd of three independent experiments. Asterisk means statistically significant difference between root length of the mutants and wild-type plants (P < 0.05). D, Image of seedlings after 14 d of growing on control media or media supplemented with 30 or 1,000 μm CdCl₂ (bar = 1 cm). Data represent one of three independent experiments showing similar results. [See online article for color version of this figure.]

Figure 6.

Analysis of the level of thiol-containing compounds in roots of snrk2.4 mutants wild-type and Arabidopsis plants. Four-week-old Arabidopsis Col-0 wild-type and mutant plants grown in hydroponic culture were treated with 20 μm CdCl₂ for 2 d. Nonprotein thiol level was measured by Ellman’s test (A). Concentration of GSH (B) and major PC species (C) in cadmium-treated plants was analyzed in roots according to method described by Wojas et al. (2008). Asterisk means statistically significant difference in the concentration of GSH and PCs between the wild type and mutants according to ANOVA one way followed by Dunnets test (P < 0.05). Values correspond to means ± sd (n = 3).

Figure 7.

SnRK2.4 is involved in regulation of ROS level in Arabidopsis seedlings exposed to Cd²⁺. Five-day-old seedlings of snrk2.4-1 and snrk2.4-2 mutants and wild-type Col-0 were incubated for 30 min in one-half Murashige and Skoog medium (control) or medium with 50 μm CdCl₂, rinsed with medium, and stained with PI (20 μg/mL) and H₂DCFDA (30 μm), as described in “Materials and Methods.” Stained roots were observed in inverted epifluorescence microscope coupled with EZ-C1 confocal laser-scanning head. Fluorescent and differential interference contrast images are z-stacks projected from 10 collected images. A, Wild type (1, 2), snrk2.4.1 (3, 4), and snrk2.4.2 (5, 6) roots incubated in control (1, 3, 5) or cadmium-containing medium (2, 4, 6), stained with H₂DCFDA (2',7'-dichlorofluorescein fluorescence in ROS presence), PI (nuclei in dead cells stained), and both images merged with differential interference contrast images. B, Total intensity of 2',7'-dichlorofluorescein fluorescence in all images rendered from z-stack projections calculated with NIS-Elements AR 3.0 software. Data represent the mean ± sd of 18 to 30 images collected from three independent experiments. Asterisk means statistically significant difference between ROS level of mutant and wild-type plants (P < 0.05).

Figure 8.

Effect of SnRK2.4 gene mutation on expression of genes involved in iron transport and homeostasis. Transcript level was monitored by qRT-PCR in roots of four-week-old plants, not treated or treated with 20 μm CdCl₂ for 48 h (+CdCl₂) and normalized against geometric mean of four housekeeping genes. In snrk2.4-1 and snrk2.4-2 knockout lines expression of each gene is plotted relative to that in nontreated wild-type plants (Col-0). sd bars represent sd of three replicates of each sample within experiment.

Figure 9.

A hypothetical model for SnRK2 role in Arabidopsis roots exposed to Cd²⁺. Cadmium ions induce NO and ROS accumulation in plant roots, which contribute to SnRK2 activation. Active SnRK2 stimulates Cd²⁺-dependent ROS accumulation in plant cells, most probably by phosphorylation and activation of NADPH oxidase(s). SnRK2 kinases contribute to the increase of the level of PCs presumably by regulating the activity or expression level of PCS. Enhanced Cd²⁺-induced PC synthesis resulting in an increased metal tolerance might, however, cause further accumulation of ROS, as a consequence of GSH depletion.