Nitric oxide contributes to cadmium toxicity in Arabidopsis by promoting cadmium accumulation in roots and by up-regulating genes related to iron uptake

Abstract

Nitric oxide (NO) functions as a cell-signaling molecule in plants. In particular, a role for NO in the regulation of iron homeostasis and in the plant response to toxic metals has been proposed. Here, we investigated the synthesis and the role of NO in plants exposed to cadmium (Cd(2+)), a nonessential and toxic metal. We demonstrate that Cd(2+) induces NO synthesis in roots and leaves of Arabidopsis (Arabidopsis thaliana) seedlings. This production, which is sensitive to NO synthase inhibitors, does not involve nitrate reductase and AtNOA1 but requires IRT1, encoding a major plasma membrane transporter for iron but also Cd(2+). By analyzing the incidence of NO scavenging or inhibition of its synthesis during Cd(2+) treatment, we demonstrated that NO contributes to Cd(2+)-triggered inhibition of root growth. To understand the mechanisms underlying this process, a microarray analysis was performed in order to identify NO-modulated root genes up- and down-regulated during Cd(2+) treatment. Forty-three genes were identified encoding proteins related to iron homeostasis, proteolysis, nitrogen assimilation/metabolism, and root growth. These genes include IRT1. Investigation of the metal and ion contents in Cd(2+)-treated roots in which NO synthesis was impaired indicates that IRT1 up-regulation by NO was consistently correlated to NO's ability to promote Cd(2+) accumulation in roots. This analysis also highlights that NO is responsible for Cd(2+)-induced inhibition of root Ca(2+) accumulation. Taken together, our results suggest that NO contributes to Cd(2+) toxicity by favoring Cd(2+) versus Ca(2+) uptake and by initiating a cellular pathway resembling those activated upon iron deprivation.

Figure 1.

NO production in Arabidopsis roots exposed to Cd²⁺. Roots of 3-week-old Arabidopsis seedlings grown on plates were loaded with 20 μm DAF-2DA for 2 h and then treated with 200 μm Cd²⁺ for 7 h in the absence or presence of 1 mm cPTIO, 5 mm l-NAME, or 1 mm PBITU. Roots were observed in phase contrast (A, D, G, J, and M), in fluorescence (B, E, H, K, and N), or both (C, F, I, L, and O). A to C, Cd²⁺-treated roots. D to F, Cd²⁺ + cPTIO-treated roots. G to I, Cd²⁺ + l-NAME-treated roots. J to L, Cd²⁺ + PBITU-treated roots. M to O, Control (water-treated) plants. Results are from one of four representative experiments.

Figure 2.

NO production in leaves of Arabidopsis plants treated by Cd²⁺ at the root level. Roots of 2-week-old Arabidopsis plants grown on plates were treated with 50 μm Cd²⁺ for 96 h. Leaves were detached, infiltrated with 20 μm DAF-2DA, and then observed in phase contrast (A) or in fluorescence (B). Results are from one of three representative experiments.

Figure 3.

NO production and origin in Cd²⁺-treated Arabidopsis leaf discs. A, NO production in Cd²⁺-treated Arabidopsis leaf discs. Leaf discs from 7-week-old Arabidopsis plants grown on soil were vacuum infiltrated with 20 μm DAF-2DA, maintained for 1 h in the dark, and then incubated with 200 μm Cd²⁺. NO production was followed by measuring DAF-2T fluorescence. Each value represents the mean ± sd of 30 measurements (10 replicates per experiment performed three times). B, Origin of Cd²⁺-induced NO production. Leaf discs from 7-week-old Arabidopsis wild-type (WT), atnoa1, and nia1 nia2 plants were treated by 200 μm Cd²⁺ in the absence or presence of 1 mm cPTIO, 5 mm l-NAME, or 500 μm PBITU. NO production measured after 20 h of treatment is expressed as a percentage of the maximal response measured in wild-type leaf discs in response to Cd²⁺. The experimental procedure was the same as described for A. Each value represents the mean ± sd of 30 measurements (10 replicates per experiment performed three times).

Figure 4.

NO involvement in Cd²⁺-triggered root growth inhibition. Arabidopsis seedlings were grown vertically for 3 weeks on plates containing 30 μm Cd²⁺ and supplemented or not with 250 μm cPTIO or 0.5 mm l-NAME. Primary root length was measured using Optimas version 6.0. The mean ± se of four independent experiments is represented. Means with a same letter are not significantly different at P = 0.05.

Figure 5.

Class I NO-regulated genes. Annotation refers to automatic Arabidopsis annotation according to Arabidopsis Genome Initiative (AGI) number from The Institute for Genomic Research. The Cd 15, Cd 30, Cd 30 + l-NAME, and l-NAME ratio columns indicate CATMA results in a log₂ ratio obtained for comparisons between 15 μm Cd²⁺-, 30 μm Cd²⁺-, 30 μm Cd²⁺ + l-NAME-, and l-NAME-treated plants and untreated plants. A statistical cutoff, P < 0.05 after Bonferroni correction (see color code), was used to determine which genes were differentially expressed. A positive ratio indicates that the gene is induced (red), and a negative ratio indicates that the gene is repressed (green).

Figure 6.

Class II NO-regulated genes. For details, see Figure 5 legend.

Figure 7.

NO production in irt1 and wild-type roots exposed to Cd²⁺ or to Cd²⁺ and/or Fe²⁺, respectively. Roots of 3-week-old Arabidopsis irt1 and wild-type seedlings grown on plates were loaded with 20 μm DAF-2DA for 2 h and then treated with 200 μm Cd²⁺ for 7 h in the presence or absence of 200 μm Fe²⁺. Sorbitol (250 mm) was used as a positive control for NO production. A, Control wild-type (WT) roots. B, Cd²⁺-treated wild-type roots. C, Fe²⁺-treated wild-type roots. D, Cd²⁺ + Fe²⁺-treated wild-type roots. E, Control irt1 roots. F, Cd²⁺-treated irt1 roots. G, Sorbitol-treated irt1 roots. Results are from one of four representative experiments.

Figure 8.

Hypothetical scheme for NO functions in Arabidopsis roots exposed to Cd²⁺. NO favors root Cd²⁺ uptake and therefore contributes to root growth inhibition by partly preventing the Cd²⁺-induced repression of the Fe starvation-responsive genes IRT1, FRO2, and FIT and by decreasing root Ca²⁺ content.