Cadmium-induced sulfate uptake in maize roots

Abstract

The effect of cadmium (Cd) on high-affinity sulfate transport of maize (Zea mays) roots was studied and related to the changes in the levels of sulfate and nonprotein thiols during Cd-induced phytochelatin (PC) biosynthesis. Ten micromolar CdCl(2) in the nutrient solution induced a 100% increase in sulfate uptake by roots. This was not observed either for potassium or phosphate uptake, suggesting a specific effect of Cd(2+) on sulfate transport. The higher sulfate uptake was not dependent on a change in the proton motive force that energizes it. In fact, in Cd-treated plants, the transmembrane electric potential difference of root cortical cells was only slightly more negative than in the controls, the external pH did not change, and the activity of the plasma membrane H(+)-ATPase did not increase. Kinetics analysis showed that in the range of the high-affinity sulfate transport systems, 10 to 250 microM, Cd exposure did not influence the K(m) value (about 20 microM), whereas it doubled the V(max) value with respect to the control. Northern-blot analysis showed that Cd-induced sulfate uptake was related to a higher level of mRNA encoding for a putative high-affinity sulfate transporter in roots. Cd-induced sulfate uptake was associated to both a decrease in the contents of sulfate and glutathione and synthesis of a large amount of PCs. These results suggest that Cd-induced sulfate uptake depends on a pretranslational regulation of the high-affinity sulfate transporter gene and that this response is necessary for sustaining the higher sulfur demand during PC biosynthesis.

Figure 1

Effects of Cd²⁺ exposure on growth of roots (A) and shoots (B) of maize plants. Plants were grown for 96 h in a complete nutrient solution supplemented (●) or not (○) with 10 μm CdCl₂. Plants were harvested at different times, blotted with paper towels, and weighed. Data points and error bars are means and se of two experiments run in quadruplicate (n = 8).

Figure 2

Time course of Cd²⁺ accumulation in roots (▪) and shoots (●) of maize plants grown in a complete nutrient solution supplemented with 10 μm CdCl₂. Plants were harvested at different times, and their Cd²⁺ contents were measured, after complete mineralization, by atomic absorption spectrophotometry. Data points and error bars are means and se of two experiment run in triplicate (n = 6).

Figure 3

Effect of Cd²⁺ exposure on the sulfate content of maize roots. Plants were grown for 24 or 48 h in a complete nutrient solution supplemented (black bars) or not (white bars) with 10 μm CdCl₂. At different times, roots were excised from shoots, rinsed with distilled water, and homogenized. The sulfate content was measured turbidimetrically. Bars and error bars are means and se of two experiments run in quadruplicate (n = 8).

Figure 4

Effects of Cd²⁺ exposure on SO₄²⁻ (A), K⁺ (B), and Pi (C) uptake in maize roots. Plants were grown for 24 or 48 h in a complete nutrient solution supplemented (black bars) or not (white bars) with 10 μm CdCl₂. The influxes were evaluated by measuring the rates of ³⁵SO₄²⁻, ⁸⁶Rb⁺, and ³²Pi absorption into roots of intact plants over a 10-min pulse. The incubation solutions contained 200 μm SO₄²⁻, 240 μm K⁺, and 40 μm Pi. Bars and error bars are means and se of three experiments in run quadruplicate (n = 12).

Figure 5

Sulfate influx isotherms in maize roots (A) and relative Lineweaver-Burk plot (B). Plants were grown for 24 h in a complete nutrient solution supplemented (●) or not (○) with 10 μm CdCl₂. The influxes were evaluated by measuring the rates of ³⁵SO₄²⁻ absorption into roots of intact plants over a 10-min pulse. Sulfate concentrations ranged from 10 to 250 μm. Data points and error bars are means and se of three experiments in quadruplicate (n = 12).

Figure 6

Northern-blot analysis of HAST expression in maize roots. Total RNA was extracted from roots of sulfur-starved (−S), control (C), and Cd-exposed (Cd) plants. Thirty micrograms of total RNA was loaded onto each lane. Blots were hybridized with ³²P-labeled HAST probe. Ribosomal RNAs were stained on the gel with ethidium bromide (Et-BR) and used to check loading.