Heavy metal stress and sulfate uptake in maize roots

Abstract

ZmST1;1, a putative high-affinity sulfate transporter gene expressed in maize (Zea mays) roots, was functionally characterized and its expression patterns were analyzed in roots of plants exposed to different heavy metals (Cd, Zn, and Cu) interfering with thiol metabolism. The ZmST1;1 cDNA was expressed in the yeast (Saccharomyces cerevisiae) sulfate transporter mutant CP154-7A. Kinetic analysis of sulfate uptake isotherm, determined on complemented yeast cells, revealed that ZmST1;1 has a high affinity for sulfate (Km value of 14.6 +/- 0.4 microm). Cd, Zn, and Cu exposure increased both ZmST1;1 expression and root sulfate uptake capacity. The metal-induced sulfate uptakes were accompanied by deep alterations in both thiol metabolism and levels of compounds such as reduced glutathione (GSH), probably involved as signals in sulfate uptake modulation. Cd and Zn exposure strongly increased the level of nonprotein thiols of the roots, indicating the induction of additional sinks for reduced sulfur, but differently affected root GSH contents that decreased or increased following Cd or Zn stress, respectively. Moreover, during Cd stress a clear relation between the ZmST1;1 mRNA abundance increment and the entity of the GSH decrement was impossible to evince. Conversely, Cu stress did not affect nonprotein thiol levels, but resulted in a deep contraction of GSH pools. Our data suggest that during heavy metal stress sulfate uptake by roots may be controlled by both GSH-dependent or -independent signaling pathways. Finally, some evidence suggesting that root sulfate availability in Cd-stressed plants may limit GSH biosynthesis and thus Cd tolerance are discussed.

Figure 1.

Phenotypic complementation of the yeast double sulfate transporter mutant CP154-7A by ZmST1;1. Yeast mutant cells expressing ZmST1;1 under the control of the inducible GAL10 promoter or harboring the empty pESC-TRP vector were grown at 28°C for 3 d on a minus-sulfur medium (−S) or on minimal media containing 0.1 mm sulfate (SO₄²⁻) or 0.1 mm homo-Cys (HCys) as sole sulfur sources.

Figure 2.

Sulfate uptake isotherm in CP154-7A cells expressing ZmST1;1. Sulfate uptake was evaluated by measuring the rate of ³⁵SO₄²⁻ absorption over a 1 min and 30 s pulse period. K_m was calculated by fitting the kinetic equation y = V_max × x/(K_m + x) to the data. Data points and error bars are means and se of two experiments run in quadruplicate (n = 8).

Figure 3.

Effect of Cd on thiol compound content (A and B), ZmST1;1 transcript level (C), and sulfate uptake capacity (D) in maize roots. Seedlings grown for 3 d in complete nutrient solutions were transferred and grown for an additional 48 h period in complete nutrient solutions supplemented with different amount of CdCl₂. NPT, total glutathione, GSH (white bars), and GSSG (black bars) levels are expressed as GSH equivalents. GSH/GSSG ratios are reported in parentheses above the histogram columns. Sulfate influxes were evaluated by measuring the rate of ³⁵SO₄²⁻ absorption into roots of intact plants over a 15 min pulse. The incubation solutions contained 0.2 mm SO₄²⁻. Bars and error bars are means and se of three experiments run in triplicate (n = 9). Different letters indicate significant differences (P < 0.05). For northern analysis, 30 μg of total RNA extracted from roots were loaded onto each lane. The blot was hybridized with ³²P-labeled ZmST1;1 probe. Ribosomal RNAs were stained on the gel with ethidium bromide (Et-Br) and used to check loading.

Figure 4.

Northern-blot analysis of ZmST1;1 expression in maize roots in response to sulfate deprivation or Cd exposure. Seedlings grown for 3 d in complete nutrient solutions were transferred and grown for additional 3, 6, and 12 h periods in complete nutrient solutions supplemented or not with 10 μm CdCl₂ or in a minus-sulfate solution. Total RNA was extracted from roots of control (C), sulfate deprived (−S), and Cd-exposed (Cd) plants. Thirty micrograms of total RNA were loaded onto each lane. Blots were hybridized with ³²P-labeled ZmST1;1 probe. Ribosomal RNAs were stained on the gel with ethidium bromide (Et-Br) and used to check loading.

Figure 5.

Effect of Zn and Cu on thiol compound content (A and B), ZmST1;1 transcript level (C), and sulfate uptake capacity (D) in maize roots. Seedlings grown for 3 d in complete nutrient solutions were transferred and grown for an additional 48 h period in complete nutrient solutions supplemented with ZnCl₂ (100 or 250 μm) or CuCl₂ (10 μm). NPT, total glutathione, GSH (white bars), and GSSG (black bars) levels are expressed as GSH equivalents. GSH/GSSG ratios are reported in parentheses above the histogram columns. Sulfate influxes were evaluated by measuring the rate of ³⁵SO₄²⁻ absorption into roots of intact plants over a 15 min pulse. The incubation solutions contained 0.2 mm SO₄²⁻. Bars and error bars are means and se of three experiments run in triplicate (n = 9). Different letters indicate significant differences (P < 0.05). For northern analysis, 30 μg of total RNA extracted from roots were loaded onto each lane. Blots were hybridized with ³²P-labeled ZmST1;1 probe. Ribosomal RNAs were stained on the gel with ethidium bromide (Et-Br) and used to check loading.

Figure 6.

Effect of sulfate availability on total glutathione levels of maize roots. Seedlings grown for 3 d in complete nutrient solutions were transferred and grown for an additional 72 h period in pregrowing solutions containing different sulfate concentrations (0, 0.2, and 2 mm). Plants were then exposed to 10 μm CdCl₂ for 48 h in the complete nutrient solution. Total glutathione levels were evaluated at the end of both pregrowing (white bars) and Cd-exposure (black bars) periods and are expressed as GSH equivalents. Bars and error bars are means and se of three experiments run in triplicate (n = 9). Different letters indicate significant differences (P < 0.05).