Differential regulation of the expression of two high-affinity sulfate transporters, SULTR1.1 and SULTR1.2, in Arabidopsis

Abstract

The molecular mechanisms regulating the initial uptake of inorganic sulfate in plants are still largely unknown. The current model for the regulation of sulfate uptake and assimilation attributes positive and negative regulatory roles to O-acetyl-serine (O-acetyl-Ser) and glutathione, respectively. This model seems to suffer from exceptions and it has not yet been clearly validated whether intracellular O-acetyl-Ser and glutathione levels have impacts on regulation. The transcript level of the two high-affinity sulfate transporters SULTR1.1 and SULTR1.2 responsible for sulfate uptake from the soil solution was compared to the intracellular contents of O-acetyl-Ser, glutathione, and sulfate in roots of plants submitted to a wide diversity of experimental conditions. SULTR1.1 and SULTR1.2 were differentially expressed and neither of the genes was regulated in accordance with the current model. The SULTR1.1 transcript level was mainly altered in response to the sulfur-related treatments. Split-root experiments show that the expression of SULTR1.1 is locally regulated in response to sulfate starvation. In contrast, accumulation of SULTR1.2 transcripts appeared to be mainly related to metabolic demand and is controlled by photoperiod. On the basis of the new molecular insights provided in this study, we suggest that the expression of the two transporters depends on different regulatory networks. We hypothesize that interplay between SULTR1.1 and SULTR1.2 transporters could be an important mechanism to regulate sulfate content in the roots.

Figure 1.

Relationship between glutathione content and accumulation of SULTR1.1 and SULTR1.2 mRNA in Arabidopsis roots. The mRNA accumulation of SULTR1.1 (white circles) and SULTR1.2 (black diamonds) is plotted against the root glutathione content. Accumulation of SULTR1.1 and SULTR1.2 mRNA is expressed relative to that of the reference genes as −ΔCt values. Each data point was obtained from the analysis of roots collected from a pool of six plants. For both SULTR1.1 and SULTR1.2, each symbol represents one of the different treatments used in this work and is the average of four to six biological repeats. The three glutathione treatments were not considered in this analysis. The two black lines represent linear regression lines corresponding to the least square adjustment of all the data obtained for either the SULTR1.1 or the SULTR1.2 gene; the corresponding Pearson's correlation coefficients (R²) are reported.

Figure 2.

Relationship between O-acetyl-Ser content and accumulation of SULTR1.1 and SULTR1.2 mRNA in Arabidopsis roots. The mRNA accumulation of SULTR1.1 (white circles) and SULTR1.2 (black diamonds) is plotted against the root O-acetyl-Ser content. Accumulation of SULTR1.1 and SULTR1.2 mRNA is expressed relative to that of the reference genes as −ΔCt values. Each data point was obtained from the analysis of roots collected from a pool of six plants. For both SULTR1.1 and SULTR1.2, each symbol represents one of the different treatments used in this work and is the average of four to six biological repeats. The O-acetyl-Ser treatment was not considered in this analysis. The two black lines represent linear regression lines corresponding to the least square adjustment of all the data obtained for either the SULTR1.1 or the SULTR1.2 gene; the corresponding Pearson's correlation coefficients (R²) are reported.

Figure 3.

Relationship between sulfate content and accumulation of SULTR1.1 and SULTR1.2 mRNA in Arabidopsis roots. The root sulfate content is plotted against the accumulation of SULTR1.1 (A) and SULTR1.2 (B) mRNA inferred from quantitative PCR analysis. Accumulation of SULTR1.1 and SULTR1.2 mRNA is expressed relative to that of the reference genes as −ΔCt values. Each symbol represents one of the different treatments used in this work and is the average of four to six biological repeats. The sulfate-free treatments were not considered in this analysis. The two black lines represent linear regression lines corresponding to the least square adjustment of all the data obtained for either the SULTR1.1 or the SULTR1.2 gene; the corresponding Pearson's correlation coefficients (R²) are reported.

Figure 4.

Effect of localized sulfate starvation on accumulation of sulfate, glutathione, O-acetyl-Ser, and SULTR1.1 and SULTR1.2 mRNA. Plants were grown hydroponically for 5 weeks, and then roots were split in two parts and maintained in complete nutrient solution for an additional week, before each part of split-roots was treated with either sulfate deficient (−S) or sufficient (+S) media. Each side of split-roots was separately harvested 3 d after the treatment, and accumulation of sulfate (black bars), glutathione (white bars; A), and O-acetyl-Ser (hatched bars; B) was determined. Abundance of SULTR1.1 (C) and SULTR1.2 (D) mRNA was normalized against their respective expression in +S/+S control conditions. Individual measurements were obtained from the analysis of roots collected from a pool of five plants. Error bars correspond to sd; biological repeats (n =6).

Figure 5.

Comparison of SULTR1.1 and SULTR1.2 mRNA accumulation. Accumulation of SULTR1.1 and SULTR1.2 mRNA is expressed relative to that of the reference genes as −ΔCt values. Each data point was obtained from the analysis of roots collected from a pool of six plants. Each symbol represents one of the different treatments used in this work and is the average of four to six biological repeats. The white symbols correspond to the glutathione treatments. The black line represents the linear regression line corresponding to the least square adjustment of all the data, whereas the dotted line represents the linear regression line calculated without considering the glutathione treatments; the corresponding Pearson's correlation coefficients (R²) are reported.

Figure 6.

Effect of individual treatments on the accumulation of SULTR1.1 and SULTR1.2 mRNA. Accumulation of SULTR1.1 and SULTR1.2 mRNA is expressed relative to that of the reference genes as −ΔCt values. The treatments to which the plants were submitted are detailed in “Materials and Methods”. Every data point was obtained from the analysis of roots collected from a pool of six plants. Error bars correspond to sd; biological repeats (4 ≤ n ≤ 6).

Figure 7.

Accumulation of SULTR1.2 mRNA during day and night. Roots of 6-week-old plants grown under an 8-h-day/16-h-night cycle from germination were collected just before switching to day, and 2, 5, and 8 h thereafter. Additionally samples were collected 5 and 10 h after switching back to night. Accumulation of SULTR1.2 mRNA is expressed relative to that of the reference genes as −ΔCt values. Every data point was obtained from the analysis of roots collected from a pool of six plants. Error bars correspond to sd; biological repeats (n =6).

Figure 8.

Schematic representation for the differential regulation of SULTR1.1 and SULTR1.2. In the model, the expression of SULTR1.2 is predominantly controlled by metabolic demand (S/N/C), while limitation in sulfate availability in conjunction with stress conditions results in the activation of SULTR1.1 expression. In this scheme, the thick and thin hashed arrows indicate the major and minor signaling networks, respectively.