A Formate Dehydrogenase Confers Tolerance to Aluminum and Low pH

Abstract

Formate dehydrogenase (FDH) is involved in various higher plant abiotic stress responses. Here, we investigated the role of rice bean (Vigna umbellata) VuFDH in Al and low pH (H(+)) tolerance. Screening of various potential substrates for the VuFDH protein demonstrated that it functions as a formate dehydrogenase. Quantitative reverse transcription-PCR and histochemical analysis showed that the expression of VuFDH is induced in rice bean root tips by Al or H(+) stresses. Fluorescence microscopic observation of VuFDH-GFP in transgenic Arabidopsis plants indicated that VuFDH is localized in the mitochondria. Accumulation of formate is induced by Al and H(+) stress in rice bean root tips, and exogenous application of formate increases internal formate content that results in the inhibition of root elongation and induction of VuFDH expression, suggesting that formate accumulation is involved in both H(+)- and Al-induced root growth inhibition. Over-expression of VuFDH in tobacco (Nicotiana tabacum) results in decreased sensitivity to Al and H(+) stress due to less production of formate in the transgenic tobacco lines under Al and H(+) stresses. Moreover, NtMATE and NtALS3 expression showed no changes versus wild type in these over-expression lines, suggesting that herein known Al-resistant mechanisms are not involved. Thus, the increased Al tolerance of VuFDH over-expression lines is likely attributable to their decreased Al-induced formate production. Taken together, our findings advance understanding of higher plant Al toxicity mechanisms, and suggest a possible new route toward the improvement of plant performance in acidic soils, where Al toxicity and H(+) stress coexist.

Figure 1.

Biochemical analysis of the recombinant VuFDH protein. A, SDS-PAGE gel showing the HisTrap FF affinity-purified His-tagged VuFDH protein stained with Coomassie Blue. Marker, molecular weight markers; VuFDH-f, His-tagged full-length VuFDH; VuFDH-a, His-tagged VuFDH lacking the signal peptide. B, Substrate specificity of VuFDH. Formate dehydrogenase activities were determined at pH 7.0, with various substrates at concentrations of 50 mm.

Figure 2.

Subcellular localization of VuFDH in N. benthamiana leaves. Constructs expressing VuFDH-GFP fusion protein were transiently expressed in N. benthamiana leaves. A mitochondria-specific fluorescence dye, TMRM, was used to stain mitochondria. Bar: 20 μm.

Figure 3.

Rice bean VuFDH expression patterns. A, Time-dependent VuFDH expression in rice bean root tips (0–1 cm). The roots were exposed to 25 μm Al for various times. B, Dose-dependent VuFDH expression in rice bean root tips (0–1 cm). The roots were exposed to various concentrations of Al for 12 h. C, pH- and metal-dependent VuFDH expression in rice bean root tips (0–1 cm). The seedlings were grown at pH 5.5 and subjected to low pH (4.5) or low pH with various metals for 12 h. D, Tissue-specific expression of VuFDH. Seedlings were exposed to 0 or 25 μm Al for 12 h. All data were normalized relative to VuFDH expression in the absence of Al at pH 4.5. The expression was determined by RT-PCR and 18S rRNA was used as an internal control. Values are means ± sd (n = 3). The asterisk indicates significant differences between treatment and control (pH 4.5 without Al stress).

Figure 4.

The effect of low pH stress on root elongation and VuFDH expression in rice bean. A, Relative root elongation. Seedlings were grown in nutrient solution with different pH values for 12 h. Root elongation was measured with a ruler before and after treatment (n = 12). B, VuFDH expression. After treatments, root tips (0–1 cm) were excised for RNA extraction and qRT-PCR analysis of VuFDH expression (n = 3). Different letters indicate significant differences between treatments.

Figure 5.

The effect of Al stress and H⁺ stress on rice bean root tip formate content. A, Al-induced formate accumulation. Seedlings were exposed to nutrient solution containing 0 or 25 μm AlCl₃ for different times. B, H⁺ stress-induced formate accumulation. Seedlings were exposed to nutrient solution with different pH values for 12 h. After treatment, the root tips were homogenized thoroughly in deionized water for formate content analysis. Data are expressed as mean ± sd (n = 3). Asterisks in (A) and different letters in (B) indicate significant differences between treatments at P < 0.05.

Figure 6.

The effect of exogenous formate on rice bean root growth, internal formate accumulation, and VuFDH expression. A, The effect of formate on rice bean root elongation. Seedlings were exposed to nutrient solution (pH 4.5) containing different concentrations of exogenous formate for 12 h. Root elongation was measured with a ruler before and after treatment (n = 12). After treatment, root tips (0–1 cm) were excised for internal formate quantification (n = 3). In a parallel experiment, root tips (0–1 cm) were excised for RNA extraction and qRT-PCR analysis of VuFDH expression (n = 3). B, Correlation between internal formate content and VuFDH expression. Different letters indicate significant differences between treatments at P < 0.05.

Figure 7.

Over-expression of VuFDH enhances Al and H⁺ tolerance. A, Detection of expression of VuFDH in the wild-type and VuFDH over-expression lines. RT-PCR analysis was performed to detect the mRNA expression of VuFDH (32 cycles) and the internal control NtACTIN (29 cycles). B, Representative seedlings showing difference in Al sensitivity between the wild-type and the over-expression lines. Seedlings were grown in the 1:30 strength Hoagland nutrient solution containing 0, 4 or 6 μm AlCl₃ at pH 5.0 for 6 d. C, Representative seedlings showing difference in H⁺ sensitivity between the wild-type and the over-expression lines. Seedlings were grown in the 1:30 strength Hoagland nutrient solution at pH 5.5 or 4.5 for 6 d. D, Relative root elongation of wild-type and the transgenic lines grown as described in (B). Data are means ± sd (n = 15). E, Relative root elongation between wild-type and the transgenic lines grown as described in (C). Data are means ± sd (n = 15). Dashed white lines in (B) and (C) indicate the root tip position at the beginning of treatment. Different letters indicate significant differences between treatments at P < 0.05. Bar: 1 cm in (B) and (C).

Figure 8.

The effect of Al and H⁺ stress on formate content in wild-type and over-expression tobacco lines. A, Al-induced accumulation of formate. The plants of wild-type and two independent transgenic lines were exposed to 1:30 strength Hoagland nutrient solution with 0 or 4 μm Al for 24 h. B, H⁺ stress-induced accumulation of formate. The plants of wild-type and two independent transgenic lines were exposed to 1:30 strength Hoagland nutrient solution with pH adjusted to either 5.5 or 4.5 for 24 h. After treatment, root tips were homogenized thoroughly in deionized water for formate content analysis. Data are mean ± sd (n = 3). Asterisk indicates significant differences between treatments at P < 0.05.

Figure 9.

The effect of Al stress on the expression of Al-tolerance gene expression in tobacco. A, NtMATE expression. B, NtALS3 expression. The plants of wild-type and independent transgenic line1 were exposed to 1:30 strength Hoagland nutrient solution containing 4 μm Al for different times. The expression was determined by RT-PCR and NtACTIN was used as an internal control. Data are means ± sd (n = 3).