Enhanced formaldehyde detoxification by overexpression of glutathione-dependent formaldehyde dehydrogenase from Arabidopsis

Abstract

The ADH2 gene codes for the Arabidopsis glutathione-dependent formaldehyde dehydrogenase (FALDH), an enzyme involved in formaldehyde metabolism in eukaryotes. In the present work, we have investigated the potential role of FALDH in detoxification of exogenous formaldehyde. We have generated a yeast (Saccharomyces cerevisiae) mutant strain (sfa1Delta) by in vivo deletion of the SFA1 gene that codes for the endogenous FALDH. Overexpression of Arabidopsis FALDH in this mutant confers high resistance to formaldehyde added exogenously, which demonstrates the functional conservation of the enzyme through evolution and supports its essential role in formaldehyde metabolism. To investigate the role of the enzyme in plants, we have generated Arabidopsis transgenic lines with modified levels of FALDH. Plants overexpressing the enzyme show a 25% increase in their efficiency to take up exogenous formaldehyde, whereas plants with reduced levels of FALDH (due to either a cosuppression phenotype or to the expression of an antisense construct) show a marked slower rate and reduced ability for formaldehyde detoxification as compared with the wild-type Arabidopsis. These results show that the capacity to take up and detoxify high concentrations of formaldehyde is proportionally related to the FALDH activity in the plant, revealing the essential role of this enzyme in formaldehyde detoxification.

Figure 1.

Analysis of the yeast sfa1Δ strain (sfa1::HIS3). A, Isoelectric focusing and FALDH activity staining on homogenates of wild-type W303D (lane 1) and sfa1::HIS3 strain transformed with (lane 2) and without (lane 3) the ADH2-pYes2 plasmid. B, Southern-blot analysis of genomic DNA digested with PstI and EcoRI from sfa1::HIS3 (lane 1) or W303D (lane 2) strain. The HIS3 gene contains an internal PstI restriction site that is not present in the SFA1 gene. C, Schema of the homologous recombination procedure used to construct the sfa1Δ yeast strain. The PstI/NcoI fragment containing the SFA1 gene (at the bottom) was used as probe for Southern analysis.

Figure 2.

Electrophoretic analysis of purified Arabidopsis FALDH and specificity of the anti-FALDH antibodies. A, Purified FALDH (1.4 μg) was analyzed by SDS-PAGE and revealed by silver staining (lane 1). B, Specificity of the antibodies. Twenty micrograms of Arabidopsis protein extracts was electrophoresed, blotted to a membrane, stained with Coomassie Blue (lane 1), and developed with the anti-FALDH antibodies (lane 2) or the pre-immune antiserum (lane 3). C, Competition experiments for the antibodies binding. Fifty nanograms of purified FALDH (lanes 1, 3, and 5) or 20 μg of Arabidopsis protein extracts (lanes 2, 4, and 6) was electrophoresed, and the blotted membranes were incubated either with the antibodies (lanes 1 and 2), the pre-immune antiserum (lanes 3 and 4), or the antibodies pre-incubated with 30 μg of the protein used for immunization (lanes 5 and 6).

Figure 3.

Growth kinetics and formaldehyde detoxification by yeast strains expressing Arabidopsis FALDH. Growth (A) and formaldehyde concentration (B) were measured in culture medium containing no added formaldehyde (circles) or 1 (triangles) or 2 (squares) mm formaldehyde. White symbols, Yeast strain overexpressing the Arabidopsis FALDH; black symbols, W303D yeast strain.

Figure 4.

Analysis of Arabidopsis transgenic lines. Western-blot analysis and FALDH-specific activity (SA) values of several independent transgenic lines overexpressing Arabidopsis FALDH (A) or bearing FALDH antisense constructs (B). Ten-day-old homozygous seedlings (T₃ progeny) grown in liquid medium were used. Schematic diagrams of the constructs used for transformation are also shown. n.d., Not determined. CaMV35S, 35S cauliflower mosaic virus promoter. p-NOS, Nopaline synthase promoter. NPTII, neomycin phosphotransferase gene-coding sequence.

Figure 5.

Kinetics of formaldehyde detoxification by Arabidopsis transgenic lines. Seedlings germinated and grown in liquid medium were subjected to formaldehyde treatment at an initial concentration of 2 (A) or 5 (B) mm, respectively. Transgenic lines are those shown in Figure 4. Lines 1 (○), 4 (▿), and 6 (▾) express high levels of FALDH. Line 13 (⋄) is a cosuppression line. Lines 5a (▪), 10a (□), and 17a (♦) are antisense lines. ▪, Wild-type Arabidopsis. Data are shown as percentage formaldehyde remaining in the liquid culture at different times after inoculation. Values are expressed as the mean of at least two separate experiments. ses of means were <12%.