T-DNA insertion mutants reveal complex expression patterns of the aldehyde dehydrogenase 3H1 locus in Arabidopsis thaliana

Abstract

The Arabidopsis thaliana aldehyde dehydrogenase 3H1 gene (ALDH3H1; AT1G44170) belongs to family 3 of the plant aldehyde dehydrogenase superfamily. The full-length transcript of the corresponding gene comprises an open reading frame of 1583 bp and encodes a protein of 484 amino acid residues. Gene expression studies have shown that this transcript accumulates mainly in the roots of 4-week-old plants following abscisic acid, dehydration, and NaCl treatments. The current study provided experimental data that the ALDH3H1 locus generates at least five alternative transcript variants in addition to the previously described ALDH3H1 mRNA. The alternative transcripts accumulated in wild-type plants at a low level but were upregulated in a mutant that carried a T-DNA insertion in the first exon of the gene. Expression of the transcript isoforms involved alternative gene splicing combined with an alternative promoter. The transcript isoforms were differentially expressed in the roots and shoots and showed developmental stage- and tissue-specific expression patterns. These data support the hypothesis that alternative isoforms produced by gene splicing or alternative promoters regulate the abundance of the constitutively spliced and functional variants.

Fig. 1.

Structural organization of the ALDH3H1 gene locus in A. thaliana. (A) Schematic representation of the protein-coding gene models of the ALDH3H1 locus. Grey boxes indicate the protein-coding regions of exons numbered from 1 to 10. The black box represents exon 2′. Open white boxes represent introns. Blue boxes and arrows represent the 5′- and 3′UTR, respectively. The black arrow labelled ATG indicates the position of the translational start codon. (B) Locations of the T-DNA insertions and the primers used for molecular analyses. Inverted red and yellow triangles indicate T-DNAs. The names above the triangles (3h1-A, 3h1-B, and 3h1-C) refer to the corresponding T-DNA insertion lines. The 5′- and 3′UTRs are shown as horizontal black lines. Arrows indicate the orientation of primers and their approximate position within the gene or the T-DNA. LB and RB indicate the left and right borders of the T-DNA fragment. See Table 1 for primer sequences.

Fig. 2.

Molecular analysis of the ALDH3H1 T-DNA insertion mutants 3h1-A, 3h1-B, and 3h1-C. (A) PCR-based genotyping of the homozygous 3h1-A, 3h1-B and 3h1-C lines. The upper and lower panels show amplicons produced using ALDH3H1 gene-specific primers and the combination of gene and T-DNA specific primers in PCR experiments using genomic DNA, respectively. See Materials and methods for details of the genotyping. WT, genomic DNA (Col-0). (B–D) Comparative analysis of ALDH3H1 transcripts in homozygous 3h1-A, 3h1-B, and 3h1-C mutants. P1, P2, P3, and P4 are the forward primers used in combination with the reverse primer P5 or E9R in the RT-PCR assays. Primer pair P1/P5 was specific to ALDH3H1-α, whereas P2/P5 and P2/E9R allowed amplification of ALDH3H1-β. Primer pairs P3/P5 and P4/P5 could not discriminate between the variants. Genomic WT DNA (Col-0) was used as control (G), while (–) denotes a control reaction without nucleic acid. M, DNA size marker. See Fig. 1B and Table 1 for details of the primers.

Fig. 3.

Agarose gel electrophoresis of amplicons derived from RT-PCR experiments using total RNA from homozygous 3h1-A plants. A1 and A2 are two independently extracted RNA samples, G denotes genomic WT DNA (Col-0) and (–) a control reaction without nucleic acid. See Fig. 1B for primer locations.

Fig. 4.

Schematic representation of the major spliced isoforms of ALDH3H1. The structure of the primary transcript and the splicing events leading to the isolated spliced transcripts are shown above each variant. Grey boxes indicate the protein-coding region of exons. The black box represents exon 2′. Open white boxes indicate introns. Horizontal black lines indicate the 5′- and 3'UTR. The 5′UTR is not shown for the alternative isoforms because it could not be determined unequivocally. Further details are provided in the text. Triangles indicate splicing events confirmed by identification of the resulting cDNAs, while question marks indicate a putative splicing event. An asterisk (*) indicates the presence of at least one stop codon in the retained intron. The black arrow labelled ATG indicates the position of the translational start codon of the predicted longest open reading frame.

Fig. 5.

Accumulation of the ALDH3H1 protein in WT, ALDH3H1 overexpressors, and T-DNA insertion mutants. Fifteen micrograms of crude protein extract from plant leaves was analysed by immunoblotting. Antiserum raised against the ALDH3H1 protein was used for the immunodetection assay. Line 3h1-B is homozygous 3h1-B, lines 3h1-A3 and 3h1-A4 are homozygous 3h1-A plants, and lines 3h1-C4 and 3h1-C9 are homozygous 3h1-C plants. S10 and S13 are two independent transgenic lines overexpressing the protein ALDH3H1-α. The extra band indicated by an asterisk (*) is intrinsic to the antiserum and is not ALDH3H1 specific because this band occurred in all samples including that from the knockout line 3h1-C, which does not produce a transcript. (This figure is available in colour at JXB online.)

Fig. 6.

RT-PCR-based expression analysis of the major and alternatively spliced ALDH3H1 isoforms under normal growth conditions. Primer pairs that were used in the RT-PCR assays are shown for each panel to refer to the major (ALDH3H1-α) and the alternative (β, γ, δ, ϵ, and ζ) isoforms. Primer pair P1/P5 was specific to ALDH3H1-α, whereas P2/P5 and P2/E9R allowed simultaneous amplification of ALDH3H1-β and the other alternative isoforms (γ, δ, ϵ, and ζ). Primer pairs P3/P5 or P4/P5 could not discriminate between the variants (see Fig. 1b for primer locations). (A) Comparative analysis of the accumulation of ALDH3H1 transcripts in WT, homozygous (3h1-AA) and heterozygous (3h1-Aa) 3h1-A mutants. (B, C) Semi-quantitative analysis of the expression of the isoforms in WT cDNA (B) or both WT and 3h1-A fully developed leaf tissues (C). R1, R2, and A in (B) represent reactions involving primer pairs P1/E9R and P2/E9R and those for Actin2, respectively. (D) Tissue-specific expression of the isoforms. Primer pairs P2/I5 and P2/I7 partially discriminated between the ALDH3H1 isoforms δ, ϵ, and ζ and did not amplify the isoforms α, β, or γ. The numbers 1 and 2 indicate amplicons 1 and 2, respectively.

Fig. 7.

In situ GUS staining of transiently transformed A. thaliana seedlings carrying the 3h1-intron::GUS gene cassette. The promoter activity of the intron 1 fragment of ALDH3H1 is shown by expression of the GUS reporter protein in roots and cotyledons. The figure shows GUS staining from three different seedlings.

Fig. 8.

Expression patterns of the ALDH3H1 transcript isoforms in response to stress conditions. The expression pattern of the major transcript (ALDH3H1-α) was compared with that of the alternatively spliced ALDH3H1 isoforms (β, γ, δ, ϵ, and ζ) following dehydration and NaCl treatments in seedlings (A) and in adult plants (B). Primer pairs that were used in the PCR assays are shown for each panel to refer to the major (ALDH3H1-α) and the alternative isoforms. Primer pair P1/P5 was specific to ALDH3H1-α whereas P2/P5 allowed simultaneous amplification of ALDH3H1-β and the other alternative isoforms. Primer pair P4/P5 could not discriminate between the variants (see Fig. 1B for the primer locations).

Fig. 9.

Root lengths, germination rates, and MDA levels of the ALDH3H1 T-DNA insertion mutants and of the ALDH3H1-α overexpressors subjected to salt or dehydration stress. (A) Root growth inhibition by salt. Root growth was measured as the increase in root length on MS agar medium supplemented with 100 mM NaCl. The growth on medium not supplemented with salt was taken as the reference for calculating the percentage increase in root length. 3h1-Aa and 3h1-AA refer to the heterozygous and homozygous knockout T-DNA insertion mutant 3h1-A, respectively. Columns with different letters (a, b) are significantly different (Student’s t-test; P <0.05). (B) Germination rate. The percentage of seeds that germinated on salt is shown for each genotype. (C) MDA contents in 2-week-old seedlings grown on MS agar supplemented with NaCl. (D, E) MDA content in adult plants grown on soil and irrigated with different concentrations of NaCl (D) or subjected to dehydration (E). Line 3h1-B is an ALDH3H1 knockout T-DNA insertion mutant, while S10 and S13 are ALDH3H1-α overexpressors. FW, fresh weight.