Characterisation of selected Arabidopsis aldehyde dehydrogenase genes: role in plant stress physiology and regulation of gene expression

Abstract

The importance of aldehyde dehydrogenase (ALDH) proteins in plant stress responses was investigated in this study by functionally analysing transgenic Arabidospsis thaliana ALDH knock-out and over-expressing plants. From the nine ALDH gene families present in Arabidopsis, four gene members of the families 10, 3 and 7 have been analysed in this work. <br /> Both ALDH10A8 (AT1G74920) and ALDH10A9 (AT3G48170) belong to the family 10 of the superfamily of ALDH proteins and, based on sequence similarity, they putatively code for betaine aldehyde dehydrogenases (BADHs), enzymes that catalyse the last step of glycine betaine biosynthesis. But, Arabidopsis is known not to be able to produce glycine betaine. The function of these two genes was therefore investigated. ALDH10A8 was found to be localized in leucoplasts whereas ALDH10A9 is targeted to peroxisomes. The ALDH10A8 and ALDH10A9 transcripts were detected in the plant and were slightly induced by stress treatments. Plants lacking ALDH10A8 transcripts were found to be drought and salt sensitive, indicating that ALDH10A8 may be involved in other pathways than the biosynthesis of glycine betaine in Arabidopsis. Using betaine aldehyde, 4-aminobutyraldehyde (ABAL) and 3-aminopropionaldehyde (APAL) as substrates, the recombinant ALDH10A9 protein showed both betaine aldehyde and aminoaldehyde dehydrogenase activities, although the affinity to the substrates was low compared to data from the literature. No enzymatic data was obtained for ALDH10A8 as it was not possible to purify sufficient amounts of the enzyme in its active form. Considering the high amino acid sequence similarity between ALDH10A8 and ALDH10A9, I propose that ALDH10A8 may be also active in vivo and likely both proteins function as aminoaldehyde dehydrogenases by detoxifying cells from metabolism-derived cytotoxic aminoaldehydes. <br /> The Arabidopsis ALDH3H1 (AT1G44170) gene belongs to the family 3 of the ALDH superfamily. Previous findings showed that ALDH3H1 transcripts mostly accumulate in roots of 4 week-old plants upon ABA, dehydration and NaCl treatments. Here, the expression analysis was extended to the protein level and in adult plants. Together with the previous observations it is found that the up-regulation of ALDH3H1 protein by salt stress mainly occurs in leaves of plants older than 4 weeks. To understand the function of ALDH3H1 in the stress response of Arabidopsis, transgenic plants over-expressing the ALDH3H1 protein were generated and analysed. It appeared that the constitutive expression of ALDH3H1 did not confer stress tolerance to the transgenic plants. However, the results indicate that the ALDH3H1 protein can help the plant to cope with stress injuries by alleviating damages from lipid peroxidation. <br /> Besides, the results from this study gives for the first time the experimental evidence that the ALDH3H1 short transcript variant (AT1G44170.3 (T3)) is expressed in Arabidopsis. It is nearly absent or expressed at a very low level in the wild type but accumulates in the 3h1-A mutant, which carries a T-DNA insertion in the first exon of the ALDH3H1 locus. The expression of the transcript T3 is shown to be directed by an alternative promoter comprised within the first intron of this gene. T3 and other ALDH3H1 transcript variants (AT1G44170.1 (T1) and AT1G44170.2 (T2)) are found to be differentially expressed in roots and shoots. Sub-cellular localisation experiments indicated that the protein T3 is targeted to the cytosol but its presence could be revealed neither in the 3h1-A mutant nor the wild type by using ALDH3H1 antibodies. Comparative analysis of the wild type and different T-DNA insertion mutants showed that the transcript T3 does not functionally compensate the lack of T1 and T2 under salt stress. The possible origin and functions of the transcript T3 are discussed. <br /> It is hypothesized that aldehydes may function as signal molecules and trigger aldehyde dehydrogenase gene expression. To test this hypothesis, transgenic plants expressing the β-glucuronidase (GUS) reporter gene driven by the ALDH7B4 (AT1G54100) gene promoter were generated (7B4-GUS). The ALDH7B4 promoter was found to be constitutively active in naturally desiccation-tolerant organs like seeds and pollen. In addition, both pentanal and trans-2-hexenal activated the promoter. The comparison of the GUS activities revealed that dehydration and NaCl induce the promoter stronger than trans-2-hexenal. To further understand the mechanism of the promoter activation by aldehydes the enzymatic activity of the GUS protein in plant extracts was compared to the accumulation pattern of malondialdehyde (MDA). Except for the methyl viologen treatment, no correlation was found between the GUS activity and the plant MDA content for the other treatments. Moreover, the in silico analysis of the ALDH7B4 promoter region revealed the presence of several stress-related cis-elements including one putative dehydration-responsive element/C-repeat – low temperature-responsive element (DRE/CRT-box) and three ACGT-boxes. The functional analysis of these elements suggested that the two proximal ACGT2 and ACGT3 boxes are the most influential ACGT-boxes involved in the salt response of the promoter. To identify factors involved in the aldehyde-induced expression of ALDH genes, a genome-wide mutagenesis approach has been chosen. Seeds from a homozygous transgenic 7B4-GUS plant were treated by the mutagen ethyl methanesulphonate (EMS). A second generation seed population has been generated.