Aldehyde dehydrogenases may modulate signaling by lipid peroxidation-derived bioactive aldehydes

ABSTRACT

Aldehyde molecules were shown to induce gene expression but because of their toxicity, the cell expresses ALDEHYDE DEHYDROGENASES (ALDH, EC 1.2.1.3) that oxidize them to carboxylic acids. To understand how the aldehydes may be both toxic and gene activators, we expressed the ALDH7B4 gene promoter fused to the β-glucuronidase reporter gene in independent transgenic lines and found that pentanal and trans-2-hexenal activated the promoter whereas trans-2-hexenal induced the ALDH7B4 protein. Paraquat led to higher amounts of malondialdehyde compared to trans-2-hexenal and H₂O₂, and only the treatment by Paraquat activated the ALDH7B4 promoter, indicating that a threshold level of aldehydes is required for gene activation. These findings suggest that ALDH activity may also serve to fine-tune gene activation by the aldehydes.

Lipid peroxidation-derived aldehydes are potentially toxic due to their extreme reactivity with the nucleophilic compounds (nucleic acids, proteins, membrane lipids) present in different cell compartments,¹ and they are cleared from the cell by ALDEHYDE DEHYDROGENASES, NAD(P)⁺ dependent enzymes (ALDH, EC 1.2.1.3) that are widely distributed in all organisms including human and plant genomes.^2,3 We previously demonstrated that significant amounts of toxic aldehydes are produced during dehydration, salt, heat, and oxidative stresses and are followed by an increased ALDH expression. Gain-of-function and loss-of-function of selected stress-responsive ALDHs were more tolerant and more sensitive to these stresses than the wild type, respectively.^1,4,5 Most recently, we found that ALDH mutants were more sensitive to heat and to the combination of heat with drought, salt, or wounding stresses than the wild type.⁶ All these previous findings indicate that ALDHs are most likely induced by intermediate signaling molecules such reactive oxygen species (ROS) and lipid peroxidation-derived aldehydes (reactive carbonyl species) common to dehydration, salt, wounding, and heat stresses. Indeed, some aldehyde molecules including malondialdehyde (MDA), trans-2-hexenal and 4-hydroxynonenal were proved to function as powerful gene activators despite their potential toxicity.^7–13 For the aldehydes to function as gene activator despite their potential toxicity, we hypothesized that a fine-tuning of their intracellular concentration might be necessary.

To understand how the aldehydes may induce gene activation besides their cytotoxicity, we have in a first step analyzed the responsiveness of the ALDH gene promoter to ROS and aldehydes. For that, we used the promoter of the ALDH7B4 gene that was previously shown to be activated by dehydration, salinity, heat, excessive light, heavy metals (Cu²⁺ and Cd²⁺), H₂O₂ and ABA treatment.^1,4,14,15 Transgenic plants expressing the β-glucuronidase (GUS) reporter gene driven by the ALDH7B4 gene promoter were generated.¹⁵ As in numerous studies reporting about the bioactivity of aldehyde molecules in animals and plants,^16–20 we found that pentanal and trans-2-hexenal activated the ALDH7B4 gene promoter in independent transgenic lines.²¹ Electrophilic aldehydes containing an α,β-unsaturated carbonyl (Michael acceptor) were shown to induce gene expression more efficiently than their aliphatic counterparts.⁸ Consistent with this, we found that the ALDH7B4 promoter activity measured by the GUS activity was induced more strongly by trans-2-hexenal than pentanal (27.0 ± 2.6 versus 11.1 ± 1.1 pmol 4-MU min⁻¹ mg protein⁻¹; test t of student, P < 0.05).²¹ The ALDH7B4 protein was monitored in parallel with the endogenous ALDH7B4 promoter activity by using protein-blot. The ALDH7B4 protein also accumulated in response to trans-2-hexenal,²¹ thus demonstrating the responsiveness of the ALDH7B4 gene to aldehydes and particularly to trans-2-hexenal. In another experiment, the ALDH7B4 promoter activity was compared to the contents of MDA, which is a Michael acceptor widely used as a marker of free-radical-catalyzed lipid peroxidation.⁸ Leaves were detached from soil-grown 4 week-old transgenic plants and incubated in water (as control), 300 mM NaCl, 30 mM H₂O₂, 5 µmol trans-2-hexenal or 50 µM Paraquat® (methyl viologen) for 8 h.²¹ The leaves were then divided into pools and used for the in situ detection of reductive aldehydes, the quantification of the GUS activity and the determination of the MDA content. There were more MDA in leaves treated by H₂O₂ than in trans 2-hexenal-treated and water-treated leaves (19.2 ± 2.5, 13.7 ± 1.0, and 33.4 ± 1.1 nmol MDA equivalents fresh weight⁻¹ for water, trans 2-hexenal, and H₂O₂, respectively; test t of student, P < 0.05) but no difference was observed for the ALDH7B4 promoter activity in these samples after 8 h.²¹ In contrast, the Paraquat® treatment led to a significantly high amounts of aldehydes (shown by the Schiff reagent assay¹⁸) and MDA (49.8 ± 5.2 nmol MDA equivalents fresh weight⁻¹, test t of student, P < 0.05), concomitantly with a significant increase of the ALDH7B4 promoter activity (172.4 ± 18.7, 163.1 ± 8.9, 126.1 ± 57.7, and 401.2 ± 64.1 pmol 4-MU min⁻¹ mg protein⁻¹, for water, H₂O₂, trans 2-hexenal, and Paraquat®, respectively; test t of student, P < 0.05).²¹ The ALDH7B4 gene thus appeared to be induced only beyond a threshold of the intracellular concentration of aldehydes and ROS. In fact, it was shown that the concentration and the localization of unbound MDA would be key factors directing its biological activities in vivo, and that about 75% of total MDA content in expanded leaves was shown to originate from trienoic fatty acids while the source of the second pool is so far unknown.⁸ Moreover, by using triple fatty acid desaturase (fad3-2fad7-2fad8) mutants deficient in the accumulation of the trienoic fatty acid, it was found that a basal MDA level is kept in plant tissues in physiological conditions, and that the biological activity of MDA mostly derives from changes in the free MDA pool that is dynamic and increases upon stress.^10,22,23 Our observation that Paraquat® led to a significant accumulation of MDA in parallel with a strong ALDH7B4 promoter activity is consistent with these previous works and underlines the correlation between the intracellular ROS level, bioactive aldehydes, and gene expression. Moreover, the induction of ALDH7B4 by direct application of aldehydes clearly indicates that ALDHs can sense and fine-tune the levels of aldehydes by reacting with the reactive carbonyls for proper gene activation. The findings that the ALDH7B4 protein was carbonylated in salt-stressed Arabidopsis leaves support this hypothesis.²⁴ Moreover, ALDHs have been co-purified in signaling complexes,^25–27 and ALDH3H1 was recently shown to interact with the Arabidopsis extra-large G proteins XLG1 and XLG3 on the plasma membrane; the interaction was confirmed in both yeast-two-hybrid and bimolecular fluorescence complementation assays.²⁸ Our data and these previous findings clearly suggest a dual role for the ALDH proteins as sensors of lipid-derived reactive carbonyl species and as modulators of gene activation by aldehydes and ROS (Fig. 1). The α,β-unsaturated aldehydes possess a thiol-reactivity that allows them to covalently modify proteins in vivo,^13,29 and it is unlikely that lipid peroxidation-derived aldehydes directly interact with the target gene promoters in the nucleus. The most obvious signal relay mechanism by aldehydes must be a covalent modification of trans-acting factors. The ALDHs would exert their role of sensors of oxidative stress or modulators of gene activation by aldehydes either by terminating the activity of aldehydes when oxidizing them into carboxylic acids, or after being themselves carbonylated, which may lead to the release of a downstream positive or negative regulator of the target genes as shown before.³⁰

Figure 1.

Modulation of the aldehyde bioactivity by ALDEHYDE DEHYDROGENASES (ALDHs). Reactive carbonyl species including aldehydes are derived from the metabolism and the peroxidation of membrane lipids by reactive oxygen species. As the aldehyde levels increase at the onset of stress or developmental cues, protein carbonylation occurs and may serve for signaling or trigger cell death if carbonylated proteins accumulate in the cells. ALDH expression is induced to control the levels of bioactive aldehydes by oxidizing them to their corresponding carboxylic acids.

Disclosure of interest

The authors report no conflict of interest.

Acknowledgments

TDM is grateful for DAAD financial support for the completion of Ph.D. thesis. TDM would like to thank Dr. Dorothea Bartels (University of Bonn, Germany) for her generosity and Ph.D. mentorship. TDM is grateful for the technical support of Dr. Kotchoni, SO (Rutgers-Camden University).