MALONDIPLANT · Origin, localization and biological function of malondialdehyde in plants

Origin, localization and biological function of malondialdehyde in plants

Project context and objectives Aerobic organisms carry out metabolic processes that require electron transfers, such as mitochondrial respiration or photosynthesis in plants. Although oxygen is an essential molecule for the existence of these organisms, it can also be harmful, since during the electron transfer processes, oxygen can undergo partial electronic reductions yielding reactive oxygen species (ROS). ROS are continuously produced in plant cells under physiological conditions as by-products of these aerobic metabolic processes. However, ROS production can be enhanced during some developmental stages and by biotic or abiotic stress. When ROS generation exceeds the antioxidant defence mechanisms of the cells, oxidative stress occurs causing oxidation and damage to cellular components such as proteins, deoxyribonucleic acid (DNA) and lipids. Lipid oxidation can be initiated in two different ways in the plant cell: - enzymatically, by the action of lipoxygenases, a-dioxygenases, etc.; - non-enzymatically, by ROS-catalysed oxidative reactions, generating a large number of molecules, generally termed as oxylipins. Plant membranes are especially rich in linoleic and linolenic fatty acids. The enzymatic oxidation of linolenic acid can result in the synthesis of the jasmonic acid-type oxylipins, whereas the non-enzymatic ROS-catalysed oxidation may yield several classes of oxylipins, including phytoprostanes and other linolenic acid fragmentation products such as malondialdehyde (MDA). The great chemical complexity of the fatty acid-oxidised products is simplified by the fact that many of them contain carbonyl groups adjacent to double bonds (a,ß-unsaturated carbonyl groups), which are termed reactive electrophile species (RES). As occurs with ROS, RES can have powerful biological activities when their production in the cell is strictly controlled. Thus, although an excess of RES production can lead to cell damage and death, lower levels may modulate the expression of some cell survival genes during severe stress. MDA is a three-carbon aldehyde that has been considered to be the exclusive by-product of the non-enzymatic oxidation of many fatty acids with three or more double bonds. However, exposure of plants to low levels of MDA up-regulated many genes involved in environmental stress responses that are different from genes activated by other RES. In Arabidopsis leaves, MDA levels are relatively high, in part explained because at cytosolic pH the molecule has a low reactivity. However, when MDA is exposed to acidic pH, as occurs in apoptosis, pathogenesis or during the disruption of vacuoles or apoplast, MDA assumes the protonated reactive form. Consequently, MDA is thought to be a latent RES, a molecule made to be mobilised rapidly in case of stress so as to activate cell survival signalling. Currently, the subcellular localisation of MDA is unknown, and may be important in order to establish a role for its compartmentalisation in the activation and function of this signalling molecule. In mature expanded Arabidopsis thaliana leaves, MDA is produced from at least two genetically independent sources. The principal source in leaves, accounting for approximately 75 % of total MDA, is polyunsaturated fatty acids, whereas the origin of a second pool of MDA is unknown. The use of fad3-2fad7-2fad8 Arabidopsis mutants, which are unable to synthesise fatty acids with more than two double bonds, confirmed the non-trienoic fatty acid origin of this second MDA pool in roots. We attempted to find out how this second pool of MDA originated. The main objective of the MALONDIPLANT project was to study the origin of the second pool of MDA in plants, its biological function and where it is localised at the subcellular level. Work performed We designed a genetic screen using approximately 7000 Arabidopsis insertion mutant plants to find any alteration to the levels of MDA in the root tips. To fulfil this objective, we used a combination of genetics and molecular and cell biology techniques. The model plant Arabidopsis offers a particularly good system to study the biology of MDA, because it has a short life cycle, its genome has been completely sequenced, and for the relative feasibility to obtain mutants in fatty acid synthesis and metabolism. To carry out the genetic screening, we grew seedlings of the mutants (and wild-type plants for comparison) vertically on agar plates for four days and then we incubated the plants with 35 mM 2-thiobarbituric acid (TBA) (or 35 mM trichloroacetic acid as negative control) at 25 °C for 90 minutes. Then, we visualised the fluorescence generated by the adduct (TBA)2MDA with a stereomicroscope equipped with a custom-made filter with excitation at 515±10 nm and emission at 555±15 nm. We identified a mutant containing a less fluorescent signal due to MDA in the root tips compared to the wild-type plants. We called this mutant mad1 (malondialdehyde downregulated 1). The mutated gene belongs to a family of three genes and its function is unknown in Arabidopsis. To elucidate if the protein encoded by mad1 could be responsible for the altered levels of MDA observed in the root tip of the mutant, we cloned the coding sequence of mad1 and produced transgenic plants overexpressing the corresponding protein. When we incubated these plants with TBA as we did with the mad1 mutant, we observed a higher generation of MDA in the root tip, indicating that mad1 could be involved in MDA generation in the plant. We confirmed the results obtained by microscopy with the use of quantitative gas chromatography/mass spectrometry techniques. Regarding the subcellular localisation of MDA, we observed that MDA in the leaves is localised inside the cell, but we have still not succeeded in finding out where MDA is localised in the roots. Main results The results obtained from this project indicate, for the first time, a putative enzymatic production of MDA in roots. Our findings would also be consistent with a biological function for MDA, not described yet, in plants. In order to establish a role for MDA, we studied the phenotype of the mad1 mutant subjected to different abiotic stresses. We grew wild-type and mad1 plants under high light intensities (250-1000 µE m-2 s-1) and low temperatures (12 ºC). There were no significant differences between the two groups of plants analysed. Nevertheless, we are in the process of repeating the same study by including this time the overexpressor plants and studying additional stress, for example infection with the pathogenic fungus Botrytis cinerea. In summary, we found an Arabidopsis mutant (mad1) with a decreased production of MDA in the roots and we provided a range of data supporting a role of this protein in the synthesis of MDA in plants. By comparing mad1 mutants, transgenic plants overexpressing the corresponding protein, and wild-type plants subjected to different growing conditions and stresses, we will establish a function for MDA in plants. Since MDA has been implicated in plant stress signalling, we think that the use of our mutant or transgenic plants will be potentially significant in plant biology, but we need to conduct some further studies regarding the improvement of plant resistance to the different environmental stresses to which plants are subjected in natural conditions.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз