Functional Characterization of Transgenic <em>Arabidopsis thaliana </em>Plants Co-over expressing Aldehyde dehydrogenases and Genes for Soluble Osmolytes

Abstract

Plant growth and productivity is largely hampered by a number of abiotic stresses. These stresses affect the physiological and metabolic pathways leading to the production of many harmful substances like aldehydes, reactive oxygen species and abscisic acid etc. Aldehyde dehydrogenases (ALDHs) are important in detoxification of potentially toxic aldehydes in many plants. They are intermediates in a variety of fundamental biochemical pathways, but they can also be generated in a variety of environmental stresses like salinity, dehydration and temperature extremes. Transgenic Arabidopsis plants containing two different aldehyde dehydrogenases (ALDH3I1 and ALDH7B4) were characterized under various stress conditions. RNA and proteins were extracted; RT-PCR and Western blots were done to confirm the expression of the genes. Western blots revealed that in almost all of the salt (200 mM NaCl) as well drought stressed plants the ALDH3I1 and ALDH7B4 specific proteins were expressed. The RT-PCR also showed higher expression levels of mRNAs, of the two aldehye dehydrogenase genes in the stressed plants as compared to the wild type (untransformed) Arabidopsis plants. In addition to molecular studies some physiological studies i.e., measurement of rate of gas exchange/photosynthesis and measurement of lipid peroxidation, were also done. After one week of stress treatment the CO2 assimilation rate was measured using GFS-3000 (Heinz WALZ Gmbh). The transformed plants showed a higher CO2 assimilation rate than wild-type plants under both drought and salt stress. Similarly, lipid peroxidation was higher in wild-type Arabidopsis plants as compared to transgenic single and double over expressor plants under stress conditions. Based on these results it was decided to transform these genes into tobacco to investigate whether the same performance of the transgenic plants was seen. Transgenic tobacco plants as well as non transgenic plants were put under various stress conditions. The constitutive or stress activated expression of the gene resulted in increased tolerance to salt, drought and oxidative stress in transgenic tobacco plants associated with improved plant growth. It is demonstrated that transgenic plants produce less reactive oxygen species (ROS) under stress conditions; therefore, less damage occurs to cell structures specially membranes. Similar results were observed for the chlorophyll content of stressed plants that ultimately resulted in more photosynthesis and gas exchange. Malondialdehyde (MDA) that results from lipid peroxidation was also found to be at lower levels in transgenic tobacco plants under stress conditions. The transgenic plants accumulated higher amounts of proline than wild-type plants under drought stress. The studies suggest that Arabidopsis ALDH overexpression generally induces higher stress tolerance in tobacco plants. In addition to experiments with ALDH genes, cloning of the genes responsible for production of the soluble osmolyte ectoine (1, 4, 5, 6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) was done. Genes of the ectoine synthesis were derived from a halophilic bacterium Marinococus halophilus. Vector was designed in a way that all the three genes responsible for ectoine production possess different promoters and terminators; eventually the three genes were cloned in the same binary vector. The genes responsible for the synthesis of the soluble osmolyte ectoine were transformed into Arabidopsis. The ectoine genes showed variable degree of expression in the transgenic plants. The plants over-expressing all the three genes also produced ectoine but the amount was very low and could not be quantified under control conditions.