The role of <em>ALDH</em> in drought stress

Abstract

The work was divided into 3 major parts. The first part is studying the effect of ALDH enzymes on the redox homeostasis during oxidative stress, and the role of ALDHs during the germination, and longevity of A. thaliana seeds. Secondly, comparing the ALDH gene family in Linderniaceae family. Finally, unveiling the ALDH family in Hordeum vulgare. and its possible roles under drought conditions. <br /> Both WT, over-expression, and knockout mutant lines were transformed with peredox sensor vector via floral dipping. Only the roots of ALDH7B4 OE mutant line showed a significant fluorescence increase compared to WT roots under normal conditions. After oxidative stress, the same mutant line matched the increase in ROS and reactive aldehyde production. This allows to conclude that the ALDH7B4 enzyme is the most effective of the enzymes tested in neutralizing the reactive aldehydes and to protect the plant facing drought and oxidative stress. <br /> Secondly, the RNA and protein expression of the ALDH7B4 gene was measured in dog1, rdo2, rdo4 (Ler), and rdo4. The protein relative expression was significantly lower in rdo2 and rdo4 lines compared to WT. Under normal conditions, the germination rates of all the lines were similar except for the dog1 mutant line, showing that ALDH genes does not affect the germination rate under normal conditions. Controlled deterioration test (CDT) was performed. After 12 days of stress, both over-expression mutant lines were performing better than the rest of the lines. Overexpression of ALDH genes in A. thaliana can help to increase seed longevity. <br /> An RNA-seq map of dehydration and rehydration cycles of L. brevidens against L. subracemosa was used to look for ALDH genes. Nineteen ALDH genes were uncovered. ALDH5 Lbr_028666 gene was the only gene to increase during the dehydration period, and decrease after rehydration. This points to its importance during the drought stress. <br /> A bioinformatic search on the promoter region of the ALDH7A1 gene in barley to identify all possible motifs. DREB2D and MYB61 motifs were identified. They are involved in dehydration responses, and the stomatal regulation to increase water-use efficiency. The relative expression of the gene was measured in all genotypes after drought stress for confirmation. The relative ALDH7A1 gene expression was significantly higher in the drought resistant genotypes. We also measured the relative expression of the ALDH7A1 gene in the vegetative and reproductive developmental stages. In both genotypes, the gene relative expression was higher in seeds-insidekernels than in tillers. It showed that the ALDH7B4 gene had the highest expression level in reproductive organs and seeds. <br /> Two measurements (RWC, MDA) were taken for the three genotypes under normal and drought conditions. The readings were not conclusive of the direct participation of the gene-of-interest in drought resistance. <br /> The ALDH7A1 promoter region was co-bombarded into the Golden Promise genotype. The transformed plants were subjected to drought, heat, cold, and salt stress. The promoter region was not activated after any of the stresses. <br /> An extensive bioinformatic heatmap was constructed for all potential ALDH gene family members. Twenty-six potential genes belonging to eight classes were found and analyzed. The focus was on the genes that showed an increase in later developmental stages and seed formation. Both HORVU6HG0519800-1, and HORVU5HG0165080-1 genes fit that description. They belong to ALDH2 and ALDH7 families respectively.