Browsing by Author "Waheed M.T."

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Isolation of the 3β-HSD promoter from Digitalis ferruginea subsp. ferruginea and its functional characterization in Arabidopsis thaliana
(2022-07-01) Aslam N.; Sameeullah M.; Yildirim M.; Baloglu M.C.; Yucesan B.; Lössl A.G.; Waheed M.T.; Gurel E.
Background: Although members of the SDR gene family (short chain dehydrogenase) are distributed in kingdom of life, they have diverse roles in stress tolerance mechanism or secondary metabolite biosynthesis. Nevertheless, their precise roles in gene expression or regulation under stress are yet to be understood. Methods: As a case study, we isolated, sequenced and functionally characterized the 3β-HSD promoter from Digitalis ferruginea subsp. ferruginea in Arabidopsis thaliana. Results: The promoter fragment contained light and stress response elements such as Box-4, G-Box, TCT-motif, LAMP element, ABRE, ARE, WUN-motif, MYB, MYC, W box, STRE and Box S. The functional analysis of the 3β-HSD promoter in transgenic Arabidopsis seedlings showed that the promoter was expressed in cotyledon and root elongation zone in 2 days’ seedlings. However, this expression was extended to hypocotyl and complete root in 6 days’ seedlings. In 20 days-old seedlings, promoter expression was distributed to the whole seedling including hydathodes aperture, vascular bundle, shoot apical meristem, trichomes, midrib, leaf primordia, hypocotyl and xylem tissues. Further, expression of the promoter was enhanced or remained stable under the different abiotic stress conditions like osmotic, heat, cold, cadmium or low pH. In addition, the promoter also showed response to methyl jasmonate (MeJA) application. The expression could not be induced in wounded cotyledon most likely due to lack of interacting elements in the promoter fragment. Conclusions: Taken together, the 3β-HSD promoter could be a candidate for the development of transgenic plants especially under changing environmental conditions.
Plastidial expression of 3β‐hydroxysteroid dehydrogenase and progesterone 5β‐reductase genes confer enhanced salt tolerance in tobacco
(2021-11-01) Sameeullah M.; Yildirim M.; Aslam N.; Baloğlu M.C.; Yucesan B.; Lössl A.G.; Saba K.; Waheed M.T.; Gurel E.
The short‐chain dehydrogenase/reductase (SDR) gene family is widely distributed in all kingdoms of life. The SDR genes, 3β‐hydroxysteroid dehydrogenase (3β‐HSD) and progesterone 5‐ β‐reductases (P5βR1, P5βR2) play a crucial role in cardenolide biosynthesis pathway in the Digitalis species. However, their role in plant stress, especially in salinity stress management, remains unex-plored. In the present study, transplastomic tobacco plants were developed by inserting 3β‐HSD, P5βR1 and P5βR2 genes. The integration of transgenes in plastomes, copy number and transgene expression at transcript and protein level in transplastomic plants were confirmed by PCR, end‐to-end PCR, qRT‐PCR and Western blot analysis, respectively. Subcellular localization analysis showed that 3β‐HSD and P5βR1 are cytoplasmic, and P5βR2 is tonoplast‐localized. Transplastomic lines showed enhanced growth in terms of biomass and chlorophyll content compared to wild type (WT) under 300 mM salt stress. Under salt stress, transplastomic lines remained greener without negative impact on shoot or root growth compared to the WT. The salt‐tolerant transplastomic lines exhibited enhanced levels of a series of metabolites (sucrose, glutamate, glutamine and proline) under control and NaCl stress. Furthermore, a lower Na+ /K+ ratio in transplastomic lines was also ob-served. The salt tolerance, mediated by plastidial expression of 3β‐HSD, P5βR1 and P5βR2 genes, could be due to the involvement in the upregulation of nitrogen assimilation, osmolytes as well as lower Na+/K+ ratio. Taken together, the plastid‐based expression of SDR genes leading to enhanced salt tolerance, which opens a window for developing saline‐tolerant plants via plastid genetic engineering.