Plastidial Expression of 3β-Hydroxysteroid Dehydrogenase and Progesterone 5β-Reductase Genes Confer Enhanced Salt Tolerance in Tobacco.

Muhammad Sameeullah,Buhara Yucesan,Mehmet Cengiz Baloğlu,Ekrem Gurel,Kiran Saba,Noreen Aslam,Muhammet Yildirim,Kiran Saba,Andreas G Lössl,Mohammad Tahir Waheed

doi:10.3390/ijms222111736

Abstract

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 unexplored. In the present study, transplastomic tobacco plants were developed by inserting the 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 observed. The salt tolerance, mediated by plastidial expression of the 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 the SDR genes leading to enhanced salt tolerance, which opens a window for developing saline-tolerant plants via plastid genetic engineering.

Highlights

Digitalis species, known as foxglove, are famous for the production of secondary metabolites known as cardiac glycosides, which have pharmaceutical importance in cardiac arrest and possess anti-cancer activities [1,2]
The final plastid expression vectors consisted of 3β-hydroxysteroid dehydrogenase (3β-HSD), P5βR1 and P5βR2 genes under control of constitutive PrrnPEP+NEP promoter
The transplastomic plants were generated by gene gunmediated DNA delivery and the transformants were selected on RMOP media containing 500 mg/L spectinomycin [30]

Summary

Introduction

Known as foxglove, are famous for the production of secondary metabolites known as cardiac glycosides, which have pharmaceutical importance in cardiac arrest and possess anti-cancer activities [1,2]. The genes 3β-hydroxysteroid dehydrogenase (3β-HSD) and progesterone 5-β-reductases (P5βR1, P5βR2) are among the important key step genes in the pathway of biosynthesis of cardenolide in Digitalis species [3,4,5]. During the last two decades, extensive studies have been conducted on the cardiac glycosides, pathways and substrates for the glycosides and recombinant protein production of the 3β-HSD, P5βR1 and P5βR2 using a bacterial heterologous expression system [4,5,6,7,8]. Extensive studies of enzymatic reactions, crystal structure and substrate specificity have been conducted, until now, the functional analysis of these genes in transgenic plant studies has not been a focus of study

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