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

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 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 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.

Correction of Frameshift Mutations in the atpB Gene by Translational Recoding in Chloroplasts of Oenothera and Tobacco

Translational recoding, also known as ribosomal frameshifting, is a process that causes ribosome slippage along the messenger RNA, thereby changing the amino acid sequence of the synthesized protein. Whether the chloroplast employs recoding is unknown. I-iota, a plastome mutant of Oenothera (evening primrose), carries a single adenine insertion in an oligoA stretch [11A] of the atpB coding region (encoding a β-subunit of the ATP synthase). The mutation is expected to cause synthesis of a truncated, non-functional protein. We report that a full-length AtpB protein is detectable in I-iota leaves, suggesting operation of a recoding mechanism. To characterize the phenomenon, we generated transplastomic tobacco lines in which the atpB reading frame was altered by insertions or deletions in the oligoA motif. We observed that insertion of two adenines was more efficiently corrected than insertion of a single adenine, or deletion of one or two adenines. We further show that homopolymeric composition of the oligoA stretch is essential for recoding, as an additional replacement of AAA lysine codon by AAG resulted in an albino phenotype. Our work provides evidence for the operation of translational recoding in chloroplasts. Recoding enables correction of frameshift mutations and can restore photoautotrophic growth in the presence of mutation that otherwise would be lethal.

Correction of frameshift mutations in the atpB gene by translational recoding in chloroplasts of Oenothera and tobacco

Translational recoding, also known as ribosomal frameshifting, is a process that causes ribosome slippage along the messenger RNA, thereby changing the amino acid sequence of the synthesized protein. Whether the chloroplast employs recoding, is unknown. I-iota, a plastome mutant of Oenothera (evening primrose), carries a single adenine insertion in an oligoA stretch of atpB (encoding a β-subunit of the ATP synthase). The mutation is expected to cause synthesis of a truncated, non-functional protein. We report that a full-length AtpB protein is detectable in I-iota leaves, suggesting operation of a recoding mechanism. To characterize the phenomenon, transplastomic tobacco lines were generated, in which the atpB reading frame was altered by insertions or deletions in the oligoA motif. We found that insertion of two adenines was more efficiently compensated than insertion of a single adenine, or deletion of one or two adenines. We further show that homopolymeric composition of the oligoA stretch is essential for recoding. Plants carrying a disrupted oligoA stretch have an albino-phenotype, indicating absence of indel correction. Our work provides evidence for the operation of translational recoding in chloroplasts. Recoding enables correction of frameshift mutations and can restore photoautotrophic growth in mutants that otherwise would be lethal.

HIV-1 P24 EXPRESSION IN TRANSPLASTOMIC TOBACCO PLANTS

Expression of HIV-1 p24 gene in chloroplasts was achieved in a tobacco variety V2 (Virginia TBE2). Through PCR and Southern blot analyses, it was demonstrated that the transgene integrated into the target site in the chloroplasts, between trnfM and trnG. Western blot results showed that HIV-1 p24 gene expressed in transplastomic tobacco plants. p24 protein accumulations were detected by ELISA in the range from 1.7% to 6.3% TSP and the high concentrations in the leaves near the top. p24 protein was purified by gel filtration chromatography demonstrated that the purification is 9.694 folds and the performance is 31.94%, however, protein p24 largely was inactive after purification.