Overexpression of wild-type and mutant Brassica juncea 3-hydroxy-3-methylglutaryl-CoA synthase1 in Solanaceae

Seeds are very important in the life cycle of the plant and they represent food sources for man and animals. Isoprenoids, including sterols are generated through the mevalonate (MVA) pathway. Dietary phytosterols have been reported to lower blood cholesterol levels and might thereby reduce the risk of heart disease. We report herein a mutant of 3-hydroxy-3-methylglutaryl-coenzyme A synthase (HMGS), the second enzyme in the MVA pathway that can promote phytosterols, plant growth and/or seed yield when overexpressed in tobacco and tomato. In Brassica juncea, four isogenes encode HMGS. Recombinant mutant BjHMGS1 S359A displayed a 10-fold higher enzyme activity. The overexpression of wt and mutant (S359A) BjHMGS1 in Arabidopsis has up-regulated several genes in sterol biosynthesis, increasing sterol content. To quickly test the effects of BjHMGS1 overexpression in a more distant species beyond the Brassicaceae, wt and mutant BjHMGS1 were initially expressed in tobacco of the family Solanaceae. Based on previous preliminary findings on transgenic tobacco overexpressing BjHMGS1, the role of ectopically-expressed BjHMGS1 in tobacco HMGS-OEs was followed up in this study. Tobacco HMGS-overexpressors (OEs) showed upregulation of native NtHMGR1, NtIPI2, NtSQS, NtSMT1-2, NtSMT2-1, NtSMT2-2 and NtCYP85A1 expression in seedlings, improving sterol content, plant growth (beyond germination) and seed yield over the vector-transformed control. Enhanced growth and seed yield in tobacco OE-S359A in comparison to OE-wtBjHNMGS1 coincided with elevations in NtSQS expression and sterol content. Thus, BjHMGS1 (S359A) holds promise in applications on phytosterol content, plant growth and seed yield enhancement in agriculture. Subsequently, an investigation on the potential application of BjHMGS1 was extended to a crop plant. Tomato (Solanum lycopersicum) was selected because it belongs to the same family as tobacco. Tomato HMGS-OEs showed up-regulation of genes associated with the biosynthesis of C10, C15 and C20 universal precursors of isoprenoids, phytosterols, BRs, dolichols, MEP, carotenoid and vitamin E. Greater expression of SlHMGR1, SlFPS1, SlCPT1, SlCPT2, SlSQS, SlSQE, SlCAS1, SlCYP85A3, SlCPT3, SlSSTLE1 and SlSSTLH3 in tomato OE-S359A than OE-wtBjHMGS1 seedlings, corresponding to significant growth enhancement evident in 63-d-old OE-S359A tomato plants. Furthermore, squalene, sterol and vitamin E (α–tocopherol) were elevated in HMGS-OE tomato fruits. Tomato OE-S359A fruits also showed higher squalene and phytosterol content than OE-wtBjMGS1, which was attributed to the higher expression of SlHMGR2, SlFPS1, SlGPS, SlSQS and SlCYP710A11 in OE-S359A. Therefore, these tomato HMGS-OEs have demonstrated potential as a new form of health-promoting food. To elucidate the effect of S359A at a molecular level, (His)6-BjHMGS1 (S359A) protein was purified for future crystallography studies. Preliminary data suggests that (His)6-BjHMGS1 (S359A) is a homodimer similar to (His)6-wtBjHMGS1. Also the mutation (S359A) did not appear to directly affect binding between HMGS and acetyl-CoA. As predicted from molecular dynamic simulation, mutant S359A displayed reduced constraint to the substrate in the absence of hydrogen bonding between Ser359 and acetyl-CoA, and this may account for its higher HMGS activity. Experiments using X-ray crystallography to study the mechanism on how S359A can participate in a more effective enzymatic reaction and the effects of this mutation on structure and function is foreseeable in the near future.published_or_final_versionBiological SciencesDoctoralDoctor of Philosoph