P Liao, X Chen, M Wang, TJ Bach, ML Chye

Plant Biotechnology Journal; Volume 16, Issue 3, March 2018, Pages 784–796

Summary

3-Hydroxy-3-methylglutaryl-coenzyme A synthase (HMGS) in the mevalonate (MVA) pathway generates isoprenoids including phytosterols. Dietary phytosterols are important because they can lower blood cholesterol levels. Previously, the overexpression of Brassica juncea wild-type (wt) and mutant (S359A) BjHMGS1 in Arabidopsis up-regulated several genes in sterol biosynthesis and increased sterol content. Recombinant S359A had earlier displayed a 10-fold higher in vitro enzyme activity. Furthermore, tobacco HMGS overexpressors (OEs) exhibited improved sterol content, plant growth and seed yield. Increased growth and seed yield in tobacco OE-S359A over OE-wtBjHMGS1 coincided with elevations in NtSQS expression and sterol content. Herein, the overexpression of wt and mutant (S359A) BjHMGS1 in a crop plant, tomato (Solanum lycopersicum), caused an accumulation of MVA-derived squalene and phytosterols, as well as methylerythritol phosphate (MEP)-derived α-tocopherol (vitamin E) and carotenoids, which are important to human health as antioxidants. In tomato HMGS-OE seedlings, genes associated with the biosyntheses of C10, C15 and C20 universal precursors of isoprenoids, phytosterols, brassinosteroids, dolichols, methylerythritol phosphate, carotenoid and vitamin E were up-regulated. In OE-S359A tomato fruits, increased squalene and phytosterol contents over OE-wtBjHMGS1 were attributed to heightened SlHMGR2, SlFPS1, SlSQS and SlCYP710A11 expression. In both tomato OE-wtBjHMGS1 and OE-S359A fruits, the up-regulation of SlGPS and SlGGPPS1 in the MEP pathway that led to α-tocopherol and carotenoid accumulation indicated cross-talk between the MVA and MEP pathways. Taken together, the manipulation of BjHMGS1 represents a promising strategy to simultaneously elevate health-promoting squalene, phytosterols, α-tocopherol and carotenoids in tomato, an edible fruit.

See http://onlinelibrary.wiley.com/doi/10.1111/pbi.12828/full

Figure 1: Characterization of representative transgenic tomato HMGS-OEs. (a, b) Western blot analysis using antibodies against BjHMGS1 to verify the expression of BjHMGS1 in tomato leaves of 5-week-old representative wild-type HMGS-OEs (OE-wtBjHMGS1) in (a) and mutant HMGS-OEs (OE-S359A) in (b). The cross-reacting HMGS band is indicated by an arrowhead. Putative tomato HMGS-OEs were designated as OE-wtBjHMGS1 lines (413, 430, 439, 442, 444 and 445) in (a) and OE-S359A lines (622, 623, 624, 625, 631 and 635) in (b). PC, positive control (tobacco BjHMGS1 OE line 402 as reported in Liao et al., 2014b); pSa13, vector (pSa13)-transformed tomato. Bottom, Coomassie Blue-stained gel of 20 μg total protein in each well. Two independent lines from each construct selected for further tests are underlined. White lines have been inserted between lanes that have been spliced together from the same original gel/blot. (c) Semiquantitative RT-PCR analysis on representative transgenic tomato plants. BjHMGS1-specific primers (ML1666 and ML1667) and tomato ACTIN (SlACTIN)-specific primers (ML1688 and ML1689) were used. The PCR bands of 147-bp BjHMGS1 and 176-bp SlACTIN are indicated. pSa13, vector (pSa13)-transformed control.