Abstract
Seeds are very important not only in the life cycle of the plant but they represent food sources for man and animals. We report herein a mutant of 3-hydroxy-3-methylglutaryl-coenzyme A synthase (HMGS), the second enzyme in the mevalonate (MVA) pathway that can improve seed yield when overexpressed in a phylogenetically distant species. In Brassica juncea, the characterisation of four isogenes encoding HMGS has been previously reported. Enzyme kinetics on recombinant wild-type (wt) and mutant BjHMGS1 had revealed that S359A displayed a 10-fold higher enzyme activity. The overexpression of wt and mutant (S359A) BjHMGS1 in Arabidopsis had up-regulated several genes in sterol biosynthesis, increasing sterol content. To quickly assess the effects of BjHMGS1 overexpression in a phylogenetically more distant species beyond the Brassicaceae, wt and mutant (S359A) BjHMGS1 were expressed in tobacco (Nicotiana tabacum L. cv. Xanthi) of the family Solanaceae. New observations on tobacco OEs not previously reported for Arabidopsis OEs included: (i) phenotypic changes in enhanced plant growth, pod size and seed yield (more significant in OE-S359A than OE-wtBjHMGS1) in comparison to vector-transformed tobacco, (ii) higher NtSQS expression and sterol content in OE-S359A than OE-wtBjHMGS1 corresponding to greater increase in growth and seed yield, and (iii) induction of NtIPPI2 and NtGGPPS2 and downregulation of NtIPPI1, NtGGPPS1, NtGGPPS3 and NtGGPPS4. Resembling Arabidopsis HMGS-OEs, tobacco HMGS-OEs displayed an enhanced expression of NtHMGR1, NtSMT1-2, NtSMT2-1, NtSMT2-2 and NtCYP85A1. Overall, increased growth, pod size and seed yield in tobacco HMGS-OEs were attributed to the up-regulation of native NtHMGR1, NtIPPI2, NtSQS, NtSMT1-2, NtSMT2-1, NtSMT2-2 and NtCYP85A1. Hence, S359A has potential in agriculture not only in improving phytosterol content but also seed yield, which may be desirable in food crops. This work further demonstrates HMGS function in plant reproduction that is reminiscent to reduced fertility of hmgs RNAi lines in let-7 mutants of Caenorhabditis elegans.
Highlights
Isoprenoids form a large and diverse group of natural products, which have promising pharmacological applications including anti-cancer, antibacterial and anti-malarial properties [1,2,3,4]
New observations from tobacco hydroxy-3-methylglutaryl-coenzyme A synthase (HMGS)-OEs Our investigations on the overexpression of HMGS in transgenic tobacco revealed new observations not previously evident in Arabidopsis HMGS-OEs including the upregulation of NtIPPI2, NtSQS and NtGGPPS2 and downregulation of NtIPPI1, NtGGPPS1, NtGGPPS3 and NtGGPPS4 (Figures 7–8)
In Arabidopsis, higher AtHMGS expression had been observed in flowers than seedlings or leaves from RT-PCR analysis [4]
Summary
Isoprenoids form a large and diverse group of natural products, which have promising pharmacological applications including anti-cancer, antibacterial and anti-malarial properties [1,2,3,4]. Some isoprenoids including gibberellic acids, abscisic acid, cytokinins, sterols and brassinosteroids (BRs) play significant roles in plant growth and development [4,5,6]. Two pathways generate isopentenyl diphosphate (IPP), which constitutes the universal precursor of all isoprenoids: the mevalonate (MVA) pathway in the cytosol, and the non-MVA, methylerythritol phosphate (MEP) pathway in plastids [1,3,12](and references cited therein), with some crosstalk between them [13,14] (Figure 1). Sterols and BRs are synthesized in the cytoplasm and thereby derive from MVA, while gibberellic acids and abscisic acid precursors, active cytokinins, carotenoids and chlorophylls are produced in plastids [1,15,16,17,18,19,20,21] and depend on the MEP pathway (Figure 1)
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