Abstract
Arabidopsis thaliana contains two genes encoding farnesyl diphosphate (FPP) synthase (FPS), the prenyl diphoshate synthase that catalyzes the synthesis of FPP from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In this study, we provide evidence that the two Arabidopsis short FPS isozymes FPS1S and FPS2 localize to the cytosol. Both enzymes were expressed in E. coli, purified and biochemically characterized. Despite FPS1S and FPS2 share more than 90% amino acid sequence identity, FPS2 was found to be more efficient as a catalyst, more sensitive to the inhibitory effect of NaCl, and more resistant to thermal inactivation than FPS1S. Homology modelling for FPS1S and FPS2 and analysis of the amino acid differences between the two enzymes revealed an increase in surface polarity and a greater capacity to form surface salt bridges of FPS2 compared to FPS1S. These factors most likely account for the enhanced thermostability of FPS2. Expression analysis of FPS::GUS genes in seeds showed that FPS1 and FPS2 display complementary patterns of expression particularly at late stages of seed development, which suggests that Arabidopsis seeds have two spatially segregated sources of FPP. Functional complementation studies of the Arabidopsis fps2 knockout mutant seed phenotypes demonstrated that under normal conditions FPS1S and FPS2 are functionally interchangeable. A putative role for FPS2 in maintaining seed germination capacity under adverse environmental conditions is discussed.
Highlights
All isoprenoids are derived from the C5 building blocks isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP)
The optimal pH range for FPS1S and FPS2 was determined. Both enzymes showed a similar pHrate profile with only minor differences (Figure 2A). Maximal activity for both enzymes was observed at a pH value of 7.0, albeit the pH-rate profile of FPS1S was slightly shifted toward more acidic pH values compared to FPS2
We investigated the effect of NaCl on the activity of FPS1S and FPS2 using concentrations in the range from 0 to 2 M
Summary
All isoprenoids are derived from the C5 building blocks isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). Pathway specialized branches starting from these prenyl diphosphates lead to the production of the astonishing number of isoprenoid end products synthesized by plants. GPP serves as a precursor for monoterpenoids, GGPP is a precursor of diterpenoids, gibberellins, carotenoids and abscisic acid, side chains of chlorophyll, phylloquinone, plastoquinone and tocopherols, and geranylgeranylated proteins, and FPP serves as a precursor for sesquiterpenoids, sterols, brassinosteroids, triterpenoids, polyprenols, side chains of ubiquinone, and farnesylated proteins [5]. It is generally accepted that the intracellular levels of these prenyl diphosphates and their precursors IPP and DMAPP must be strictly controlled to avoid deleterious effects on the metabolic flux through the pathway branches competing for these intermediates [6]. Overexpression of FPP synthase (FPS) in Arabidopsis [7], [8], [9] and phytoene synthase in tomato [10] results in altered levels of specialized isoprenoid end products that negatively affect plant performance
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