Characterization of Arabidopsis FPS Isozymes and FPS Gene Expression Analysis Provide Insight into the Biosynthesis of Isoprenoid Precursors in Seeds

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.

Suppressing Farnesyl Diphosphate Synthase Alters Chloroplast Development and Triggers Sterol-Dependent Induction of Jasmonate- and Fe-Related Responses

“…Such a regulatory response also has been reported in Arabidopsis seeds with reduced levels of FPS activity (Closa et al, 2010;Keim et al, 2012). HMGR activity measurements in amiFPSa, amiFPSb, and EV plants grown with and without MFZ revealed that down-regulation of FPS activity at the early stages of plant development triggers a similar response.…”

“…FPS activity was measured as described by Arró et al (2014) and is reported as picomoles of IPP incorporated into acid-labile products per minute per milliliter of protein extract at 37°C. Immunoblot analysis was performed as described by Keim et al (2012) in the same protein extracts used for enzyme activity assays. Protein samples corresponding to 7 and 2 mL of each of the extracts analyzed for FPS and HMGR activity levels, respectively, were loaded into gel lanes.…”

“…FPS1 encodes isoforms FPS1L and FPS1S, which differ by an N-terminal extension of 41 amino acids that targets FPS1L into mitochondria (Cunillera et al, 1997;Manzano et al, 2006), whereas FPS1S, like FPS2, localizes to the cytosol ( Fig. 1A; Keim et al, 2012). The characterization of Arabidopsis mutants with either gain or loss of function of specific FPS genes revealed that FPS1 and FPS2 have highly overlapping but not completely redundant functions in isoprenoid biosynthesis.…”

A Single Arabidopsis Gene Encodes Two Differentially Targeted Geranylgeranyl Diphosphate Synthase Isoforms

“…The long IDI1 isoform is targeted to plastids, the long IDI2 isoform is transported to mitochondria, and both short isoforms are sorted to peroxisomes. The two genes encoding FPPS in Arabidopsis produce three isoforms (Cunillera et al, 1997;Manzano et al, 2006;Keim et al, 2012). FPS1 encodes a long isoform targeted to mitochondria (FPP1L) and a short one lacking the N-terminal end that remains in the cytosol, whereas FPS2 only produces a cytosolic enzyme (Fig.…”

Overexpression of an Isoprenyl Diphosphate Synthase in Spruce Leads to Unexpected Terpene Diversion Products That Function in Plant Defense

“…However, little is known about how IDS control flux into the different branches. A few recent publications have addressed the importance of FDP synthases (Chen et al, 2000;Masferrer et al, 2002;Manzano et al, 2004Manzano et al, , 2006Han et al, 2006;Banyai et al, 2010;Closa et al, 2010;Keim et al, 2012), but only one focuses on a GDP synthase (Lange et al, 2011) and one on a GGDP synthase (Kai et al, 2011).…”