Nerolidol production in agroinfiltrated tobacco: Impact of protein stability and membrane targeting of strawberry (Fragraria ananassa) NEROLIDOL SYNTHASE1.

The sesquiterpene alcohol nerolidol, synthesized from farnesyl diphosphate (FDP), mediates plant-insect interactions across multiple trophic levels with major implications for pest management in agriculture. We compared nerolidol engineering strategies in tobacco using agroinfiltration to transiently express strawberry (Fragraria ananassa) linalool/nerolidol synthase (FaNES1) either at the endoplasmic reticulum (ER) or in the cytosol as a soluble protein. Using solid phase microextraction and gas chromatography-mass spectrometry (SPME-GCMS), we have determined that FaNES1 directed to the ER via fusion to the transmembrane domain of squalene synthase or hydroxymethylglutaryl - CoA reductase displayed significant improvements in terms of transcript levels, protein accumulation, and volatile production when compared to its cytosolic form. However, the highest levels of nerolidol production were observed when FaNES1 was fused to GFP and expressed in the cytosol. This SPME-GCMS method afforded a limit of detection and quantification of 1.54 and 5.13 pg, respectively. Nerolidol production levels, which ranged from 0.5 to 3.0 µg/g F.W., correlated more strongly to the accumulation of recombinant protein than transcript level, the former being highest in FaNES-GFP transfected plants. These results indicate that while the ER may represent an enriched source of FDP that can be exploited in metabolic engineering, protein accumulation is a better predictor of sesquiterpene production.

Complex interplays between phytosterols and plastid development.

Isoprenoids comprise the largest class of natural compounds and are found in all kinds of organisms. In plants, they participate in both primary and specialized metabolism, playing essential roles in nearly all aspects of growth and development. The enormous diversity of this family of compounds is extensively exploited for biotechnological and biomedical applications as biomaterials, biofuels or drugs. Despite their variety of structures, all isoprenoids derive from the common C5 precursor isopentenyl diphosphate (IPP). Plants synthesize IPP through two different metabolic pathways, the mevalonic acid (MVA) and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathways that operate in the cytosol-RE and plastids, respectively. MEP-derived isoprenoids include important compounds for chloroplast function and as such, knock-out mutant plants affected in different steps of this pathway display important alterations in plastid structure. These alterations often lead to albino phenotypes and lethality at seedling stage. MVA knock-out mutant plants show, on the contrary, lethal phenotypes already exhibited at the gametophyte or embryo developmental stage. However, the recent characterization of conditional knock-down mutant plants of farnesyl diphosphate synthase (FPS), a central enzyme in cytosolic and mitochondrial isoprenoid biosynthesis, revealed an unexpected role of this pathway in chloroplast development and plastidial isoprenoid metabolism in post-embryonic stages. Upon FPS silencing, chloroplast structure is severely altered, together with a strong reduction in the levels of MEP pathway-derived major end products. This phenotype is associated to misregulation of genes involved in stress responses predominantly belonging to JA and Fe homeostasis pathways. Transcriptomic experiments and analysis of recent literature indicate that sterols are the cause of the observed alterations through an as yet undiscovered mechanism.

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

Farnesyl diphosphate synthase (FPS) catalyzes the synthesis of farnesyl diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate. Arabidopsis (Arabidopsis thaliana) contains two genes (FPS1 and FPS2) encoding FPS. Single fps1 and fps2 knockout mutants are phenotypically indistinguishable from wild-type plants, while fps1/fps2 double mutants are embryo lethal. To assess the effect of FPS down-regulation at postembryonic developmental stages, we generated Arabidopsis conditional knockdown mutants expressing artificial microRNAs devised to simultaneously silence both FPS genes. Induction of silencing from germination rapidly caused chlorosis and a strong developmental phenotype that led to seedling lethality. However, silencing of FPS after seed germination resulted in a slight developmental delay only, although leaves and cotyledons continued to show chlorosis and altered chloroplasts. Metabolomic analyses also revealed drastic changes in the profile of sterols, ubiquinones, and plastidial isoprenoids. RNA sequencing and reverse transcription-quantitative polymerase chain reaction transcriptomic analysis showed that a reduction in FPS activity levels triggers the misregulation of genes involved in biotic and abiotic stress responses, the most prominent one being the rapid induction of a set of genes related to the jasmonic acid pathway. Down-regulation of FPS also triggered an iron-deficiency transcriptional response that is consistent with the iron-deficient phenotype observed in FPS-silenced plants. The specific inhibition of the sterol biosynthesis pathway by chemical and genetic blockage mimicked these transcriptional responses, indicating that sterol depletion is the primary cause of the observed alterations. Our results highlight the importance of sterol homeostasis for normal chloroplast development and function and reveal important clues about how isoprenoid and sterol metabolism is integrated within plant physiology and development.

Characterization of Arabidopsis FPS isozymes and FPS gene expression analysis provide insight into the biosynthesis of isoprenoid precursors in seeds.

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.