The Small Subunit of Snapdragon Geranyl Diphosphate Synthase Modifies the Chain Length Specificity of Tobacco Geranylgeranyl Diphosphate Synthase in Planta

Abstract

Geranyl diphosphate (GPP), the precursor of many monoterpene end products, is synthesized in plastids by a condensation of dimethylallyl diphosphate and isopentenyl diphosphate (IPP) in a reaction catalyzed by homodimeric or heterodimeric GPP synthase (GPPS). In the heterodimeric enzymes, a noncatalytic small subunit (GPPS.SSU) determines the product specificity of the catalytic large subunit, which may be either an active geranylgeranyl diphosphate synthase (GGPPS) or an inactive GGPPS-like protein. Here, we show that expression of snapdragon (Antirrhinum majus) GPPS.SSU in tobacco (Nicotiana tabacum) plants increased the total GPPS activity and monoterpene emission from leaves and flowers, indicating that the introduced catalytically inactive GPPS.SSU found endogenous large subunit partner(s) and formed an active snapdragon/tobacco GPPS in planta. Bimolecular fluorescence complementation and in vitro enzyme analysis of individual and hybrid proteins revealed that two of four GGPPS-like candidates from tobacco EST databases encode bona fide GGPPS that can interact with snapdragon GPPS.SSU and form a functional GPPS enzyme in plastids. The formation of chimeric GPPS in transgenic plants also resulted in leaf chlorosis, increased light sensitivity, and dwarfism due to decreased levels of chlorophylls, carotenoids, and gibberellins. In addition, these transgenic plants had reduced levels of sesquiterpene emission, suggesting that the export of isoprenoid intermediates from the plastids into the cytosol was decreased. These results provide genetic evidence that GPPS.SSU modifies the chain length specificity of phylogenetically distant GGPPS and can modulate IPP flux distribution between GPP and GGPP synthesis in planta.

Chrysanthemyl Diphosphate Synthase Operates in Planta as a Bifunctional Enzyme with Chrysanthemol Synthase Activity

“…The small subunits are usually inactive in the in vitro assays, and the big subunits are either inactive (35,49) or function as well as homodimeric geranylgeranyl diphosphate synthases (39,47,48). Two of four geranylgeranyl diphosphate synthase-like enzymes of tobacco could interact in tobacco with the small subunit from snapdragon (Antirrhinum majus) (48). Therefore, we propose that CDS may possibly also form heterodimers with geranylgeranyl diphosphate synthaselike large subunits of tobacco (and pyrethrum) that significantly promote its activity.…”

“…13), can be estimated to be around 30 M, assuming a regression coefficient of 1 g ml Ϫ1 for leaf fresh weight to leaf volume (44), a chloroplast volume of 20% of total cell volume in mature leaf cells (45), and 60% of DMAPP to be found in the chloroplast (46). Third, inactive GDSs have been shown before to be activated by the formation of a heterodimer with geranylgeranyldiphospate synthase (39,47,48). The small subunits are usually inactive in the in vitro assays, and the big subunits are either inactive (35,49) or function as well as homodimeric geranylgeranyl diphosphate synthases (39,47,48).…”

“…Third, inactive GDSs have been shown before to be activated by the formation of a heterodimer with geranylgeranyldiphospate synthase (39,47,48). The small subunits are usually inactive in the in vitro assays, and the big subunits are either inactive (35,49) or function as well as homodimeric geranylgeranyl diphosphate synthases (39,47,48). Two of four geranylgeranyl diphosphate synthase-like enzymes of tobacco could interact in tobacco with the small subunit from snapdragon (Antirrhinum majus) (48).…”

An alternative pathway contributes to phenylalanine biosynthesis in plants via a cytosolic tyrosine:phenylpyruvate aminotransferase

“…The Arabidopsis TYDC coding region was PCR-amplified using forward primer 5 0 -CCATGGATGGAATTTGGT ACCGGCAACG-3 0 and reverse primer 5 0 -TCCAAATTTACACGCAACGACC NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3833 ARTICLE ATTATTAACTGCAG-3 0 and subcloned into the pGEMT easy vector (Invitrogen, Carlsbad, CA, USA). After sequence verification, the fragment was subcloned into the NcoI/PstI site of the pEF1-LIS binary vector 47 and transient AtTYDC overexpression was performed as described above.…”

Volatile terpenoids: multiple functions, biosynthesis, modulation and manipulation by genetic engineering

“…Two different and independent pathways are involved in the formation of five carbon isoprene building units, the plastidial MEP and cytosolic MVA pathways; however, the MEP pathway is also disseminated in the endoplasmic reticulum and peroxisomes (Mahmoud and Croteau 2002;Dudareva and Pichersky 2008;Lange and Ahkami 2013), whereas these compartmentally separated isoprenoid biosynthetic pathways are interconnected by a metabolic "crosstalk" (Flügge and Gao 2005;Orlova et al 2009). Mevalonate-5-diphosphate is produced in Arabidopsis thaliana by the MVA pathway through phosphorylation, and the entire reaction is catalyzed by mevalonate kinase and phosphomevalonate kinase (Lluch et al 2000;Yu and Utsumi 2009).…”