Shuiqin Wu

Redirection of cytosolic or plastidic isoprenoid precursors elevates terpene production in plants

Terpenes constitute a distinct class of natural products1 that attract insects, defend against phytopathogenic microbes and combat human diseases. However, like most natural products, they are usually made by plants and microbes in small amounts and as complex mixtures. Chemical synthesis is often costly and inefficient, and may not yield enantiomerically pure terpenes, whereas large-scale microbial production requires expensive feedstocks. We engineered high-level terpene production in tobacco plants by diverting carbon flow from cytosolic or plastidic isopentenyl diphosphate through overexpression in either compartment of an avian farnesyl diphosphate synthase and an appropriate terpene synthase. Isotopic labeling studies suggest little, if any, metabolite exchange between these two subcellular compartments. The strategy increased synthesis of the sesquiterpenes patchoulol and amorpha-4,11-diene more than 1,000-fold, as well as the monoterpene limonene 10–30 fold, and seems equally suited to generating higher levels of other terpenes for research, industrial production or therapeutic applications.

Metabolic engineering of natural products in plants; tools of the trade and challenges for the future.

Plant natural products play essential roles in plant survivability and many of them are used as nutrients, colorants, flavors, fragrances, and medicines. Genetic engineering of plants for natural products can help alleviate the demands for limited natural resources. Successes in enhancing production capacities have included manipulating blocks of genes coding for segments of pathways, over-expression of putative rate-limiting steps in pathways, expression of transcription factors regulating the entire metabolic pathways, and the construction of novel branch pathways capable of diverting carbon to the biosynthesis of unique metabolites in unexpected intracellular compartments. Further enhancements are likely if more efficient pathways can be constructed, providing for the efficient channeling of intermediates to final products, and if the means for sequestering natural products in planta can be accomplished.

Surrogate Splicing for Functional Analysis of Sesquiterpene Synthase Genes

A method for the recovery of full-length cDNAs from predicted terpene synthase genes containing introns is described. The approach utilizes Agrobacterium-mediated transient expression coupled with a reverse transcription-polydeoxyribonucleotide chain reaction assay to facilitate expression cloning of processed transcripts. Subsequent expression of intronless cDNAs in a suitable prokaryotic host provides for direct functional testing of the encoded gene product. The method was optimized by examining the expression of an intron-containing β-glucuronidase gene agroinfiltrated into petunia (Petunia hybrida) leaves, and its utility was demonstrated by defining the function of two previously uncharacterized terpene synthases. A tobacco (Nicotiana tabacum) terpene synthase-like gene containing six predicted introns was characterized as having 5-epi-aristolochene synthase activity, while an Arabidopsis (Arabidopsis thaliana) gene previously annotated as a terpene synthase was shown to possess a novel sesquiterpene synthase activity for α-barbatene, thujopsene, and β-chamigrene biosynthesis.

Doubly deuterium-labeled patchouli alcohol from cyclization of singly labeled [2-2H1]farnesyl diphosphate catalyzed by recombinant patchoulol synthase

Incubations of isotopically pure [2-(2)H(1)](E,E)-farnesyl diphosphate with recombinant patchoulol synthase (PTS) from Pogostemon cablin afforded a 65:35 mixture of monodeuterated and dideuterated patchoulols as well as numerous sesquiterpene hydrocarbons. Extensive NMR analyses ((1)H and (13)C NMR, (1)H homodecoupling NMR, HMQC, and (2)H NMR) of the labeled patchoulol mixture and comparisons of the spectra with those of unlabeled alcohol led to the conclusion that the deuterium label was located at positions (patchoulol numbering system) C5 (both isotopomers, ca. 100%) and C12 (minor isotopomer, 30-35%), that is, an approximately 2:1 mixture of [5-(2)H(1)]- and [5,12-(2)H(2)]-patchoulols. Low-resolution FIMS analyses and isotope ratio calculations further corroborated the composition of the mixture as mainly one singly deuterated and one doubly deuterated patchoulol. From a mechanistic point of view, the formation of [5,12-(2)H(2)]patchoulol is rationalized through the intermediacy of an unknown exocyclic [7,10:1,5]patchoul-4(12)-ene (15-d(1)), which could incorporate a deuteron at the C-12 position on the pathway to doubly labeled patchoulol. The corresponding depletion of deuterium content observed in the hydrocarbon coproducts, beta-patchoulene and alpha-guaiene (55% d(0)), identified the source of the excess label found in patchoulol-d(2). Comparison of the PTS amino acid sequence with those of other sesquiterpene synthases, and examination of an active site model, suggested that re-orientation of leucine 410 side chain in PTS might facilitate the creation of a 2-pocket active site where the observed deuteron transfers could occur. The retention of deuterium at C5 in the labeled patchoulol and its absence at C4 rule out an alternative mechanism involving two consecutive 1,2-hydride shifts and appears to confirm the previously proposed occurrence of a 1,3-hydride shift across the 5-membered ring. A new, semisystematic nomenclature is presented for the purpose of distinguishing the three different skeletal structures of the patchoulane sesquiterpenes.

Method for annual cropping of Pelargonium graveolens for rose scented geranium essential oil in subtropical agroclimates

Summary A method is described for the multiplication of planting material of rose scented geranium Pelargonium graveolens ‘Bipuli’, which yields Reunion Island quality essential oil, under the agroclimatic conditions of the north Indian plains. The plantlets generated from stem cuttings were planted in the field blocks using crop densities varying from 17,000 to 125,000 plants per hectare in February. The top shoots were harvested in May and the ratoon crop was fully harvested at the end of June. Two harvests from treatments in which there were 71,000 plants per hectare gave about 95.kg essential oil per hectare in the ratio of 1.7:1 in the first and second harvests. The quality of essential oil produced had a rhodinol content of 70% in which the citronellol to geraniol ratio is 1.7. These results demonstrate that the adoption of geranium cultivation will allow crop rotations such as rice/pigeon pea-potato-geranium for large economic gains per hectare, in subtropical agroclimates.

KELCH F-BOX protein positively influences Arabidopsis seed germination by targeting PHYTOCHROME-INTERACTING FACTOR1

Significance The completion of seed germination is an irrevocable event for plants, determining, for most plants, the site of the remainder of their life cycle. One environmental cue important to the completion of seed germination is light, which, in Arabidopsis thaliana, can influence a host of transcription factors, including PHYTOCHROME-INTERACTING FACTOR1 (PIF1), a negative regulator of the completion of germination and seedling de-etiolation. The KELCH F-BOX protein COLD TEMPERATURE GERMINATING10 (CTG10) can recognize and bind to PIF1, negatively influencing PIF1 stability, stimulating the completion of germination, and promoting a de-etiolated seedling morphology. PIF1, in turn, can downregulate CTG10 expression, revealing a complex coregulation orchestrated by light presence and quality that dictates whether the seed completes germination. Seeds employ sensory systems that assess various environmental cues over time to maximize the successful transition from embryo to seedling. Here we show that the Arabidopsis F-BOX protein COLD TEMPERATURE-GERMINATING (CTG)-10, identified by activation tagging, is a positive regulator of this process. When overexpressed (OE), CTG10 hastens aspects of seed germination. CTG10 is expressed predominantly in the hypocotyl, and the protein is localized to the nucleus. CTG10 interacts with PHYTOCHROME-INTERACTING FACTOR 1 (PIF1) and helps regulate its abundance in planta. CTG10-OE accelerates the loss of PIF1 in light, increasing germination efficiency, while PIF1-OE lines fail to complete germination in darkness, which is reversed by concurrent CTG10-OE. Double-mutant (pif1 ctg10) lines demonstrated that PIF1 is epistatic to CTG10. Both CTG10 and PIF1 amounts decline during seed germination in the light but reaccumulate in the dark. PIF1 in turn down-regulates CTG10 transcription, suggesting a feedback loop of CTG10/PIF1 control. The genetic, physiological, and biochemical evidence, when taken together, leads us to propose that PIF1 and CTG10 coexist, and even accumulate, in the nucleus in darkness, but that, following illumination, CTG10 assists in reducing PIF1 amounts, thus promoting the completion of seed germination and subsequent seedling development.