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ToWards the Identification of Sesquiterpene TransportERs

Periodic Reporting for period 1 - TWISTER (ToWards the Identification of Sesquiterpene TransportERs)

Reporting period: 2015-08-01 to 2017-07-31

In plants, trichomes are small protrusions of the epidermis present on the surface of aerial organs. Some of them, namely glandular trichomes, stand for a chemical barrier against pathogens, synthesizing and excreting large amounts of secondary (or specialized) metabolites such as terpenes. Because of their biochemical properties, some of these metabolites are used by humans in pharmaceutical, fragrance, and food additive industries.

In Artemisia annua, glandular trichomes have been extensively studied since they are known to produce the sesquiterpene lactone, artemisinin, whose derivatives are extremely potent anti-malarial agents and appear to be effective drugs against other diseases including cancers. Besides, A. annua bears another type of trichomes, which so far has received little attention. These T-shaped trichomes are involved in the synthesis and the secretion of another sesquiterpene, ß-caryophyllene, which is part of the volatile cocktail playing roles in recruiting natural enemies of herbivores. Moreover, ß-caryophyllene was shown to be involved in the modulation of inflammatory and neuropathic pain responses.

Despite the immense benefits of these compounds for plant defense and human health, molecular mechanisms leading to sesquiterpene secretion out of the cell are still undeciphered. However, it was recently shown that some Pleiotropic Drug Resistance (PDR) transporters, which belong to the ATP-Binding Cassette family, are involved in transporting diterpenes across the plasma membrane, leading to their excretion from Nicotiana tabacum and N. plumbaginifolia trichomes. Thus, other PDR transporters are expected to transport other types of terpenes such as sesquiterpenes. In A. annua, two PDR transporters, namely AaPDR1 and AaPDR2, have been shown to be specifically expressed in glandular and T-shaped trichomes. In this context, the overall objectives of this project were to investigate the putative role of AaPDR1 and AaPDR2 in sesquiterpene transport and decipher the functions of T-shaped and glandular trichomes in A. annua.
Thus, to characterize putative sesquiterpene transporters, stable N. tabacum BY-2 cell lines expressing AaPDR1 or AaPDR2 were generated. Their ability to transport different types of terpenes, including sesquiterpenes, was then investigated through toxicity and accumulation assays in whole cells, as well as direct transport assays in inverted plasma membrane vesicles.
Because Lipid Transfer Proteins (LTP) were previously shown in N. tabacum to facilitate diterpene transport in the apoplast, transient expression combining AaPDRs and AaLTPs together with the artemisinin biosynthesis pathway was performed in Nicotiana benthamiana leaves (collaboration with Wageningen University). Significant increase of artemisinin and dihydroartemisinic acid (the precursor of artemisinin) secretion was observed in plants expressing both AaPDR2 and AaLTP3 in addition to the artemisinin biosynthesis pathway. These results indicate that AaPDR2 is able to transport at least the dihydroartemisinic acid. Further work has to be done to check whether AaPDR1 can also mediate dihydroartemisinic acid transport.

In the previous experiments, expression of the artemisinin synthesis and transport pathway was performed in the whole leaf. We wished to amplify these results by specifically expressing the pathway in trichomes where terpenoid precursors required for artemisinin synthesis are available in large amounts. To this purpose, we searched for trichome-specific transcription promoters in N. tabacum. Comparative proteomics was performed between trichomes and all other tissues of the plant to identify proteins that are specific to trichomes. Several were found and their specificity was confirmed at the RNA level. The corresponding gene promoters were retrieved and are being used to express the entire artemisinin biosynthesis and transport pathway in trichomes. Surprisingly, one of the trichome-specific proteins identified is a small subunit of Rubisco (RbcS), the enzyme involved in CO2 fixation in the chloroplasts. The biochemical characterization of the trichome Rubisco revealed higher catalytic activity, higher Km for CO2, and a more acidic activity versus profile than Rubisco commonly found in tobacco leaves. We made the hypothesis that this particular Rubisco is involved in refixation of CO2 released by the abundant specialized metabolism taking place in trichomes. Phylogenetic analysis showed that a trichome-type RbcS was present in many species, including ancient phyla such as bryophytes. This finding opens a new area of research.

A last objective of this project was to improve our understanding of the specific metabolism occurring in each of the two types of A. annua trichomes. Large-scale purification of T-shaped and glandular trichomes was performed prior quantitative proteomic comparison by mass spectrometry. Crude data have been obtained and are currently analyzed. This approach should provide new knowledge on the trichome functions, particularly on the poorly studied T-shaped trichomes.
In conclusion, this project has led to a wealth of knowledge in basic sciences regarding the transport of sesquiterpenes, the characterization of ABC transporters and LTP as well as the trichome functions and the trichome carbon management. Moreover, the outcomes are expected to outreach the basic science frontiers by providing new applied perspectives in medicine, agriculture and environment. Many attempts to increase the artemisinin production by bioengineering strategies have been undertaken. However, none of them reached the expected yield. Transport of dihydroartemisinic acid through AaPDR2 and AaLTP3 should increase the total production by avoiding cell self-toxicity, interference with the endogenous metabolism, and retro-inhibition. Moreover, depleting the intracellular concentration of the final product should also contribute to increasing the rate of its synthesis. Because terpene precursors are mainly available in trichomes and because these organs are no essential for plants, specific expression of the entire artemisinin biosynthesis and transport pathway should lead to an even higher artemisinin production and thus economic and societal benefits. Moreover, sesquiterpenes are known to play roles in the plant defense against herbivores and pathogens. It is expected that their overproduction might be an approach to increase plant resistance. Finally, as Rubisco is often the limiting step in photosynthesis, the discovery of the particular RbcS leading to a higher carboxylase activity could be of interest to improve catalytic properties of Rubisco, and therefore the biomass production in a context of increasing atmospheric CO2.
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