Novel routes towards bioplastics from plants: elucidation of the methylperillate biosynthesis pathway from Salvia dorisiana trichomes

Plants produce a large variety of highly functionalized terpenoids. Functional groups such as partially unsaturated rings and carboxyl groups provide handles to use these compounds as feedstock for biobased commodity chemicals. For instance, methylperillate, a monoterpenoid found in Salvia dorisiana, may be used for this purpose, as it carries both an unsaturated ring and a methylated carboxyl group. The biosynthetic pathway of methylperillate in plants is still unclear. In this work, we identified glandular trichomes from S. dorisiana as the location of biosynthesis and storage of methylperillate. mRNA from purified trichomes was used to identify four genes that can encode the pathway from geranyl diphosphate towards methylperillate. This pathway includes a (–)-limonene synthase (SdLS), a limonene 7-hydroxylase (SdL7H, CYP71A76), and a perillyl alcohol dehydrogenase (SdPOHDH). We also identified a terpene acid methyltransferase, perillic acid O-methyltransferase (SdPAOMT), with homology to salicylic acid OMTs. Transient expression in Nicotiana benthamiana of these four genes, in combination with a geranyl diphosphate synthase to boost precursor formation, resulted in production of methylperillate. This demonstrates the potential of these enzymes for metabolic engineering of a feedstock for biobased commodity chemical

Development of CarbonTracker Europe-CH4 – part 1 : system set-up and sensitivity analyses

CarbonTracker Europe-CH4 (CTE-CH4) inverse model versions 1.0 and 1.1 are presented. The model optimizes global surface methane emissions from biosphere and anthropogenic sources using an ensemble Kalman filter (EnKF) based optimization method, using the TM5 chemistry transport model as an observation operator, and assimilating global in-situ atmospheric methane mole fraction observations. In this study, we examine sensitivity of our CH4 emission estimates on the ensemble size, covariance matrix, prior estimates, observations to be assimilated, assimilation window length, convection scheme in TM5, and model structure in the emission estimates by performing CTE-CH4 with several set-ups. The analyses show that the model is sensitive to most of the parameters and inputs that were examined. Firstly, using a large enough ensemble size stabilises the results. Secondly, using an informative covariance matrix reduces uncertainty estimates. Thirdly, agreement with discrete observations became better when assimilating continuous observations. Finally, the posterior emissions were found sensitive to the choice of prior estimates, convection scheme and model structure, particularly to their spatial distribution. The distribution of posterior mole fractions derived from posterior emissions is consistent with the observations to the extent prescribed in the various covariance estimates, indicating a satisfactory performance of our system.peer-reviewe

The seco-iridoid pathway from Catharanthus roseus

The (seco)iridoids and their derivatives, the monoterpenoid indole alkaloids (MIAs), form two large families of plant-derived bioactive compounds with a wide spectrum of high-value pharmacological and insect-repellent activities. Vinblastine and vincristine, MIAs used as anticancer drugs, are produced by Catharanthus roseus in extremely low levels, leading to high market prices and poor availability. Their biotechnological production is hampered by the fragmentary knowledge of their biosynthesis. Here we report the discovery of the last four missing steps of the (seco)iridoid biosynthesis pathway. Expression of the eight genes encoding this pathway, together with two genes boosting precursor formation and two downstream alkaloid biosynthesis genes, in an alternative plant host, allows the heterologous production of the complex MIA strictosidine. This confirms the functionality of all enzymes of the pathway and highlights their utility for synthetic biology programmes towards a sustainable biotechnological production of valuable (seco)iridoids and alkaloids with pharmaceutical and agricultural applications

Global methane emission estimates for 2000–2012 from CarbonTracker Europe-CH4 v1.0

We present a global distribution of surface methane (CH4) emission estimates for 2000–2012 derived using the CarbonTracker Europe-CH4 (CTE-CH4) data assimilation system. In CTE-CH4, anthropogenic and biospheric CH4 emissions are simultaneously estimated based on constraints of global atmospheric in situ CH4 observations. The system was configured to either estimate only anthropogenic or biospheric sources per region, or to estimate both categories simultaneously. The latter increased the number of optimizable parameters from 62 to 78. In addition, the differences between two numerical schemes available to perform turbulent vertical mixing in the atmospheric transport model TM5 were examined. Together, the system configurations encompass important axes of uncertainty in inversions and allow us to examine the robustness of the flux estimates. The posterior emission estimates are further evaluated by comparing simulated atmospheric CH4 to surface in situ observations, vertical profiles of CH4 made by aircraft, remotely sensed dry-air total column-averaged mole fraction (XCH4) from the Total Carbon Column Observing Network (TCCON), and XCH4 from the Greenhouse gases Observing Satellite (GOSAT). The evaluation with non-assimilated observations shows that posterior XCH4 is better matched with the retrievals when the vertical mixing scheme with faster interhemispheric exchange is used. Estimated posterior mean total global emissions during 2000–2012 are 516 ± 51 Tg CH4 yr−1 , with an increase of 18 Tg CH4 yr−1 from 2000–2006 to 2007–2012. The increase is mainly driven by an increase in emissions from South American temperate, Asian temperate and Asian tropical TransCom regions. In addition, the increase is hardly sensitive to different model configurations (< 2 Tg CH4 yr−1 difference), and much smaller than suggested by EDGAR v4.2 FT2010 inventory (33 Tg CH4 yr−1 ), which was used for prior anthropogenic emission estimates. The result is in good agreement with other published estimates from inverse modelling studies (16–20 Tg CH4 yr−1 ). However, this study could not conclusively separate a small trend in biospheric emissions (−5 to +6.9 Tg CH4 yr−1 ) from the much larger trend in anthropogenic emissions (15–27 Tg CH4 yr−1 ). Finally, we find that the global and North American CH4 balance could be closed over this time period without the previously suggested need to strongly increase anthropogenic CH4 emissions in the United States. With further developments, especially on the treatment of the atmospheric CH4 sink, we expect the data assimilation system presented here will be able to contribute to the ongoing interpretation of changes in this important greenhouse gas budget.peer-reviewe

Reconstitution of the Costunolide Biosynthetic Pathway in Yeast and Nicotiana benthamiana

The sesquiterpene costunolide has a broad range of biological activities and is the parent compound for many other biologically active sesquiterpenes such as parthenolide. Two enzymes of the pathway leading to costunolide have been previously characterized: germacrene A synthase (GAS) and germacrene A oxidase (GAO), which together catalyse the biosynthesis of germacra-1(10),4,11(13)-trien-12-oic acid. However, the gene responsible for the last step toward costunolide has not been characterized until now. Here we show that chicory costunolide synthase (CiCOS), CYP71BL3, can catalyse the oxidation of germacra-1(10),4,11(13)-trien-12-oic acid to yield costunolide. Co-expression of feverfew GAS (TpGAS), chicory GAO (CiGAO), and chicory COS (CiCOS) in yeast resulted in the biosynthesis of costunolide. The catalytic activity of TpGAS, CiGAO and CiCOS was also verified in planta by transient expression in Nicotiana benthamiana. Mitochondrial targeting of TpGAS resulted in a significant increase in the production of germacrene A compared with the native cytosolic targeting. When the N. benthamiana leaves were co-infiltrated with TpGAS and CiGAO, germacrene A almost completely disappeared as a result of the presence of CiGAO. Transient expression of TpGAS, CiGAO and CiCOS in N. benthamiana leaves resulted in costunolide production of up to 60 ng.g−1 FW. In addition, two new compounds were formed that were identified as costunolide-glutathione and costunolide-cysteine conjugates

Comparison of expression profiles for Nicotiana benthamiana agroinfiltrated with aryophyllene synthase and SNARE-RNAi [Nicotiana benthamiana]

Here we show for the first time that vesicle fusion is one of multiple pathways in plant cells involved in terpene emissions. We demonstrate a positive feedback on terpene production when vesicle fusion is inhibited in cells producing high levels of terpenes, which relates to an induced proteasome malfunctio

Comparison of expression profiles for Nicotiana benthamiana agroinfiltrated with aryophyllene synthase and SNARE-RNAi [Nicotiana benthamiana]

Here we show for the first time that vesicle fusion is one of multiple pathways in plant cells involved in terpene emissions. We demonstrate a positive feedback on terpene production when vesicle fusion is inhibited in cells producing high levels of terpenes, which relates to an induced proteasome malfunctio

Secrets of the world's most popular bedding plant unlocked

Boosted by next-generation sequencing technology, there is now an ever-growing list of fully sequenced plant genomes. Recent additions to this list are two presumed ancestors of Petunia hybrida, the most popular bedding plant worldwide. These genome sequences provide new information on a species at a key position in plant phylogeny, and support the use of petunia as a research model plant species