Final Report Summary - METAPRO (The development of tools and effective strategies for the optimisation of useful secondary METAbolite PROduction in planta)
Executive Summary:
Isoprenoids are classical secondary metabolites being synthesised in specialised tissues and/or cellular compartments. Their abundance is typically low but can be dramatically increased under stress conditions or defined stages in development. Most isoprenoids of commercial interest are used as fragrances in cosmetics and flavours, colorants and nutritional supplements in foods and feeds (e.g. ketocarotenoids) and numerous important medicinal properties. Their industrial relevance also means they are compounds of high value with global markets in the range of $ 1 billion per annum. A contributing factor to the high cost of these molecules resides in the fact that they are produced in low yields by slow growing plant species that are not readily amenable to agricultural production. In the METAPRO project we aimed to optimise the production of several useful isoprenoids to demonstrate the tools and strategies developed for the generic production of useful secondary metabolites in plants. To demonstrate our genetic engineering approaches two examples, astaxanthin formation and crocin production where used. The host platforms to be used in the proposed project will be tomato and potato. These crops are the most common fruit and vegetables produced worldwide and thus amenable to modern agricultural practices and from a GM perspective can be produced in contained growth facilities if necessary. These two crops also have specialised storage (sink) tissues containing cellular structures amenable to isoprenoid formation/accumulation without disruption of essential plant processes. It is these specific tissues and specialised cellular structures (plastids) that will be exploited in the METAPRO project.
Most of the fundamental reactions involved in isoprenoid formation have been established and rudimentary engineering of the pathway performed. One of the aims of the METAPRO project was to build on these applied foundations and advance our generic knowledge base to a new era in our understanding and engineering of plant metabolism for high-value secondary metabolites. The key objectives for the project are:
(i) Develop a more comprehensive understanding of regulation at an integrative systems level not just in the target pathway.
(ii) Identification and elucidation of mechanisms associated with the storage, deposition and sequestration of isoprenoids.
(iii) Reveal stability parameters of newly synthesised compounds in the cell.
(iv) Demonstrate tools and strategies for high-level production of useful isoprenoids such as ketocarotenoids and apocarotenoids.
During the project our integrative approach to characterisation revealed that it is difficult to view secondary metabolism as a separate entity, but as a component linked to primary and intermediary metabolism and often associated with re-programming of developmental/environmental related metabolism. Transcriptomic RNA –Seq approaches became the method of choice during the project and were successful in revealing to candidate genes. It is also apparent that biosynthesis and sequestration are interlinked and changes in metabolite composition can alter/induce cellular structures used for deposition. Our findings on stability transcend isoprenoids and our chosen hosts. Project partners have revealed both enzymatic and non-enzymatic degradation processes operate. To overcome these phenomena a down-stream process involving encapsulation has been developed that facilitates product stability. Through the implementation of our engineering strategies exceptionally high levels of isoprenoids have been generated. Some pleotropic limitations have been experienced which has highlighted the need for more controlled production. The prototypes are now available to perform production, technical and economic feasibility studies with a wide range of ketocarotenoid producing varieties.
The METAPRO project has disseminating its findings at the earliest opportunity to the scientific community, general public, schools and government agencies. For example to date thirty peer reviewed publications have been generated several in high impact journals such as Science, PNAS and Plant Cell. Over 100 dissemination activities have been delivered and at these three patents generated, that least three permanent jobs have also been created through the projects activities. The work has strengthened the position and sustainable foundations of an SME partner, which in turn has contributed to rural development. The project website www.isoprenoid.com has also been successful as a focal point for information and will continue beyond the RTD activity of the project. Two informative promotional video of the project have been generated and one with high school pupils. A highlight of the outreach and training activities during the project has been the metabolite profiling training school carried out in association with a related COST ACTION and the numerous training visits for early stage researchers (ESRs).
Project Context and Objectives:
Plant secondary metabolites are defined as compounds that are non-essential to fundamental plant processes, but do have an important role in the plant’s interaction with its environment. Secondary metabolites can confer agronomic traits such as quality, resistance and stress tolerance. Many secondary metabolites are also essential components of the human diet, are utilised as phytomedicines and routinely used as industrial raw materials and high-value fine chemicals. Chemically, secondary metabolites exhibit an enormous chemical diversity and complexity, which in most cases precludes chemical synthesis as an economically feasible means of production. On the basis of their biosynthetic origins, plant secondary metabolites can be structurally divided into five major groups; polyketides, alkaloids, phenylpropanoids, flavonoids and isoprenoids. In the METAPRO project we have optimise the production of several useful isoprenoids to demonstrate the tools and strategies developed for the generic production of useful secondary metabolites in plants.
In the majority of cases isoprenoids are classical secondary metabolites being synthesised in specialised tissues and/or cellular compartments. Their abundance is typically low but can be dramatically increased under stress conditions or defined stages in development. Most isoprenoids of commercial interest are used as fragrances in cosmetics and flavours, colorants and nutritional supplements in foods and feeds (e.g. ketocarotenoids) and numerous important medicinal properties. Their industrial relevance also means they are compounds of high value with global markets in the range of $ 1 billion per annum. A contributing factor to the high cost of these molecules resides in the fact that they are produced in low yields by slow growing plant species that are not readily amenable to agricultural production (for example, crocins produced in the stigmas of purple crocus flowers). Alternative production systems such as plant cell culture have in the main not proven to be feasible. It is therefore not surprising that total or semi-synthetic chemical synthesis is presently the method of choice for obtaining many of these isoprenoid molecules. However their structural complexity makes chemical synthesis expensive and difficult. Given the high value and utility of these isoprenoids as well as the lack of amenable plant based sources, a genetic engineering (GE) approach provides a logical solution to the creation of renewable bio-sources of these molecules with improved economic and environmental potential. In the METAPRO project we will demonstrate our genetic engineering approaches using two examples, astaxanthin formation and crocin production. Astaxanthin is a carotenoid (ketocarotenoid) while the crocins are derived from a carotenoid cleavage product (apocarotenoid) and require transport from their site of synthesis to the vacuole where they accumulate. They are typical secondary metabolites where optimisation in their native plant sources does not provide an optimal production platform.
The host platforms to be used in the proposed project will be tomato and potato. These crops are the most common fruit and vegetables produced worldwide and thus amenable to modern agricultural practices and from a GM perspective can be produced in contained growth facilities if necessary. Genetic and Genomic resources are well developed in these two Solanaceae with a vast collection of genetic diversity available and the sequencing of both genomes underway. Advanced transcriptomic, metabolomic and proteomic platforms have also been established and customised for these crop plants. These two crops also have specialised storage (sink) tissues containing cellular structures amenable to isoprenoid formation/accumulation without disruption of essential plant processes. It is these specific tissues and specialised cellular structures (plastids) that have been exploited in the METAPRO project.
Most of the fundamental reactions involved in isoprenoid formation have been established and rudimentary engineering of the pathway performed. One of the aims of the METAPRO project was to build on these applied foundations and advance our generic knowledge base to a new era in our understanding and engineering of plant metabolism for high-value secondary metabolites. The key objectives for the project are:
(i) Develop a more comprehensive understanding of regulation at an integrative systems level not just in the target pathway.
(ii) Identification and elucidation of mechanisms associated with the storage, deposition and sequestration of isoprenoids.
(iii) Reveal stability parameters of newly synthesised compounds in the cell.
(iv) Demonstrate tools and strategies for high-level production of useful isoprenoids such as ketocarotenoids and apocarotenoids.
Numerous tomato and potato varieties with perturbed isoprenoid content have been subjected to concurrent multi-level omic based analyses over the life time of the project. For example tomato genotypes with the simultaneous elevation of multiple high-value isoprenoids and other antioxidants have been analysed using an integrative omic based approach. Through the integration of transcriptomic and metabolomic datasets the consortium have revealed that the progenitor of elevated isorprenoids is enhanced core metabolism which may directly or indirectly result in the presence of a greater plastid area per cell (Enfissi et al., 2010. Plant cell, 22, 1190). Using high precursor tomato genotypes similar multi-level “omic” analyses have revealed a strong association between increased isoprenoid (beta-carotene) content and ripening related processes. In potato correlation network analysis have identified a number of regulatory HUBs existing within the pathway of interest (Diretto, et al., 2010, Plant Physiol., 154, 899). These approaches have illustrated the potential of modern systems based approaches in delivering new potential regulatory HUBs and have been important in characterising metabolic changes and the reprogramming that exists during secondary metabolite production. Over the course of the project techniques advanced with RNA-SEq replacing microarrays as the transcriptomic method of choice. The advent of modern faster Mass Spectrometers enabled development of label-free protein quantification procedures and the incorporation of proteomic data into our parallel multi-omic datasets (Mora et al, (2013) Proteomics). The inclusion of proteomic data was highly informative as it showed the different levels of cellular regulation and how translation is a key component of the process. A number of correlation (co-response) models have been generated. From these models it is clear that secondary metabolism is associated with core metabolism and the latter is frequently the progenitor of secondary metabolite formation. It is interesting that the Calvin-Benson cycle is at the core of the network in ripening fruit, as this process is typically associated with photosynthetic tissues. Although tomato fruit are photosynthetically active during fruit development it is not a key aspect of their metabolism and recently the role of photosynthesis in fruit development has been questioned with the belief that photosynthate is derived from vegetative material. The integrative characterisation of transgenic tomato plants possessing fruit specific down regulation of DETIOLATED1 is another example where core metabolism particularly carbon fixation appears to be the progenitor of down-stream secondary or in this case “tertiary” pathways in tomato fruit. The metabolite of the Calvin cycle displaying the greatest connections was D- glyceralde-3-phosphate. This metabolite is common to both the Calvin cycle and MEP pathway. Collectively these findings suggest that the plasticity of Calvin cycle components in tomato fruit warrant further investigation and the utilisation of photosynthate/carbon close to its source without further resource allocation could be a valuable strategy to explore as part of a combinatory engineering approach. Other metabolic links between isoprenoid formation and photosynthetic carbon flow have also been revealed through metabolomic analysis of hemiterpenoid glycosides under nutrient deprivation. Finally, cofactors/reductants appear to have an important influence over multiple pathways presumably by direct action on biosynthetic enzymes or perhaps via the modulation of redox states.
In addition perturbations in the isoprenoid content appear to have a direct link to changes in phytohormone balance, which can alter developmental and responses to environmental cues. A number of HUBs in the models have been identified and functional validation performed. Such an approach has revealed new transcriptional regulator. However, a transcription factor acting on isoprenoids solely remains elusive. Instead it is common that they act on other developmental processes or sectors of metabolism and isoprenoids an associate of a more fundamental process. Never the less a number of transcription factors that can alter pigments have been identified and one patent generated.
The METAPRO project addressed product sequestration/storage of products, several important insights have been revealed. For example, localisation experiments and conventional biochemical fractionation suggest that biosynthesis and accumulation of products utilise discrete functional inter cellular and intra organelle compartments. It would appear that the specialisation of organelles can also result from high-level production of certain metabolites. Finally through the screening of natural variation new potential alleles that can confer improved product sequestration have been identified. Our data also suggest that lipid formation (or plastid lipids) is concurrent with carotenoid formation. Traditionally the focus of metabolic engineering has been towards increased biosynthesis capacity but the experimental data generated shows biosynthesis and sequestration is linked and must be addressed with equal importance. Sub-plastid organelle fractionation revealed cellular structures for the first time associated with storage. These data also revealed several fundamental aspects in that perturbation in metabolites can alter and induce new sub-organelle structures. From correlation analysis and the screening of natural variation new transcriptional regulators have been isolated and a mechanisms demonstrated that shows how modulating Abscisic acid (ABA) levels can alter plastid levels.
The potential of altering enzymatic carotenoid degradation has been illustrated (Campbell et al., 2010 Plant Physiol., 154, 656) within the project. Further characterisation of these classes of enzymes is has been performed including the elucidation of carotenoid cleavage activities involved in strigolactone formation a new class of plant hormones. Unfortunately no QTL for carotenoid cleavage enzymes were found. In certain tissues such as leaf material it would appear that newly formed carotenoids are preferentially degraded. Storage and down-stream processing where found to be key areas where enzymatic and n0n-enzymatic degradation arose. In order to overcome these difficulties an extraction procedure that does not allow the material in question to dry was developed and an encapsulation procedure developed and implemented. These are key technologies that will make feasibility and translation possible. A highlight in the catabolism aspect of the project has been the implementation of RNA-Seq transcriptomic procedures to elucidate the apocarotenoid pathway in Saffron. This approach has enabled the dissection of the pathway and subsequent functional characterisation. These studies have now established a consensus overcoming the misleading data in the literature. As a result of these data the tools are now available to engineer the pathway and this approach has been performed in the METAPRO project to the primary transformant (T0) stage.
Significant advances have been made in the production of ketocarotenoids, which are one of the projects demonstration molecules, both in tomato and potato tubers. Firstly a plant variety was generated that expressed an optimised astaxanthin biosynthetic pathway. This approach benefitted from translational enhancement, plastid transformation technologies and suitable host platforms for high level production. To these varieties optimised precursors for the target pathways were delivered, improved sequestration through the modulation of plastid parameters performed, and the combining traits performed. As a result attaining targeted levels has not been a problem with high-levels in the 20 to 25 mg/gDW achieved for tomato. The issues have been pleiotopic effects on plant development. For example levels achieved with plastid transformation were so high that the plants cannot be regenerated. Ripening and development in general are effected which delays the growth cycle. However, despite these difficult the METAPRO project has delivered phenotypically stable prototypes with very high levels that will enable feasibility studies to be carried out.
The METAPRO project has disseminated its findings at the earliest opportunity to the scientific community, general public, schools and government agencies. For example to date thirty peer reviewed publications have been generated several in high impact journals such as Science, PNAS and Plant Cell. These outputs have advanced our scientific knowledge in the field. Over 100 dissemination activities have been delivered and at these three patents generated; to date at least three permanent jobs have also been created through the projects activities. The work has strengthened the position and sustainable foundations of an SME partner, which in turn has contributed to rural development. Through the work associated with the METAPRO project national funding has been awarded to several of the partners. The project website www.isoprenoid.com has also been successful as a focal point for information and will continue beyond the RTD activity of the project. Two informative promotional video of the project have been generated and one with high school pupils utilising new technologies. A highlight of the outreach and training activities during the project has been the metabolite profiling training school carried out in association with a related COST ACTION and the numerous training visits for early stage researchers (ESRs).
Project Results:
The RTD activities of METAPRO were divided into four work packages 2 to 5. Concerned with regulation, storage, stability and optimised production respectively. Work was performed to specific task which led to the completion of the projects objectives.
1. Work package 2. Identification of regulatory mechanisms involved in the synthesis of isoprenoids (WP leader P4 SCRI).
1.2. WP2 Objectives
O2.1. Identification of regulatory mechanisms involved in the formation of isoprenoids/carotenoids through characterisation at the gene, transcript, protein, enzyme activity and metabolite levels.
O2.2. Identify potential protein components of isoprenoid biosynthetic complexes.
O2.3. Determine changes to the metabolome and transcriptome associated with isoprenoid formation and/or perturbation.
O2.4. Integrate transcriptomic and metabolomic datasets to identify putative regulatory HUBs and candidate genes.
O2.5. Compile meta and experimental datasets, statistical analysis, integration and display of data over metabolic pathways.
O2.6. Utilise the methodologies established in WP2 as an enabling technologies platform to characterise genotypes generated and investigated in WP3, 4 and 5.
1.3. RTD activities performed in WP2
WP2 activities have utilised germplasm available within the consortium. These genotypes include genetically modified plants (GMPs) and mutants with elevated precursors for the production of ketocarotenoids and crocetin, and pleiotropic effects resulting from the perturbation of isoprenoid content. In accordance with Task 2.1 this material has been generated and distributed to respective partners for analysis. The consortium’s main focus has been (i) tomato and potato genotypes with increased precursors such as the high -carotene and xanthophylls varieties generated by P7, (ii) tomato varieties with altered plastid parameters constituting a cross workpackage activity with WP3 (P1) and (iii) potato genotypes producing high-value ketocarotenoids (P4). Sampling of tomato and potato material has been carried out on defined tissues over development to create dynamic dataset. Metabolomic analysis has utilised targeted isoprenoid determinations by HPLC-PDA, UPLC-PDA and LC-MS (Task 2.2). Direct infusion Mass spectrometry has been performed to generate chemical fingerprints and ascertain global compositional changes. In addition quantitative metabolite profiling using GC-MS and LC-MS for intermediary metabolites have been performed (Task 2.6). The robustness of these procedures has been validated by small scale inter-laboratory ring testing approaches (P1 and P8).
In order to complement the metabolomic analysis performed, transcriptomics has been carried out with either the tomato TOM2 microarray or potato POCI (Task 2.5). Verification of changes across the transcriptome and within the target pathway(s) has then been carried out by QPCR. Where appropriate selected analysis using specific enzyme assays (Task 2.3) and proteomic analysis (Task 2.4) has been performed. Of the omic level approaches proteomics has been the most challenging, especially in a quantitative manner. Allow quantitative profiles have been obtained using iTRAQ approaches the coverage is poor (e.g. 100 proteins detected at 99% confidence) and the representation of membrane proteins is low. To date only a limited number of isoprenoid/carotenoid related biosynthetic enzymes have been identified these include CRTISO and isopentenyl diphosphate isomerase (iDI). In addition no carotenoid bisosynthetic enzymes have been identified within protein complexes tested, although biosynthetic enzymes involved in the formation of prenyl lipids such as GGPP have been found associated with photosynthetic complexes. The proteomic approach was subsequently improved (P1) with the development of a routine quantitative label-free method for proteomic analysis. The label-free procedure uses internal standards for relative quantification and facilitates good coverage of membrane protein through the use of SDS-PAGE prior to reverse phase nano-LC. A manuscript describing the procedure and its development has been accepted in the Proteomics journal (Mora, Bramley and Fraser).This procedure has been very effective when combining activities with WP3, using enriched organelle and sub-plastidial fractions which have revealed new regulatory mechanisms, whereby the enzymes are in a different location to the precursors and products (Objectives 2.1 2.2 2.5 & 2.6).
In order to complement the metabolomics and proteomic analysis performed, transcriptomics has been carried out with either the tomato TOM2 microarray or potato POCI (Task 2.5). Verification of changes across the transcriptome and within the target pathway(s) has then been carried out by QPCR.
1.4. Examples of case studies
“Omic” characterisation of
(i) Tomato varieties with increased plastid content (P1)- The multi-level omic based characterisation of the DET1 down regulated tomato varieties has revealed the close association of secondary metabolism with core metabolic processes. This would suggest that future systems based engineering approaches that do not solely affect the pathway of the interest should be explored (Enfissi et al., 2010. Plant cell, 22, 1190). The proteomic studies indicated the importance of studying all levels of cellular regulation as elevations in transcripts were qualitatively and quantitatively not aligned with protein levels. Thus indicating the importance of post-transcriptional regulation. Organisation and integration of large-scale datasets and associated metadata has been successfully carried out predominantly using a large-scale metabolomic dataset for tomato introgression lines with modulated isoprenoid levels. The data has been subjected to co-regulation analysis and a model created for isoprenoid biosynthesis. An important aspect of these data is that the different sections of isoprenoid metabolism cluster in a biosynthetic intuitive manner but interestingly all relate back to primary metabolism especially the Calvin cycle, which suggests that defining metabolism into primary and secondary metabolism perhaps is not the correct approach. If the fixation of carbon could be exploited increased secondary metabolite formation could be significantly increased. The datasets have been deposited on the SGN website (www.Solgenomics.net) for the scientific community with a disclaimer describing its use.
(ii) High beta-carotene and xanthophyll precursor tomato and potato lines (P3 and P7)- Fruits of tomato plants overexpressing the LYCOPENE BETA CYCLASE (LCY-b) and/or BETA-CAROTENE HYDROXYLASE (CHY) transgenes and golden potato tubers where generated. The EU-TOM3 Affymetrix microarray, the transcriptome of tomato fruits overexpressing the LCY-b and CHY transgenes and of tomato mutants affected in fruit pigmentation (at, r, t, Beta). A full phenotypic characterization (ethylene and ABA production, cell wall and cuticle composition, post-harvest characteristics) was conducted on the former. The data indicate that beta-carotene overproduction in fruits triggers a complex syndrome, resulting in decreased ethylene production and enhanced self-life of the fruits. The transcriptome of the high-carotenoid DM potato line has been characterized using Illumina RNA-Seq (Massa et al, 2011). Network analysis performed on potato genotypes with elevated precursors illustrated how regulatory HUBs in the target pathway can change upon pathway engineering and that those components having the greatest quantitative changes are not always responsible for regulatory control within the pathway. (Diretto, et al., 2010, Plant Physiol., 154). The glasshouse evaluation at the Metapontum Agrobios research facilities in Metaponto (Italy) was aimed at the generation and characterization of fruit samples from the HighCaro (HC) LycB experessor, UO expressing a beta-carotene hydroxylase (CrtRb2) and their hybrid (HU) as well as from the control Red Setter (RS) parent line. Carotenoid contents of fruits of the four tomato lines (RS, HC, UO, HU), harvested at four stages (IG, MG, TR, RR), clearly showed that the overexpression of the biosynthetic gene encoded by the transgene was the determinant of the patterns of carotenoid biosynthesis and accumulation in tomato fruit. In the HC fruits, almost all lycopene was converted to b-carotene while a consistent amount of xanthophylls, mainly violaxanthin, was present in fruits of plant carrying th Research activities of the second year were aimed at: performing the expression analysis by RT-qPCR of the most important genes involved in biosynthesis of carotenoids, ethylene and ABA on fruit samples of RS, UO, HC and HU. Expression analysis of eleven genes (PSY1 , LCYb, CrtRb2, ZEP, CCD1A, CCD1B, CCD4B, NCED1, ACS2, ACS4, ACO1) was carried out on the fruit samples (4 sampling stages) of the high precursor and control lines (RS, UO, HC, HU) using RT-qPCR analysis. This dataset (11 genes x 16 samples) was combined with the datasets of HPLC determinations (16 compounds) and Metabolomics (42 metabolites, performed by P1 RHUL) which were derived on the same tissue samples. The final dataset was made, therefore, of 69 variables determined on 16 objects (fruit samples) for a total of 1104 data points each one derived from the analysis of at least 3 (up to 5) biological replicates. Data analysis was performed on single datasets as well as on combinations of two of them and on the whole dataset using univariate and multivariate statistical methods.
Results of RT-qPCR analysis on fruit samples of four high precursor and control lines clearly showed that none of the carotenoid biosynthetic genes was up-regulated by the Lcyb and CrtRb2 over-expression. Therefore, the reduction of the total carotenoid content in ripe fruits of the transgenic plants was not due to differential expression of biosynthetic genes. Conversely, the HU line showed higher contents of CCD1B and CCD4B transcripts, while in HC fruits the contents of NCED1, ACO1, ACS2, ACS4 gene transcript were higher at TR stage and the ABA content was higher (2-fold) at RR stage. PCA (Principal Component Analysis) on single datasets clearly showed that RT-qPCR and HPLC variables have similar discriminative power because both clustered objects according to fruit stage and genotype. Metabolomics data were also highly predictive of the stage of maturation since fruit samples collected at the same stage tended to cluster together irrespective of genotype.
(iii) Ketocarotenoid producing potato lines (P1 and P4)- Metabolomic and transcriptomic analysis was perfomed on potato varieties synthesising high levels of ketocarotenoids but with the exception of reduced starch content and altered flavonoids very few significant changes occurred.
1.5. Significant results, conclusions and recommendations
• WP2 illustrated the utility of an enabling technology platform for the characterisation of existing varieties and outputs from WP3 and 5 (objective 2.6).
• The multi-level omic based characterisation of the DET1 down regulated tomato varieties has revealed the close association of secondary metabolism with core metabolic processes. This would suggest that future systems based engineering approaches that do not solely affect the pathway of the interest should be explored (Enfissi et al., 2010. Plant cell, 22, 1190: Objectives 2.1 2.3 2.4 and 2.5).
• Network analysis performed on potato genotypes with elevated precursors illustrated how regulatory HUBs in the target pathway can change upon pathway engineering and that those components having the greatest quantitative changes are not always responsible for regulatory control within the pathway. (Diretto, et al., 2010, Plant Physiol., 154: Objective 2.1 2.4 and 2.5).
• The breakdown of network correlation strengthens between transcripts and metabolites compared to analysis between transcript and metabolite solely highlights the importance of post-transcriptional regulation. An area that has not been fully explored. (Objective 2.1 2.3 2.4 2.5 and 2.6).
• The engineering of non-endogenous products into the isoprenoid pathway to generate high levels had a surprising minimal impact on the global transcriptome and metabolome. In contrast engineering existing pathway components yield a plethora changes across the metabolome and transcriptome (objective 2.1 2.3 2.4 2.5 and 2.6).
• A model of isoprenoid biosynthesis has been created shows intuitive clustering of the isoprenoid classes on a biosynthetic basis with key common intermediates at the HUBs. The model also reveals that the Calvin/Benson Cycle is the progenitor of the formation and thus primary and secondary metabolism are linked. This suggests that modulating the fixation of carbon could also elevate secondary metabolites.
• The data and its metadata has been organised and as published deposited on the SGN website (www.Solgenomics.net) for the scientific community with a disclaimer describing its use.
1.6. Deviations and resources
There has been a problem keeping up with technology as the Affy microarray chip has now been surpassed and RNA-Seq is the method of choice. The latter approach is being finally used in the METAPRO. In addition the use of fast ion-trap MS platforms facilitated the use of label-free approaches.
1.7. Future work
Subsequent activities will be directed to characterising the transgenic lines produced in WP3 and 5 using transcriptomic and metabolomics analysis (objective 2.6.) as well as proteomic analysis. A key aspect will be the use of RNA-Seq approaches for candidate gene/allele discovery among biodiversity. The combined analysis of the transcriptome, proteome and metabolome are key to the understanding of cellular regulation.
2. Work package 3. Optimising the cellular storage of carotenoids (WP leader P5, HUJ)
2.1. WP3 Objectives
O3.1. Exploitation of natural variation to identify traits associated with altered plastid biogenesis.
O3.2. Elucidate if enhancing the plastid volume per cell increases carotenoid content by improving storage and/or synthesis.
O3.3. Identify further regulators of plastid biogenesis with the potential to optimise isoprenoid secondary metabolite production.
O3.4. Advance our understanding of metabolite induced plastid differentiation.
O3.5. Identify further regulators of plastid specialisation.
O3.6. Determine the role/essentially of concurrent lipid synthesis for the sequestration of carotenoids and other hydrophobic metabolites in the plastid.
O3.7. Identification of mechanisms and tools associated with carotenoid esterfication in plants as a means of storage.
O3.8. Characterisation of potato tuber plastid parameters and their role in isoprenoid/carotenoid synthesis and storage.
2.2. RTD activities performed in WP3.
Tomato population representing diverse natural variation have been screened for altered plastid biogenesis. This activity has been performed on one crop to date, using both molecular, biochemical and cellular based diagnostic assays. Genotypes have been identified with altered plastid biogenesis await confirmation in year two using crop2 (summer 2011). A large-scale re-screening of tomato mutant collection (http://zamir.sgn.cornell.edu/mutants/) was carried out in the field (Rehovot) in order to find new mutants with high-pigment phenotype. Several candidates were identified based on dark-green fruit phenotype during early development and higher lycopene concentration in ripe fruit. However, all of these mutants were found to be alleles of the known HIGH-PIGMENT 2 (hp2) locus. The screening revealed new alleles of tangerine and yellow-flesh as well as other pigmentation mutations. One of them displayed a typical phenotype of LUTESCENT, which is manifested by bleached fruit during the “green” stages of development and normal lycopene accumulation during ripening. The mutation was identified as LUTESCENT 2 and mapped on chromosome 10. This mutant was further characterized with the group of Dr. J Giovannoni (Cornell). Ppositional cloning revealed that the locus l2 encodes a chloroplast-targeted zinc metalloprotease of the M50 family that is homologous to the Arabidopsis gene ETHYLENE-DEPENDENT GRAVITROPISM DEFICIENT AND YELLOW-GREEN1. This study suggested a role for the chloroplast in mediating the onset of fruit ripening in tomato and indicated that chromoplast development in fruit does not depend on functional chloroplasts (Barry et al., 2012). In addition from the network biology approach used in WP2 P1 (& collaborators in Nottingham University) has identified a transcriptional activator that can alter plastid parameters so that there is a large plastid volume per cell. This has the effect of increasing isoprenoids/carotenoids.
In WP2 characterisation of varieties with increased plastid parameters has been performed. In addition WP3, has embarked upon the delivery of more genotypes with altered plastid biogenesis for increased cellular storage capacity. The direction used here in tomato is the manipulation of Abscisic acid (ABA) formation, in a fruit-specific manner building on P5’s earlier discovery of hp-3 and its increased plastid biogenesis derived from a defective zeaxanthin epoxidase and subsequent affects on ABA. In potato work is advanced in the generation of potato genotypes harbouring the Or gene, which converts amyloplasts to chromoplasts in potato tubers, e.g. the alteration of plastid specialisation. Further details of the later are provided in WP5.
Enhanced PSY protein levels in Arabidopsis results in strongly increased carotenoid amounts in non-green tissues such as callus and roots. Mainly beta-carotene accumulates in the form of crystals, similar to the curd of the cauliflower OR mutant. We performed western blot analysis using curd tissue from a white cauliflower cultivar and the OR mutant using antibodies directed against Arabidopsis PSY. Signal intensities were close to detection limit, but suggested no strikingly different PSY protein levels in both tissues. Roots and seed-derived callus were generated from Arabidopsis wild type (wt) and one 35S::AtPSY line. Curds from one white-colored cauliflower cultivar and the cauliflower OR mutant were used. Our antibody reactions and yeast two hydrid data suggest that PSY-1 and OR interact.
Molecular tools to enhance total carotenoids and potentially other plastid metabolites in tomato fruit through increasing the plastid number or plastid compartment size in fruit cells, has utilised the knowledge that in tomato and Arabidopsis that reduced levels of abscisic acid (ABA) lead to increase in the plastid compartment size. Since ABA plays crucial roles in stress physiology, mutations the abrogate ABA or manipulations of ABA synthesis and signal transduction decrease the adaptation of plants to stresses and thus reduce yield. To enhance carotenoids in fruit while avoiding deleterious effects of ABA deficiency on the whole plant, we created transgenic tomato plants in which the ABA-activated SnRK2 Protein Kinase family was suppressed specifically in fruits. To this end, a fruit-specific promoter, PPC2, was used with RNA interference (RNAi) of the gene SnRK2. The transcript levels of the SnRK2C and SnRK2.4 in the transgenic fruit were down-regulated to 5%-20% of control fruit. The number of plastids per cell was determined in green fruit using confocal microscopy and found to be higher in the transgenic plants. The reduction in the signal transduction of ABA led to an increase in the amount of fruit carotenoids by 30%-60%, while leaving the carotenoid composition unchanged, i.e. the main species was lycopene. Physiological experiments in the greenhouse indicated that drought tolerance was not compromised in the transgenic plants. However, no field test has been conducted. These results confirm the previous hypothesis that ABA is involved in the regulation of plastid number in the cell. Moreover, since no ABA deficiency phenotype has been observed at the whole plant level, the results suggest a novel approach to obtain high-pigment fruit phenotype by means of genetic engineering.
To confirm the important role of ABA in regulation of fruit ripening, the slZep (zeaxanthin epoxidase) gene was suppressed in tomato plants by transformation with a RNA interference (RNAi) construct, driven by the fruit-specific PPC2 promoter. Fruit of all RNAi lines displayed deep red coloration and increased lycopene levels compared to control fruit. However, silencing of the tomato Zep gene also damaged seed production. These results demonstrate the possibility to increase carotenoids in fruit through manipulation of the signal transduction of ABA in a fruit-specific manner.
Further work have been carried out by P1 and P6 showing how the enhancement of certain metabolites can alter plastid type and internal plastid structures. For example P6 has demonstrated crystal formation as a result of carotenoid hyperproduction while P1 has shown chromoplast induction upon the high-level production of the ketocarotenoid demonstration molecules. Comparison between subcompartments of the fruit chromoplasts from control and transgenic lines with increased beta-carotene lines showed a number of important differences. Firstly, an increased number of beta-carotene and lycopene crystal-like structures arose in the thylakoid-like membrane fractions of the high beta carotene line. It seems that this sequestration mechanism has been upregulated in the transgenic lines containing increased carotenoids.
Secondly, the membranes of the chromoplast (envelope and thylakoid-like membrane) also appeared to play an important role. The inner envelope of the high beta carotene chromoplasts seemed to be actively producing vesicles (membranous sacs), which were visible by electron micrographs of the high beta carotene line chromoplasts. The thylakoid-like membranes appeared in greater quantity and electron density in the high beta carotene chromoplasts compared to those in AC. The darker and thicker membranes could be caused by a high number of lipids, proteins and carotenoids or complexes of these components. The phytoene synthase and desaturase introduced by transgenesis were found in the same submembrane compartment, which strengthens the hypothesis that they can interact with each other and have a synergistic effect on the carotenoid production. However, the endogenous phytoene synthase (PSY-1) was mainly found in the stroma, whereas its product phytoene was predominantly found in the membranes and in the plastoglobules. The location of PSY-1 within the chromoplasts confirmed results found in the literature. The presence of phytoene in plastoglobules means that a significant quantity of the substrate for phytoene desaturase (and carotene formation) is partitioned away from the enzyme, possibly causing it to be metabolically inert, unless it is re-incorporated into the membrane enzyme complex. Collectively, the data illustrate that synthesis and sequestration are two processes
which are important for engineering carotenoid in plants, with the latter requiring further investigation.
P4 used an alternative approach to address the cellular location of synthesis versus accumulation. Excellent progress has been made in generating transgenic potato lines with biosynthetic enzymes phytoene synthase (PSY) and beta-carotene hdroxylase (BCH) possessing GFP and RFP tags for visualisation using microscopy. The resulting data to date shows that phytoene synthase and beta carotene hydroxylase are localised to the plastid but appear to reside in different structures within the plastid. a manuscript has been submitted (Pasare et al., 2012, J.Exp. Bot.) for publication.
In order to demonstrate the effects of concurrent lipid formation with carotenoids, transplastomic gentotypes amplifying the AccD component of the ACC fatty acid biosynthetic complex are at an advanced stage. P2 has generated Transplastomic tomato plants overexpressing the key (plastid-encoded) enzyme of fatty acid biosynthesis were generated. These lines have been purified to homoplasmy and their fruits were analysed with respect to total carotenoid accumulation. Preliminary data indicate a 10-20% increase in carotenoid accumulation suggesting that indeed, fatty acid and lipid biosynthesis limit carotenoid sequestration. Thus, fatty acid biosynthesis and lipid biosynthesis represent important target pathways for future metabolic engineering efforts that are directed towards increasing the carotenoid content of crop plants.
During the first reporting period P4 reported putative candidate genes for acyltransferase(s) capable of forming carotenoid esters. Unfortunately these gene products appear not to be the underlying candidates and the mechanism of these high carotenoid QTLs still awaits elucidation. However, microarray experiments were carried out on samples from a segregating diploid population bulked according to tuber carotenoid content and individual carotenoid components. A high proportion (47 out of 155) of the differentially expressed genes mapped directly under major QTL for tuber carotenoid content with 13 mapping to the novel chromosome 9 QTL for lutein content which is the predominant carotenoid in designated tubers. These genes became new candidates for the control of tuber carotenoid content.
2.3. Significant results, conclusions and recommendations
• Candidate genes for plastid biogenesis and carotenoid sequestration have been identified from the association of mQTL and eQTL (objective 3.1 3.3 and 3.7).
• Genetic intervention resources are being generated to ascertain how ABA acts as the progenitor of plastid biogenesis (objective 3.2)
• Sequestration mechanisms associated with depositing non-endogenous high value molecules have been revealed (objective 3.6 and 3.7).
• Synthesis of carotenoids and their deposition are not necessarily co-localised (Objective 3.8).
• Changes in metabolite composition can influence and alter cellular structures, especially at the sub plastid level (objective 3.4).
• The HFP gene has been identified, isolated and characterised as a tool to increase plastid biogenesis and core metabolism in tomato fruit. An additional homolog has also been identified (objective 3.1 3.3 and 3.7).
• Cell biology and biochemical (proteomics) have shown that the synthesis of carotenoids and their deposition are not necessarily co-localised (objective 3.2 and 3.8). This has led to the elucidation of new regulator mechanisms
2.4. Deviations and resources
No major deviations have occurred; the introduction of proteomics approaches has greatly helped in understanding the mechanisms and assigning putative functions to unknown gene products.
2.5. Future work
The resources developed for improved storage (WP3) will be combined with outputs from WP5 (objective 3.1 3.2 3.3 and 3.6). The mechanisms of carotenoid sequestration and metabolite induced plastid differentiation require further investigation further (objective 3.5 and 3.8). A greater understanding of the plastid and how their internal structures form with changes in metabolites is an area of important fundamental interest that impacts directly on the application of metabolic engineering and Synthetic Biology.
3. Work package 4. Understanding mechanisms of carotenoid degradation for (i) improved stability properties and (ii) the biosynthesis and transport of high-value cleavage products.
3.1. WP4 objectives.
O4.1. Identification and characterisation of genes and their products involved in the catabolism of carotenoids.
O4.2. Development of methods to monitor non-enzymatic degradation of carotenoids.
O4.3. Determine the relative contributions of catabolism and non-enzymatic processes to carotenoid degradation in planta and during bioprocessing.
O4.4. Functional characterisation of the biosynthetic and transport components involved in the formation of crocetin and its derivatives.
O4.5. Develop robust, efficient, high yielding extraction and purification procedures for ketocarotenoids and crocetin derivatives from tomato and potato.
O4.6. Assessment of “greener” chemistries extraction procedures for the targeted isoprenoids.
O4.7. Preparation of a ketocarotenoid “meal” from plant tissues and its formulation into aquaculture feed.
O4.8. Assess the physico-chemical properties of crocetin derivatives and ketocarotenoids produced in engineered systems with those extracted from natural sources and produced by chemical synthesis.
3.2. RTD activities performed in WP4
The role of carotenoid cleavage dioxygenases in the breakdown of carotenoid/isoprenoid pigments has been demonstrated, (i) in vitro, (ii) in transgenic plants and (iii) in vivo have been investigated. For example transgenic potato genotypes in which the CCD4 enzyme has been down-regulated contained elevated carotenoid levels P4. The potato CAROTENOID CLEAVAGE DIOXYGENASE 4 (CCD4) gene was identified as a candidate gene for regulation of tuber carotenoid content based on its expression profile in white and yellow tuber-fleshed potato cultivars, revealed by transcriptomic analysis.
Using an RNAi approach, down-regulation in transgenic tubers resulted in an increased carotenoid content, two- to five-fold higher than in control plants (Campbell et al., 2010. Plant Physiol., 154, 656). Tubers from 4 CCD4 RNAi lines were provided to P2 for more detailed elucidation of the CCD4 substrate carotenoid. The CCD4 construct was also provided to partners 1 and 5 for transformation other Solanaceous species. A second full length potato CCD gene, CCD8, was cloned, a fragment transferred into an RNAi vector and transformed in Desiree. A total of 20 independent transgenic lines were generated and grown in the glasshouse.
Following expression and purification in recombinant hosts such as E.coli the activity of these CCD’s appears to be very promiscuous and they can act on a variety of carotenoids (and carotenoid-like) precursors. The activity of both CCD1 from tomato (Solanum lycopersicum; SlCCD1A and SlCCD1B) was investigated to gain insight into their potentially different cleavage pattern and their capability of producing volatile isoprenoids correlating with carotenoid content and pattern of the fruits. A role of CCD1 enzymes, particularly of CCD1B, in producing carotenoid-derived volatiles in tomato fruits is indicated by the high abundance of the corresponding transcripts in this tissue. The cDNAs encoding SlCCD1A and SlCCD1B were cloned and inserted into inducible protein expression plasmids pBAD-THIO and pGEX), which enable the investigation of the enzymatic activity in vivo and in vitro. In vivo assays were then performed using engineered carotenoid accumulating E. coli strains, and in vitro assays with heterologously expressed, and affinity chromatography purified protein obtained using the pGEX-epression system.
Our results confirm literature data showing that both CCD1 enzymes catalyze the cleavage of the C9-C10/C9´-C10´ double bond in various bicyclic substrates and also the C5-C6 double bond of lycopene. In addition, our work revealed the C7-C8 double bond as a new cleavage sites in lycopene and apolycopenals giving rise to the formation of geranial and neral, two major tomato volatiles of hitherto unknown origin. This activity resembles that of the rice CCD1. P6 also demonstrated that the two tomato enzymes, particularly SlCCD1B, cleave the C13-C14 and the C11-C12 double bonds, two novel cleavage sites. The cleavage of the C13-C14 double bond leads in case of phytoene and phytofluene to farnesyl acetone, an additional tomato volatile of unknown origin. The cleavage of the C11-C12 site was only observed with 9-cis-β-carotene yielding -apo-13-carotenone, a compound previously shown to be biologically active affecting root hair growth by interfering with auxin transport.
The in vitro results generated by P6 indicate that almost all of tomato volatiles correlating in their abundance with the carotenoid content can be produced by SlCCD1A and/or CCD1B. To confirm this activity in planta, we made binary vectors carrying the corresponding cDNA either in sense, anti-sense orientation or equipped with a sequence encoding a plastid transit peptide. These constructs were then delivered to Partner 1 who performed transient transformation of tomato fruits. However, we did not observe any alteration in carotenoid pattern or content. However, P4 did successfully generated antisense CCD4 transgenic plants, which revealed increased lycopene contents.
P3 performed deep sequencing and transcriptomic approaches, to acquire ESTs from saffron stigmas have been carried out. Bioinformatic interrogation of these sequences has revealed putative biosynthetic enzymes involved in crocetin synthesis, and modifying enzymes involved in sequestration and transporters associated with the accumulation into the vacuole of the cell. Characterisation of these candidates has been the focus, transcriptomic along with complementation in E.coli and transgenic plants have been used as approaches. The transgenic plants expressing the crocus zeaxanthin cleavage dioxygenase has yielded a detectable cleavage product from zeaxanthin but better characterisation is required. It is also apparent that some of the data published are inconsistent with the findings and unambiguous characterisation would be helpful. Several candidate crocin biosynthetic genes (7 CCDs, 7 AlDHs, 2 Glucosyltransferases, 8 ABC and MATE transporters have been cloned in appropriate expression vectors. All CCDs have been transformed in tomato plants and in E. coli. The biochemical characterisation of CCDs in collaboration with P6 has revealed a lack of 7,8,7’,8’ cleavage activity for the ZCD gene published previously (Bouvier et al. Plant Cell 15: 47-62) and the presence of such activity in a different CCDs.
In addition to enzymatic degradation non-enzymatic degradation of pigments was studied. Analytical methods have been developed to identify products potentially derived from the non-enzymatic degradation of carotenoids. Further characterisation is underway and required to fully elucidate products and underlying mechanism involved in their formation. It is clear however that in dry tissues that non-enzymatic degradation is a crucial factor. The degradation would appear to arise from radicals generated through lipid perioxidation. The initiator of the radicals is likely to be the action of super oxide dismutase (SOD) without the coordinated action of catalase.
The non-enzymatic degradation of pigments in drying tissues has had important implications to down-stream processing. P8 has tested the stability of a ketocarotenoid containing “meal” from tomato fruit and potato tuber material evaluated in different conditions of temperature, humidity, light. The ability of these rich-ketocarotenoid powders to be incorporated into feed for aquaculture was evaluated, by mixing it with vegetable-based meal (commercial fish pelleted meal) and their stability was tested. Physico-chemical properties for ketocarotenoids derivatives prepared from the GM material developed. As a consequence of these studies P8 has developed a down-stream processing approach that eliminates the use of organic solvents, enriching carotenoid complexes instead. The extracts have also been encapsulated for stability with non-toxic polymers and release into aqueous solution, this work has represented a major breakthrough and delivered a means of stabilising the pigments.
3.3. Significant results, conclusions and recommendations
• The down-regulation of the carotenoid cleavage dioxygenase (ccd4) increased carotenoid content in potato tubers and tomato fruit. The carotenoids increased are precursors for ketocarotenoid and crocin formation (objective 4.1).
• EST sequence analysis and transcriptomics of saffron stigma has identified crocin biosynthetic, sequestration and accumulation (objective 4.4).
• It appears that the enzymatic cleavage of ketocarotenoids can occur in vegetative tissues.
• The contribution by non-enzymatic mechanisms to the breakdown of carotenoids and ketocarotenoids is a major contributor in dried tissues. This finding has major implications for cereal crops such as rice, maize and wheat (objective 4.1 4.2 and 4.3).
• Procedures have been developed that facilitate “green” extraction without solvents, an encapsulation method that enables stability as well as chemical and physical compound integrity (objective 4.5 4.6 4.7 and 4.8).
3.4. Deviations and resources
No major deviations have occurred.
4. Work package 5. The development of tools and implementation of strategies for the efficient engineering of useful isoprenoids.
4.1. WP5 objectives.
O5.1. Optimisation of the tomato plastid transformation system.
O5.2. Suite of novel plastid expression cassettes conferring improved biosafety characteristics.
O5.3. Extend the range of valuable tomato and potato varieties amenable to routine efficient transformation.
O5.4. Generation of transformation vectors enabling the simultaneous introduction of multiple cDNAs for mini-pathway engineering.
O5.5. Transplastomic tomato plants producing ketocarotenoids.
O5.6. Illustrate the potential of translational optimised biosynthetic genes in optimising secondary metabolite levels.
O5.7. Demonstrate how, in combination, pathway engineering and improved storage properties can increase the yields of useful isoprenoids (ketocarotenoids).
O5.8. Show the potential of reducing carotenoid catabolism as a means of increases ketocarotenoid yield in defined plant tissues.
4.2. RTD activities performed in WP5
The tomato plastid transformation system remains labour intensive but it has been improved especially with respect to its robustness and diversity of varieties that can be transformed (objectives 5.1 and 5.2). During the METAPRO project P2 has improved of plastid transformation technology for tomato and the identification of highly regenerable green-fruited commercial tomato varieties. Plastid transformation for two green-fruited commercial tomato varieties was accomplished (published in PNAS 2013). In addition, varieties amenable to conventional transformation have been developed to include the high pigment (hp) mutants where it was found necessary to alter the phytohormone complement, especially a reduction in auxin supplementation (objectives 5.2).
To aid the engineering of pathways a collection of vectors for plastid (Ruf and Bock, 2011. Methods Mol. Biol. In press) and conventional transformation had been modified and developed (objective 5.4). Building up on the vector construction work and plastid transformation experiments performed resulted in a breakthrough with achieving high-level transgene expression in non-green plastids. By combining promoters of plastid genes that show high mRNA accumulation in non-leafy tissues with 5’ untranslated regions (harbouring the cis-elements for translational regulation) that are active in non-photosynthetic tissues, chimeric expression elements could be identified that confer high-level expression in root amyloplasts and fruit chromoplasts (published in two papers in Plant Journal in 2012 and 2013). These novel expression elements pave the way to efficient metabolic pathway engineering in non-green storage organs, such as potato tubers and tomato fruits.
Strategies for the translational enhancement of gene products have been developed. Firstly P6 has identified inhibitory regions in the untranslated regions (UTR’s) of carotenoid biosynthetic genes. Removal of these regions confers the high level production of active protein. Using resources provided by the projects international advisor (Prof. Misawa) transferable sequences for enhanced translational have also been incorporated upstream to target gene sequence.
In order to investigate the translation-inhibitory function of the 5’UTR of Arabidopsis PSY in more details, two systems were developed:
(i) Transient expression of reporter genes in tobacco leaves. We intended a rapid and reliable analysis of 5’UTR sequence motifs involved in the regulation. For this we combined 5’UTR-controlled translation of GUS with an internal ribosome entry side (IRES)-controlled translation of luciferase in bicistronic transcripts. Although it was shown that IRES-driven luciferase levels can be used to determine levels of bicistronic transcripts, quantification of 5’UTR-controlled GUS levels revealed that GUS protein behaved non-linear compared with transgene expression. This suggested a limitation of the translation capacity. The system was unsuitable for the intended studies.
(ii) Stable expression of 35S::5’UTR-GUS. For investigations of the regulatory function of the AtPSY 5’UTR, we generated Arabidopsis lines overexpressing 403 bp, 330 bp and 280 bp of AtPSY 5’UTR, respectively, followed by GUS as reporter gene.RNA electrophoretic mobility shift assays (REMSAs) were established to identify putative cytoplasmic AtPSY 5’UTR-binding proteins. Results indicated the formation of specific protein-RNA complexes with cytoplasmatic proteins, subfractionated by ion exchange chromatography. Affinity columns were constructed to allow the identification of the bound proteins.
Ratios between GUS to transgene transcript were determined for callus and leaves of homozygous 35S::5’UTR-GUS lines with 403, 330 and 280 nt of 5’UTR. In comparison with lines carrying the corresponding constructs with AtPSY, GUS lines showed only a weak translation inhibition in leaves. One essential difference between 5UTR-GUS and 5’UTR-AtPSY lines is an increased carotenoid flux in the latter. Therefore, the different responses are considered as indication for an involvement of carotenoid degradation products in 5’UTR-mediated translation inhibition. Carotenoid-derived signalling compounds in leaves are known while pronounced carotenoid degradation in callus was previously demonstrated. The GUS lines generated will be further used to investigate this effect in details. Affinity columns were constructed which contained the bound 5´-UTR and which is based on the strptomycin aptamer. Cytoplasmic proteins, pre-purified by FPLC (see above) were applied and the resulting eluted protein bands subjected to Mass-Spectrometric identification. So far, no promising candidates were obtained.
The production of ketocarotenoids in plant tissues was a key objective of the METAPRO project. Plastid transformation was one approach used. The initial work was the demonstration that efficient engineering of the carotenoid pathway in tomato fruits can be achieved by expression of biosynthetic enzymes from the plastid genome. The work, published in Plant Physiology, showed that expression of a plant lycopene cyclase gene resulted in conversion of lycopene into provitamin A (β-carotene) and, at the same time, led to a substantial increase in total carotenoid content. Furthermore, the principles of synthetic operon design for efficient multigene engineering in plastids were worked out using the tocopherol metabolic pathway as an example. Application of these design principles to astaxanthin biosynthesis (combining a lycopene cylase with a ketolase and a hydroxylase in a three-cistron operon) induced high-level astaxanthin production in transplastomic tobacco plants. ketocarotenoids represents 90% of the total carotenoid have been generated. This resource technology has now been transferred to tomato.The work on synthetic operon design and its application to engineering of the tocopherol pathway was completed by transferring the optimized operon into the two green-fruited tomato varieties for which plastid transformation protocols had been developed. The astaxanthin operon was transferred into tomato plastids and triggered even higher astaxanthin synthesis in tomato than it did in tobacco (for unknown reasons). While the astaxanthin-producing transplastomic tobacco plants grow autotrophically in soil, the transplastomic tomato plants expressing the same operon require synthetic medium to grow, due to extreme accumulation levels of astaxanthin.
In addition to plastid transformation, conventional nuclear based transformation has been used to deliver high-level ketocarotenoid production (1 to 5 % dry weight) has been achieved in tobacco tree, tomato and potato. Here transcriptional and translational enhanced gene(s)/products have been used. P1 developed to primary lines containing the astaxanthin biosynthetic operon (crtZ and W from the marine bacterium Brevundimonas) under 35S CaMV constitutive control with translation enhancers. The evaluation of progeny was performed in two sites Italy (P7) and London (P1). In addition P7 and P1 created genetic crosses high-precursor lines. The screening of the transgenic segregating progeny was firstly based on the analysis of the phenotype of the most important vegetative and reproductive organs (cotyledons, leaves, steams, flowers, and fruits) at several developmental stages. Secondly, the transgenic plants were characterised at molecular level through PCR and Southern analyses. Following this screening two plants, ZW10-6 and ZW13-8 were selected as the best parents and were crossed to lines HC (high beta-carotene) and UO (high xanthophyll) carrying, respectively, the transgene encoding the lycopene beta-cyclase (Lcyb) and the transgene encoding the carotene beta-hydroxylase 2 (CrtRb2). The two hybrids were named HZW and UZW. Carotenoid contents of fruits of the four tomato lines (RS, HC, UO, HU), harvested at four stages (IG, MG, TR, RR), clearly showed that the overexpression of the biosynthetic gene encoded by the transgene was the determinant of the patterns of carotenoid biosynthesis and accumulation in tomato fruit. In the HC fruits, almost all lycopene was converted to -carotene while a consistent amount of xanthophylls, mainly violaxanthin, was present in fruits of plant carrying the CrtRB2 transgene, particularly in HU fruits. Two transgenic ZW plants, ZW10-6 and ZW13-8, were chosen as the best parents since they were shown to carry a single insertion of the transgene CrtZ/CrtW whose expression resulted in the synthesis of ketocarotenoids in all analysed tissues. In particular, leaves were brown while the flowers were deep red. Fruits were pink during the IG and MG stages while later turned red. However, the synthesis of ketocarotenoids was higher in hemizygous plants than in homozygous plants where the transgene appeared to be silenced. Homozygous plants showed, in fact, normal flowers and fruits while the leaves were with brown veins and green inter-vein sectors. The double hybrids (UZW, HZW) and the ZWn parent lines were grown in glasshouse and the carotenoid contents of leaves, flowers and fruits were determined by HPLC. UZW double hybrid plants were crossed to homozygous HC line to combine in a single line all three transgenes (Lcyb, CrtRb2, CrtZ/W). The derived triple hybrid line was named HUZW. These populations have been grown over several growth cycles to judge inheritability of the phenotype. One during the summer of 2012, a number of fruit samples of the two most interesting hybrids, HZW and HUZW, as well as of the control ZW13-8 line were collected and sent to partner P8 (Proplanta) for the production of microcapsules. Moreover, a forth breeding nursery for the production of seeds to be used for possible future activities aimed at the exploitation of the final products of METAPRO project. In this respect, the best set of parental line would be the one: including the lower number of lines to be maintained; requiring the lower number of crosses to produce the best performing lines, and; that do not necessitate the screening of the progeny, either visual or molecular.
Analysis of the HZW and HUZW ripe fruits contained 2.2- and 1.7-fold more carotenoids than the fruits of ZW line and UZW hybrid. Most of carotenoids in the former fruits were ketocarotenoids while in the latter fruits the lycopene was the most represented carotenoid. More precisely, ketocarotenoid content in HZW and HUZW fruits accounted for the 88% and the 96%, respectively, of all carotenoids of the fruits while in the parent ZW line the ketocarotenoids were less than the 8%. For these reasons, the HZW and HUZW ripe fruits contained, respectively, 24- and 20-times more ketocarotenoids than the ZW parent line. Most of the ketocarotenoids in HZW and HUZW fruits were present in the form of esters. Roughly, the 52% and the 76% of all ketocarotenoids were present as esters in HZW and HUZW fruit extracts, respectively. As expected, in the chromatograms of HUZW fruit extracts both mono- and di-esters were present while the mono-esters were the most important fraction of esters detectable in the chromatograms of the HZW fruit extracts. This finding represents a clear proof that the esterification process in the tomato fruit chromoplast is in no way a limitation to the accumulation of high amounts of xanthophylls or other hydroxylated compounds. A de-esterification treatment was applied to fruit extracts of HZW and HUZW lines before the HPLC analysis in order to estimate the level of hydroxylation of ketocarotenoids. Results of this analysis showed that in HUZW fruits the esters were made of astaxanthin for the 56% and of adonixanthin and canthaxanthin for less than 20%. Conversely, in HZW fruits less than 20% of esters were made of astaxanthin while adonixanthin and canthaxanthin accounted for almost the 73 % of all esters. P1 has obtained MS data for all the ketocarotenoids formed. In addition to the crosses performed by P7, P1 has introgressed the astaxanthin biosynthetic operon into a high--carotene background. The IL line has exceptionally high -carotene content 100 fold more than the wild type and altered cellular structures (dense plastoglobuli) to accumulate carotenoids. In addition this variety of tomato is heat and drought tolerant and capable of growing on marginal soils. P1 has also carried out transcript analysis over ripening and found a delay in the expression of the carotenoid transcripts. Improved fruit shelf-life has also been observed.
All the candidate biosynthetic enzymes and putative transporters identified in WP3 for crocin biosynthesis have been transformed into tomato by P3 some of the products await elucidation.
The generation of transgenic plants with combined traits such as plastid capacity, plastid specialization has also been pefomed. Transgenic potato plants expressing the β-carotene and ketolase genes isolated from the marine bacterium Brevundimonas sp. SD212, obtained from partner 1, were transformed into the cultivar Desiree. P1 has then developed these plants over multiple generations and analysed the pigments and the metabolome. Transcriptomic analysis using the potato microarray was carried outby P4. Additional transformations were carried out in the high tuber carotenoid cultivar Mayan Gold and 01H15 57 which contains high levels of the astaxanthin precursor, zeaxanthin. Putative transgenic lines were then re-transformed with the cauliflower mutant Or gene to improve the carotenoid storage capacity. A number of transgenic lines expressing either the constitutive crtZW construct or tuber targeted mutant cauliflower Or gene were successfully produced by Agrobacterium mediated transformation. Preliminary analysis of carotenoids present in various tissues (in collaboration with partner 1) showed Mayan Gold based crtZW lines accumulated both keto-lutein and astaxanthin, whereas lines based on the high zeaxanthin line, 01H15, accumulated almost entirely astaxanthin. Lines expressing only the cauliflower Or mutant accumulated over 75% more tuber carotenoids compared to WT. The best transgenic lines expressing only the crtZW construct were then re-transformed with the Or construct to produce co-transformant lines.
In tomato P1 has crossed and transformed the high plastid lines with the astaxanthin producers as a result a synergistic effect on the carotenoid content has been observed.
The METAPRO project has been successful and prototype genotype (summarised below) have been generated that produce high levels of ketocarotenoids. These can be utilised in feasibility/translational studies. However a number of unforeseen scientific circumstances have occurred that were unpredictable during the conception of the description of work (DOW). Predominantly, these reside from the high ketocarotenoid contents in vegetative, fruit and tuber material approaching 10 % DW. As a result the vigour of the plants and development has been affected this can in some cases delay the development of the plant and its sink tissues by up six months. We presume this is due to alterations in ABA content. Alternatively, the effects can be lethal in some cases. The latter is particularly evident in the case of plastid transformation. These initial effects have delayed the production and development of plants whereby the traits for the optimised biosynthetic pathway has been combined with augmenting traits such as increased plastid related sequestration, a supportive lipid matrix and precursor supply. For example Transformation of potato genotypes particularly the Solanum phureja genotypes Mayan Gold and O1H15 is considerably slower than for standard test genotypes (Solanum tuberosum cv Desiree) taking 5 months rather than 3 months to regenerate plants. Additionally transformation with the CrtZW construct (that confers high tuber ketocarotenoid content) appears to cause delayed tuberisation (by up to 2 months). Thus generation of some transgenic tubers is taking 4-6 months longer than anticipated.
4.3. Significant results, conclusions and recommendations
• Improved plastid transformation procedures (objective 5.1).
• Extending transformation protocols to varieties with enhanced traits (objective 5.3).
• Utilisable vectors for improved mini-pathway production (objective 5.2)
• The production of very high level ketocarotenoids in tobacco, tomato (leaf and fruit) and potato (leaf and tuber), (objective 5.4 5.5 and 5.6). Prototypes available for feasibility studies are:
(i) Nicotiana glauca- High ketocarotenoid containing leaves (0.5% DW) and high ketocarotenoid flowers (10% DW).This renewable source is a nicotine free variety, but enrichment and purification will be required prior to application (P1).
(ii) Transplastomic tobacco lines- High astaxanthin (1 to 5% DW) leaf material purification will be required prior to use. Grafting needs to be assessed to reduce nicotine content (P2).
(iii) High zeaxanthin potatoes- varieties delivering high levels of zeaxanthin in their tubers, this material could be used as an addmix or purification (P4).
(iv) High astaxanthin potatoes- varieties delivering high levels of astaxanthin in their tubers, this material could be used as an addmix or purification (P4 &1).
(v) High canthaxanthin tomato fruit- Due to altered expression levels in a background with high beta carotene, canthaxanthin is the predominant product (P1 and 7).
(vi) High ketocarotenoid tomato fruit- This variety has a range of rare and valuable ketocarotenoids (P1 and 7).
(vii) High lycopene tomato fruit- The presence of CrtZ/W at a low expression level results in the metabolism of beta-carotene. This removes the feedback inhibition in the pathway and lycopene is over produced to a level of 15 to 20 mg/gDW (P1).
(viii) High phytoene and phytofluene tomato fruit- While engineering precursors for the ketaocarotenoid pathway P5 revealed a strategy to produce very high phytoene/phytofluene containing fruit, with the need for GM approaches.
(ix) High beta carotene tomato fruit- Natural varieties producing high levels of beta carotene have been identified and developed.
(x) High 4-ketozeaxanthin tomato fruit- Varieties producing the intermediate 4-ketozeaxanthin have been generated.
(xi) High astaxanthin tomato fruit- Fruit containing up to 25mg/gDW Astaxanthin in a mono-esterified form have been generated and the phenotype stable.
4.4. Deviations and resources
No major deviations or changes to resources beyond everyday practical and logistical resources have occurred.
4.5. Future work
The prototype tomato, tobacco and potato varieties are available producing high levels of canthaxanthin, astaxanthin, beta carotene and phytoene. It is now important to carryout production, technical and economic feasibility studies.
Although adequate prototypes are in place the pleiotropic effects arising should not be ignored. One way round these effects would be inducible or very precise tissue specific promoters. This aspect is of particular pertinence to plastid transformation approaches.
5. Final considerations
The METAPRO project delivered vast knowledge of the optimisation of secondary metabolites in planta it is difficult to address the entire scope of the project. However, in summary the salient points are provided below for each WP executed.
WP2- The experimental approach illustrated the potential and necessity to use systems level characterisation. The data revealed the multiple levels of regulation that must be considered and exploited. Evidence was produced to illustrate that secondary metabolites are intrinsically linked to primary metabolism, or developmental processes, or environmental responses/conditions and engineering or Synthetic biology strategies should/must consider the system in general not just the targeted pathway.
WP3- Traditionally metabolic engineering and even marker assisted selection has focussed on biosynthetic capacity. The experimental data clearly illustrates that sequestration must be considered concurrently.
Our focus on the plastid organelle revealed new regulatory mechanisms, new sub-plastid structures and demonstrated the fundamental point that changes in metabolite composition can alter organelle and sub-organelle structures.
WP4- Newly synthesised and endogenous pigments are subject to enzymatic and non-enzymatic degradation. The non-enzymatic radical attack is potentially the most potent. This has important ramifications for cereal crops in developing countries. The METAPRO project has developed a procedure facilitating low environmental impact of processing and stability of product.
The other highlight delivered by WP4 is the elucidation of apocarotenoid formation in Saffron.
WP5- Implementing strategies associated with effector gene optimisation, translational enhancement, improved sequestration and elevated precursor levels unprecedented levels of ketocarotenoids have been generated in tobacco leaves, tomato fruit and potato tubers. This resource will enable us to answer fundamental aspects of metabolism and cell biology. In addition the varieties will enable progression to technical, production and economic feasibility studies on route to commercialisation.
6. The final partner reports are attached on-line. These reports provide clear visual representation of some of the data generated.
Potential Impact:
The outputs from the METAPRO project will impact directly on a number of key strategic areas including; improved sustainable bio-production and processing, enhanced quality of life, important socio-economic factors, the knowledge based economy, increased competitiveness and prosperity, international development as well as advancing fundamental science and the exploitation of knowledge. Examples are provided below. 1. Scientific advancements and the exploitation of knowledge. Important underpinning fundamental knowledge will be acquired from the project. This information will benefit the scientific community and contribute to the progression of science as well as further societal and commercial exploitation. The research will advance the present state of the art in the following areas: (i). Improved fundamental knowledge of plant metabolic pathways and their regulation for the generic production of useful secondary metabolites and other commodities. The isolation, identification and characterisation of the genes encoding the biosynthetic enzymes involved in Saffron derived apocarotenoids is an example of how METAPRO has improved our fundamental knowledge of biochemical pathways responsible for high-value phytochemicals. The utilisation of systems biology and parallel “omics” approaches in METAPRO advanced our fundamental knowledge of metabolism in general. It has become clear that primary and secondary metabolism is closely associated and systems engineering is necessary for optimised levels of secondary metabolites. (ii). An improved understanding of how secondary metabolites are stored in specific tissues within specialised organelles. The METAPRO project revealed that metabolite composition can alter or induce cellular structures this is a fundamental scientific advance with generic scientific implications that transcend the discipline. In addition new cellular based regulatory mechanisms were revealed and the finding that plastoglobules are not inert storage compartments is an important development. This sub-plastid structure requires further investigations. (iii). Advance our understanding of the mechanisms involved in catabolism and non-enzymatic degradation of isoprenoid derived secondary metabolites in the cell and during bioprocessing. METAPRO activities have illustrated the key role played by non-enzymatic degradation probably lipid peroxidation and how it can be overcome by using aqueous non-toxic enrichment and encapsulation. (iv). Deliver the tools and expertise necessary to engineer high value isoprenoids (e.g. crocetin derivatives and ketocarotenoids) into amenable plant based hosts. The characterisation of enzymatic processes for carotenoid catabolism has delivered a suite of new genes from Saffron derived apocarotenoid formation this represents a major scientific advancement. New vectors for plastid and conventional transformation, with transcriptional and translational enhancers have been generated and distributed in the scientific community. Clear effective metabolic engineering strategies have been developed and validated. (v). Demonstrate how efficient engineering strategies incorporating synergistic traits such as optimised storage and stability can complement pathway engineering to achieve the production of useful natural products at economically favourable quantities. The METAPRO project is one of the first examples were augmenting traits have been combine to deliver optimised yields. (vi). Development of new transfection technologies/procedures. Through activities in the METAPRO project tomato plastid transformation is now a routine laboratory practice and the numbers of transformable varieties have been extended. (vii). Rapid transient expression systems. Optimised transient transformation systems have been developed to assess new genes independently and in combination. (viii). Characterisation of promoters and other expression elements. Several new promoters predominantly for expression in tomato fruit have been characterised, while translation enhancers have been identified and developed., for both conventional and plastid transformation. (ix). Improve and extend the application of metabolomics and transcriptomics. During the METAPRO project the very latest developemnts in trranscriptomics and metabolomics were implemented into the work programme. The utility of RNA-Seq was demonstrated through the identification of transcripts associated with apocarotenoid formation in Saffron. Accurate mass metabolomics approaches have been developed and used to identify and assign putative formula to unknowns. Thus the METAPRO project has developed the use of these enabling technologies. (x). A repository of meta and experimental datasets relating to the optimisation or perturbation of metabolic pathways for the production of useful fine chemicals. Large datasets of high quality have been generated in the METAPRO project. These can and will be utilised by the scientific community in the future. To facilitate community used the data is deposited at www.phenome-netwroks.com and www.solgnomics.net. A dedicated database for Solanaceae has greatly benefited the data basing aspect of the work. (xi). Isolation of important rare authentic standards. The lack of available authentic standards is often a key limiting factor that hinders scientific research. METAPRO has been responsible for generating unique sources of carotenoids that have the potential to act as analytical standards and be used as new chemical entities in biological activity assays. (xii). Further demonstrate the potential of natural variation and extend genetic resources in Solanaceae. Several biodiversity agreements have been developed by METAPRO. Through these MTAs diversity collections have been screened for altered plastid related parameters and isoprenoids. In addition, new the genotypes have been generated within the project and will be made available to scientific community after capturing potential IPR thus adding to our existing genetic resources in Solanaceae. (xiii). Initiate the process of field evaluation. Advances in the METAPRO project now have resulting in the consortium submitting and gaining authorisation to grow prototypes in the environment, e.g. ISR and UK (under polytunnel conditions). (xv). Utilisation of potential semi-synthetic approaches. It is an increasing occurrence to utilise intermediates/products derived from plants as molecular scaffolds for synthesis of end-products or derivatives. The outputs from the project will provide further foundations for such approaches using improved renewable sources. (xvi). Create a supportive environment for sharing knowledge (know-how), information, expertise and innovation (Knowledge Transfer). METAPRO mobilised critical mass in the field not just through project specific activities but in association with the concurrent COST ACTIONs and projects e.g. FP7-TiMET. This aspect was of particular benefit to the SMEs giving them direct access to developments in fundamental research for example P8 benefited from the exchange of metabolomic expertise. The METAPRO project will clearly advance our fundamental scientific knowledge and highlight potential exploitation. 2. Improved sustainable bioproduction. The METAPRO project has illustrated how chemicals typically generated by chemical synthesise; using petrochemical by-products as precursors, with down-stream processing approaches that have adverse environmental impact or derived from low yielding plant/microbial sources not amenable to agricultural production, can potentially be produced in renewable biosources at competitive levels. Feasibility studies are now required to demonstrate this on a production scale. The technologies and resources generated by the proposed project are generic and can potentially be exploited to impact directly on creating sustainable renewable bio-resources for most high-value plant secondary metabolites. In the case of ketocarotenoids, such as astaxanthin which was one of the projects demonstration molecules there is a clear environmental and economic impact a more sustainable bioproduction platform. For example over the past decade the world’s fish stocks have been heavily depleted, with Salmon production declining to a level that is only just sustainable. To supply demand fish farming is increasing rapidly. However, astaxanthin/canthaxanthin like essential oils are an integral costly part of the aquaculture process, without which aquaculture will be unsustainable. The prototypes from the METAPRO project can now provide us with this opportunity to show the feasibility of these sustainable sources. The use of Solanaceae has added a biorefining aspect to the project as it is generally estimated that 20 -40% of the raw vegetable or fruit material ends up as waste during processing. In the METAPRO we have identified Solanesol as a high value compound that can generated from solanaceae waste to create added-value co-products. In summary the outputs of the METAPRO project offer a means of producing high value plant derived products using cheap renewable bio-sources that have a reduced impact on the environment. These are important factors that will impact directly on society in the coming years. As society moves from chemical to biorefining processes. 3. Increasing competitiveness and prosperity. The tools, resources, plant varieties and training generated and provided by the METAPRO project will contribute to ensuring European enterprises working in the field or related services are competitive. In this way the potential has been developed for European industry to compete with their US and Japan counterparts or licence technologies to these established and emerging global partners. It is hoped that the renewable and cheaper means of production developed in the MEATPRO project will facilitate expansion of existing, and the creation of new, markets and companies, increasing European prosperity. For example, the high-value demonstration molecules targeted in the project offer an opportunity to upgrade plant based raw materials to high value co-products. Such approaches offer an important alternative market for European agriculture in a period where subsidised production of bulk products is being reduced significantly. With the increased energy costs, environmental implications as well as the fossil fuel based production systems, the markets for high-value natural products like those targeted in the present project are likely to expand rapidly. In addition such products have a high export value. 4. Socio-economic impact. In 2050 it is estimated that there will be more elderly people than children in several parts of the world, particularly in Europe. Maintaining the health and well-being in an ageing population will have important economic implications on national health systems. Reducing the production costs of useful natural products such as those targeted in the METAPRO project would greatly reduce the costs of pharmaceuticals, nutraceuticals, cosmoceutricals and over the counter (OTC) medicines in which they are used. In addition, reduced costs will stimulate wider and more accessible use. The utilisation of renewable bio-resources will also enable the consumer to have a natural choice instead of a reliance on the synthetic market. In Europe the present chemical synthesis mode of production is often linked to the petro-chemical industry for the supply of precursors and thus prone to price fluctuations. The use of plant based production platforms would improve self-sustainability and dependence on volatile markets. Thus, through its objective of reducing production costs and improving sustainability the outputs from METAPRO will have direct economic implications in future years. The project has illustrated how the chemical industry can be replaced by reliable and competitive biosources. 5. Training. The impact EU networks have on training is often overlooked and credit for this achievement not promoted to the level it deserves. The METAPRO project has provided an unprecedented opportunity for the training of EU based scientists to the very highest standards. Over the course of the project over five postgraduate students have been trained, six targeted training missions have been carried out and metabolite training school run which attracted 140 applicants for 14 places. To highlight the PhD students trained students from France and Romania were trained in the UK acquiring University of London degrees, this unprecedented opportunity could not have been achieved without the METPRO activities. The process will create a workforce with high technical expertise and leadership that can be utilised in European scientific industries and related businesses. A workforce with these skills will be essential to achieve the EC objective to build the world’s most competitive knowledge-based economy. METAPRO has contributed to the EC objective to build the world’s most competitive knowledge-based economy. 6. Rural development. The METAPRO has provided opportunities for rural development (e.g. Romania, southern Italy and north east Scotland). Expertise developed has in each case remained local. This approach is in line with the policy of involving rural areas into the globalisation process by ensuring an active economic role. 7. Improved consumer quality and safety. The METAPRO project has delivered the tools and resource necessary to provide the consumer with a choice of a natural product over a synthetic product derived from the chemical industry. There is also potential for a wider consumer choice, greater availability and cheaper costs. In additional to the improved quality the increasing evidence of health benefits conferred by crocetin and ketocarotenoids will lead to incorporation of these metabolites into a wider range of products. This will impact both on quality and potential health benefits which have direct implications on the health of the nation, well-being and prosperity. The utilisation of bio-resources instead of chemical synthesis means safety aspects are generally improved. There is less chance of chemical contamination so the working environment is improved for employees and the process platform will have less impact on the environment. Feasibility studies will enable us to acquire precise data for the evaluation of these potential beneficial impacts to human and animal health, as well as conferring quality to food and feedstuffs. 8. Industrial policy and employment. The scientific and technological advances will potentially impact on European competitiveness creating new and increased markets leading to increased economic growth and job creation. Industrial partners including SMEs are actively involved in the programme fostering industrial cooperation and capacity building at a European level. To date the METAPRO project has created four permanent jobs in the public sector and 15 fixed term employment opportunities. 9. European cohesion. The METAPRO project has through the transfer of technology and joint efforts of selected experts with complementary skills and industrial cooperation created European cohesion and capacity building at a European level. In addition further global links have been established which has resulted in METAPRO activities contributing to capability building in developing countries. 10. Contribution to policy developments. METAPRO partners have presented and contributed data to National agencies and the EC. In addition the coordinator has been involved in EU/US taskforce activities associated with Plant Biotechnology-added value crops. These events contribute to policy especially in the case of reducing chemical refining and the promoting of biorefining procedures. 11. Ethical issues. Participants in the METAPRO have at all times complied with current legislation and regulations in the countries where the research has been carried out. We have performed ethical reviews on the use of GM periodically throughout the project. With the generation of plant varieties with such high ketocarotenoid content and the potential they offer the conclusion is that it is unethical not to pursue their utilization. 12. Contributions to standards. METAPRO implemented procedures for to ensure accuracy, traceability and reliability of the data and procedures, to health and safety standards, and to staff development and training. The consortium worked to community standards relating to the reporting of “omic” datasets. The consortium has and will continue to engage in data sharing embracing the policies of National funding agencies. Data management has been addressed throughout the project. Metabolomic experiments have been performed in a MIAMET (Minimum Information About a Metabolomic Experiment) compliant manner, while transcriptomic datasets will be MIAME (Minimum Information About a Microarray Experiment) compliant. The contextual information or documentation (“metadata”) is essential for the project partners and future secondary users. The consortium agreement will ensure access to all datasets and accompanying metadata for the project participants. After publication and intellectual property rights have been secured, which we predict to be within three years after the completion of the project, the metadata and datasets will made publicity available through third party databases. Depending on the level of usage and collaborative nature the project partners expect joint authorship or acknowledgements to the data originator. All outputs from the scientific activities shall adhere to the principles of good laboratory practice. The aim in this instance is to provide technical assistance, capacity building and technology transfer for developing countries. 13. Dissemination. The METAPRO programme has disseminated its findings and activities through a number of verifiable format for example, (i) scientific papers submitted to journals and presented at conferences, (ii) through press releases, (iii) through meetings organised and/or attended by partners, (iv) public engagement including local meetings, school and college visits and open days, web TV/ podcast, public facing websites and newsletters, (v) articles in technical journals, (vi) presentations at technical conferences and exhibitions, (vii) articles at market orientated journals, (viii) patents, PhD and MSc theses and (ix) through international workshops and (x) through the websites. Thus the dissemination activities have been extensive. Four different examples will be highlighted; (i) Dissemination of METAPRO to Early Stage Researchers. The metabolite profiling training school, its associated mini-symposium and short training missions have been a highlight of the METAPRO dissemination and training activities. Working with the COST ACTION active in this area 140 applicants applied for 14 places on the training school held in London (RHUL). These 14 participants came from 14 different countries across Europe. The course covered sample preparation to data analysis. Presentations on METAPRO activities were made at the start of the course and during the mini-symposium. In addition the technique demonstrated first hand during the course was developed in METAPRO. There was also an opportunity for the course participants to discuss activities with staff engaged in METAPRO activities. Finally, a trip to Syngenta was carried out during the course to acquire an industrial perspective. To complement these activities several short training mission were carried out for consortium partners and early stage researchers from developing countries. (ii) Dissemination to the scientific community. Already METAPRO has generated a number of peer reviewed scientific publications in high impact journals such as PNAS, Plant Cell, and Science. We anticipate more publications of this quality to follow. Another highlight for the scientific community was presentations made at high profile international conferences and COST ACTION meetings. For example, at the International Plant Molecular Biology Conference in Korea, 2012, within the plant secondary metabolite section two of the five speakers described METAPRO activities. Presentations have also been given at the Plant and Animal Genome (PAG) conference 2012, and International Metabolomics conference. At the annual COST ACTION meeting in Murcia 2011, METAPRO participated in the event that brought together several networks funded through the EUFP7 KBBE programme. This activity forged future links and interactions between projects. For example the transfer of resources such as specific plant varieties, constructs and authentic standards. (iii) Interaction with industry. METAPRO outputs have been disseminated to industry through individual seminars and also a highlight being the participation in an industrial exploiation workshop held in Japan. It is through the latter activity that a potential route to market was forged. (iv) Dissemination to the general public. The website has acted as a focal point for interested parties from the general public. In addition, METAPRO dissemination material has been provided at a number of science open days to the public. The highlight is however the promotional videos of the project, these are of the highest quality describing the project and its outputs with interviews with Early stage researchers. We will maintain the project website after the end of RTD activities and the promotional videos placed on CommNET (www.commnet.eu) as accessible archives.
List of Websites:
www.isoprenoid.com
Dr Paul D. Fraser, Centre for Systems and Synthetic Biology, School of Biological Sciences, Royal Holloway University of London, Egham Hill, Egham, Surrey, TW20 OEX.UK. Tel: 01784 443894, E-mail: p.fraser@rhul.ac.uk Publications whereby the on-line system will not let entry: 1. Lu, Y., Rijzani, H., Karcher, D., Ruf, S. and Bock, R. (2013). Efficient metabolic pathway engineering in transgenic tobacco and tomato plastids with synthetic multigene operons. Proc.Natl. Acad. Sci. USA, 110, e623-e632. 2. Mora, L., Bramley, P.M. and Fraser, P.D. (2013). Developemnt and optimisation of a lable-free quantitaive proteomic procedure and its application in the assessment of genetically modified tomto fruit. Protemics, Doi:10.1002/pmic.200790091.
Patents (whereby the on-line system would not permit entry).
1. Jones, M.O. Bramley, P.M. Fraser, P.D. (2010). Increased phytochemical production in plants. GB1008551.2.
2. Fraser, P.D. and Mortimer, C.L. (2011). Hydrocarbons from Plants. GB1108436.5
3. Baxter, C., Bradley, G., Pan, Y., Ball, G., Hodgman, T.C. Seymour, G.B. Wood, A and Fraser .P (2011). Modulation of tomato fruit ripening. International Patent Application submitted end September 2011, awaiting Publication Number. Supersedes European Patent Application of same filed 2009, Application number 10183748.2
Isoprenoids are classical secondary metabolites being synthesised in specialised tissues and/or cellular compartments. Their abundance is typically low but can be dramatically increased under stress conditions or defined stages in development. Most isoprenoids of commercial interest are used as fragrances in cosmetics and flavours, colorants and nutritional supplements in foods and feeds (e.g. ketocarotenoids) and numerous important medicinal properties. Their industrial relevance also means they are compounds of high value with global markets in the range of $ 1 billion per annum. A contributing factor to the high cost of these molecules resides in the fact that they are produced in low yields by slow growing plant species that are not readily amenable to agricultural production. In the METAPRO project we aimed to optimise the production of several useful isoprenoids to demonstrate the tools and strategies developed for the generic production of useful secondary metabolites in plants. To demonstrate our genetic engineering approaches two examples, astaxanthin formation and crocin production where used. The host platforms to be used in the proposed project will be tomato and potato. These crops are the most common fruit and vegetables produced worldwide and thus amenable to modern agricultural practices and from a GM perspective can be produced in contained growth facilities if necessary. These two crops also have specialised storage (sink) tissues containing cellular structures amenable to isoprenoid formation/accumulation without disruption of essential plant processes. It is these specific tissues and specialised cellular structures (plastids) that will be exploited in the METAPRO project.
Most of the fundamental reactions involved in isoprenoid formation have been established and rudimentary engineering of the pathway performed. One of the aims of the METAPRO project was to build on these applied foundations and advance our generic knowledge base to a new era in our understanding and engineering of plant metabolism for high-value secondary metabolites. The key objectives for the project are:
(i) Develop a more comprehensive understanding of regulation at an integrative systems level not just in the target pathway.
(ii) Identification and elucidation of mechanisms associated with the storage, deposition and sequestration of isoprenoids.
(iii) Reveal stability parameters of newly synthesised compounds in the cell.
(iv) Demonstrate tools and strategies for high-level production of useful isoprenoids such as ketocarotenoids and apocarotenoids.
During the project our integrative approach to characterisation revealed that it is difficult to view secondary metabolism as a separate entity, but as a component linked to primary and intermediary metabolism and often associated with re-programming of developmental/environmental related metabolism. Transcriptomic RNA –Seq approaches became the method of choice during the project and were successful in revealing to candidate genes. It is also apparent that biosynthesis and sequestration are interlinked and changes in metabolite composition can alter/induce cellular structures used for deposition. Our findings on stability transcend isoprenoids and our chosen hosts. Project partners have revealed both enzymatic and non-enzymatic degradation processes operate. To overcome these phenomena a down-stream process involving encapsulation has been developed that facilitates product stability. Through the implementation of our engineering strategies exceptionally high levels of isoprenoids have been generated. Some pleotropic limitations have been experienced which has highlighted the need for more controlled production. The prototypes are now available to perform production, technical and economic feasibility studies with a wide range of ketocarotenoid producing varieties.
The METAPRO project has disseminating its findings at the earliest opportunity to the scientific community, general public, schools and government agencies. For example to date thirty peer reviewed publications have been generated several in high impact journals such as Science, PNAS and Plant Cell. Over 100 dissemination activities have been delivered and at these three patents generated, that least three permanent jobs have also been created through the projects activities. The work has strengthened the position and sustainable foundations of an SME partner, which in turn has contributed to rural development. The project website www.isoprenoid.com has also been successful as a focal point for information and will continue beyond the RTD activity of the project. Two informative promotional video of the project have been generated and one with high school pupils. A highlight of the outreach and training activities during the project has been the metabolite profiling training school carried out in association with a related COST ACTION and the numerous training visits for early stage researchers (ESRs).
Project Context and Objectives:
Plant secondary metabolites are defined as compounds that are non-essential to fundamental plant processes, but do have an important role in the plant’s interaction with its environment. Secondary metabolites can confer agronomic traits such as quality, resistance and stress tolerance. Many secondary metabolites are also essential components of the human diet, are utilised as phytomedicines and routinely used as industrial raw materials and high-value fine chemicals. Chemically, secondary metabolites exhibit an enormous chemical diversity and complexity, which in most cases precludes chemical synthesis as an economically feasible means of production. On the basis of their biosynthetic origins, plant secondary metabolites can be structurally divided into five major groups; polyketides, alkaloids, phenylpropanoids, flavonoids and isoprenoids. In the METAPRO project we have optimise the production of several useful isoprenoids to demonstrate the tools and strategies developed for the generic production of useful secondary metabolites in plants.
In the majority of cases isoprenoids are classical secondary metabolites being synthesised in specialised tissues and/or cellular compartments. Their abundance is typically low but can be dramatically increased under stress conditions or defined stages in development. Most isoprenoids of commercial interest are used as fragrances in cosmetics and flavours, colorants and nutritional supplements in foods and feeds (e.g. ketocarotenoids) and numerous important medicinal properties. Their industrial relevance also means they are compounds of high value with global markets in the range of $ 1 billion per annum. A contributing factor to the high cost of these molecules resides in the fact that they are produced in low yields by slow growing plant species that are not readily amenable to agricultural production (for example, crocins produced in the stigmas of purple crocus flowers). Alternative production systems such as plant cell culture have in the main not proven to be feasible. It is therefore not surprising that total or semi-synthetic chemical synthesis is presently the method of choice for obtaining many of these isoprenoid molecules. However their structural complexity makes chemical synthesis expensive and difficult. Given the high value and utility of these isoprenoids as well as the lack of amenable plant based sources, a genetic engineering (GE) approach provides a logical solution to the creation of renewable bio-sources of these molecules with improved economic and environmental potential. In the METAPRO project we will demonstrate our genetic engineering approaches using two examples, astaxanthin formation and crocin production. Astaxanthin is a carotenoid (ketocarotenoid) while the crocins are derived from a carotenoid cleavage product (apocarotenoid) and require transport from their site of synthesis to the vacuole where they accumulate. They are typical secondary metabolites where optimisation in their native plant sources does not provide an optimal production platform.
The host platforms to be used in the proposed project will be tomato and potato. These crops are the most common fruit and vegetables produced worldwide and thus amenable to modern agricultural practices and from a GM perspective can be produced in contained growth facilities if necessary. Genetic and Genomic resources are well developed in these two Solanaceae with a vast collection of genetic diversity available and the sequencing of both genomes underway. Advanced transcriptomic, metabolomic and proteomic platforms have also been established and customised for these crop plants. These two crops also have specialised storage (sink) tissues containing cellular structures amenable to isoprenoid formation/accumulation without disruption of essential plant processes. It is these specific tissues and specialised cellular structures (plastids) that have been exploited in the METAPRO project.
Most of the fundamental reactions involved in isoprenoid formation have been established and rudimentary engineering of the pathway performed. One of the aims of the METAPRO project was to build on these applied foundations and advance our generic knowledge base to a new era in our understanding and engineering of plant metabolism for high-value secondary metabolites. The key objectives for the project are:
(i) Develop a more comprehensive understanding of regulation at an integrative systems level not just in the target pathway.
(ii) Identification and elucidation of mechanisms associated with the storage, deposition and sequestration of isoprenoids.
(iii) Reveal stability parameters of newly synthesised compounds in the cell.
(iv) Demonstrate tools and strategies for high-level production of useful isoprenoids such as ketocarotenoids and apocarotenoids.
Numerous tomato and potato varieties with perturbed isoprenoid content have been subjected to concurrent multi-level omic based analyses over the life time of the project. For example tomato genotypes with the simultaneous elevation of multiple high-value isoprenoids and other antioxidants have been analysed using an integrative omic based approach. Through the integration of transcriptomic and metabolomic datasets the consortium have revealed that the progenitor of elevated isorprenoids is enhanced core metabolism which may directly or indirectly result in the presence of a greater plastid area per cell (Enfissi et al., 2010. Plant cell, 22, 1190). Using high precursor tomato genotypes similar multi-level “omic” analyses have revealed a strong association between increased isoprenoid (beta-carotene) content and ripening related processes. In potato correlation network analysis have identified a number of regulatory HUBs existing within the pathway of interest (Diretto, et al., 2010, Plant Physiol., 154, 899). These approaches have illustrated the potential of modern systems based approaches in delivering new potential regulatory HUBs and have been important in characterising metabolic changes and the reprogramming that exists during secondary metabolite production. Over the course of the project techniques advanced with RNA-SEq replacing microarrays as the transcriptomic method of choice. The advent of modern faster Mass Spectrometers enabled development of label-free protein quantification procedures and the incorporation of proteomic data into our parallel multi-omic datasets (Mora et al, (2013) Proteomics). The inclusion of proteomic data was highly informative as it showed the different levels of cellular regulation and how translation is a key component of the process. A number of correlation (co-response) models have been generated. From these models it is clear that secondary metabolism is associated with core metabolism and the latter is frequently the progenitor of secondary metabolite formation. It is interesting that the Calvin-Benson cycle is at the core of the network in ripening fruit, as this process is typically associated with photosynthetic tissues. Although tomato fruit are photosynthetically active during fruit development it is not a key aspect of their metabolism and recently the role of photosynthesis in fruit development has been questioned with the belief that photosynthate is derived from vegetative material. The integrative characterisation of transgenic tomato plants possessing fruit specific down regulation of DETIOLATED1 is another example where core metabolism particularly carbon fixation appears to be the progenitor of down-stream secondary or in this case “tertiary” pathways in tomato fruit. The metabolite of the Calvin cycle displaying the greatest connections was D- glyceralde-3-phosphate. This metabolite is common to both the Calvin cycle and MEP pathway. Collectively these findings suggest that the plasticity of Calvin cycle components in tomato fruit warrant further investigation and the utilisation of photosynthate/carbon close to its source without further resource allocation could be a valuable strategy to explore as part of a combinatory engineering approach. Other metabolic links between isoprenoid formation and photosynthetic carbon flow have also been revealed through metabolomic analysis of hemiterpenoid glycosides under nutrient deprivation. Finally, cofactors/reductants appear to have an important influence over multiple pathways presumably by direct action on biosynthetic enzymes or perhaps via the modulation of redox states.
In addition perturbations in the isoprenoid content appear to have a direct link to changes in phytohormone balance, which can alter developmental and responses to environmental cues. A number of HUBs in the models have been identified and functional validation performed. Such an approach has revealed new transcriptional regulator. However, a transcription factor acting on isoprenoids solely remains elusive. Instead it is common that they act on other developmental processes or sectors of metabolism and isoprenoids an associate of a more fundamental process. Never the less a number of transcription factors that can alter pigments have been identified and one patent generated.
The METAPRO project addressed product sequestration/storage of products, several important insights have been revealed. For example, localisation experiments and conventional biochemical fractionation suggest that biosynthesis and accumulation of products utilise discrete functional inter cellular and intra organelle compartments. It would appear that the specialisation of organelles can also result from high-level production of certain metabolites. Finally through the screening of natural variation new potential alleles that can confer improved product sequestration have been identified. Our data also suggest that lipid formation (or plastid lipids) is concurrent with carotenoid formation. Traditionally the focus of metabolic engineering has been towards increased biosynthesis capacity but the experimental data generated shows biosynthesis and sequestration is linked and must be addressed with equal importance. Sub-plastid organelle fractionation revealed cellular structures for the first time associated with storage. These data also revealed several fundamental aspects in that perturbation in metabolites can alter and induce new sub-organelle structures. From correlation analysis and the screening of natural variation new transcriptional regulators have been isolated and a mechanisms demonstrated that shows how modulating Abscisic acid (ABA) levels can alter plastid levels.
The potential of altering enzymatic carotenoid degradation has been illustrated (Campbell et al., 2010 Plant Physiol., 154, 656) within the project. Further characterisation of these classes of enzymes is has been performed including the elucidation of carotenoid cleavage activities involved in strigolactone formation a new class of plant hormones. Unfortunately no QTL for carotenoid cleavage enzymes were found. In certain tissues such as leaf material it would appear that newly formed carotenoids are preferentially degraded. Storage and down-stream processing where found to be key areas where enzymatic and n0n-enzymatic degradation arose. In order to overcome these difficulties an extraction procedure that does not allow the material in question to dry was developed and an encapsulation procedure developed and implemented. These are key technologies that will make feasibility and translation possible. A highlight in the catabolism aspect of the project has been the implementation of RNA-Seq transcriptomic procedures to elucidate the apocarotenoid pathway in Saffron. This approach has enabled the dissection of the pathway and subsequent functional characterisation. These studies have now established a consensus overcoming the misleading data in the literature. As a result of these data the tools are now available to engineer the pathway and this approach has been performed in the METAPRO project to the primary transformant (T0) stage.
Significant advances have been made in the production of ketocarotenoids, which are one of the projects demonstration molecules, both in tomato and potato tubers. Firstly a plant variety was generated that expressed an optimised astaxanthin biosynthetic pathway. This approach benefitted from translational enhancement, plastid transformation technologies and suitable host platforms for high level production. To these varieties optimised precursors for the target pathways were delivered, improved sequestration through the modulation of plastid parameters performed, and the combining traits performed. As a result attaining targeted levels has not been a problem with high-levels in the 20 to 25 mg/gDW achieved for tomato. The issues have been pleiotopic effects on plant development. For example levels achieved with plastid transformation were so high that the plants cannot be regenerated. Ripening and development in general are effected which delays the growth cycle. However, despite these difficult the METAPRO project has delivered phenotypically stable prototypes with very high levels that will enable feasibility studies to be carried out.
The METAPRO project has disseminated its findings at the earliest opportunity to the scientific community, general public, schools and government agencies. For example to date thirty peer reviewed publications have been generated several in high impact journals such as Science, PNAS and Plant Cell. These outputs have advanced our scientific knowledge in the field. Over 100 dissemination activities have been delivered and at these three patents generated; to date at least three permanent jobs have also been created through the projects activities. The work has strengthened the position and sustainable foundations of an SME partner, which in turn has contributed to rural development. Through the work associated with the METAPRO project national funding has been awarded to several of the partners. The project website www.isoprenoid.com has also been successful as a focal point for information and will continue beyond the RTD activity of the project. Two informative promotional video of the project have been generated and one with high school pupils utilising new technologies. A highlight of the outreach and training activities during the project has been the metabolite profiling training school carried out in association with a related COST ACTION and the numerous training visits for early stage researchers (ESRs).
Project Results:
The RTD activities of METAPRO were divided into four work packages 2 to 5. Concerned with regulation, storage, stability and optimised production respectively. Work was performed to specific task which led to the completion of the projects objectives.
1. Work package 2. Identification of regulatory mechanisms involved in the synthesis of isoprenoids (WP leader P4 SCRI).
1.2. WP2 Objectives
O2.1. Identification of regulatory mechanisms involved in the formation of isoprenoids/carotenoids through characterisation at the gene, transcript, protein, enzyme activity and metabolite levels.
O2.2. Identify potential protein components of isoprenoid biosynthetic complexes.
O2.3. Determine changes to the metabolome and transcriptome associated with isoprenoid formation and/or perturbation.
O2.4. Integrate transcriptomic and metabolomic datasets to identify putative regulatory HUBs and candidate genes.
O2.5. Compile meta and experimental datasets, statistical analysis, integration and display of data over metabolic pathways.
O2.6. Utilise the methodologies established in WP2 as an enabling technologies platform to characterise genotypes generated and investigated in WP3, 4 and 5.
1.3. RTD activities performed in WP2
WP2 activities have utilised germplasm available within the consortium. These genotypes include genetically modified plants (GMPs) and mutants with elevated precursors for the production of ketocarotenoids and crocetin, and pleiotropic effects resulting from the perturbation of isoprenoid content. In accordance with Task 2.1 this material has been generated and distributed to respective partners for analysis. The consortium’s main focus has been (i) tomato and potato genotypes with increased precursors such as the high -carotene and xanthophylls varieties generated by P7, (ii) tomato varieties with altered plastid parameters constituting a cross workpackage activity with WP3 (P1) and (iii) potato genotypes producing high-value ketocarotenoids (P4). Sampling of tomato and potato material has been carried out on defined tissues over development to create dynamic dataset. Metabolomic analysis has utilised targeted isoprenoid determinations by HPLC-PDA, UPLC-PDA and LC-MS (Task 2.2). Direct infusion Mass spectrometry has been performed to generate chemical fingerprints and ascertain global compositional changes. In addition quantitative metabolite profiling using GC-MS and LC-MS for intermediary metabolites have been performed (Task 2.6). The robustness of these procedures has been validated by small scale inter-laboratory ring testing approaches (P1 and P8).
In order to complement the metabolomic analysis performed, transcriptomics has been carried out with either the tomato TOM2 microarray or potato POCI (Task 2.5). Verification of changes across the transcriptome and within the target pathway(s) has then been carried out by QPCR. Where appropriate selected analysis using specific enzyme assays (Task 2.3) and proteomic analysis (Task 2.4) has been performed. Of the omic level approaches proteomics has been the most challenging, especially in a quantitative manner. Allow quantitative profiles have been obtained using iTRAQ approaches the coverage is poor (e.g. 100 proteins detected at 99% confidence) and the representation of membrane proteins is low. To date only a limited number of isoprenoid/carotenoid related biosynthetic enzymes have been identified these include CRTISO and isopentenyl diphosphate isomerase (iDI). In addition no carotenoid bisosynthetic enzymes have been identified within protein complexes tested, although biosynthetic enzymes involved in the formation of prenyl lipids such as GGPP have been found associated with photosynthetic complexes. The proteomic approach was subsequently improved (P1) with the development of a routine quantitative label-free method for proteomic analysis. The label-free procedure uses internal standards for relative quantification and facilitates good coverage of membrane protein through the use of SDS-PAGE prior to reverse phase nano-LC. A manuscript describing the procedure and its development has been accepted in the Proteomics journal (Mora, Bramley and Fraser).This procedure has been very effective when combining activities with WP3, using enriched organelle and sub-plastidial fractions which have revealed new regulatory mechanisms, whereby the enzymes are in a different location to the precursors and products (Objectives 2.1 2.2 2.5 & 2.6).
In order to complement the metabolomics and proteomic analysis performed, transcriptomics has been carried out with either the tomato TOM2 microarray or potato POCI (Task 2.5). Verification of changes across the transcriptome and within the target pathway(s) has then been carried out by QPCR.
1.4. Examples of case studies
“Omic” characterisation of
(i) Tomato varieties with increased plastid content (P1)- The multi-level omic based characterisation of the DET1 down regulated tomato varieties has revealed the close association of secondary metabolism with core metabolic processes. This would suggest that future systems based engineering approaches that do not solely affect the pathway of the interest should be explored (Enfissi et al., 2010. Plant cell, 22, 1190). The proteomic studies indicated the importance of studying all levels of cellular regulation as elevations in transcripts were qualitatively and quantitatively not aligned with protein levels. Thus indicating the importance of post-transcriptional regulation. Organisation and integration of large-scale datasets and associated metadata has been successfully carried out predominantly using a large-scale metabolomic dataset for tomato introgression lines with modulated isoprenoid levels. The data has been subjected to co-regulation analysis and a model created for isoprenoid biosynthesis. An important aspect of these data is that the different sections of isoprenoid metabolism cluster in a biosynthetic intuitive manner but interestingly all relate back to primary metabolism especially the Calvin cycle, which suggests that defining metabolism into primary and secondary metabolism perhaps is not the correct approach. If the fixation of carbon could be exploited increased secondary metabolite formation could be significantly increased. The datasets have been deposited on the SGN website (www.Solgenomics.net) for the scientific community with a disclaimer describing its use.
(ii) High beta-carotene and xanthophyll precursor tomato and potato lines (P3 and P7)- Fruits of tomato plants overexpressing the LYCOPENE BETA CYCLASE (LCY-b) and/or BETA-CAROTENE HYDROXYLASE (CHY) transgenes and golden potato tubers where generated. The EU-TOM3 Affymetrix microarray, the transcriptome of tomato fruits overexpressing the LCY-b and CHY transgenes and of tomato mutants affected in fruit pigmentation (at, r, t, Beta). A full phenotypic characterization (ethylene and ABA production, cell wall and cuticle composition, post-harvest characteristics) was conducted on the former. The data indicate that beta-carotene overproduction in fruits triggers a complex syndrome, resulting in decreased ethylene production and enhanced self-life of the fruits. The transcriptome of the high-carotenoid DM potato line has been characterized using Illumina RNA-Seq (Massa et al, 2011). Network analysis performed on potato genotypes with elevated precursors illustrated how regulatory HUBs in the target pathway can change upon pathway engineering and that those components having the greatest quantitative changes are not always responsible for regulatory control within the pathway. (Diretto, et al., 2010, Plant Physiol., 154). The glasshouse evaluation at the Metapontum Agrobios research facilities in Metaponto (Italy) was aimed at the generation and characterization of fruit samples from the HighCaro (HC) LycB experessor, UO expressing a beta-carotene hydroxylase (CrtRb2) and their hybrid (HU) as well as from the control Red Setter (RS) parent line. Carotenoid contents of fruits of the four tomato lines (RS, HC, UO, HU), harvested at four stages (IG, MG, TR, RR), clearly showed that the overexpression of the biosynthetic gene encoded by the transgene was the determinant of the patterns of carotenoid biosynthesis and accumulation in tomato fruit. In the HC fruits, almost all lycopene was converted to b-carotene while a consistent amount of xanthophylls, mainly violaxanthin, was present in fruits of plant carrying th Research activities of the second year were aimed at: performing the expression analysis by RT-qPCR of the most important genes involved in biosynthesis of carotenoids, ethylene and ABA on fruit samples of RS, UO, HC and HU. Expression analysis of eleven genes (PSY1 , LCYb, CrtRb2, ZEP, CCD1A, CCD1B, CCD4B, NCED1, ACS2, ACS4, ACO1) was carried out on the fruit samples (4 sampling stages) of the high precursor and control lines (RS, UO, HC, HU) using RT-qPCR analysis. This dataset (11 genes x 16 samples) was combined with the datasets of HPLC determinations (16 compounds) and Metabolomics (42 metabolites, performed by P1 RHUL) which were derived on the same tissue samples. The final dataset was made, therefore, of 69 variables determined on 16 objects (fruit samples) for a total of 1104 data points each one derived from the analysis of at least 3 (up to 5) biological replicates. Data analysis was performed on single datasets as well as on combinations of two of them and on the whole dataset using univariate and multivariate statistical methods.
Results of RT-qPCR analysis on fruit samples of four high precursor and control lines clearly showed that none of the carotenoid biosynthetic genes was up-regulated by the Lcyb and CrtRb2 over-expression. Therefore, the reduction of the total carotenoid content in ripe fruits of the transgenic plants was not due to differential expression of biosynthetic genes. Conversely, the HU line showed higher contents of CCD1B and CCD4B transcripts, while in HC fruits the contents of NCED1, ACO1, ACS2, ACS4 gene transcript were higher at TR stage and the ABA content was higher (2-fold) at RR stage. PCA (Principal Component Analysis) on single datasets clearly showed that RT-qPCR and HPLC variables have similar discriminative power because both clustered objects according to fruit stage and genotype. Metabolomics data were also highly predictive of the stage of maturation since fruit samples collected at the same stage tended to cluster together irrespective of genotype.
(iii) Ketocarotenoid producing potato lines (P1 and P4)- Metabolomic and transcriptomic analysis was perfomed on potato varieties synthesising high levels of ketocarotenoids but with the exception of reduced starch content and altered flavonoids very few significant changes occurred.
1.5. Significant results, conclusions and recommendations
• WP2 illustrated the utility of an enabling technology platform for the characterisation of existing varieties and outputs from WP3 and 5 (objective 2.6).
• The multi-level omic based characterisation of the DET1 down regulated tomato varieties has revealed the close association of secondary metabolism with core metabolic processes. This would suggest that future systems based engineering approaches that do not solely affect the pathway of the interest should be explored (Enfissi et al., 2010. Plant cell, 22, 1190: Objectives 2.1 2.3 2.4 and 2.5).
• Network analysis performed on potato genotypes with elevated precursors illustrated how regulatory HUBs in the target pathway can change upon pathway engineering and that those components having the greatest quantitative changes are not always responsible for regulatory control within the pathway. (Diretto, et al., 2010, Plant Physiol., 154: Objective 2.1 2.4 and 2.5).
• The breakdown of network correlation strengthens between transcripts and metabolites compared to analysis between transcript and metabolite solely highlights the importance of post-transcriptional regulation. An area that has not been fully explored. (Objective 2.1 2.3 2.4 2.5 and 2.6).
• The engineering of non-endogenous products into the isoprenoid pathway to generate high levels had a surprising minimal impact on the global transcriptome and metabolome. In contrast engineering existing pathway components yield a plethora changes across the metabolome and transcriptome (objective 2.1 2.3 2.4 2.5 and 2.6).
• A model of isoprenoid biosynthesis has been created shows intuitive clustering of the isoprenoid classes on a biosynthetic basis with key common intermediates at the HUBs. The model also reveals that the Calvin/Benson Cycle is the progenitor of the formation and thus primary and secondary metabolism are linked. This suggests that modulating the fixation of carbon could also elevate secondary metabolites.
• The data and its metadata has been organised and as published deposited on the SGN website (www.Solgenomics.net) for the scientific community with a disclaimer describing its use.
1.6. Deviations and resources
There has been a problem keeping up with technology as the Affy microarray chip has now been surpassed and RNA-Seq is the method of choice. The latter approach is being finally used in the METAPRO. In addition the use of fast ion-trap MS platforms facilitated the use of label-free approaches.
1.7. Future work
Subsequent activities will be directed to characterising the transgenic lines produced in WP3 and 5 using transcriptomic and metabolomics analysis (objective 2.6.) as well as proteomic analysis. A key aspect will be the use of RNA-Seq approaches for candidate gene/allele discovery among biodiversity. The combined analysis of the transcriptome, proteome and metabolome are key to the understanding of cellular regulation.
2. Work package 3. Optimising the cellular storage of carotenoids (WP leader P5, HUJ)
2.1. WP3 Objectives
O3.1. Exploitation of natural variation to identify traits associated with altered plastid biogenesis.
O3.2. Elucidate if enhancing the plastid volume per cell increases carotenoid content by improving storage and/or synthesis.
O3.3. Identify further regulators of plastid biogenesis with the potential to optimise isoprenoid secondary metabolite production.
O3.4. Advance our understanding of metabolite induced plastid differentiation.
O3.5. Identify further regulators of plastid specialisation.
O3.6. Determine the role/essentially of concurrent lipid synthesis for the sequestration of carotenoids and other hydrophobic metabolites in the plastid.
O3.7. Identification of mechanisms and tools associated with carotenoid esterfication in plants as a means of storage.
O3.8. Characterisation of potato tuber plastid parameters and their role in isoprenoid/carotenoid synthesis and storage.
2.2. RTD activities performed in WP3.
Tomato population representing diverse natural variation have been screened for altered plastid biogenesis. This activity has been performed on one crop to date, using both molecular, biochemical and cellular based diagnostic assays. Genotypes have been identified with altered plastid biogenesis await confirmation in year two using crop2 (summer 2011). A large-scale re-screening of tomato mutant collection (http://zamir.sgn.cornell.edu/mutants/) was carried out in the field (Rehovot) in order to find new mutants with high-pigment phenotype. Several candidates were identified based on dark-green fruit phenotype during early development and higher lycopene concentration in ripe fruit. However, all of these mutants were found to be alleles of the known HIGH-PIGMENT 2 (hp2) locus. The screening revealed new alleles of tangerine and yellow-flesh as well as other pigmentation mutations. One of them displayed a typical phenotype of LUTESCENT, which is manifested by bleached fruit during the “green” stages of development and normal lycopene accumulation during ripening. The mutation was identified as LUTESCENT 2 and mapped on chromosome 10. This mutant was further characterized with the group of Dr. J Giovannoni (Cornell). Ppositional cloning revealed that the locus l2 encodes a chloroplast-targeted zinc metalloprotease of the M50 family that is homologous to the Arabidopsis gene ETHYLENE-DEPENDENT GRAVITROPISM DEFICIENT AND YELLOW-GREEN1. This study suggested a role for the chloroplast in mediating the onset of fruit ripening in tomato and indicated that chromoplast development in fruit does not depend on functional chloroplasts (Barry et al., 2012). In addition from the network biology approach used in WP2 P1 (& collaborators in Nottingham University) has identified a transcriptional activator that can alter plastid parameters so that there is a large plastid volume per cell. This has the effect of increasing isoprenoids/carotenoids.
In WP2 characterisation of varieties with increased plastid parameters has been performed. In addition WP3, has embarked upon the delivery of more genotypes with altered plastid biogenesis for increased cellular storage capacity. The direction used here in tomato is the manipulation of Abscisic acid (ABA) formation, in a fruit-specific manner building on P5’s earlier discovery of hp-3 and its increased plastid biogenesis derived from a defective zeaxanthin epoxidase and subsequent affects on ABA. In potato work is advanced in the generation of potato genotypes harbouring the Or gene, which converts amyloplasts to chromoplasts in potato tubers, e.g. the alteration of plastid specialisation. Further details of the later are provided in WP5.
Enhanced PSY protein levels in Arabidopsis results in strongly increased carotenoid amounts in non-green tissues such as callus and roots. Mainly beta-carotene accumulates in the form of crystals, similar to the curd of the cauliflower OR mutant. We performed western blot analysis using curd tissue from a white cauliflower cultivar and the OR mutant using antibodies directed against Arabidopsis PSY. Signal intensities were close to detection limit, but suggested no strikingly different PSY protein levels in both tissues. Roots and seed-derived callus were generated from Arabidopsis wild type (wt) and one 35S::AtPSY line. Curds from one white-colored cauliflower cultivar and the cauliflower OR mutant were used. Our antibody reactions and yeast two hydrid data suggest that PSY-1 and OR interact.
Molecular tools to enhance total carotenoids and potentially other plastid metabolites in tomato fruit through increasing the plastid number or plastid compartment size in fruit cells, has utilised the knowledge that in tomato and Arabidopsis that reduced levels of abscisic acid (ABA) lead to increase in the plastid compartment size. Since ABA plays crucial roles in stress physiology, mutations the abrogate ABA or manipulations of ABA synthesis and signal transduction decrease the adaptation of plants to stresses and thus reduce yield. To enhance carotenoids in fruit while avoiding deleterious effects of ABA deficiency on the whole plant, we created transgenic tomato plants in which the ABA-activated SnRK2 Protein Kinase family was suppressed specifically in fruits. To this end, a fruit-specific promoter, PPC2, was used with RNA interference (RNAi) of the gene SnRK2. The transcript levels of the SnRK2C and SnRK2.4 in the transgenic fruit were down-regulated to 5%-20% of control fruit. The number of plastids per cell was determined in green fruit using confocal microscopy and found to be higher in the transgenic plants. The reduction in the signal transduction of ABA led to an increase in the amount of fruit carotenoids by 30%-60%, while leaving the carotenoid composition unchanged, i.e. the main species was lycopene. Physiological experiments in the greenhouse indicated that drought tolerance was not compromised in the transgenic plants. However, no field test has been conducted. These results confirm the previous hypothesis that ABA is involved in the regulation of plastid number in the cell. Moreover, since no ABA deficiency phenotype has been observed at the whole plant level, the results suggest a novel approach to obtain high-pigment fruit phenotype by means of genetic engineering.
To confirm the important role of ABA in regulation of fruit ripening, the slZep (zeaxanthin epoxidase) gene was suppressed in tomato plants by transformation with a RNA interference (RNAi) construct, driven by the fruit-specific PPC2 promoter. Fruit of all RNAi lines displayed deep red coloration and increased lycopene levels compared to control fruit. However, silencing of the tomato Zep gene also damaged seed production. These results demonstrate the possibility to increase carotenoids in fruit through manipulation of the signal transduction of ABA in a fruit-specific manner.
Further work have been carried out by P1 and P6 showing how the enhancement of certain metabolites can alter plastid type and internal plastid structures. For example P6 has demonstrated crystal formation as a result of carotenoid hyperproduction while P1 has shown chromoplast induction upon the high-level production of the ketocarotenoid demonstration molecules. Comparison between subcompartments of the fruit chromoplasts from control and transgenic lines with increased beta-carotene lines showed a number of important differences. Firstly, an increased number of beta-carotene and lycopene crystal-like structures arose in the thylakoid-like membrane fractions of the high beta carotene line. It seems that this sequestration mechanism has been upregulated in the transgenic lines containing increased carotenoids.
Secondly, the membranes of the chromoplast (envelope and thylakoid-like membrane) also appeared to play an important role. The inner envelope of the high beta carotene chromoplasts seemed to be actively producing vesicles (membranous sacs), which were visible by electron micrographs of the high beta carotene line chromoplasts. The thylakoid-like membranes appeared in greater quantity and electron density in the high beta carotene chromoplasts compared to those in AC. The darker and thicker membranes could be caused by a high number of lipids, proteins and carotenoids or complexes of these components. The phytoene synthase and desaturase introduced by transgenesis were found in the same submembrane compartment, which strengthens the hypothesis that they can interact with each other and have a synergistic effect on the carotenoid production. However, the endogenous phytoene synthase (PSY-1) was mainly found in the stroma, whereas its product phytoene was predominantly found in the membranes and in the plastoglobules. The location of PSY-1 within the chromoplasts confirmed results found in the literature. The presence of phytoene in plastoglobules means that a significant quantity of the substrate for phytoene desaturase (and carotene formation) is partitioned away from the enzyme, possibly causing it to be metabolically inert, unless it is re-incorporated into the membrane enzyme complex. Collectively, the data illustrate that synthesis and sequestration are two processes
which are important for engineering carotenoid in plants, with the latter requiring further investigation.
P4 used an alternative approach to address the cellular location of synthesis versus accumulation. Excellent progress has been made in generating transgenic potato lines with biosynthetic enzymes phytoene synthase (PSY) and beta-carotene hdroxylase (BCH) possessing GFP and RFP tags for visualisation using microscopy. The resulting data to date shows that phytoene synthase and beta carotene hydroxylase are localised to the plastid but appear to reside in different structures within the plastid. a manuscript has been submitted (Pasare et al., 2012, J.Exp. Bot.) for publication.
In order to demonstrate the effects of concurrent lipid formation with carotenoids, transplastomic gentotypes amplifying the AccD component of the ACC fatty acid biosynthetic complex are at an advanced stage. P2 has generated Transplastomic tomato plants overexpressing the key (plastid-encoded) enzyme of fatty acid biosynthesis were generated. These lines have been purified to homoplasmy and their fruits were analysed with respect to total carotenoid accumulation. Preliminary data indicate a 10-20% increase in carotenoid accumulation suggesting that indeed, fatty acid and lipid biosynthesis limit carotenoid sequestration. Thus, fatty acid biosynthesis and lipid biosynthesis represent important target pathways for future metabolic engineering efforts that are directed towards increasing the carotenoid content of crop plants.
During the first reporting period P4 reported putative candidate genes for acyltransferase(s) capable of forming carotenoid esters. Unfortunately these gene products appear not to be the underlying candidates and the mechanism of these high carotenoid QTLs still awaits elucidation. However, microarray experiments were carried out on samples from a segregating diploid population bulked according to tuber carotenoid content and individual carotenoid components. A high proportion (47 out of 155) of the differentially expressed genes mapped directly under major QTL for tuber carotenoid content with 13 mapping to the novel chromosome 9 QTL for lutein content which is the predominant carotenoid in designated tubers. These genes became new candidates for the control of tuber carotenoid content.
2.3. Significant results, conclusions and recommendations
• Candidate genes for plastid biogenesis and carotenoid sequestration have been identified from the association of mQTL and eQTL (objective 3.1 3.3 and 3.7).
• Genetic intervention resources are being generated to ascertain how ABA acts as the progenitor of plastid biogenesis (objective 3.2)
• Sequestration mechanisms associated with depositing non-endogenous high value molecules have been revealed (objective 3.6 and 3.7).
• Synthesis of carotenoids and their deposition are not necessarily co-localised (Objective 3.8).
• Changes in metabolite composition can influence and alter cellular structures, especially at the sub plastid level (objective 3.4).
• The HFP gene has been identified, isolated and characterised as a tool to increase plastid biogenesis and core metabolism in tomato fruit. An additional homolog has also been identified (objective 3.1 3.3 and 3.7).
• Cell biology and biochemical (proteomics) have shown that the synthesis of carotenoids and their deposition are not necessarily co-localised (objective 3.2 and 3.8). This has led to the elucidation of new regulator mechanisms
2.4. Deviations and resources
No major deviations have occurred; the introduction of proteomics approaches has greatly helped in understanding the mechanisms and assigning putative functions to unknown gene products.
2.5. Future work
The resources developed for improved storage (WP3) will be combined with outputs from WP5 (objective 3.1 3.2 3.3 and 3.6). The mechanisms of carotenoid sequestration and metabolite induced plastid differentiation require further investigation further (objective 3.5 and 3.8). A greater understanding of the plastid and how their internal structures form with changes in metabolites is an area of important fundamental interest that impacts directly on the application of metabolic engineering and Synthetic Biology.
3. Work package 4. Understanding mechanisms of carotenoid degradation for (i) improved stability properties and (ii) the biosynthesis and transport of high-value cleavage products.
3.1. WP4 objectives.
O4.1. Identification and characterisation of genes and their products involved in the catabolism of carotenoids.
O4.2. Development of methods to monitor non-enzymatic degradation of carotenoids.
O4.3. Determine the relative contributions of catabolism and non-enzymatic processes to carotenoid degradation in planta and during bioprocessing.
O4.4. Functional characterisation of the biosynthetic and transport components involved in the formation of crocetin and its derivatives.
O4.5. Develop robust, efficient, high yielding extraction and purification procedures for ketocarotenoids and crocetin derivatives from tomato and potato.
O4.6. Assessment of “greener” chemistries extraction procedures for the targeted isoprenoids.
O4.7. Preparation of a ketocarotenoid “meal” from plant tissues and its formulation into aquaculture feed.
O4.8. Assess the physico-chemical properties of crocetin derivatives and ketocarotenoids produced in engineered systems with those extracted from natural sources and produced by chemical synthesis.
3.2. RTD activities performed in WP4
The role of carotenoid cleavage dioxygenases in the breakdown of carotenoid/isoprenoid pigments has been demonstrated, (i) in vitro, (ii) in transgenic plants and (iii) in vivo have been investigated. For example transgenic potato genotypes in which the CCD4 enzyme has been down-regulated contained elevated carotenoid levels P4. The potato CAROTENOID CLEAVAGE DIOXYGENASE 4 (CCD4) gene was identified as a candidate gene for regulation of tuber carotenoid content based on its expression profile in white and yellow tuber-fleshed potato cultivars, revealed by transcriptomic analysis.
Using an RNAi approach, down-regulation in transgenic tubers resulted in an increased carotenoid content, two- to five-fold higher than in control plants (Campbell et al., 2010. Plant Physiol., 154, 656). Tubers from 4 CCD4 RNAi lines were provided to P2 for more detailed elucidation of the CCD4 substrate carotenoid. The CCD4 construct was also provided to partners 1 and 5 for transformation other Solanaceous species. A second full length potato CCD gene, CCD8, was cloned, a fragment transferred into an RNAi vector and transformed in Desiree. A total of 20 independent transgenic lines were generated and grown in the glasshouse.
Following expression and purification in recombinant hosts such as E.coli the activity of these CCD’s appears to be very promiscuous and they can act on a variety of carotenoids (and carotenoid-like) precursors. The activity of both CCD1 from tomato (Solanum lycopersicum; SlCCD1A and SlCCD1B) was investigated to gain insight into their potentially different cleavage pattern and their capability of producing volatile isoprenoids correlating with carotenoid content and pattern of the fruits. A role of CCD1 enzymes, particularly of CCD1B, in producing carotenoid-derived volatiles in tomato fruits is indicated by the high abundance of the corresponding transcripts in this tissue. The cDNAs encoding SlCCD1A and SlCCD1B were cloned and inserted into inducible protein expression plasmids pBAD-THIO and pGEX), which enable the investigation of the enzymatic activity in vivo and in vitro. In vivo assays were then performed using engineered carotenoid accumulating E. coli strains, and in vitro assays with heterologously expressed, and affinity chromatography purified protein obtained using the pGEX-epression system.
Our results confirm literature data showing that both CCD1 enzymes catalyze the cleavage of the C9-C10/C9´-C10´ double bond in various bicyclic substrates and also the C5-C6 double bond of lycopene. In addition, our work revealed the C7-C8 double bond as a new cleavage sites in lycopene and apolycopenals giving rise to the formation of geranial and neral, two major tomato volatiles of hitherto unknown origin. This activity resembles that of the rice CCD1. P6 also demonstrated that the two tomato enzymes, particularly SlCCD1B, cleave the C13-C14 and the C11-C12 double bonds, two novel cleavage sites. The cleavage of the C13-C14 double bond leads in case of phytoene and phytofluene to farnesyl acetone, an additional tomato volatile of unknown origin. The cleavage of the C11-C12 site was only observed with 9-cis-β-carotene yielding -apo-13-carotenone, a compound previously shown to be biologically active affecting root hair growth by interfering with auxin transport.
The in vitro results generated by P6 indicate that almost all of tomato volatiles correlating in their abundance with the carotenoid content can be produced by SlCCD1A and/or CCD1B. To confirm this activity in planta, we made binary vectors carrying the corresponding cDNA either in sense, anti-sense orientation or equipped with a sequence encoding a plastid transit peptide. These constructs were then delivered to Partner 1 who performed transient transformation of tomato fruits. However, we did not observe any alteration in carotenoid pattern or content. However, P4 did successfully generated antisense CCD4 transgenic plants, which revealed increased lycopene contents.
P3 performed deep sequencing and transcriptomic approaches, to acquire ESTs from saffron stigmas have been carried out. Bioinformatic interrogation of these sequences has revealed putative biosynthetic enzymes involved in crocetin synthesis, and modifying enzymes involved in sequestration and transporters associated with the accumulation into the vacuole of the cell. Characterisation of these candidates has been the focus, transcriptomic along with complementation in E.coli and transgenic plants have been used as approaches. The transgenic plants expressing the crocus zeaxanthin cleavage dioxygenase has yielded a detectable cleavage product from zeaxanthin but better characterisation is required. It is also apparent that some of the data published are inconsistent with the findings and unambiguous characterisation would be helpful. Several candidate crocin biosynthetic genes (7 CCDs, 7 AlDHs, 2 Glucosyltransferases, 8 ABC and MATE transporters have been cloned in appropriate expression vectors. All CCDs have been transformed in tomato plants and in E. coli. The biochemical characterisation of CCDs in collaboration with P6 has revealed a lack of 7,8,7’,8’ cleavage activity for the ZCD gene published previously (Bouvier et al. Plant Cell 15: 47-62) and the presence of such activity in a different CCDs.
In addition to enzymatic degradation non-enzymatic degradation of pigments was studied. Analytical methods have been developed to identify products potentially derived from the non-enzymatic degradation of carotenoids. Further characterisation is underway and required to fully elucidate products and underlying mechanism involved in their formation. It is clear however that in dry tissues that non-enzymatic degradation is a crucial factor. The degradation would appear to arise from radicals generated through lipid perioxidation. The initiator of the radicals is likely to be the action of super oxide dismutase (SOD) without the coordinated action of catalase.
The non-enzymatic degradation of pigments in drying tissues has had important implications to down-stream processing. P8 has tested the stability of a ketocarotenoid containing “meal” from tomato fruit and potato tuber material evaluated in different conditions of temperature, humidity, light. The ability of these rich-ketocarotenoid powders to be incorporated into feed for aquaculture was evaluated, by mixing it with vegetable-based meal (commercial fish pelleted meal) and their stability was tested. Physico-chemical properties for ketocarotenoids derivatives prepared from the GM material developed. As a consequence of these studies P8 has developed a down-stream processing approach that eliminates the use of organic solvents, enriching carotenoid complexes instead. The extracts have also been encapsulated for stability with non-toxic polymers and release into aqueous solution, this work has represented a major breakthrough and delivered a means of stabilising the pigments.
3.3. Significant results, conclusions and recommendations
• The down-regulation of the carotenoid cleavage dioxygenase (ccd4) increased carotenoid content in potato tubers and tomato fruit. The carotenoids increased are precursors for ketocarotenoid and crocin formation (objective 4.1).
• EST sequence analysis and transcriptomics of saffron stigma has identified crocin biosynthetic, sequestration and accumulation (objective 4.4).
• It appears that the enzymatic cleavage of ketocarotenoids can occur in vegetative tissues.
• The contribution by non-enzymatic mechanisms to the breakdown of carotenoids and ketocarotenoids is a major contributor in dried tissues. This finding has major implications for cereal crops such as rice, maize and wheat (objective 4.1 4.2 and 4.3).
• Procedures have been developed that facilitate “green” extraction without solvents, an encapsulation method that enables stability as well as chemical and physical compound integrity (objective 4.5 4.6 4.7 and 4.8).
3.4. Deviations and resources
No major deviations have occurred.
4. Work package 5. The development of tools and implementation of strategies for the efficient engineering of useful isoprenoids.
4.1. WP5 objectives.
O5.1. Optimisation of the tomato plastid transformation system.
O5.2. Suite of novel plastid expression cassettes conferring improved biosafety characteristics.
O5.3. Extend the range of valuable tomato and potato varieties amenable to routine efficient transformation.
O5.4. Generation of transformation vectors enabling the simultaneous introduction of multiple cDNAs for mini-pathway engineering.
O5.5. Transplastomic tomato plants producing ketocarotenoids.
O5.6. Illustrate the potential of translational optimised biosynthetic genes in optimising secondary metabolite levels.
O5.7. Demonstrate how, in combination, pathway engineering and improved storage properties can increase the yields of useful isoprenoids (ketocarotenoids).
O5.8. Show the potential of reducing carotenoid catabolism as a means of increases ketocarotenoid yield in defined plant tissues.
4.2. RTD activities performed in WP5
The tomato plastid transformation system remains labour intensive but it has been improved especially with respect to its robustness and diversity of varieties that can be transformed (objectives 5.1 and 5.2). During the METAPRO project P2 has improved of plastid transformation technology for tomato and the identification of highly regenerable green-fruited commercial tomato varieties. Plastid transformation for two green-fruited commercial tomato varieties was accomplished (published in PNAS 2013). In addition, varieties amenable to conventional transformation have been developed to include the high pigment (hp) mutants where it was found necessary to alter the phytohormone complement, especially a reduction in auxin supplementation (objectives 5.2).
To aid the engineering of pathways a collection of vectors for plastid (Ruf and Bock, 2011. Methods Mol. Biol. In press) and conventional transformation had been modified and developed (objective 5.4). Building up on the vector construction work and plastid transformation experiments performed resulted in a breakthrough with achieving high-level transgene expression in non-green plastids. By combining promoters of plastid genes that show high mRNA accumulation in non-leafy tissues with 5’ untranslated regions (harbouring the cis-elements for translational regulation) that are active in non-photosynthetic tissues, chimeric expression elements could be identified that confer high-level expression in root amyloplasts and fruit chromoplasts (published in two papers in Plant Journal in 2012 and 2013). These novel expression elements pave the way to efficient metabolic pathway engineering in non-green storage organs, such as potato tubers and tomato fruits.
Strategies for the translational enhancement of gene products have been developed. Firstly P6 has identified inhibitory regions in the untranslated regions (UTR’s) of carotenoid biosynthetic genes. Removal of these regions confers the high level production of active protein. Using resources provided by the projects international advisor (Prof. Misawa) transferable sequences for enhanced translational have also been incorporated upstream to target gene sequence.
In order to investigate the translation-inhibitory function of the 5’UTR of Arabidopsis PSY in more details, two systems were developed:
(i) Transient expression of reporter genes in tobacco leaves. We intended a rapid and reliable analysis of 5’UTR sequence motifs involved in the regulation. For this we combined 5’UTR-controlled translation of GUS with an internal ribosome entry side (IRES)-controlled translation of luciferase in bicistronic transcripts. Although it was shown that IRES-driven luciferase levels can be used to determine levels of bicistronic transcripts, quantification of 5’UTR-controlled GUS levels revealed that GUS protein behaved non-linear compared with transgene expression. This suggested a limitation of the translation capacity. The system was unsuitable for the intended studies.
(ii) Stable expression of 35S::5’UTR-GUS. For investigations of the regulatory function of the AtPSY 5’UTR, we generated Arabidopsis lines overexpressing 403 bp, 330 bp and 280 bp of AtPSY 5’UTR, respectively, followed by GUS as reporter gene.RNA electrophoretic mobility shift assays (REMSAs) were established to identify putative cytoplasmic AtPSY 5’UTR-binding proteins. Results indicated the formation of specific protein-RNA complexes with cytoplasmatic proteins, subfractionated by ion exchange chromatography. Affinity columns were constructed to allow the identification of the bound proteins.
Ratios between GUS to transgene transcript were determined for callus and leaves of homozygous 35S::5’UTR-GUS lines with 403, 330 and 280 nt of 5’UTR. In comparison with lines carrying the corresponding constructs with AtPSY, GUS lines showed only a weak translation inhibition in leaves. One essential difference between 5UTR-GUS and 5’UTR-AtPSY lines is an increased carotenoid flux in the latter. Therefore, the different responses are considered as indication for an involvement of carotenoid degradation products in 5’UTR-mediated translation inhibition. Carotenoid-derived signalling compounds in leaves are known while pronounced carotenoid degradation in callus was previously demonstrated. The GUS lines generated will be further used to investigate this effect in details. Affinity columns were constructed which contained the bound 5´-UTR and which is based on the strptomycin aptamer. Cytoplasmic proteins, pre-purified by FPLC (see above) were applied and the resulting eluted protein bands subjected to Mass-Spectrometric identification. So far, no promising candidates were obtained.
The production of ketocarotenoids in plant tissues was a key objective of the METAPRO project. Plastid transformation was one approach used. The initial work was the demonstration that efficient engineering of the carotenoid pathway in tomato fruits can be achieved by expression of biosynthetic enzymes from the plastid genome. The work, published in Plant Physiology, showed that expression of a plant lycopene cyclase gene resulted in conversion of lycopene into provitamin A (β-carotene) and, at the same time, led to a substantial increase in total carotenoid content. Furthermore, the principles of synthetic operon design for efficient multigene engineering in plastids were worked out using the tocopherol metabolic pathway as an example. Application of these design principles to astaxanthin biosynthesis (combining a lycopene cylase with a ketolase and a hydroxylase in a three-cistron operon) induced high-level astaxanthin production in transplastomic tobacco plants. ketocarotenoids represents 90% of the total carotenoid have been generated. This resource technology has now been transferred to tomato.The work on synthetic operon design and its application to engineering of the tocopherol pathway was completed by transferring the optimized operon into the two green-fruited tomato varieties for which plastid transformation protocols had been developed. The astaxanthin operon was transferred into tomato plastids and triggered even higher astaxanthin synthesis in tomato than it did in tobacco (for unknown reasons). While the astaxanthin-producing transplastomic tobacco plants grow autotrophically in soil, the transplastomic tomato plants expressing the same operon require synthetic medium to grow, due to extreme accumulation levels of astaxanthin.
In addition to plastid transformation, conventional nuclear based transformation has been used to deliver high-level ketocarotenoid production (1 to 5 % dry weight) has been achieved in tobacco tree, tomato and potato. Here transcriptional and translational enhanced gene(s)/products have been used. P1 developed to primary lines containing the astaxanthin biosynthetic operon (crtZ and W from the marine bacterium Brevundimonas) under 35S CaMV constitutive control with translation enhancers. The evaluation of progeny was performed in two sites Italy (P7) and London (P1). In addition P7 and P1 created genetic crosses high-precursor lines. The screening of the transgenic segregating progeny was firstly based on the analysis of the phenotype of the most important vegetative and reproductive organs (cotyledons, leaves, steams, flowers, and fruits) at several developmental stages. Secondly, the transgenic plants were characterised at molecular level through PCR and Southern analyses. Following this screening two plants, ZW10-6 and ZW13-8 were selected as the best parents and were crossed to lines HC (high beta-carotene) and UO (high xanthophyll) carrying, respectively, the transgene encoding the lycopene beta-cyclase (Lcyb) and the transgene encoding the carotene beta-hydroxylase 2 (CrtRb2). The two hybrids were named HZW and UZW. Carotenoid contents of fruits of the four tomato lines (RS, HC, UO, HU), harvested at four stages (IG, MG, TR, RR), clearly showed that the overexpression of the biosynthetic gene encoded by the transgene was the determinant of the patterns of carotenoid biosynthesis and accumulation in tomato fruit. In the HC fruits, almost all lycopene was converted to -carotene while a consistent amount of xanthophylls, mainly violaxanthin, was present in fruits of plant carrying the CrtRB2 transgene, particularly in HU fruits. Two transgenic ZW plants, ZW10-6 and ZW13-8, were chosen as the best parents since they were shown to carry a single insertion of the transgene CrtZ/CrtW whose expression resulted in the synthesis of ketocarotenoids in all analysed tissues. In particular, leaves were brown while the flowers were deep red. Fruits were pink during the IG and MG stages while later turned red. However, the synthesis of ketocarotenoids was higher in hemizygous plants than in homozygous plants where the transgene appeared to be silenced. Homozygous plants showed, in fact, normal flowers and fruits while the leaves were with brown veins and green inter-vein sectors. The double hybrids (UZW, HZW) and the ZWn parent lines were grown in glasshouse and the carotenoid contents of leaves, flowers and fruits were determined by HPLC. UZW double hybrid plants were crossed to homozygous HC line to combine in a single line all three transgenes (Lcyb, CrtRb2, CrtZ/W). The derived triple hybrid line was named HUZW. These populations have been grown over several growth cycles to judge inheritability of the phenotype. One during the summer of 2012, a number of fruit samples of the two most interesting hybrids, HZW and HUZW, as well as of the control ZW13-8 line were collected and sent to partner P8 (Proplanta) for the production of microcapsules. Moreover, a forth breeding nursery for the production of seeds to be used for possible future activities aimed at the exploitation of the final products of METAPRO project. In this respect, the best set of parental line would be the one: including the lower number of lines to be maintained; requiring the lower number of crosses to produce the best performing lines, and; that do not necessitate the screening of the progeny, either visual or molecular.
Analysis of the HZW and HUZW ripe fruits contained 2.2- and 1.7-fold more carotenoids than the fruits of ZW line and UZW hybrid. Most of carotenoids in the former fruits were ketocarotenoids while in the latter fruits the lycopene was the most represented carotenoid. More precisely, ketocarotenoid content in HZW and HUZW fruits accounted for the 88% and the 96%, respectively, of all carotenoids of the fruits while in the parent ZW line the ketocarotenoids were less than the 8%. For these reasons, the HZW and HUZW ripe fruits contained, respectively, 24- and 20-times more ketocarotenoids than the ZW parent line. Most of the ketocarotenoids in HZW and HUZW fruits were present in the form of esters. Roughly, the 52% and the 76% of all ketocarotenoids were present as esters in HZW and HUZW fruit extracts, respectively. As expected, in the chromatograms of HUZW fruit extracts both mono- and di-esters were present while the mono-esters were the most important fraction of esters detectable in the chromatograms of the HZW fruit extracts. This finding represents a clear proof that the esterification process in the tomato fruit chromoplast is in no way a limitation to the accumulation of high amounts of xanthophylls or other hydroxylated compounds. A de-esterification treatment was applied to fruit extracts of HZW and HUZW lines before the HPLC analysis in order to estimate the level of hydroxylation of ketocarotenoids. Results of this analysis showed that in HUZW fruits the esters were made of astaxanthin for the 56% and of adonixanthin and canthaxanthin for less than 20%. Conversely, in HZW fruits less than 20% of esters were made of astaxanthin while adonixanthin and canthaxanthin accounted for almost the 73 % of all esters. P1 has obtained MS data for all the ketocarotenoids formed. In addition to the crosses performed by P7, P1 has introgressed the astaxanthin biosynthetic operon into a high--carotene background. The IL line has exceptionally high -carotene content 100 fold more than the wild type and altered cellular structures (dense plastoglobuli) to accumulate carotenoids. In addition this variety of tomato is heat and drought tolerant and capable of growing on marginal soils. P1 has also carried out transcript analysis over ripening and found a delay in the expression of the carotenoid transcripts. Improved fruit shelf-life has also been observed.
All the candidate biosynthetic enzymes and putative transporters identified in WP3 for crocin biosynthesis have been transformed into tomato by P3 some of the products await elucidation.
The generation of transgenic plants with combined traits such as plastid capacity, plastid specialization has also been pefomed. Transgenic potato plants expressing the β-carotene and ketolase genes isolated from the marine bacterium Brevundimonas sp. SD212, obtained from partner 1, were transformed into the cultivar Desiree. P1 has then developed these plants over multiple generations and analysed the pigments and the metabolome. Transcriptomic analysis using the potato microarray was carried outby P4. Additional transformations were carried out in the high tuber carotenoid cultivar Mayan Gold and 01H15 57 which contains high levels of the astaxanthin precursor, zeaxanthin. Putative transgenic lines were then re-transformed with the cauliflower mutant Or gene to improve the carotenoid storage capacity. A number of transgenic lines expressing either the constitutive crtZW construct or tuber targeted mutant cauliflower Or gene were successfully produced by Agrobacterium mediated transformation. Preliminary analysis of carotenoids present in various tissues (in collaboration with partner 1) showed Mayan Gold based crtZW lines accumulated both keto-lutein and astaxanthin, whereas lines based on the high zeaxanthin line, 01H15, accumulated almost entirely astaxanthin. Lines expressing only the cauliflower Or mutant accumulated over 75% more tuber carotenoids compared to WT. The best transgenic lines expressing only the crtZW construct were then re-transformed with the Or construct to produce co-transformant lines.
In tomato P1 has crossed and transformed the high plastid lines with the astaxanthin producers as a result a synergistic effect on the carotenoid content has been observed.
The METAPRO project has been successful and prototype genotype (summarised below) have been generated that produce high levels of ketocarotenoids. These can be utilised in feasibility/translational studies. However a number of unforeseen scientific circumstances have occurred that were unpredictable during the conception of the description of work (DOW). Predominantly, these reside from the high ketocarotenoid contents in vegetative, fruit and tuber material approaching 10 % DW. As a result the vigour of the plants and development has been affected this can in some cases delay the development of the plant and its sink tissues by up six months. We presume this is due to alterations in ABA content. Alternatively, the effects can be lethal in some cases. The latter is particularly evident in the case of plastid transformation. These initial effects have delayed the production and development of plants whereby the traits for the optimised biosynthetic pathway has been combined with augmenting traits such as increased plastid related sequestration, a supportive lipid matrix and precursor supply. For example Transformation of potato genotypes particularly the Solanum phureja genotypes Mayan Gold and O1H15 is considerably slower than for standard test genotypes (Solanum tuberosum cv Desiree) taking 5 months rather than 3 months to regenerate plants. Additionally transformation with the CrtZW construct (that confers high tuber ketocarotenoid content) appears to cause delayed tuberisation (by up to 2 months). Thus generation of some transgenic tubers is taking 4-6 months longer than anticipated.
4.3. Significant results, conclusions and recommendations
• Improved plastid transformation procedures (objective 5.1).
• Extending transformation protocols to varieties with enhanced traits (objective 5.3).
• Utilisable vectors for improved mini-pathway production (objective 5.2)
• The production of very high level ketocarotenoids in tobacco, tomato (leaf and fruit) and potato (leaf and tuber), (objective 5.4 5.5 and 5.6). Prototypes available for feasibility studies are:
(i) Nicotiana glauca- High ketocarotenoid containing leaves (0.5% DW) and high ketocarotenoid flowers (10% DW).This renewable source is a nicotine free variety, but enrichment and purification will be required prior to application (P1).
(ii) Transplastomic tobacco lines- High astaxanthin (1 to 5% DW) leaf material purification will be required prior to use. Grafting needs to be assessed to reduce nicotine content (P2).
(iii) High zeaxanthin potatoes- varieties delivering high levels of zeaxanthin in their tubers, this material could be used as an addmix or purification (P4).
(iv) High astaxanthin potatoes- varieties delivering high levels of astaxanthin in their tubers, this material could be used as an addmix or purification (P4 &1).
(v) High canthaxanthin tomato fruit- Due to altered expression levels in a background with high beta carotene, canthaxanthin is the predominant product (P1 and 7).
(vi) High ketocarotenoid tomato fruit- This variety has a range of rare and valuable ketocarotenoids (P1 and 7).
(vii) High lycopene tomato fruit- The presence of CrtZ/W at a low expression level results in the metabolism of beta-carotene. This removes the feedback inhibition in the pathway and lycopene is over produced to a level of 15 to 20 mg/gDW (P1).
(viii) High phytoene and phytofluene tomato fruit- While engineering precursors for the ketaocarotenoid pathway P5 revealed a strategy to produce very high phytoene/phytofluene containing fruit, with the need for GM approaches.
(ix) High beta carotene tomato fruit- Natural varieties producing high levels of beta carotene have been identified and developed.
(x) High 4-ketozeaxanthin tomato fruit- Varieties producing the intermediate 4-ketozeaxanthin have been generated.
(xi) High astaxanthin tomato fruit- Fruit containing up to 25mg/gDW Astaxanthin in a mono-esterified form have been generated and the phenotype stable.
4.4. Deviations and resources
No major deviations or changes to resources beyond everyday practical and logistical resources have occurred.
4.5. Future work
The prototype tomato, tobacco and potato varieties are available producing high levels of canthaxanthin, astaxanthin, beta carotene and phytoene. It is now important to carryout production, technical and economic feasibility studies.
Although adequate prototypes are in place the pleiotropic effects arising should not be ignored. One way round these effects would be inducible or very precise tissue specific promoters. This aspect is of particular pertinence to plastid transformation approaches.
5. Final considerations
The METAPRO project delivered vast knowledge of the optimisation of secondary metabolites in planta it is difficult to address the entire scope of the project. However, in summary the salient points are provided below for each WP executed.
WP2- The experimental approach illustrated the potential and necessity to use systems level characterisation. The data revealed the multiple levels of regulation that must be considered and exploited. Evidence was produced to illustrate that secondary metabolites are intrinsically linked to primary metabolism, or developmental processes, or environmental responses/conditions and engineering or Synthetic biology strategies should/must consider the system in general not just the targeted pathway.
WP3- Traditionally metabolic engineering and even marker assisted selection has focussed on biosynthetic capacity. The experimental data clearly illustrates that sequestration must be considered concurrently.
Our focus on the plastid organelle revealed new regulatory mechanisms, new sub-plastid structures and demonstrated the fundamental point that changes in metabolite composition can alter organelle and sub-organelle structures.
WP4- Newly synthesised and endogenous pigments are subject to enzymatic and non-enzymatic degradation. The non-enzymatic radical attack is potentially the most potent. This has important ramifications for cereal crops in developing countries. The METAPRO project has developed a procedure facilitating low environmental impact of processing and stability of product.
The other highlight delivered by WP4 is the elucidation of apocarotenoid formation in Saffron.
WP5- Implementing strategies associated with effector gene optimisation, translational enhancement, improved sequestration and elevated precursor levels unprecedented levels of ketocarotenoids have been generated in tobacco leaves, tomato fruit and potato tubers. This resource will enable us to answer fundamental aspects of metabolism and cell biology. In addition the varieties will enable progression to technical, production and economic feasibility studies on route to commercialisation.
6. The final partner reports are attached on-line. These reports provide clear visual representation of some of the data generated.
Potential Impact:
The outputs from the METAPRO project will impact directly on a number of key strategic areas including; improved sustainable bio-production and processing, enhanced quality of life, important socio-economic factors, the knowledge based economy, increased competitiveness and prosperity, international development as well as advancing fundamental science and the exploitation of knowledge. Examples are provided below. 1. Scientific advancements and the exploitation of knowledge. Important underpinning fundamental knowledge will be acquired from the project. This information will benefit the scientific community and contribute to the progression of science as well as further societal and commercial exploitation. The research will advance the present state of the art in the following areas: (i). Improved fundamental knowledge of plant metabolic pathways and their regulation for the generic production of useful secondary metabolites and other commodities. The isolation, identification and characterisation of the genes encoding the biosynthetic enzymes involved in Saffron derived apocarotenoids is an example of how METAPRO has improved our fundamental knowledge of biochemical pathways responsible for high-value phytochemicals. The utilisation of systems biology and parallel “omics” approaches in METAPRO advanced our fundamental knowledge of metabolism in general. It has become clear that primary and secondary metabolism is closely associated and systems engineering is necessary for optimised levels of secondary metabolites. (ii). An improved understanding of how secondary metabolites are stored in specific tissues within specialised organelles. The METAPRO project revealed that metabolite composition can alter or induce cellular structures this is a fundamental scientific advance with generic scientific implications that transcend the discipline. In addition new cellular based regulatory mechanisms were revealed and the finding that plastoglobules are not inert storage compartments is an important development. This sub-plastid structure requires further investigations. (iii). Advance our understanding of the mechanisms involved in catabolism and non-enzymatic degradation of isoprenoid derived secondary metabolites in the cell and during bioprocessing. METAPRO activities have illustrated the key role played by non-enzymatic degradation probably lipid peroxidation and how it can be overcome by using aqueous non-toxic enrichment and encapsulation. (iv). Deliver the tools and expertise necessary to engineer high value isoprenoids (e.g. crocetin derivatives and ketocarotenoids) into amenable plant based hosts. The characterisation of enzymatic processes for carotenoid catabolism has delivered a suite of new genes from Saffron derived apocarotenoid formation this represents a major scientific advancement. New vectors for plastid and conventional transformation, with transcriptional and translational enhancers have been generated and distributed in the scientific community. Clear effective metabolic engineering strategies have been developed and validated. (v). Demonstrate how efficient engineering strategies incorporating synergistic traits such as optimised storage and stability can complement pathway engineering to achieve the production of useful natural products at economically favourable quantities. The METAPRO project is one of the first examples were augmenting traits have been combine to deliver optimised yields. (vi). Development of new transfection technologies/procedures. Through activities in the METAPRO project tomato plastid transformation is now a routine laboratory practice and the numbers of transformable varieties have been extended. (vii). Rapid transient expression systems. Optimised transient transformation systems have been developed to assess new genes independently and in combination. (viii). Characterisation of promoters and other expression elements. Several new promoters predominantly for expression in tomato fruit have been characterised, while translation enhancers have been identified and developed., for both conventional and plastid transformation. (ix). Improve and extend the application of metabolomics and transcriptomics. During the METAPRO project the very latest developemnts in trranscriptomics and metabolomics were implemented into the work programme. The utility of RNA-Seq was demonstrated through the identification of transcripts associated with apocarotenoid formation in Saffron. Accurate mass metabolomics approaches have been developed and used to identify and assign putative formula to unknowns. Thus the METAPRO project has developed the use of these enabling technologies. (x). A repository of meta and experimental datasets relating to the optimisation or perturbation of metabolic pathways for the production of useful fine chemicals. Large datasets of high quality have been generated in the METAPRO project. These can and will be utilised by the scientific community in the future. To facilitate community used the data is deposited at www.phenome-netwroks.com and www.solgnomics.net. A dedicated database for Solanaceae has greatly benefited the data basing aspect of the work. (xi). Isolation of important rare authentic standards. The lack of available authentic standards is often a key limiting factor that hinders scientific research. METAPRO has been responsible for generating unique sources of carotenoids that have the potential to act as analytical standards and be used as new chemical entities in biological activity assays. (xii). Further demonstrate the potential of natural variation and extend genetic resources in Solanaceae. Several biodiversity agreements have been developed by METAPRO. Through these MTAs diversity collections have been screened for altered plastid related parameters and isoprenoids. In addition, new the genotypes have been generated within the project and will be made available to scientific community after capturing potential IPR thus adding to our existing genetic resources in Solanaceae. (xiii). Initiate the process of field evaluation. Advances in the METAPRO project now have resulting in the consortium submitting and gaining authorisation to grow prototypes in the environment, e.g. ISR and UK (under polytunnel conditions). (xv). Utilisation of potential semi-synthetic approaches. It is an increasing occurrence to utilise intermediates/products derived from plants as molecular scaffolds for synthesis of end-products or derivatives. The outputs from the project will provide further foundations for such approaches using improved renewable sources. (xvi). Create a supportive environment for sharing knowledge (know-how), information, expertise and innovation (Knowledge Transfer). METAPRO mobilised critical mass in the field not just through project specific activities but in association with the concurrent COST ACTIONs and projects e.g. FP7-TiMET. This aspect was of particular benefit to the SMEs giving them direct access to developments in fundamental research for example P8 benefited from the exchange of metabolomic expertise. The METAPRO project will clearly advance our fundamental scientific knowledge and highlight potential exploitation. 2. Improved sustainable bioproduction. The METAPRO project has illustrated how chemicals typically generated by chemical synthesise; using petrochemical by-products as precursors, with down-stream processing approaches that have adverse environmental impact or derived from low yielding plant/microbial sources not amenable to agricultural production, can potentially be produced in renewable biosources at competitive levels. Feasibility studies are now required to demonstrate this on a production scale. The technologies and resources generated by the proposed project are generic and can potentially be exploited to impact directly on creating sustainable renewable bio-resources for most high-value plant secondary metabolites. In the case of ketocarotenoids, such as astaxanthin which was one of the projects demonstration molecules there is a clear environmental and economic impact a more sustainable bioproduction platform. For example over the past decade the world’s fish stocks have been heavily depleted, with Salmon production declining to a level that is only just sustainable. To supply demand fish farming is increasing rapidly. However, astaxanthin/canthaxanthin like essential oils are an integral costly part of the aquaculture process, without which aquaculture will be unsustainable. The prototypes from the METAPRO project can now provide us with this opportunity to show the feasibility of these sustainable sources. The use of Solanaceae has added a biorefining aspect to the project as it is generally estimated that 20 -40% of the raw vegetable or fruit material ends up as waste during processing. In the METAPRO we have identified Solanesol as a high value compound that can generated from solanaceae waste to create added-value co-products. In summary the outputs of the METAPRO project offer a means of producing high value plant derived products using cheap renewable bio-sources that have a reduced impact on the environment. These are important factors that will impact directly on society in the coming years. As society moves from chemical to biorefining processes. 3. Increasing competitiveness and prosperity. The tools, resources, plant varieties and training generated and provided by the METAPRO project will contribute to ensuring European enterprises working in the field or related services are competitive. In this way the potential has been developed for European industry to compete with their US and Japan counterparts or licence technologies to these established and emerging global partners. It is hoped that the renewable and cheaper means of production developed in the MEATPRO project will facilitate expansion of existing, and the creation of new, markets and companies, increasing European prosperity. For example, the high-value demonstration molecules targeted in the project offer an opportunity to upgrade plant based raw materials to high value co-products. Such approaches offer an important alternative market for European agriculture in a period where subsidised production of bulk products is being reduced significantly. With the increased energy costs, environmental implications as well as the fossil fuel based production systems, the markets for high-value natural products like those targeted in the present project are likely to expand rapidly. In addition such products have a high export value. 4. Socio-economic impact. In 2050 it is estimated that there will be more elderly people than children in several parts of the world, particularly in Europe. Maintaining the health and well-being in an ageing population will have important economic implications on national health systems. Reducing the production costs of useful natural products such as those targeted in the METAPRO project would greatly reduce the costs of pharmaceuticals, nutraceuticals, cosmoceutricals and over the counter (OTC) medicines in which they are used. In addition, reduced costs will stimulate wider and more accessible use. The utilisation of renewable bio-resources will also enable the consumer to have a natural choice instead of a reliance on the synthetic market. In Europe the present chemical synthesis mode of production is often linked to the petro-chemical industry for the supply of precursors and thus prone to price fluctuations. The use of plant based production platforms would improve self-sustainability and dependence on volatile markets. Thus, through its objective of reducing production costs and improving sustainability the outputs from METAPRO will have direct economic implications in future years. The project has illustrated how the chemical industry can be replaced by reliable and competitive biosources. 5. Training. The impact EU networks have on training is often overlooked and credit for this achievement not promoted to the level it deserves. The METAPRO project has provided an unprecedented opportunity for the training of EU based scientists to the very highest standards. Over the course of the project over five postgraduate students have been trained, six targeted training missions have been carried out and metabolite training school run which attracted 140 applicants for 14 places. To highlight the PhD students trained students from France and Romania were trained in the UK acquiring University of London degrees, this unprecedented opportunity could not have been achieved without the METPRO activities. The process will create a workforce with high technical expertise and leadership that can be utilised in European scientific industries and related businesses. A workforce with these skills will be essential to achieve the EC objective to build the world’s most competitive knowledge-based economy. METAPRO has contributed to the EC objective to build the world’s most competitive knowledge-based economy. 6. Rural development. The METAPRO has provided opportunities for rural development (e.g. Romania, southern Italy and north east Scotland). Expertise developed has in each case remained local. This approach is in line with the policy of involving rural areas into the globalisation process by ensuring an active economic role. 7. Improved consumer quality and safety. The METAPRO project has delivered the tools and resource necessary to provide the consumer with a choice of a natural product over a synthetic product derived from the chemical industry. There is also potential for a wider consumer choice, greater availability and cheaper costs. In additional to the improved quality the increasing evidence of health benefits conferred by crocetin and ketocarotenoids will lead to incorporation of these metabolites into a wider range of products. This will impact both on quality and potential health benefits which have direct implications on the health of the nation, well-being and prosperity. The utilisation of bio-resources instead of chemical synthesis means safety aspects are generally improved. There is less chance of chemical contamination so the working environment is improved for employees and the process platform will have less impact on the environment. Feasibility studies will enable us to acquire precise data for the evaluation of these potential beneficial impacts to human and animal health, as well as conferring quality to food and feedstuffs. 8. Industrial policy and employment. The scientific and technological advances will potentially impact on European competitiveness creating new and increased markets leading to increased economic growth and job creation. Industrial partners including SMEs are actively involved in the programme fostering industrial cooperation and capacity building at a European level. To date the METAPRO project has created four permanent jobs in the public sector and 15 fixed term employment opportunities. 9. European cohesion. The METAPRO project has through the transfer of technology and joint efforts of selected experts with complementary skills and industrial cooperation created European cohesion and capacity building at a European level. In addition further global links have been established which has resulted in METAPRO activities contributing to capability building in developing countries. 10. Contribution to policy developments. METAPRO partners have presented and contributed data to National agencies and the EC. In addition the coordinator has been involved in EU/US taskforce activities associated with Plant Biotechnology-added value crops. These events contribute to policy especially in the case of reducing chemical refining and the promoting of biorefining procedures. 11. Ethical issues. Participants in the METAPRO have at all times complied with current legislation and regulations in the countries where the research has been carried out. We have performed ethical reviews on the use of GM periodically throughout the project. With the generation of plant varieties with such high ketocarotenoid content and the potential they offer the conclusion is that it is unethical not to pursue their utilization. 12. Contributions to standards. METAPRO implemented procedures for to ensure accuracy, traceability and reliability of the data and procedures, to health and safety standards, and to staff development and training. The consortium worked to community standards relating to the reporting of “omic” datasets. The consortium has and will continue to engage in data sharing embracing the policies of National funding agencies. Data management has been addressed throughout the project. Metabolomic experiments have been performed in a MIAMET (Minimum Information About a Metabolomic Experiment) compliant manner, while transcriptomic datasets will be MIAME (Minimum Information About a Microarray Experiment) compliant. The contextual information or documentation (“metadata”) is essential for the project partners and future secondary users. The consortium agreement will ensure access to all datasets and accompanying metadata for the project participants. After publication and intellectual property rights have been secured, which we predict to be within three years after the completion of the project, the metadata and datasets will made publicity available through third party databases. Depending on the level of usage and collaborative nature the project partners expect joint authorship or acknowledgements to the data originator. All outputs from the scientific activities shall adhere to the principles of good laboratory practice. The aim in this instance is to provide technical assistance, capacity building and technology transfer for developing countries. 13. Dissemination. The METAPRO programme has disseminated its findings and activities through a number of verifiable format for example, (i) scientific papers submitted to journals and presented at conferences, (ii) through press releases, (iii) through meetings organised and/or attended by partners, (iv) public engagement including local meetings, school and college visits and open days, web TV/ podcast, public facing websites and newsletters, (v) articles in technical journals, (vi) presentations at technical conferences and exhibitions, (vii) articles at market orientated journals, (viii) patents, PhD and MSc theses and (ix) through international workshops and (x) through the websites. Thus the dissemination activities have been extensive. Four different examples will be highlighted; (i) Dissemination of METAPRO to Early Stage Researchers. The metabolite profiling training school, its associated mini-symposium and short training missions have been a highlight of the METAPRO dissemination and training activities. Working with the COST ACTION active in this area 140 applicants applied for 14 places on the training school held in London (RHUL). These 14 participants came from 14 different countries across Europe. The course covered sample preparation to data analysis. Presentations on METAPRO activities were made at the start of the course and during the mini-symposium. In addition the technique demonstrated first hand during the course was developed in METAPRO. There was also an opportunity for the course participants to discuss activities with staff engaged in METAPRO activities. Finally, a trip to Syngenta was carried out during the course to acquire an industrial perspective. To complement these activities several short training mission were carried out for consortium partners and early stage researchers from developing countries. (ii) Dissemination to the scientific community. Already METAPRO has generated a number of peer reviewed scientific publications in high impact journals such as PNAS, Plant Cell, and Science. We anticipate more publications of this quality to follow. Another highlight for the scientific community was presentations made at high profile international conferences and COST ACTION meetings. For example, at the International Plant Molecular Biology Conference in Korea, 2012, within the plant secondary metabolite section two of the five speakers described METAPRO activities. Presentations have also been given at the Plant and Animal Genome (PAG) conference 2012, and International Metabolomics conference. At the annual COST ACTION meeting in Murcia 2011, METAPRO participated in the event that brought together several networks funded through the EUFP7 KBBE programme. This activity forged future links and interactions between projects. For example the transfer of resources such as specific plant varieties, constructs and authentic standards. (iii) Interaction with industry. METAPRO outputs have been disseminated to industry through individual seminars and also a highlight being the participation in an industrial exploiation workshop held in Japan. It is through the latter activity that a potential route to market was forged. (iv) Dissemination to the general public. The website has acted as a focal point for interested parties from the general public. In addition, METAPRO dissemination material has been provided at a number of science open days to the public. The highlight is however the promotional videos of the project, these are of the highest quality describing the project and its outputs with interviews with Early stage researchers. We will maintain the project website after the end of RTD activities and the promotional videos placed on CommNET (www.commnet.eu) as accessible archives.
List of Websites:
www.isoprenoid.com
Dr Paul D. Fraser, Centre for Systems and Synthetic Biology, School of Biological Sciences, Royal Holloway University of London, Egham Hill, Egham, Surrey, TW20 OEX.UK. Tel: 01784 443894, E-mail: p.fraser@rhul.ac.uk Publications whereby the on-line system will not let entry: 1. Lu, Y., Rijzani, H., Karcher, D., Ruf, S. and Bock, R. (2013). Efficient metabolic pathway engineering in transgenic tobacco and tomato plastids with synthetic multigene operons. Proc.Natl. Acad. Sci. USA, 110, e623-e632. 2. Mora, L., Bramley, P.M. and Fraser, P.D. (2013). Developemnt and optimisation of a lable-free quantitaive proteomic procedure and its application in the assessment of genetically modified tomto fruit. Protemics, Doi:10.1002/pmic.200790091.
Patents (whereby the on-line system would not permit entry).
1. Jones, M.O. Bramley, P.M. Fraser, P.D. (2010). Increased phytochemical production in plants. GB1008551.2.
2. Fraser, P.D. and Mortimer, C.L. (2011). Hydrocarbons from Plants. GB1108436.5
3. Baxter, C., Bradley, G., Pan, Y., Ball, G., Hodgman, T.C. Seymour, G.B. Wood, A and Fraser .P (2011). Modulation of tomato fruit ripening. International Patent Application submitted end September 2011, awaiting Publication Number. Supersedes European Patent Application of same filed 2009, Application number 10183748.2