Progress 09/01/08 to 08/31/12
Outputs
OUTPUTS: We have obtained several GPPS-SSU transgenic tomato lines that produce high levels of geraniol, geranial, neral, citronellol and citronellal in fruits (all transgenes in this study are expressed under the control of a strong fruit-ripening specific promoter). Ripe fruits of these plants are also less red than those of control plants, indicating that some substrate is diverted from carotenoid biosynthesis to GPP formation. Evidently, with the lack of expression of endogenous TPS genes, the produced GPP is hydrolyzed by non-specific phosphatases to geraniol, which then becomes converted to the other observed products by endogenous reductases and alcohol dehydrogenases. Combining these GPPS-SSU plants with transgenic tomato lines overexpressing geraniol synthase (GES) resulted in plants that produce very large amounts of geraniol and related products in their fruits (a more than 3-fold increase compared to the GES parental line). Co-expression of GPPS-SSU with ZIS, a cytosolic TPS using preferentially FPP as substrate and only to a minor extent GPP, resulted in the increased formation of the ZIS monoterpene products in tomato fruits. We also obtained transgenic tomato lines expressing NDPS1 (Gutensohn et al., to be submitted). The fruits of these NDPS lines accumulate large amounts of nerol, the hydrolysis product of NPP, suggesting the absence of endogenous TPS enzymes in tomato fruits capable of using this substrate. NDPS tomato fruits also have severely reduced carotenoid contents indicating a redirection of the metabolic flux from carotenoid towards NPP formation. To investigate availability of produced NPP for monoterpene formation, tomato lines were constructed expressing beta-phellandrene synthase (PHS). PHS expression alone in tomato fruits caused production of only little ocimene and myrcene, indicating the presence of a small endogenous GPP but no NPP pool in fruits. Its co-expression with GPPS-SSU strongly increased formation of these monoterpenes, confirming the ability of PHS to also accept GPP as substrate. Co-expression of NDPS and PHS resulted in the production of very large amounts of beta-phellandrene and several other monoterpenes similar to those detected in tomato trichomes. This blend of monoterpenes formed from NPP by PHS in the NDPS/PHS coexpressing tomato fruits had a negative effect on the feeding of tomato fruitworms (Helicoverpa zea). PARTICIPANTS: Dr. Vasiliki Falara - Post-doc (U. of Michigan): Examining the genomic organization of the terpene synthase (TPS) familiy in tomato and the expression of these genes in fruits. Dr. Thuong Nguyen - Post-doc (U. of Michigan) - preparing tomato fruits and RNA samples for RT-PCR for examing the expression of the TPS genes. Dr. Tariq Akhtar - Post-doc (U. of Michigan) - Examing the expression of the TPS genes in tomato fruit. Dr. Yuki Matsuba - Post-doc (University of Tokyo) - isolated and enzymatically assayed terpene synthases. Richard Xu - undergraduate (U. of Michigan) - preparing vector constructs for tomato transformation. Dr. Michael Gutensohn - Post-doc (Purdue University) - Generating and crossing transgenic tomato plants, analysis of transgene expression (RNA, protein, enzyme activities) and metabolic profiling (carotenoids, emission and internal pools of mono-/sesquiterpenes). Christine Kish - technician (Purdue University) - genetic characterization of > 500 transgenic plants (genomic DNA isolation, screening for presence of transgenes by PCR). TARGET AUDIENCES: The target was the scientific community of plant researchers, plant breeders, flavor scientists. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The project has used recent high-through sequencing approaches to analyze the complete genetic system responsible for synthesis of terpenes in tomato. Tomato is now the most comprehensive system to study terpene biosynthesis, especially in fruits, and has therefore become the model system of choice. Several breakthrough results have been obtained, notably the use of unusual substrates by various terpene synthases, and the transport of various terpene precursors across organellar boundries. In addition, a large number of students and post-docs were trained in various plant chemical and biochemical techniques and acquired expertise in areas that are in great demand in academia and industry.
Publications
- Chen F, D Tholl, J Bohlmann, E Pichersky. The family of terpene synthase in plants: A mid-size family that is highly diversified throughout the kingdom. Plant J 66:212-229 (2011). Falara V, T Akhtar, TTH Nguyen, EA Spyropoulou, PM Bleeker, I Schauvinhold, Y Matsuba, ME Bonini, AL Schilmiller, RL Last, R Schuurink, E Pichersky. The tomato (Solanum lycopersicum) terpene synthase gene family. Plant Physiol 157:770-789 (2011). Falara V, E Pichersky. Plant volatiles and other specialized metabolites: synthesis, storage, emission, and function. In: Secretions and Exudates in Biological Systems (J Vivanco, Frantisek Baluska, eds), Springer, pp 109-123 (2012). Heinig U, Gutensohn M, Dudareva N, Aharoni A. The challenges of cellular compartmentalization in plant metabolic engineering. Curr. Opin. Biotechnol., http://dx.doi.org/10.1016/j.copbio.2012.11.006 (2012). Gutensohn M, Nagegowda DA, Dudareva N. Involvement of compartmentalization in monoterpene and sesquiterpene biosynthesis in plants. In: Bach T.J. and Rohmer M. (eds) Isoprenoid synthesis in plants and microorganisms: New concepts and experimental approaches, Springer, New York, pp 155-169 (2013). Gutensohn M, Orlova I, Nguyen TTH, Davidovich-Rikanati R, Feruzzi M, Sitrit Y, Lewinsohn E, Pichersky E, Dudareva N. Cytosolic monoterpene biosynthesis is supported by plastid-generated geranyl diphosphate substrate in transgenic tomato fruits. Plant J (submitted). Davidovich-Rikanati R, Y Sitrit, Y Tadmor, E Pichersky, N Dudareva and E Lewinsohn. Tomato Aroma: Biochemistry and Biotechnology. In Biotechnology in Flavor Production, (eds N Dudai and D Havkin-Frenkel). (submitted) Gutensohn M, Nguyen T, Kaplan I, Pichersky E, Dudareva N. In planta product formation of the tomato terpene synthase PHS1 depends on the precursor provided by neryl diphosphate synthase or geranyl diphosphate synthase. (to be submitted)
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Progress 09/01/10 to 08/31/11
Outputs
OUTPUTS: With the tomato genome sequence recently (end of 2009) becoming available, we were able to identify and characterize the entire set of TPS genes in tomato. We showed that tomato has 29 functional TPS genes, and several of them are expressed, albeit at very low levels, in ripening fruit (Falara et al., 2011). Since we had previously noted that GES transgenic fruits make myrcene and limonene even though GES is not able to make such products, we examined the expression of all endogenous TPS genes in GES transgenic plants and identified one gene whose expression is induced in the transgenic line. Subsequent biochemical work showed that this enzyme indeed encodes a myrcene/limonene synthase. This is an example of how the introduction of a transgene can affect the activity of an endogenous gene. When we started this project it was assumed that all momoterpene synthases use geranyl diphosphate (GPP) as the substrate. After this project begun, we discovered in a parallel project that tomato has an enzyme, called NDPS1, that makes neryl diphosphate, the cis-isomer of GPP (but from the same precursors that GGP synthase uses), and that a monoterpene synthase called PHS1 (β-phellandrene synthase) uses NPP rather than GPP. NDPS1 and PHS1 are normally expressed in the leaf and stem trichomes, not in the fruit. Since carotene biosynthesis uses GPP as an intermediate but not NPP, we have set out to make transgenic tomatoes that overexpress the small subunit of GGPS (which is sufficient to make more GPPS holoenzyme, since the large subunit of GPPS is present in the fruit at high levels) or NDPS2 and determine which approach is better for increasing terpenes in the fruit. Once we are able to increase substrate availability for TPSs, we intend to combine such lines with a second transgenic line that overexpresses an introduced TPS. We have now obtained several GPPS-SSU transgenic tomato lines that produce high levels of geraniol, geranial and neral (the latter two are the oxidation products of geraniol). These plants are also less red than control plants, indicating that some substrate is diverted from carotene biosynthesis to GPP biosynthesis. Evidently, with the lack of expression of endogenous TPS genes, the accumulated GPP is hydrolyzed by non-specific phosphatases to geraniol. We have also obtained NDPS1-transgenic tomatoes (all transgenes in this study are fused to a strong fruit-ripening promoter). These fruits too are much paler than control, and they accumulate large amounts of nerol. Again, NPP is synthesized but in the absence of a TPS enzyme that can use it as a substrate it is hydrolyzed to nerol. It therefore appears that increasing availability of both GPP and NPP in the fruit is feasible. We have also obtained transgenic lines that overexpress PHS1 in the fruit. They do not produced β -phellandrene, indicating that NPP is not normally available in the fruit. Combining PHS1 and NDPS1-transgnic lines should result in plants that produce high levels of β-phellendrene in their fruits. PARTICIPANTS: Training: Dr. Vasiliki Falara - Post-doc (U. of Michigan): Examining the genomic organization of the terpene synthase (TPS) familiy in tomato and the expression of these genes in fruits. Dr. Thuong Nguyen - Post-doc (U. of Michigan) - preparing tomato fruits and RNA samples for RT-PCR for examing the expression of the TPS genes. Dr. Tariq Akhtar - Post-doc (U. of Michigan) - Examing the expression of the TPS genes in tomato fruit. Yuki Matsuba - Post-doc (University of Tokyo) - isolkated and enzymaticall assayed terpene synthases. Richard Xu - undergraduate (U. of Michigan) - preparing vector constructs for tomato transformation. Dr. Michael Gutensohn - Post-doc (Purdue University) - Generating transgenic tomato plants and analysis of expression of transgenes and metabolic profiling of fruit volatiles. Christine Kish - technician (Purdue University) - preparing genomic DNA from 520 transgenic plants to screen for the presence of transgenes by PCR. Collaborations: In this project we have collaborated with a group in Israel led by Dr. Efraim Lewinsohn and a group in England led by Drs. Peter Bramley and Paul Fraser, sharing transgenic tomato lines with them and the results of metabolic profiling. We are also collaborating with a group led by Drs. Haring and Schuurink in The Netherlands on the biochemical characterization of TPS enzymes. TARGET AUDIENCES: Publications in scientific journals are aimed at the research community and industry. Presentation at scientific meetings were aimed at same audience plus undergraduates. PROJECT MODIFICATIONS: The research program proceeded as planned.
Impacts
The project has used recent high-through sequencing approaches to analyze the complete genetic system responsible for synthesis of terpenes in tomato. Tomato is now the most comprehensive system to study terpene biosynthesis, especially in fruits, and gas therefore become the model system of choice. Several breakthrough results have been obtained, notably the use of unusual substrates by various terpene synthases, and the transport of various terpene precursors across organellar boundries. In addition, a large number of students and post-docs were trained in various plant chemical and biochemical techniques and acquired expertise in areas that are in great demand in academia and industry.
Publications
- Chen F, D Tholl, J Bohlmann, E Pichersky. The family of terpene synthase in plants: A mid-size family that is highly diversified throughout the kingdom. Plant J 66:212-229 (2011).
- Falara V, T Akhtar, THT Nguyen, EA Spyropoulou, PM Bleeker, I Schauvinhold, Y Matsuba, ME Bonini, AL Schilmiller, RL Last, R Schuurink, E Pichersky. The tomato (Solanum lycopersicum) terpene synthase gene family. Plant Physiol 157:770-789 (2011).
- Falara V, E Pichersky. Plant volatiles and other specialized metabolites: synthesis, storage, emission, and function. In: Secretions and Exudates in Biological Systems (J Vivanco, Frantisek Baluska, eds), Springer, pp 109-123 (2012).
- Bleeker PM, EA Spyropoulou, PJ Diergaarde, H Volpin, MTJ De Both, P Zerbe, J Bohlmann, V Falara, Y Matsuba, E Pichersky, MA Haring, RC Schuurink. Sesquiterpene synthases in S. lycopersicum and S. habrochaites trichomes: Discovery by RNA-Seq and intra- and interspecific variation in expression and activity. Plant Mol Biol 77:323-336 (2011).
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Progress 09/01/09 to 08/31/10
Outputs
OUTPUTS: So far we have concentrated on the first two goals. Transgenic tomato plants expressing various TPS genes or related genes in the pathway have been generated: GES (geraniol synthase), ZIS (zingeberene synthase), PHS1 (β-phellandrene synthase), NDPS1 (neryl-PP synthase, synthesizes the substrate of PHS1), GPPS-SSU (subunit of the enzyme that makes geranyl-PP, the substrate of many monoterpene synthases), NES/LIS1 (cytosolic enzyme catalyzing the formation of the sesquiterpene nerolidol from farnesyl-PP and the monoterpene linalool from GPP), NES/LIS2 (plastidic enzyme with the same terpene synthase activities as NES/LIS1), all under the control of the fruit ripening-specific PG promoter, and a bacterial DXS gene (encoding a key enzyme in the MEP pathway providing IPP and DMAPP substrate for GPP and NPP biosynthesis) under the control of fruit ripening-specific fibrillin. After selection and regeneration of plants in tissue culture, the putative transgenic tomato plants were adapted to greenhouse conditions. A total of 520 transgenic plants were generated. All putative transgenic lines were analyzed by PCR on genomic DNA using primers specific for each respective transgene. For 99 of 203 putative GPPS-SSU lines, 29 of 44 putative NES/LIS1 lines, 56 of 77 putative NES/LIS2 lines, 26 of 30 putative NDPS1 lines and 97 of 98 putative PHS1 lines we were able to verify the presence of the respective transgene. From the first transformation using the bacterial DXS construct we had obtained 7 putative transgenic lines but only for 2 the transgene could be confirmed. Therefore a second transformation was initiated and recently resulted in 20 additional putative transgenic lines which will be further analyzed. Positively tested transgenic lines were grown to maturity to obtain fruits for subsequent analyses and were also propagated by stem cuttings. The fruits obtained from these plants were used for first analyses of transgene expression and/or production of volatiles, in particular terpenes. Our preliminary results with these transgenic lines are reported in the next section ("Outcomes"). PARTICIPANTS: Dr. Vasiliki Falara - Post-doc (U. of Michigan): Examining the genomic organization of the terpene synthase (TPS) familiy in tomato and the expression of these genes in fruits. Dr. Thuong Nguyen - Post-doc (U. of Michigan) - preparing tomato fruits and RNA samples for RT-PCR for examing the expression of the TPS genes. Dr. Tariq Akhtar - Post-doc (U. of Michigan) - Examing the expression of the TPS genes in tomato fruit. Richard Xu - undergraduate (U. of Michigan) - preparing vector constructs for tomato transformation. Dr. Michael Gutensohn - Post-doc (Purdue University) - Generation of transgenic tomato plants and analysis of expression of transgenes and metabolic profiling of fruit volatiles. Christine Kish - technician (Purdue University) - preparing genomic DNA from 520 transgenic plants to screen for the presence of transgenes by PCR. TARGET AUDIENCES: The target audience is other researchers in the field (academia, plant breeding industry) and the companies themselves engaged in biotechnology. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
1) Among the confirmed GPPS-SSU transgenic tomato lines we observed several lines producing ripe fruits with a red color similar to the untransformed MP-1 control as well as a large number of lines producing ripe fruits with an orange color. A first non-quantitative RT-PCR performed for a subset of lines indicated that the GPPS-SSU transgene is expressed in red and orange fruit producing lines. We have also analysed the accumulation of volatiles in ripe fruits of a subset of the GPPS-SSU lines using closed-loop stripping (as described by Dudareva et al., 2005), solid-phase micro extraction (SPME) and methyl tert-butyl ether (MTBE) extraction. Analysis of the collected volatiles by GC-MS indicated that red GPPS-SSU fruits produce linalool while this monoterpene was absent in MP-1 fruits and orange GPPS-SSU fruits. The latter contained citronellal, neral and geranial which were either not detected or detected in lower amounts in the MP-1 control. 2) Analysis of the volatiles present in ripe fruits of 16 of the confirmed NES/LIS1 tomato lines using SPME indicated that fruits of 5 lines accumulated the sesquiterpene nerolidol which was absent in ripe fruits of the untransformed MP-1 line. An identical SPME analysis of volatiles performed with confirmed NES/LIS2 lines showed that ripe fruits from 9 of 10 tested lines accumulated the monoterpene linalool which is not found in fruits of the MP-1 control. These results are consistent with the cytosolic (NES/LIS1) and plastidic (NES/LIS2) localization of the two nerolidol/linalool synthases (Nagegowda et al., 2008). 3) SPME analysis of fruit volatiles for 10 confirmed NDPS1 transgenic lines revealed that 5 of those lines produced nerol, which was not found in the MP-1 control. However, further analysis of NDPS1 transcript levels are required to confirm that the observed changes in the fruit volatile profile correlate with the transgene expression in these lines. 4) We have also examined the expression of the entire set of terpene synthase (TPS) genes in the the fruit of the GES transgenic lines. We had previously reported that these plants have, in addition to increased synthesis of geraniol, also show increased synthesis of several other monoterpenes that are not likely to be the product of the geraniol synthase transgene. With the sequence of the tomato genome now available, we prepared primers to check the expression of all TPS genes in the fruit, and carried out such an analysis by RT-PCR. Our preliminary results are that there are several TPS genes that whose expression is increased in the GES transgenic line. We are currently analyzing the biochemical activities of the enzymes encoded by these genes.
Publications
- Chen F, D Tholl, J Bohlmann, E Pichersky. The family of terpene synthase in plants: A mid size family that is highly diversified throughout the kingdom. Plant J, in press (2011).
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Progress 09/01/08 to 08/31/09
Outputs
OUTPUTS: Generation of tomato plants expressing various genes involved in biosynthesis of flavour terpenes in fruits: We have made constructs to express some newly discovered genes in tomato under the control of the ripening fruit-specific promoter PG: PHS1 (α-phellandrene synthase, a new discovered tomato gene usually expressed in trichomes), NDPS1 (neryl-PP synthase, a new discovered tomato gene usually expressed in trichomes whose encoded protein synthesizes the substrate of PHS1), GPPS-SSU (a snapdragon gene that encodes a critical subunit of the enzyme that make geranyl-PP, the substrate of many monoterpene synthases), NES/LIS1 (a snapdragon gene encoding a cytosolic enzyme that catalyzes the formation of esquiterpene nerolidol from farnesyl-PP and monoterpene linalool from GPP), NES/LIS2 (a snapdragon gene encoding a plastidial enzyme with the same terpene synthase activities as that for NES/LIS1). Finally, we have obtained a construct of a bacterial DXS gene (encoding a key enzyme in the MEP pathway that provides IPP and DMAPP substrate for GPP and NPP biosynthesis) under the control of the fruit-ripening-specific fibrillin promoter. Transformation of tomato plants with all these constructs is now in progress, after growing tomato lines to increase the number of seeds, since current transformation procedures require a lot of seeds. In addition, we have already obtained transgenic plants from past experiments that express the linalool synthase (LIS), geraniol synthase (GES), and zingiberene synthase (ZIS; zingiberene is a sesquiterpene) under the PG promoter; ZIS expressed under the 35 S promoter; and DXS under the fibrillin promoter. The PG -GES and ZIS transgenic plants have been crossed to obtain F1 plants that express both. Crosses with the DXS transgenic plants are currently on their way. Measuring the levels of expression of endogenous terpene synthases in leaves and fruits of transgenic tomato plants We grew plants of the currently available transgenic lines - pgGES, pg-ZIS, 35S-ZIS, fDXS- and their controls and harvested fruit at 4 different stages: mature green (MG), braker (Br), braker +3 days (Br+3) and ripe red (R) for RNA extraction. We are momentarily harvesting fruits of these 4 ripening stages from a second set of GES-, ZIS- and MP-1 tomato plants. A third set of these 3 tomato lines is growing in the greenhouse right now and will produce fruits soon. The RNA obtained from these three independent biological samples will be used for large scale expression analysis with a DNA chip containing 44,000 probes representing all tomato ESTs. Likewise, these RNAs will be used for quantitative RT-PCR analysis to determine expression levels of selected genes involved in the MEP and MVA pathways. PARTICIPANTS: In addition to the PD Eran Pichersky, and the Co-PD Natalia Dudareva, two post-doctoral associates are involved in carrying out the experiments they are being trained in molecular biology methods, metabolomics, and analytical chemistry. They are Drs. Thuong Nguyen and Michael Gutensohn. TARGET AUDIENCES: The scientific communitit studying flavor and aroma. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
In bioinfromatic searches we have identified the extent of tomato terpene synthase gene family members for which some DNA sequence information is available. These genes or putative genes include a total of five putative monoterpene synthases and five sesquiterpene synthases. We are designing primers for testing their expression in tomato fruit by qRT-PCR.
Publications
- No publications reported this period
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