BioMatNet Item: FAIR-CT97-3628 - Improving production and quality of essential oil from aromatic plants by genetic engineering

FAIR-CT97-3628
Improving production and quality of essential oil from aromatic plants by genetic engineering

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Biotechnology : Essential Oil : FAIR Area 1.2 - Green Chemicals and Polymers Chain : Fine Chemicals : Flavours/Fragrances : Pharmaceuticals/Cosmetics : Plant Genetics

	Proposal No:	FAIR-CT97-3628
	Date Prepared:	March 1999
	Source:	First Annual Progress Report Abstract & Executive Summary

First Annual Progress Report Abstract

Objectives

The overall aim is to determine the molecular basis regulating terpenoid production by plant cells in a process that applies genetic engineering to improve rose essential oil yield. Genetic engineering will be applied to alter cell concentration of key regulatory enzymes of terpenoid production as well as the levels of components of the signal transduction pathway that activate the genes for such enzymes. There is also an effort to elucidate aspects of the molecular mechanism regulating essential oil production. The role of trans-acting proteins, activating regulatory genes of terpenoid production in rose genotypes and transformants will be examined by isolating their genes and relating their transcriptional activity and availability of their active products to essential oil yield. It is hoped to uncouple terpenoid production from cell differentiation and thus to enable future large-scale terpenoid synthesis in bioreactor like facilities.

Activities

It is most probable that the limiting factor in essential oil synthesis is the availability of the enzyme HMGR produced by the gene hmgr. Heterologous constitutively expressed hmgr will be introduced into rose plants to increase the cell levels of HMGR and therefore to achieve higher rate of terpenoid synthesis and to enhance essential oil yield. For the same reason, stimulation of the endogenous hmgr expression will be attempted by altering the cell concentration of peroxides and free radicals that affect transcription of hmgr. This will be done by using antisense constructs to down-regulate the genes cat and apx that are involved in removing such second messengers. Trans-acting factor genes will be isolated from cDNA libraries by screening with hmgr cis elements or using the yeast hybrid trap system. Their involvement in regulating terpenoid production in response to stress factors will be examined

Progress

Thirteen rose varieties have been put into in vitro callus culturing conditions to prepare them for transformation by the selected genes. Three of them have produced embryos. One of them has also successfully produced regenerated non-transformed plants and can be used in transformation experiments. Gene constructs for sense hmgr, cat, and apx, as well as for antisense cat have been made and have been transformed into A. tumefaciens ready for the transformation process. Methodology for the isolation of rose leaf and callus terpenoids from small amounts of tissue have been developed. Isolation of pure rose DNA and its digestion to inserts of proper size has been done. Efforts are still under way to construct a nuclear DNA library.

Achievements

The establishment of rose callus culturing and regeneration conditions and the production of the gene constructs to be used in the transformation experiments were the two milestones for the first year of the project. Both tasks were accomplished in the expected time frame.

Future activities

Two main technical accomplishments are due next year. The first is the production of transgenic roses. It is a milestone. The second milestone due is the isolation of the rose hmgr cis-elements and of any corresponding transcription factor genes. This requires prior construction of DNA and cDNA libraries. A very important part of the project would be the examination of the effect of the introduced genes on the callus tissue. That includes analysis of any terpenoids or other secondary metabolites synthesised in the callus, checking the presence of transcript and the coded protein corresponding to the transgene, and examination of the regenerability and growth rate of the callus tissue.

First Annual Progress Report Executive Summary

Introduction

The overall objective is to probe into the mechanism of regulation of the plant terpenoid synthesis pathway in order to device a general way of manipulating it in the great variety of plant species. Rose plants were chosen as the experimental material because of the simplicity of their terpenoid pathway that terminates in a few simple monoterpenes, because of the preliminary successful in vitro culturing and transformation work done by other labs, and because of the economic importance of the species. The great majority of the available literature points to the enzymatic reaction catalysed by hydroxymethyl coenzyme A reductase (HMGR) as the main and most upstream terpenoid synthesis regulating step determining the final total amount of terpenoid content in plant tissues.

Downstream regulatory steps encountered in species with more lengthy pathways share with HMGR the characteristic that their regulation is at the transcriptional level and responds to stress conditions of the biotic or abiotic type. The majority of the data on the mode of stress signal transduction to transcriptional activation of secondary metabolism genes, including hmgr, support the involvement of active oxygen species and free radicals in the process.

We have attempted in this project to combine the production of transgenic plants of increased terpenoid production to a more in-depth look into the role of the free radical system in plant secondary metabolism. More specifically the following specific objectives were set:

To transform rose plants with heterologous constitutively expressed hmgr in an effort to modify the cell concentration of active enzyme and to speed up terpenoid synthesis. Uncoupling of terpenoid synthesis from cell differentiation could also be one of the results since the introduced gene would not carry the normal promoter regulated by internal cell transcription factors. If successful, this would open the field of in vitro secondary metabolite production in bioreactor of other type of facilities.
To transform rose plants with constitutively expressed sense and antisense constructs of the active oxygen and free radical scavenging system genes for catalase (cat) and ascorbate peroxidase (apx). Down-regulation of these genes by their antisense copies or by co-suppression should result in increased cell concentrations of active oxygen species, lipid peroxides and free radicals. These potential second messengers in the signal transduction pathway activating secondary metabolism genes could activate the endogenous hmgr and other regulatory genes resulting, among others, in higher terpenoid synthesis by a sort of genetically imposed oxidative stress
To isolate and characterise the regulatory cis-elements of the rose hmgr gene and to use it for the isolation of the corresponding transcription factor genes regulating hmgr transcription. This is an effort to further elucidate upstream the signal transduction path leading from stress perception to secondary metabolism gene activation. It enlarges the possibilities or fine tuning the regulation of these and other responding genes in future projects.

This reporting period is for the first year of the project. The work done to accomplish the specific objectives is as follows:

Thirteen rose varieties have been put into in vitro callus culturing conditions to prepare them for transformation by the selected genes. Three of them produced embryos. One of them has also successfully produced regenerated non-transformed plants and can been used in transformation and raising of transgenics.
Gene constructs for sense hmgr , cat, and apx, as well as for antisense cat have been made and have been transformed into A. tumefaciens ready for the transformation process.
Methodology for the isolation of pure rose DNA and its digestion to inserts of proper size, for the isolation of intact rose flower RNA, and for the isolation of rose leaf and callus terpenoids have been adapted. Efforts were done to construct a nuclear DNA library.

Results

Task 1: Analysis of hmgr trans-acting factor/cis-elements involvement in essential oil synthesis. Sub-task 1: Construction of rose nuclear DNA and cDNA libraries. There were some technical difficulties with obtaining pure rose DNA. This species is apparently very rich in DNA-oxidising phenolics and mucopolysaccharides contaminants. Adjusting the concentration of antioxidants and phenol-binding components in the homogenisation medium solved the problem of the phenolics. Removal of the polysaccharides was impossible and isolation of DNA from a crude nuclei pellet was employed instead. Inserts of proper size were produced but repeated efforts to produce a nuclear library were not successful. Only a few thousand clones per microgram of phage DNA were obtained. We reckon that the ligation reaction must be optimised and the dephosphorylation of the insert must be re-examined. RNA was not available because the plants were not in the flowering season. A small amount of flowering was artificially induced using temperature stress but the tissue was used to optimise the RNA isolation procedure. It was enough for isolating mRNA.

Sub-task 1.2: Isolation of hmgr cis-elements. Preliminary PCR experiments with primers to conserved regions of the hmgr gene was performed. Several bands were obtained and the major ones were sequenced and one more is still at the sequencing stage. Their use would be in the screening of the rose DNA library to isolate clones carrying the hmgr gene. However, non of the sequenced PCR products seems to be derived from the hmgr gene. The use of the available heterologous Artemisia annua probe seems the most probable alternative.

Sub-task 1.3: Isolation and characterisation of trans-acting factor gene(s). Work on this subtask has not started yet. Requires the construction of a cDNA library.

Sub-task 1.4: Analysis of trans-acting factor gene expression in natural rose genotypes and transformants of high and low essential oil yield. Work on this subtask does not start until rose genes for transcription factors regulating hmgr expression have been isolated.

Task 2: Production of transgenic roses. To avoid the possibility of having plants that can not be regenerated, a large number of varieties had to be tested avoiding difficulties of transferring from France to England enough callus cultures of varieties used and also in order to double for safety reasons the efforts of producing transgenic roses.

Sub-task 2.1: Establishment of rose tissue cultures Thirteen rose varieties were put into embryogenic callus cultures, one of which has been shown to regenerate successfully. This is a milestone that has been accomplished within the expected time frame. There were no particular technical problems, but the need to test a large number of genotypes moves back the time of production of trangenics by about six months and puts strict time limits on the task of testing them under greenhouse conditions.

Sub-task 2.2: Production of hmgr, cat, and apx constructs. Sense hmgr, cat and apx, as well as antisense cat, constructs have been made. These have been transformed into Agrobacterium strain LBA 4404 and are ready to be used in rose transformation.This is also a milestone accomplished in the expected time frame. The use of antisense apx is under reconsideration since a similar role is expected to be played by the antisense cat.

Sub-task 2.3: Transformation and regeneration of transgenics. Only recently started.

Sub-task 2.4: Analysis of transgene expression. Does not start until transgenic plants or at least transgenic callus is available. According the technical annex this is after the 18 th month of the project.(around month 18 of the project).

Task 3: Analysis of terpenoids and growth performance of transgenics

Sub-task 3. 1: Analysis of terpenoid production. This subtask requires transgenic tissue to be available. Methodology for extraction of terpenoids from small amounts of rose flowers as well as preliminary GC-MS analysis of the extracts has been developed.

Sub-task 3.2: Greenhouse testing. Starts when transgenic roses are available and most probably around the beginning of the third year of the project.

Sub-task 3.3: Connection to the industry. Most of the rose producers, perfumers, essential oil producers, and plant raw material suppliers have been contacted to determine their interest in being included in a European Group of Economic Interest. This may be formulated next year when the work on the generation of transgenics has proceeded further.

Discussion

During the first year of the project preliminary work has been carried out. Results of real scientific importance are not expected until transgenic plants or callus is available. The part of the project concerning the production of transgenics is proceeding well. The fact that establishing the in vitro culturing and regeneration conditions as well as the production of the gene constructs has been accomplished is in favour of a successful transformation and regeneration of transgenic plants follow-up. On the basis of the results obtained up to now, no significant technical difficulties are anticipated. There is however some delay of about six months in the time schedule of regeneration of transgenic. That became unavoidable because of the need to test several plant varieties. Although the transformation is expected to be accomplished, the subtask concerning the testing of the transgenic performance under greenhouse conditions is put under time restrictions. In the best case there is only a year to test them. Considering that the production of flowers by the transgenics might take even longer, testing the expression of the introduced genes in that tissue and analysing their effect on terpenoid production becomes even more difficult to be accomplished in the set time frame of 36 months. This does not mean that important results will not be obtained before the end of the project.

One of the goals was to attempt to uncouple terpenoid production from tissue differentiation and to open thus the way for a more successful in vitro production of natural products using bioreactor facilities. Transgenic callus will be available much earlier that the regeneration of transgenic plants and the expression of the introduced genes and their effect on secondary metabolism, regenerability, and callus growth rate can be examined before the end of the project. It is probable that a connection exists between the callus stage and the concentration of free radicals, a situation also found in cancer tissue. In this sense, the behaviour of transgenic callus carrying catalase and ascorbate peroxidase would be most interesting as it is expected to affect even the rate of regeneration of differentiated plants.

It should be noted that it is possible that the introduction of cat and apx, with the associated oxidative stress they are expected to effect, could induce a much earlier flowering in the regenerated transgenics and thus to reduce the delay in their testing.

As it was mentioned in the original proposal, there is the possibility that a second, not HMGR-related path of terpenoid production might be operating in at least some plants. The project was designed to address this point as well by examining the effect of the introduction of constitutively expressed hmgr on the over-all terpenoid metabolism. Up to now the available literature does diminish the role of HMGR as the principal regulatory enzyme and we see no reason to abandon the task of transformation with this gene. Besides, the whole part concerning the examination of cis-element/trans-factor involvement concerns hmgr and it is by itself of primary importance in elucidating the stress signal transmission pathway if plants. The use of other genes, such as limonene synthase, in transformation as proposed, would not give us the opportunity to examine the signal transduction path. Besides, these genes are further downstream from the point were the terpenoid synthesis path stops in roses. Their use could have no effect or it could alter the quality of the essential oil to the point that it is no more rose oil. Recent literature is even more supportive of an involvement of active oxygen, peroxides, and free radicals in regulating gene transcription. A year after its commencement, this project acquires even greater scientific importance in this respect since it addresses one of the hottest points of plant biochemistry.

The part concerning the isolation of cis-elements/trans-factor genes is the most interesting scientifically and the most demanding technologically. Unfortunately it is also the one that suffered the greatest delays for reasons not related to the ability of the involved scientists to carry out the experiments. Its greatest part, the isolation of the cis- elements and the transcription factor genes, can still be accomplished if a better co-operation and the involvement of all partners are put into effect. In the opinion of the scientific co-ordinator the last part, the examination of the expression of the trans- factor genes in various rose genotypes, is impossible to be accomplished in the present time frame. Although it does not affect the delivery of transgenic roses at the end of the project, its failure would take away an opportunity to go deeper into manipulating plant secondary metabolism in future projects and would hinder European research to run into the cutting edge of scientific progress.

Future activities

Two main technical accomplishments are due next year. The first is the production of transgenic roses. From the experience we have gained during the past year on the tissue culturing and regeneration details we see no reasons why this part of the project should not succeed. The gene constructs and the tissue cultures are ready and the transformation procedure has started already.

A very important part of the project would be the examination of the effect of the introduced genes on the callus tissue. That includes analysis of any terpenoids or other secondary metabolites synthesised in the callus, checking the presence of transcript and the coded protein corresponding to the transgene, and examination of the regenerability and growth rate of the callus tissue. The only one of the above points, that needs special attention, is the timely production of antibodies to HMGR. Raising antibodies to a synthetic oligopeptide, fraction of the HMGR protein, will be attempted but other sources of anti-HMGR antibodies will also be sought among other European or American labs. The second milestone due is the isolation of the rose hmgr cis-elements and of any corresponding transcription factor genes. This requires prior construction of DNA and cDNA libraries. This has not been accomplished to date, for non-technical reasons, that have to be resolved.