WO2005080580A2

WO2005080580A2 - Use of the myb4 transcriptional factor from rice to increase the production of secondary metabolites by transformed plants

Info

Publication number: WO2005080580A2
Application number: PCT/IB2005/000135
Authority: WO
Inventors: Immacolata Coraggio; Monica Mattana; Franca Locatelli; Marcella Bracale; Candida Vannini
Original assignee: Consiglio Nazionale Delle Ricerche; Universita' Degli Studi Dell'insubria
Priority date: 2004-01-23
Filing date: 2005-01-20
Publication date: 2005-09-01
Also published as: WO2005080580A3; ITMI20040091A1

Abstract

The invention relates to the use of rice cDNA coding for Myb4 and/or of the rice transcription factor Myb4 and/or the functional homologues thereof for the production of products of chemical, pharmaceutical and phytopharmaceutical interest, in particular of metabolites generated by plants transformed with said factor Myb4, the use of the plants overexpressing said transcription factor for the production of products of interest and also the use of cDNA coding for Myb4 and/or of the transcription factor Myb4 to confer tolerance to the herbicide glyphosate to transformed plants.

Description

"Use of the MYB₄ trαnscriptionαl factor from rice to increase the production of secondary metabolites in transformed plants"

SUMMARY OF THE INVENTION The present invention relates to the use of the rice cDNA coding for Myb₄ and/or of the rice transcription factor Myb4 and/or the functional homologues thereof for the production of products of chemical, pharmaceutical and phytopharmaceutical interest, in particular of metabolites generated by transformed plants with said factor Myb₄. The invention also relates to the use of cDNA coding for My >4 and/or of the transcription factor Myb4 (and/or the functional homologues thereof) to confer tolerance to the herbicide glyphosate to transformed plants. TECHNICAL BACKGROUND In a pending patent application, the applicants have characterized a rice cDNA Osmyb4 (access number Y1 1414 (EMBL)) coding for a transcription factor of Myb type, of which we have shown, by the analysis of transgenic plants, the capacity to increase tolerance to stress. Its overexpression in Arobidopsis fhaliana, makes this plant model much more tolerant to biotic (viruses, bacteria and fungi) and abiotic (cold, freezing, salt, dehydration, UV, ozone) stresses. The fact that the gene Osmyb4 isolated in a monocotyledon (rice) is capable of activating metabolic pathways as a response to stress in a dicotyledon {Arabidopsis) , indicates high preservation of the regulation pathways in which it is involved and, therefore, the possibility to transfer the results obtained for the model plant also to plants of officinal interest.

The ability of plants to synthesize a wide range of chemical compounds, called secondary metabolites, in response to external stimuli is known. These compounds, belonging to several chemical families including alkaloids and flavonoids, perform protective functions against pathogens, herbivores and ionizing radiations. They also have important roles in attracting pollinating insects and in proficuous interactions with other organisms.

Many of these secondary metabolites are known for their therapeutic properties (Kauffman, P. B., Cseke, L. J., Warber, S., Duke, J. A., Brielmann, H. L. (eds) 1999 Natural Products from Plants, CRC Press, Boca Raton, pp. 343). In fact, they are widely used both in popular medicine and to produce essential oils and aromatizing agents (Penso, G., 1980 Inventory of Medicinal Plants Used in the Different Countries. World Health Organization, DPM 80-3, Geneva, p.596, Lawrence, B.M., 1992 In: Advances in Labiate Science, ed. R. M. Harley and T. Reynolds. The Royal Botanic Gardens, Kew, U.K., p. 399, Reineccius, G.,(ed.) 1994 In: Source Book of Flavors, 2nd edn, Chapman and Hall, New York, p. 326).

One of the classes of well-characterized compounds includes isoprenoids, which exhibit antioxidant, antibacterial, anti-fungal, antiphlogistic and analgesic activities (Luis, J.G., 1991 In: Proceedings of Phytochemical Society of Europe: Ecological Chemistry and Biochemistry of Plant Terpenoids, vol.31 , ed. J. B. Harborne and F. A. Tomas-Barberan. Clarendon Press, Oxford, p. 63, Amaro-Luis, J. M., Ramon Herrera, J., Luis, J. M., 1998 Phytochemistry 47 (5): 895-897). Flavonoids have antioxidant, antiinflammatory, antiallergic, hepatoprotective, antithrombotic, antiviral and antitumoral activities (Middleton, E. et al. 2000 Pharmaceutical Rev. 52, 673-751 , Nijveldt, R.J. et al 2001 Am. J. Clin. Nutr. 74, 418-425).

It was recently shown that condensed tannins inhibit synthesis of endothelin-1 and thus protect against vascular damage and atherosclerosis (Corder, R. et al 2001 Nature 414, 863-864.). The pharmacological properties of alkaloids are known. Morphine and codeine are potent analgesics, vinblastine and taxol have antitumoral activities, colchicine is an antigout agent, tubocurarine is a myorelaxant, sanguinarine is an antibiotic and scopolamine is a sedative (Facchini, P.J. 2001 Annu. Rev. Plant Physiol. Plant Mol. Biol. 52, 29-66, Memelink, J. et al 2001 Trends Plant Sci. 6, 212-219) . Although in recent years there has been an increase in the interest in natural products as sources for new biochemical compounds for pharmacological, chemical and agronomic use, the use of plants as producers of active substances in in vivo plants is limited by drawbacks of various nature.

By way of an example, the factors that obstruct the use of plants to produce substances of industrial interest are a) production instability, b) complexity of product mixtures and greater difficulty in extraction and purification, c) difficulty in recovering substances secreted into the environment, d) the long growth cycle of some plant species before reaching the optimal stage for production of the desired' substance, e) lack of adequate agronomic techniques for large-scale cultivation, f) excessive exploitation of plant genetic resources in danger of extinction in their original habitats.

It is thus evident that it would be extremely useful to increase biosynthesis of active substances naturally produced by plants as secondary metabolites, both for production aimed at extracting and isolating them from the plant and for use of the plant as food rich in these substances ("nutriceuticals"). DETAILED DESCRIPTION OF THE INVENTION

It has now surprisingly been found that transgenic plants overexpressing the rice factor Myb4 (hereunder "Mytu") exhibit increased constitutive induction of several genes involved in the synthesis pathway of various substances, among which phenylpropanoids, a large group of secondary metabolites comprising, among others, anthocyans, flavones and condensed tannins. As is known, these compounds have enormous pharmacological, chemical, cosmetic and agronomic interest, for example as pesticides.

Therefore, according to one aspect thereof, the invention relates to the use of cDNA coding for Myb₄ and/or of the transcription factor Myt>4 (and/or the functional homologues thereof) to induce production of secondary metabolites in transformed plants. More specifically, the object of the invention is the use of cDNA coding for Myb4 and/or of the transcription factor Myb₄ (and/or the functional homologues thereof) to genetically transform plants in order to increase the production of substances of chemical, pharmaceutical, phytopharmaceutical, cosmetic or food interest. According to another aspect thereof, the invention relates to a method to increase the production of secondary metabolites in plants which comprises genetically transforming said plants with cDNA coding for Myb4 and/or the transcription factor Myb (and/or the functional homologues thereof).

The terms "transformed plants", "genetically transformed plants" or "transgenic plants", which are similar expressions, as used in the present invention, are intended as genetically transformed plants overexpressing the factor Myb4, in particular plants transformed with cDNA coding for Myb₄ and/or with the transcription factor Myb4 (and/or the functional homologues thereof).

In the present invention, even when not expressly indicated, "use of the transcription factor Myb4 is intended as the use of said factor, but also the use of cDNA coding for said factor and the use of the functional homologues thereof.

The term "functional homologues" as used in the present invention, is intended as the polynucleotide sequences that exert in the plants a function analogous to the one exerted by the sequence which codes for Myt>4 in the rice plant and the transcription factors functionally analogous to Myb4 which derive from said sequence. Preferably, said homologues are polynucleotide sequences that exhibit a sequence homology of at least 70% with the sequence coding for Myb4, advantageously of at least 80%, for example 90% or even greater. "Secondary metabolites", as used in the present invention, are intended as chemical compounds naturally produced by the plants following external stimuli such as pathogens, herbivores, radiations, etc.. It has in fact been observed that in transformed plants according to the invention the synthesis of mRNA coding for glucose-6-phosphate- dehydrogenase is greatly induced, the induction of which is known to occur subsequent to wounds or attacks by pathogens, to increase the precursors of the shikimate pathway. This subsequently leads to the synthesis of aromatic amino acids and the subsequent synthesis of phenylpropanoids.

The substances deriving from activation of the synthesis pathways of phenylpropanoids and of aromatic amino acids are therefore included in the secondary metabolites according to the invention. Therefore, according to a preferred aspect, the invention relates to the use of the transcription factor Myb4 to induce the production of substances deriving from activation of the synthesis pathways of phenylpropanoids and of aromatic amino acids in transformed plants. Examples of classes of representative substances deriving from the metabolic pathways activated according to the invention include alkaloids, lignans and flavonoids and relative compounds. Illustrative examples of lignans are matairesinol, secoisolariciresinol, lariciresinol, pinoresinolo and syringaresinol. Illustrative examples of flavonoids and relative compounds are arbutin, curarin, genistein, 5-methyl-7-methyl-isoflavone, alpha- naphthoflavone, naringin, quercitin, syringin and vitexin. Illustrative examples of alkaloids are berberine, caffeine, carnosine, guanosine, humulene, palmitin, phenylpropanolamine, piperine, sarcosine and tetrahydropalmitine.

As stated, in the plants transformed with Myt>4 many genes are overexpressed, including, among others, those belonging properly to the phenylpropanoid pathway: seven of the eight genes coding for enzymes involved in the synthesis of aromatic amino acids (through the chorismate pathway) (3-deoxy-arabino-heptulosonate-7- phosphate (DAHP), dehydroquinate synthase, dehydroquinate dehydratase, shikimate 5-dehydrogenase, 5-enolpyruvylshikimate-3- phosphate (EPSP) synthase, chorismate synthase, chorismate mutase. The PAL (phenylalanine ammonia-lyase) gene is induced around 13 times. PAL acts in a branching point for three pathways: one leads to synthesis of isoflavonoids and flavonoids, the second to synthesis of PR1 , mediated by salicylic acid, the third leads to other classes of secondary metabolites such as lignins, pigments and phytoalexins. In plants transformed with Myb4 genes belonging to all three pathways are constitutively overexpressed (coumarate CoA ligase, chalcone synthase, flavon-3-hydroxylase and isoflavone reductase, in the first; transcription factors induced by salicylic acid and PR1 , in the second; cinnamate-4-hydroxylase, 4-coumarate:CoA ligase, caffeoyl-CoA 3-0- methyltransferase and cinnamoyl-CoA reductase, in the third). According to another aspect thereof, the invention also relates to a method for increasing the expression of dehydroquinate synthase, of dehydroquinate dehydratase, of shikimate 5-dehydrogenase, of 5- enolpyruvylshikimate-3-phosphate (EPSP) synthase, of chorismate synthase, of chorismate mutase and of phenylalanine ammonia-lyase (PAL) which comprises genetic transformation of said plants with cDNA coding for Myt>4 and/or the transcription factor Myb and/or the functional homologues thereof. The plants transformed according to the invention exhibit increased production of many secondary metabolites which represent substances of considerable interest in various fields, for example in the pharmaceutical, phytopharmaceutical, cosmetic, and agronomic fields, in the chemical industry in general, in the food field, etc.. These substances, conspicuously produced by the plants transformed according to the invention, can be extracted from the plant, isolated and/or purified and used for humans, for animals or for plants. Alternatively, transformed plants of edible type containing the substances of interest can be used as nutraceutic foods for humans and for animals. The use of plants overexpressing the factor Myb₄ to produce the aforesaid substances of interest also forms part of the invention. It was also observed that the sixth enzyme of the chorismate pathway, EPSP, is the target of the herbicide glyphosate. EPSP synthase is present in plants, algae, bacteria and fungi, but not in animals. Its overexpression in plastids confers tolerance to glyphosate (Padgette et al., 1995). Induction of the gene coding for EPSP synthase, thus provides a potential tolerance of plants overexpressing Myb to the herbicide glyphosate. Consequently, according to another aspect thereof, the invention relates to the use of the transcription factor Myb4 to prepare transformed plants tolerating the herbicide glyphosphate. Yet another object of the invention is a method for the production of plants tolerating the herbicide glyphosate which comprises the transformation thereof with the transcription factor Myb₄ (or with its coding gene Osmyb4).

Plants transformed according to the invention can be produced according to conventional methods in use in the genetic engineering field. Transformed plants can conveniently be used in cultures in vitro, in vitro cultures and new molecular techniques in fact allow greater control of the production of bio-molecules both through empirical changes of the constituents of the substrate and of the culture conditions, and through targeted approaches (use of inhibitors or elicitors, manipulation of gene expression).

The in vitro culture techniques can be applied at undifferentiated and differentiated level (Walton, N. J., Alfermann, A.W, Rhodes, M. J. C, 1999 In: Functions of Plant Secondary Metabolites and their Exploitation in Biotechnology, Sheffield Academic Press, Sheffield, pp. 31 1-345). In the first case, cultures of calluses or cells can be obtained using as explant source the organs that normally accumulate the product of interest. In these cultures, the accumulation of compounds normally produced in specific tissues of the plant is generally low; nonetheless, in various cases by changing the chemical composition of the culture substrate and/or the environmental conditions or by selecting more productive cell clones, acceptable production levels were obtained. On the other hand, one of the greatest advantages of cells in suspension is the large scale adaptability to the culture in fermentors or bioreactors. At differentiated level, shoots and/or roots can be cultivated in controlled conditions, directing and promoting growth with the addition of growth regulators (auxin and cytokinin) to the substrate (Canto-Canche, B., Loyola-Vargas, V. M., 1999 In: Chemicals via Higher Plant Bioengineering, Kluwer Academic/Plenum Publishers, New York, pp. 235-275; Bajaj, Y. P. S., Ishimaru, K., 1999 In: Biotechnology in Agriculture and Forestry Vol. 45 Transgenic Medicinal Plants, Springer, Berlin, pp. 1-29; Walton, N. J., Alfermann, A.W, Rhodes, M. J. C, 1999 In: Functions of Plant Secondary Metabolites and their Exploitation in Biotechnology, Sheffield Academic Press, Sheffield, pp. 31 1-345).

Cell and molecular biology techniques in recent years have allowed the development of systems for genetic transformation of plant cells (Bajaj et al, supra). Among the various procedures available, the production of "hairy root" cultures through transformation with Agrobacteriυm rhizogenes is particularly interesting (Canto-Canche et al; Bajaj et al; Walton et al; supra). These show several advantages, among which: vigorous growth without hormones added to the substrate, efficient accumulation of secondary metabolites, release of the substances produced in the culture medium and genetic and biochemical stability.

Alternatively, when the compound of interest is produced in the aerial part of the plant, the transformation appears more interesting for example with Agrobacteriυm tumefaciens and the in vitro culture of teratomas. Examples of the production of transgenic plants and verification of induction of the synthesis of secondary metabolites are indicated in the experimental section below. Experimental Section EXAMPLE 1 Preparation of transgenic plants overexpressing Mvb₄ The cDNA of the gene deposited with access number Y11414 (EMBL) was placed under the CaMV35S promoter and upstream of the terminator of the gene Nos; the expression cassette thus obtained was inserted into the binary vector (E. coli - agrobacterium) PGA470. The latter was introduced by electroporation into the GV3101 strain of Agrobacterium tυmefaciens, which was then used for transforming Arabidopsis thaliana (cv Wassilewskija) plants with the "floral dip" method. EXAMPLE 2

Verification of overproduction of secondary metabolites.

1. The computer-based analysis of the results obtained by transcriptome comparison (using microarray analysis) indicated in transgenic Arabidopis plants compared with the WT the constitutive induction of several genes involved in the synthesis pathway of phenylpropanoids; in particular synthesis of the mRNA coding for glucose-6-phosphate-dehydrogenase proved to be strongly induced.

2. Overexpression of the secondary metabolites was verified by observing the following functionalities of the plant: a) increase in root exudate (the components of which we have not yet identified) identifiable in transformed seedlings of A. thaliana and Osteospermum ecklonis grown in vitro; b) strong fluorescence present in the roots of transformed plants exposed to UV light (associable with high concentrations of some aromatic compounds such as sinapyl malate), c) NMR analysis of extracts of transgenic A thaliana that identifies an increase in numerous aromatic metabolites and in particular a dramatic increase of sinapyl malate (one of the final metabolites of the phenylpropanoid pathway).

Claims

1. Use of the rice cDNA coding for Myb4 αnd/or of the rice transcription factor Myb4 and/or the functional homologues thereof, to induce production of secondary metabolites in transformed plants.

2. Use as claimed in claim 1 , to genetically transform plants in order to increase the production of substances of chemical, pharmaceutical, phytopharmaceutical, cosmetic or food interest.

3. Use as claimed in claim 1 or 2, to induce the synthesis of mRNA coding for glucose-ό-phosphate-dehydrogenase in transformed plants.

4. Use as claimed in any one of the previous claims, to induce the production of substances deriving from activation of the synthesis pathways of phenylpropanoids and/or of aromatic amino acids in transformed plants.

5. Use as claimed in any one of the previous claims, wherein said secondary metabolites are chosen from alkaloids, lignans and flavonoids.

6. Method to increase the production of secondary metabolites in plants which comprises genetically transforming said plants with rice cDNA coding for Myb4 and/or the transcription factor Myb4 and/or the functional homologues thereof.

7. Method to increase the expression of genes belonging properly to the pathways of phenylpropanoids and/or of aromatic amino acids in plants which comprises genetically transforming said plants with cDNA coding for Myb4 and/or the transcription factor Myb4 and/or the functional homologues thereof.

8. Method as claimed in claim to increase the expression of dehydroquinate synthase, of dehydroquinate dehydratase, of shikimate 5-dehydrogenase, of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, of chorismate synthase, of chorismate mutase and of phenylalanine ammonia-lyase (PAL) in plants, which comprises genetically transforming said plants with cDNA coding for My >4 and/or the transcription factor Myb₄ and/or the functional homologues thereof.

9. Method to increase the expression of 5-enolpyruvylshikimate- 3-phosphate (EPSP) synthase in plants, which comprises genetically transforming said plants with rice cDNA coding for Myb4 and/or the rice transcription factor Myb4 and/or the functional homologues thereof.

10. Method to confer tolerance to the herbicide glyphosate which comprises genetically transforming said plants with rice cDNA coding for Myb4 and/or the rice transcription factor My >4 and/or the functional homologues thereof.

11. Use of plants transformed with rice cDNA coding for My >4 and/or the rice transcription factor Myb₄ and/or the functional homologues thereof for the production of secondary metabolites.

12. Use of plants overexpressing the factor Myb4 for the production of secondary metabolites.

13. Nutraceutic food constituted by plants transformed with rice cDNA coding for Myb4 and/or the rice transcription factor Myb4 and/or the functional homologues thereof.

14. Nutraceutic food as claimed in claim 13 which comprises a high content of secondary metabolites.