WO2010055186A1

WO2010055186A1 - Procedure for the production of transgenic plants presenting high antioxidant compound content, high antioxidant capacity and resistance to browning

Info

Publication number: WO2010055186A1
Application number: PCT/ES2009/070496
Authority: WO
Inventors: Francisco José MUÑOZ PÉREZ; Miren Edurne BAROJA FERNÁNDEZ; Francisco Javier Pozueta Romero
Original assignee: Iden Biotechnology, S.L.
Priority date: 2008-11-13
Filing date: 2009-11-11
Publication date: 2010-05-20
Also published as: ES2339094B1; ES2339094A1

Abstract

The present invention protects the procedure for the production of transgenic plants together with the plants themselves presenting a high antioxidant compound content (such as polyphenols and carotenoids), high antioxidant capacity and resistance to browning through an increase in UDP-glucose:d-fructose-2-glucosyltransferase (UGFT) activity producing UDPGlucose in plants. In this invention it is demonstrated that UGFT is involved in the production of an important pool of UDPGlucose necessary for the production of highly stable glycosylated forms of polyphenols and carotenoids having high antioxidant capacity.

Description

PROCEDURE FOR THE PRODUCTION OF TRANSGENIC PLANTS THAT

PRESENT HIGH CONTENT IN ANTIOXIDANT COMPOUNDS, HIGH

ANTIOXIDANT CAPACITY AND RESISTANCE TO PRESERVE.

FIELD OF THE INVENTION

The present invention relates to a process for the production of transgenic plants that have a high antioxidant compound content, high antioxidant capacity and resistance to browning, as well as to the transgenic plants themselves. Therefore, the present invention can be encompassed within the field of engineering genetics, plant physiology, food technology and nutraceuticals

STATE OF THE ART

The UDP-glucose D-fructose-2-glucosyl transferase (UGFT) is highly regulated and catalyzes the production of UDPGlucose (UDPG) and fructose from the sucrose and UDP (3-6) UDPGlucosa is the glucose donor molecule for the transglycosylation reactions involved in the biosynthesis of ghcolipids, glycoproteins and cell wall polysaccharides such as cellulose and callose (1, 7-9) (Figure 14) In addition, tests carried out in vitro have shown that UDPGlucose acts as a glucose donor for the glycosylation reactions of antioxidant substances such as polyphenols and carotenoids These reactions are determinants for stability, conjugation with other molecules and prevention of the oxidation of polyphenols that give rise to the browning effect of vegetables (10) Polyphenols and carotenoids are responsible for the coloring of flowers and senescent leaves, and also protect the plant against microbial agents and against cell wall degrading enzymes (11) In addition, these compounds possess antioxidant and anti-inflammatory activity and are known for their properties against cancer and the accumulation of cholesterol in blood (12-15) Organs such as potato tubers characterized by accumulating large amounts of polyphenols and carotenoids (16-18), and constitute an important source of antioxidant substances in the diets of many societies (19)

The glycosylation reactions of polyphenols from UDPGlucosa take place in the cytosol (20) Although UGFT is an important source of cytosolic UDPGlucosa and that this is important in the production and stability of antioxidant substances m vitro,

SUBSTITUTE SHEET (RULE 26) to date there is no evidence that it is described that the increase in UDPGlucose producing UGFT activity is involved in the production and accumulation of antioxidant substances in vivo. There is also no experimental evidence, nor scientific works that suggest the implication of the UGFT activity producing UDPGlucosa, in the production of antioxidant substances in the cell. There is also no evidence in which the intracellular levels of UDPGlucosa are altered as a strategy to alter the levels of antioxidant substances in vivo. In this sense, the present invention describes for the first time the production of plants with (a) high content of UDPGlucose, (b) high content of polyphenols and carotenoids, (c) high antioxidant capacity and (d) low browning effect (browning effect) as a consequence of the UGFT activity producing UDPGlucosa.

DESCRIPTION OF THE INVENTION

Brief description of the invention

The present invention relates to a process for the production of transgenic plants that have a high content of antioxidant compounds, high antioxidant capacity and resistance to browning, by increasing the UGFT activity producing UDPGlucose in plants. Furthermore, the present invention relates to the transgenic plants themselves characterized by said properties.

The technical effects shown in the present invention can be extrapolated to any type of organ of the plant such as: tubers, leaves, fruits and seeds, as well as to any type of plant such as: potato, tobacco, tomato, rice, barley, wheat and corn. The results shown in the present invention were achieved, both by constitutively expressing UGFT under the control of the 35S promoter (Figure 1 A, B and C), and by expressing UGFT under the control of a tuber-specific promoter (B33 promoter of the patatin gene ) (Figure 2). It should be noted that the constitutive expression was particularly preferred since with this it was possible to analyze changes in the antioxidant activity in various organs of the plant such as leaves and tubers. The results shown in the present invention were obtained after over-expressing any isoform and UGFT sequence (UGFTX variant (SEQ ID NO: 3)) being particularly preferred. That is, any promoter expressible in plants that produces the over-expression of UGFT, as well as any isoform of UGFT with activity producing UDPGlucosa, would be included in the present invention. For the purposes of the present invention, the following terms are stated:

• Transgenic plant: plant whose genome has been modified by genetic engineering with the aim of achieving different biological characteristics and / or improved compared to those of the wild plant (WT). • Transformed plant cell: they are plant cells that present a genetic alteration resulting from the introduction and expression of external genetic material in their genome.

• Antioxidant compounds: These are substances capable of retarding or preventing the oxidation of other molecules. They are responsible for capturing the free radicals produced in the oxidation reactions of the metabolism. These compounds are widely used in the treatment of cerebrovascu- lar and neurodegenerative diseases, as well as dietary supplements, food preservatives, cosmetics and for the prevention of the degradation of rubber and gasoline. In the present invention, the antioxidant compounds are selected among polyphenols and carotenoids.

• Polyphenols: are chemical substances present in plants and characterized by the presence of more than one phenol group (group -OH linked to an aromatic ring) per molecule. The presence of these phenolic rings confers the ability to act as antioxidants since they can pick up oxygen radicals. • Carotenoids: are organic pigments that are found naturally in plants and other photosynthetic organisms. Most of the carotenoids are tetraterpenoids, composed of 40 carbon atoms formed by eight isoprenoid units and contain rings at one or both ends of the molecule. They can function as antioxidants, protecting against auto-oxidation. • Anthocyanins: polyphenolic organic pigments that provide coloration to many plants and especially to many flowers. Its chemical structure consists of a flavial group formed by a benzopyran ring attached to a phenolic ring. They have antioxidant properties avoiding the production of free radicals.

• Over-expression of the UDPGlucosa producing UGFT enzyme: it is understood that the UDPGlucose producing UGFT enzyme is over-expression when the total activity of said enzyme is significantly higher than that existing in wild plants or controls (WT) grown in them conditions and at the same time. By way of illustration in Figure 3 it is observed that the UGFT activity producing UDPGlucose in the tubers of the control potato plants (WT) is 590-710 milli units (mU) / g (fresh weight), while the UGFT activity Producer of UDPGlucose in the tubers of potato plants that express constitutively UGFT is 730-1300 mU / g fresh weight. Therefore, the present invention refers to the over-expression of the UGFT enzyme, by way of example, when the production of UDPGlucose in the tubers of potato plants is greater than 710 mU / g (fresh weight) (FIG. 3). • High content in UDPGlucosa: as used in the present invention, this expression is directly related to a statistically significant value, higher than the values observed in WT plants grown under the same conditions and at the same time. By way of illustration, in Figure 4 it is observed that the average content in UDPGlucosa in the tubers of the potato plants controls is 95-105 nmol / g (fresh weight), while the tubers of transgenic potato plants that express UGFT constitutively they accumulate 135-160 nmol / g (fresh weight) of UDPGlucosa. Therefore, the present invention refers to "high content in UDPGlucosa", by way of example, when this is higher than 105 nmol / g (fresh weight) (Figure 4). • High content of total polyphenols, anthocyanins and carotenoids: as used in

The present invention, this expression is directly referred to a statistically significant value, higher than the values observed in WT plants grown under the same conditions and at the same time. Thus, the present invention refers to "high content of total polyphenols", by way of illustration, when this is higher than 0.19 mg GAE / g (fresh weight) in the fleshy part of the tubers of the transgenic potato plants that express UGFT constitutively and superior to 0.52 mg GAE / g (fresh weight) in the skin of the tubers of the transgenic potato plants that express UGFT constitutively (Figure 5 and 6). Analogously, the present invention refers to "high anthocyanin content", by way of illustration, when this is higher than 4.0 mg / g

(fresh weight) in the skin of the tubers of transgenic potato plants that express UGFT constitutively (Figure 7). On the other hand, the present invention refers to "high content of carotenoids" when it is higher, for example, of 1.0 μg / g fresh weight in tubers of transgenic potato plants that express UGFT constitutively (Figure 8).

• High antioxidant capacity: as used in the present invention, this expression is directly related to a statistically significant value, higher than the values observed in WT plants grown under the same conditions and at the same time. Thus, the present invention refers to "high antioxidant capacity", by way of illustration, when this is higher than 6.8 μmol Trolox eq / g fresh weight in the fleshy part of the tubers of transgenic potato plants that express UGFT constitutively (Figure 9) and superior to 12.4 μmol Trolox eq / g fresh weight in the skin of the tubers of transgenic potato plants that express UGFT constitutively (Figure 10).

Description of the figures

Figure 1. Construction steps of the expression plasmid pBIN35S-UGFT-NOS.

Figure 2. Construction steps of the expression plasmid pKAN-B33-UGFT.

Figure 3. UGFT activity in tubers of wild potato plants or controls (WT), in tubers of potato plants transformed with an antisense vector of UGFT (anti-UGFT) (21) and in tubers of potato plants that express UGFT constitutively after integrating the 35S-UGFT-NOS construct into its genome after having been transformed with the expression vector Agrobacterium tumefaciens CECT: 5851. The activity is referred to in milliunits (mU) per gram of fresh weight (y axis). The unit is defined as the amount of UGFT needed to produce a micromole of UDPGlucose per minute.

Figure 4. Content in UDPGlucose (nmol / g fresh weight) in tubers of wild potato plants or controls (WT), in tubers of potato plants transformed with an antisense vector of UGFT (anti-UGFT) (21) and in tubers of plants that constitutively express UGFT after integrating the 35S-UGFT-NOS construct into their genome after having been transformed with the expression vector Agrobacterium tumefaciens CECT: 5851.

Figure 5. Content of total polyphenols (in mg equivalents of gallic acid (GAE) / g fresh weight) in the fleshy part of tubers of wild potato plants or controls (WT), in tubers of potato plants transformed with an antisense vector of UGFT (anti-UGFT) (21) and in tubers of potato plants that constitutively express UGFT after integrating the 35S-UGFT-NOS construct into their genome after having been transformed with the expression vector Agrobacterium tumefaciens CECT: 5851.

Figure 6. Content of total polyphenols (in mg GAE / g fresh weight) in the skin of tubers of wild potato plants or controls (WT) and in the skin of tubers of potato plants that constitutively express UGFT after integrating into their genome Ia 35S-UGFT-NOS construction after having been transformed with the expression vector Agrobacterium tumefaciens CECT: 5851.

Figure 7. Content of total anthocyanins (in mg / g fresh weight) in the skin of tubers of wild potato plants or controls (WT) and in the skin of tubers potato plants that constitutively express UGFT after integrating the 35S construction into their genome -UGFT- NOS after having been transformed with the expression vector Agrobacterium tumefaciens CECT: 5851.

Figure 8. Content of total carotenoids (in μg equivalents of beta-carotene (βCE) / g fresh weight) in tubers of wild potato plants or controls (WT) and potato plants that constitutively express UGFT after integrating the construction into their genome 35S-UGFT-NOS (by action of the Agrobacterium tumefaciens strain CECT: 5851). Figure 9. Antioxidant capacity (referred to μmol equivalent of Trolox / g fresh weight) in the fleshy part of tubers of wild potato plants or controls (WT) and in the fleshy part of tubers of potato plants constitutively expressing UGFT after integrating in its genome Ia construction 35S-UGFT-NOS after having been transformed with the expression vector Agrobacterium tumefaciens CECT: 5851.

Figure 10. Antioxidant capacity (referred to μmol equivalent of Trolox / g fresh weight) in the skin of tubers of wild potato plants or controls (WT) and in the skin of tubers of potato plants constitutively expressing UGFT after integrating into their genome Ia construction 35S-UGFT-NOS after having been transformed with the expression vector Agrobacterium tumefaciens CECT: 5851.

Figure 11. Antioxidant capacity (referred to μmol equivalent of Trolox / g fresh weight) in leaves of wild potato plants or controls (WT) and potato plants constitutively expressing UGFT after integrating into their genome the construction 35S-UGFT-NOS after having been transformed with the expression vector Agrobacterium tumefaciens CECT.5851.

Figure 12. Polyphenol oxidase activity (in Units / g fresh weight) (A) in the fleshy part and (B) in the skin of tubers of wild potato plants or controls (WT) and in the fleshy part and in the skin of tubers of potato plants that constitutively express UGFT after integrating the 35S-UGFT-NOS construct into their genome after having been transformed with the expression vector Agrobacterium tumefaciens CECT: 5851. Figure 13. Effect of browning on tubers of wild potato plants or controls (WT) and on tubers of potato plants constitutively expressing UGFT after mechanical cutting and exposure to air for 28 hours.

Figure 14. Proposed mechanisms for the conversion of sucrose into cell wall polysaccharides, glycoproteins and glycosylated polyphenols in plants.

Detailed description of the invention

Obtaining transgenic potato plants that overexpress constitutively UGFT

SUBSTITUTE SHEET (RULE 26) In the present invention UGFT has been expressed constitutively by the action of the 35S constitutive promoter of tobacco mosaic virus. To do this, the 35S-UGFT-NOS construction was introduced into the genome of the potato (Solanum tuberosum) whose design is detailed below. Once the nucleotide sequence coding for the UGFT4 isoform of wild potato UGFT (22) was known, two specific primers corresponding to the 5 ^' and 3 ^' ends of the gene were created, whose sequences are SEQ ID NO: 1 and SEQ ID NO: 2, respectively. Using these primers, a 2418 base pair DNA fragment, designated with UGFTX, was amplified by conventional PCR methods from a potato leaf cDNA library. Such a PCR fragment was introduced in the plasmid pSK Bluescript (Stratagene) giving rise to the pUGFT construction, which was amplified in the host bacterium XL1 Blue. The nucleotide sequence of UGFTX is SEQ ID NO: 3. The sequential introduction in pUGFT of the 35S promoter and the NOS terminator in the 5 ^' and 3 ^' regions of UGFTX, gave rise to the production of the plasmid p35S-UGFT-NOS whose map of Restriction is represented in Figure 1A.

In order to transfer this construction to the plant genome via Agrobacterium tumefaciens, it must first be cloned in a binary plasmid. For this, p35S-UGFT-NOS was digested sequentially with the enzymes Notl, T4 DNA polymerase and HindIII and was cloned into the binary plasmid pBIN20 (Figure 1B) (23) that had previously been digested sequentially with the enzymes EcoRI, T4 DNA polymerase and Hindlll. The plasmid thus obtained was designated pBIN35S-UGFT-NOS (Figure 1C), which was introduced by electroporation in the A. tumefaciens strain suitable for transforming plants according to conventional protocols. For example, for the production of transgenic potato plants pBIN35S-UGFT-NOS was introduced by electroporation into A. tumefaciens with deposit number CECT 5851, which acted as transfer vector of the 35S-UGFT-NOS construct to the genome nuclear potato when used by conventional methods of genetic transformation of potatoes (24).

Obtaining transgenic potato plants that overexpress UGFT in tubers

In the present invention UGFT has been expressed specifically in the potato tuber by the action of the B33 promoter that governs the expression of a gene coding for the patatin (24). For this, the B33-UGFT construction was introduced into the potato genome (Solanum tuberosum), the design of which is detailed below. Using primers SEQ ID NO: 4 and SEQ ID NO: 5, a DNA fragment of 2418 base pairs was amplified by PCR from the p35S-UGFT-NOS plasmid. Such a PCR fragment was introduced into the pDONR / Zeo plasmid (Invitrogen) by a Gateway BP recombination reaction giving rise to the pDONR-UGFT construct, which was amplified in the host bacterium TOP 10. On the other hand, the PCR was amplified by PCR. B33 promoter of the patatin using the specific primers corresponding to the 5 ^' and 3 ^' ends of the promoter whose sequences are SEQ ID NO: 6 and SEQ ID NO: 7, respectively. The PCR fragment obtained contains 1551 base pairs and was introduced into the plasmid pDONR / P4-P1 R (Invitrogen) by a recombination reaction Gateway BP giving rise to the construction pDONR-B33, which was amplified in the host bacterium TOP 10. Using a Gateway LR recombination reaction, the B33 promoter and UGFT were transferred to the expression plasmid pKAN R4-R2 to produce plasmid pKAN-B33-UGFT (also designated pKAN-B33-SuSy). The plasmid thus obtained was introduced by electroporation in the A. tumefaciens strain suitable for transforming plants according to conventional protocols. For example, for the production of transgenic potato plants B33-UGFT was introduced by electroporation into A. tumefaciens with the deposit number DSM 22756, which acted as transfer vector of the B33-UGFT construction to the potato nuclear genome at be used by conventional methods of genetic transformation of potato (24).

Cultivation, processing and estimation of the characteristics of transgenic potato plants with altered levels of UGFT

Controlled potato plants (WT) not transformed and lines 4, 5, 6 and 12 constitutively expressing UGFT were used as a result of the integration in their genome of the construction 35S-UGFT-NOS. Transgenic potato plants with reduced UGFT activity were also used (21). The plants were cultivated between May and September

2006 and 2007 in a plot of the term 25 of Sartaguda (Navarra, Spain). The plants were randomly distributed in plots of 50 square meters, making use of 30 plants per line. The separation between lanes was 90 cm. The separation between plant and plant of the same lane was 35 cm. The tubers were collected, those weighing approximately 100 grams were selected and cylinders were obtained that crossed the entire tuber using a cylindrical steel perforator (2 cm in diameter). Such cylinders were frozen in liquid nitrogen and pulverized using a mortar. For the measurement of the UGFT activity, the powder resulting after the homogenization of the fleshy part and / or the skin of the tuber with liquid nitrogen was resuspended at 4 ⁰ C in 100 mM HEPES (pH 7.5), 2 mM EDTA and 5 mM dithiothreitol (5 mL / gram powder). After dialyzing the extracts, the UGFT activity was measured as described in the literature (25). Briefly, the plant homogenate was incubated for 5 minutes at 37 ⁰ C in 50 mM HEPES (pH 7.0), 1 mM MgCl _2, 15 mM KCI, 50 mM sucrose and 2 mM UDP. After stopping the reaction at 100 ⁰ C the amount of UDPglucose produced was determined using an HPLC system Waters ^Associates' s adjusted to a Partisil SAX-10-column.

The UDPGlucose in the potato tuber powder obtained after homogenization with liquid nitrogen was extracted and measured as described in the literature (25). For this, 0.5 g of the frozen powder was resuspended in 0.4 ml_ of 1.4 M HCIO ₄ , left at 4 ⁰ C for 1 hour and centrifuged at 10,000 xg for 30 minutes. The supernatant was neutralized with K ₂ CO ₃ and centrifuged at 10,000 xg for 30 minutes. The UDPGlucose existing in the supernatant was measured using a Waters Associate ^' s HPLC system fitted to a Partisil-10-SAX column. The total polyphenols existing in the tuber powder obtained after homogenization in liquid nitrogen were extracted and measured essentially using the modified Folin-Ciocalteu method described in the literature (26).

The total carotenoids existing in the tuber powder obtained after homogenization in liquid nitrogen were extracted and measured essentially using the method described in (27), measuring the absorbance of the organic extract at 450 nm using a calibration curve of β-carotene.

The total anthocyanins existing in the tuber powder obtained after homogenization in liquid nitrogen were extracted and measured using the pH-differential method (28). The antioxidant capacity of the tuber powder obtained after homogenization in liquid nitrogen was measured using the TEAC (Trolox equivalent antioxidant capacity) method as described in the literature (29) using Trolox as standard and 2,2 ^' -acinobis- ( 3-ethylbenzothiazolin-6-sulfonate (ABTS).

The polyphenol oxidase activity of the tuber powder obtained after homogenization in liquid nitrogen was measured according to the method described in (30).

Thus, the first object of the present invention refers to a process for the production of transgenic plants with a high content of antioxidant compounds, high antioxidant capacity and resistance to browning, in relation to the phenotype of the wild plant (WT), which comprises the transformation of the wild plant (WT) with an expression vector that over-expresses the UGFT enzyme. In a preferred embodiment of the present invention the method is characterized in that the antioxidant compounds are preferably selected from among polyphenols and carotenoids, without excluding the possibility that other antioxidant substances have been affected by the over-expression of the UGFT.

In another preferred embodiment, the method of the invention is characterized in that the expression vectors used for the transformation of the plants are preferably selected from Agrobacterium tumefaciens CECT 5851 and Agrobacterium tumefaciens DSM 22756, which comprise the plasmids pBIN35S-UGFT-NOS and pKAN-B33 -UGFT respectively.

Another object of the present invention relates to the use of the expression vector Agrobacterium tumefaciens CECT 5851 comprising the plasmid pBIN35S-UGFT-NOS for obtaining transgenic plants that have a high content of antioxidant compounds, high antioxidant capacity and resistance to browning in relationship to the phenotype of the wild plant. The antioxidant compounds are preferably selected among polyphenols and carotenoids, without excluding the possibility that other antioxidant substances have been affected by the over-expression of the UGFT. A further object of the present invention relates to the use of the expression vector Agrobacterium tumefaciens CECT 5851 comprising the plasmid pBIN35S-UGFT-NOS for the production of transformed plant cells which have a high content of antioxidant compounds and a high antioxidant capacity in relation to the untransformed cells As mentioned above, the antioxidant compounds are preferably selected from polyphenols and carotenoids, without excluding the possibility that other antioxidant substances have been affected by the over-expression of the UGFT.

A further object of the present invention refers to the expression vector Agrobacterium tumefaciens DSM 22756, characterized in that it comprises the plasmid pKAN-B33-UGFT that over-expresses the UGFT enzyme, as well as its use for the production of transformed plant cells that have high content in antioxidant compounds and high antioxidant capacity in relation to untransformed cells. The antioxidant compounds are preferably selected among polyphenols and carotenoids, without excluding the possibility that other antioxidant substances have been affected by the over-expression of the UGFT. In another preferred embodiment of the invention, the expression vector Agrobacterium tumefaciens DSM 22756 is used for obtaining transgenic plants that have a high content of antioxidant compounds, high antioxidant capacity and resistance to browning in relation to the phenotype of the wild plant. The antioxidant compounds are preferably selected among polyphenols and carotenoids, without excluding the possibility that other antioxidant substances have been affected by the over-expression of the UGFT. Another object of the present invention relates to the transgenic plant transformed with an expression vector that over-expresses the UGFT enzyme, characterized by having a high content of antioxidant compounds, high antioxidant capacity and resistance to browning with respect to the non-transformed wild plant. The antioxidant compounds are preferably selected among polyphenols and carotenoids, without excluding the possibility that other antioxidant substances have been affected by the over-expression of the UGFT.

In a preferred embodiment of the invention, the transgenic plant is characterized in that the expression vectors used are preferably selected from Agrobacterium tumefaciens CECT 5851 and Agrobacterium tumefacien DSM 22756, comprising the plasmids pBIN35S-UGFT-NOS and pKAN-B33-UGFT, respectively . In another preferred embodiment of the invention, the transgenic plant is characterized in that it is selected from any of the following species: potato, rice, tomato, corn and barley. Another object of the present invention relates to plant cells transformed with an expression vector that over-expresses the UGFT enzyme, characterized by having a high content of antioxidant compounds and high antioxidant capacity compared to untransformed cells. The antioxidant compounds are preferably selected among polyphenols and carotenoids, without excluding the possibility that other antioxidant substances have been affected by the over-expression of the UGFT. In a preferred embodiment, the plant cell is characterized in that the expression vectors used are preferably selected from Agrobacterium tumefaciens CECT 5851 and Agrobacterium tumefacien DSM 22756, which comprise the plasmids pBIN35S-UGFT-NOS and pKAN-B33-UGFT, respectively. In another preferred embodiment, the plant cell is characterized in that it is selected from any of the following species: potato, rice, tomato, corn and barley.

In addition another object of the present invention refers to the use of the transgenic plants, mentioned above for obtaining high concentrations of UDPGlucosa and high levels of antioxidant compounds. The antioxidant compounds are preferably selected among polyphenols and carotenoids, without excluding the possibility that other antioxidant substances have been affected by the over-expression of the UGFT.

The last object of the present invention refers to the use of the transformed plant cells, mentioned above, for obtaining high concentrations of UDPGlucose and high levels of antioxidant compounds. The antioxidant compounds are preferably selected among polyphenols and carotenoids, without excluding the possibility that other antioxidant substances have been affected by the over-expression of the UGFT. DEPOSIT OF MICROORGANISMS ACCORDING TO THE BUDAPEST TREATY

The microorganisms used in the present invention were deposited in the Spanish Type Culture Collection (CECT), located in the Research Building of the University of Valencia, Burjassot Campus, Burjassot 46100 (Valencia, Spain) with deposit number CECT 5851 and in the "German National Resource Center for Biological Material", located in the DMSZ, Mascheroder Weg 1 b D-38124 (Braunschweig, Germany) with deposit number DSM 22756.

EXAMPLES OF EMBODIMENT OF THE INVENTION

Examples are described below in which the procedure for obtaining transgenic potato plants with a high content of antioxidants, high antioxidant capacity and resistance to browning is shown in detail, as a consequence of the increase of the UGFT activity producing UDPGlucosa, as well as as the values obtained for each of the aforementioned characteristics in the transgenic plants with respect to those obtained in non-transformed potato plants. The examples set forth below are intended to illustrate the invention without limiting the scope thereof.

Example 1. Process for obtaining transgenic potato plants with a high content of antioxidants, high antioxidant capacity and resistance to browning as a consequence of the increase of the UGFT activity producing UDPGlucosa

In the present invention UGFT has been expressed constitutively by the action of the 35S constitutive promoter of tobacco mosaic virus. To do this, the 35S-UGFT-NOS construction was introduced into the genome of the potato (Solanum tuberosum) whose design is detailed below.

Once the nucleotide sequence coding for the UGFT4 isoform of wild potato UGFT (22) was known, two specific primers corresponding to the 5 ^' and 3 ^' ends of the gene were created, whose sequences are SEQ ID NO: 1 and SEQ ID NO: 2, respectively. Using these primers, a 2418 base pair DNA fragment, designated with UGFTX, was amplified by conventional PCR methods from a DNA cDNA library. potato leaf. Such a fragment of PCR was introduced in the plasmid pSK Bluescript (Stratagene) giving rise to the pUGFT construction, which was amplified in the host bacterium XL1 Blue. The nucleotide sequence of UGFTX is SEQ ID NO: 3 which is different from UGFTX (access number in GenBank U24087). The sequential introduction in pUGFT of the 35S promoter and the NOS terminator in the 5 ^' and 3 ^' regions of UGFTX, gave rise to the production of the p35S-UGFT-NOS plasmid whose restriction map is shown in Figure 1A. On the other hand, the introduction of UGFT in pDONR / Zeo and the subsequent recombination with pDONR-B33 gave rise to the production of the plasmid pKAN-B33-UGFT, whose restriction map and way of obtaining it is represented in Figure 2.

In order to transfer the 35S-UGFT-NOS construct to the plant genome via Agrobacterium tumefaciens, it must first be cloned in a binary plasmid. For this, p35S-UGFT-NOS was digested sequentially with the enzymes Notl, T4 DNA polymerase and HindIII and was cloned into the binary plasmid pBIN20 (Figure 1B) (23) that had previously been digested sequentially with the enzymes EcoRI, T4 DNA polymerase and Hindlll. The plasmid thus obtained was designated pBIN35S-UGFT-NOS (Figure 1C), which was introduced by electroporation in the A. tumefaciens strain suitable for transforming plants according to conventional protocols. For example, for the production of transgenic potato plants that overexpress constitutively UGFT, pBIN35S-UGFT-NOS was introduced by electroporation into A. tumefaciens with deposit number CECT 5851, which acted as transfer vector of the construction 35S-UGFT-NOS to the nuclear potato genome when used by conventional methods of genetic transformation of potatoes (24). On the other hand, for the production of transgenic potato plants that over-express UGFT exclusively in the tubers, pKAN-B33-UGFT was introduced by electroporation into A. tumefaciens with the deposit number DSM 22756, which acted as vector of transfer of the B33-UGFT construct to the nuclear genome of the potato.

Example 2. Cultivation and processing of transgenic potato plants with high content of antioxidants, high antioxidant capacity and resistance to browning as a consequence of the increase of the UGFT activity producing UDPGlucosa

Controlled potato plants (WT) not transformed and lines 4, 5, 6 and 12 constitutively expressing UGFT were used as a result of the integration in their genome of the construction 35S-UGFT-NOS. Transgenic potato plants were also used reduced UGFT activity (21). The plants were cultivated between May and September of 2006 and of 2007 in a plot of the term 25 of Sartaguda (Navarra, Spain). The plants were randomly distributed in plots of 50 square meters, making use of 30 plants per line. The separation between lanes was 90 cm. The separation between plant and plant of the same lane was 35 cm. Leaves and tubers were collected. From the collected tubers, those that had an approximate weight of 100 grams were selected and cylinders were obtained that crossed the entire tubercle using a cylindrical steel perforator (2 cm in diameter). Such cylinders were frozen in liquid nitrogen and pulverized using a mortar.

Example 3. Measurement of the UGFT activity and the concentration of UDPGlucose in tubers of transgenic potato plants with high UGFT activity producing UDPGlucose with respect to potato plants not transformed.

For the measurement of the UGFT activity, the powder resulting after the homogenization of the leaf and the fleshy part and / or the skin of the tuber with liquid nitrogen was resuspended at 4 ⁰ C in 100 mM HEPES (pH 7.5), 2 mM EDTA and 5 mM dithiothreitol (5 mL / gram powder). After dialyzing the extracts, the UGFT activity was measured as described in the literature (25). Briefly, the plant homogenate was incubated for 5 minutes at 37 ⁰ C in 50 mM HEPES (pH 7.0), 1 mM MgCl _2, 15 mM KCI, 50 mM sucrose and 2 mM UDP. After stopping the reaction at 100 ⁰ C the amount of UDPglucose produced was determined using an HPLC system Waters ^Associates' s adjusted to a Partisil SAX-10-column. It is important to note that the estimated concentration of cytosolic UDPglucose in potato tubers (WT) is 0.8 mM, but the range of Km values for the UDPglucose of many polyphenol glycosyltransferase-type enzymes range between 1 and 7 mM. Therefore, it is inferred that:

(a) Cytosolic UDPglucose is a limiting substrate for many polyphenol glycosyltransferases and

(b) The alteration of the cytosolic levels of UDPglucose can exert an impact on the accumulation and glycosylation of polyphenols and carotenoids.

As illustrated in Figure 3, the UGFT activity in the tubers of any of the plants that express UGFT constitutively is 1.5-2 times higher than that existing in the same organ of a control plant (WT) grown under the same conditions and at the same time. On the contrary, the tubers of the anti-UGFT plants showed an activity 3-4 times lower than that observed in tubers of the WT plants grown under the same conditions and at the same time. The tubers of plants that constitutively express UGFT accumulate levels of UDPGlucosa (135-160 nmol / g fresh weight) significantly higher than those observed in tubers of WT plants (95-105 nmol / g fresh weight) (Figure 4) grown under the same conditions and at the same time. In contrast, potato tubers of UGFT antisense plants accumulate levels of UDPGlucose (15-30 nmol / g fresh weight) significantly lower than those observed in tubers of WT plants grown under the same conditions and at the same time.

Example 4. Measurement of the concentration of total polyphenols, total carotenoids and anthocyanins in the tubers of transgenic potato plants with high UGFT activity producing UDPGlucose with respect to non-transformed wild plants.

The total polyphenols existing in the tuber powder obtained after homogenization in liquid nitrogen were extracted and measured essentially using the modified Folin-Ciocalteu method described in the literature (26). The fleshy part of the tubers of plants that express UGFT constitutively accumulate levels of polyphenols (0.2-0.25 mg GAE / g fresh weight) significantly higher than those observed in tubers of WT plants (0.17-0.19 GAE / g fresh weight), grown in the same conditions and at the same time (Figure 5). On the contrary, the fleshy part of potato tubers of UGFT antisense plants accumulate polyphenol levels (0.14-0.15 nmol / g fresh weight) significantly lower than those observed in tubers of WT plants grown under the same conditions and in the same conditions. moment. Therefore, the content of total polyphenols present in the fleshy part of the tubers of potato plants that express UGFT constitutively is 25-35% higher than in the tubers of WT plants, and 45% higher than in the tubers of the UGFT antisense plants. The skin of the tubers of plants that express UGFT constitutively accumulates levels of polyphenols (0.58-0.72 mg GAE / g fresh weight) significantly higher than those observed in the skin of tubers of WT plants (0.48-0.52 mg GAE / g fresh weight) (Figure 6) grown under the same conditions and at the same time. The total carotenoids existing in the tuber powder obtained after homogenization in liquid nitrogen were extracted and measured essentially using the method described in (27), measuring the absorbance of the organic extract at 450 nm using a calibration curve of β-carotene.

The total anthocyanins existing in the tuber powder obtained after homogenization in liquid nitrogen were extracted and measured using the pH-differential method (28). The skin of the tubers of plants that express UGFT constitutively accumulates levels of anthocyanins (4.1-5.5 mg / g fresh weight) higher than those observed in the skin of tubers of WT plants (3.8-4.0 mg / g fresh weight) (Figure 7) grown under the same conditions and at the same time.

The tubers of plants that express UGFT constitutively accumulate levels of carotenoids (1.1-1.6 μg βCE / g fresh weight) significantly higher than those observed in tubers of WT plants (0.8-1.0 μg βCE / g fresh weight) (Figure 8) grown in the same conditions and at the same time.

Example 5. Measurement of the antioxidant capacity in leaves and tubers (fleshy part and skin) of transgenic potato plants with high UGFT activity producing UDPGlucose with respect to non-transformed wild plants.

The antioxidant capacity of the leaf and tuber powder (fleshy part and skin) obtained after homogenization in liquid nitrogen was measured using the TEAC (Trolox equivalent antioxidant capacity) method as described in the literature (29) using Trolox as standard and 2 , 2 ^'-acino-bis- (3-ethyl-6-sulfonate (ABTS).

The fleshy part of the tubers of plants that express UGFT constitutively have values of antioxidant capacity (7.6-9.0 μmol of Trolox / g fresh weight) significantly higher than those observed in the fleshy part of tubers of WT plants (6.5-6.8 μmol equivalent) of Trolox / g fresh weight) grown under the same conditions and at the same time (Figure 9).

The skin of the tubers of plants that express UGFT constitutively have values of antioxidant capacity (14.2-16.2 μmol equivalent of Trolox / g fresh weight) significantly higher than those observed in the skin of tubers of WT plants (12.0- 12.4 μmol equivalent) of Trolox / g fresh weight) grown under the same conditions and at the same time (Figure 10).

The leaves of plants that express UGFT constitutively have values of antioxidant capacity (75-100 μmol equivalent of Trolox / g fresh weight) significantly higher than those observed in the leaves of WT plants (40-50 μmol of Trolox equivalent / g weight fresh) grown under the same conditions and at the same time (Figure 11).

Polyphenols and carotenoids are the major determinants of the antioxidant capacity of cells. The increase in the content of polyphenols and carotenoids induced by the ectopic expression of enzymes involved in the biosynthesis of these compounds leads to the increase of the antioxidant activity in tubers of potato plants. In this sense, the constitutive expression of UGFT in potato plants by the procedure described in the present invention, led to an increase of between 20-30% of the total antioxidant activity in the tubers of the same. This result is due to the higher content of polyphenols and carotenoids presented by said plants and not to the increase of other antioxidant compounds, such as ascorbic acid, since the concentration of said acid in the tubers of plants that constitutively express UGFT remains unchanged. in relation to the concentration present in the WT plants.

Example 6. Measurement of the polyphenol oxidase activity in the fleshy part and in the skin of the tubers of transgenic potato plants with high UGFT activity producing UDPGlucose with respect to non-transformed wild plants.

The polyphenol oxidase activity of the tuber powder obtained after homogenization in liquid nitrogen was measured according to the method described in (30). The polyphenol oxidase activity of the fleshy part of potato tubers that express UGFT in a constitutive manner is normal when compared with the one existing in tubers of WT plants grown under the same conditions and at the same time (Figure 12A). The polyphenol oxidase activity of the skin of potato tubers that express UGFT constitutively has a tendency to be lower than that observed in the tubers of WT plants grown under the same conditions and at the same time (Figure 12B).

Example 7. Measurement of the resistance to browning in the tubers of transgenic potato plants with high UGFT activity producing UDPGlucose with respect to non-transformed WT plants.

The tubers of plants that overexpress UGFT are more resistant to the browning effect than the tubers of WT plants grown under the same conditions and at the same time, after suffering mechanical damage and exposure to air (Figure 13). This fact is due, at least in part, to the high content of total polyphenols and carotenoids present in the plants that express UGFT and not to the decrease in the activity of polyphenol oxidase, since the maximum polyphenol oxidase activity in tubers of plants that express constitutively UGFT was normal compared to the tubers of the WT plants. All these data indicate that the increase in UGFT activity determines the intracellular levels of substances (mainly polyphenols and stable glycosylated carotenoids) that have a high antioxidant capacity, by providing a large pool of UDPglucose to the glycosylating enzymes. BIBLIOGRAPHY

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Claims

one . Process for obtaining transgenic plants with high content of antioxidant compounds, high antioxidant capacity and resistance to browning, in relation to the phenotype of the wild plant, which comprises the transformation of the wild plant with an expression vector that over-expresses the enzyme UGFT.

2. Process according to claim 1, characterized in that the antioxidant compounds are selected among polyphenols and carotenoids.

3. Method according to any of claims 1 or 2, characterized in that the expression vector is Agrobacterium tumefaciens CECT 5851 comprising the plasmid pBIN35S-UGFT-NOS.

4. Use of the expression vector Agrobacterium tumefaciens CECT 5851 comprising the plasmid pBIN35S-UGFT-NOS for obtaining transgenic plants that have a high content of antioxidant compounds, high antioxidant capacity and resistance to browning in relation to the phenotype of the wild plant .

5. Use, according to claim 4, characterized in that the antioxidant compounds are selected among polyphenols and carotenoids.

6. Use of the expression vector Agrobacterium tumefaciens CECT 5851 comprising the plasmid pBIN35S-UGFT-NOS for the production of transformed plant cells that have a high content of antioxidant compounds and high antioxidant capacity in relation to untransformed cells.

7. Use according to claim 6, characterized in that the antioxidant compounds are selected among polyphenols and carotenoids.

Transgenic plant, transformed with an expression vector that over-expresses the UGFT enzyme, characterized by high content of antioxidant compounds, high antioxidant capacity and resistance to browning compared to the non-transformed wild plant.

9. Transgenic plant, according to claim 8, characterized in that the antioxidant compounds are selected from polyphenols and carotenoids.

Transgenic plant, according to any of claims 8 or 9, characterized in that the expression vector is Agrobacterium tumefaciens CECT 5851 comprising the plasmid

PBIN35S-UGFT-NOS.

Transgenic plant, according to any of claims 8 to 10, characterized in that it is selected from any of the following species: potato, rice, tomato, corn and barley.

12. Plant cell transformed with an expression vector that over-expresses the UGFT enzyme, characterized by having a high content of antioxidant compounds and high antioxidant capacity compared to untransformed cells.

13. Transformed plant cell according to claim 12, characterized in that the antioxidant compounds are selected among polyphenols and carotenoids.

14. Transformed plant cell according to any of claims 12 or 13, characterized in that the expression vector is Agrobacterium tumefaciens CECT 5851 comprising the plasmid pBIN35S-UGFT-NOS.

15. Transformed plant cell according to any of claims 12 to 14, characterized in that it is selected from any of the following species: potato, rice, tomato, corn and barley

16. Use of the transgenic plants of any of claims 8 to 11, for obtaining high concentrations of UDPGlucosa and high levels of antioxidant compounds.

17. Use according to claim 16, characterized in that the antioxidant compounds are selected among polyphenols and carotenoids.

18. Use of the transformed plant cells of any of claims 12 to 15 for obtaining high concentrations of UDPGlucose and high levels of antioxidant compounds.

19. Use according to claim 18, characterized in that the antioxidant compounds are selected among polyphenols and carotenoids.

20. Method according to any of claims 1 or 2, characterized in that the expression vector is Agrobacterium tumefaciens DSM22756 comprising the plasmid pKAN-B33-UGFT.

21. Expression vector Agrobacterium tumefaciens DSM 22756 characterized by comprising the plasmid pKAN-B33-UGFT.

22. Use of the expression vector, according to claim 8, for obtaining transformed plant cells that have a high content of antioxidant compounds and a high antioxidant capacity in relation to the untransformed cells.

23. Use of the expression vector, according to claim 8, for obtaining transgenic plants that have a high content of antioxidant compounds, high antioxidant capacity and resistance to browning in relation to the phenotype of the wild plant.

24. Use according to claim 10, characterized in that the antioxidant compounds are preferably selected from polyphenols and carotenoids.

25. A transgenic plant according to any of claims 8 or 9, characterized in that the expression vector is Agrobacterium tumefaciens DSM 22756 comprising the plasmid pKAN-B33-UGFT.

26. A transgenic plant according to any of claims 8 to 10, characterized in that it is selected from any of the following species: potato, rice, tomato, corn and barley.

27. Transformed plant cell according to any of claims 12 or 13, characterized in that the expression vector is Agrobacterium tumefaciens DSM 22756 comprising the plasmid pKAN-B33-UGFT.

28. Transformed plant cell according to any of claims 12 to 14, characterized in that it is selected from any of the following species: potato, rice, tomato, corn and barley.

29. Use of the transgenic plants of any of claims 25 or 26, for obtaining high concentrations of UDPGlucose and high levels of antioxidant compounds.

30. Use according to claim 29, characterized in that the antioxidant compounds are selected among polyphenols and carotenoids.

31. Use of the transformed plant cells of any of claims 27 or 28 for obtaining high concentrations of UDPGlucose and high levels of antioxidant compounds.

32. Use according to claim 31, characterized in that the antioxidant compounds are selected from polyphenols and carotenoids.