UV-B absorbing compounds isolated from plants

Abstract

The present invention relates to compounds according to general formula (I) with enhanced absorption of UV-B irradiation. The present invention also relates to an UV-B tolerant plant and a method for enhanced production of compounds according to general formula (I) in a plant or plant cell. Furthermore, the invention relates to a nucleic acid sequence SEQ-ID No. 1 encoding FPT2 catalyzing the production of compounds according to general formula (I). The invention further relates to compositions comprising compounds according to general formula (I). Furthermore, the invention relates to a method of conferring UV-B tolerance to a plant as well as an UV-B tolerant plant comprising the nucleic acid sequence SEQ-ID No. 1 encoding FPT2 catalyzing the production of compounds according to general formula (I).

Claims

1. A transgenic UV-B tolerant Arabidopsis plant cell, Arabidopsis plant tissue or Arabidopsis plant comprising an exogenous nucleic acid molecule consisting of the cDNA encoding a flavonol phenylacyltransferase 2 protein having at least 95% sequence identity to SEQ-ID NO:2, wherein said exogenous nucleic acid molecule has been introduced by genetic manipulation into a plant that does not naturally express said nucleic acid molecule, wherein said exogenous nucleic acid molecule is operably linked to a promoter, wherein said transgenic plant cell, plant tissue or plant has enhanced tolerance to UV-B irradiation relative to a native plant cell, plant tissue or plant of the same species that does not naturally express said nucleic acid molecule, and wherein said enhanced UV-B tolerance is assessed after UV-B irradiation treatment for at least 2 hours per day for 28 days with 2000 mW/m.sup.2.

2. The transgenic UV-B tolerant plant cell, plant tissue or plant according to claim 1, wherein said flavonol phenylacyltransferase 2 protein synthesizes a compound of general formula (I) ##STR00021## wherein R.sup.1, R.sup.2 and R.sup.3 are independently of each other selected from: —H, —OH, and —OCH.sub.3; R.sup.4 is selected from —H, —OH, and —OCH.sub.3; R.sup.5 represents —H, ##STR00022## and enantiomers, mixtures of enantiomers, diastereoisomers, mixtures of diastereoisomers, hydrates and solvates thereof.

3. The transgenic UV-B tolerant plant cell, plant tissue or plant according to claim 1, wherein said flavonol phenylacyltransferases 2 protein synthesizes a compound of general formula (II) ##STR00023## wherein R.sup.4 is selected from —H, —OH, and —OCH.sub.3; R.sup.5 represents —H or ##STR00024## and enantiomers, mixtures of enantiomers, diastereoisomers, mixtures of diastereoisomers, hydrates and solvates thereof.

4. The transgenic UV-B tolerant plant cell, plant tissue or plant according to claim 1, wherein said flavonol phenylacyltransferases 2 protein synthesizes a compound of general formula (III) ##STR00025## wherein R.sup.4 is selected from —H, —OH, and —OCH.sub.3; R.sup.5 represents —H or ##STR00026## and enantiomers, mixtures of enantiomers, diastereoisomers, mixtures of diastereoisomers, hydrates and solvates thereof.

5. The transgenic UV-B tolerant plant cell, plant tissue or plant according to claim 1, wherein said flavonol phenylacyltransferases 2 protein synthesizes a compound of general formula (IV) ##STR00027## wherein R.sup.4 is selected from —H, —OH, and —OCH.sub.3; R.sup.5 represents —H or ##STR00028## and enantiomers, mixtures of enantiomers, diastereoisomers, mixtures of diastereoisomers, hydrates and solvates thereof.

6. The transgenic UV-B tolerant plant cell, plant tissue or plant according to claim 1, wherein said flavonol phenylacyltransferases 2 protein synthesizes a compound selected from the group consisting of Saiginol A*, Saiginol B*, Saiginol C*, Saiginol G*, Saiginol H*, Saiginol I*, Saiginol M*, Saiginol N*, Saiginol O*, Saiginol A, Saiginol B, Saiginol C, Saiginol D, Saiginol E, Saiginol F, Saiginol G, Saiginol H, Saiginol I, Saiginol J, Saiginol K, Saiginol L, Saiginol M, Saiginol N, Saiginol O, Saiginol P, Saiginol Q, and Saiginol R.

7. The transgenic UV-B tolerant plant cell, plant tissue or plant according to claim 1, wherein said exogenous nucleic acid molecule consists of a cDNA encoding a flavonol phenylacyltransferase 2 protein, wherein said nucleic acid molecule has at least 95% sequence identity to SEQ-ID NO:1.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1: Tissue specificity of the accumulation of saiginols. The relative ratio was estimated by using total peak area of general flavonol-glycosides and phenylacylated flavonol-glycosides.

(2) FIG. 2: Structural analysis of saiginol. A computational estimation of the most stable stereochemical structure of saiginol A is shown. The estimation was performed by MMFF94 of Marvin.

(3) FIG. 3: Spectrometric analysis of saiginol. HPLC-PDA profiling of a) kaempferol-3-O-Glc-2″-O-Rha-7-O-Rha, b) saiginol A (kaempferol-3-O-Glc-2″-O-Rha-6″-sinapoyl-7-O-Rha) and c) sinapoyl-Glc in the spectral range between 260 nm and 450 nm.

(4) FIG. 4: Chromosomal mapping of metabolite Quantitative Trait Loci (mQTL) for saiginol production. The chromosomal region on chromosome 2 indentified to be responsible for saiginol production (At2g22230-At2g3160) is delimited by a frame.

(5) FIG. 5: Saiginol A production in near isogenic lines (NIL) of Col0 and C24 accessions. Chromatograms of gain-of-function and loss-of-function near isogenic lines (NIL) obtained from mQTL analysis of reciprocal crosses between Col0 and C24 are shown. N and M lines are Col0 and C24 background introgression lines, respectively. Chromatograms show ion extracted chromatograms for saiginol A (945m/z), B & I (901m/z), C (885m/z) and D (799m/z), respectively.

(6) FIG. 6: mQTL analysis of saiginol A in near isogenic lines (NIL) of Col0 and C24 accessions. In total 45 M lines (C24 background) and 69 N lines (Col0 background) were used for saiginol targeted mQTL analysis. The heatmap shows values of saiginol A production displaying on a log.sub.2 scale divided by the average of all values.

(7) FIG. 7: shows a microarray analysis of gene expression of the genes encoded in the genomic region of chromosome 2 encoding the mQTL in five producing accessions (C24, Cvi, Da, Rsch and RLD) and five non-producing accessions (Col0, La-er, Ws, Sap and Stw). Duplicate hybridisations were carried out for Col0 and C24 for all other accessions a single hybridization was performed. Intensity indicates fold change compared to the expression level in Col0.

(8) FIG. 8: In vivo functional characterization of FPT1 and 2 by metabolite profiling of transgenic plants. All transgenic lines shown have a Col0 background. FPT1(Col), FPT1(C24), FPT2-1(Col0), FPT2-2(Col0) and FPT2(C24) indicate FPT1 cloned from Col0 and C24, FPT2 cloned from Col0 (FPT2-1 is shortest transcript and FPT2-1 is longest transcript) and C24 respectively. The chromatograms show ion extracted chromatogram for saiginol A (945m/z), B & I (901m/z), D (799m/z), G (961m/z) and J (815m/z), respectively.

(9) FIG. 9: Functional characterization of the role of saiginols in protection against UV-B irradiation. a) Representative photographs of the phenotypic changes of genotypes exhibiting modified flavonol contents following UV-B irradiation treatment (2h per day for 28 days with 1000 mW/m2 UV-B light irradiation) b) Seed yield after long-term (2 h per day for 28 days) UV-B treatment (1000 and 2000 mW/m2). Seeds were collected on a single plant basis but three individual plants per genotype were measured. Data is presented as mean+SE. *P<0.05. c) Germination rate using seeds obtained after UV-B irradiation (2h per day for 28 days with 2000 mW/m.sup.2 UV-B light irradiation).

(10) FIG. 10: Rate of silique production under UV-B irradiation after long-term (2 h per day) UV-B treatment (1 W/m.sup.2) using the detached immature inflorescences of first bolting. Error bars indicate the SE of six biological replicates. Data is presented as mean±S.E.M. *P<0.05. Abbreviation foe indication of plant lines is as written in example 10. OX-1 and OX-2 designate independent overexpressing lines using the same construct.

(11) FIG. 11: AtFPT2 overexpressing transgenic tobbaco plants. Three metabolites (805, 821, 835 m/z), which are annotated phenyacylated flavonol-glycosides were observed in AtFPT2-OX tobacco transgenic plants. This figure shows results of example 13.

EXAMPLES

(12) Plant Material

(13) Accessions of Arabidopsis thaliana used in the present application were obtained from NASC the European Arabidopsis Stock Centre. Plants were cultured on agar plates in a growth chamber under normal long day light conditions (16 h day, 140-160 μmol photons m−2 −s 1, 20° C.; 8 h night, 16° C.) for 14 days and transferred to 10 soil. Flower materials were harvested from individual plants, immediately frozen in liquid nitrogen, and stored at −80° C. until further use.

Example 1: Secondary Metabolite Profiling by LC-MS

(14) Given that considerable evidence has accumulated concerning the UV-B protective function of various phenylpropanoids during the processes of flower development, pollination and seed production assessment of levels of various phenylpropanoids in flowers in a set of 72 Arabidopsis ecotypes, which provided good coverage of the overall natural variability of the species, was performed by the inventors. Application of liquid chromatography-mass spectrometry (LC-MS) resulted in the detection of a total of 67 peaks consisting of 21 flavonoids, 12 glucosinolates, five hydroxycinnamates, eight polyamines, three putative lipid derivatives and 18 peaks of unknown chemical structure. All data were processed using Xcalibur 2.1 software (Thermo Fisher Scientific, Waltham, USA). Peak identification and annotation were performed in a combination approach using standard chemical confirmation, MSMS profiling, retention time profiling, mutant analysis, literature survey. In order to carry out mutant analysis for flavonoid derivatives, 14 mutants; ugt78d2 mutant (flavonoid-3-O-glucoside-less), tt7 mutant (quercetin and isorhamnetin derivatives-less), ugt78d1 mutant (flavonol-3-O-rhamnosides-less), ugt78d3 mutant (flavonol-3-O-arabinosides-less), omt1 mutant (isorhamnetin-derivatives-less), ugt89c1 mutant (flavonol-7-O-rhamnosides-less), tt4 mutant (all flavonoid-less), and papl-D mutant (anthocyanin-overaccumulator), and La-er background tt mutant series obtained from NASC (tt3, tt4, tt5, tt6) were used.

Example 2: Procedure of Purification and Characterization of Saiginol A

(15) MS-MS fragmentation studies suggested that these 18 peaks of unknown identity from Example 1 are novel flavonol derivatives. In order to characterize the chemical structure of these peaks, large amounts of the Arabidopsis accession C24, which accumulated these compounds, were re-grown and processed.

(16) Column chromatography was carried out over ODS (Nacalai Tesque, Cosmosil 75C18-OPN). HPLC analysis was carried out on an Atlantis® (ϕ4.6×150 mm, Waters) at a flow rate of 0.5 ml min.sup.1. Preparative HPLC was performed on a LC 10A system (Shimadzu) using an Inertsil® ODS-EP 5 m ($6.0×150 mm) at 30° C. and monitoring was accomplished by PDA (200-600 nm). HPLC-PDA-ESI-MS (HPLC/photodiode array detection/electrospray ionization mass spectrometry) was performed on a Finnigan LCQ-DECA mass spectrometer (ThermoQuest, San Jose, Calif., USA) and an Agilent HPLC 1100 series (Agilent Technologies, Palo Alto, Calif., USA). HR-ESI-MS was performed on an Exactive™ mass spectrometer (ThermoQuest, San Jose, Calif., USA). Optical rotations were determined on a JASCO P-1020. UV spectra were recorded on a JASCO V-560. NMR data were recorded on JEOL JNM ECP-600. The deuterated solvent CD3OD was used for peak 4. Coupling constants are expressed in Hz.

(17) Plant Materials, Extraction and Purification

(18) Plant samples (26.72 g fresh weight) were harvested and immediately frozen in liquid nitrogen, the whole of which was immediately extracted with methanol. After concentration, MeOH liquid extraction was extracted with n-hexane, CHCl.sub.3 to remove low polarity metabolites. After the liquid-liquid partition and concentration, MeOH soluble fraction was obtained and was dissolved with H.sub.2O. After liquid-liquid partition with n-BuOH, the n-BuOH fraction was obtained (256.9 mg).

(19) This fraction was applied to ODS column (4 3.5×7 cm), and roughly separated by eluting with a gradient of H.sub.2O as solvent A and CH.sub.3CN as solvent B and the following elution profile [fraction 1: 0% CH.sub.3CN, fraction 2: 10% CH.sub.3CN, fraction 3: 20% CH.sub.3CN, fraction 4: 30% CH.sub.3CN, fraction 5: 40% CH.sub.3CN, fraction 6: 50% CH.sub.3CN, fraction 7: 60% CH.sub.3CN, fraction 8: 70% CH.sub.3CN, fraction 9 80% CH.sub.3CN and fraction 10: 100% CH.sub.3CN (elution solvent: 70 ml/fraction)] to give 10 fractions. After LC-MS analysis for trace of peak 4, fraction 2 to 4 was assembled (fraction A). Fraction A was applied to ODS column (ϕ3.5×7 cm) again, and separated by eluting with a gradient of H.sub.2O as solvent A and CH.sub.3CN as solvent B and the following elution profile [fraction A-1 and A-2: 0% CH.sub.3CN, fraction A-3 and A-4: 10% CH.sub.3CN, fraction A-5 to A-7: 20% CH.sub.3CN, fraction A-8 to A-11: 30% CH.sub.3CN and fraction A-12: 100% CH.sub.3CN (elution solvent: 30 ml (fraction A-1 to A-5), 15 ml (fraction A-6 to A-12) to give 12 fractions.

(20) After LC-MS analysis for trace of peak 4, fraction A-10 and A-11 were assembled (fraction B). Fraction B (12.0 mg) was applied to preparative HPLC using an isocratic elution (20% CH.sub.3CN in H.sub.2O) at a flow rate of 4 ml/min to give peak 4 (3.5 mg).

(21) Characterization of Saiginol A

(22) Kaempferol-3-O-[2-O-(α-L-rhamnopyranosyl)-6-O-(sinapoyl)-β-D-glucopyranoside]-7-O-α-L-rhamnopyranoside

(23) Yellow amorphous solid. [α].sub.D.sup.20 −54.1° (c 0.09, MeOH). UV (MeOH): λ.sub.max (logs): 201.5 (3.47), 223.0 (3.61), 266.5 (3.37), 331.5 (3.47). HRESI-MS: m/z 969.2637 ([M+Na].sup.+, calcd. for C.sub.44H.sub.50O.sub.23Na, 969.2635). .sup.1H NMR (CD.sub.3OD) δ: 6.25 (1H, br.s, H-6), 6.41 (1H, br.s, H-8), 7.90 (2H, d, J=8.8 Hz, H-2′ and H-6′), 6.85 (2H, d, J=8.8 Hz, H-3′ and H-5′) (kaempferol; H-6, 8, H-2′ to 6′), 5.75 (1H, d, J=7.2 Hz, H-1), 3.63 (1H, m, H-2), 3.58 (1H, m, H-3), 3.32 (1H, m, H-4), 3.51 (1H, m, H-5), 4.26 (1H, m, H-6a), 4.40 (1H, m, H-6b) (glucose; H-1 to H-6a,b), 5.23 (1H, br.s, H-1), 3.99 (1H, m, H-2), 3.75 (1H, m, H-3), 3.32 (1H, m, H-4), 3.97 (1H, m, H-5), 0.90 (1H, m, H-6) (rhamnose 1; H-1 to H-6), 6.66 (2H, s, H-2), 6.06 (1H, d, J=16.0, H-7a), 7.28 (1H, d, J=16.0, H-813), 3.87 (6H, s, —OCH.sub.3) (sinapic acid; H-2, H-7c, H-8P and —OCH.sub.3), 5.33 (1H, br.s, H-1), 3.47 (1H, m, H-2), 4.04 (1H, m, H-3), 3.76 (1H, m, H-4), 3.47 (1H, m, H-5), 1.18 (1H, m, H-6) (rhamnose 2; H-1 to H-6). .sup.13C NMR (CD.sub.3OD) δ: 161.7, 134.5, 179.7, 159.7, 99.9, 163.2, 96.4, 157.9, 107.5, 123.2, 132.5, 116.4, 163.0, 116.4, 132.5 (kaempferol; C-2 to C-10 and C-1′ to C-6′); 100.2, 80.2, 79.2, 74.3, 76.1, 64.6 (glucose; C-1 to C-6); 102.8, 70.4, 72.5, 72.6, 72.6, 17.2 (rhamnose 1; C-1 to C-6), 12.75, 107.0, 148.5, 139.5, 149.5, 107.0, 115.6, 147.0, 168.7 (sinapic acid; C1- to C-9), 99.9, 71.4, 71.8, 72.3, 73.9, 19.3 (rhamnose 2; C-1 to C-6). HMBC correlations from the anomeric proton (Rha1H-1, 6 5.23) to C-2 of glucose, the protons (glucose H-6a,b, 6 4.26 and 4.40) to C-9 of sinapic acid and the anomeric proton (Rha2H-1, 6 5.33) to C-7 of kaempferol were observed.

Example 3: UV-B Absorption Profile of Saiginol A

(24) UV-VIS spectra in the range between 260 nm and 450 nm were recorded and analyzed via HPLC-PDA profiling for kaempferol-3-Glc-2″-Rha-7Rha (FIG. 3A), saiginol A (kaempferol-3-Glc-2″-Rha-6″-sinapoyl-7Rha, FIG. 3B) and sinapoyl-glucose (FIG. 3C). HPLC-PDA (HPLC/photodiode array detection) was performed on Dionex Ultimate 3000 system (San Jose, Calif., USA). Column chromatography was carried out on a Luna® (ϕ 2.00×150 mm, phenomenex) at a flow rate of 0.2 ml min.sup.−1 and monitoring was accomplished by PDA (200-600 nm).

(25) The flavonol-glycoside kaempferol-3-Glc-2″-Rha-7Rha, a precursor of saiginol A, appeared to be much less efficient in UV-B absorption than the phenylacylated saiginol A (kaempferol-3-Glc-2″-Rha-6″-sinapoyl-7Rha)(see FIG. 3A, B). The UV-VIS spectra of a Saiginol A revealed a Amax at 201.5 (3.47), 223.0 (3.61), 266.5 (3.37) and 331.5 nm (3.47).

(26) The substances kaempferol-3-O-Glc-2″-O-Rha-7-O-Rha and sinapoyl-Glc were purified and obtained from Arabidopsis plant extracts (Nakabayashi et al., 2008).

Example 4: Tissue Specificity of Saiginol Accumulation

(27) Plant Material

(28) Arabidopsis C24 plants were cultured on agar plates in a growth chamber under normal long day light conditions (16 h day, 140-160 μmol photons m.sup.−2 s.sup.−1, 20° C.; 8 h night, 16° C.) for 14 days and transferred to soil. Tissue materials were harvested and immediately frozen in liquid nitrogen, and stored at −80° C. until further use. Relative amount was compared and evaluated using detected peak area normalized by fresh weight material.

(29) The inventive saiginols were predominantly present in floral tissues but also to a lesser extent in the stem, silique and the cauline leaf but are essentially, or maybe even totally, absent in rosette leaves, senescent leaves and the root (FIG. 4) suggesting that they accumulate in the tissues which are highly exposed to light irradiation and most important with respect to facility. Further detailed spatial analysis of flavonol profiles revealed that the inventive saiginols predominantly accumulate in the floral petal and to a lesser extent in the stamen and pistil, but are probably absent in pollen.

Example 5: Metabolite Quantitative Trait Loci (mQTL) Analysis for Saiginols

(30) The inventors aimed to narrow down the genomic basis for the capability of accession C24 to produce the inventive saiginols, compared to the accession Col0 that is not capable to produce the inventive saiginols. Therefore, near Isogenic Lines (NILs) were obtained from the saiginol-producing accession C24 and the non-producing accession Col0 by reciprocal introgression as described previously (Torjek et al., 2008, J. Hered. 99, 396-406).

(31) In Brief:

(32) In order to create a desired NIL, a plant having the phenotype of interest (here accession C24 capable of saiginol production, also called “donor”) is crossed with an accession not capable of saiginol production (here Col0, also called “background”). The result is a F1 generation having a genome that consists of 50% of the genome of C24 and 50% of the genome of Col0. During gametogenesis of this F1 generation, crossing-over occurs leading to Col0-derived chromosomes with substitutions from C24 and C24-derived chromosomes having substitutions from Col0. Several generations of backcrossing with the Col0 parent line (also called background) leads to daughter generations with a more and more reduced amount of genomic segments from C24 within the Col0 background. This process is also called reciprocal introgression. Here, Arabidopsis lines derived from Col0 containing genomic fragments of C24 were named “N lines”. Vice versa, it is also possible to backcross the F1 generation with the C24 line leading to daughter generations with a more and more reduced amount of genomic segments from Col0 within the C24 background. In this case, the accession C24 would be the background line, and the accession Col0 the donor. Here, Arabidopsis lines derived from C24 containing genomic fragments of Col0 were named “M lines”.

(33) In total, 45 M lines (C24 background) and 69 N lines (Col0 background) were used for saiginol targeted mQTL analysis. The presence or absence of the inventive saiginols in the NILs was assessed by LC-MS (compare Example 1 and 2).

(34) Intriguingly, the inventors found a loss-of-function line in the NILs having a C24 background, i.e. a M line that is not capable of saiginol production (see NIL M16, FIG. 5 and FIG. 6 b), wherein the substituted genome segment (provided by Col0) mapped to chromosome 2 (At2g22230-At2g31610) as shown in FIG. 4.

(35) Interestingly a genomic substitution (provided by C24) in the same chromosomal segment led to a gain-of-function line within the NILs having a Col0 background, i.e. a N line that is now capable of saiginol production (see NIL N09, FIG. 5 and FIG. 6 a). Thus it can be concluded that a gene of accession C24 residing on chromosome 2 (At2g22230-At2g31610) is responsible to the production of the inventive saiginols.

(36) O. Torjek et al. (2008): Construction and analysis of 2 reciprocal Arabidopsis introgression line populations. J. Hered. 99, 396-406.

Example 6: Transcript Profiling Via Microarray Analysis

(37) The inventors further performed transcript profiling experiments in several accessions producing the inventive saiginols (among them C24) and in several accession not producing the inventive saiginols (among them Col0) in order to compare the gene expression between producers and non-producers in the region of chromosome 2 assigned to be relevant for saiginol production (see Example 2) to narrow down the genetic basics for saiginol production

(38) Transcriptome analysis was carried out using ATH1 microarrays as described previously. The experiment was performed with five producing accessions (C24, Cvi, Da, Rsch and RLD) and five non-producing accessions (Col0, La-er, Ws, Sap and Stw).

(39) In brief, the total RNA was extracted from frozen plant materials (see above) of each accession used by using the RNeasy Plant Mini Kit (Qiagen) according to the manufacturer's instructions.

(40) Subsequently, labelled target cRNA was prepared according to the manufacturer's instructions of the Arabidopsis Genome ATH1 DNA array (Affymetrix, Santa Clara, Calif., USA). For this, first double-stranded cDNA was prepared from 40 μg of total RNA by use of the SuperScript Choice System (invitrogen), again according to the manufacturer's instructions. Said cDNA was then transcribed in vitro using BioArray High Yield RNA Transcript Kit (Enzo, New York, N.Y., USA) into labelled cRNA.

(41) Following purification and fragmentation, the labeled cRNA was hybridized to Arabidopsis Genome ATH1 GeneChip array in a Hybridization Oven model 640 (Affymetrix). Washing and staining of the GeneChips were carried out using GeneChip Fluidics Station model 400. Scanning of the GeneChips was carried out with gene Array Scanner (Agilent Technologies). Duplicate hybridisations were carried out for Col0 and C24, while for all other accessions a single hybridization was performed.

(42) As a result, the expression levels were generally very similar between the genotypes although a number of genes were significantly different between producing and non-producing accessions including several transposable elements. However, only two genes were dramatically altered between Col0 and C24, whereas the remaining 827 genes, which were localized to the chromosomal segment of interest exhibited regular expression (see FIG. 7). The genes altered in their expression between Col0 and C24 were renamed as putative flavonol phenylacyltransferases 1 and 2 (FPT1 and FPT2). The gene transcripts for FPT1 and FPT2 were more than 17.7fold and 6.7fold higher in accession C24 than in accession Col0.

Example 7: Cloning of Full-Length cDNA of FPT1 and FPT2 Genes from Col0 and C24 Arabidopsis

(43) The C24 and Col0 alleles of both genes (FPT1 and FPT2) were cloned and the transcript production was evaluated. For this, first the total RNA was extracted from frozen plant materials (see above) of both accessions Col0 and C24 by using the RNeasy Plant Mini Kit (Qiagen©) according to the manufacturer's instructions.

(44) The mRNA contained in the total RNA that was used as template, was transcribed into oligo(dT)-primed cDNA by using using SuperScript III Reverse Transcriptase (Invitrogen©, Germany) according to the manufacturer's protocol. After digestion of the remaining RNA by an RNase H treatment, 1/10 of the cDNA reaction volume was used for PCR with gene-specific primers for FPT2 (SEQ-ID No. 5 & 6) or with gene-specific primers for FPT1 (SEQ-ID No. 19 & 20).

(45) The obtained PCR products were purified, cloned into a bacterial vector, transformed into E. coli, expressed in E. coli and purified. A cDNA segment was subcloned into GATEWAY pENTR Dual Selection vector (Invitrogen©, Germany). Then, 50 ng of the resulting plasmid expressing are mixed with 50 μl of Escherichia co/i One Shot® TOP10 Chemically Competent E. coli cells (Invitrogen©, Germany) and incubated on ice for 10 min. The mix is subsequently heated to 42° C. for 45 seconds followed by 5 min incubation in ice. 1 ml of Lysogeny Broth (LB) medium is added before incubation at 37° C. for 1h. Bacteria transformed in such a way are then plated on LB-agar plates containing 50 μg/ml of kanamycin for selection of positive clones. A positive clone was grown in LB medium over night. Afterwards the cells were pelleted and the expressed plasmids containing the cloned cDNA fragment were isolated using the kit NucleoSpin Plasmid (Macherey-Nagel, Germany). Finally, the fragments were sequenced.

(46) As a result, FPT1 transcript was invariant between Col0 and C24 (see SEQ-ID No. 3). However, the FPT2-Col0 allele encoded four different transcripts: FPT2-1(Col0) (737 bp in length, SEQ-ID No. 7), FPT2-2(Col0) (651 bp in length, SEQ-ID No. 8), FPT2-3(Col0) (631 bp in length, SEQ-ID No. 9) and FPT2-4(Col0) (560 bp in length, SEQ-ID No. 10). In contrast, FPT2-C24 encoded a single but considerably longer transcript (1305 bp, see SEQ-ID No. 1).

Example 8: Genomic Sequence of FPT2 Gene in Arabidopsis Accessions

(47) Primers used for amplification and sequencing of FPT2 are described below. Four primer sets for both producing/non-producing accessions, two primer sets for only producing accessions were used. DNA fragments amplified by sets of primers were sequenced by ABI PRISM® 3700 Genetic Analyzer (Applied Biosystems, USA)

(48) TABLE-US-00001 Set Forward Primer Reverse Primer 1 TGGACTAGTACGAGAATTGCAA ACCGGGAAGAAACTTGACGA AG (TT1f v2F; (TT1f v2R; SEQ-ID No. 11) SEQ-ID No. 12) 2 CGTGGAGCGACCAGTGAT CTTCACGTACCCTCTCGTCA (TT2f v4F; (TT2f v4R; SEQ-ID No. 13) SEQ-ID No. 14) 3 CTTATGCAGGGCTACCACAGT ACCACTGTTTACCAAAAACCGC (TT3f v2F; (TT3f v2R; SEQ-ID No. 15) SEQ-ID No. 16) 4 ATATCATATGAATAACAGCATC GCATAGGGCATCATCATCTC (TT4f v1F; (TT4f v1R; SEQ-ID No. 17) SEQ-ID No. 18)

(49) Genomic sequence analyses of FPT2 revealed a large gene deletion (˜2279 bp) in Col0, spanning the region corresponding to the second to tenth exons of FPT2-C24. This analysis indicated that the four transcripts of FPT2-Col0 described above (see Example 6) are alternative splicing variants.

Example 9: Overexpression of FPT2 and FPT1 in Col0 Plants

(50) In order to experimentally test the function of FPT1 and FPT2 in vivo, the inventors performed complementation assays in Col0 accessions by introducing an expression vector containing one of the transcripts described in Example 6 and over-expressing these transcripts.

(51) FPT1(Col0) and FPT1(C24), indicate FPT1 cloned from Col0 and C24, respectively.

(52) FPT2-1(Col0), FPT2-2(Col0) and FPT2(C24) indicate FPT2 cloned from Col0 (FPT2-1 is shortest transcript and FPT2-2 is longest transcript) and C24, respectively.

(53) A cDNA segment was subcloned into GATEWAY pK2GW7 vector (Invitrogen©, Germany). Then, obtained plasmids were transformed to Agrobacterium tumefaciens strain GV3101 pmp 90. Positive clones, which were checked by kanamycin selection, were suspended in 20 μl of sterile water and plated out on YEB petridishes with gentamycin (25 mg/l), rifampicin (100 mg/l) and the bacterial resistance marker kanamycin (50 mg/l). After incubation for 48 hours at 28° C., the solution was mixed with transformation medium (5% sucrose medium) for flower dipping transformation. T1 seedling plants have been selected by MS-agar plates (Murashige-Skoog-Medium agar plates) containing 50 μg/ml of kanamycin for selection of positive clones. These antibiotics selection were repeated for T2 and T3 generation seedling to obtain T3 homozygote transgenic plants.

(54) In order to determine the saiginol production in the transformed plants, flower and leaf materials of Col-0, NIL N09, tt4 mutant and transgenic lines (FPT2(C24)-OX, FPT2-1(Col0)-OX, FPT1(C24)-OX) were harvested for saiginol profile by chromatographic methods as described previously.

(55) As a result, overexpression of FPT1, irrespective if derived from C24 (FPT1(C24)) or Col0 (FPT1(Col0)), did not lead to a production of saiginols in Col0 plants (see FIG. 8) The same result was obtained for the overexpression of the shortest transcript (FPT2-1(Col0)), or the longest transcript (FPT2-2(Col0)) of FPT2 derived from Col0 (FIG. 8). Only the overexpression of FPT2 derived from C24 (FPT2(C24)) led to the production of saiginols in the otherwise non-producing accession Col0 (FIG. 8).

Example 10: UV-B Irradiation Experiment

(56) In order to test the effect of saiginol production with respect to UV-B irradiation in vivo, the following plants were tested in this experiment:

(57) (1) Col0 as a representative for a non-producing accession

(58) (2) NIL N09—the gain-of-function line (C24 donor, Col0 background, Example 5)

(59) (3) FPT2(C24)-OX—transgenic Col0 plant overexpressing FPT2(C24) (Example 8)

(60) (4) FPT2-1(Col0)-OX—transgenic Col0 plant overexpressing FPT2-1(Col0) (Example 8)

(61) (5) FPT1 (C24)-OX—transgenic Col0 plant overexpressing FTP1(C24) (Example 8)

(62) (6) tt4—a mutant Arabidopsis line lacking flavonoids

(63) Plants were cultured on agar plate in a growth chamber under normal long day light conditions (16 h day, 140-160 μmol photons m.sup.−2 s.sup.−1, 20° C.; 8 h night, 16° C.) for 14 days and transferred to soil.

(64) One week after transferring the plants to the soil and before plant bolting, plants were started to be irradiated with UV-B light in addition to the normal light regimen. UV-B light was irradiated 2h per day during midday time (stated 5-7h after starting normal light). The intensities of UV-B light tested in two trials were 1000 mW/m.sup.2 and 2000 mW/m.sup.2.

(65) After bolting of the plants, the seeds were collected and both the seed yield and the seed germination rate were determined using 2 weeks old seedling.

(66) The seed yield was calculated as ratio between the seed yield (in mg) of an UV-B radiated plant and the seed yield (in mg) of a control plant without additional UV-B irradiation.

(67) Not surprisingly, the flavonoid less tt4 mutant line was especially prone to UV-B irradiation and showed the lowest seed yield and seed germination rate (FIG. 9b, c).

(68) The seed yield of the non-producing accession Col0 was reduced in comparison to not UV-B irradiated Col0 plants, especially with higher UV-B intensity (FIG. 9b). The Col0 lines overexpressing FPT1(C24) or FPT2-1(Col0) were indistinguishable from regular Col0 (FIG. 9b).

(69) However, the seed yield in the gain-of-function line NIL N09 and the Col0 line overexpressing FPT2(C24) was considerably higher compared to normal Col0 plants (FIG. 9b). Moreover, the germination rate of the seeds was also doubled in NIL N09 and FPT2(C24)-OX compared to Col0 (FIG. 9c), revealing that the production of germinable seeds was almost five times higher in the gain-of-function (NIL N09) line and the C24-FPT2 overexpressing line than in lines that do not produce the inventive saiginols.

(70) In addition, the vitality of the NIL N09 gain-of-function line and the FPT2-C24 overexpressing line was reflected in the number of leaves and inflorescences these lines produced compared to Col0 and tt4 (FIG. 9a). Moreover, NIL N09 and FPT2-C24 overexpressing line was considerably larger than tt4.

(71) For silique production experiment (result shown in FIG. 10), inflorescences from the primary bolts of 5-week-old Arabidopsis plants were used. The detached inflorescences were irradiated with UV-B light (1 W m2) for 2 h per day during midday time (5-7 h after the onset of normal light) for 14 days by placing their cut ends in wells of a 96-well microtiter plate containing water.

Example 11: Sunscreen Formulation

(72) A suitable sunscreen composition contains the following components with listed percentages based on total weight of the composition:

(73) Active Ingredients: titanium oxide (20.0%), saiginol A (7.5%),

(74) Inactive Ingredients: ethanol (59.9%), hyaluronic acid (2.5%), grape seed oil (5.0%), sesame oil (2.5%), sunflower seed oil (2.5%), and tetrahexyldecyl ascorbate (0.1%).

Example 12: Transformation of Tobacco and Tomato Plants

(75) A cDNA segment was subcloned into GATEWAY pK2GW7 vector (Invitrogen©, Germany). Then, obtained plasmids were transformed to Agrobacterium tumefaciens strain GV2260 pmp 90. Positive clones, which were checked by spectinomycin selection, were suspended in 20 μl of sterile water and plated out on YEB petridishes with gentamycin (25 mg/l), rifampicin (100 mg/l) and the bacterial resistance marker spectinomycin (50 mg/l). After incubation for 24 hours at 28° C., the solution was mixed with transformation medium (5% sucrose medium) for transformation. Tobacco or tomato leaf disc (1×1 cm) which was injured by metal blade was used for injection. After dipping into agrobacterium solution, leaf discs were incubated on MS-agar plate for 48 h under darkness. After this treatment, leaf disc has been transferred to MS-agar plate (Murashige-Skoog-Medium agar plates) containing 50 μg/ml of kanamycin for selection of positive clones and 6-Benzylaminopurine (BAP) and indole-3-acetic acid (IAA) for induction of callus root.

Example 13: Secondary Metabolite Profiling of Transgenic Tobacco Plants by LC-MS

(76) Application of liquid chromatographyÐmass spectrometry (LC-MS) using the method described in Example 2 resulted in the detection of a total of 3 peaks annotated as putative phenyacylated flavonol-glycosides in tobacco. All data were processed using Xcalibur 2.1 software (Thermo Fisher Scientific, Waltham, USA). This result indicates that FPT2 gene can be employed to metabolic engineering in other plant species. Since function of FPT2 has a broader function with lower substrate specificity, it can be used to transform other plant species than Arabidopsis and improve their UV-B resistance.

(77) TABLE-US-00002 SEQUENCE LIST SEQ-ID No. 1: cDNA of C24 FPT2 ATGAGAACTTTTTCACCCAAGTTGCTGCTTCTTCTTTTACTTGTTTTAAGACATCATGCTGAATCTGGCTCTA TCGTCAAGTTTCTTCCCGGTTTTGAAGGCCCTCTTCCTTTCGAACTTGAAACCGGGTACATCGGTATTGGTGA AGAAGAAGAACTGCAATCGTTTTACTATTTCATTAAGTCTGAGAAGAATCCAAAAGAAGATCCTCTTCTTCTT TGGATATCTGGAGGACCTGGTTGCTCTTCTATTTCTGCTCTTCTTTTTGAGAATGGACCTGTGGCTCTAAAGT TCGAGGTTTACAATGAAACTCTCCCTTATTTGGTCTCTACTACATATTCATGGACCAAGATGACGAACGTATT ATTCTTGGATCAGCCTATTGGAGTTGGCTTCTCCTACAAAAGAACTCCAAATCTTGATAAATCGAGTGACACA ATAGAAGTATTGCGGATATACGAATTTCTTCAGAAGTGGCTAGGTGAACATCCTGAGTTTTTCTCCAACACTT TTTACGTAGGAGGAGATTCTTATTCCGGTAAGATTGTTCCAGCTATCGTTGATAAAATCTCACAAGAAAATTA TTTGTGCTGCAAACCTCCAATAAATCTTCAGGGTTATGTTCTCGGAAACCCAATAACAAATTTGGAATCTGAT TCTAACTATCGTATTCCATATGCTCATGGAATGGCATTAATTTCTGATGAGCTCTACGAATCCCTGAAGAGAA ACTGCAAAGGAAGATATAAAACCGTGGATCCATCTAACAAAAAATGTTTGAAACTTGTTGAAAAATACAATAA GTGTTCTGATAAAATATTTAGAGAACTAATATTATTACCACAGTGTGATGAAAGATCTCCACTCTGCTGGGGC TACCACAGTACACTAGCTAAATATTGGGCCAATGACGAGAGGGTACGTGAAGCTCTTCAAATAAGAAAGGGAA GTATAGGAAAATGGATACGATGTAATACGAATATACATTACGGTGACGACATTATTAGCAGCATACCATATCA TATGAATAACAGCATCAACGGATACCGATCTCTCATTTACAGTGGTGATCACGATATGGAGGTACCTTTCCTT GCAACTGAAGCTTGGATAAGATCTCTCAATTATCCTATTATTGATGATTGGAGGCCTTGGATAATAAACAATC AGATTTCAGGATACACGATGACCTATGCCAATAAGATGACATATGCTACTATCAAGGGAGGTGGACACACTGC AGAGTATAAACCAGCGGAGAGCTTTATCATGTTCCAACGATGGATCAGTGGCCAGTCTCTGTAA SEQ-ID No. 2: amino acid sequence coded by SEQ-ID No. 1 MRTFSPKLLLLLLLVLRHHAESGSIVKFLPGFEGPLPFELETGYIGIGEEEELQSFYYFIKSEKNPKEDPLLL WISGGPGCSSISALLFENGPVALKFEVYNETLPYLVSTTYSWTKMTNVLFLDQPIGVGFSYKRTPNLDKSSDT IEVLRIYEFLQKWLGEHPEFFSNTFYVGGDSYSGKIVPAIVDKISQENYLCCKPPINLQGYVLGNPITNLESD SNYRIPYAHGMALISDELYESLKRNCKGRYKTVDPSNKKCLKLVEKYNKCSDKIFRELILLPQCDERSPLCWG YHSTLAKYWANDERVREALQIRKGSIGKWIRCNTNIHYGDDIISSIPYHMNNSINGYRSLIYSGDHDMEVPFL ATEAWIRSLNYPIIDDWRPWIINNQISGYTMTYANKMTYATIKGGGHTAEYKPAESFIMFQRWISGQSL SEQ-ID No. 3: cDNA of C24 FPT1 ATGAAATCAACACTAAAATTGCTGCTTCTGCTTCTGTTTATGTTAAACCATCATGTTGATTCTGGCTCTATCG TCAAGTTTCTTCCCGGCTTTGAAGGCCCTCTTCCTTTCGAACTCGAAACCGGGTACATTGGTATTGGTGAGGA AGAGGAAGTACAGTTGTTCTACTACTTTATAAAGTCTGAGAGAAATCCAAAAGAAGACCCTCTTCTTCTCTGG TTAAGTGGAGGACCTGGATGTTCATCTATCACTGGCCTTCTTTTCGAGAATGGACCTTTGGCTTTGAAGTCCG AGGTTTACAATGGAAGTGTCCCTTCTTTGGTCTCTACTACATATTCGTGGACAAAGACGGCGAACATAATATT CTTGGATCAGCCTATTGGAGCTGGCTTCTCCTACTCAAGAATCCCACTTATTGATACGCCTAGTGACACAGGC GAAGTTAAGAATATCCATGAGTTTCTCCAAAAGTGGTTAAGCAAGCATCCACAGTTTTCTTCCAATCCTTTCT ATGCTAGCGGAGATTCTTATTCCGGTATGATTGTTCCAGCCCTCGTTCAAGAAATTTCGAAAGGAAATTATAT ATGTTGCAAACCTCCTATAAATCTACAGGGCTATATACTCGGGAACCCAATAACATATTTTGAAGTCGACCAA AACTATCGCATTCCATTTTCTCATGGAATGGCACTTATTTCAGATGAACTATACGAGTCAATTAGGAGAGACT GCAAAGGAAATTATTTCAACGTGGATCCACGTAACACAAAATGTTTGAAACTTGTTGAAGAATACCATAAGTG TACCGACGAACTAAATGAATTCAATATATTATCACCAGATTGCGACACGACATCTCCTGATTGCTTTGTATAT CCATATTATCTCCTTGGCTACTGGATCAACGACGAGAGCGTTCGCGATGCTCTTCATGTTAATAAGAGCAGTA TTGGAAAATGGGAGCGATGTACTTATCAAAATAGAATCCCATACAACAAAGACATCAATAACAGCATACCATA CCATATGAATAACAGTATTAGTGGCTACCGATCTCTCATCTACAGTGGTGATCATGATTTGGTGGTTCCTTTC CTTGCAACTCAAGCCTGGATAAAATCTCTAAATTACTCCATCATTCATGAATGGAGACCTTGGATGATTAAAG ATCAAATCGCTGGGTATATAATATATTTTTGTGTTATACACGAGAACTTATTCCAATAA SEQ-ID No. 4: amino acid sequence coded by SEQ-ID No. 3 MKSTLKLLLLLLFMLNHHVDSGSIVKFLPGFEGPLPFELETGYIGIGEEEEVQLFYYFIKSERNPKEDPLLLW LSGGRGCSSITGLLFENGPLALKSEVYNGSVPSLVSTTYSWTKTANIIFLDQPIGAGFSYSRIPLIDTRSDTG EVKNIHEFLQKWLSKHPQFSSNPFYASGDSYSGMIVPALVQEISKGNYICCKPPINLQGYILGNPITYFEVDQ NYRIPFSHGMALISDELYESIRRDCKGNYFNVDPRNTKCLKLVEEYHKCTDELNEFNILSPDCDTTSPDCFVY PYYLLGYWINDESVRDALHVNKSSIGKWERCTYQNRIPYNKDINNSIPYHMNNSISGYRSLIYSGDHDLVVPF LATQAWIKSLNYSIIHEWRPWMIKDQIAGYIIYFCVIHENLFQ SEQ-ID No. 5: forward primer for amplification of FPT2 cDNA (At2g22980) ATGAGAACTTTTTCACCCAAGTT SEQ-ID No. 6: reverse primer for amplification of FPT2 cDNA (At2g22960) TCATCATCTCTTATTATTACAGA SEQ-ID No. 7: cDNA of Co10 FPT2-1 ATGAGAACTTTTTCACCCAAGTTGCTGCTTCTTCTTTTACTTGTTTTAAGACATCATGCTGAATCTGGCTCTA TCGTCAAGTTTCTTCCCGGTTTTGAAGGCCCTCTTCCTTTCGAACTTGAAACCGGGGCTACCACAGTACACTA GCTAAATATTGGGCCAATGACGAGAGGGTACGTGAAGCTCTTCAAATAAGAAAGGGAAGTATAGGAAAATGGA TACGATGTAATTCGAATATACATTACGATGACGACATTATTAGCAGCATACCATATCATATGAATAACAGCAT CAACGGATACCGATCTCTTATTTACAGGTTTGATTAAAATTACATTTTAAAGAATCGATCTAGTTCTCTATAC AAACTATGCGGTTTTTGGTAAACAGTGGTGATCACGATATGGAGGTACCTTTCCTTGCAACTGAAGCTTGGAT AAGATCTCTTAATTATCCTATTATTGATGATTGGAGGCCTTGGATAATAAACAATCAGATTGCTGGGTGAATA ATTTTTACAATTTTTTTTTTTGCTTCTTTATTCATTAACTCAGTCGAATCATGAGCTCATTAGTCATTTTGAA ATCCCTCTTACGATTTTCTTGGACAGATACACGATGACCTATGCCAATAAGATGACATATGCTACTATCAAGG GAGGTGGACACACTGCAGAGTATAAACCAGCGGAGAGCTTTATCATGTTCCAACGATGGATCAGTGGCCAGCC TCTGTAA SEQ-ID No. 8: cDNA of Co10 FPT2-2 ATGAGAACTTTTTCACCCAAGTTGCTGCTTCTTCTTTTACTTGTTTTAAGACATCATGCTGAATCTGGCTCTA TCGTCAAGTTTCTTCCCGGTTTTGAAGGCCCTCTTCCTTTCGAACTTGAAACCGGGGCTACCACAGTACACTA GCTAAATATTGGGCCAATGACGAGAGGGTACGTGAAGCTCTTCAAATAAGAAAGGGAAGTATAGGAAAATGGA TACGATGTAATTCGAATATACATTACGATGACGACATTATTAGCAGCATACCATATCATATGAATAACAGCAT CAACGGATACCGATCTCTTATTTACAGTGGTGATCACGATATGGAGGTACCTTTCCTTGCAACTGAAGCTTGG ATAAGATCTCTTAATTATCCTATTATTGATGATTGGAGGCCTTGGATAATAAACAATCAGATTGCTGGATACA CGATGACCTATGCCAATAAGATGACATATGCTACTATCAAGGCAAGTTTTTTTTTTTTGTTCTTACTCTTAAT GTTTTTTATTACACAAATCATTTTGTTTCTCAGTAACTGTTTTGAGGGGTTTACTATAGGGAGGTGGACACAC TGCAGAGTATAAACCAGCGGAGAGCTTTATCATGTTCCAACGATGGATCAGTGGCCAGCCTCTGTAA SEQ-ID No. 9: cDNA of Col0 FPT2-3 ATGAGAACTTTTTCACCCAAGTTGCTGCTTCTTCTTTTACTTGTTTTAAGACATCATGCTGAATCTGGCTCTA TCGTCAAGTTTCTTCCCGGTTTTGAAGGCCCTCTTCCTTTCGAACTTGAAACCGGGGCTACCACAGTACACTA GCTAAATATTGGGCCAATGACGAGAGGGTACGTGAAGCTCTTCAAATAAGAAAGGGAAGTATAGGAAAATGGA TACGATGTAATTCGAATATACATTACGATGACGACATTATTAGCAGCATACCATATCATATGAATAACAGCAT CAACGGATACCGATCTCTTATTTACAGGTTTGATTAAAATTACATTTTAAAGAATCGATCTAGTTCTCTATAC AAACTATGCGGTTTTTGGTAAACAGTGGTGATCACGATATGGAGGTACCTTTCCTTGCAACTGAAGCTTGGAT AAGATCTCTTAATTATCCTATTATTGATGATTGGAGGCCTTGGATAATAAACAATCAGATTGCTGGATACACG ATGACCTATGCCAATAAGATGACATATGCTACTATCAAGGGAGGTGGACACACTGCAGAGTATAAACCAGCGG AGAGCTTTATCATGTTCCAACGATGGATCAGTGGCCAGCCTCTGTAA SEQ-ID No. 10: cDNA of Co10 FPT2-4 ATGAGAACTTTTTCACCCAAGTTGCTGCTTCTTCTTTTACTTGTTTTAAGACATCATGCTGAATCTGGCTCTA TCGTCAAGTTTCTTCCCGGTTTTGAAGGCCCTCTTCCTTTCGAACTTGAAACCGGGGCTACCACAGTACACTA GCTAAATATTGGGCCAATGACGAGAGGGTACGTGAAGCTCTTCAAATAAGAAAGGGAAGTATAGGAAAATGGA TACGATGTAATTCGAATATACATTACGATGACGACATTATTAGCAGCATACCATATCATATGAATAACAGCAT CAACGGATACCGATCTCTTATTTACAGTGGTGATCACGATATGGAGGTACCTTTCCTTGCAACTGAAGCTTGG ATAAGATCTCTTAATTATCCTATTATTGATGATTGGAGGCCTTGGATAATAAACAATCAGATTGCTGGATACA CGATGACCTATGCCAATAAGATGACATATGCTACTATCAAGGGAGGTGGACACACTGCAGAGTATAAACCAGC GGAGAGCTTTATCATGTTCCAACGATGGATCAGTGGCCAGCCTCTGTAA SEQ-ID No. 11: forward primer of set 1 for amplification of FPT2 genome sequence TGGACTAGTACGAGAATTGCAAAG SEQ-ID No. 12: reverse primer of set 1 for amplification of FPT2 genome sequence ACCGGGAAGAAACTTGACGA SEQ-ID No. 13: forward primer of set 2 for amplification of FPT2 genome sequence CGTGGAGCGACCAGTGAT SEQ-ID No. 14: reverse primer of set 2 for amplification of FPT2 genome sequence CTTCACGTACCCTCTCGTCA SEQ-ID No. 15: forward primer of set 3 for amplification of FPT2 genome sequence CTTATGCAGGGCTACCACAGT SEQ-ID No. 16: reverse primer of set 3 for amplification of FPT2 genome sequence ACCACTGTTTACCAAAAACCGC SEQ-ID No. 17: forward primer of set 4 for amplification of FPT2 genome sequence ATATCATATGAATAACAGCATC SEQ-ID No. 18: reverse primer of set 4 for amplification of FPT2 genome sequence GCATAGGGCATCATCATCTC SEQ-ID  No. 19: forward primer for amplification of FPT1 cDNA (At2g22920) ATGAAATCAACACCAAAAT SEQ-ID No. 20: reverse primer for amplification of FPT1 cDNA (At2g22920) ATTTAGGTAAGGTCATTCAAGTA SEQ-ID No. 21: amino acid (aa) sequence of a variant of BLOCK 1 GPGCSSxxxL (x = random amino acid) SEQ-ID No. 22: amino acid (aa) sequence of a variant of BLOCK 2 GDSYSG