US 2013 O305408A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2013/0305408 A1 ROMMENS et al. (43) Pub. Date: Nov. 14, 2013 (54) AUREUSIDIN-PRODUCING TRANSGENIC Publication Classification PLANTS (51) Int. Cl. (71) Applicant: J.R. SIMPLOT COMPANY, Boise, ID CI2N 5/82 (2006.01) (US) (52) U.S. Cl. CPC .................................... CI2N 15/825 (2013.01) (72) Inventors: Caius M. ROMMENS, Boise, ID (US); USPC ......................... 800/278; 800/298; 435/320.1 Roshani SHAKYA, Boise, ID (US); Jingsong YE, Boise, ID (US) (57) ABSTRACT Aurone, including aureusidin-6-O-glucoside, are known to (21) Appl. No.: 13/829,691 have antioxidant properties. The compounds are produced in (22) Filed: Mar 14, 2013 the flowers Snapdragon (e.g., Antirrhinum majus) and have 9 been suggested for potential medicinal use. The present meth O O ods use recombinant and genetic methods to produce aurone Related U.S. Application Data in plants and plant NS In particular, i. present meth (60) Provisional application No. 61/646,020, filed on May ods have resulted in the production of aureusidin-6-O-gluco 11, 2012. side in the leaves of various plants. Patent Application Publication Nov. 14, 2013 Sheet 1 of 18 US 2013/0305408A1 +———L- Patent Application Publication Nov. 14, 2013 Sheet 2 of 18 US 2013/0305408A1 Patent Application Publication Nov. 14, 2013 Sheet 3 of 18 US 2013/0305408A1 | Patent Application Publication Nov. 14, 2013 Sheet 4 of 18 US 2013/0305408A1 ^^k-oxo~~~ W. 6 s & a i Patent Application Publication Nov. 14, 2013 Sheet 5 of 18 US 2013/0305408A1 i Patent Application Publication Nov. 14, 2013 Sheet 6 of 18 US 2013/0305408A1 Patent Application Publication Nov. 14, 2013 Sheet 7 of 18 US 2013/0305408 A1 Patent Application Publication Nov. 14, 2013 Sheet 8 of 18 US 2013/0305408A1 i 8 oov ose S. Patent Application Publication Nov. 14, 2013 Sheet 9 of 18 US 2013/0305408A1 §§§ 6ºunãIA 3 3 Patent Application Publication Nov. 14, 2013 Sheet 10 of 18 US 2013/0305408A1 Patent Application Publication Nov. 14, 2013 Sheet 11 of 18 US 2013/0305408A1 i Patent Application Publication Nov. 14, 2013 Sheet 12 of 18 US 2013/0305408A1 ########'$$$* Patent Application Publication Nov. 14, 2013 Sheet 13 of 18 US 2013/0305408A1 Figure 13 Bg i (;) CaMv35S polyA T Border (L) '': am H (597) Sac II (13822) A Sac I (1090) x . \ , ; ; Sina I ( ) Kanamycin(R) AmA$1 ps R322 or ^ \| Sac II (1751) 883 : Sac I (2190) HindIII (2834) Not I (11595) Ubi3T of 8 e pSIM1251 Pst I (3O83) s Sal (321O) 14347 bp Not I (10305) PV - Sac I (4834) Not I (8773) . : N Sph (7894) . N Nos T-border(R) Kpn I (5117) NOS polyA Bam HI (57O3) Bst EII (7489} . ; : , , , , ... Nco I (5707) Kpn I (7486) , , , , , Sac II (59oo) Nco I (7478) is . Sac II (5911) Apa I (778) Sima (6224) Sal I (7455) Xba I (6299) Pst I (7328) Xio (6360) 3. i HindIII (6379) specios3) 'CG Patent Application Publication Nov. 14, 2013 Sheet 14 of 18 US 2013/0305408A1 Figure 14 3xx: { EcoRI 3.333 ic:8: {so}}: A. f 35s Promoter A EcoRises Sixx: {}:33: . pt &:::::33:33 Xix: {33 a W38S xxiya 8:::::::33: &" pSIM1252 {}}}} p Karanycin (R - pBR322 or pBR322 bom 3: 8:3 Patent Application Publication Nov. 14, 2013 Sheet 15 of 18 US 2013/0305408A1 Figure 15 Kpn I (1) NA (L) FVV HindIII (16660) X s Sac I (1080) UbiST Sna I (1560) Spe I (16046) Nco I (14976) . AmAS1 Bgt II (14748) . ipt Sac 1 (218o) Xba I (14462) y U3Psix ‘. Hillin dIII (2824) Xba I (14268) U 3. Xba I (14O76) Sal I (32OO) Kanamycin (R) pSIM1257 CMPSP pBR322 ori pBR322 bom 16856 bp ... Sac I (4824) Not I (11585) Nos - Kpn I (5107) x888; Not I (10295) FMV Y. Nco I (5697) Pvs1 Sta Sna I (6214) Not (8763) f ', XitsXba I (635)(6289) Sph (7884) f , , , HindIII (6369) : -border(R) 4'CGT NOS polyAp ya, A.S. SO(3)3. Kpn I (7476) Sal I (7445) Nco (7468). Apa (7468) Patent Application Publication Nov. 14, 2013 Sheet 16 of 18 US 2013/0305408A1 Figure 16 Kpn I (11) FMV Bgi II (1) | Bam HI (597) Cai V35S poiy A Sac I (1990) Sca I (14225) Sinai (157O) T Border (L) AmAS1 Sac II (13868) . Sacsci II (1751) (60) pBR322 or N PstIbis I (3083) per322 born Sal I (321o) Not I (11641) E. w8 ep pSIM1610 35S Xba I (4062) Not I (10351) 14393 bp Bam HI (4068) Sma I (4O75) pVS1 Sta is. Pst I (4261) Not I (8819) -- not *... R (4613) 's Neo I (4640) Sph I (7940) - Sac I (4879) -border(R) ; : A '. \ w x s Nost NOS polyA / , , , , , , , , , Sma I (5157) Bst EII (7535) . Kipn I (5163) Kpn I (7532) ‘. Apa $75,(7524) . , . , . , . , . , NcoBan.HI I (5753) (5749) Still (750) , , , Sac II (596) PstUbi3 I (7374) . SingSac II(676) (59.7) Spei (729) ES:45 M'CG Hin dIII (64.25) Patent Application Publication Nov. 14, 2013 Sheet 17 of 18 US 2013/0305408A1 Figure 17 Caivass polypolyA ".FMV Sca I (9002) Eco RI (531) TBorder () Ban H. (397) Sac i (864.5) \ - R x8 Nico I (1358) Kanamycin(R) DFR EcoRV (7768) Spei (1906) EcoRV (2079) :::::::::::: -- 8 & pSIM1618 3 :38: 888 9170 bp Pstitd. (2151)o r-, Sal (2278) .. Apa i (230) Not (6418) Nco I (2301) x8: 8: Kpn (2309) ABst EII (232) Noti (528) ANOS polyA Not (512 pWS1 Sta\ liberder-border R Not (3596) Sca I (2748) Patent Application Publication Nov. 14, 2013 Sheet 18 of 18 US 2013/0305408A1 Figure 18 Nos eritator 883: {333333 & 838: Ubiz Promoter 8:::::::::::::: ::::::::::8: Bg::::::::: Ubit$8::::::::::::8:3 intron 388: 8:33 xxxx pSIM646 38:::383: 33-3-3 is ap is:::::33:3 CaMv35s polyA border(R) *:::::::::::: S: 38: s: R. 8 8 pvs1 Sta US 2013/0305408 A1 Nov. 14, 2013 AUREUSDN-PRODUCING TRANSGENIC antioxidants that are not normally expressed or produced in PLANTS the plant, or are expressed or produced at low levels in the plant. In one embodiment, the modification encompasses FIELD OF THE INVENTION expression at least one of a chalcone 4'-O-glucosyltransferase 0001. The present inventive technology concerns genetic gene (Ama"CGT) and an aureusidin synthase (AmAS1) gene modifying the aurone biosynthetic pathway of crop plants. in plants that do not normally express either gene. 0007 Another aspect of the present invention is a plant BACKGROUND comprising in its genome at least one of a chalcone 4'-O- 0002 Aurones are flavonoids with a 5-membered C-ring glucosyltransferase gene (Ama"CGT) and an aureusidin Syn that provide a bright yellow color to the petals of some vari thase (AmAS1) gene, wherein the plant genome does not eties of Snapdragon (Antirrhinum), morning glory (Ipomoea), naturally comprise the Am4'CGT or AmAS1 gene. Dahlia and Coreopsis (Saito, 1990; Iwashina, 2000). An 0008. In another embodiment, the plant genome does analysis of flower color variation in natural populations of comprise at least one of the Am4'CGT or AmAS1 gene but Snapdragon Suggests that aurones play a role in fertilization either does not express these genes or expresses these genes at and seed set by attracting pollinators (Whibley et al., 2006). low levels. The present inventive methods disclosed herein Indeed, the patterning of aurone pigmentation is thought to encompass operably linking one or both of the Am4'CGT or provide a nectar guide for pollinating bumblebees (Harborne AmAS1 genes to a promoter functional in plants and intro and Smith 1978, Lunau et al. 1996). In addition to this role in ducing the resultant construct into the plant, wherein the pigmentation, aurones have been described as phytoalexins promoter expresses the Am4'CGT and/or AmAS1 gene in the that are used by the plant as defense agents against various plant to which it is operably linked and changes leaf color and pathogens; they were found to exhibit antiviral, antiparasitic, antioxidant production, compared to an untransformed plant. and antifungal activities (Boumendjel, 2003). 0009. In one embodiment, the expression of one or both of 0003 Previously, a two-step mechanism involving the the Am4'CGT or AmAS1 genes in the transformed plant is oxidation of isoliduiritigenin by a hydrogen peroxide (H2O)- transient. In another embodiment, the expression of one or dependent peroxidase (PRX), followed by dehydration of the both of the Am4'CGT or AmAS1 genes in the transformed intermediate compound to formaurone 4,6-dihydroxyaurone plant is constitutive. In another embodiment, the expression was proposed for aurone biosynthesis in soybean (Soja his of one or both of the Am4'CGT or AmAS1 genes in the pida) seedlings (Wong E, 1966; Rathmell and Bendall, 1972). transformed plant is inducible. In Snapdragon, the aurone aureusidin-6-O-glucoside (AOG) 0010. One aspect of the present invention comprises trans is produced by glucosylation of 2',4',6'4-tetrahydroxychal forming a plant with a construct that comprises (i) a promoter cone (naringenin chalcone), which facilitates transport of this functional in plant tissue, operably linked to a nucleotide compound from cytoplasm to vacuole (Ono et al., 2006), sequence encoding either or both of (ii) an Am4'CGT protein, followed by cyclization of the carbon bridge. The proteins or (iii) an AmAs1 protein, wherein the color of the trans involved in these reactions are chalcone 4'-O-glucosyltrans formed plants leaves are different than that of an untrans ferase (Ama"CGT) and the copper-containing glycoprotein formed plant of the same species, and/or the leaves of the aureusidin synthase (AmAS1) (Nakayama et al., 2000), transformed plant comprise higher Super oxide dismutase respectively.