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WO 2016/016826 Al 4 February 2016 (04.02.2016) P O P C T

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WO 2016/016826 Al 4 February 2016 (04.02.2016) P O P C T (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2016/016826 Al 4 February 2016 (04.02.2016) P O P C T (51) International Patent Classification: (74) Agent: xyAJ PARK; Level 22, State Insurance Tower, 1 A01H 1/06 (2006.01) C12N 15/61 (2006.01) Willis Street, Wellington (NZ). C12N 15/29 (2006.01) A01H 5/08 (2006.01) (81) Designated States (unless otherwise indicated, for every C12N 15/113 (2010.01) kind of national protection available): AE, AG, AL, AM, (21) International Application Number: AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, PCT/IB2015/055743 BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (22) International Filing Date: HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, 30 July 2015 (30.07.2015) KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, (25) Filing Language: English MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, (26) Publication Language: English SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, (30) Priority Data: TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. 6282 14 1 August 2014 (01.08.2014) NZ (84) Designated States (unless otherwise indicated, for every (72) Inventors; and kind of regional protection available): ARIPO (BW, GH, (71) Applicants : DARE, Andrew Patrick [NZ/NZ]; 40 Jef GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, ferson Street, Glendowie, Auckland, 1071 (NZ). GREEN¬ TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, WOOD, David R [NZ/NZ]; Plant & Food Research, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, Private Bag 92169, Auckland Mail Centre, Auckland, 1142 DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, (NZ). HELLENS, Roger Paul [AU/AU]; Center for Trop LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, ical Crops and Biocommodities, Queensland University of SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, Technology (QUT), GPO Box 2434, Brisbane, Queensland GW, KM, ML, MR, NE, SN, TD, TG). 4001 (AU). Published: — with international search report (Art. 21(3)) [Continued on next page] (54) Title: METHODS AND MATERIALS FOR MANIPULATING PHLORIDZIN PRODUCTION (57) Abstract: The invention provides materials and Figure 12 methods from producing plants, or parts thereof, with altered levels of phloridzin, by altering levels of chal- cone isomerase (CHI) in the plants, or parts thereof. The invention also provides plants, and parts thereof, with altered levels of phloridzin, as a result of having altered levels of chalcone isomerase (CHI). The in vention also provides methods of selecting and breed ing plants with altered levels of phloridzin, as a result of having altered levels of chalcone isomerase (CHI). Preferably the plants, or parts thereof, have increased levels of phloridzin, as a result of having decreased levels of chalcone isomerase (CHI). w o 2016/016826 Λ Ι llll II II 11III III III III 111III III II I II — with sequence listing part of description (Rule 5.2(a)) METHODS AND MATERIALS FOR MANIPULATING PHLORIDZIN PRODUCTION TECHNICAL FIELD The present invention relates to compositions and methods for producing plants with altered altered phloridzin content. BACKGROUND ART The dihydrochalcone phlordizin (phloretin 2'-glucoside, see Fig. 1) is the major phenolic glucoside found in apple trees. Phlorizin has a bitter taste that contributes to the characteristic flavour of cider, and its dimerised oxidation products contribute to the colour of apple juices. However, since it was isolated from the bark of the apple tree in 1835, phlorizin has attracted most scientific interest through its use as a pharmaceutical and tool for physiology research. The principal pharmacological action of phlordizin is to produce renal glycosuria and block glucose transportation by inhibition of the sodium-linked glucose transporters. Phloridzin and its derivatives have also been shown to be extremely effective antioxidants in vitro, and to have a range of bioactive functions such as inhibition of lipid peroxidation, prevention of bone loss, enhancement of memory, and inhibition of cancer cell growth. Until recently phlorizin was believed to exist only in Malus species. However, phloretin glycosides have been reported in the leaves of Australian native sarsaparilla (Smilax glyciphylla), sweet tea (Lithocarpus polystachyus) and at very low levels in strawberry fruit. In apple trees, phlorizin is found primarily in the young shoots, roots, leaves and bark. In fruit, phloridzin is most abundant in the seeds, with intermediate levels in both the core and the skin, and the lowest level in the cortex. Variation has been assessed within apple trees, between orchards, between different cultivars and among mutants). Despite this information, relatively little is known of the in planta function of phloridzin in apple tree physiology, although it has been suggested that it might act in apple tree growth and development or be an inhibitor of bacterial or fungal growth. The molecular basis for production of phloridzin in planta has not been fully described. Phloretin is a product of the phenylpropanoid pathway, with conversion to its glucoside, phlorizin, likely to be catalysed by the action of a uridine diphosphate (UDP) glycosyltransferase (UGT). UGTs mediate the transfer of a sugar residue from an activated nucleotide sugar to acceptor molecules (aglycones). Plants contain large families of UGTs with over 100 genes being described in Arabidopsis. These genes have a common signature motif of 42 amino acids thought to be involved in binding of the UDP moiety of the activated sugar. A phylogenetic analysis established the presence of distinct Groups (A-N) and Families (UGT71-92) of UGT genes in Arabidopsis and this facilitated the characterisation of many new activities. Although initially thought to be promiscuous enzymes, recent evidence suggests that their broad substrate specificity is limited by regio-specificity, and in some cases UGTS have been shown to be highly specific. Using a functional genomics approach Judge et al (FEBS J. 2008 Aug; 275 (15): 3804-14) identified and characterised a UGT from apple belonging to the previously uncharacterised UGT Family 88. The authors established that MpUGT88Al mediates the glycosylation of the dihydrochalcone phloretin to phlorizin. I n spite of this research, however, comaparatively little is known about which steps in the phloridzin production pathway could potentially be manipulated to modulate production of phloridizin in plants or parts thereof. It would be beneficial to have a further means to increase phloridzin levels in plants. It is an object of the invention to provide novel compositions and methods for modulating phloridzin content in plants or at least to provide the public with a useful choice. SUMMARY OF THE INVENTION METHODS Altering phloridzin by altering CHI In one aspect the invention provides a method for producing a plant, or part thereof, with altered levels of phloridzin, the method comprising altering expression of at least one chalcone isomerase (CHI) protein in the plant or part thereof. In one embodiment expression of chalcone isomerase (CHI) protein is increased and the level of phloridzin is decreased. In a preferred embodiment expression of chalcone isomerase (CHI) protein is decreased and the level of phloridzin is increased. In one embodiment the method comprises introducing a construct into the plant, or part thereof, to affect the altering expression of the at least one chalcone isomerase (CHI) protein. In a further embodiment the method comprises introducing a construct into the plant or part thereof to affect the reducing expression of the at least one chalcone isomerase (CHI) protein. In a further embodiment the construct contains a promoter sequence operably linked to at least part of a chalcone isomerase (CHI) gene, wherein the part of the gene is in an antisense orientation relative to the promoter sequence. In one embodiment the part of the gene is at least 2 1 nucleotides in length. In one embodiment the construct is an antisense construct. In a further embodiment the construct is an RNA interference (RNAi) construct. In a further embodiment the construct is a CRISPR-CAS construct. In one aspect the invention provides a plant, or part thereof, produced by the method of the invention. In one embodiment the plant, or part thereof, comprises the construct. Reducing or eliminating CHI In one aspect the invention provides a method for producing a plant, or part thereof, with increased levels of phloridzin, the method comprising reducing, or eliminating, expression of a chalcone isomerase (CHI) protein in the plant or part thereof. Non-GM selection method for reduced or eliminated chalcone isomerase (CHI) In a further aspect the invention provides a method for identifying a plant with a genotype indicative of producing increased levels of phloridzin, the method comprising testing a plant for at least one of: a) reduced, or eliminated, expression of at least one chalcone isomerase (CHI) protein, b) reduced, or eliminated, expression of at least one polynucleotide encoding an chalcone isomerase (CHI) protein, c) presence of a marker associated with reduced expression of at least one chalcone isomerase (CHI) protein, and d) presence of a marker associated with reduced expression of at least one polynucleotide encoding an chalcone isomerase (CHI) protein. In one embodiment presence of any of a) to d) indicates that the plant will produce increased levels of phloridzin. Methods for breeding plants with increased levels of phloridzin In a further aspect the invention provides a method for producing a plant that produces with increased levels of phloridzin, the method comprising crossing one of: a) a plant of the invention, b) a plant produced by a method of the invention, and c) a plant selected by a method of the invention with another plant, wherein the off-spring produced by the crossing is a plant that produces increased levels of phloridzin.
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