US 2010.0199386A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0199386 A1 Bhaskar et al. (43) Pub. Date: Aug. 5, 2010 (54) CONTROL OF COLD-INDUCED Related U.S. Application Data SWEETENING AND REDUCTION OF (60) Provisional application No. 61/149,397, filed on Feb. ACRYLAMIDE LEVELS IN POTATO OR 3, 2009, provisional application No. 61/241,876, filed SWEET POTATO on Sep. 12, 2009. Publication Classification (75) Inventors: Pudota Bala Bhaskar, Madison, (51) Int. Cl W SS Jiming Jiang, Fitchburg, AOIH I/00 (2006.01) C7H 2L/04 (2006.01) CI2N 5/82 (2006.01) Correspondence Address: AOIH 5/00 (2006.01) Lisa Mueller A2.3L I/26 (2006.01) c/o Polsinelli Shughart PC (52) U.S. Cl. ..................... 800/284:536/23.1: 435/320.1; 161 N. Clark Street, Suite 4200 800/298; 800/278; 426/637; 800/317.2 Chicago, IL 60601 (US) (57) ABSTRACT The present invention is directed to methods and composi (73) Assignee: WISCONSINALUMNI tions to eliminate cold storage-induced Sweetening of potato RESEARCH FOUNDATION, or Sweet potato. The invention is accomplished in part by Madison, WI (US) silencing the vacuolar acid invertase gene using RNAi tech nology. The resulting potatoes withstand cold storage without (21) Appl. No.: 12/698,275 significant hexogenesis, producing potatoes or Sweet pota toes that have reduced Maillard reactions when fried in hot oil. The fried products accumulate significantly lower levels (22) Filed: Feb. 2, 2010 of acrylamide compared to controls. 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Of 8122 & 1 & 1 & YY8 Figure 11 Patent Application Publication Aug. 5, 2010 Sheet 12 of 13 US 2010/019938.6 A1 35000 30000 as 25000 s 2. 20000 s , 15000 d 1. 10000 5000 - * , Y& X w x k k O 8 8:8:8 : & s Ps s R | s w w w s w w w w y w y w w w s s s s s s c r ge w ge s d w e e e e O O is is S is g Figure 12 Patent Application Publication Aug. 5, 2010 Sheet 13 of 13 US 2010/019938.6 A1 6000 SOOO 4000 - 3000 - 2000 - 1000 - Figure 13 US 2010/O 19938.6 A1 Aug. 5, 2010 CONTROL OF COLD-INDUCED Swedish National Food Administration reported alarmingly SWEETENING AND REDUCTION OF high levels of acrylamide in carbohydrate-rich heated foods ACRYLAMIDE LEVELS IN POTATO OR (products from potato tubers, wheat flour, and coffee beans) SWEET POTATO (Tareke et al. 2002). While the potential carcinogeneticity of low levels of acrylamide in humans is still being investigated, RELATED APPLICATION INFORMATION (Pelucchi et al. 2003; Granath and Tomcqvist, 2003), a great deal of research has focused on understanding the mechanism 0001. This application claims the benefit of U.S. Ser. No. (s) of acrylamide formation in food as well as elimination/ 61/149,397 filed on Feb. 3, 2009 and to U.S. Ser. No. 61/241, reduction strategies to minimize possible human health risk. 876 filed on Sep. 12, 2009, the contents of each of which are In 2005 (Food Navigator report) several lawsuits were filed herein incorporated by reference. by the state of California against major food companies regarding acrylamide levels in potato-processed foods. As a GOVERNMENT SUPPORT result, several food companies agreed to Substantially reduce 0002 This invention was made with support from the the acrylamide levels in fried potato products over the follow National Science Foundation, Grant No. DBI-0218166 and ing 3-5 years (San Francisco Chronicle article, 2008). How USDA/CSREES 08-CRHF-0-6055. The US government ever, the mechanisms regulating Sugar accumulation in the may have certain rights in this invention. cold remain poorly understood (Keijbets, 2008; Sowokinos, 2007), and a need remains for methods to reduce acrylamide FIELD OF THE INVENTION levels in fried potato products. 0003. The present invention is generally directed to the 0008 Potato Carbohydrate Metabolism inhibition of Sugar conversion in potato or Sweet potato dur 0009 Carbohydrate metabolism is complex in the potato ing cold storage (2-12°C., especially 2-4°C.). Specifically, (Sowokinos, 2007) and is thought to be a quantitative genetic the invention is directed to silencing the vacuolar acid inver trait (Menendez et al., 2002). The actual concentration of free tase gene using RNAi to inhibit the conversion of Sucrose to Sugar in potatoes involves the interaction of several pathways fructose and glucose in potato tubers and to reduce the acry of carbohydrate metabolism, including starch synthesis/deg lamide levels in fried edible potato products or sweet potato radation, glycolysis, respiration and Sweetening. These path products. ways are controlled at many levels, including hormonal, membrane structure and function, compartmentalization and SEQUENCE LISTING concentration of enzymes, key ions, and Substrate; and of course, enzyme expression levels and activity (Sowokinos, 0004 The instant application contains a Sequence Listing 2007). which has been submitted via EFS-Web and is hereby incor 0010. Sucrose is synthesized from chloroplast-derived porated by reference in its entirety. Said ASCII copy, created trosephosphate in a source leaf. After entering the apoplastic on Feb. 1, 2010, is named WARFPOTA.txt, and is 20,397 space around the phloem, a Sucrose proton Symporteractively bytes in size. takes the Sucrose into the phloem. In a sink tissue. Such as a tuber, Sucrose is symplastically unloaded and/or released into COMPACT DISC FOR SEQUENCE LISTINGS the apoplast. From there, it can either be taken up by a Sucrose AND TABLES proton symporter, or hydrolyzed by cell wall invertase to 0005. Not applicable. glucose and fructose. Within the sink cells, Sucrose can either (1) be converted by sucrose synthase to uridine diphospho BACKGROUND OF THE INVENTION glucose (UDPG) and fructose, or (2) hydrolyzed by a cyto Solic invertase. After entering vacuoles. Sucrose can also be Cold-Storage Induced Sweetening split into fructose and glucose by vacuolar invertase. Hexoki 0006 Potato tubers (Solanum tuberosum) are stored at low nases phosphorylate the simple Sugars, resulting in hexoses temperatures ((47-50 F. (8-10° C.)) to prevent sprouting, that can enterrespiration. In the sink tissue, cell wall invertase reduce respiration and minimize disease losses (Rausch and and vacuolar invertase can be regulated post-translationally Greiner, 2004). However, these temperatures are not ideal; by inhibitors of B-fructocidases (Rausch and Greiner, 2004). colder storage temperatures are more preferable (2-4° C.) 0011. A holy grail in industrial agriculture pertaining to because colder temperatures would reduce (1) the need to use potatoes, especially those to be processed into crisps, chips, fungicides and bactericides in storage; (2) the loss of Solids and French fries, has been to control cold storage-induced through respiration; (3) the need for chemical sprout Suppres Sweetening of potatoes, a long-felt need for the potato pro sants; and (4) would help to increase the marketing window cessing industry (Keijbets, 2008). Solving the problem of (Sowokinos, 2007). In the cold, however, starch, a polysac cold-induced Sweetening is particularly difficult because charide, is converted into the simple reducing Sugars glucose Sugar content is affected by a multitude of factors, including and fructose; a phenomenon recognized as cold-induced (1) starch synthesis, (2) starch breakdown, (3) glycolysis, (4) sweetening (CIS) (Dale and Bradshaw, 2003; Keijbets, 2008; mitochondrial respiration (in which the tuber is rich); and (5) Rausch and Greiner, 2004, Sowokinos, 2001; Sowokinos, hexogensis (Dale and Bradshaw, 2003). The importance of 2007). The carbonyl groups of these sugars react with the this goal is more easily understood when the statistics Sur amino group of free amino acids (a Maillard type reaction) as rounding potato processing is understood.