US00724777OB2
(12) United States Patent (10) Patent No.: US 7,247,770 B2 Goddijn et al. (45) Date of Patent: Jul. 24, 2007
(54) REGULATING METABOLISM BY WO 974.2327 11, 1997 MODIFYING THE LEVEL OF TREHALOSE-6-PHOSPHATE OTHER PUBLICATIONS (75) Inventors: Oscar Johannes Maria Goddijn, Ln Vogel et al The Plant Journal 1998, 13:673-683.* Leider (NL); Jan Pen, Js Leiden (NL); Romero et al Planta, 1997 201:293-297.* Josephus Christianus Maria Heuer et al Plant Physiology 2001 127:33-45.* Smeekens, Ae Driebergen (NL) Goddijn, O.J.M. et al. “Transgenic Tobacco Plants as a Model System for the Production of Trehalose' Plant Physiology, vol. 108, (73) Assignee: Syngenta Mogen B.V. (NL) No. 2 (Jun. 1, 1995), p. 149. Jang, J. C. et al. “Sugar Sensing in Higher Plants' Plant Cell, vol. (*) Notice: Subject to any disclaimer, the term of this 6 (Nov. 19940, pp. 1665-1679. patent is extended or adjusted under 35 Lu, X. et al. “Differentiation of HT-29 Human Colonic Adenocarcinoma Cells Correlates with Increased Expression of U.S.C. 154(b) by 469 days. Mitochondrial RNA: Effects of Trehalose on Cell Growth and Maturation.” Cancer Research, vol. 52 (Jul 1992), pp. 3718-3725. (21) Appl. No.: 10/682,456 Holmstrom, K.O. et al. “Drought tolerance in plants' Nature, vol. 379 (Feb. 22, 1996), pp. 683-684. (22) Filed: Oct. 9, 2003 Blacquez, M. A. et al. “Trehalose-6-phosphate, a new regulator of yeast glycolysis that inhibits hexokinases' Febs Letters, vol. 329, (65) Prior Publication Data No. 1, 2 (1993), pp. 51-54. US 2004/0093641 A1 May 13, 2004 Hohmann, S. et al. “Evidence for trehalose-6-phosphate-dependent and -independent mechanisms in the control of Sugar influx into yeast glycolysis.” Molecular Microbiology 20 (5) (1996), pp. 981 Related U.S. Application Data 991. (62) Division of application No. 09/171.937, filed as appli Luyten, K. "Functional Analysis of the Yeast Genes GGS1/TPS1, cation No. PCT/EP97/02497 on May 2, 1997, now Encoding a Subunit of the Trehalose Synthase Complx, and FPS1, Pat. No. 6,833,490. Encoding a Glycerol Facilitator (Saccharomyces cerevisiae)” Dis sertation Abstracts international, vol. 58, No. 1C (1996), p. 105. (30) Foreign Application Priority Data Order No. AARC537180. Abstract. Kaasen, I. et al. “Analysis of the otsBA operon for osmoregulatory May 3, 1996 (NL) ...... 962O1225 trehalose synthesis in Escherichia coli and homology of the OtSA Jul. 26, 1996 (NL) ...... 962O2128 and OtsB proteins to the yeast trehalose-6-phsphate synthase? Aug. 29, 1996 (NL) ...... 962O2395 phosphatase complex' Gene, vol. 145 (1994), pp. 9-15. Newman, T. et al. “11536 Arabidopsis thaliana cDNA clone (51) Int. Cl. 151A11T7” EMBL Sequence Database, Release 43 (Mar. 25, 1995) CI2N 5/82 (2006.01) Accession No. T76758. CI2N 9/10 (2006.01) Newman, T. et al. “13527 Arabidopsis thaliana cDNA clone CI2N 15/29 (2006.01) 170D15T7” EMBL Sequence Database, Release 44 (Jun. 4, 1995) AOIH 5/00 (2006.01) Accession No. R65023. Newman, T. et al. “11168 Arabidopsis thaliana cDNA clone (52) U.S. Cl...... 800/284; 800/278; 800/286; 149K12T7” EMBL Sequence Database, Release 44 (Jun. 4, 1995) 800/290: 435/468; 435/320.1536/23.1536/23.2: Accession No. T76390. 536/23.6:536/24.5 Newman, T. et al. “15707 Arabidopsis thaliana cDNA clone (58) Field of Classification Search ...... None 183N13T7” EMBL Sequence Database, Release 44 (Jul 27, 1995) See application file for complete search history. Accession No. H37578. Newman, T. et al. “6714 Arabidopsis thaliana cDNA clone (56) References Cited 117M5T7” EMBL Sequence Database, Release 42 (Feb. 3, 1995) U.S. PATENT DOCUMENTS Accession No. T43451. Newman, T. et al. “15723 Arabidospis thaliana cDNA clone 5,422.254. A * 6/1995 Londesborough et al. .... 435/97 183P18T7” EMBL Sequence Database, Release 44 (Jul 27, 1995) 6,133,034 A 10/2000 Strom et al...... 435/419 Accession No. H37594. 6,833,490 B1* 12/2004 Goddijn et al...... 800,284 (Continued) FOREIGN PATENT DOCUMENTS Primary Examiner David T. Fox EP O438904 7, 1991 Assistant Examiner Brent T Page EP O442592 8, 1991 (74) Attorney, Agent, or Firm Michael E. Yates; Edouard EP O451896 10, 1991 G. Lebel EP O530978 3, 1993 EP 0577915 1, 1994 EP O784095 7/1997 (57) ABSTRACT WO 93.17.093 9, 1993 WO 9501446 1, 1995 WO 95.244.87 9, 1995 Method for the inhibition of carbon flow in the glycolytic WO 96.00789 1, 1996 direction in a cell by increasing the intracellular availability WO 96.17069 6, 1996 of trehalose-6-phosphate. WO 96.21030 T 1996 WO 9726357 7/1997 3 Claims, 42 Drawing Sheets US 7,247,770 B2 Page 2
OTHER PUBLICATIONS complex in Saccharomyces cerevisiae, causes accumulation of trehalose-6-phosphate phosphatase activity” Eur: J. Biochem... vol. Minobe, Y. et al. “Rice cDNA, partial sequence (C10408-1A)” EMBL Sequence Database, Release 37 (Nov. 27, 1993) Accession 212 (1993), pp. 315-323. No. D22143. Foyer, C.H.et al. “Modifications in Carbon Assimilation, Carbon Sasaki, T. et al. “Rice cDNA, partial sequence (S1776-1A)” EMBL Partitioning and Total Biomass as a Result of Over-Expression of Sequence Database, Release 41 (Nov. 13, 1994) Accession No. Sucrose Phosphates Synthase in Transgenic Tomato Plants' Plant D40048. Physiology, vol. 105, No. 1 (May 1, 1994), p. 23. Minobe, Y. et al. “Rice cDNA, partial sequence (C10773-1A)” EMBL Sequence Database, Release 37(Nov. 27, 1993) Accession Kossmann, J. et al. “Reduction of the Chloroplastic Fructose-1, No. D22344. 6-Bisphosphatase in Transgenic Potato Plants Impairs Photosyn Zentella, R. etal. “Molecular Characterization of a cDNA Encoding thesis and Plant Growth' Plant Journal, vol. 6, No. 5 (Nov. 1, trehalose-6-Phosphate Lepidophylla Synthases/phosphatase from 1994), pp. 637-650. the resurrection plant selaginella' Plant Physiology, vol. 111, No. 2 Hajirezaei. M. et al. “Transgenic potato plants with strongly (Jun. 1996), p. 47. decreased expression of pyrophosphate: fructose-6-phosphate phos Gancedo, C. et al. “A. thaliana mRNA for trehalose-6-phosphate photransferase show no visible phenotype and only minor changes synthase” EMBL Sequence Database, Release 51 (Mar. 1, 1997), in metabolic fluxes in their tubers.” Plant, vol. 192(1994), pp. 16-30. Accession No. YO8568. Trethewey, R. et al. “Transgenic approaches to improving the flux Goddijn, O.J.M. et al. "Inhibition of trehalase activity enhances trehalose accumulation in transgenic plants.” Plant Physiology of carbohydrate into potato tubers.” International Conference on the (Rockville) 113 (1) (1997) pp. 181-190. Transport of Photoassimilates, Canterbury, England, UK, Aug. Thevelein, J.M. “Trehalose synthase: guard to the gate of glycolysis 13-17, 1995. Journal of Experimantal Botany 47 (Spec. Issue) in yeast?” Trends in Biochemical Sciences, vol. 20, No. 1 (Jan. (1996). 1995), pp. 3-10. Vogel, et al., Plant Journal vol. 13(5), (1998), pp. 673-683. De Virgillio, C. et al. “Disruption of TPS2, the gene encoding the 100-kDa subunit of the trehalose-6-phosphate synthase phosphatase * cited by examiner U.S. Patent Jul. 24, 2007 Sheet 1 of 42 US 7,247,770 B2
10889 bps
Fig. 1 U.S. Patent Jul. 24, 2007 Sheet 2 of 42 US 7,247,770 B2
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U.S. Patent Jul. 24, 2007 Sheet S of 42 US 7,247,770 B2
U.S. Patent Jul. 24, 2007 Sheet 8 of 42 US 7,247,770 B2
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U.S. Patent Jul. 24, 2007 Sheet 9 of 42 US 7,247,770 B2
99IIGI WN1 JIX GZIýz9 09I
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Fig. 8 U.S. Patent Jul. 24, 2007 Sheet 10 of 42 US 7,247,770 B2 S. tuberosum plMOG1027
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Fig. 9 U.S. Patent Jul. 24, 2007 Sheet 11 of 42 US 7,247,770 B2
Potato tuber xtract Substrates Fructose
O 5 1 O 15 20 25 3O 35 Time (min)
Fig. 10 U.S. Patent Jul. 24, 2007 Sheet 12 of 42 US 7,247,770 B2 Potato tuber extract Substrate = Fructose
C u
O
O 2 3 4. 5 6 7 Time (min) Potato tuber extract
0.4 Substrate = Glucose
0.35 O.3 O. O.25 ver 3 0.2 O O. 15 O. 1 O.05 O O 2 4 6 8 1 O Time (min) Fig. 11 U.S. Patent Jul. 24, 2007 Sheet 13 of 42 US 7,247,770 B2
Tobacco leaf extract Substrate - Fructose 0.4
s 0.08 ------
Time (min) Tobacco leaf extract
O.15 Substrate Glucose
O. 1
3 .. 4 Time (min)
Fig. 12 U.S. Patent Jul. 24, 2007 Sheet 14 of 42 US 7,247,770 B2
Tobacco leaf extract Substrate = glucose
-o-Tobacco eaf -O---- TM TSP 0.04 -- a-- 0.125 UHKII yeast
O 2O 40 60 80 100
Fig. 13 U.S. Patent Jul. 24, 2007 Sheet 15 of 42 US 7,247,770 B2
Ric leaf extract Substrate = FructOSe
O 2 4. 6 8 10 12 Time (min) Rice leaf extract
O.6 Substrate E Glucose
O, 5
O.4 O us cro O.3 ?a O O.2 -o- 5 mM T6P 0.1 ""--s-- no T6P - - O - .. 5 nM T6P
O 5 1 O 15 2O 25 Time (min) Fig. 14 U.S. Patent Jul. 24, 2007 Sheet 16 of 42 US 7,247,770 B2 Maize leaf extract Substrate = Fructose
O V () a O
Time (min) Maize leaf extract
Substrate = Glucose
O V C O
O 2 4 6 8 10 2 14 16 Time (min) Fig. 15 U.S. Patent Jul. 24, 2007 Sheet 17 of 42 US 7,247,770 B2
DARK TREATMENT LIGHT TREAMENT
1 1
0.9 O.9
0.8 0.8 0.7 0.7 0.6 0.6 0.5 0.5 0.4 0.3 0.4 0.2 0.3 0.1 0.2 O 0.1
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GHT TREATMENT 1. 1
0.9 0.9 0.8 0.8 0.7 O.7 0.6 0.6
0.5 0.5
0.4 0.4 0.3 0.3
0.2 0.2
0.1 0.1 O O
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Fig. 16 U.S. Patent Jul. 24, 2007 Sheet 18 of 42 US 7,247,770 B2
NET (ACCUMULATION (% PER DAY) O 3
SHOOHIP
RNFAMINE, D GROWNGEAF FEAST, D OFAMINE, D+N MATURELEAF (7 FEAS, D+N
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(DSRBION BEYEEN PLANT PARIS (% OFTOTALIN PLANT)
AAA SHOGIP E (FAMINE, D FEAST, D GROWINGLEAF it FAMINE, D+N UPPERSTEM, AAAAAA 7 FEAST, D+N LABELED BARKLEAF b)Fr. All II STEM bit LLITILITIrLIII III (BLDEAF I , ODEST EAF filliliult
Fig. 18 U.S. Patent Jul. 24, 2007 Sheet 20 of 42 US 7,247,770 B2
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s CD C. ad U.S. Patent Jul. 24, 2007 Sheet 23 of 42 US 7,247,770 B2
U.S. Patent Jul. 24, 2007 Sheet 24 of 42 US 7,247,770 B2 B. Vulgaris PC-TPS
D B C C A A A A PC-PSineS Controls Leaf-size of independent transformants B. vulgaris PC-TPS
2
1 2 3 4 5 6 1 2 3 4 5 6 7 Controls PC-TPS line 2862 Fig. 22 U.S. Patent Jul. 24, 2007 Sheet 25 of 42 US 7,247,770 B2
S. tuberosum 2 5 O
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1 OO
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pMOG 799 transgenic lines Fig. 23
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S. tuberosum 2OO
wild-type lines
Fig. 25 U.S. Patent Jul. 24, 2007 Sheet 27 of 42 US 7,247,770 B2
Wild-type KARDAL
140
120 1 OO
Wild-type leaf size
pMOG 1093: PC TPS
2OO
SO
O O
. . . . . B B B C C C D D D, E E E F F F F F F G PC-TPS leaf size
Fig. 26 U.S. Patent Jul. 24, 2007 Sheet 28 of 42 US 7,247,770 B2
Potato 140 2O OO 8O 60 40 20
B B B C C C Pat TPS leaf size Potato
40
120
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80
60
B B B C Wild-type leaf size
Fig. 27 US 7.247,770 B2
pMGG129(845-1 l) Fig. 28A
S.TUBEROSUM
Fig. 28B U.S. Patent Jul. 24, 2007 Sheet 30 of 42 US 7,247,770 B2
S.UBEROSUM
ar. -- r or -i- da - a s p us un- a me- a his dra- a rve - ur r ------a- % %
ACAV.- %( % An pMOG1129(845-28) Fig. 28C U.S. Patent Jul. 24, 2007 Sheet 31 of 42 US 7,247,770 B2
UPPER EPIDERS -1 PALSADE MESOPHYLL
SPONGYMESOPHY N OWER EPIDERMS
TPSTRANSGENICTOBACCO EAF Fig. 29A
UPPER EPIDERMS / PALSADE MESOPHYL. SPONGY MESOPHY N LOWER EPIDERMS
Af TPPTRANSGENCTOBACCO LEAF Fig.29B U.S. Patent Jul. 24, 2007 Sheet 32 of 42 US 7,247,770 B2
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S. tuberosum tubers (cv. Kardal) sugar profile after harvesting O 8 SUCOS6 -20C fructose -2OC Q N. glucose 20C (MB/%)JefinS U.S. Patent Jul. 24, 2007 Sheet 33 of 42 US 7,247,770 B2
. U.S. Patent Jul. 24, 2007 Sheet 34 of 42 US 7,247,770 B2
NABACUM 0.003
0.0025
0.002 I
GLUCOSE - - - FRUCTOSE ------W - // SU(ROSE ? i 0.5 ------M ".% it.t St. In?mism,... St.. I 0.05 CIPIEEE , O ASRS ACC.W\lm
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Fig. 32D U.S. Patent Jul. 24, 2007 Sheet 36 of 42 US 7,247,770 B2
S. tuberosum
, pMOG1027 pMOG1027(845 pMOG1027(845 pMOG1027(845-28)i -11) -22) Fig. 33 U.S. Patent Jul. 24, 2007 Sheet 37 of 42 US 7,247,770 B2 S. tuberosum arr-M.---- coN,ÇO?O<+C\,)CN• OQCo wild-typelines pMOG1027(845 pMOG1027(845 pMOG1027(845 -11) -22) -28) Fig. 34 U.S. Patent Jul. 24, 2007 Sheet 38 of 42 US 7,247,770 B2 S.TUBEROSUM No.,! u ;ESSSSSSSSSSSSSSSSSSS |SSSSSSSSSSSSSS: |SSSSSSSSSSSSSS| ;SSSSSSSSS :SSSSSSSSS ;SSSSSSSSS :ESSSSSSS SSSG SSSSSSSSSSSSSSSS: SSSSSSSSSSSSSSSS }§§§§§§§§§§ +SSSSSSSSSSSSS|}sSSSSSSSSS !SSSSSSSSSSSSS SSSSSSSSSSS, SSSSSSSSSSSÈ SSSSSSSSSSS !SSSSSS SSSSSSSSSSSSSSSSSSSSS ;SSSSSSSSSSSSSSSSSSS}SSSSSSSSSSSSSSSSSSS| SSSSSSSSSSSSSSSSS: SSSSSSSSSSSSSSSSSS SSSSSSSSSSSSSSSSS: SSSSSSSSSSSSSSS! SSSSSSSSSSSSSSSÈSSSSSSSSSSSS5 :;SSSSSSSSSSSSSSSS S.TUBEROSUM WILD-TYPELINES Fig. 35B U.S. Patent Jul. 24, 2007 Sheet 39 of 42 US 7,247,770 B2 S.TUBEROSUM 6 W %,h 1 a a P - are a ae . 1028(845-22) Fig. 35C S.UBEROSUM W...... --...... (IYIELD : im. l (AAEO B. h II. Fig. 35D U.S. Patent Jul. 24, 2007 Sheet 40 of 42 US 7,247,770 B2 S.TUBEROSUM In7 " | W ( 6 a - a r w as - w is a his a 4-wr - - is ...... 7 % i % air - - or war . . . A - 2f %. %%in % . | . % .... . % ..., War. 1028(845-28) Fig. 35E U.S. Patent Jul. 24, 2007 Sheet 41 of 42 US 7.247,770 B2 S. tuberosum pMOG 1092 Fig. 36 U.S. Patent Jul. 24, 2007 Sheet 42 of 42 US 7,247,770 B2 S. tuberosum pMOG 1130 Fig. 37 US 7,247,770 B2 1. 2 REGULATING METABOLISM BY distribution of assimilation products and consequently the MODIFYING THE LEVEL OF formation of sinks and sources is yet unknown. The mecha TREHALOSE-6-PHOSPHATE nism is believed to be located somewhere in the carbohy drate metabolic pathways and their regulation. This application is a divisional of application number 5 In the recent research it has become apparent that hex 09/171,937 filed on Apr. 28, 1999 now U.S. Pat. No. okinases may play a major role in metabolite signalling and 6,833,490, which is a 371 of PCT/EP97/02497 filed May 2, control of metabolic flow. A number of mechanisms for the 1997, which designated the U.S., claims the benefit thereof regulation of the hexokinase activity have been postulated and incorporates the same by references. (Graham et al. (1994), The Plant Cell 6: 761; Jang & Sheen 10 (1994), The Plant Cell 6, 1665; Rose et al. Eur. J. FIELD OF THE INVENTION Biochem. 199, 511-518, 1991; Blazquez et al. (1993), FEBS 329, 51; Koch, Annu. Rev. Plant Physiol. Plant. Mol. Glycolysis has been one of the first metabolic processes Biol. (1996) 47, 509; Jang et al. (1997), The Plant Cell 9, 5, described in biochemical detail in the literature. Although one of these theories of hexokinase regulation, postulated in the general flow of carbohydrates in organisms is known and 15 yeast mentions trehalose and its related monosaccharides although all enzymes of the glycolytic pathway(s) are elu (Thevelein & Hohmann (1995). TIBS 20, 3). However, it is cidated, the signal which determines the induction of hard to see that this would be an universal mechanism, as metabolism by stimulating glycolysis has not been unrav trehalose synthesis is believed to be restricted to certain elled. Several hypotheses, especially based on the situation species. in yeast have been put forward, but none has been proven beyond doubt. Thus, there still remains a need for the elucidation of the Influence on the direction of the carbohydrate partitioning signal which can direct the modification of the development does not only influence directly the cellular processes of and/or composition of cells, tissue and organs in vivo. glycolysis and carbohydrate storage, but it can also be used SUMMARY OF THE INVENTION to influence secondary or derived processes such as cell 25 division, biomass generation and accumulation of Storage It has now been found that modification of the develop compounds, thereby determining growth and productivity. ment and/or composition of cells, tissue and organs in vivo Especially in plants, often the properties of a tissue are is possible by introducing the enzyme trehalose-6-phosphate directly influenced by the presence of carbohydrates, and the synthase (TPS) and/or trehalose-6-phosphatase phosphate steering of carbohydrate partitioning can give Substantial 30 (TPP) thereby inducing a change in metabolic pathways of differences. the saccharide trehalose-6-phosphate (T-6-P) resulting in an The growth, development and yield of plants depends on alteration of the intracellular availability of T-6-P. Introduc the energy which Such plants can derive from CO-fixation tion of TPS thereby inducing an increase in the intracellular during photosynthesis. concentration of T-6-P causes inhibition of carbon flow in Photosynthesis primarily takes place in leaves and to a 35 the glycolytic direction, stimulation of the photosynthesis, lesser extent in the stem, while other plant organs such as inhibition of growth stimulation of sink-related activity and roots, seeds or tubers do not essentially contribute to the an increase in storage of resources. Introduction of TPP photoassimilation process. These tissues are completely thereby introducing a decrease in the intracellular concen dependent on photosynthetically active organs for their tration of T-6-P causes stimulation of carbon flow in the growth and nutrition. This then means that there is a flux of 40 products derived from photosynthesis (collectively called glycolytic direction, increase in biomass and a decrease in “photosynthate') to photosynthetically inactive parts of the photosynthetic activity. plants. The levels of T-6-P may be influenced by genetic engi neering of an organism with gene constructs able to influ The photosynthetically active parts are denominated as ence the level of T-6-P or by exogenously (orally, topically, “sources” and they are defined as net exporters of photo 45 synthate. The photosynthetically inactive parts are denomi parenterally etc.) Supplying compounds able to influence nated as “sinks' and they are defined as net importers of these levels. photosynthate. The gene constructs that can be used in this invention are It is assumed that both the efficiency of photosynthesis, as constructs harbouring the gene for trehalose phosphate Syn well as the carbohydrate partitioning in a plant are essential. 50 thase (TPS) the enzyme that is able to catalyze the reaction Newly developing tissues like young leaves or other parts from glucose-6-phosphate and UDP-glucose to T-6-P. On like root and seed are completely dependent on photosyn the other side a construct coding for the enzyme trehalose thesis in the sources. The possibility of influencing the phosphate phosphatase (TPP) which catalyzes the reaction carbohydrate partitioning would have great impact on the from T-6-P to trehalose will, upon expression, give a decrease of the amount of T-6-P. phenotype of a plant, e.g. its height, the internodium dis 55 tance, the size and form of a leaf and the size and structure Alternatively, gene constructs harbouring antisense TPS of the root system. or TPP can be used to regulate the intracellular availability Furthermore, the distribution of the photoassimilation of T-6-P. products is of great importance for the yield of plant biomass Furthermore, it was recently reported that an intracellular and products. An example is the development in wheat over 60 phospho-alpha-(1,1)-glucosidase, Trea, from Bacillus sub the last century. Its photosynthetic capacity has not changed tilis was able to hydrolyse T-6-P into glucose and glucose considerably but the yield of wheat grain has increased 6-phosphate (Schöck et al., Gene, 170, 77-80, 1996). A substantially, i.e. the harvest index (ratio harvestable biom similar enzyme has already been described for E. coli asS/total biomass) has increased. The underlying reason is (Rimmele and Boos (1996), J. Bact. 176 (18), 5654-). that the sink-to-source ratio was changed by conventional 65 For overexpression heterologous or homologous gene breeding, such that the harvestable sinks, i.e. seeds, portion constructs have to be used. It is believed that the endogenous increased. However, the mechanism which regulates the T-6-P forming and/or degrading enzymes are under allos US 7,247,770 B2 3 4 teric regulation and regulation through covalent modifica carbohydrates Such as glycogen are formed, but also a tion. This regulation may be circumvented by using heter large amount of energy rich carbon compounds is ologous genes. transferred into fat and lipids. Alternatively, mutation of heterologous or homologous Biomass is the total mass of biological material. genes may be used to abolish regulation. The invention also gives the ability to modify source-sink DESCRIPTION OF THE FIGURES relations and resource allocation in plants. The whole carbon economy of the plant, including assimilate production in FIG. 1. Schematic representation of plasmid pVDH275 Source tissues and utilization in source tissues can be modi harbouring the neomycin-phosphotransferase gene (NPTII) fied, which may lead to increased biomass yield of harvested 10 flanked by the 35S cauliflower mosaic virus promoter products. Using this approach, increased yield potential can (P35S) and terminator (T35S) as a selectable marker; an be realized, as well as improved harvest index and product expression cassette comprising the pea plastocyanin pro quality. These changes in source tissues can lead to changes moter (ppCpea) and the nopaline synthase terminator in sink tissues by for instance increased export of photo (Tnos); right (RB) and left (LB) T-DNA border sequences synthase. Conversely changes in sink tissue can lead to 15 and a bacterial kanamycin resistance (KanR) marker gene. change in Source tissue. FIG. 2. Northern blot analysis of transgenic tobacco Specific expression in a cell organelle, a tissue or other plants. Panel A depicts expression of otSA mRNA in leaves part of an organism enables the general effects that have of individual pMOG799 transgenic tobacco plants. The been mentioned above to be directed to specific local control lane “C” contains total RNA from a non-transformed applications. This specific expression can be established by N. tabacum plant. placing the genes coding for TPS, TPP or the antisense genes FIGS. 3A and 3B. Lineup of plant derived TPS encoding for TPS or TPP under control of a specific promoter. sequences compared with the TPS, sequence using the Specific expression also enables the simultaneous expres Wisconsin GCG sequence analysis package (Devereux et al. sion of both TPS and TPP enzymes in different tissues (1984) A comprehensive set of sequence analysis programs thereby increasing the level of T-6-P and decreasing the level 25 of the VAX. Nucl. Acids Res., 12, 387). of T-6-P locally. FIG. 4. Alignment of PCR amplified tobacco TPS cDNA By using specific promoters it is also possible to construct fragments with the TPS encoding yeast TPS1 gene. Boxes a temporal difference. For this purpose promoters can be indicate identity between amino-acids of all four listed used that are specifically active during a certain period of the Sequences. organogenesis of the plant parts. In this way it is possible to 30 FIG. 5. Alignment of PCR amplified tobacco TPP cDNA first influence the amount of organs which will be developed fragments with the TPP encoding yeast TPS2 gene. Boxes and then enable these organs to be filled with storage indicate identity between amino-acids of all four listed material like starch, oil or proteins. Sequences. Alternatively, inducible promoters may be used to selec FIG. 6. Alignment of a fragment of the PCR amplified tively switch on or off the expression of the genes of the 35 sunflower TPS/TPP bipartite cDNA (SEQID NO: 24) with invention. Induction can be achieved by for instance patho the TPP encoding yeast TPS2 gene. Boxes indicate identity gens, stress, chemicals or light/dark Stimuli. between amino-acids of both sequences. FIG. 7. Alignment of a fragment of the Arabidopsis TPS1 DEFINITIONS and Rice EST clones with the TPS encoding yeast TPS1 40 gene. Boxes indicate identity between amino-acids of all Hexokinase activity is the enzymatic activity found in three sequences. cells which catalyzes the reaction of hexose to hexose FIG. 8. Alignment of a fragment of the PCR amplified 6-phosphate. Hexoses include glucose, fructose, galac human TPS cDNA (SEQID NO: 10) with the TPS encoding tose or any other C. Sugar. It is acknowledged that there yeast TPS1 gene. Boxes indicate identity between amino are many isoenzymes which all can play a part in said 45 biochemical reaction. By catalyzing this reaction hex acids of both sequences. okinase forms a key enzyme in hexose (glucose) sig FIG. 9. Trehalose accumulation in tubers of pMOG1027 nalling. (35S astrehalase) transgenic potato plants. Hexose signalling is the regulatory mechanism by which FIG. 10. Hexokinase activity of a wild-type potato tuber a cell senses the availability of hexose (glucose). 50 (Solanum tuberosum cv. Kardal) extract with and without Glycolysis is the sequence of reactions that converts the addition of trehalose-6-phosphate. glucose into pyruvate with the concomitant production FIG. 11. Hexokinase activity of a wild-type potato tuber of ATP. (Solanum tuberosum cv. Kardal) extract with and without Cold Sweetening is the accumulation of Soluble Sugars in the addition of trehalose-6-phosphate. Fructose or glucose is potato tubers after harvest when stored at low tempera 55 used as Substrate for the assay. tures. FIG. 12. Hexokinase activity of a wild-type tobacco leaf Storage of resource material is the process in which the extract (Nicotiana tabacum cv. SR1) with and without the primary product glucose is metabolized into the addition of trehalose-6-phosphate. Fructose or glucose is molecular form which is fit for storage in the cell or in used as Substrate for the assay. a specialized tissue. These forms can be divers. In the 60 FIG. 13. Plot of a tobacco hexokinase activity measure plant kingdom storage mostly takes place in the form of ment. Data series 1: Tobacco plant extract Data series 2: carbohydrates and polycarbohydrates such as starch, Tobacco plant extract +1 mM trehalose-6-phosphate Data fructan and cellulose, or as the more simple mono- and series 3: Commercial hexokinase extract from yeast (1/8 di-saccharides like fructose. Sucrose and maltose; in the unit) form of oils such as arachic or oleic oil and in the form 65 FIG. 14. Hexokinase activity of a wild-type rice leaf of proteins such as cruciferin, napin and seed storage extract (Oryza sativa) extract with and without the addition proteins in rapeseed. In animal cells also polymeric of trehalose-6-phosphate. Experiments have been performed US 7,247,770 B2 5 6 in duplicate using different amounts of extracts. Fructose or FIG. 30. HPLC-PED analysis of tubers transgenic for glucose is used as Substrate for the assay. TPS. before and after storage at 4° C. Kardal C, F, B, G FIG. 15. Hexokinase activity of a wild-type maize leaf and H are non-transgenic control lines. extract (Zea mais) extract with and without the addition of FIG. 31. Scanned images showing leaf morphology, color trehalose-6-phosphate. Fructose or glucose is used as Sub 5 and size of tobacco lines transgenic for 35S TPS (upper strate for the assay. leaf), wild-type (middle leaf) and transgenic for 35S TPP FIG. 16. Fluorescence characteristics of wild-type (tri (bottom leaf). angle), PC-TPS (square) and 35S-TPP (cross) tobacco FIGS. 32 A-D. Metabolic profiling of 35S TPS leaves. The upper two panels show the electron transport (pMOG799), 35S TPP (pMOG1010), wild-type (WT), PC efficiency (ETE) at the indicated light intensities (PAR). 10 TPS (pMOG1177) and PC-TPP (pMOG1124) transgenic Plants were measured after a dark-period (upper-left panel) tobacco lines. Shown are the levels of trehalose, soluble and after a light-period (upper-right panel). The bottom sugars (FIGS. 32A and B), starch and chlorophyll FIGS. 32C panels show reduction of fluorescence due to assimilate and D). accumulation (non-photochemical quenching). Left and FIG. 33. Tuber yield of pMOG1027 (35S as-trehalase) right panel as above. 15 and pMOG1027(845-11/22/28) (35S as-trehalase pat TPS) FIG. 17. Relative sink-activity of plant-parts of PC-TPS transgenic potato lines in comparison to wild-type potato (Famine) and 35S-TPP (Feast) transgenic tobacco plants. lines. Indicated is the nett C-accumulation expressed as percentage FIG. 34. Starch content of pMOG1027 (35S as-trehalase) of total C-content, for various plant-parts after a period of and pMOG1027(845-11/22/28) (35S as-trehalase pat TPS) light (D) or light--dark (D+N). transgenic potato lines in comparison to wild-type potato FIG. 18. Actual distribution of carbon in plant-parts of lines. The sequence of all lines depicted is identical to FIG. PC-TPS (Famine) and 35S-TPP (Feast) transgenic tobacco 33. plants. Indicated is the nett C-accumulation expressed as FIGS. 35A-E. Yield of pMOG1028 (pat as-trehalase) and percentage of total daily accumulated new C for various pMOG1028(845-11/22/28) (pat as-trehalase pat TPS) trans plant-parts after a period of light (D) or light--dark (D+N). 25 genic potato lines in comparison to wild-type potato lines. FIG. 19. Scanned images showing reduced and enhanced FIG. 36. Yield of pMOG1092 (PC as-trehalase) trans bolting in transgenic lettuce lines expressing PC-TPS or genic potato lines in comparison to wild-type potato lines as PC-TPP compared to wild-type plants. The lower panel depicted in FIGS. 35 A-E. shows leaf morphology and color. FIG. 37. Yield of pMOG 1130 (PC as-trehalase PC TPS) FIGS. 20. A-D Profile of soluble sugars FIGS. 20A and B) 30 transgenic potato lines in comparison to wild-type potato in extracts of transgenic lettuce (upper panel) and transgenic lines as depicted in FIGS. 35 A-E. beet (lower panel) lines. In the upper pannel controls are GUS-trangenic lines which are compared to lines trangenics DETAILED DESCRIPTION OF THE for PC-TPS and PC-TPP. In lower panel all transgenic are INVENTION PC-TPS. Starch profiles are depicted in FIGS. 20B and C. 35 FIG. 21. Scanned image showing plant and leaf morphology The invention is concerned with the finding that metabo of transgenic sugarbeet lines expressing PC-TPS (TPS) or lism can be modified in vivo by the level of T-6-P. A decrease PC-TPP (TPP) compared to wild-type plants (Control). TPS of the intracellular concentration of T-6-P stimulates glyco A-type has leaves which are comparable to wild-type while lytic activity. On the contrary, an increase of the T-6-P TPS D-type has clearly smaller leaves. The leaves of the TPP 40 concentration will inhibit glycolytic activity and stimulate transgenic line have a lighter green color, a larger petiole and photosynthesis. an increased size compared to the control. These modifications established by changes in T-6-P FIG. 22. Taproot diameter of transgenic sugarbeet lines levels are most likely a result of the signalling function of (PC-TPS). In the upper panel A, B, C and D indicate hexokinase, which activity is shown to be regulated by decreasing leaf sizes as compared to control (A). In the 45 T-6-P. An increase in the flux through hexokinase (i.e. an lower panel individual clones of control and PC-TPS line increase in the amount of glucose) that is reacted in glucose 286-2 are shown. 6-phosphate has been shown to inhibit photosynthetic activ FIG. 23. Tuber yield of pMOG799 (35S TPS) transgenic ity in plants. Furthermore, an increase in the flux through potato lines. hexokinase would not only stimulate the glycolysis, but also FIG. 24. Tuber yield of pMOG1010 (35S TPP) and 50 cell division activity. pMOG1124 (PC-TPP) transgenic potato lines. Theory of Trebalose-6-Phosphate Regulation of FIG. 25. Tuber yield of 22 independent wild-type S. Carbon Metabolism tuberosum clones. FIG. 26. Tuber yield of pMOG1093 (PC-TPS) transgenic 55 In a normal plant cell formation of carbohydrates takes potato lines in comparison to wild-type. B, C, D, E, F, G place in the process of photosynthesis in which CO, is fixed indicate decreasing leaf sizes as compared to wild-type and reduced to phosphorylated hexoses with Sucrose as an (B/C). end-product. Normally this sucrose is transported out of the FIG. 27. Tuber yield of pMoG845 (Pat-TPS) transgenic cell to cells or tissues which through uptake of this Sucrose potato lines (FIG. 27-1) in comparison to wild-type (FIG. 60 can use the carbohydrates as building material for their 27-2). B, C indicate leaf sizes. metabolism or are able to store the carbohydrates as e.g. FIGS. 28 A-C. Tuber yield of pMOG 1129 (845-11/22/28) starch. In this respect, in plants, cells that are able to transgenic potato lines. photosynthesize and thus to produce carbohydrates are FIGS. 29 A-B. Scanned images showing cross section denominated as sources, while cells which consume or store through leaves of TPP (FIG. 29B) and TPS (FIG. 29A) 65 the carbohydrates are called sinks. transgenic tobacco plants. Additional cell layers and In animal and most microbial cells no photosynthesis increased cell size are visible in the TPS cross section. takes place and the carbohydrates have to be obtained from US 7,247,770 B2 7 8 external sources, either by direct uptake from Saccharides On a molecular level we have data that indicate that next (e.g. yeasts and other micro-organisms) or by digestion of to Selaginella also trehalose synthesizing genes are present carbohydrates (animals). Carbohydrate transport usually in Arabidopsis, tobacco, rice and Sunflower. Using degen takes place in these organisms in the form of glucose, which erated primers, based on conserved sequences between is actively transported over the cell membrane. 5 TPS, and TPS, we have been able to identify genes After entrance into the cell, one of the first steps in the encoding putative trehalose-6-phosphate generating metabolic pathway is the phosphorylation of glucose into enzymes in Sunflower and tobacco. Sequence comparison glucose-6-phosphate catalyzed by the enzyme hexokinase. It revealed significant homology between these sequences, the has been demonstrated that in plants Sugars which are TPS genes from yeast and E. coli, and EST (expressed phosphorylated by hexokinase (HXK) are controlling the 10 sequences tags) sequences from Arabidopsis and rice (see expression of genes involved in photosynthesis (Jang & also Table 6b which contains the EST numbers of homolo Sheen (1994), The Plant Cell 6, 1665). Therefor, it has been gous EST's found). proposed that HXK may have a dual function and may act Recently an Arabidopsis gene has been elucidated (dis as a key sensor and signal transmitter of carbohydrate closed in GENBANKAcc. No. YO8568, depicted in SEQ ID mediated regulation of gene-expression. It is believed that 15 NO:39) that on basis of its homology can be considered as this regulation normally signals the cell about the availabil a bipartite enzyme. These data indicate that, in contrast to ity of starting product, i.e. glucose. Similar effects are current beliefs, most plants do contain genes which encode observed by the introduction of TPS or TPP which influence trehalose-phosphate-synthases enabling them to synthesize the level of T-6-P. Moreover, it is shown that in vitro T-6-P T-6-P. As proven by the accumulation of trehalose in TPS levels affect hexokinase activity. By increasing the level of 20 expressing plants, plants also contain phosphatases, non T-6-P, the cell perceives a signal that there is a shortage of specific or specific, able to dephosphorylate the T-6-P into carbohydrate input. Conversely, a decrease in the level of trehalose. The presence of trehalase in all plants may be to T-6-P results in a signal that there is plenty of glucose, effectuate turnover of trehalose. resulting in the down-regulation of photosynthesis: it signals Furthermore, we also provide data that T-6-P is involved that Substrate for glycolysis and consequently energy Supply 25 in regulating carbohydrate pathways in human tissue. We for processes as cell growth and cell division is Sufficiently have elucidated a human TPS gene (depicted in SEQID NO: available. This signalling is thought to be initiated by the 10) which shows homology with the TPS genes of yeast, E. increased flux through hexokinase (J. J. Van Oosten, public coli and plants. Furthermore, we show data that also the lecture at RijksUniversiteit Utrecht dated Apr. 19, 1996). activity of hexokinase is influenced in mammalian (mouse) The theory that hexokinase signalling in plants can be 30 tissue. regulated through modulation of the level of trehalose-6- Generation of the "plenty signal by decreasing the intra phosphate would imply that all plants require the presence of cellular concentration of trehalose-6-phosphate through an enzyme system able to generate and break-down the expression of the enzyme TPP (or inhibition of the enzyme signal molecule trehalose-6-phosphate. Although trehalose TPS) will signal all cell systems to increase glycolytic is commonly found in a wide variety of fungi, bacterial, 35 carbon flow and inhibit photosynthesis. This is nicely shown yeasts and algae, as well as in Some invertebrates only a very in the experimental part, where, for instance in Experiment limited range of vascular plants have been proposed to be 2 transgenic tobacco plants are described in which the able to synthesize this sugar (Elbein (1974), Adv. Carboh. enzyme TPP is expressed having increased leaf size, Chem. Biochem. 30, 227). A phenomenon which was not increased branching and a reduction of the amount of understood until now is that despite the apparent lack of 40 chlorophyll. However, since the "plenty, signal is generated trehalose synthesizing enzymes, all plants do seem to con in the absence of Sufficient Supply of glucose, the pool of tain trehalases, enzymes which are able to break down carbohydrates in the cell is rapidly depleted. trehalose into two glucose molecules. Thus, assuming that the artificial "plenty signal holds on, Indirect evidence for the presence of a metabolic pathway the reduction in carbohydrates will finally become limiting for trehalose is obtained by experiments presented herein 45 for growth and cell division, i.e. the cells will use up all their with trehalase inhibitors such as Validamycin A or transfor storage carbohydrates and will be in a "hunger-stage. Thus, mation with anti-sense trehalase. leaves are formed with a low amount of stored carbohy Production of trehalose would be hampered if its inter drates. On the other hand, plants that express a construct mediate T-6-P would influence metabolic activity too much. with a gene coding tor TPS, which increases the intracellular Preferably, in order to accumulate high levels of trehalose 50 amount of T-6-P, showed a reduction of leaf size, while also without affecting partitioning and allocation of metabolites the leaves were darker green, and contained an increased by the action of trehalose-6-phosphate, one should overex amount of chlorophyll. press a bipartite TPS/TPP enzyme. Such an enzyme would In yeast, a major role of glucose-induced signalling is to resemble a genetic constitution as found in yeast, where the Switch metabolism from a neogenetic/respirative mode to a TPS2 gene product harbours a TPS and TPP homologous 55 fermentative mode. Several signalling pathways are region when compared with the E. coli ots A and otsB gene involved in this phenomenon (Thevelein and Hohmann, (Kaasen et al. (1994), Gene 145, 9). Using such an enzyme, (1995) TIBS 20, 3). Besides the possible role of hexokinase trehalose-6-phosphate will not become freely available to signalling, the RAS-cyclic-AMP (cAMP) pathway has been other cell components. Another example of Such a bipartite shown to be activated by glucose. Activation of the RAS enzyme is given by Zentella & Iturriaga (Plant Physiol. 60 cAMP pathway by glucose requires glucose phosphoryla (1996), 111 Abstract 88) who isolated a 3.2 kb cDNA from tion, but no further glucose metabolism. So far, this pathway Selaginella lepidophylla encoding a putative trehalose-6- has been shown to activate trehalase and 6-phosphofructo phosphate synthase/phosphatase. It is also envisaged that 2-kinase (thereby stimulating glycolysis), while fructose-1, construction of a truncated TPS-TPP gene product, whereby 6-bisphosphatase is inhibited (thereby preventing gluconeo only the TPS activity would be retained, would be as 65 genesis), by cAMP-dependent protein phosphorylation. This powerful for synthesis of T-6-Pas the otSA gene of E. coli, signal transduction route and the metabolic effects it can also when used in homologous systems. bring about can thus be envisaged as one that acts in US 7,247,770 B2 9 10 parallels with the hexokinase signalling pathway, that is The invention also encompasses nucleic acid sequences shown to be influenced by the level of trehalose-6-phos which have been obtained by modifying the nucleic acid phate. sequence represented in SEQ ID NO: 1 by mutating one or As described in our invention, transgenic plants express more codons so that it results in amino acid changes in the ing as-trehalase reveal similar phenomena, like dark-green encoded protein, as long as mutation of the amino acid leaves, enhanced yield, as observed when expressing a TPS sequence does not entirely abolish trehalose phosphate Syn gene. It also seems that expression of as-trehalase in double thase activity. constructs enhances the effects that are caused by the expres According to another embodiment of the invention the sion of TPS. Trehalase activity has been shown to be present trehalose-6-phosphate in a cell can be converted into treha in e.g. plants, insects, animals, fungi and bacteria while only 10 lose by trehalose phosphate phosphatase encoding genes in a limited number of species, trehalose is accumulated. under control of regulatory elements necessary for the Up to now, the role of trehalase in plants is unknown expression of DNA in cells. A preferred open reading frame although this enzyme is present in almost all plant-species. according to the invention is one encoding a TPP-enzyme as It has been proposed to be involved in plant pathogen represented in SEQ ID NO: 4 (Kaasen et al. (1994) Gene, interactions and/or plant defense responses. We have iso 15 145, 9). It is well known that more than one DNA sequence lated a potato trehalase gene and show that inhibition of may encode an identical enzyme, which fact is caused by the trehalase activity in potato leaf and tuber tissues leads to an degeneracy of the genetic code. If desired, the open reading increase in tuber-yield. Fruit-specific expression of as-tre frame encoding the trehalose phosphate phosphatase activity halase in tomato combined with TPS expression dramati may be adapted to codon usage in the host of choice, but this cally alters fruit development. is not a requirement. According to one embodiment of the invention, accumu The isolated nucleic acid sequence represented by SEQ lation of T-6-P is brought about in cells in which the capacity ID NO: 3, may be used to identify trehalose phosphate of producing T-6-P has been introduced by introduction of phosphatase genes in other organisms and Subsequently an expressible gene construct encoding trehalose-phosphate isolating and cloning them, by PCR techniques and/or by synthase (TPS). Any trehalose phosphate synthase gene 25 hybridizing DNA from other sources with a DNA- or RNA under the control of regulatory elements necessary for fragment obtainable from the E. coli gene. Preferably, such expression of DNA in cells, either specifically or constitu DNA sequences are screened by hybridizing under more or tively, may be used, as long as it is capable of producing a less stringent conditions (influenced by factors such as trehalose phosphate synthase capable of T-6-P production in temperature and ionic strength of the hybridization mixture). said cells. One example of an open reading frame according 30 Whether or not conditions are stringent also depends on the to the invention is one encoding a TPS-enzyme as repre nature of the hybridization, i.e. DNA:DNA, DNA:RNA, sented in SEQ ID NO: 2. Other examples are the open RNA:RNA, as well as the length of the shortest hybridizing reading frames as represented in SEQID NO’s: 10, 18-23, fragment. Those of skill in the art are readily capable of 41 and 45-53. As is illustrated by the above-mentioned establishing a hybridization regime stringent enough to sequences it is well known that more than one DNA 35 isolate TPP genes, while avoiding aspecific hybridization. sequence may encode an identical enzyme, which fact is As genes involved in trehalose synthesis from other sources caused by the degeneracy of the genetic code. If desired, the become available these can be used in a similar way to open reading frame encoding the trehalose phosphate Syn obtain an expressible trehalose phosphate phosphatase gene thase activity may be adapted to codon usage in the host of according to the invention. More detail is given in the choice, but this is not a requirement. 40 experimental section. The isolated nucleic acid sequence represented by for instance SEQ ID NO: 2, may be used to identify trehalose Sources for isolating trehalose phosphate phosphatase phosphate synthase genes in other organisms and Subse activities include microorganisms (e.g. bacteria, yeast, fungi), plants, animals, and the like. Isolated DNA quently isolating and cloning them, by PCR techniques sequences encoding trehalose phosphate phosphatase activ and/or by hybridizing DNA from other sources with a DNA 45 or RNA fragment obtainable from the E. coli gene. Prefer ity from other sources may be used likewise. ably, such DNA sequences are screened by hybridizing The invention also encompasses nucleic acid sequences under more or less stringent conditions (influenced by which have been obtained by modifying the nucleic acid factors such as temperature and ionic strength of the hybrid sequence represented in SEQ ID NO:3 by mutating one or ization mixture). Whether or not conditions are stringent 50 more codons so that it results in amino acid changes in the also depends on the nature of the hybridization, i.e. DNA: encoded protein, as long as mutation of the amino acid DNA, DNA:RNA, RNA:RNA, as well as the length of the sequence does not entirely abolish trehalose phosphate phos shortest hybridizing fragment. Those of skill in the art are phatase activity. readily capable of establishing a hybridization regime strin Other enzymes with TPS or TPP activity are represented gent enough to isolate TPS genes, while avoiding non 55 by the so-called bipartite enzymes. It is envisaged that the specific hybridization. As genes involved in trehalose Syn part of the sequence which is specifically coding for one of thesis from other sources become available these can be the two activities can be separated from the part of the used in a similar way to obtain an expressible trehalose bipartite enzyme coding for the other activity. One way to phosphate synthase gene according to the invention. More separate the activities is to insert a mutation in the sequence detail is given in the experimental section. 60 coding for the activity that is not selected, by which muta Sources for isolating trehalose phosphate synthase activi tion the expressed protein is impaired or deficient of this ties include microorganisms (e.g. bacteria, yeast, fungi), activity and thus only performs the other function. This can plants, animals, and the like. Isolated DNA sequences be done both for the TPS- and TPP-activity coding encoding trehalose phosphate synthase activity from other sequence. Thus, the coding sequences obtained in Such a Sources may be used likewise in a method for producing 65 way can be used for the formation of novel chimaeric open T-6-Paccording to the invention. As an example, genes for reading frames capable of expression of enzymes having producing T-6-P from yeast are disclosed in WO 93/17093. either TPS or TPP activity. US 7,247,770 B2 11 12 According to another embodiment of the invention, espe effect, or co-suppression. Details of the procedure of cially plants can be genetically altered to produce and enhancing Substrate availability are provided in the accumulate the above-mentioned enzymes in specific parts Examples of WO 95/01446, incorporated by reference of the plant. Preferred sites of enzyme expression are leaves herein. and storage parts of plants. In particular potato tubers are Host cells can be any cells in which the modification of considered to be suitable plant parts. A preferred promoter to hexokinase-signalling can be achieved through alterations in achieve selective TPS-enzyme expression in microtubers the level of T-6-P. Thus, accordingly, all eukaryotic cells are and tubers of potato is obtainable from the region upstream Subject to this invention. From an economic point of view of the open reading frame of the patatin gene of potato. the cells most Suited for production of metabolic compounds Another suitable promoter for specific expression is the 10 are most Suitable for the invention. These organisms are, plastocyanin promoter, which is specific for photoassimilat amongst others, plants animals, yeast, fungi. However, also ing parts of plants. Furthermore, it is envisaged that specific expression in specialized animal cells (like pancreatic beta expression in plant parts can yield a favourable effect for cells and fat cells) is envisaged. plant growth and reproduction or for economic use of said Preferred plant hosts among the Spermatophytae are the plants. Promoters which are useful in this respect are: the 15 Angiospermae, notably the Dicotyledoneae, comprising E8-promoter (EP 0 409 629) and the 2A11-promoter (van inter alia the Solanaceae as a representative family, and the Haaren and Houck (1993), Plant Mol. Biol., 221, 625) which Monocotyledoneae, comprising inter alia the Gramineae as are fruit-specific; the cruciferin promoter, the napin pro a representative family. Suitable host plants, as defined in the moter and the ACP promoter which are seed-specific; the context of the present invention include plants (as well as PAL-promoter; the chalcon-isomerase promoter which is parts and cells of said plants) and their progeny which flower-specific; the SSU promoter, and ferredoxin promoter, contain a modified level of T-6-P, for instance by using which are leaf-specific; the TobRb7 promoter which is recombinant DNA techniques to cause or enhance produc root-specific, the RolC promoter which is specific for tion of TPS or TPP in the desired plant or plant organ. Crops phloem and the HMG2 promoter (Enjuto et al. (1995). Plant according to the invention include those which have flowers Cell 7, 517) and the rice PCNA promoter (Kosugi et al. 25 Such as cauliflower (Brassica oleracea), artichoke (Cynara (1995), Plant J. 7, 877) which are specific for meristematic scolymus), cut flowers like carnation (Dianthus caryophyl tissue. lus), rose (Rosa spp), Chrysanthemum, Petunia, Alstromeria, Another option under this invention is to use inducible Gerbera, Gladiolus, lily (Lilium spp), hop (Humulus lupu promoters. Promoters are known which are inducible by lus), broccoli, potted plants like Rhododendron, AZalia, pathogens, by stress, by chemical or light/dark stimuli. It is 30 Dahlia, Begonia, Fuchsia, Geranium etc.; fruits such as envisaged that for induction of specific phenoma, for apple (Malus, e.g. domesticus), banana (Musa, e.g. Acumi instance sprouting, bolting, seed setting, filling of storage nata), apricot (Prunus armeniaca), olive (Oliva sativa). tissues, it is beneficial to induce the activity of the genes of pineapple (Ananas Comosus), coconut (Cocos nucifera), the invention by external stimuli. This enables normal mango (Mangifera indica), kiwi, avocado (Persea ameri development of the plant and the advantages of the induc 35 cana), berries (such as the currant, Ribes, e.g. rubrum), ibility of the desired phenomena at control. Promoters which cherries (such as the Sweet cherry, Prunus, e.g. avium), qualify for use in Such a regime are the pathogen inducible cucumber (Cucumis, e.g. sativus), grape (Vitis, e.g. vin promoters described in DE 4446342 (fungus and auxin ifera), lemon (Citrus limon), melon (Cucumis melo), mus inducible PRP-1), WO 96/28561 (fungus inducible PRP-1), tard (Sinapis alba and Brassica nigra), nuts (such as the EP0586 612 (nematode inducible), EP0712273 (nematode 40 Walnut, Juglans, e.g. regia; peanut, Arachis hypogeae), inducible), WO 96/34949 (fungus inducible), PCT/EP96/ orange (Citrus, e.g. maxima), peach (Prunus, e.g. persica). 02437 (nematode inducible), EP 0 330 479 (stress induc pear (Pyra, e.g. Communis), pepper (Solanum, e.g. capsi ible), U.S. Pat. No. 5,510,474 (stress inducible), WO cum), plum (Prunus, e.g. domestica), Strawberry (Fragaria, 96/12814 (cold inducible), EP0494724 (tetracycline induc e.g. moschata), tomato (Lycopersicon, e.g. esculentum); ible), EP 0 619 844 (ethylene inducible), EP 0 337 532 45 leaves, such as alfalfa (Medicago sativa), cabbages (such as (salicylic acid inducible), WO95/24491 (thiamine induc Brassica oleracea), endive (Cichoreum, e.g. endivia), leek ible) and WO 92/19724 (light inducible). Other chemical (Allium porruin), lettuce (Lactuca sativa), spinach (Spinacia inducible promoters are described in EP 0 674 608, EP 637 oleraceae), tobacco (Nicociana tabacum), grasses like Fes 339, EP 455 667 and U.S. Pat. No. 5,364,780. tuca, Poa, rye-grass (such as Lolium perenne, Lolium mul According to another embodiment of the invention, cells 50 tiflorum and Arrenatherum spp.), amenity grass, turf, sea are transformed with constructs which inhibit the function of weed, chicory (Cichorium intybus), tea (Thea Sinensis), the endogenously expressed TPS or TPP. Inhibition of celery, parsley (Pecroselinum crispum), chevil and other undesired endogenous enzyme activity is achieved in a herbs; roots, such as arrowroot (Maranta arundinacea), beet number of ways, the choice of which is not critical to the (Beta vulgaris), carrot (Daucus Carota), cassaya (Manihot invention. One method of inhibition of gene expression is 55 esculenta), ginseng (Panax ginseng), turnip (Brassica rapa). achieved through the so-called antisense approach Herein radish (Raphanus sativus), yam (Dioscorea esculenta), a DNA sequence is expressed which produces an RNA that Sweet potato (Ipomoea batatas), taro; seeds, such as beans is at least partially complementary to the RNA which (Phaseolus vulgaris), pea (Pisum sativum), soybean (Glycin encodes the enzymatic activity that is to be blocked. It is max), wheat (Triticum aestivum), barley (Hordeum vulgare), preferred to use homologous antisense genes as these are 60 corn (Zea mays), rice (Oryza sativa), bush beans and broad more efficient than heterologous genes. beans (Vicia faba), cotton (Gossypium spp.), coffee (Coffea An alternative method to block the synthesis of undesired arabica and C. Canephora); tubers, such as kohlrabi (Bras enzymatic activities is the introduction into the genome of sica oleraceae), potato (Solanum tuberosum); bulbous the plant host of an additional copy of an endogenous gene plants as onion (Allium cepa), Scallion, tulip (Tulipa spp.), present in the plant host. It is often observed that such an 65 daffodil (Narcissus spp.), garlic (Allium sativum); stems additional copy of a gene silences the endogenous gene: this Such as cork-oak, Sugarcane (Saccharum spp.), sisal (Sisal effect is referred to in the literature as the co-suppressive spp.), flax (Linum vulgare), jute; trees like rubber tree, oak US 7,247,770 B2 13 14 (Quercus spp.), beech IBetula spp.), alder (Alnus spp.), DNA transfer by Agrobacterium strains (vide WO94/00977, ashtree (Acer spp.), elm (Ulmus spp.), palms, ferns, ivies EP 0 159 418 B1; Gould et al. (1991) Plant. Physiol. 95, and the like. 426-434). Transformation of yeast and fungal or animal cells can be To obtain transgenic plants capable of constitutively done through normal state-of-the art transformation tech expressing more than one chimeric gene, a number of niques through commonly known vector systems like pblue alternatives are available including the following: script, puC and viral vector systems like RSV and SV40. A. The use of DNA, e.g. a T-DNA on a binary plasmid, with The method of introducing the expressible trehalose a number of modified genes physically coupled to a second phosphate synthase gene, the expressible trehalose-phos 10 selectable marker gene. The advantage of this method is that phate-phosphatase gene, or any other sense orantisense gene the chimeric genes are physically coupled and therefore into a recipient plant cell is not crucial, as long as the gene migrate as a single Mendelian locus. is expressed in said plant cell. B. Cross-pollination of transgenic plants each already Although some of the embodiments of the invention may capable of expressing one or more chimeric genes, prefer not be practicable at present, e.g. because some plant species 15 ably coupled to a selectable marker gene, with pollen from are as yet recalcitrant to genetic transformation, the prac a transgenic plant which contains one or more chimeric ticing of the invention in Such plant species is merely a genes coupled to another selectable marker. Afterwards the matter of time and not a matter of principle, because the seed, which is obtained by this crossing, maybe selected on amenability to genetic transformation as such is of no the basis of the presence of the two selectable markers, or on relevance to the underlying embodiment of the invention. the basis of the presence of the chimeric genes themselves. Transformation of plant species is now routine for an The plants obtained from the selected seeds can afterwards impressive number of plant species, including both the be used for further crossing. In principle the chimeric genes are not on a single locus and the genes may therefore Dicotyledoneae as well as the Monococyledoneae. In prin segregate as independent loci. ciple any transformation method may be used to introduce 25 chimeric DNA according to the invention into a suitable C. The use of a number of a plurality chimeric DNA ancestor cell. Methods may suitably be selected from the molecules, e.g. plasmids, each having one or more chimeric calcium/polyethylene glycol method for protoplasts (Krens genes and a selectable marker. If the frequency of co et al. (1982), Nature 296, 72: Negrutiu et al. (1987), Plant transformation is high, then selection on the basis of only Mol. Biol. 8, 363, electroporation of protoplasts (Shillito et 30 one marker is Sufficient. In other cases, the selection on the al. (1985) Bio/Technol. 3, 1099), microinjection into plant basis of more than one marker is preferred. material (Crossway et al. (1986), Mol. Gen. Genet. 202), (DNA or RNA-coated) particle bombardment of various D. Consecutive transformation of transgenic plants already plant material (Klein at al. (1987), Nature 327, 70), infection containing a first, second, (etc), chimeric gene with new with (non-integrative) viruses, in planta Agrobacterium chimeric DNA, optionally comprising a selectable marker tumefaciens mediated gene transfer by infiltration of adult 35 gene. As in method B, the chimeric genes are in principle not plants or transformation of mature pollen or microspores (EP on a single locus and the chimeric genes may therefore 0301 316) and the like. A preferred method according to the segregate as independent loci. invention comprises Agrobacterium-mediated DNA transfer. E. Combinations of the above mentioned strategies. Especially preferred is the use of the so-called binary vector 40 The actual strategy may depend on several considerations technology as disclosed in EP A 120516 and U.S. Pat. No. as maybe easily determined such as the purpose of the 4,940,838). parental lines (direct growing, use in a breeding programme, Although considered somewhat more recalcitrant towards use to produce hybrids) but is not critical with respect to the genetic transformation, monocotyledonous plants are ame described invention. nable to transformation and fertile transgenic plants can be 45 It is known that practically all plants can be regenerated regenerated from transformed cells or embryos, or other from cultured cells or tissues. The means for regeneration plant material. Presently, preferred methods for transforma vary from species to species of plants, but generally a tion of monocots are microprojectile bombardment of Suspension of transformed protoplasts or a petri plate con embryos, explants or Suspension cells, and direct DNA taining transformed explants is first provided. Shoots may be uptake or (tissue) electroporation (Shimamoto et al. (1989), 50 induced directly, or indirectly from callus via organogenesis Nature 338, 274-276). Transgenic maize plants have been or embryogenesis and Subsequently rooted. Next to the obtained by introducing the Screptomyces hygroscopicus selectable marker, the culture media will generally contain bar-gene, which encodes phosphinothricin acetyltransferase various amino acids and hormones, such as auxin and (an enzyme which inactivates the herbicide phosphinothri cytokinins. It is also advantageous to add glutamic acid and cin), into embryogenic cells of a maize suspension culture 55 proline to the medium, especially for Such species as corn by microprojectile bombardment (Gordon-Kamm (1990), and alfalfa. Efficient regeneration will depend on the Plant Cell, 2, 603). The introduction of genetic material into medium, on the genotype and on the history of the culture. aleurone protoplasts of other monocot crops such as wheat If these three variables are controlled regeneration is usually and barley has been reported (Lee (1989), Plant Mol. Biol. reproducible and repeatable. After stable incorporation of 13, 21). Wheat plants have been regenerated from embryo 60 the transformed gene sequences into the transgenic plants, genic Suspension culture by selecting embryogenic callus for the traits conferred by them can be transferred to other plants the establishment of the embryogenic Suspension cultures by sexual crossing. Any of a number of Standard breeding (Vasil (1990) Bic/Technol. 8, 429). The combination with techniques can be used, depending upon the species to be transformation systems for these crops enables the applica crossed. tion of the present invention to monocots. 65 Suitable DNA sequences for control of expression of the Monocotyledonous plants, including commercially plant expressible genes (including marker genes). Such as important crops such as rice and corn are also amenable to transcriptional initiation regions, enhancers, non-transcribed US 7,247,770 B2 15 16 leaders and the like, may be derived from any gene that is like hemp, sisal, flax which are used for the production of expressed in a plant cell. Also intended are hybrid promoters rope and linen; crops like bamboo and Sugarcane; rubber combining functional portions of various promoters, or tree, cork-oak, for the prevention of flattening in crops or synthetic equivalents thereof. Apart from constitutive pro crop parts, like grains, corn, legumes and strawberries. moters, inducible promoters, or promoters otherwise regu A third phenomenon is increased bleaching of the leaves lated in their expression pattern, e.g. developmentally or (caused by a decrease of photosynthetic activity). Less cell-type specific, may be used to control expression of the colourful leaves are preferred for crops such as chicory and expressible genes according to the invention. asparagus. Also for cut flowers bleaching in the petals can be To select or screen for transformed cells, it is preferred to desired, for instance in Alstromeria. include a marker gene linked to the plant expressible gene 10 An overall effect is the increase in biomass resulting from according to the invention to be transferred to a plant cell. an increase in metabolic activity. This means that the bio The choice of a suitable marker gene in plant transformation mass consists of metabolized compounds Such as proteins is well within the scope of the average skilled worker; some and fats. Accordingly, there is an increased protein/carbo examples of routinely used marker genes are the neomycin hydrate balance in mature leaves which is an advantage for phosphotransferase genes conferring resistance to kanamy 15 crops like Silage maize, and all fodder which can be ensi cin (EP-B 131 623), the glutathion-S-transferase gene from laged. A similar increased protein/carbohydrate balance can rat liver conferring resistance to glutathione derived herbi be established in fruits, tubers and other edible plant parts. cides (EP-A256 223), glutamine synthetase conferring upon Outside the plant kingdom an increased metabolism overexpression resistance to glutamine synthetase inhibitors would be beneficial for protein production in microorgan such as phosphinothricin (WO 87/05327), the acetyl trans isms or eukaryotic cell cultures. Both production of endog ferase gene from Streptomyces viridochromogenes confer enous but also of heterologous proteins will be enhanced ring resistance to the selective agent phosphinothricin (EP-A which means that the production of heterologous proteins in 275957), the gene encoding a 5-enolshikimate-3-phosphate cultures of yeast or other unicellular organisms can be synthase (EPSPS) conferring tolerance to N-phosphonom enhanced in this way. For yeast this would give a more ethylglycine, the bar gene conferring resistance against 25 efficient fermentation, which would result in an increased Bialaphos (e.g. WO 91/02071) and the like. The actual alcohol yield, which of course is favourable in brewery choice of the marker is not crucial as long as it is functional processes, alcohol production and the like. (i.e. selective) in combination with the plant cells of choice. In animals or human beings it is envisaged that diseases The marker gene and the gene of interest do not have to caused by a defect in metabolism can be overcome by stable be linked, since co-transformation of unlinked genes (U.S. 30 expression of TPP or TPS in the affected cells. In human Pat. No. 4,399.216) is also an efficient process in plant cells, the increased glucose consumption of many tumour transformation. cells depends to a large extent on the overexpression of Preferred plant material for transformation, especially for hexokinase (Rempeletal. (1996) FEBS Lett. 385,233). It is dicotyledonous crops are leaf-discs which can be readily envisaged that the flux of glucose into the metabolism of transformed and have good regenerative capability (Horsch 35 cancer cells can be influenced by the expression of treha et al. (1985), Science 227, 1229). lose-6-phosphate synthesizing enzymes. It has also been Specific use of the invention is envisaged in the following shown that the hexokinase activation is potentiated by the ways: as can be seen from the Examples the effects of the cAHP/PKA (protein kinase A pathway). Therefore, inacti expression of TPP (which causes a decrease in the intrac Vation of this signal transduction pathway may affect glu ellular T-6-P concentration) are an increased leaf size, 40 cose uptake and the proliferation of neoplasias. Enzyme increased branching leading to an increase in the number of activities in mammalian cells able to synthesize trehalose leaves, increase in total leaf biomass, bleaching of mature 6-phosphate and trehalose and degrade trehalose have been leaves, formation of more small flowers and sterility. These shown in e.g. rabbit kidney cortex cells (Sacktor (1968) effects are specifically useful in the following cases: Proc. Natl. Acad. Sci. USA 60, 1007). increased leaf size (and increase in the number of leaves) is 45 economically important for leafy vegetables such as spin Another example can be found in defects in insulin secretion ach, lettuce, leek, alfalfa, Silage maize; for ground coverage in pancreatic beta-cells in which the production of glucose and weed control by grasses and garden plants; for crops in 6-phosphate catalyzed by hexokinase is the predominant which the leaves are used as product, Such as tobacco, tea, reaction that couples rises in extracellular glucose levels to hemp and roses (perfumes); for the matting up of cabbage 50 insulin secretion (Efrat et al. (1994), TIBS 19, 535). An like crops such as cauliflower. increase in hexokinase activity caused by a decrease of An additional advantage of the fact that these leaves are intracellular T-6-P then will stimulate insulin production in stimulated in their metabolic activity is that they tend to burn cells which are deficient in insulin secretion. all their intracellular resources, which means that they are Also in transgenic animals an increased protein/carbohy low in starch-content. For plants meant for consumption a 55 drate balance can be advantageous. Both the properties of on reduction in starch content is advantageous in the light of the increased metabolism and an enhanced production of pro present tendency for low-calorie foodstuffs. Such a reduc teins are of large importance in farming in which animals tion in starch content also has effects on taste and texture of should gain in flesh as soon as possible. Transformation of the leaves. An increase in the protein/carbohydrate balance the enzyme TPP into meat-producing animals like chickens, as can be produced by the expression of TPP is especially 60 cattle, sheep, turkeys, goats, fish, lobster, crab, shrimps, important for leafy crops as Silage maize. Snails etc., will yield animals that grow faster and have a Increased branching, which is accompanied by a tendency more proteinaceous meat. to have stems with a larger diameter, can be advantageous in In the same way this increased metabolism means an crops in which the stem is responsible for the generation of increase in the burn rate of carbohydrates and it thus an economically attractive product. Examples in this cat 65 prevents obesity. egory are all trees for the increased production of wood, More plant-specific effects from the decrease of intracel which is also a starting material for paper production; crops lular T-6-P concentration are an increase in the number of US 7,247,770 B2 17 18 flowers (although they do not seem to lead co the formation Loss of apical dominance also causes formation of mul of seed). However, an increase in the number of flowers is tiple shoots which is of economic importance for instance in advantageous for cutflower plants and pot flower plants and alfalfa. also for all plants suitable for horticulture. A reduction in growth is furthermore desired for the A further effect of this flowering phenomenon is sterility, industry of "veggie Snacks, in which vegetables are con because the plants do not produce seed. Sterile plants are sidered to be consumed in the form of snacks. Cherry advantageous in hybrid breeding. tomatoes is an example of reduced size vegetables which are Another economically important aspect is the prohibiting Successful in the market. It can be envisaged that also other of bolting of culture crops such as lettuce, endive and both vegetables like cabbages, cauliflower, carrot, beet and Sweet recreational and fodder grasses. This is a beneficial property 10 potato and fruits like apple, pear, peach, melon, and several because it enables the crop to grow without having to spend tropical fruits like mango and banana would be marketable metabolic efforts to flowering and seed production. More on miniature size. over, in crops like lettuce, endive and grasses the commer Reduced growth is desired for all cells that are detrimental cial product/application is non-bolted. to an organism, such as cells of pathogens and cancerous Specific expression of TPP in certain parts (sinks) of the 15 cells. In this last respect a role can be seen in regulation of plant can give additional beneficial effects. It is envisaged the growth by changing the level of T-6-P. An increase in the that expression of TPP by a promoter which is active early T-6-P level would reduce growth and metabolism of cancer in e.g. seed forming enables an increased growth of the tissue. One way to increase the intracellular level of T-6-P is developing seed. A similar effect would be obtained by to knock-out the TPP gene of such cells by introducing a expressing TPP by a flower-specific promoter. To put it specific recombination event which causes the introduction shortly: excessive growth of a certain plant part is possible of a mutation in the endogenous TPP-genes. One way in if TPP is expressed by a suitable specific promoter. In fruits which this could be done is the introduction of a DNA specific expression can lead to an increased growth of the sequence able of introducing a mutation in the endogenous skin in relation to the flesh. This enables improvement of the gene via a cancer cell specific internalizing antibody. 25 Another way is targeted microparticle bombardment with peeling of the fruit, which can be advantageous for auto said DNA. Thirdly a cancer cell specific viral vectors having matic peeling industries. said DNA can be used. Expression of TPP during the process of germination of The phenomenon of a darker green colour seen with an oil-storing seeds prevents oil-degradations. In the process of increased concentration of T-6-P, is a property which is germination, the glyoxylate cycle is very active. This meta 30 desirable for pot flower plants and, in general, for species in bolic pathway converts acetyl-CoA via malate into Sucrose horticulture and for recreational grasses. which can be transported and used as energy source during Increase in the level of T-6-Palso causes an increase in the growth of the seedling. Key-enzymes in this process are storage carbohydrates such as starch and Sucrose. This then malate synthase and isocitrate lyase. Expression of both would mean that tissues in which carbohydrates are stored enzymes is Supposed to be regulated by hexokinase signal 35 would be able to store more material. This can be illustrated ling. One of the indications for this regulation is that both by the Examples where it is shown that in plants increased 2-deoxyglucose and mannose are phosphorylated by hex biomass of storage organs Such as tubers and thickened roots okinase and able to transduce their signal, being reduction of as in beets (storage of Sucrose) are formed. malate synthase and isocitrate lyase expression, without Crops in which this would be very advantageous are being further metabolised. Expression of TPP in the seed, 40 potato, Sugarbeet, carrot, chicory and Sugarcane. thereby decreasing the inhibition of hexokinase, thereby An additional economically important effect in potatoes is inhibiting malate synthase and isocitrate lyase maintains the that after transformation with DNA encoding for the TPS storage of oil into the seeds and prevents germination. gene (generating an increase in T-6-P) it has been found that In contrast to the effects of TPP the increase in T-6-P the amount of Soluble Sugars decreases, even after harvest caused by the expression of TPS causes other effects as is 45 and storage of the tubers under cold conditions (4° C.). illustrated in the Examples. From these it can be learnt that Normally even colder storage would be necessary to prevent an increase in the amount of T-6-P causes dwarfing or early sprouting, but this results in excessive Sweetening of stunted growth (especially at high expression of TPS), the potatoes. Reduction of the amount of reducing Sugars is formation of more lancet-shaped leaves, darker colour due to of major importance for the food industry since Sweetened an increase in chlorophyll and an increase in starch content. 50 potato tuber material is not suitable for processing because As is already acknowledged above, the introduction of an a Maillard reaction will take place between the reducing anti-sense trehalase construct will also stimulate similar Sugars and the amino-acids which results in browning. effects as the introduction of TPS. Therefore, the applica In the same way also inhibition of activity of invertase can tions which are shown or indicated for TPS will equally be be obtained by transforming Sugarbeets with a polynucle established by using as-trehalase. Moreover, the use of 55 otide encoding for the enzyme TPS. Inhibition of invertase double-constructs of TPS and as-trehalase enhances the activity in Sugarbeets after harvest is economically very effects of a single construct. important. Dwarfing is a phenomenon that is desired in horticultural Also in fruits and seeds, storage can be altered. This does plants, of which the Japanese bonsai trees are a proverbial not only result in an increased storage capacity but in a example. However, also creation of mini-flowers in plants 60 change in the composition of the stored compounds. Crops like allseed, roses, Amaryllis, Hortensia, birch and palm will in which improvements in yield in seed are especially have economic opportunities. Next to the plant kingdom important are maize, rice, cereals, pea, oilseed rape, Sun dwarfing is also desired in animals. It is also possible to flower, soybean and legumes. Furthermore, all fruitbearing induce bolting in culture crops such as lettuce. This is plants are important for the application of developing a beneficial because it enables a rapid production of seed. 65 change in the amount and composition of stored carbohy Ideally the expression of TPS for this effect should be under drates. Especially for fruit the composition of stored prod control of an inducible promoter. ucts gives changes in Solidity and firmness, which is espe US 7,247,770 B2 19 20 cially important in soft fruits like tomato, banana, Isolation of a Patatin Promoter/Construction of pMOG546 Strawberry, peach, berries and grapes. A patatin promoter fragment is isolated from chromo In contrast to the effects seen with the expression of TPP, somal DNA of Solanu tuberosum cv. Bintje using the the expression of TPS reduces the ratio of protein/carbohy polymerase chain reaction. A set of oligonucleotides, drate in leaves. This effect is of importance in leafy crops complementary to the sequence of the upstream region of Such as fodder grasses and alfalfa. Furthermore, the leaves the pat?1 patatin gene (Bevan et al. (1986) Nucl. Acids have a reduced biomass, which can be of importance in Res. 14, 5564), is synthesized consisting of the following amenity grasses, but, more important, they have a relatively Sequences: increased energy content. This property is especially ben 10 eficial for crops as onion, leek and silage maize. 5' AAG CTT ATG TTG CCA TAT AGA GTA G 3' (SEQIDNO:5) Furthermore, also the viability of the seeds can be influ Pat 33.2 enced by the level of intracellularly available T-6-P. 5 GTA GTT GCC ATG GTG CAA ATG TTC 3' (SEQIDNO: 6) Combinations of expression of TPP in one part of a plant PatATG-2 and TPS in an other part of the plant can synergize to 15 increase the above-described effects. It is also possible to These primers are used to PCR amplify a DNA fragment of express the genes sequential during development by using 1123 bp, using chromosomal DNA isolated from potato cv. specific promoters. Lastly, it is also possible to induce Bintje as a template. The amplified fragment shows a high expression of either of the genes involved by placing the degree of similarity to the Wpat21 patatin sequence and is coding the sequence under control of an inducible promoter. cloned using EcoRI linkers into a puC18 vector resulting in It is envisaged that combinations of the methods of appli plasmid pMOG546. cation as described will be apparent to the person skilled in Construction of pMDCG45. the art. Construction of pMOG845 is described in WO 96/21030. The invention is further illustrated by the following 25 Construction of pVDH318, Plastocvanin-TPS examples. It is stressed that the Examples show specific Plasmid pMOG79B (described in WO95/01446) is embodiments of the inventions, but that it will be clear that digested with HindIII and ligated with the oligonucleotide variations on these examples and use of other plants or duplex TCV11 and TCV12 (see construction of pNOG845). expression systems are covered by the invention. The resulting vector is digested with PstI and HindIII 30 followed by the insertion of the PotPill terminator resulting Experimental in pTCV118. Plasmid pTCV118 is digested with SmaI and HindIII yielding a DNA fragment comprising the TPS DNA Manipulations coding region and the PotPill terminator. BglII linkers were added and the resulting fragment was inserted in the plant All DNA procedures (DNA isolation from E. coli, restric 35 binary expression vector pVDH275 (FIG. 1) digested with tion, ligation, transformation, etc.) are performed according BamHI, yielding pVDH318. PVDH275 is a derivative of to standard protocols (Sambrook et al. (1989) Molecular pMOG23 (Simons et al. (1990), Bio/Technol. 8, 217) Cloning: a laboratory manual, 2nd ed. Cold Spring Harbor harbouring the NPTII selection marker under control of the Laboratory Press, CSH, New York). 35S CaMV promoter and an expression cassette comprising Strains 40 the pea plastocyanin (PC) promoter and nos terminator sequences. The plastocyanin promoter present in pVDH275 In all examples E. coli K-12 strain DH5a is used for cloning. The Agrobacterium tumefaciens strains used for has been described by Pwee & Gray (1993) Plant J. 3, 437. plant transformation experiments are EHA 105 and MOG This promoter has been transferred to the binary vector 101 (Hood et al. (1993) Trans. Research 2, 208). using PCR amplification and primers which contain suitable 45 cloning sites. Construction of Agrobacterium Strain MOG101 Cloning of the E. coli otsB Gene and Construction of Construction of Agrobacterium strain MOG101 is pMOG100 (35S CaMV TPP) described in WO 96/21030. A set of oligonucleotides, TPP I (5' CTCAGATCTGGC CACAAA 3')(SEQ ID NO. 56) and TPP II (5' Cloning of the E. coli ots A Gene and Construction of 50 GTGCTCGTCTGCAGGTGC 3')(SEQ ID NO. 57), was pMOG799 synthesized complementary to the sequence of the E. coli In E. coli trehalose phosphate synthase (TPS) is encoded TPP gene (SEQID NO:3). These primers were used to PCR by the otSA gene located in the operon otsBA. The cloning amplify a DNA fragment of 375 bp harbouring the 3' part of and sequence determination of the otSA gene is described in the coding region of the E. coli TPP gene, introducing a PstI detail in Example I of WO95/01446, herein incorporated by 55 site 10 bp down-stream of the stop codon, using pMOG748 reference. To effectuate its expression in plant cells, the open (WO95/01446) as a template. This PCR fragment was reading frame has been linked to the transcriptional regula digested with BglII and PstI and cloned into pNOG445 (EP tory elements of the CaMV 35S RNA promoter, the trans O 449 376 A2 example 7a) and linearized with BglII and lational enhancer of the ALMV leader, and the transcrip PstI. The resulting vector was digested with PstI and HindIII tional terminator of the nos-gene, as described in greater 60 and a PotPill terminator was inserted (see construction detail in Example I of WO95/01446, resulting in pMOG799. pMOGS845). The previous described vector was digested A sample of an E. coli strain harbouring pMOG799 has been with BglII and HindIII, the resulting 1325 bp fragment was deposited under the Budapest Treaty at the Centraal Bureau isolated and cloned together with the 5'TPPPCRed fragment voor Schimmelcultures, Oosterstraat 1, P.O. Box 273, 3740 digested with SmaI and BglII into puC18 linearized with AG Baarn, The Netherlands, on Monday 23 Aug. 1993: the 65 SmaI and HindIII. The resulting vector was called Accession Number given by the International Depositary pTCV124. This vector was linearized with EcoRI and SmaI Institution is CBS 430.93. and used to insert the 35S CaMV promoter (a 850 bp US 7,247,770 B2 21 22 EcoRI-NcoI (the NcoI site was made blunt by treatment indole acetic acid (IAA). After two days, discs were trans with mungbean nuclease) fragment isolated from pMOG 18 ferred to fresh PM medium with 200 mg/icefotaxim and 100 containing the 355 CaMV double enhancer promoter). This mg/l Vancomycin. Three days later, the tuber discs were vector was called pTCV127. From this vector a 2.8kb transferred to shoot induction medium (SIM) which con EcoRI-HindIII fragment was isolated containing the com sisted of PM medium with 250 mg/l carbenicillin and 100 plete 35S TPP expression cassette and cloned in binary mg/l kanamycin. After 4-8 weeks, shoots emerging from the vector pMOG800 resulting in vector pMOG1010. discs were excised and placed on rooting medium Construction of pVDH321, Plastocyanin (PC) TPP (MS30R3-medium with 100 mg/l cefotaxim, 50 mg/l van The BamHI site of plasmid pTCV124 was removed by comycin and 50 mg/l kanamycin). The shoots were propa BamHI digestion, filling-in and Subsequent religation. Sub 10 gated axenically by meristem cuttings. sequent digestion with HindIII and EcoRI yields a DNA Transformation of Lettuce fragment comprising the TPP coding region and the PotPill Transformation of lettuce, Lattuca sativa cV. Evola was terminator. BamHI linkers were added and the resulting performed according to Curtis et al. (1994) J. Exp. Bot. 45, 1441. fragment was inserted in the plant binary expression vector 15 pVDH275 (digested with BamHI) yielding pVDH321. Transformation of Sugarbeet Construction of a Patatin TPP Expression Vector Transformation of Sugarbeet, Beta vulgaris (maintainer Similar to the construction of the patatin TPS expression population) was performed according to Fry et al. (1991) vector (see construction of pMOG845), a patatin TPP Third International Congress of ISPMB, Tucson USA expression vector was constructed yielding a binary vector Abstract No. 384, or according to Krens et al. (1996), Plant (pMOG 1128) which, after transformation, can effectuate Sci. 116, 97. expression of TPP in a tuber-specific manner. Transformation of Lycopersicon Esculentum Construction of Other Expression Vectors Tomato transformation was performed according to Van Similar to the construction of the above mentioned vec Roekel et al. (1993) Plant Cell Rep. 12, 644. tors, gene constructs can be made where different promoters 25 are used, in combination with TPS, TPP or trehalase using Transformation of Arabidopsis binary vectors with the NPTII gene or the Hygromycin Transformation of Arabidopsis thaliana was carried out resistance gene as selectable marker gene. A description of either by the method described by Clarke et al. (1992) Plant. binary vector pMOG22 harbouring a HPT selection marker Mol. Biol. Rep. 10, 178 or by the method described by is given in Goddijn et al. (1993) Plant J. 4, 863. Valvekens et al. (1988) Proc. Natl. Acad. Sci. USA, 85, 5536. Triparental Matings The binary vectors are mobilized in triparental matings Induction of Micro-Tubers with the E. coli strain HB101 containing plasmid pRK2013 Stem segments of in vitro potato plants harbouring an (Ditta et al. (1980) Proc. Natl. Acad. Sci. USA 77,7347) into 35 auxiliary meristem were transferred to micro-tuber inducing Agrobacterium tumefaciens strain MOG101 or EHA105 and medium. Micro-tuber inducing medium contains 1xMS used for transformation. salts supplemented with R3 vitamins, 0.5 g/l MES (final pH 5.8, adjusted with KOH) and solidified with 8 g/1 Transformation of Tobacco (Nicotiana tabacum cv. SR1 or Daichinagar, 60 g/l sucrose and 2.5 mg/l kinetin. After 3 to cv. Samsun NN) 40 5 weeks of growth in the dark at 24°C., micro-tubers were Tobacco was transformed by cocultivation of plant tissue formed. with Agrobacterium tumefaciens strain MOG101 containing the binary vector of interest as described. Transformation Isolation of Validamycin A was carried out using cocultivation of tobacco leaf disks as Validamycin A has been found to be a highly specific described by Horschet al. (1985) Science 227, 1229. Trans inhibitor of trehalases from various sources ranging from genic plants are regenerated from shoots that grow on 45 (ICs) 10M to 10'M (Asano et al. (1987).J. Antibiot. 40, selection medium containing kanamycin, rooted and trans 526; Kameda et al. (1987) J. Antibiot 40, 563). Except for ferred to soil. trehalase, it does not significantly inhibit any Cl- or B-gly cohydrolase activity. Validamycin A was isolated from Sola Transformation of Potato col, a commercial agricultural formulation (Takeda Chem. Potato (Solanum tuberosum cv. Kardal) was transformed 50 Indust., Tokyo) as described by Kendall et al. (1990) Phy with the Agrobacterium strain EHA 105 containing the tochemistry 29, 2525. The procedure involves ion-exchange binary vector of interest. The basic culture medium was chromatography (QAE-Sephadex A-25 (Pharmacia), bed MS30R3 medium consisting of MS salts (Murashige and vol. 10 ml, equilibration buffer 0.2 mM Na-Pi pH 7) from a Skoog (1962) Physiol. Plant. 14, 473), R3 vitamins (Ooms 3% agricultural formulation of Solacol. Loading 1 ml of et al. (1987) Theor. Appl. Genet. 73,744), 30 g/l sucrose, 0.5 55 Solacol on the column and eluting with water in 7 fractions, g/l MES with final pH 5.8 (adjusted with KOH) solidified practically all Validamycin was recovered in fraction 4. when necessary with 8 g/l Daichinagar. Tubers of Solanum Based on a 100% recovery, using this procedure, the con tuberosum cv. Kardal were peeled and surface sterilized by centration of Validamycin A was adjusted to 1.10 M in burning them in 96% ethanol for 5 seconds. The flames were MS-medium, for use in trehalose accumulation tests. Alter extinguished in Sterile water and cut slices of approximately 60 natively, Validamycin A and B may be purified directly from 2 mm thickness. Disks were cut with a bore from the Streptomyces hygroscopicus var. limoneus, as described by vascular tissue and incubated for 20 minutes in MS30R3 Iwasa et al. (1971) J. Antibiot. 24, 119, the content of which medium containing 1-5x10 bacteria/ml of Agrobacterium is incorporated herein by reference. EHA 105 containing the binary vector. The tuber discs were washed with MS30R3 medium and transferred to solidified 65 Carbohydrate Analysis postculture medium (PM). PM consisted of M30R3 medium Carbohydrates were determined quantitatively by anion supplemented with 3.5 mg/l Zeatin riboside and 0.03 mg/1 exchange chromatography with pulsed electrochemical US 7,247,770 B2 23 24 detection. Extracts were prepared by extracting homog EXAMPLE 1. enized frozen material with 80% EtOH. After extraction for 15 minutes at room temperature, the soluble fraction is Expression of the E. coli ots A Gene (TPS) in evaporated and dissolved in distilled water. Samples (25 ul) Tobacco and Potato were analyzed on a Dionex DX-300 liquid chromatograph 5 equipped with a 4x250 mm Dionex 35391 carbopac PA-1 column and a 4x50 mm Dionex 43096 carbopac PA-1 Transgenic tobacco plants were generated barbouring the precolumn. Elution was with 100 mM NaOH at 1 ml/min otsA gene driven by the de35SCaMV promoter (pMOG799) followed by a NaAc gradient. Sugars were detected with a or the plastocyanin promoter (pVDH318). pulsed electrochemical detector (Dionex, PED). Commer 10 cially available carbohydrates (Sigma) were used as a stan Transgenic potato plants were generated harbouring the dard. =A gene driven by the potato tuber-specific patatin promoter (pMOG845). Starch Analysis Starch analysis was performed as described in: Aman et Tobacco leaf discs were transformed with the binary al. (1994) Methods in Carbohydrate Chemistry, Volume X 15 vector pMOG799 using Agrobacterium tumefaciens. Trans (eds. BeMiller et al.), pp 111-115. genic shoots were selected on kanamycin. Expression Analysis Leaves of Some soil-grown plants did not fully expand in The expression of genes introduced in various plant lateral direction, leading to a lancet-shaped morphology species was monitored using Northern blot analysis. (FIG. 31). Trehalose-6-Phosnhate Phosphatase Assay Furthermore, apical dominance was reduced resulting in TPP was assayed at 37° C. by measuring the production stunted growth and formation of several axillary shoots. of ''Citrehalose from "Ctrehalose-6-phosphate (Londes Seven out of thirty-two plants showed severe growth reduc borough and Vuorio (1991).J. of Gen. Microbiol. 137,323). 25 tion, reaching plant heights of 4-30 cm at the time of Crude extracts were prepared in 25 mM Tris, HCl pH 7.4, flowering (Table 1). containing 5.5 mM MgCl2. Samples were diluted to a protein concentration of 1 mg/ml in extraction buffer con TABLE 1. taining 1 mg/ml BSA. Standard assay mixtures (50 ul final 30 volume) contained 27.5 mM Tris, HCl pH 7.4, 5.5 mM Trehalose accumulation in leaf samples of otSA transgenic tobacco plants and their plant length at the time of MgCl, 1 mg/ml BSA and 0.55 mMT-6-P (specific activity flowering. 854 cpm/nmol). Reactions were initiated by the addition of 5 ul enzyme and terminated after 1 hour by heating for 5 plant-line trehalose mg.g' fresh weight height cm 35 minutes in boiling water. AG1-X8 (formate) anion-exchange controls O.OO 60-70 resin (BioRad) was added and the reaction mixtures were 799-1 O.04 ND centrifuged after 20 minutes of equilibration at room tem 799-3 O.O2 10 perature. The radioactivity in the Supernatant of the samples 799-5 O.08 4 (400 ul) was measured by liquid Scintillation counting. 799-15 0.055 30 40 799-24 O.O2 12 Preparation of Plant Extracts for Hexokinase Assays 799-26 O.OS 25 Frozen plant material was grinded in liquid nitrogen and 799-32 0.055 30 homogenized for 30 seconds with extraction buffer (EB: 100 799-40 O.11 25 mM HEPES pH7.0 (KOH), 1% (w/v) PVP 5 mM MgCl, ND: not determined 1.5 mM EDTA, 0.1% v/v B-MeOH) including Proteinase 45 Inhibitors Complete (Boehringer Mannheim). After cen Control plants reached lengths of 60-70 cm at the time of trifugation, proteins in the Supernatant were precipitated flowering. Less seed was produced by transgenic lines with using 80% ammoniumsulphate and dissolved in Tris-HCl the stunted growth phenotype. Northern blot analysis con pH 7.4 and the extract was dialyzed overnight against 100 50 firmed that plants having the stunted growth phenotype mM Tris-HCl pH 7.4. Part of the sample was used in the expressed the otSA gene from E. coli (FIG. 2). In control hexokinase assay. plants no transcript could be detected. The functionality of Hexokinase Assay the introduced gene was proven by carbohydrate analyses of Hexokinase activity was measured in an assay containing 55 leaf material from 32 transgenic greenhouse-grown tobacco 0.1 M Hepes-KOH pH 7.0, 4 MM MgCl, 5 mM ATP, 0.2 plants, revealing the presence of 0.02 to 0.12 mg.g-1 fresh mM NADP", 10 U/ml Creatine Phosphate Kinase (dissolved weight trehalose in plants reduced in length (table 1) indi in 50% glycerol, 0.1% BSA, 50 mM Hepes pH 7.0), 3.5 mM cating that the product of the TPS-catalyzed reaction is Creatine Phosphate, 7 U/ml Glucose-6-Phosphate Dehydro dephosphorylated by plant phosphatases. Further proof for genase and 2 mM Glucose by measuring the increase in OD 60 at 340 nm at 25° C. the accumulation of trehalose in tobacco was obtained by When 2 mM Fructose was used instead of glucose as treating crude extracts with porcine trehalase. Prolonged substrate for the hexokinase reaction, 3.8 U/ml Phosphoglu incubation of a tobacco leaf extract with trehalase resulted in cose Isomerase was included. Alternatively, a hexokinase 65 complete degradation of trehalose (data not shown). Treha assay as described by Gancedo et al. (1977) J. Biol. Chem. lose was not detected in control plants or transgenic tobacco 252, 4443 was used. plants without an aberrant phenotype. US 7,247,770 B2 25 26 TABLE 1 a Primary PC-TPS tobacco transformants Dry Leaf Leaf Plant Fw matter Plant- fw 88 No. of height Leaf Axilliary 808 Dry 808 line (g) cm branches cm colour shoots g/cm matter % g/cm’ ctrl. 1 8.18 34937 1 Wt O.O23 7.21 O.OO17 ctrl. 2 1O.S 418.89 1 Wt O.O2S 9.52 O.OO24 ctrl. 3 9.99 373.87 1 Wt O.O27 2.91 O.OO3S ctrl. 4 9.91 362.92 1 Wt O.O27 9.59 O.OO26 ctrl. 5 9.82 393.84 1 Wt O.O2S 1.51 O.OO29 average O.O2S4 O.148 OOO26 2 8.39 290 2 O5 wt O.O29 2.16 O.OO3S 3 9.34 296 1 23 wit O.O32 2.21 O.OO39 4 8.36 254 2 30 wit many O.O33 O.OS O.OO33 6 2.28 106 5 90 wit O.O22 140 O.OO2S 8 S.21 133 4 OO dark many O.O39 7.49 O.OO29 10 8.08 258 2 65 8K many O.O31 2.25 O.OO38 11 2.61 64 12 95 8K many O.O41 9.20 O.OO38 13 2.83 92 SO dark many O.O31 8.48 O.OO26 16 5.86 209 3 30 dark many O.O28 O.S8 O.OO3O 17 S.1S 224 2 SS w O.O23 1.65 O.OO27 18 17.2 547 33 w O.O31 O.35 O.OO33 19 2.13 63 4 80 dark many O.O34 1.74 O.OO40 2O 3.44 113 4 90 wt + Da many O.O3O 8.14 O.OO2S 21 9.88 246 05 8K many O.040 8. SO O.OO34 22 13.1 409 3S w O.O32 O.68 O.OO34 23 2.50 73 6 55 8K many O.O34 8.8O O.OO3O 24 8.76 286 2 30 w O.O31 5.07 O.OO46 27 7.91 219 24 w O.036 4.41 O.OOS2 28 1O.O 269 2 17 dark many O.O38 8.62 O.OO32 29 4.17 142 85 8K many O.O29 O.O7 O.OO3O 30 10.2 343 60 W O.O3O 9.56 O.OO29 32 1.9S 61 3 75 8K many O.O32 8.21 O.OO26 33 2.85 96 5 95 wt + Da many O.O3O 1.23 O.OO33 34 8.38 244 23 w O.O34 3.60 O.OO47 35 5.59 173 3 26 w O.O32 4.49 O.OO47 36 3.28 84 3 OO dark many O.O39 1.28 O.OO44 37 7.80 222 25 wt + Da many O.O3S 1.28 O.OO40 39 3.70 131 2 23 w O.O28 7.84 O.OOSO 40 240 68.5 3 08 dark many O.O3S 9.58 O.OO34 average O.O32 1.00 O.OO3S Transgenic pVDH318 transgenic tobacco plants devel oped stunted growth and development of small leaves which TABLE 1 b-continued were darker green and slightly thicker than control leaves, a 45 phenotype similar to the pMOG799 transgenic plants (table Chlorophyll content of N. tabacum leaves (To) transgenic 1a). Further analysis of these leaves showed an increased for PC-TPS fresh and dry weight per leaf-area compared to the controls Sample Chlorophyll (mgg leaf) (table la and 2). The dark green leaves indicate the presence of more chlorophyll in the transgenic leaves (table 1b). 50 PC TPS 19 O.64 Plants transgenic for pNOG799 (35STPS) and pMCG1177 PC TPS 37 O.96 (PCTPS) were analyzed on soluble carbohydrates, chloro phyll, trehalose and starch (FIG. 32). pMOG 1177 is func Note: tionally identical to pVDH318. light conditions during growth will influence the determined levels of 55 chlorophyll significantly. The calculated amounts of chlorophyll may thus only be compared between plants harvested and analyzed within one TABLE 1b. experiment Chlorophyll content of N. tabacum leaves (To) transgenic for PC-TPS TABLE 2 60 Sample Chlorophyll (mg/g leaf) Fresh weight and dry weight data of leaf material transgenic for plastocyanin-TPSE. coli control 1 O.S9 N. tabacum cv. Samsun NN transgenic for PC-TPS PC TPS 10-1 0.75 PC TPS 10-2 O.8O Transgene Control PC TPS 11 O.6O PC TPS 13 O.81 65 Fresh weight (g) O.83 O.78 PC TPS 16 O.90 Dry weight (g) O.O72 O.O79 US 7,247,770 B2 27 28 TABLE 3-continued TABLE 2-continued Fresh weight and dry weight data of leaf material Tobacco plants (cv. Samsun NN) transgenic for pVDH318 transgenic for plastocyanin-TPSE. coli N. tabacum cv. Samsun NN transgenic for PC-TPS Transformant Length (cm) Width (cm) Ratio I?w Transgene Control % dry matter 8.70% 10.10% 1318-24 27 17 1.59 FW area 39 (139%) 28 (100%) 1318-13 15 7 2.14% DW area 3.46 (12.1%) 2.87 (100%) 10 area (units) 208 275 1318-17 24 16 1...SO 1318-18 23 16.5 1.39 Calculation of the ratio between the length and width of the developing leaves clearly indicate that leaves of plants *typical TPS phenotypes Ratio 1/w average of controls is 1.50 transgenic for PC-TPS are more lancet-shaped (table 3). TABLE 4 Potato Soianum tuberosum cv. Xardal tuber discs were transformed with Agrobacterium tumefaciens EHA105 har Trehalose (% fresh weight bouring the binary vector pMOG845, transgenics were obtained with transformation frequencies comparable to +Validamycin A -Validamycin A empty vector controls. All plants obtained were phenotypi 845-2 O.O16 cally indistinguishable from wild type plants indicating that 845-4 use of a tissue specific promoter prevents the phenotypes 845-8 O.OS1 observed in plants where a constitutive promoter drives the 845-11 O.O15 TPS gene. Micro-tubers were induced on stem segments of 845-13 O.O11 25 845-22 O112 transgenic and wild-type plants cultured on microtuber 845-25 O.OO2 inducing medium supplemented with 10 M. Validamycin 845-28 O.109 A. As a control, microtubers were induced on medium wild-type Kardal O.OO1 without Validamycin A. Microtubers induced on medium with Validamycin A showed elevated levels of trehalose in comparison with microtubers grown on medium without 30 Validamycin A (table 4). The presence of small amounts of EXAMPLE 2 trehalose in wild-type plants indicates the presence of a functional trehalose biosynthetic pathway. Expression of the E. coli otaB Gene (TPP) in TABLE 3 Tobacco 35 Tobacco plants (cv. Samsun NN) transgenic for pVDH318 Transgenic tobacco plants were generated harbouring the Transformant Length (cm) Width (cm) Ratio Iw otsb gene driven by the double enhanced 35SCaMV pro control 1 12 8 SO control 2 13 8.5 53 moter (pMOG 1010) and the plastocyanin promoter 40 control 3 12 7.5 60 (pVDH321). control 4 15 9 .67 control 5 25 6 S6 Tobacco plants (cv. Samsun NN) transformed with control 6 24 6.5 45 pMOG1010 revealed in the greenhouse the development of control 7 28 2O 40 control 8 25 6 S6 very large leaves (leaf area increased on average up to control 9 26 9 37 45 approximately 14.0%) which started to develop chlorosis control 10 21 5 40 318-28 6 8.5 88: when fully developed (FIG. 31). Additionally, thicker stems 318-29 1 6.5 69 318-30 9 4 36 were formed as compared to the controls, in some instances 318-35 9 2 S8 leading to bursting of the stems. In some cases, multiple 318-39 21 6.5 27 50 stems were formed (branching) from the base of the plant 318-40 4 7 2.00: 318-34 21 3 62 (table 5). Leaf samples of plants developing large leaves 318-36 3.5 7 93: revealed 5-10 times enhanced trehalose-6-phosphate phos 318-37 7 9 89* 318-4 2O.S 2 .71 phatase activities compared to control plants proving func 31.8-23 4 4.5 3.78% 55 tionality of the gene introduced. The dry and fresh weight/ 318-22 27 8 SO 318-19 9 4 2.25% cm of the abnormal large leaves was comparable to control 318-2 27 9 42 318-15 1 5 2.20% leaves, indicating that the increase in size is due to an 318-10 2O 3 54 increase in dry matter and not to an increased water content. 318-3 25 8 39 318-21 7 8.5 2.00: 60 The inflorescence was also affected by the expression of 318-16 2O O 2.00: TPP. Plants which had a stunted phenotype, probably caused 318-6 9 O.S 81 by the constitutive expression of the TPP gene in all plant 318-2O 3 5 2.60: 318-33 2 5 2.40: parts, developed many small flowers which did not fully 318-27 23 2O 1S 318-11 2 5 2.40: 65 mature and fell off or necrotized. The development of 318-8 8.5 6.5 2.85: flowers and seed setting seems to be less affected in plants which were less stunted. US 7,247,770 B2 31 32 TABLE 6-continued Primary PC-TPP tobacco transformants Leaf Leaf Plant Dry Plant- fiv 88 No. of height Leaf Fw Dry matter line (g) cm? branches cm colour Bleaching 808 matter 90 88 39 14.9 S60 1 112 wit bleaching 0.0267 6.08 O.OO16 40 24.5 843 O.O292 9.8O O.OO29 41 8.86 343 1 115 wt O.O2S8 2.93 O.OOO8 42 6.93 289 1 Wt O.O240 3.32 O.OOO8 43 11.3 433 136 135 wt. O.O261 6.73 O.OO18 44 1O.O 341 2 135 wt. O.O294 6.49 O.OO19 45 9.40 327 2 135 wt. O.O287 8.51 O.0024 46 9.18, 284 2 115 wt O.O323 15.69 O.OOS1 average O.O27 9.60 O.OO2S wt = wild-type Tobacco plants (cv. Samsun NN) transformed with Degenerated primers used (IUB code): pVDH321 revealed in the greenhouse a pattern of develop ment comparable to pMOG1010 transgenic plants (table 6). TPSdeg1: Plants transgenic for pMOG1010 (35S-TPP) and GAY ITI ATI TGG RTI CAY GAY TAY CA (SEQ ID NO: 7) pMOG1124 (PC-TPP) were analyzed on carbohydrates, TPSdeg2: chlorophyll, trehalose and starch (FIGS. 32 A-D). For chlo 25 TIG GIT KIT TYY TIC AYA YIC CIT TYC C (SEQ ID NO:8) rophyll data see also Table 6a. TPSdeg5: TABLE 6a GYI ACI ARR TTC ATI CCR TCI C (SEQ ID NO:9) Chlorophyll content of N. tabacum leaves (To) PCR fragments of the expected size were cloned and transgenic for PC-TPP 30 sequenced. Since a large number of homologous sequences Chlorophyll were isolated, Southern blot analysis was used to determine Sample (mgg leaf) Leaf phenotype which clones hybridized with Selaginella genomic DNA. Two clones were isolated, clone 8 of which the sequence is control 1 1.56 wild-type given in SEQID NO: 42 (PCR primer combination 1/5) and control 2 140 wild-type 35 control 3 1.46 wild-type clone 43 of which the sequence is given in SEQID NO: 44 control 4 1.56 wild-type (PCR primer combination 2/5) which on the level of amino control 5 1.96 wild-type acids revealed regions with a high percentage of identity to PC TPP 12 0.79 bleaching PC TPP 22 O.76 bleaching the TPS genes from E. coli and yeast. PC TPP 25 1.30 wild-type 40 One TPS gene fragment was isolated from Helianthus PC TPP 37 O.86 wild-type annuus (Sunflower) using primer combination TPSdeg 2/5 in PC TPP 38 O.74 bleaching a PCR amplification with genomic DNA of H. annuus as a Note: template. Sequence and Southern blot analysis confirmed light conditions during growth will influence the determined levels of the homology with the TPS genes from E.coli, yeast and chlorophyll significantly. The calculated amounts of chlorophyll may thus Selaginella. Comparison of these sequences with EST only be compared between plants harvested and analyzed within one 45 sequences (expressed sequence tags) from various organ experiment isms, see Table 6b and SEQID NOS 45-53 and 41, indicated the presence of highly homologous genes in rice and Ara EXAMPLE 3 bidopsis, which Supports our invention that most plants 50 contain TPS homologous genes (FIGS. 3A and 3B). Isolation of Gene Fragments Encoding Trehalose-6-Phosphate Synthases from Selaginella TABLE 6b legidophylla and Helianthus annuus Genbank dbEST ID. Accession No. Organism Function 55 Comparison of the TPS protein sequences from E. coli 35567 D22143 Oryza sativa TPS S81.99 D35348 Caenorhabditis elegans TPS and S. cerevisiae revealed the presence of several conserved 60O2O D36432 Caenorhabditis elegans TPS regions. These regions were used to design degenerated 87366 T36750 Saccharomyces cerevisiae TPS 35991 D22344 Oryza sativa TPS primers which were tested in PCR amplification reactions 57576 D34725 Caenorhabditis elegans TPS using genomic DNA of E. coli and yeast as a template. A 60 298.273 H37578 Arabidopsis thaliana TPS PCR program was used with a temperature ramp between 298.289 H37594 Arabidopsis thaliana TPS 315344 T76390 Arabidopsis thaliana TPS the annealing and elongation step to facilitate annealing of 315675 T76758 Arabidopsis thaliana TPS the degenerate primers. 317475 R65023 Arabidopsis thaliana TPS 71710 D4004.8 Oryza sativa TPS PCR amplification was performed using primer sets TPS 65 4O1677 D67869 Caenorhabditis elegans TPS deg 1/5 and TPSdeg 2/5 using cDNA of Selaginella lepido 322639 TZ13451 Arabidopsis thaliana TPS phylla as a template. US 7,247,770 B2 33 34 EXAMPLE 5 TABLE 6b-continued Isolation of a Bipartite TPS/TPP Gene from Genbank Helianthus Annuus and Nicotiana Tabacum dbEST ID. Accession No. Organism Function 5 76O27 D41954 Oryza sativa Using the sequence information of the TPS gene fragment 3. E. 4.CE being TE from sunflower (Helianthus annuus), a full length sunflower 30O237 T21695 TCE. C TPS clone was obtained using RACE-PCR technology. 3721.19 U37923 Oryza sativa Sequence analysis of this full length clone and alignment 680701 AAOS4930 Brugia malayi 88Se. 10 with TPS2 from yeast (FIG. 6) and TPS and TPP encoding 693476 C12818 Caenorhabditis elegans renalase sequences indicated the isolated clone encodes a TPS/TPP 914O6831.1652 T21173AA273O90 ArabidopsisBrugia malayi thaliana XX88Se. bi1partite enzyme ( SEOQ ID NO 24,s 26 andal 28).). Thee bibipartite 43328 T17578 Saccharomyces cerevisiae TPP clone isolated (pMOG 1192) was deposited at the Central 267495 HO761S Brassica naptis 88Se. Bureau for Strain collections under the rules of the Budapest 317331 R648SS Arabidopsis thaliana 15 treaty with accession number CBS692.97 at Apr. 21, 1997. 367171SOO8 TOO368D23329 CaenorhabditisOryza sativa elegans renalaseXX Subsequently,s we investigated if other plant species also 716SO D39988 Oryza sativa contain TPS/TPP bipartite clones. A bipartite TPS/TPP 147057 D49134 Oryza sativa cDNA was amplified from tobacco. A DNA product of the as: Ris 84 crisis elegans N. expected size (i.e. 1.5 kb) was detected after PCR with 694414 C13756 CE. elegans 88Se. 20 primers TPS degl/TRE-TPP-16 and nested with TPS deg2/ 871371 AA231986 Brugia malayi 88Se. TRE-TPP-15 (SEQID NO:33). An identical band appeared 894468 AA253544 Brugia malayi 88Se. with PCR with TPS degl/TRE-TPP-6 (SEQID NO. 34) and 86985 T36369 Saccharomyces cerevisiae TPP nested with TPS deg2/TRE-TPP-15. The latter fragment was shown to hybridize to the sunflower bipartite cDNA in a 25 Southern blot experiment. Additionally, using computer EXAMPLE 4 database searches, an Arabidopsis bipartite clone was iden tified (SEQ ID NO:39) Isolation of Plant TPS ad TPP Genes from Nicotiana Tabacum EXAMPLE 6 30 Fragments of plant TPS- and TPP-encoding cDNA were Expression of Plant Derived TPS Genes in Plants isolated using PCR on c)NA derived from tobacco leaf total RNA preparations. The column “nested in table 7 indicates Further proof for the function of the TPS genes from if a second round of PCR amplification was necessary with Sunflower and Selaginella lepidophylla was obtained by primer set 3 and 4 to obtain the corresponding DNA frag- 35 isolating their corresponding full-length cDNA clones and ment. Primers have been included in the sequence listing Subsequent expression of these clones in plants under con (table 7). Subcloning and Subsequent sequence analysis of trol of the 35S CaMV promoter. Accumulation of trehalose the DNA fragments obtained with the primer sets mentioned by expression of the Seliganella enzyme has been reported revealed substantial homology to known TPS genes (FIGS. by Zentella and Iturriaga (1996) (Plant Physiol. 111. 4 & 5). Abstract 88). TABLE 7 Amplification of plant derived TPS and TPP cDNAs TPS-cDNA primer 1 primer 2 nested primer 3 primer 4 “825 bp Tre-TPS-14 Deg 1 No SEQ ID. NO SEQ ID NO 30 SEQ ID NO 7 22 & 23 “840 bp Tre-TPS-14 Tre-TPS-12 Yes Tre-TPS-13 Deg 5 SEQ ID NO SEQ ID NO 30 SEQ ID NO 31 SEQ ID NO 32 SEQ ID NO 9 18 & 19 “630 bp Tre-TPS-14 Tre-TPS-12 Yes Deg 2 Deg 5 SEQ ID NO SEQ ID NO 30 SEQ ID NO 31 SEQ ID NO 8 SEQ ID NO 9 20 & 21 TPP-cDNA primer 1 primer 2 nested “723 bp Tre-TPP-5 Tre-TPP-16 No SEQ ID NO 16 & 17 SEQ ID NO 35 SEQ ID NO 38 “543 bp Tre-TPP-7 Tre-TPP-16 No SEQ ID NO 14 SEQ ID NO 36 SEQ ID NO 38 “447 bp Tre-TPP-11 Tre-TPP-16 No SEQ ID NO 12 SEQ ID NO 37 SEQ ID NO 38 US 7,247,770 B2 35 36 EXAMPLE 7 mulation was never detected in any control plants cultured without inhibitor. Similar data were obtained with wild-type Genes Encoding TPS and TPP from Monocot microtubers cultured in the presence of Validamycin A. Ten Species out of seventeen lines accumulated on average 0.001% A computer search in Genbank sequences revealed the trehalose (fw) (table 4). No trehalose was observed in presence of several rice EST-sequences homologous to microtubers which were induced on medium without Val TPS1 and TPS2 from yeast (FIG. 7) which are included in idamycin A. the sequence listing (SEQ ID NO; 41, 51, 52 and 53). 10 EXAMPLE 12 EXAMPLE 8 Trehalose Accumulation in Potato Plants Isolation Human TPS Gene Transgenic for Astrehalase A TPS gene was isolated from human cDNA. A PCR 15 Further proof for the presence of endogenous trehalose reaction was performed on human cDNA using the degen biosynthesis genes was obtained by transforming wild-type erated TPS primers deg2 and deg5. This led to the expected TPS fragment of 0.6 kb. Sequence analysis (SEQID NO.10) potato plants with a 35S CaMV anti-sense trehalase con and comparison with the TPSyeast sequence indicated that struct (SEQ ID NO:54 and 55, pMOG1027; described in isolated sequence encodes a homologous TPS protein (FIG. WO 96/21030). A potato shoot transgenic for pMOG1027 showed to accumulate trehalose up to 0.008% on a fresh 8). weight basis. The identity of the trehalose peak observed EXAMPLE 9 was confirmed by specificly breaking down the accumulated trehalose with the enzyme trehalase. Tubers of some Inhibition of Endogenous TPS Expression by 25 pMOG1027 transgenic lines showed to accumulate small Anti-Sense Inhibition amounts of trehalose (FIG. 9). The expression of endogenous TPS genes can be inhibited EXAMPLE 13 by the anti-sense expression of a homologous TPS gene under control of promoter sequences which drive the expres 30 Inhibition of Plant Hexokinase Activity by sion of such an anti-sense TPS gene in cells or tissue where Trehalose-6-Phosphate the inhibition is desired. For this approach, it is preferred to use a fully identical sequence to the TPS gene which has to To demonstrate the regulatory effect of trehalose-6-phos be suppressed although it is not necessary to express the phate on hexokinase activity, plant extracts were prepared entire coding region in an anti-sense expression vector. 35 Fragments of Such a coding region have also shown to be and tested for hexokinase activity in the absence and pres functional in the anti-sense inhibition of gene-expression. ence of trehalose-6-phosphate. Alternatively, heterologous genes can be used for the anti Potato tuber extracts were assayed using fructose (FIG. sense approach when these are Sufficiently homologous to 10, FIG. 11) and glucose (FIG. 11) as substrate. The potato the endogenous gene. 40 tuber assay using 1 mMT-6-P and fructose as substrate was Binary vectors similar to pMOG845 and pMOG1010 can performed according to Gancedo et al. (1997) J. Biol. Chem. be used ensuring that the coding regions of the introduced 252, 4443. The following assays on tobacco, rice and maize genes which are to be suppressed are introduced in the were performed according to the assay described in the reverse orientation. All promoters which are suitable to drive section experimental. expression of genes in target tissues are also suitable for the 45 Tobacco leaf extracts were assayed using fructose (FIG. anti-sense expression of genes. 12) and glucose (FIG. 12, FIG. 13) as substrate. EXAMPLE 10 Rice leaf extracts were assayed using fructose and glucose (FIG. 14) as substrate. Inhibition of Endogenous TPP Expression by 50 Maize leaf extracts were assayed using fructose and Anti-Sense Inhibition glucose (FIG. 15) as substrate. Similar to the construction of vectors which can be used EXAMPLE 1.4 to drive anti-sense expression of tps in cells and tissues (Example 9), vectors can be constructed which drive the 55 Inhibition of Hexokinase Activity in Animal Cell anti-sense expression of TPP genes. Cultures by Trehalose-6-Phosphate EXAMPLE 11 To demonstrate the regulation of hexokinase activity in Trehalose Accumulation in Wild-Type Tobacco and 60 animal cells, total cell extracts were prepared from mouse Potato Plants Grown on Validamycin A hybridoma cell cultures. A hexokinase assay was performed using glucose or fructose as Substrate under conditions as Evidence for the presence of a trehalose biosynthesis described by Gancedo et al. (see above). Mouse hybridoma pathway in tobacco was obtained by culturing wild-type cells were subjected to osmotic shock by exposing a cell plants in the presence of 10M of the trehalase inhibitor 65 pellet to 20% sucrose, followed by distilled water. This Validamycin A. The treated plants accumulated very small crude protein extract was used in the hexokinase assay (50 amounts of trehalose, up to 0.0021% (fw). Trehalose accu ul extract corresponding to ca.200 ug protein). US 7,247,770 B2 37 38 TABLE 8 TABLE 9-continued Inhibition of animal hexokinase activity by T-6-P Rate of photosynthesis and respiration, STD is standard deviation Concen- 5 tration T6P Vo V Inhibition 350 ppm 950 ppm Substrate (mM) (mM) (ODU/min) (ODU/min) (%) micromol/m’s STD micromol/m/s STD Glucose 2 O.83 O.O2O4 O.O133 35 Glucose 2O O.83 O.O214 O.O141 35 O10-5 Glucose 1OO O.83 O.O188 O.O12S 34 10 Fructose 2O O.23 O.O2O7 O.O2OS 1 RD O.873 O.O60 1.014 O.134 Fructose 2O O.43 O.O267 O.O197 26 EFF O.059 O.OO2 O.O90 O.007 Fructose 2O O.83 O.O234 O.O151 35 AMAX 12.083 1546 18.651 1941 Fructose 2O 1.67 O.O246 O.O133 46 318-10 15 RD O.974 O.O76 1.078 O.108 The data obtained clearly showed that hexokinase activity EFF O.064 O.OO3 O.O88 O.OO8 in mouse cell extracts is inhibited- - - - - by trehalose-6-phosphate. AMAX318-37 16.261 2.538 24.154 1854 The T-6-P concentration range in which this effect is noted is comparable to what has been observed in crude plant RD E. S. 8. 9. extracts. No difference is noted in the efficiency of hexoki- 20 XMAx 16.sis 2.36s 25.174 2093 nase inhibition by trehalose-6-phosphate using glucose or Lower leaf fructose as substrate for the enzyme. Wild-type RD O.0438 O.O79 O.S26 O. 112 EXAMPLE 1.5 EFF O.O68 O.OO2 O.O85 O.004 25 AMAX 6.529 1.271 11489 1841 Photosynthesis and Respiration of TPS and TPP O10-5 Expressing Tobacco Plants RD O.4S5 O.O68 O.S62 O.118 EFF O.064 O.OO2 O.O85 O.OO6 Using tobacco plants transgenic for 35S-TPP (1010-5), so AMAXso 8.527 0.770 13.181 1.038 PC-TPS (1318-10 and 1318-37) and wild-type Samsun NN plants, effects of expression of these genes on photosynthe- RD C. 8. 9. 9. sis and respiration were determined in leaves. AMAX 11.562 1778 20.031 1826 Measurements were performed in a gas exchange-experi- 318-37 mental set-up. Velocities of gas-exchange were calculated 35 RD 0.767 O.O33 O.918 O.O99 on the basis of differences in concentration between ingoing EFF O.O73 O.OO6 O.103 O.004 and outgoing air using infra-red gas-analytical equipment. AMAX 13.467 1818 19.587 1681 Photosynthesis and respiration were measured from identi cal leaves. From each transgenic plant, the youngest, fully matured leaf was used (upper-leaf) and a leaf that was 3-4 40 TABLE 10 leaf-"stores' lower (lower-leaf). Photosynthesis was measured as a function of the photo- Respiration during 12 hour dark period (mmol CO) synthetic active light intensity (PAR) from 0-975 umol.m STD is standard deviation 2.s' (200 Watt m'), in four-fold at CO-concentrations of 45 Upper leaf STD Lower leaf STD 350 vpm and 950 vpm. Wild-type 25.17 O.82 13.19 1.98 Respiration was measured using two different time-scales. 1010-5 30.29 S.09 13.08 1.52 Measurements performed during a short dark-period after 1318-10 28.37 4SO 20.47 O.87 the photosynthesis experiments are coded RD in table 9. 1318-37 32.53 2.01 17.7 1.03 These values reflect instantaneous activity since respiration so varies substantially during the dark-period. Therefor, the In contrast to the respiration in the upper-leaves, in lower values for the entire night-period were also summed as leaves the respiration of TPS transgenic plants is signifi shown in table 10 (only measured at 350 vpm CO). cantly higher than for wild-type and TPP plants (table 10) indicating a higher metabolic activity. The decline in respi TABLE 9 55 ration during aging of the leaves is significantly less for TPS Rate of photosynthesis and irati transgenic plants. e Pry e 1On, Also, the photosynthetic characteristics differed signifi cantly between on the one hand TPS transgenic plants and 350 ppm 950 ppm on the other hand TPP transgenic and wild-type control l/m/ STD l/m/ STD 60 plants. The AMAX values (maximum of photosynthesis at COOLS COOLS light saturation), efficiency of photosynthesis (EFF) and the Upper leaf respiration velocity during a short dark-period after the Wild-type photosynthetic measurements (RD) are shown in table 9. On RD O.O826 O.048 1.016 O.142 average, the upper TPS leaves had a 35%0A, 1.higher AMAX EFF O.O60 O.OO)4 O.O87 O.OO)4 65 value compared to the TPP and wild-type leaves. The lower AMAX 11.596 O.S88 19:215 O.942 leaves show even a higher increased rate of photosynthesis (88%). US 7,247,770 B2 39 40 To exclude that differences in light-absorption were caus EXAMPLE 17 ing the different photosynthetic rates, absorption values were measured with a SPAD-502 (Minolta). No significant Export and Allocation of Assimilates in TPS and differences in absorption were measured (table 11). RPP Expressing Tobacco Plants Using tobacco plants transgenic for 35S-TPP (1010-5) TABLE 11 and PC-TPS (1318-37), 1) the export of carbon-assimilates from a fully grown leaf Absorbtion values of transgenic lines (indicating “relative source activity”, Koch (1996) Annu. 10 Rev. Plant Physiol. Plant. Mol. Biol. 47, 509 and Absorbtion (%) Upper-leaf Lower-leaf 2) the net accumulation of photo-assimilates in sinks (“rela tive sink activity'), during a light and a dark-period, were Wild-type Samson NN 84 83 determined. 1010-5 84 82 15 Developmental stage of the plants: flowerbuds just vis 1318-10 85 86 ible. Labelling technique used: Steady-state high abundance 1318-37 86 86 13C-labelling of photosynthetic products (De Visser et al. (1997) Plant Cell Environ 20, 37). Of both genotypes, 8 plants, using a fully grown leaf, were labelled with 5.1 atom % CO, during a light-period (10 hours), when appropriate EXAMPLE 16 followed by a dark-period (14 hours). After labelling, plants were split in: 1) shoot-tip, 2) young growing leaf. 3) young Chlorophyll-Fluorescence of TPS and TPP fully developed leaf (above the leaf being labelled), 4) Expressing Tobacco Plants young stem (above the leaf being labelled), 5) labelled leaf, 25 6) petiole and base of labelled leaf, 7) old, senescing leaf. 8) other and oldest leaves lower than the labelled leaf. 9) stem Using tobacco plants transgenic for 35S-TPP (1010-5), lower than the labelled leaf. 10) root-tips. Number, fresh and PC-TPS (1318-10 and 1318-37) and wild-type Samsun NN dry weight and 'C percentage (atom% 'C) of carbon were plants, effects of expressing these genes were determined on determined. Next to general parameters as biomass, dry chlorophyll fluorescence of leaf material. Two characteris 30 matter and number of leaves, calculated were: 1) Export of tics of fluorescence were measured: C out of the labelled leaf; 2) the relative contribution of imported C in plant parts; 3) the absolute amount of 1) ETE (electron transport efficiency), as a measure for the imported C in plant parts; 4) the relative distribution of electron transport Velocity and the generation of reducing imported C during a light period and a complete light and power, and 35 dark-period. The biomass above soil of the TPP transgenics was 27% 2) Non-photochemical quenching, a measure for energy larger compared to the TPS transgenics (P<0.001); also the dissipation caused by the accumulation of assimilates. root-system of the TPP transgenics were better developed. Plants were grown in a greenhouse with additional light of The TPP plants revealed a significant altered dry matter 40 distribution, +39% leaf and +10% stem biomass compared 100 umol. m°.s' (04:00-20:00 hours). Day/night T=21° to TPS plants. TPS plants had a larger number of leaves, but C./18° C. R.H.75%. During a night-period preceding the a smaller leaf-area per leaf. Total leafarea per TPS plant was measurements (duration 16 hours), two plants of each geno comparable with wild-type (0.4 m plant-') type were transferred to the dark and two plants to the light Relative Source Activity of a Fully Developed Leaf (+430 umol m°.s", 20° C., R.H.70%). The youngest fully 45 The net export rate of photosynthates out of the labelled matured leaf was measured. The photochemical efficiency of leaf is determined by the relative decrease of the '% “new C. PSII (photosystem II) and the “non-photochemical quench during the night (for TPP 39% and for TPS 56%) and by the ing parameters were determined as a function of increasing, total fixated amount present in the plant using the amount of light intensity. At each light intensity, a 300 sec. stabilisation “new C' in the plant (without the labelled leaf) as a measure. time was taken. Measurements were performed at 5, 38,236, 50 After a light period, TPP leaves exported 37% compared to 51% for TPS leaves (table 11). In a following dark-period, 422 and 784 umol m.s' PAR with a frequency of 3 this percentage increased to respectively 52% and 81%. light-flashes min-', 350 ppm CO, and 20% O. Experiments Both methods support the conclusion that TPS transgenic were replicated using identical plants, reversing the pretreat plants have a significantly enhanced export rate of photo ment from dark to light and vice versa. The fluorescence 55 synthetic products compared to the TPP transgenic plants. characteristics are depicted in FIG. 16. Absolute Amount of “New C, in Plant Parts The decrease in electron-transport efficiency (ETE) was Export by TPS transgenics was significantly higher com comparable between TPP and wild-type plants. TPS plants pared to TPP transgenics. Young growing TPS leaves import clearly responded less to a increase of light intensity. This C stronger compared to young growing TPP leaves. 60 Relative Increase of “New C' in Plant Parts, Sink difference was most clear in the light pretreatment. These Strength observations are in agreement with the “non-photochemi The relative contribution of “new C to the concerning cal quenching data. TPS plants clearly responded less to the plant part is depicted in FIG. 17. This percentage is a additional supply of assimilates by light compared to TPP measure for the sink-strength. A significant higher sink and wild-type plants. In the case of TPS plants, the negative 65 strength was present in the TPS transgenics, especially in the regulation of accumulating assimilates on photosynthesis shoot-top, the stem above and beneath the labelled leaf and was significantly reduced. the petiole of the labelled leaf. US 7,247,770 B2 41 42 (FIG. 21). Taproot diameter was determined for all plants TABLE 11 after ca. 8 weeks of growth in the greenhouse. Some PC-TPS transgenic lines having a leaf size similar to the control Source activity of a full grown labelled leaf: C accumulation and export. Nett daily accumulation and export plants showed a significant larger diameter of the tap-root of C-assimilates in labelled leaf and the whole plant (above (FIG. 22). PC-TPP transgenic lines formed a smaller taproot soil) after steady-state 13-labelling during a light period compared to the non-transgenic controls. Leaf extracts of (day). N = 4: LSD values indicated the Smallest significant transgenic Sugarbeet lines were analyzed for Sugars and differences for P < 0.05 starch (FIGS. 20 A-D). Source activity grown leaf 10 EXAMPLE 20 nett C new C in new C in export Arabidopsis source leaf nett C export source leaf to plant Time (% of total during night (% of new C (% of total (end of) Transgene C in leaf) % of “Day' in plant new C) Performance of Arabidopsis Plants Transgenic for 15 PC-TPS and PC-TPP Day TPS 17.8 48.7 51 TPP 22.6 63.0 37 Day + TPS 7.8 56 16.6 81 Constructs used in Arabidopsis transformation experi Night TPP 13.8 39 48.4 52 ments: PC-TPS and PC-TPP. The phenotypes of both TPS LSD. O.OS 2.4 6.1 and TPP transgenic plants were clearly distinguishable from wild-type controls: TPS transgenic plants had thick, dark Relative Distribution, Within the Plant, of “New C. green leaves and TPP transgenic plants had larger, bleaching Between the Plant Parts: Relative Sink Strength leaves when compared to wild-type plants. Plants with high The distribution of fixed carbon between plant organs levels of TPP expression did not set seed. (FIG. 18) confirmed the above mentioned conclusions. TPS transgenic plants revealed a relative large export of assimi 25 EXAMPLE 21 lates to the shoot-top, the young growing leaf (day) and even the oldest leaf (without axillary meristems), and to the young Potato and old stem. Performance of Solanum Tuberosum Plants EXAMPLE 1.8 30 Transgenic for TPS and TPP Constructs Lettuce Construct: 35S-TPS pMOG799 Plants transgenic for pMOG799 were grown in the green Performance of Lettuce Plants Transgenic for house and tuber-yield was determined (FIG. 23). The major PC-TPS and PC-TPP 35 ity of the transgenic plants showed Smaller leaf sizes when compared to wild-type controls. Plants with smaller leaf Constructs used in lettuce transformation experiments: sizes yielded less tuber-mass compared to control lines (FIG. PC-TPS and PC-TPP, PC-TPS transgenics were rescued 25). during regeneration by culturing explants on 60 g/l Sucrose. Construct: 35S-TPP pMOG1010 and PC-TPP The phenotypes of both TPS and TPP transgenic plants are 40 pMOG1124 clearly distinguishable from wild-type controls: TPS trans Plants transgenic for PMOG 1010 and pMOG1124 were genic plants have thick, dark-green leaves and TPP trans grown in the greenhouse and tuber-yield was determined. genic plants have light-green leaves with a Smoother leaf Tuber-yield (FIG. 24) was comparable or less than the edge when compared to wild-type plants. wild-type control lines (FIG. 25). The morphology of the leaves, and most prominent the 45 Construct: PC-TPS pMOG1093 leaf-edges, was clearly affected by the expression of TPS Plants transgenic for pMOG1093 were grown in the and TPP. Leaves transgenic for PC-TPS were far more greenhouse and tuber-yield was determined. A number of “notched than the PC-TPP transgenic leaves that had a transgenic lines having leaves with a size comparable to more smooth and round morphology (FIG. 19). Leaf extracts wild-type (B-C) and that were slightly darker green in colour of transgenic lettuce lines were analyzed for Sugars and 50 yielded more tuber-mass compared to control plants (FIG. starch (FIGS. 20 A-D). 26). Plants with leaf sizes smaller (D-G) than control plants yielded less tuber-mass. EXAMPLE 19 Construct: Pat-TPP pMOG1128 Microtubers were induced in vitro on explants of pat-TPP Sugarbeet 55 transgenic plants. The average fresh weight biomass of the microtubers formed was substantially lower compared to the Performance of Sugarbeet Plants Transgenic for control lines. PC-TPS and PC-TPP Construct: Pat-TPS pMOG845 Plants transgenic for pMOG 845 were grown in the Constructs used in Sugarbeet transformation experiments: 60 greenhouse and tuber-yield was determined. Three Pat-TPS PC-TPS and PC-TPP. Transformation frequencies obtained lines produced more tuber-mass compared to control lines with both the TPS and the TPP construct were comparable (FIG. 27). to controls. The phenotypes of both TPS and TPP transgenic Construct: PC TPS Pat TPS; pMOG 1129(845-11/22/28) plants were clearly distinguishable from wild-type controls; Plants expressing PC TPS and Pat-TPS simultaneously TPS transgenic plants had thick, dark-green leaves and TPP 65 were generated by retransforming Pat-TPS lines (resistant transgenic plants had light-green coloured leaves with against kanamycin) with construct pMOG1129, harbouring slightly taller petioles when compared to wild-type plants a PC TPS construct and a hygromycin resistance marker US 7,247,770 B2 43 44 gene, resulting in genotypes pMOG1129(845-11), pMOG1129(845-22) and pMOG1129(845-28). Tuber-mass TABLE 12-continued yield varied between almost no yield up to yield comparable or higher then control plants (FIGS. 28 A-C). Germination of transgenic 35S-TPP seeds Rel. EXAMPLE 22 Seedlot Bleaching (TPPmRNA) Germination Tobacco 1010-22 -- 8.8 not 1010-23 4.5 normal 1010-24 10.2 delayed Performance of A. Tabacum Plants Transgenic for 10 1010-25 4.7 delayed (less) tPS and TPP Constructs 1010-27 4.8 normal 1010-28 -- 22.1 delayed 101O-31 -- 9.4 delayed (less) Root System 101O-32 O.3 delayed (less) Tobacco plants transgenic for 35S TPP (pMOG1010) or 101O-33 -- 14.7 delayed 35S TPS (pMOG799) were grown in the greenhouse. Root 15 size was determined just before flowering. Lines transgenic for pMOG1010 revealed a significantly smaller/larger root Influence of Expressing TPS and/or TPP on Seed Yield size compared to pMOG795 and non-transgenic wild-type Seed-yield was determined for S1 plants transgenic for tobacco plants. pMOG1010-5. On average, pMOG1010-5 yielded 4.9 g, Influence of Expressing TPS and/or TPP on Flowering seed/plant (n=8) compared to 7.8 g. seed/plant (n=8) for Tobacco plants transgenic for 35S-TPS, PC-TPS, 35S wild-type plants. The “1000-grain' weight is 0.06 g for line TPP or PC-TPP were cultured in the greenhouse. Plants pMOG1010-5 compared to 0.08 g for wild-type Samsun expressing high levels of the TPS gene revealed significantly NN. These data can be explained by a reduced export of slower growth rates compared to wild-type plants. Flower 25 carbohydrates from the Source leaves, leading to poor devel ing and senescence of the lower leaves was delayed in these plants resulting in a stay-green phenotype of the normally opment of seed "sink’ tissue. senescing leaves. Plants expressing high levels of the TPP Influence of TPS and TPP expression on leaf morpholo gene did not make any flowers or made aberrant, not fully gySegments of greenhouse grown PC-TPS transgenic, PC developing flower buds resulting in sterility. 30 TPP transgenic and non-transgenic control tobacco leaves Influence of Expressing TPS and/or TPP on Seed Setting were fixed, embedded in plastic and coupes were prepared Tobacco plants transgenic for 35S-TPS, PC-TPS, 35S to study cell structures using light-microscopy. Cell struc TPP or PC-TPP were cultured in the greenhouse. Plants tures and morphology of cross-sections of the PC-TPP expressing high levels of the TPP gene revealed poor or no 35 transgenic plants were comparable to those observed in development of flowers and absence of seed-setting. control plants. Cross-sections of PC-TPS transgenics Influence of Expressing TPS and/or TPP on Seed Germi revealed that the Spongy parenchyme cell-layer constituted nation of 7 layers of cells compared to 3 layers in wild-type and Tobacco plants transgenic for 35STPP (pMOG11010) or 40 TPP transgenic plants FIGS. 29A and B. This finding agrees PC TPP were grown in the greenhouse. Some of the trans with our observation that TPS transgenic plant lines form genic lines, having low expression levels of the transgene, thicker and more rigid leaves compared to TPP and control did flower and set seed. Upon germination of S1 seed, a plants. significantly reduced germination frequency was observed (or germination was absent) compared to S1 seed derived 45 EXAMPLE 23 from wild-type plants (table 12). Inhibition of Cold-Sweetening by the Expression of TABLE 12 Trehalose Phosphate Synthase 50 Sermination of transgenic 35S-TPP seeds Transgenic potato plants (Solanum tuberosum cv. Kardal) Rel. were generated harbouring the TPS gene under control of the Seedlot Bleaching (TPPmRNA) Germination potato tuber-specific patatin promoter (pMOG845; Example O1O-2 -- 15.8 delayed 1). Transgenic plants and wild-type control plants were O1O-3 5.3 delayed 55 O1O-4 -- 4.2 delayed grown in the greenhouse and tubers were harvested. O10-5 -- 5.2 delayed Samples of tuber material were taken for Sugar analysis O1O-6 -- 3.9 delayed O1 O-7 2.8 delayed directly after harvesting and after 6 months of storage at 4 O10-8 -- 6.5 delayed C. Data resulting from the HPLC-PED analysis are depicted O1O-9 -- 4.6 delayed in FIG. 30. O1O-10 1.9 normal 60 O10-11 5.7 normal What is clearly shown is that potato plants transgenic for O1O-12 -- 1.4 normal O1O-14 O.1 normal TPS have a lower amount of total Sugar (glucose., O10-15 O.3 normal fructose and Sucrose) accumulating in tubers directly after O1O-18 -- S.6 delayed O1O-2O -- 6.4 delayed 65 harvesting. After a storage period of 6 months at 4°C., the O1O-21 -- 9.5 delayed increase in Soluble Sugars is significantly less in the trans genic lines compared to the wild-type control lines. US 7,247,770 B2 45 46 EXAMPLE 24 day (32 d after the onset of treatment for the wildtype plants), as harvesting the transgenic plants at a later stage Improved Performance of 358 TPS 358 TPP would complicate the comparison of the plant lines. At the (pMOG851) Transgenic Tobacco Plants Under Drought Stress time of harvest the total leaf area was measured using a Delta-T Devices leaf area meter (Santa Clara, Calif.). In Transgenic tobacco plants were engineered harbouring addition, the fresh weight and dry weight of the leaves, both the TPS and TPP gene from E. coli under control of the stems and roots was determined. 35S CaMV promoter. The expression of the TPS and TPP A second experiment was done essentially in the same genes was verified in the lines obtained using Northern blot 10 way, to analyze the osmotic potential of the plants. After 35 and enzyme activity measurements. pMOG851-2 was days of drought stress, samples from the youngest mature shown to accumulate 0.008 mg trehalose.g' fow and leaves were taken at the beginning of the light period (n-3). pMOGB51-5 accumulated 0.09 mg trehalose.g' fow. Air-Drying of Detached Leaves Expression of both genes had a pronounced effect on plant 15 morphology and growth performance under drought stress. The water loss from air-dried detached leaves was mea When grown under drought stress imposed by limiting water sured from well-watered, four-week old pMOG851-2, supply, the two transgenic tobacco lines tested, pMOGS51-2 pMOG851-5 and wildtype plants. Per plant line, five plants and pMOG851-5, yielded total dry weights that were 28% were used, and from each plant the two youngest mature (P<0.01) and 39% (P<0.001) higher than those of wild-type leaves were detached and airdried at 25% relative humidity. tobacco. These increases in dry weight were due mainly to The fresh weight of each leaf was measured over 32 hours. increased leaf production: leaf dry weights were up to 85% At the time of the experiment samples were taken from higher for pMOG851-5 transgenic plants. No significant comparable, well-watered leaves, for osmotic potential mea differences were observed under well-watered conditions. 25 Surements and determination of Soluble Sugar contents. Drought Stress Experiments Osmotic Potential Measurements F1 seeds obtained from self-fertilization of primary trans Leaf samples for osmotic potential analysis were imme formants pMOG851-2 and pMOG851-5 (Goddijn et al. diately stored in capped 1 ml syringes and frozen on dry ice. (1997) Plant Physiol. 113, 181) were used in this study. 30 Just before analysis the leaf sap was Squeezed into a small Seeds were sterilized for 10 minutes in 20% household vial, mixed, and used to saturate a paper disc. The osmotic bleach, rinsed five times in sterile water, and sown on potential was then determined in Wescor C52 chambers, half-strength Murashige and Skoog medium containing 10 using a Wescor HR-33T dew point microvolt meter. g.L. Sucrose and 100 mg.L. kanamycin. Wildtype SR1 Chlorophyl Fluorescence seeds were sown on plates without kanamycin. After two 35 weeks seedlings from all lines were transferred to soil Chlorophyll fluorescence of the wildtype, pMOG851-2 (Sandy loam), and grown in a growth chamber at 22°C. at and pMOG851-5 plants was measured for each plant line approximately 100 uE.m light intensity, 14h.d. All plants after 20 days of drought treatment, using a pulse modulation were grown in equal amounts of soil, in 3.8 liter pots. The (PAM) fluorometer (Walz, Effeltrich, Germany). Before the plants were watered daily with half-strength Hoaglands 40 measurements, the plants were kept in the dark for two nutrient solution. The seedlings of pMOG851-2 and hours, followed by a one-hour light period. Subsequently, pMOG851-5 grew somewhat slower than the wildtype seed the youngest mature leaf was dark-adapted for 20 minutes. lings. Since we considered it most important to start the At the beginning of each measurement, a small (0.05 umol experiments at equal developmental stage, we initiated the 45 m’s' modulated at 1.6 KHz) measuring light beam was drought stress treatments of each line when the seedlings turned on, and the minimal fluorescence level (Fo) was were at equal height (10 cm), at an equal developmental measured. The maximal fluorescence level (F) was then stage (4-leaves), and at equal dry weight (as measured from measured by applying a: Saturation light pulse of 4000 umol two additional plants of each line). This meant that the onset m’s', 800 ms in duration. After another 20s, when the of pMOG851-2 treatment was two days later than wildtype, 50 signal was relaxed to near Fo, brief saturating pulses of and that of pMOG851-5 seven days later than wildtype. actinic light (800 ms in length, 4000 umol m°s') were From each line, six plants were subjected to drought stress, given repetitively for 30 s with 2 s dark intervals. The while four were kept under well-watered conditions as photochemical (q) and non-photochemical (q) quenching controls. The wildtype tobacco plants were droughted by 55 components were determined from the fluorescence/time maintaining them around the wilting point: when the lower curve according to Bolhar-Nordenkampf and Oquist (1993). half of the leaves were wilted, the plants were given so much At the moment of measurement, the leaves in question were nutrient solution that the plants temporarily regained turgor. not visibly wilted. Statistical data were obtained by one-way In practice, this meant Supplying 50 ml of nutrient Solution analysis of variance using the program Number Cruncher every three days; the control plants were watered daily to 60 Statistical System (Dr. J. L. Hintze, 865 East 400 North, keep them at field capacity. The pMOG851-2 and Kaysville, Utah 84.037, USA). pMOG851-5 plants were then watered in the exact same Chlorophyll fluorescence analysis of drought-stressed way as wildtype, i.e., they were Supplied with equal amounts plants showed a higher photochemical quenching (q) and a of nutrient Solution and after equal time intervals as wild 65 higher ratio of variable fluorescence over maximal fluores type. The stem height was measured regularly during the cence (F/F) in pMOG851-5, indicating a more efficiently entire study period. All plants were harvested on the same working photosynthetic machinery (table 13). US 7,247,770 B2 47 48 TABLE 13 Chlorophyll fluorescence parameters of wild-type (wt) and trehalose-accumulating (pMOG851-2, pMOG851-5) transgenic tobacco plants. P (probability) values were obtained from ANOVA tests analyzing differences per plant line between plants grown under well-watered (control) or dry conditions, as well as differences between each of the transgenic lines and WT, grown under well-watered or dry conditions. F: maximal fluorescence; F: variable fluorescence (F-Fo): qo: photochemical quenching: qE: non-photochemical quenching. F, F are expressed in arbitrary units (chart mm). WT pMOG851-1 pMOG851-5 8-51-2/WT 815-S Fm control 1744 1804 175.6 S dry 151.5 155.7 167.8 S P (ctrl.dry) OOOO4 OOOOO S F. control 134.6 143.3 142.8 S dry 118.4 1221 135.6 S P (ctrl.dry) O.OO6 OOOOO S F/F control 0.771 O.794 O.813 O.O59 dry O.782 O.784 O.809 S P (ctrl.dry) S S S CE control 15.2 23.8 29.9 O.259 dry 25.4 21.6 23.5 S P (ctrl.dry) O.048 S S 9Q control 91.3 92.4 90.4 S S dry 73.69 78.5 92.75 S O.OOOS P (ctrl.dry) O.OOS O.OO6 S Carbohydrate Analysis 30 after 32 h leaves of pMOG851-5 and pMOG851-2 had 44% At the time of harvest, pMOG851-5 plants contained 0.2 and 41% of their fresh weight left, respectively, compared to mgg dry weight trehalose, whereas in pMOG851-2 and 30% for wildtype. At the time of the experiment samples wildtype the trehalose levels were below the detection limit, were taken from comparable, well-watered leaves for under both stressed and unstressed conditions. The trehalose osmotic potential determination and analysis of trehalose, content in pMOG851-5 plants was comparable in stressed 35 Sucrose, glucose and fructose. The two transgenic lines had and unstressed plants (0.19 and 0.20 mg.g' dry weight, lower osmotic potentials than wildtype (P<0.05), with respectively). Under well-watered conditions, the levels of pMOG851-5 having the lowest water potential (-0.63+0.03 glucose and fructose were twofold higher in pMOG851-5 Mpa), wildtype the highest (-0.51+0.02 Mpa) and plants than in wildtype. Leaves of stressed pMOG851-5 pMOG851-2 intermediate (-0.57+0.04 Mpa). The levels of plants contained about threefold higher levels of each of the 40 all Sugars tested were significantly higher in leaves of four nonstructural carbohydrates starch, Sucrose, glucose pMOG851-5 plants than for wildtype leaves resulting in a and fructose, than leaves of stressed wildtype plants. In threefold higher level of the four sugars combined pMOG851-2 leaves, carbohydrate levels, like chlorophyll (P=0.002). pMOG851-2 plants contained twofold higher fluorescence values, did not differ significantly from those in levels of the four sugars combined (P=0.09). The trehalose wildtype. Stressed plants of all lines contained increased 45 levels were 0.24+0.02 mg.g. DW in pMOG851-5 plants, levels of glucose and fructose compared to unstressed and below detection in pMOG851-2 and wildtype. plants. EXAMPLE 25 Osmotic Potential of Drought Stressed and Control Plants During a second, similar experiment under greenhouse Performance of TPS and TPP Transgenic Lettuce conditions, the transgenic plants showed the same pheno 50 Plant Lines Under Drought Stress types as described above, and again the pMOG851-5 plants showed much less reduction in growth under drought stress Primary TPS and TPP transformants and wild-type con than pMOG851-2 and wildtype plants. The osmotic poten trol plants were subjected to drought-stress. Lines transgenic tial in leaves of droughted pMOG851-5 plants (-1.77+0.39 for TPP reached their wilting point first, then control plants, Mpa) was significantly lower (P=0.017) than in wildtype 55 followed by TPS transgenic plants indicating that TPS leaves (-1,00+0.08 Mpa); pMOG851-2 showed intermedi transgenic lines, as observed in other plant species, have a ate values (-1.12+0.05 Mpa). Similarly, under well-watered clear advantage over the TPP and wild-type plants during conditions the osmotic potential of pMOG851-5 plants drought stress. (-0.79+0.05 Mpa) was significantly lower (P=0.038) than 60 EXAMPLE 26 that of wildtype leaves (-0.62+0.03 Mpa), with pMOG851-2 having intermediate values (-0.70+0.01 Mpa). Bolting of Lettuce Plants is Affected in Plants Airdrying of Detached Leaves Transgenic for PC-TPS or PC-TPP Leaves of pMOG851-2, pMOG851-5 and wildtype were detached and their fresh weight was measured over 32 hours 65 Bolting of lettuce is reduced in plants transgenic for of airdrying. Leaves of pMOG851-2 and pMOG851-5 plants PC-TPP (table 14). Plant lines transgenic for PC-TPS show lost significantly less water (P<0.05) than wildtype leaves: enhanced bolting compared to wild-type lettuce plants. US 7,247,770 B2 49 50 (pMOG845-11/22/28). The fresh weight biomass of micro TABLE 1.4 tubers obtained from lines transgenic for pMOG1027 only was slightly higher then wild-type control plants. Resulting Bolting of lettuce plants plants were grown in the greenhouse and tuber yield was 5. Com 5 determined (FIG. 33). Lines transgenic for 35S as-trehalase Total 1. 2. 3. 4. pletely or a combination of 35S as-trehalase and pat-TPS yielded PC-TPP # of Normal Reduced Visible Possible vege significantly more tuber-mass compared to control lines. lines plants bolting bolting inflorescence fasciation tative Starch determination revealed no difference in starch content 1A 4 4 of tubers produced by plant lines having a higher yield (FIG. 2A 3 1 2 10 34). A large number of the 1027(845-11/22/28) lines pro 3A 2 2 4A 5 1 1 1 2 duced tubers above the soil out of the axillary buds of the SA 5 1 1 3 leaves indicating a profound influence of the constructs used 7A 1 1 on plant development. Plant lines transgenic for 35S as 8A 5 4 1 trehalase only did not form tubers above the soil. 9A 5 5 15 10A 3 1 2 Constructs: Pat as-trehalase (pMOG1028) and Pat as 11A 5 2 3 trehalase Pat TPS (pMOG1028(845-11/22/28)) 12A 4 4 Plants expressing Pat as-trehalase and Pat-TPS simulta Control 5 5 neously were generated by retransforning Pat-TPS lines (resistant against kanamycin) with construct pMOG1028, harbouring the Pat as-trehalase construct and a hygromycin resistance marker gene, resulting in genotypes pMOG1028 EXAMPLE 27 (845-11), pMOG1028(845-22) and pMOG1028(845-28). Performance of Tomato Plants Transgenic for TPS Plants were grown in the greenhouse and tuber yield was and TPP determined (FIGS. 35A-E). A number of pMOG1028 trans 25 genic lines yielded significantly more tuber-mass compared to control lines. Individual plants transgenic for both Pat Constructs used in tomato transformation experiments: TPS and Patas-trehalase revealed a varying tuber-yield from 35S TPP, PC-TPS, PC-TPS as-trehalase, PC-TPP, E8-TPS, almost no yield up to a yield comparable to or higher then E8-TPP, E8TPS E8as-trehalase. Plants transgenic for the the control-lines (FIGS. 35 A-E). TPP gene driven by the plastocyanin promoter and 35S Construct: PC as-trehalase (pMOG1092) promoter revealed phenotypes similar to those observed in Plants transgenic for pMOG1092 were grown in the other plants: bleaching of leaves, reduced formation of greenhouse and tuber-yield was determined. Several lines flowers or absent flower formation leading to small fruits or formed darker-green leaves compared to controls. Tuber absence of fruits. A small number of 35S-TPP transgenic yield was significantly enhanced compared to non-trans lines generated extreme large fruits. Those fruits revealed 35 genic plants (FIG. 36). enhanced outgrow of the pericarp. Plants transgenic for the Construct: PC as-trehalase PC-TPS (PMOG 1130) TPS gene driven by the plastocyanin promoter and 35S Plants transgenic for pMOG 1130 were grown in the promoter did not form Small lancet shaped leaves. Some greenhouse and tuber-yield was determined. Several trans severely stunted plants did form Small dark-green leaves. genic lines developed Small dark-green leaves and severely Plants transgenic for PC-TPS and PC-as-trehalase did form 40 stunted growth indicating that the phenotypic effects Smaller and darker green leaves as compared to control observed when plants are transformed with TPS is more plants. severe when the as-trehalase gene is expressed simulta The colour and leaf-edge of the 35S or PC driven TPS and neously (see Example 21). Tuber-mass yield varied between TPP transgenic plants were clearly distinguishable similar to almost no yield up to significantly more yield compared to what is observed in other crops. 45 control plants (FIG. 37). Plants harbouring the TPS and TPP gene under control of the fruit-specific E8 promoter did not show any phenotypical EXAMPLE 29 differences compared to wild-type fruits. Plants transgenic for E8 TPS E8 astrehalase produced aberrant fruits with a Overexpression of a Potato Trehalase cDNA in N. yellow skin and incomplete ripening. 50 Tabacum Construct: de35S CaMV trehalase (pMOG1078) EXAMPLE 28 Primary tobacco transformants transgenic for pMOG 1078 revealed a phenotype different from wild-type tobacco, Performance of Potato Plants Transgenic For Some transgenics have a dark-green leaf colour and a thicker As-Trehalase and/or TPS 55 leaf (the morphology of the leaf is not lancet-shaped) indicating an influence of trehalase gene-expression on plant Constructs: 35S as-trehalase (pMOG1027) and 35S as metabolism. Seeds of selfed primary transformants were trehalase Pat TPS (PMOG1027(845-11/22/28). Sown and selected on kanamycin. The phenotype showed to Plants expressing 35S as-trehalase and pat-TPS simulta segregate in a mendelian fashion in the S1 generation. neously were generated by retransforming pat-TPS lines 60 (resistant against kanamycin) with construct pMOG1027, Deposits harbouring the 35S as-trehalase construct and a hygromycin resistance marker gene, resulting in genotypes pMOG1027 The following deposits were made under the Budapest (845-11), pMOG1027(845-22) and pMOG1027(845-28). Treaty. The clones were deposited at the Centraal Bureau Microtubers were induced in vitro and fresh weight of the 65 voor Schimnelcultures, Oosterstraat 1, P.O. Box 273, 3740 microtubers was determined. The average fresh weight yield AG Baarn, The Netherlands on Apr. 21, 1997 and received was increased for transgenic lines harbouring pMOG1027 the following numbers: US 7,247,770 B2 51 52 pMOG800 Derivative of pMOG402 with restored KipnI site in polylinker Escherichia coi DH5alpha/pMOG 1192 CBS 692.97 2. TPS/TPP Expression Constructs DH5alpha/pMOG1241DH5alpha/pMOG1240 CBS 694.97693.97 5 pMOG 799 35S-TPS-3'nosTDSRSI PA4OG 810 idem with Hyg DH5alpha/pMOG 1242 CBS 695.97 marker DH5alpha/pMOG 1243 CBS 696.97 All constructs harbour the NPTII selection marker unless noted DH5alpha/pMOG1244 CBS 697.97 otherwise DH5alpha/pMOG1245 CBS 698.97 pMOG 845 Pat-TPS-3'PotPill pMOG 925 idem with Hyg 10 marker Deposited Clones: pMOG 851 35S-TPS-3'nos 35S-TPP(atg) pMOG1 192 harbors the Helianchus annuus TPS/TPP ° Two types of TPP constructs have been used as described in Goddijn bipartite cDNA inserted in the multi-copy vector at al. (1997) Plant Physiol. 113, 181. PGEM-T (Promega). pMOG 1010 de35S CaMV anv leader TPP(gtg), PotPill pMOG 1240 harbors the tobacco TPS “825” bp cDNA 15 pMOG 1142 idem with Hyg marker fragment inserted in pCRscript (Stratagene). pMOG 1093 Plastocyanin-TPS-3'nos pMOG 1129 idem PMragment 1nsertedNES." 1n p (SSEE- I (Fromega). se bp cDNA with Hyg marker a pMOG1242 harbors the tobacco TPS “630 bp cDNA pMOG 1177 Plastocyanin-TPS-3'PotPill 3'nos fragment inserted in pGEM-T (Promega). 20 pVDH318 Identical to pMOG1177 Functionally identical pMOG 1243 harbors the tobacco TPP “543, bp cDNA to pMOG1093 fragment inserted in pGEM-T (Promega). pMOG 1124 Plastocyanin-TPP(gtg) 3'PotPiII 3'nos PMO,ragment 1nserted 1nhe a ps plasm1d.E2 bp cDNA pVDH 321 Identical to pMOG1124 pMOG 1245 harbors the tobacco TPP “447 bp fragment 2 pMOG 1128 Patatin TPP(gtg) 3'PotPiLI inserted in PGEM-T (Promega). pMOG 1140 E8-TPS-3'nos List of Relevant pMOG# and pVDHHH! Clones pMOG 1141 E8-TPP(gtg)-3"PotPill MES SC tor (ca. 10 Kb) harboring the NPTII 3. Trehalase Constructs p Selection R O Ca. arboring une 30 pMOG 1028 Patatin as-trehalase 3"PotPill, Hygromycin pMOG22 Derivative of pMOG23, the NPTII-gene has resistance marker been replaced by the HPT-gene which confers resis- pMOG 1078 de35S CaMVamv leader trehalase 3'nos tance to hygromycine pMOG 1090 de35S CaMVamv leader as-trehalase 3'nos pVDH 275 Binary vector derived from pMOG23, harbors 35 pMOG 1027 idem with Hyg marker a stocyanin promoter-nos terminator expression cas- pMOG 1092 Plastocyanin-as trehalase-3'nos pMOG 402 Derivative of pMOG23, a point-mutation in pMOG 1130 Plastocyanin-as trehalase-3'nos Plastocya the NPTII-gene has been restored, no KpnI restriction nin-TPS-3'noS site present in the polylinker pMOG 1153 E8-TPS-3'nos E8-as trehalase-3'PotPill SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 57 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1450 base pairs (B) TYPE : nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (ix) FEATURE: (A) NAME/KEY : CDS (B) LOCATION: 21... 1450 (xi). SEQUENCE DESCRIPTION: SEQ ID NO: 1: ATAAAACTCT CCCCGGGACC ATG ACT ATG AGT CGT TTA GTC GTA GTA TCT 50 Met Thr Met Ser Arg Leu Val Val Val Ser 1 5 10 US 7,247,770 B2 55 56 -continued 315 320 325 330 GGC TGG ACG CCG CTT TAT TTG AAT CAG CAT TTT GAC CG TTA Gly Trp Thr Pro Teu Telu Asn Gln His Phe Asp Arg Lys Telu 335 340 345 CTG ATG ATA TTC CGC TCT GAC GTG GGC TTA GTG ACG CCA CTG 106 Teu Met Ile Phe Arg Ser Asp Val Gly Teu Wall Thr Pro Telu 350 355 360 CGT GAC GGG ATG AAC CTG GTA GCA GAG TAT GTT GCT GC CAG GAC 154 Arg Asp Gly Met Asn Teu Wall Ala Glu Tyr Wall Ala Ala Glin Asp 365 370 375 CCA GCC AAT CCG GGC GTT CTT GTT CTT TCG CAA TTT GCG GGA GCG GCA Pro Ala Asn Pro Gly Wall Teu Wall Telu Ser Glin Phe Ala Gly Ala Ala 38O 385 390 AAC GAG TTA ACG TCG GCG TTA ATT GTT AAC CCC TAC GAT CG GAC GAA 25 O Asn Glu Telu Thr Ser Ala Teu Ile Wall Asn. Pro Asp Arg Asp Glu 395 400 405 410 GTT GCA GCT GCG CTG GAT CGT GCA TTG ACT ATG TCG CTG GCG GAA CGT 298 Wall Ala Ala Ala Teu Asp Arg Ala Telu Thr Met Ser Teu Ala Glu Arg 415 420 425 ATT TCC CGT CAT GCA GAA ATG CTG GAC GTT ATC GTG AAC GAT ATT 346 Ile Ser Arg His Ala Glu Met Telu Asp Wall Ile Wall Asn Asp Ile 430 435 4 4 O AAC CAC TGG CAG GAG TTC ATT AGC GAC CTA AAG CAG ATA GTT CCG 394 Asn His Trp Glin Glu Phe Ile Ser Asp Lieu Glin Ile Wall Pro 445 450 455 CGA AGC GCG GAA AGC CAG CAG CGC GAT AAA GTT GCT ACC TT CCA AAG 442 Arg Ser Ala Glu Ser Glin Glin Arg Asp Lys Val Ala Thr Phe Pro Lys 460 465 470 CTC GC AG 450 Teu 475 (2) INFORMATION FOR SEQ ID NO 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 476 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi). SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Thr Met Ser Arg Teu Wal Wal Wal Ser Asn Arg Ile Ala Pro Pro 5 10 15 Asp Glu His Ala Ala Ser Ala Gly Gly Leu Ala Wall Gly Ile Telu Gly 2O 25 3O Ala Telu Lys Ala Ala Gly Gly Telu Trp Phe Gly Trp Ser Gly Glu Thr 35 40 45 Gly Asn Glu Asp Glin Pro Teu Lys Wall Lys Lys Gly Asn Ile Thr 5 O 55 60 Trp Ala Ser Phe Asn Teu Ser Glu Glin Asp Lieu Asp Glu Asn 65 70 75 Glin Phe Ser Asn Ala Wall Teu Trp Pro Ala Phe His Arg Telu Asp 85 90 95 Teu Wall Glin Phe Glin Arg Pro Ala Trp Asp Gly Tyr Teu Arg Wall Asn 100 105 110 Ala Telu Telu Ala Teu Telu Pro Telu Teu Glin Asp Asp Asp Ile 115 120 125 Ile Trp Ile His Asp His Telu Telu Pro Phe Ala His Glu Telu US 7,247,770 B2 57 58 -continued 130 135 1 4 0 Lys Arg Gly Wall Asn Asn Ile Gly Phe Phe Teu His Ile Pro Phe 145 15 O 155 160 Pro Thr Pro Glu Ile Phe Asn Ala Leu Pro Thr Tyr Asp Thr Telu Telu 1.65 170 175 Glu Glin Telu Cys Asp Tyr Telu Leu Gly Phe Glin Thr Glu Asn Asp 18O 185 19 O Arg Telu Ala Phe Leu Asp Cys Telu Ser Asn Teu Thr Arg Wall Thr Thr 195 200 Arg Ser Ala Lys Ser His Thr Ala Trp Gly Lys Ala Phe Arg Thr Glu 210 215 220 Wall Pro Ile Gly Ile Glu Pro Lys Glu Ile Ala Lys Glin Ala Ala 225 230 235 240 Gly Pro Telu Pro Pro Lys Teu Ala Gln Leu Lys Ala Glu Telu Lys Asn 245 250 255 Wall Glin Asn Ile Phe Ser Wall Glu Arg Lieu Asp Ser Lys Gly Telu 260 265 27 O Pro Glu Arg Phe Leu Ala Tyr Glu Ala Leu Teu Glu Lys Pro Glin 275 280 285 His His Gly Lys Ile Tyr Thr Glin Ile Ala Pro Thr Ser Arg Gly 29 O 295 Asp Wall Glin Ala Tyr Glin Asp Ile Arg His Glin Teu Glu Asn Glu Ala 305 310 315 320 Gly Arg Ile Asin Gly Lys Tyr Gly Gln Leu Gly Trp Thr Pro Telu 325 330 335 Telu Asn Gln His Phe Arg Lys Lieu Teu Met Lys Ile Phe Arg 340 345 35 O Ser Asp Val Gly Teu Wall Thr Pro Leu Arg Asp Gly Met Asn Telu 355 360 365 Wall Ala Glu Tyr Wall Ala Ala Glin Asp Pro Ala Asn Pro Gly Wall 370 375 Teu Wall Telu Ser Glin Phe Ala Gly Ala Ala Asn Glu Teu Thr Ser Ala 385 390 395 400 Teu Ile Wall Asn. Pro Tyr Arg Asp Glu Wall Ala Ala Ala Telu Asp 405 410 415 Arg Ala Telu Thr Met Ser Teu Ala Glu Arg Ile Ser His Ala Glu 420 425 43 O Met Telu Asp Wall Ile Wall Lys Asn Asp Ile Asn His Trp Glin Glu 435 4 40 4 45 Phe Ile Ser Asp Leu Lys Glin Ile Wall Pro Arg Ser Ala Glu Ser Glin 450 455 460 Glin Arg Asp Lys Val Ala Thr Phe Pro Lys Teu 465 470 475 (2) INFORMATION FOR SEQ ID NO: 3. (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 835 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (ix) FEATURE: US 7,247,770 B2 61 62 -continued (A) LENGTH: 266 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi). SEQUENCE DESCRIPTION: SEQ ID NO: 4: Met Thr Glu Pro Leu Thr Glu Thr Pro Glu Lieu Ser Ala Tyr Ala 5 10 15 Trp Phe Phe Asp Leu Asp Gly. Thir Lieu Ala Glu Ile Pro His Pro 2O 25 Asp Glin Val Val Val Pro Asp Asin Ile Telu Glin Gly Teu Glin Telu Telu 35 40 45 Ala Thir Ala Ser Asp Gly Ala Lieu Ala Telu Ile Ser Gly Arg Ser Met 5 O 55 60 Wall Glu Lieu. Asp Ala Leu Ala Lys Pro Arg Phe Pro Telu Ala Gly 65 70 75 Wall His Gly Ala Glu Arg Arg Asp Ile Asn Gly Thr His Ile Wall 85 90 95 His Leu Pro Asp Ala Ile Ala Arg Asp Ile Ser Wall Glin Telu His Thr 100 105 110 Wall Ile Ala Glin Tyr Pro Gly Ala Glu Telu Glu Ala Lys Gly Met Ala 115 120 125 Phe Ala Lieu. His Tyr Arg Glin Ala Pro Glin His Glu Asp Ala Telu Met 130 135 1 4 0 Thr Leu Ala Glin Arg Ile Thr Glin Ile Trp Pro Glin Met Ala Telu Glin 145 15 O 155 160 Glin Gly Lys Cys Val Val Glu Ile Lys Pro Arg Gly Thr Ser Lys Gly 1.65 170 175 Glu Ala Ile Ala Ala Phe Met Glin Glu Ala Pro Phe Ile Gly Arg Thr 18O 185 19 O Pro Val Phe Leu Gly Asp Asp Lieu. Thr Asp Glu Ser Gly Phe Ala Wall 195 200 Wall Asn Arg Lieu Gly Gly Met Ser Val Ile Gly Thr Gly Ala Thr 210 215 220 Glin Ala Ser Trp Arg Lieu Ala Gly Val Pro Asp Wall Trp Ser Trp Telu 225 230 235 240 Glu Met Ile Thir Thr Ala Leu Glin Glin Lys Arg Glu Asn Asn Arg Ser 245 250 255 Asp Asp Tyr Glu Ser Phe Ser Arg Ser Ile 260 265 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (xi). SEQUENCE DESCRIPTION: SEQ ID NO: 5: AAGCTTATGT TGCCATATAG AGTAGAT 27 (2) INFORMATION FOR SEQ ID NO: 6: US 7,247,770 B2 63 -continued (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (xi). SEQUENCE DESCRIPTION: SEQ ID NO: 6: GTAGTTGCCA TGGTGCAAAT GTTCATATG (2) INFORMATION FOR SEQ ID NO: 7 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: (A) NAME/KEY: modified base, Inosine (B) LOCATION: 4 (D) OTHER INFORMATION: /note= N is Inosine (ix) FEATURE: (A) NAME/KEY: modified base, Inosine (B) LOCATION: 6 (D) OTHER INFORMATION: /note= N is Inosine (ix) FEATURE: (A) NAME/KEY: modified base, Inosine (B) LOCATION: 9 (D) OTHER INFORMATION: /note= N is Inosine (ix) FEATURE: (A) NAME/KEY: modified base, Inosine (B) LOCATION: 15 (D) OTHER INFORMATION: /note= N is Inosine (xi). SEQUENCE DESCRIPTION: SEQ ID NO : 7 : GAYNTNATAT GGRTNCAYGA YTAYCA (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: (A) NAME/KEY: modified base, Inosine (B) LOCATION: 2 (D) OTHER INFORMATION: /note= N is Inosine (ix) FEATURE: (A) NAME/KEY: modified base, Inosine (B) LOCATION: 5 (D) OTHER INFORMATION: /note= N is Inosine (ix) FEATURE: US 7,247,770 B2 65 66 -continued (A) NAM E/KEY: modified base Inosine (B) LOCATION: 8 (D) OTH ER INFORMATION: /note= N is Inosine (ix) FEATU RE (A) NAM E/KEY: modified base Inosine (B) LOCATION 14 (D) OTH ER INFORMATION: /note= N is Inosine (ix) FEATU RE (A) NAM E/KEY: modified base Inosine (B) LOCATION: 20 (D) OTH ER INFORMATION: /note= N is Inosine (ix) FEATU RE (A) NAM E/KEY: modified base Inosine (B) LOCATION: 23 (D) OTH ER INFORMATION: /note= N is Inosine (xi). SEQUENCE DESCRIPTION: SEQ ID NO: 8: TNGGNTKNTT YYTNCAYAYN CCNTTYCC (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOT HETICAL NO (iv) ANTI-SENSE: NO (ix) FEATU RE (A) NAM E/KEY: modified base Inosine (B) LOCATION: 3 (D) OTH ER INFORMATION: /note= N is Inosine (ix) FEATU RE (A) NAM E/KEY: modified base Inosine (B) LOCATION: 6 (D) OTH ER INFORMATION: /note= N is Inosine (ix) FEATU RE (A) NAM E/KEY: modified base Inosine (B) LOCATION: 15 (D) OTH ER INFORMATION: /note= N is Inosine (ix) FEATU RE (A) NAM E/KEY: modified base Inosine (B) LOCATION: 21 (D) OTH ER INFORMATION: /note= N is Inosine (xi). SEQUENCE DESCRIPTION: SEQ ID NO: 9: GYNACNARRT TCATNCCRTC. NC (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 743 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens US 7,247,770 B2 67 68 -continued (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1. .. 743 (D) OTHER INFORMATION: /partial (xi). SEQUENCE DESCRIPTION: SEQ ID NO : 10: GAC GTG ATG TGG ATG CAC GAC TAC CAT TTG ATG GTG TTG CC ACG TTC 48 Asp Wall Met Trp Met His Asp Tyr His Telu Met Wall Teu Pro Thr Phe 5 10 15 TTG. AGG AGG CGG TTC AAT CGT TTG AGA ATG GGG TTT TTC CT CAC AGT 96 Leu Arg Arg Arg Phe Asn Arg Telu Arg Met Gly Phe Phe Telu His Ser 2O 25 3O CCA TTT CCC TCA TCT GAG ATT TAC AGG ACA CTT CCT GTT AGA GAG GAA 144 Pro Phe Pro Ser Ser Glu Ile Tyr Arg Thr Teu Pro Wall Arg Glu Glu 35 40 45 ATA CTC AAG GCT TTG CTC TGT GCT GAC ATT GTT GGA TTC CAC ACT TTT 192 Ile Leu Ala Lel Teu Cys Ala Asp Ile Wall Gly Phe His Thr Phe 55 60 GAC TAC GCG AGA CAC TTC CTC TCT AGT CGG ATG TTG GGT TTA 240 Asp Tyr Ala Arg His Phe Teu Ser Ser Arg Met Telu Gly Telu 65 70 75 8O GAG TAT CAG TCT AGA GGT TAT ATA GGG TTA GAA TAC TAT GGA CGG 288 Glu Tyr Glin Ser Arg Gly Ile Gly Teu Glu Gly Arg 90 95 ACA GTA GGC ATC ATT ATG CCC GTC GGG ATA CAT ATG GGT CAT ATT 336 Thir Wall Gly Ile Ile Met Pro Wall Gly Ile His Met Gly His Ile 100 105 110 GAG TCC ATG AAG CTT GCA GCG GAG TTG ATG CTT AAG GCG CTA 384 Glu Ser Met Teu Ala Ala Glu Teu Met Teu Ala Telu 115 120 125 AAG CAG CAA TTT GAA GGG ACT GTG TTG CTT GGT GCC GAT GAC CTG 432 Glin Glu Gly Lys Thr Wall Telu Teu Gly Ala Asp Asp Telu 130 135 1 4 0 GAT ATT TTC GGT ATA AAC TTA AAG CTT CTA GCT ATG GAA CAG ATG 480 Asp Ile Phe Gly Ile Asn Telu Telu Teu Ala Met Glu Glin Met 145 15 O 155 160 CTC AAA CAG CAC CCC AAG TGG CAA GGG CAG GCT GTG TTG GTC CAG ATT 528 Leu Lys Glin His Pro Trp Glin Gly Glin Ala Wall Teu Wall Glin Ile 1.65 170 175 GCA AAT CCT ACG AGG GGT GGA GTA GAT TTT GAG GAA ATA CAG GCT 576 Ala Asn Pro Thr Arg Gly Gly Wall Asp Phe Glu Glu Ile Glin Ala 18O 185 19 O GAG ATA TCG GAA AGC TGT AGA ATC AAT AAG CAA TTC GGC AAG CCT 624 Glu Ile Ser Glu Ser Arg Ile Asn Glin Phe Gly Pro 195 200 2O5 GGA TAT GAG CCT ATA GTT TAT ATT GAT AGG CCC GTG TCA AGC AGT GAA 672 Gly Tyr Glu Pro Ile Wall Tyr Ile Asp Arg Pro Wall Ser Ser Ser Glu 210 215 220 CGC ATG GCA TAT TAC AGT ATT GCA GAA TGT GTT GTT GTC ACG GCT GTG 720 Arg Met Ala Tyr Tyr Ser Ile Ala Glu Cys Wall Wall Wall Thr Ala Wall 225 230 235 240 AGC GAC GGC ATG AAC TTC GTC TC 743 Ser Asp Gly Met Asn Phe Wall 245 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 247 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear US 7,247,770 B2 69 -continued (ii) MOLECULE TYPE: protein (xi). SEQUENCE DESCRIPTION: SEQ ID NO : 11: Asp Val Met Trp Met His Asp Tyr His Leu Met Val Leu Pro Thr Phe 1 5 10 15 Leu Arg Arg Arg Phe Asn Arg Lieu Arg Met Gly Phe Phe Lieu. His Ser 2O 25 3O Pro Phe Pro Ser Ser Glu Ile Tyr Arg Thr Leu Pro Val Arg Glu Glu 35 40 45 Ile Leu Lys Ala Leu Lleu. Cys Ala Asp Ile Val Gly Phe His Thr Phe 5 O 55 60 Asp Tyr Ala Arg His Phe Leu Ser Cys Cys Ser Arg Met Leu Gly Lieu 65 70 75 8O Glu Tyr Glin Ser Lys Arg Gly Tyr Ile Gly Lieu Glu Tyr Tyr Gly Arg 85 90 95 Thr Val Gly Ile Lys Ile Met Pro Val Gly Ile His Met Gly His Ile 100 105 110 Glu Ser Met Lys Lys Lieu Ala Ala Lys Glu Lieu Met Lieu Lys Ala Lieu 115 120 125 Lys Glin Glin Phe Glu Gly Lys Thr Val Lieu Lieu Gly Ala Asp Asp Lieu 130 135 1 4 0 Asp Ile Phe Lys Gly Ile Asn Lieu Lys Lieu Lieu Ala Met Glu Gln Met 145 15 O 155 160 Leu Lys Gln His Pro Lys Trp Gln Gly Glin Ala Val Leu Val Glin Ile 1.65 170 175 Ala Asn Pro Thr Arg Gly Lys Gly Val Asp Phe Glu Glu Ile Glin Ala 18O 185 19 O Glu Ile Ser Glu Ser Cys Lys Arg Ile Asn Lys Glin Phe Gly Lys Pro 195 200 2O5 Gly Tyr Glu Pro Ile Val Tyr Ile Asp Arg Pro Val Ser Ser Ser Glu 210 215 220 Arg Met Ala Tyr Tyr Ser Ile Ala Glu Cys Val Val Val Thr Ala Val 225 230 235 240 Ser Asp Gly Met Asn Phe Val 245 (2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 395 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA ill-liii). HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Nicotiana tabacum. (B) STRAIN: Samsun NN (F) TISSUE TYPE : Leaf (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1. .395 (D) OTHER INFORMATION: /partial (xi). SEQUENCE DESCRIPTION: SEQ ID NO: 12: US 7,247,770 B2 -continued GCG AAA CCG GTG ATG AAA CTT TAC AGG GAA GCA ACT GAC GGA TCA. TAT 48 Ala Lys Pro Wal Met Lys Lieu. Tyr Arg Glu Ala Thr Asp Gly Ser Tyr 1 5 10 15 ATA GAA. ACT AAA GAG AGT, GCA TTA. GTG TGG CAC CAT CAT GAT G.C.A. GAC 96 Ile Glu Thir Lys Glu Ser Ala Lieu Val Trp His His His Asp Ala Asp 2O 25 3O CCT GAC TTT GGC. TCC TGC CAG GCA AAG GAA TTG TTG GAT CAT TTG GAA 144 Pro Asp Phe Gly Ser Cys Glin Ala Lys Glu Lieu Lleu. Asp His Leu Glu 35 40 45 AGC GTA. CTT, GCA AAT GAA CCT, GCA GTT GTT AAG AGG GGC CAA CAT ATT 192 Ser Val Lieu Ala Asn. Glu Pro Ala Val Val Lys Arg Gly Gln His Ile 5 O 55 60 GTT GAA GTC AAG CCA CAA. GGT, GTG ACC AAA. GGA TTA GTT TCA GAG AAG 240 Val Glu Val Lys Pro Gln Gly Val Thr Lys Gly Leu Val Ser Glu Lys 65 70 75 8O GTT CTC. TCG ATG ATG GTT GAT AGT GGG AAA CCG CCC GAT TTT GTT ATG 288 Val Leu Ser Met Met Val Asp Ser Gly Lys Pro Pro Asp Phe Val Met 85 90 95 TGC ATT GGA. GAT GAT AGG TCA. GAC GAA. GAC ATG TTT GAG AGC ATA TTA. 336 Cys Ile Gly Asp Asp Arg Ser Asp Glu Asp Met Phe Glu Ser Ile Leu 100 105 110 AGC ACC GTA TCC AGT CTG TCA GTC ACT, GCT, GCC CCT GAT GTC TTT GCC 384 Ser Thr Val Ser Ser Leu Ser Val Thr Ala Ala Pro Asp Val Phe Ala 115 120 125 TGC ACC GTC GG 395 Cys Thr Val 130 (2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 131 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi). SEQUENCE DESCRIPTION: SEQ ID NO : 13: Ala Lys Pro Wal Met Lys Lieu. Tyr Arg Glu Ala Thr Asp Gly Ser Tyr 1 5 10 15 Ile Glu Thir Lys Glu Ser Ala Lieu Val Trp His His His Asp Ala Asp 2O 25 3O Pro Asp Phe Gly Ser Cys Glin Ala Lys Glu Lieu Lleu. Asp His Leu Glu 35 40 45 Ser Val Lieu Ala Asn. Glu Pro Ala Val Val Lys Arg Gly Gln His Ile 5 O 55 60 Val Glu Val Lys Pro Gln Gly Val Thr Lys Gly Leu Val Ser Glu Lys 65 70 75 8O Val Leu Ser Met Met Val Asp Ser Gly Lys Pro Pro Asp Phe Val Met 85 90 95 Cys Ile Gly Asp Asp Arg Ser Asp Glu Asp Met Phe Glu Ser Ile Leu 100 105 110 Ser Thr Val Ser Ser Leu Ser Val Thr Ala Ala Pro Asp Val Phe Ala 115 120 125 Cys Thr Val 130 (2) INFORMATION FOR SEQ ID NO: 14: US 7,247,770 B2 73 -continued (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 491 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Nicotiana tabacum. (B) STRAIN: Samsun NN (F) TISSUE TYPE : Leaf (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1. . 491 (D) OTHER INFORMATION: /partial (xi). SEQUENCE DESCRIPTION: SEQ ID NO: 14: GGG CTG TCG GCG GAA CAC GGC. TAT TTC TTG. AGG ACG. AGT CAA. GAT GAA 48 Gly Lieu Ser Ala Glu His Gly Tyr Phe Leu Arg Thr Ser Glin Asp Glu 1 5 10 15 GAA TGG GAA ACA TGT, GTA. CCA. CCA GTG GAA TGT TGT TGG AAA GAA. ATA 96 Glu Trp Glu Thr Cys Val Pro Pro Val Glu Cys Cys Trp Lys Glu Ile 2O 25 3O GCT GAG CCT GTT ATG CAA CTT TAC ACT GAG ACT ACT GAT GGA TCA GTT 144 Ala Glu Pro Val Met Gln Leu Tyr Thr Glu Thir Thr Asp Gly Ser Val 35 40 45 ATT GAA. GAT. AAG GAA ACA TCA ATG GTC TGG TCT TAC GAG GAT GCG GAT 192 Ile Glu Asp Lys Glu Thir Ser Met Val Trp Ser Tyr Glu Asp Ala Asp 5 O 55 60 CCT GAT TTT GGA TCA TGT CAG GCT. AAG GAA CTT. CTT GAT CAC CTA GAA 240 Pro Asp Phe Gly Ser Cys Glin Ala Lys Glu Lieu Lleu. Asp His Leu Glu 65 70 75 8O AGT, GTA CTA GCT. AAT GAA CCG GTC ACT GTC AGG AGT GGA CAG AAT ATA 288 Ser Val Leu Ala Asn Glu Pro Val Thr Val Arg Ser Gly Glin Asn Ile 85 90 95 GTG GAA GTT AAG CCC CAG GGT GTA TCC AAA. GGG CTT, GTT GCC AAG CGC 336 Val Glu Wall Lys Pro Glin Gly Val Ser Lys Gly Lieu Val Ala Lys Arg 100 105 110 CTG CTT. TCC GCA ATG CAA GAG AAA GGA ATG TCA CCA GAT TTT GTC CTT 384 Leu Lleu Ser Ala Met Glin Glu Lys Gly Met Ser Pro Asp Phe Val Lieu 115 120 125 TGC ATA GGA. GAT GAC CGA TCG GAT GAA. GAC ATG TTC GAG GTG ATC. ATG 432 Cys Ile Gly Asp Asp Arg Ser Asp Glu Asp Met Phe Glu Val Ile Met 130 135 1 4 0 AGC TCG ATG TCT GGC CCG TCC ATG GCT CCA ACA GCT GAA GTC TTT GCC 480 Ser Ser Met Ser Gly Pro Ser Met Ala Pro Thr Ala Glu Val Phe Ala 145 15 O 155 160 TGC ACC GTC GG 491 Cys Thr Val (2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 163 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi). SEQUENCE DESCRIPTION: SEQ ID NO: 15: US 7,247,770 B2 75 76 -continued Gly Telu Ser Ala Glu His Gly Tyr Phe Leu Arg Thr Ser Glin Asp Glu 10 15 Glu Trp Glu Thr Cys Val Pro Pro Val Glu Cys Cys Trp Lys Glu Ile 25 Ala Glu Pro Val Met Gln Leu Tyr Thr Glu Thr Thr Asp Gly Ser Wall 35 40 45 Ile Glu Asp Lys Glu Thir Ser Met Val Trp Ser Tyr Glu Asp Ala Asp 5 O 55 60 Pro Asp Phe Gly Ser Cys Glin Ala Lys Glu Teu Teu Asp His Leu Glu 65 70 75 8O Ser Wall Telu Ala Asn. Glu Pro Wall Thr Wall Arg Ser Gly Glin Asn. Ile 85 90 95 Wall Glu Wall Lys Pro Gln Gly Val Ser Lys Gly Teu Wall Ala 100 105 110 Teu Telu Ser Ala Met Glin Glu Lys Gly Met Ser Pro Asp Phe Wall Leu 115 120 125 Ile Gly Asp Asp Arg Ser Asp Glu Asp Met Phe Glu Wall Ile Met 130 135 1 4 0 Ser Ser Met Ser Gly Pro Ser Met Ala Pro Thr Ala Glu Wall Phe Ala 145 15 O 155 160 Thr Wall (2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: A) LENGTH: 361 base pairs B) TYPE: nucleic acid C) STRANDEDNESS: double D) TOPOLOGY: linear (ii) MOLECULE TYPE cDNA to mRNA (iii) HYPOTHETICAL NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Nicotiana tabacum. ( B) STRAIN: Samsun. NN (F) TISSUE TYPE : Leaf (xi). SEQUENCE DESCRIPTION: SEQ ID NO: 16: TTTGATTATG ATGGGACGCT GCTGTCGGAG GAGAGTGTGG ACAAAACCCC GAGTGAAGAT 60 GACATCTCAA TTCTGAATGG TTTATGCAGT, GATCCAAAGA ACGTAGTCT TATCGTGAGT 120 GGCAGAGGAA AGGATACACT TAGCAAGTGG TCTCTCCGT GTCCGAGACT CGGCCTATCA 18O GCAGAACATG GATATTTCAC TAGGTGGAGT AAGGATTCCG AGTGGGAATC TCGTCCATAG 240 CTGCAGACCT TGACTGGAAA AAAATAGTGT TGCCTATTAT GGAGCGCTAC ACAGAGCACA GATGGTTCGT CGATAGAACA GAAGGAAACC TCGTGTTGGC TCATCAAATG CTGGCCCCGA 360 A. 361 (2) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS: A) LENGTH: 118 base pairs B) TYPE: nucleic acid C) STRANDEDNESS: double D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA US 7,247,770 B2 77 -continued (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Nicotiana tabacum. (B) STRAIN: Samsun NN (F) TISSUE TYPE : Leaf (xi). SEQUENCE DESCRIPTION: SEQ ID NO: 17: GGAAACCCAC AGGATGTAAG CAAAGTTTTA GTTTTTGAGA TCTCTTGGCA TCAAGCAAAG 60 TAGAGGGAAG TCACCCGATT CGTGCTGTGC GTAGGGATGA CAGATCGGAC GACTTAGA 118 (2) INFORMATION FOR SEQ ID NO: 18 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 417 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Nicotiana tabacum. (B) STRAIN: Samsun NN (F) TISSUE TYPE : Leaf (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: TTGTGGCCGA TGTTCCACTA CATGTTGCCG TTCTCACCTG ACCATGGAGG CCGCTTTGAT 60 CGCTCTATGT. GGGAAGCATA TGTTTCTGCC AACAAGTTGT TTTCACAAAA AGTAGTTGAG 120 GTTCTTAATC CTGAGGAGA CTTTGTCTGG ATTCATGATT ATCATTGAT GGTGTTGCCA 18O ACGTTCTTGA. GGAGGCGGTT CAATCGTTTG. AGAATGGGGT TTTTCCTTCA CAGCCATTC 240 CTTCATCTGA GATTTACAGG ACACTTCCTG TTAGAGAGGA AATACTCAAG GCTTTGCTCT GTGCTGACAT TGTTGGATTC CACACTTTTG ACTACGCGAG ACACTTCCTC. TCTTGTTGCA 360 GTCGATTTTG GGTAGAGTAC AGTCTAAAAA AAGTTATATT GGGTTAAAAT ACTATGG 417 (2) INFORMATION FOR SEQ ID NO: 19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 411 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Nicotiana tabacum. (B) STRAIN: Samsun NN (F) TISSUE TYPE : Leaf (xi). SEQUENCE DESCRIPTION: SEQ ID NO : 19: GGGTCATATT GATCCATGAA GAAATTGCAG CGAAAGAGTG ATGCTTTAAT GCGTAAAGCA 60 GCAATTTGAA. GGGAAAACTG TGTTGTTAGG TGCCGATGAC CTGGATATTT TCAAAGGTAT 120 GAACTTAAAG CTTCTAGCTA TGGAACAGAT GCTCAAACAT CACCCCAAGT GGCAAGGGCA US 7,247,770 B2 79 80 -continued GGCTGTGTTG GTCCAAGATT GCAAATCCTA CGAGGGGTAA AGGAGTAGAT TTTGACGAAA 240 TACGGCTGAG ACATCGGAAA GCTGTAAGAG AATCAATAAG CAATTCGGCA AGCCTGGATA TGAGCCTATA GTTTATATTG ATAGGCCCGT GTCAAGCAGT, GAACGCATGG CATATTACAG 360 TATTGCAGGA TGTGTTGTGG TCACGCTGTG. AGCGATGGCA TGAATCTGTT C. 411 (2) INFORMATION FOR SEQ ID NO: 2O: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 405 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Nicotiana tabacum. (B) STRAIN: Samsun NN (F) TISSUE TYPE : Leaf (xi). SEQUENCE DESCRIPTION: SEQ ID NO: 2O: TGGGGGGTT CCTGCATACG CCGTTTCCTT. CTTCTGAGA AATAAAACT TTGCCTATTC 60 GCGAAAGATC TTACAGCTCT CTTGAATTCA ATTTGATTGG GTTCCACACT TTTGACTATG 120 CAGGCACTTC CTCTCGTGTT GCAGTCGGAT GTTAGGTATT TCTTATGATC AAAAAGGGGT 18O TACATAGGCC. TCGATATTAT GGCAGGACTG TAATATAAAA ATTCTGCCAG CGGGTATTCA 240 TATGGGGCAG CTTCAGCAAG TCTTGAGTCT TCCTGAAACG GAGGCAAAAT CTCGGAACTC GTGCAGCATT TAATCATCAG GGGGAGGACA TTGTTGCTGG GATTGATGAC TGGACATATT 360 TAAAGGCTCA TTTGAATTTA TTACCATGGA ACAACTCTAT TGCAC 405 (2) INFORMATION FOR SEQ ID NO: 21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 427 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Nicotiana tabacum. (B) STRAIN: Samsun NN (F) TISSUE TYPE : Leaf (xi). SEQUENCE DESCRIPTION: SEQ ID NO: 21: ATCATATGGG GCAGCTTCAG CAATCTTGAT. CTTCCTGAAA CGGAGGCAAA AGTCTTCGGA 60 ACTCGGCAGC AGTTTAATCA TCAGGGGAGG ACATTGTTGC TGGGAGTTGA TGACATGGAC 120 ATATTTAAAG GCATCAGTTT GAAGTTATTA GCAATGGAAC AACTTCTATT GCAGCACCCG 18O GAGAAGCAGG GGAAGGTTGT TTTGGTGCAG ATAGCCAATC CTGCTAGAGG CAAAGGAAAA 240 GATGTCAAAG AAGTGCAGGA, AGAAACTCAT. TGACGGTGAA. GCGAATTAAT GAAGCATTTG GAAGACCTGG GTACGAACCA GTTATCTTGA TTGATAAGCC ACTAAAGTTT TATGAAAGGA 360 TTGCTTATTA TGTTGTTGCA GAGTGTTGCC TAGTCACTGC TGTCAGCGAT GGCATGAACC 420 US 7,247,770 B2 81 82 -continued TCGTCTC 427 (2) INFORMATION FOR SEQ ID NO: 22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 315 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Nicotiana tabacum. (B) STRAIN Samsun. NN (F) TISSUE TYPE Leaf (xi). SEQUENCE DESCRIPTION: SEQ ID NO: 22: GATGTGGATG CATGACTACC AATCCAAGAG. GGGGTATATT GGTCTTGACT ATTATGGTAA 60 ACTGTGACCA TTAAAATCCT. TCCAGTTGGT ATTCACATGG GACAACTCCA AAATGTTATG 120 TCACTACAGA CACGGGAAAG AAAGCAAAGG AGTTGAAAGA AAAATATGAG GGGAAAATTG 18O TGATGTTAGG TATTGATGAT ATGGACATGT, TTAAAGGAAT TGGTCTAAAG TTTCTGGCAA 240 TGGGGAGGCT, TCTAGATGAA. AACCCTGTCT TOGAGGGGTAA, AGTGGTATTG GTTCAATCAC CAGGCCTGGA. AATTA. 315 (2) INFORMATION FOR SEQ ID NO: 23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 352 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Nicotiana tabacum. (B) STRAIN Samsun. NN (F) TISSUE TYPE Leaf (xi). SEQUENCE DESCRIPTION: SEQ ID NO: 23: AGAAGTAAAG GGAGTGAGTC CCCGAGGTTC AAAAAGAGGT CAACAGAATT GCAGTGAAAT 60 TAATAAAAAA. TATGGCAAAC CGGGGTACAA. GCCGATTGTT TGTATCAATG GTCCAGTTTC 120 GACACAAGAC AAGATTGCAC ATTATGCGGT CTTGAGTGTG TTGTTGTTAA TGCTGTTAGA 18O GATGGGATGA ACTTGGTGCC TTATGAGTAT ACGGTCTTTA. GGCAGGGCAG CGATAATTTG 240 GATAAGGCCT TGCAGCTAGA TGGTCCTACT GCTTCCAGAA AGAGTGTGAT TATTGTCTTG AATTCGTTGG GTGCTCGCCA TCTTAGTGG CGCCATCCGC GTCAACCCCT GG 352 (2) INFORMATION FOR SEQ ID NO: 24 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2640 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS double (D) TOPOLOGY: linear US 7,247,770 B2 83 -continued (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Helianthus annuus (F) TISSUE TYPE : Leaf (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 171. .2508 (ix) FEATURE: (A) NAME/KEY: unsure (B) LOCATION: replace (2141 . .2151, "ccatninnitta") (ix) FEATURE: (A) NAME/KEY: unsure (B) LOCATION: replace (2237 . . 2243, "actnaaa") (xi). SEQUENCE DESCRIPTION: SEQ ID NO: 24 : GGATCCTGCG GTTTCATCAC ACAATATGAT ACTGTTACAT. CTGATGCCCC TTCAGATGTC 60 CCAAATAGGT TGATTGTCGT ATCGAATCAG TTACCCATAA TCGCTAGGCT. AAGACTAACG 120 ACAATGGAGG GTCCTTTTGG GATTTCACTT GGGACGAGAG. TTCGATTTAC ATG CAC 176 Met His 1 ATC AAA GAT GCA TTA. CCC GCA GCC GTT GAG GTT TTC TAT GTT GGC GCA 224 Ile Lys Asp Ala Leu Pro Ala Ala Val Glu Val Phe Tyr Val Gly Ala 5 10 15 CTA AGG GCT GAC GTT GGC. CCT ACC GAA CAA. GAT GAC GTG TCA AAG ACA 272 Leu Arg Ala Asp Val Gly Pro Thr Glu Glin Asp Asp Wal Ser Lys Thr 20 25 30 TTG CTC GAT AGG TTT AAT GC GT, GCG GT TTT GTC CCT AC TCA AAA 320 Leu Leu Asp Arg Phe Asn Cys Val Ala Val Phe Val Pro Thr Ser Lys 35 40 45 50 TGG GAC CAA TAT TAT CAC TGC TTT TGT AAG CAG TAT TTG TGG CCG ATA 368 Trp Asp Glin Tyr Tyr His Cys Phe Cys Lys Glin Tyr Leu Trp Pro Ile 55 60 65 TTT CAT TAC AAG GTT CCC GCT, TCT GAC GTC AAG AGT, GTC CCG AAT AGT 416 Phe His Tyr Lys Val Pro Ala Ser Asp Val Lys Ser Val Pro Asn Ser 70 75 8O CGG GAT TCA TGG AAC GCT TAT GTT CAC GTG AAC AAA GAG TTT TCC CAG 464 Arg Asp Ser Trp Asn Ala Tyr Val His Val Asn Lys Glu Phe Ser Glin 85 9 O 95 AAG GTG ATG GAG GCA GTA ACC AAT, GCT AGC AAT TAT GTA TGG ATA CAT 512 Lys Val Met Glu Ala Val Thr Asn Ala Ser Asn Tyr Val Trp Ile His 100 105 110 GAC TAC CAT TTA ATG ACG CTA CCG ACT TTC TTG. AGG CGG GAT TTT TGT 560 Asp Tyr His Leu Met Thr Leu Pro Thr Phe Leu Arg Arg Asp Phe Cys 115 120 125 130 CGT TTT AAA ATC GGT TTT T.T.T. CTG CAT AGC CCG TTT CCT, TCC TCG GAG 608 Arg Phe Lys Ile Gly Phe Phe Leu. His Ser Pro Phe Pro Ser Ser Glu 135 140 145 GTT TAC AAG ACC CTA. CCA. ATG AGA. AAC GAG CTC TTG AAG GGT CTG TTA. 656 Val Tyr Lys Thr Lieu Pro Met Arg Asn. Glu Lieu Lleu Lys Gly Lieu Lieu 150 155 16 O AAT, GC GAT. CTT, ATC. GGG TC CAT ACA. TAC GAT TAT GCC CGT CAT TTT O4 Asn Ala Asp Lieu. Ile Gly Phe His Thr Tyr Asp Tyr Ala Arg His Phe 1.65 17 O 175 CTA ACG TGT TGT AGT CGA ATG TTT GGT TTG GAT CAT CAG TTG AAA AGG 752 Lieu. Thir Cys Cys Ser Arg Met Phe Gly Lieu. Asp His Glin Leu Lys Arg US 7,247,770 B2 89 90 -continued (A) LENGTH: 779 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi). SEQUENCE DESCRIPTION: SEQ ID NO: 25: Met His Ile Lys Asp Ala Leu Pro Ala Ala Val Glu Wall Phe Tyr Wall 5 10 15 Gly Ala Telu Arg Ala Asp Wall Gly Pro Thr Glu Glin Asp Asp Wall Ser 25 3O Lys Thr Telu Telu Asp Arg Phe Asn Cys Wall Ala Wall Phe Wall Pro Thr 35 40 45 Ser Lys Trp Asp Glin Tyr His Cys Phe Lys Glin Telu Trp 5 O 55 60 Pro Ile Phe His Tyr Lys Wall Pro Ala Ser Asp Wall Ser Wall Pro 65 70 75 Asn. Ser Asp Ser Trp Asn Ala Tyr Wall His Wall Asn Glu Phe 85 90 95 Ser Glin Lys Wall Met Glu Ala Wall Thr Asn Ala Ser Asn Tyr Wall Trp 100 105 110 Ile His Asp Tyr His Teu Met Thr Telu Pro Thr Phe Teu Arg Arg Asp 115 120 125 Phe Cys Arg Phe Lys Ile Gly Phe Phe Telu His Ser Pro Phe Pro Ser 130 135 1 4 0 Ser Glu Wall Lys Thr Teu Pro Met Arg Asn Glu Teu Telu Gly 145 15 O 155 160 Teu Telu Asn Ala Asp Teu Ile Gly Phe His Thr Asp Ala Arg 1.65 170 175 His Phe Telu Thr Cys Ser Met Phe Gly Teu Asp His Glin Telu 18O 185 19 O Gly Tyr Ile Phe Teu Glu Tyr Asn Gly Ser Ile Glu Ile 195 200 Lys Ala Ser Gly Ile His Wall Gly Arg Met Glu Ser Telu 210 215 220 Ser Glin Pro Asp Thr Arg Teu Glin Wall Glin Glu Teu Lys Arg Phe 225 230 235 240 Glu Gly Lys Ile Wall Teu Teu Gly Wall Asp Asp Teu Asp Ile Phe 245 250 255 Gly Val Asn Phe Lys Wall Teu Ala Telu Glu Teu Teu Lys Ser His 260 265 27 O Pro Ser Trp Glin Gly Wall Wall Telu Wall Glin Ile Teu Asn Pro Ala 275 280 285 Arg Ala Arg Cys Glin Asp Wall Asp Glu Ile Asn Ala Glu Ile Arg Thr 29 O 295 Val Cys Glu Ile Asn Asn Glu Telu Gly Ser Pro Gly Tyr Glin Pro 305 310 315 320 Wal Wall Telu Ile Asp Gly Pro Wall Ser Telu Ser Glu Lys Ala Ala 325 330 335 Tyr Ala Ile Ala Asp Met Ala Ile Wall Thr Pro Teu Asp Gly Met 340 345 35 O Asn Lieu Ile Pro Tyr Glu Tyr Wall Wall Ser Arg Glin Ser Wall Asn Asp 355 360 365 Pro Asn Pro Asn Thr Pro Lys Ser Met Teu Wall Wall Ser Glu Phe 370 375 38O US 7,247,770 B2 91 92 -continued Ile Gly Cys Ser Teu Ser Leu. Thr Gly Ala Ile Arg Wall Asn Pro Trp 385 390 395 400 Asp Glu Telu Glu Thr Ala Glu Ala Telu Tyr Asp Ala Teu Met Ala Pro 405 410 415 Asp Asp His Lys Glu Thr Ala His Met Lys Glin Tyr Glin Tyr Ile Ile 420 425 43 O Ser His Asp Wall Ala Asn Trp Ala Arg Ser Phe Phe Glin Asp Telu Glu 435 4 40 4 45 Glin Ala Cys Ile His Ser Arg Lys Arg Met Asn Telu Gly Phe 450 455 460 Gly Telu Asp Thr Wall Wall Leu Phe Asp Glu Phe Ser Telu 465 470 475 480 Asp Ile Asp Wall Teu Glu Asn Ala Tyr Ser Met Ala Glin Asn Arg Ala 485 490 495 Ile Telu Telu Asp Tyr Gly Thr Wall Thr Pro Ser Ile Ser Ser 5 OO 505 Pro Thr Glu Ala Wall Ile Ser Met Ile Asn Teu Cys Asn Asp Pro 515 52O 525 Asn Met Wall Phe Ile Wal Ser Gly Ser Arg Glu Asn Telu Gly 530 535 540 Ser Trp Phe Gly Ala Cys Glu Lys Pro Ala Ile Ala Ala Glu His Gly 545 550 555 560 Phe Ile Trp Ala Gly Asp Glin Glu Trp Glu Thr Ala Arg 565 570 575 Asn Asn Wall Gly Trp Met Glu Met Ala Glu Pro Wall Met Asn Telu 58O 585 59 O Thr Glu Thr Thr Gly Ser Tyr Ile Glu Lys Glu Thr Ala 595 600 605 Met Wall Trp His Tyr Glu Asp Ala Asp Asp Teu Gly Telu Glu Glin 610 615 62O Ala Glu Telu Teu Asp His Lieu Glu Asn Wall Teu Ala Asn Glu Pro 625 630 635 640 Wall Glu Wall Arg Gly Gln Tyr Ile Wall Glu Wall Pro Glin Wall 645 650 655 Pro His Gly Telu Pro Ser Asp Ile His Arg His Arg Phe Wall 660 665 67 O Glu Ser Phe Asn Teu Asn Phe Phe Lys Glu Asn Arg Gly 675 680 685 Ser Telu Lys Gly Ile Wall Ala Glu Lys Ile Phe Ala Phe Met Ala Glu 69 O. 695 7 OO Lys Gly Lys Glin Ala Asp Phe Wall Telu Ser Wall Gly Asp Asp Arg Ser 705 710 715 720 Asp Glu Asp Met Phe Wall Ala Ile Gly Asp Gly Ile Gly Arg 725 730 735 Ile Thr Asn Asn Asn Ser Wall Phe Thr Wall Wall Gly Glu Pro 740 745 750 Ser Ala Ala Glu Tyr Phe Leu Asp Glu Thr Asp Wall Ser Met Met 755 760 765 Teu Glu Lys Telu Gly Cys Teu Ser Asn Glin Gly 770 775 (2) INFORMATION FOR SEQ ID NO 26 US 7,247,770 B2 93 -continued (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2130 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Helianthus annuus (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 171 . .2130 (D) OTHER INFORMATION: /partial (xi). SEQUENCE DESCRIPTION: SEQ ID NO: 26: GGATCCTGCG GTTTCATCAC ACAATATGAT ACTGTTACAT. CTGATGCCCC TTCAGATGTC 60 CCAAATAGGT TGATTGTCGT ATCGAATCAG TTACCCATAA TCGCTAGGCT. AAGACTAACG 120 ACAATGGAGG GTCCTTTTGG GATTTCACTT GGGACGAGAG. TTCGATTTAC ATG CAC 176 Met His 1 ATC AAA GAT GCA TTA. CCC GCA GCC GTT GAG GTT TTC TAT GTT GGC GCA 224 Ile Lys Asp Ala Leu Pro Ala Ala Val Glu Val Phe Tyr Val Gly Ala CTA AGG GCT GAC GTT GGC. CCT ACC GAA CAA. GAT GAC GTG TCA AAG ACA 272 Leu Arg Ala Asp Val Gly Pro Thr Glu Glin Asp Asp Val Ser Lys Thr 20 25 30 TTG CTC GAT AGG TTT AAT GC GT, GCG GT TTT GTC CCT AC TCA AAA 320 Leu Leu Asp Arg Phe Asn Cys Val Ala Val Phe Val Pro Thr Ser Lys 35 40 45 50 TGG GAC CAA TAT TAT CAC TGC TTT TGT AAG CAG TAT TTG TGG CCG ATA 368 Trp Asp Glin Tyr Tyr His Cys Phe Cys Lys Glin Tyr Leu Trp Pro Ile 55 60 65 TTT CAT TAC AAG GTT CCC GCT, TCT GAC GTC AAG AGT, GTC CCG AAT AGT 416 Phe His Tyr Lys Val Pro Ala Ser Asp Val Lys Ser Val Pro Asn Ser 70 75 8O CGG GAT TCA TGG AAC GCT TAT GTT CAC GTG AAC AAA GAG TTT TCC CAG 464 Arg Asp Ser Trp Asn Ala Tyr Val His Val Asn Lys Glu Phe Ser Glin 85 9 O 95 AAG GTG ATG GAG GCA GTA ACC AAT, GCT AGC AAT TAT GTA TGG ATA CAT 512 Lys Val Met Glu Ala Val Thr Asn Ala Ser Asn Tyr Val Trp Ile His 100 105 110 GAC TAC CAT TTA ATG ACG CTA CCG ACT TTC TTG. AGG CGG GAT TTT TGT 560 Asp Tyr His Leu Met Thr Leu Pro Thr Phe Leu Arg Arg Asp Phe Cys 115 120 125 130 CGT TTT AAA ATC GGT TTT T.T.T. CTG CAT AGC CCG TTT CCT, TCC TCG GAG 608 Arg Phe Lys Ile Gly Phe Phe Leu. His Ser Pro Phe Pro Ser Ser Glu 135 140 145 GTT TAC AAG ACC CTA. CCA. ATG AGA. AAC GAG CTC TTG AAG GGT CTG TTA. 656 Val Tyr Lys Thr Lieu Pro Met Arg Asn. Glu Lieu Lleu Lys Gly Lieu Lieu 150 155 16 O AAT, GC GAT. CTT, ATC. GGG TC CAT ACA. TAC GAT TAT GCC CGT CAT TTT O4 Asn Ala Asp Lieu. Ile Gly Phe His Thr Tyr Asp Tyr Ala Arg His Phe 1.65 17 O 175 CTA ACG TGT TGT AGT CGA ATG TTT GGT TTG GAT CAT CAG TTG AAA AGG 752 Lieu. Thir Cys Cys Ser Arg Met Phe Gly Lieu. Asp His Glin Leu Lys Arg 18O 185 190 GGG TAC ATT TTC TTG GAA. TAT. AAT GGA AGG AGC ATT GAG ATC AAG ATA 800 US 7,247,770 B2 95 96 -continued Gly Ile Phe Teu Glu Asn Gly Arg Ser Ile Glu Ile Lys Ile 195 200 2O5 210 GCG AGC GGG ATT CAT GTT GGT CGA ATG GAG TCG TAC TTG AGT CAG 848 Ala Ser Gly Ile His Wall Gly Arg Met Glu Ser Telu Ser Glin 215 220 225 GAT ACA AGA TTA CAA GTT CAA GAA CTA CGT TTC GAA GGG 896 Asp Thr Arg Teu Glin Wall Glin Glu Telu Lys Arg Phe Glu Gly 230 235 24 O ATC GTG CTA CTT GGA GTT GAT GAT TTG GAT ATA TTC GGT GTG 944 Ile Wall Telu Teu Gly Wall Asp Asp Telu Asp Ile Phe Gly Wall 245 25 O 255 AAC TTC AAG GTT TTA GCG TTG GAG AAG TTA CTT TCA CAC CCG AGT 992 Asn Phe Wall Teu Ala Teu Glu Telu Teu Lys Ser His Pro Ser 260 265 270 TGG CAA GGG CGT GTG GTT TTG GTG CAA ATC TTG AAT CCC GC CGC GCG O4. O Trp Glin Gly Arg Wall Wall Teu Wall Glin Ile Teu Asn Pro Ala Arg Ala 275 280 285 290 CGT CAA GAC GTC GAT GAG ATC AAT GCC GAG ATA AGA ACA GTC TGT Arg Glin Asp Wall Asp Glu Ile Asn Ala Glu Ile Arg Thr Wall Cys 295 3OO 305 GAA AGA ATC AAT AAC GAA CTG GGA AGC CCG GGA TAC CAG CCC GTT GTG 136 Glu Arg Ile Asn Asn Glu Teu Gly Ser Pro Gly Glin Pro Wall Wall 310 315 32O TTA ATT GAT GGG CCC GTT TCG TTA AGT GAA GCT GCT TAT TAT GCT 184 Teu Ile Asp Gly Pro Wall Ser Telu Ser Glu Ala Ala Ala 325 330 335 ATC GCC GAT ATG GCA ATT GTT ACA CCG TTA CGT GAC GGC ATG AAT CTT 232 Ile Ala Asp Met Ala Ile Wall Thr Pro Telu Arg Asp Gly Met Asn Telu 34 O 345 350 ATC CCG TAC GAG GTC GTT TCC CGA CAA AGT GTT AAT GAC CCA AAT 280 Ile Pro Glu Wall Wall Ser Arg Glin Ser Wall Asn Asp Pro Asn 355 360 365 370 CCC AAT ACT CCA AAG AGC ATG CTA GTG GTC TCC GAG TTC ATC GGG 328 Pro Asn Thr Pro Ser Met Telu Wall Wall Ser Glu Phe Ile Gly 375 38O 385 TCA CTA TCT TTA ACC GGG GCC ATA CGG GTC AAC CCA TGG GAT GAG 376 Ser Telu Ser Teu Thr Gly Ala Ile Arg Wall Asn Pro Trp Asp Glu 390 395 4 OO TTG GAG ACA GCA GAA GCA TTA TAC GAC GCA CTC ATG GCT CC GAT GAC 424 Teu Glu Thr Ala Glu Ala Teu Tyr Asp Ala Teu Met Ala Pro Asp Asp 405 410 415 CAT GAA ACC GCC CAC ATG CAG TAT CAA TAC ATT ATC TCC CAT 472 His Lys Glu Thr Ala His Met Glin Tyr Glin Tyr Ile Ile Ser His 420 4.25 430 GAT GTA GCT AAC TGG GCT CGT AGC TTC TTT CAA GAT TTA GAG CAA GCG Asp Wall Ala Asn Trp Ala Arg Ser Phe Phe Glin Asp Teu Glu Glin Ala 435 4 40 4 45 450 ATC GAT CAT TCT CGT CGA ATG AAT TTA GGA TT GGG TTA 568 Ile Asp His Ser Arg Arg Met Asn Teu Gly Phe Gly Telu 455 460 465 GAT ACT AGA GTC GTT CTT TTT GAT GAG AAG TTT AGC AAG TTG GAT ATA 616 Asp Thr Arg Wall Wall Teu Phe Asp Glu Lys Phe Ser Telu Asp Ile 470 475 48O GAT GTC TTG GAG AAT GCT TAT TCC ATG GCT CAA AAT CGG GCC ATA CTT 664 Asp Wall Telu Glu Asn Ala Ser Met Ala Glin Asn Arg Ala Ile Telu 485 490 495 TTG GAC TAT GAC GGC ACT GTT ACT CCA TCT ATC AGT TC CCA ACT 12 Teu Asp Asp Gly Thr Wall Thr Pro Ser Ile Ser Ser Pro Thr 5 OO 505 510 US 7,247,770 B2 97 98 -continued GAA GCT GTT ATC TCC ATG ATC AAC CTG GC AAT GAT CCA AAG AAC 760 Glu Ala Wall Ile Ser Met Ile Asn Telu Cys Asn Asp Pro Lys Asn 515 52O 525 530 ATG GTG TTC ATC GTT AGT GGA CGC AGT AGA GAA AAT CTT GGC AGT TGG 808 Met Wall Phe Ile Wall Ser Gly Arg Ser Arg Glu Asn Teu Gly Ser Trp 535 540 545 TTC GGC GCG TGT GAG CCC GCC ATT GCA GCT GAG CAC GGA TAC TTT 856 Phe Gly Ala Cys Glu Pro Ala Ile Ala Ala Glu His Gly Phe 550 555 560 ATA AGG TGG GCG GGT GAT CAA GAA TGG GAA ACG GCA CG GAG AAT 904 Ile Arg Trp Ala Gly Asp Glin Glu Trp Glu Thr Ala Arg Glu Asn 565 570 575 AAT GTC GGG TGG ATG GAA ATG GCT GAG CCG GTT ATG AAT CT TAT ACA 952 Asn Wall Gly Trp Met Glu Met Ala Glu Pro Wall Met Asn Telu Thr 58O 585 590 GAA ACT ACT GAC GGT TCG TAT ATT GAA AAG GAA ACT GCA ATG GTT 2OOO Glu Thr Thr Asp Gly Ser Ile Glu Lys Glu Thr Ala Met Wall 595 600 605 610 TGG CAC TAT GAA GAT GCT GAT GAT CTT GGG TTG GAG CAG GCT AAG 2O48 Trp His Glu Asp Ala Asp Asp Telu Gly Teu Glu Glin Ala Lys 615 62O 625 GAA CTG TTG GAC CAT CTT GAA AAC GTG CTC GCT AAT GAG CCC GTT GAA 2.096 Glu Telu Telu Asp His Teu Glu Asn Wall Telu Ala Asn Glu Pro Wall Glu 630 635 64 O GTG CGA GGT CAA TAC ATT GTA GAA GTT 21.30 Wall Arg Gly Glin Ile Wall Glu Wall Lys 645 650 (2) INFORMATION FOR SEQ ID NO : 27 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 653 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi). SEQUENCE DESCRIPTION: SEQ ID NO: 27: Met His Ile Lys Asp Ala Teu Pro Ala Ala Wall Glu Wall Phe Tyr Wall 5 10 15 Ala Telu Arg Ala Asp Wall Gly Pro Thr Glu Glin Asp Asp Wall Ser 25 3O Thr Telu Telu Asp Arg Phe Asn Wall Ala Wall Phe Wall Pro Thr 35 40 45 Ser Lys Trp Asp Glin Tyr His Phe Cys Lys Glin Telu Trp 5 O 55 60 Pro Ile Phe His Tyr Lys Wall Pro Ser Asp Wall Ser Wall Pro 65 70 75 Asn Ser Arg Asp Ser Trp Asn Ala Wall His Wall Asn Glu Phe 85 90 95 Ser Glin Wall Met Glu Ala Wall Thr Asn Ala Ser Asn Tyr Wall Trp 100 105 110 Ile His Asp His Teu Met Thr Telu Pro Thr Phe Teu Arg Arg Asp 115 120 125 Phe Cys Arg Lys Ile Gly Phe Phe Telu His Ser Pro Phe Pro Ser 130 135 1 4 0 Ser Glu Wall Lys Thr Teu Pro Met Arg Asn Glu Teu Telu Gly 145 15 O 155 160 Teu Telu Asn Ala Teu Ile Gly Phe His Thr Asp Ala Arg US 7,247,770 B2 99 100 -continued 1.65 170 175 His Phe Telu Thr Cys Cys Ser Met Phe Gly Teu Asp His Glin Telu 18O 185 19 O Arg Gly Tyr Ile Phe Teu Glu Tyr Asn Gly Ser Ile Glu Ile 195 200 2O5 Ile Ala Ser Gly Ile His Wall Gly Arg Met Glu Ser Tyr Telu 210 215 220 Ser Glin Pro Asp Thr Arg Teu Glin Wall Glin Glu Teu Arg Phe 225 230 235 240 Glu Gly Lys Ile Wall Teu Teu Gly Wall Asp Asp Teu Asp Ile Phe 245 250 255 Gly Wall Asn Phe Lys Wall Teu Ala Telu Glu Teu Teu Lys Ser His 260 265 27 O Pro Ser Trp Glin Gly Wall Wall Telu Wall Glin Ile Teu Asn Pro Ala 275 280 285 Arg Ala Arg Glin Wall Asp Glu Ile Asn Ala Glu Ile Thr 29 O 295 Wall Glu Arg Ile Asn Asn Glu Telu Gly Ser Pro Gly Tyr Glin Pro 305 310 315 320 Wall Wall Telu Ile Asp Gly Pro Wall Ser Telu Ser Glu Ala Ala 325 330 335 Ala Ile Ala Asp Met Ala Ile Wall Thr Pro Teu Arg Asp Gly Met 340 345 35 O Asn Telu Ile Pro Tyr Glu Tyr Wall Wall Ser Arg Glin Ser Wall Asn Asp 355 360 365 Pro Asn Pro Asn Thr Pro Lys Lys Ser Met Teu Wall Wall Ser Glu Phe 370 375 Ile Gly Cys Ser Teu Ser Teu Thr Gly Ala Ile Arg Wall Asn Pro Trp 385 390 395 400 Asp Glu Telu Glu Thr Ala Glu Ala Telu Tyr Asp Ala Teu Met Ala Pro 405 410 415 Asp Asp His Lys Glu Thr Ala His Met Lys Glin Glin Tyr Ile Ile 420 425 43 O Ser His Asp Wall Ala Asn Trp Ala Arg Ser Phe Phe Glin Asp Telu Glu 435 4 40 4 45 Glin Ala Ile His Ser Arg Lys Met Asn Telu Gly Phe 450 455 460 Gly Telu Asp Thr Wall Wall Telu Phe Glu Phe Ser Lys Telu 465 470 475 480 Asp Ile Asp Wall Teu Glu Asn Ala Tyr Ser Met Ala Glin Asn Arg Ala 485 490 495 Ile Telu Telu Asp Tyr Gly Thr Wall Thr Pro Ser Ile Ser Lys Ser 5 OO 505 Pro Thr Glu Ala Wall Ile Ser Met Ile Asn Teu Cys Asn Asp Pro 515 525 Asn Met Wall Phe Ile Wall Ser Gly Arg Ser Arg Glu Asn Telu Gly 530 535 540 Ser Trp Phe Gly Ala Cys Glu Lys Pro Ala Ile Ala Ala Glu His Gly 545 550 555 560 Phe Ile Arg Trp Ala Gly Glin Glu Trp Glu Thr Ala Arg 565 570 575 Glu Asn Asn Wall Gly Trp Met Glu Met Ala Glu Pro Wall Met Asn Telu 58O 585 59 O US 7,247,770 B2 101 102 -continued Thr Glu Thr Thr Asp Gly Ser Tyr Ile Glu Lys Lys Glu Thr Ala 595 600 605 Met Wall Trp His Tyr Glu Asp Ala Asp Lys Asp Leu Gly Lieu Glu Glin 610 615 Ala Lys Glu Lieu Lleu. Asp His Telu Glu Asn Wall Teu Ala Asn Glu Pro 625 630 635 640 Wall Glu Wall Lys Arg Gly Glin Tyr Ile Wall Glu Wall Lys 645 650 (2) INFORMATION FOR SEQ ID NO : 28 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 390 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS double (D) TOPOLOGY linear (ii) MOLECULE TYPE cDNA to mRNA (iii) HYPOTHETICAL NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Helianthus annuus (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 3.258 (D) OTHER INFORMATION: /partial (xi). SEQUENCE DESCRIPTION: SEQ ID NO: 28: TT GCA GAG AAG ATT TTT GCG TTC ATG GCT GAA. AAG GGA AAA CAG GCT 47 Ala Glu Lys Ile Phe Ala Phe Met Ala Glu Lys Gly Lys Glin Ala 1 5 10 15 GAT TTC GTG TTG. AGC GTT GGA GAT GAT AGA AGT GAT GAA GAC ATG TTT 95 Asp Phe Wall Teu Ser Wall Gly Asp Asp Arg Ser Asp Glu Asp Met Phe 2O 25 30 GTG GCC ATT GGG GAT GGA ATA AAG GGT CGG ATA ACT AAC AAC AAT 1 4 3 Wall Ala Ile Gly Asp Gly Ile Lys Gly Arg Ile Thr Asn Asn. Asn 35 40 45 TCA GTG TTT ACA TGC GTA GTG GGA GAG AAA CCG AGT GCA GC GAG TAC 191 Ser Wall Phe Thr Cys Wall Wall Gly Glu Lys Pro Ser Ala Ala Glu Tyr 5 O 55 60 TTT TTA GAC GAG ACG AAA GAT GTT TCA ATG ATG CTC GAG AAG CTC. GGG 239 Phe Telu Asp Glu Thr Lys Asp Wall Ser Met Met Teu Glu Lys Leu Gly 65 70 75 CTC AGC AAC CAA GGA T GATGATCCGG AAGCTTCTCG TGATCTTTAT 288 Telu Ser Asn Glin Gly 85 GAGTTAAAAG TTTTCGACTT TTTCTTCATC AAGATTCATG GGAAAGTTGT, TCAATATGAA 348 CTTGGTTTC TTGGTTCTGG ATTTTAGGGA GTCTAGGAT CC 39 O. (2) INFORMATION FOR SEQ ID NO: 29 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 85 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi). SEQUENCE DESCRIPTION: SEQ ID NO : 29: Ala Glu Lys Ile Phe Ala Phe Met Ala Glu Lys Gly Lys Glin Ala Asp US 7,247,770 B2 103 104 -continued 5 10 15 Phe Wall Leu Ser Val Gly Asp Asp Arg Ser Asp Glu Asp Met Phe Wall 25 Ala Ile Gly Asp Gly Ile Lys Lys Gly Arg Ile Thr Asn Asn Asn. Ser 35 40 45 Wall Phe Thr Cys Val Val Gly Glu Lys Pro Ser Ala Ala Glu Tyr Phe 5 O 55 60 Leu Asp Glu Thir Lys Asp Val Ser Met Met Leu Glu Lys Telu Gly Cys 65 70 75 8O Teu Ser Asn Glin Gly 85 (2) INFORMATION FOR SEQ ID NO: 30: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE cDNA (iii) HYPOTHETICAL NO (iv) ANTI-SENSE: NO (ix) FEATURE: (A) NAM E/KEY: modified base Inosine (B) LOCATION: 4 (D) OTH ER INFORMATION: /note= N is Inosine (ix) FEATURE: (A) NAM E/KEY: modified base Inosine (B) LOCATION: 10 (D) OTH ER INFORMATION: /note= N is Inosine (ix) FEATURE: (A) NAM E/KEY: modified base Inosine (B) LOCATION: 13 (D) OTH ER INFORMATION: /note= N is Inosine (ix) FEATURE: (A) NAM E/KEY: modified base Inosine (B) LOCATION: 19 (D) OTH ER INFORMATION: /note= N is Inosine (ix) FEATURE: (A) NAM E/KEY: modified base Inosine (B) LOCATION: 22 (D) OTH ER INFORMATION: /note= N is Inosine (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 30: CCANGGRTTN ACNCK DNTNG CNCC 24 (2) INFORMATION FOR SEQ ID NO: 31: (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE cDNA (iii) HYPOTHETICAL NO (iv) ANTI-SENSE: NO (ix) FEATURE: (A) NAME/KEY: modified base, Inosine (B) LOCATION: 6 US 7,247,770 B2 105 106 -continued (D) OTHER INFORMATION: /note= A is Inosine (ix) FEATURE: (A) NAME/KEY: modified base, Inosine (B) LOCATION: 9 (D) OTHER INFORMATION: /note= A is Inosine (ix) FEATURE: (A) NAME/KEY: modified base, Inosine (B) LOCATION: 12 (D) OTHER INFORMATION: /note= A is Inosine (ix) FEATURE: (A) NAME/KEY: modified base, Inosine (B) LOCATION: 18 (D) OTHER INFORMATION: /note= A is Inosine (ix) FEATURE: (A) NAME/KEY: modified base, Inosine (B) LOCATION: 21 (D) OTHER INFORMATION: /note= A is Inosine (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 31: ATHGTNGTNW SNAAYMRNYT NCC 23 (2) INFORMATION FOR SEQ ID NO: 32: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLEC ULE TYPE cDNA (iii) HYPOT HETICAL NO (iv) ANTI SENSE: NO (ix) FEATU RE (A) NAME/KEY: modified base Inosine (B) LOCATION: 3 (D) OTHER INFORMATION: Wnote= N is Inosine (ix) FEATU RE (A) NAME/KEY: modified base Inosine (B) LOCATION: 9 (D) OTHER INFORMATION: Wnote= N is Inosine (ix) FEATU RE (A) NAME/KEY: modified base Inosine (B) LOCATION: 12 (D) OTHER INFORMATION: Wnote= N is Inosine (xi). SEQUENCE DESCRIPTION: SEQ ID NO: 32: YTNTGGCCNA TNTTYCAYTA 20 (2) INFORMATION FOR SEQ ID NO: 33: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLEC ULE TYPE cDNA (iii) HYPOT HETICAL NO (iv) ANTI SENSE: NO (ix) FEATU RE (A) NAME/KEY: modified base Inosine (B) LOCATION 6 (D) OTHER INFORMATION: Wnote= N is Inosine US 7,247,770 B2 107 108 -continued (ix) FEATURE: (A) NAME/KEY: modified base, Inosine (B) LOCATION: 9 (D) OTHER INFORMATION: /note= N is Inosine (ix) FEATURE: (A) NAME/KEY: modified base, Inosine (B) LOCATION: 18 (D) OTHER INFORMATION: /note= N is Inosine (xi). SEQUENCE DESCRIPTION: SEQ ID NO : 33: TGRTCNARNA RYCYTNGC 20 (2) INFORMATION FOR SEQ ID NO: 34: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE cDNA (iii) HYPOTHETICAL NO (iv) ANTI-SENSE: NO (ix) FEATURE: (A) NAME/KEY: modified base, Inosine (B) LOCATION: 6 (D) OTHER INFORMATION: /note= N is Inosine (ix) FEATURE: (A) NAME/KEY: modified base, Inosine (B) LOCATION: 18 (D) OTHER INFORMATION: /note= N is Inosine (xi). SEQUENCE DESCRIPTION: SEQ ID NO: 34: TCRTCNGTRA ARTCRTCNCC 20 (2) INFORMATION FOR SEQ ID NO: 35: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE cDNA (iii) HYPOTHETICAL NO (iv) ANTI-SENSE: NO (ix) FEATURE: (A) NAME/KEY: modified base, Inosine (B) LOCATION: 15 (D) OTHER INFORMATION: /note= N is Inosine (ix) FEATURE: (A) NAME/KEY: modified base, Inosine (B) LOCATION: 18 (D) OTHER INFORMATION: /note= N is Inosine (xi). SEQUENCE DESCRIPTION: SEQ ID NO: 35: TTYGAYTAYG AYGGNACNYT 20 (2) INFORMATION FOR SEQ ID NO: 36: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid US 7,247,770 B2 109 110 -continued (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE cDNA (iii) HYPOTHETICAL NO (iv) ANTI-SENSE: NO (ix) FEATURE: (A) NAME/KEY: modified base, Inosine (B) LOCATION: 3 (D) OTHER INFORMATION: /note= N is Inosine (ix) FEATURE: (A) NAME/KEY: modified base, Inosine (B) LOCATION: 6 (D) OTHER INFORMATION: /note= N is Inosine (ix) FEATURE: (A) NAME/KEY: modified base, Inosine (B) LOCATION: 12 (D) OTHER INFORMATION: /note= N is Inosine (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 36: GGNYTNWBNG CNGARCAYGG 20 (2) INFORMATION FOR SEQ ID NO: 37: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLEC ULE TYPE cDNA (iii) HYPOT HETICAL NO (iv) ANTI SENSE: NO (ix) FEATU RE (A) NAME/KEY: modified base Inosine (B) LOCATION: 3 (D) OTHER INFORMATION: Wnote= N is Inosine (ix) FEATU RE (A) NAME/KEY: modified base Inosine (B) LOCATION: 6 (D) OTHER INFORMATION: Wnote= N is Inosine (ix) FEATU RE (A) NAME/KEY: modified base Inosine (B) LOCATION: 12 (D) OTHER INFORMATION: Wnote= N is Inosine (ix) FEATU RE (A) NAME/KEY: modified base Inosine (B) LOCATION: 15 (D) OTHER INFORMATION: Wnote= N is Inosine (xi). SEQUENCE DESCRIPTION: SEQ ID NO : 37: ATNGCNAARC CNGTNATGAA 20 (2) INFORMATION FOR SEQ ID NO: 38: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO US 7,247,770 B2 111 112 -continued (iv) ANTI-SENSE: NO (ix) FEATURE: (A) NAME/KEY: modified base, Inosine (B) LOCATION: 3 (D) OTHER INFORMATION: /note= N is Inosine (ix) FEATURE: (A) NAME/KEY: modified base, Inosine (B) LOCATION: 6 (D) OTHER INFORMATION: /note= N is Inosine (ix) FEATURE: (A) NAME/KEY: modified base, Inosine (B) LOCATION: 12 (D) OTHER INFORMATION: /note= N is Inosine (ix) FEATURE: (A) NAME/KEY: modified base, Inosi le (B) LOCATION: 18 (D) OTHER INFORMATION: /note= N is Inosine (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 38 CCNACNGTRC ANGCRAANAC 20 (2) INFORMATION FOR SEQ ID NO: 39: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2982 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE cDNA to mRNA (iii) HYPOTHETICAL NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Arabidopsis thaliana (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 64. .2889 (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 39 ATAAACTTCC TCGCGGCCGC CAGGGAGT AATTTAGTTT TGGTTCTGTT TTGGGGAG 60 CGT ATG CCT GGA AAT AAG TAC AAC TGC AGT, TCT TCT CAT ATC CCA CTC 108 Met Pro Gly Asn Lys Tyr Asn. Cys Ser Ser Ser His Ile Pro Leu 1 5 10 15 TCT CGA ACA GAA CGC CTC TTG. AGA. GAT AGA. GAG CTT AGA GAG AAG AGG 15 6 Ser Arg Thr Glu Arg Lieu Lleu Arg Asp Arg Glu Teu Arg Glu Lys Arg 2O 25 30 AAG AGC AAC CGA GCT CGT AAT CCT AAT GAC GTT GCT GGC AGT TCC GAG 204 Ser Asn Arg Ala Arg Asn Pro Asn Asp Wall Ala Gly Ser Ser Glu 35 40 45 AAC TCT GAG AAT GAC TTG CGT TTA. GAA. GGT GAC AGT TCA AGG CAG TAT 252 Asn Ser Glu Asn Asp Leu Arg Lieu Glu Gly Asp Ser Ser Arg Gln Tyr 5 O 55 60 GTT GAA CAG TAC TTG GAA. GGG GCT, GCT, GCT, GCA ATG GCG CAC GAT GAT Wall Glu Glin Tyr Lieu Glu Gly Ala Ala Ala Ala Met Ala His Asp Asp 65 70 75 GCG GAG. AGG CAA. GAA GTT AGG CCT TAT. AAT AGG CAA CGA CTA CTT 348 Ala Glu Arg Glin Glu Val Arg Pro Tyr Asn Arg Glin Arg Telu Telu 8O 85 90 95 GTA GTG GCT. AAC AGG CTC CCA GTT T.C.T CCC GTG AGA AGA GGT GAA. GAT 396 Wall Wall Ala Asn Arg Leu Pro Val Ser Pro Val Arg Arg Gly Glu Asp